July 2023
Volume 64, Issue 10
Open Access
Lens  |   July 2023
ErbBs in Lens Cell Fibrosis and Secondary Cataract
Author Affiliations & Notes
  • Judy K. VanSlyke
    Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, Oregon, United States
  • Bruce A. Boswell
    Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, Oregon, United States
  • Linda S. Musil
    Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, Oregon, United States
  • Correspondence: Linda S. Musil, Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, 3181 Southwest Sam Jackson Park Road, Portland, OR, USA; musill@ohsu.edu
Investigative Ophthalmology & Visual Science July 2023, Vol.64, 6. doi:https://doi.org/10.1167/iovs.64.10.6
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      Judy K. VanSlyke, Bruce A. Boswell, Linda S. Musil; ErbBs in Lens Cell Fibrosis and Secondary Cataract. Invest. Ophthalmol. Vis. Sci. 2023;64(10):6. https://doi.org/10.1167/iovs.64.10.6.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: TGFβ-induced epithelial-to-myofibroblast transition (EMyT) of lens cells has been linked to the most common vision-disrupting complication of cataract surgery—namely, posterior capsule opacification (PCO; secondary cataract). Although inhibitors of the ErbB family of receptor tyrosine kinases have been shown to block some PCO-associated processes in model systems, our knowledge of ErbB signaling in the lens is very limited. Here, we investigate the expression of ErbBs and their ligands in primary cultures of chick lens epithelial cells (dissociated cell-derived monolayer cultures [DCDMLs]) and how TGFβ affects ErbB function.

Methods: DCDMLs were analyzed by immunofluorescence microscopy and Western blotting under basal and profibrotic conditions.

Results: Small-molecule ErbB kinase blockers, including the human therapeutic lapatinib, selectively inhibit TGFβ-induced EMyT of DCDMLs. Lens cells constitutively express ErbB1 (EGFR), ErbB2, and ErbB4 protein on the plasma membrane and release into the medium ErbB-activating ligand. Culturing DCDMLs with TGFβ increases soluble bioactive ErbB ligand and markedly alters ErbBs, reducing total and cell surface ErbB2 and ErbB4 while increasing ErbB1 expression and homodimer formation. Similar, TGFβ-dependent changes in relative ErbB expression are induced when lens cells are exposed to the profibrotic substrate fibronectin. A single, 1-hour treatment with lapatinib inhibits EMyT in DCDMLs assessed 6 days later. Short-term exposure to lower doses of lapatinib is also capable of eliciting a durable response when combined with suboptimal levels of a mechanistically distinct multikinase inhibitor.

Conclusions: Our findings support ErbB1 as a therapeutic target for fibrotic PCO, which could be leveraged to pharmaceutically preserve the vision of millions of patients with cataracts.

The lens consists of a monolayer of epithelial cells on the anterior face of the organ and the highly elongated, crystallin-rich lens fiber cells that differentiate from them.1 The cells of the lens are encased in the acellular lens capsule,2 which has been referred to as the thickest basement membrane in the body.3 Cataracts, defined as a loss of transparency of the lens, remain a leading cause of blindness worldwide.46 The only treatment for most cataracts, including those associated with older age, is surgery. During this procedure, the cells of the cloudy natural lens are disrupted by ultrasonication, aspirated from the lens capsule, and replaced with an artificial lens (IOL) that is implanted within the capsule. The most common vision-disrupting complication of this operation is posterior capsule opacification (PCO; also referred to as secondary cataract), which results from incomplete removal of the lens epithelial cells during cataract surgery.712 Surviving cells can migrate to the posterior of the lens capsule and undergo abnormal differentiation into either myofibroblasts or lens fiber–like cells. Accumulation of these light-scattering elements in the visual axis interferes with transmission of light to the retina. Children are particularly susceptible to PCO, which can result in irreversible loss of vision due to the development of amblyopia.13,14 Despite extensive efforts over more than 30 years, no pharmacologic approach to prevent the development of PCO has entered clinical use.15 
The growth factor most closely linked to the etiology of PCO is TGFβ, the signaling of which is increased shortly after cataract surgery as part of the wounding response.1618 Addition of TGFβ to primary lens epithelial cell systems from several species, including mouse, rat, chicken, and humans, induces an epithelial-to-myofibroblast transition (EMyT) to a myofibroblastic phenotype.19 These systems include dissociated cell-derived monolayer cultures (DCDMLs), which consist of serum-free, unpassaged lens epithelial cells prepared from E10 chick embryos and plated onto laminin, a major component of the natural lens capsule.20 TGFβ induces other lens epithelial cells in DCDMLs to undergo differentiation into lens fiber–like cells.21,22 Exogenous expression of active TGFβ in the rodent lens in vivo also causes some lens epithelial cells to become myofibroblasts and others in the same region to acquire markers of lens fiber cells.23,24 Although several types of physiologically relevant growth factors can stimulate lens fiber cell differentiation in primary lens epithelial cells (e.g., FGF, BMP, IGF1), TGFβ and its downstream effector, the matricellular protein connective tissue growth factor (CCN2, CTGF), are the only factors known to induce EMyT in lens epithelial cells.19 
The ErbB family of cell surface integral membrane proteins includes three functional kinases—ErbB1 (EGFR), ErbB2 (also known as HER2), and ErbB4—that form homo- or heterodimers. Each type of ErbB dimer can mediate distinct biological functions due in part to differences in the ligands to which they bind, their ability to engage different downstream signaling effectors, and their sensitivity to ligand-induced internalization and/or degradation.2530 The ErbB family kinase inhibitors AG1478, erlotinib, and gefitinib have been shown to reduce cell coverage on the posterior capsule of explanted human lens capsular bags.3133 There is also a report that small interfering RNA knockdown of ErbB1 has a similar effect in an in vivo rat PCO model.34 More recently, the ErbB kinase inhibitor PD153035 was reported to block induction of EMyT by TGFβ in primary explants of postnatal rat lens epithelium.35 
In addition to being essential for normal development, ErbBs are important therapeutic targets for several forms of cancer.3638 The widespread clinical use of ErbB inhibitors and the lack of ocular toxicity of a least one such drug (lapatinib) raise the possibility that such proven therapeutics could be repurposed to combat PCO. A major obstacle toward attaining this goal is the general lack of knowledge of ErbBs in the lens. In this study, we use our DCDML system to study ErbB kinase expression and signaling in lens epithelial cells to investigate the role of individual ErbB family members in lens cell fibrosis. Our findings provide new insights into how the TGFβ and ErbB pathways interact and how the latter could best be leveraged to block the development of fibrotic PCO. 
Methods
Materials
Recombinant human TGFβ1, TGFα, HB-EGF, NRG1(EGF domain; #396-HB-050), and bovine FGF2 were from R&D Systems (Minneapolis, MN, USA). R3IGF-1, an analogue of human insulin-like growth factor 1, was from GroPep (Adelaide, Australia). Mouse laminin (#23017015) and bovine plasma fibronectin (#33010018) were from Invitrogen (Carlsbad, CA, USA). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): anti–phospho-p44/42 MAP kinase (#9106), anti–total p44/42 MAPK (#9102), anti–phospho-p38 (#9211), and anti–phospho (Ser473) AKT (#9275). The following antibodies against the cytoplasmic tail domains were used to detect ErbBs in DCDMLs: for ErbB1, rabbit SC-03 from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the rat monoclonal 20.3.639 (the latter a kind gift of Dr. Michael Hayman, Stonybrook University); for ErbB4, C18 (Santa Cruz Biotechnology); and for ErbB2, A0485 from Agilent (Santa Clara, CA, USA). Two antibodies specific for the tyrosine 1068 autophosphorylated, activated form of ErbB1 were used, a rabbit polyclonal (#2234) and a mouse monoclonal (#2236), both from Cell Signaling Technology. Antibodies used to detect ErbBs only in mammalian cells include mouse monoclonal anti-ErbB2 (clone 3B5 from EMD Millipore) and CPTC-EGFR1 (deposited to the Developmental Studies Hybridoma Bank by Clinical Proteomics Technologies for Cancer). Other commercial antibodies used in this study included the following: for luciferase, G745A from Promega Corp. (Madison, WI, USA); for green fluorescent protein (GFP), JL-8 (Clonetech, Mountainview, CA, USA); for phospho-Smad3, ab51451 from Abcam (Cambridge, MA, USA); for total p38, sc-535 from Santa Cruz Biotechnology; for α-tubulin, T5168 from Sigma (St. Louis, MO, USA); for αSMA, clone 1A4 from Agilent; for β-actin, clone C4 (MilliporeSigma, Billerica, MA, USA); for anti-phosphotyrosine, clone 4G10 (MilliporeSigma); and for chick fibronectin, B3/D6 (from D. Fambrough, Johns Hopkins University; Developmental Studies Hybridoma Bank, University of Iowa). Rabbit anti-mouse CP49 polyclonal serum (#899 or #900) was a generous gift of Paul FitzGerald, University of California, Davis, as was the rabbit anti-CP115 antiserum (#76). Rabbit anti-chicken AQP0 antibodies were from Ross Johnson, University of Minnesota. Lapatinib and erlotinib were from LC Labs (Woburn, MA, USA), and SB-431542 was from Calbiochem (La Jolla, CA, USA). Rebastinib (DCC-2036) was from Selleckchem (Houston, TX, USA). 
DCDML Cell Culture and Treatments
DCDML cultures were prepared from E10 chick lenses as previously described.40 During this process, cells exterior to the lens capsule are removed and mature lens fiber cells die, leaving a preparation of purified lens epithelial cells. Unless indicated otherwise, cells were plated at a subconfluent density (0.9 × 105 cells/well) onto laminin-coated 96-well tissue culture plates and cultured in the absence of serum in 200 µL M199 medium, penicillin G, and streptomycin at 37°C in a 5% CO2 incubator. In the experiments shown in Figures 1A and 1B, Supplementary Figure S1, and Figure 2, 100 µL/well medium was supplemented with BOTS (2.5 µg/mL bovine serum albumin, 25 µg/mL ovotransferrin, 300 nM selenium) to increase the extent of fiber cell differentiation. Cells were fed every 2 days with fresh medium. We refer to these cultures as DCDMLs to distinguish them from related, but functionally distinct, systems such as central epithelial explants and immortalized lens-derived cell lines.20 Where indicated, DCDMLs were plated on 12.5 µg/mL bovine plasma fibronectin instead of laminin.22 Unless indicated otherwise, drugs were used at the following final concentrations: 4 µM lapatinib, 5 µM erlotinib, 3 µM SB-431542, and 0.5 µM rebastinib. 
Figure 1.
 
TGFβ-induced upregulation of EMyT in lens DCDMLs requires ErbB signaling. (A–C) DCDML cultures of primary lens epithelial cells preincubated for 1 hour with DMSO (vehicle control), the ErbB kinase inhibitor lapatinib or erlotinib, or the specific TGFβ receptor inhibitor SB-431542 (SB4) as indicated were cultured from days 1 to 7 in the presence of TGFβ. Controls were cultured in DMSO only. (A) Cells were then processed for immunofluorescence detection of αSMA and AQP0. Hoechst staining of nuclei is also shown. Typical of four experiments. (B, C) Western blotting of whole-cell lysates of DCDMLs cultured in either 100 µL/well M199/BOTS medium (B) or 200 µL/well M199 medium (C) for FN or αSMA. (D) Results from experiments shown in B and C were quantitated as fold inhibition relative to DMSO + TGFβ controls in the same experiment. For all conditions, P = 0.000. (E) DCDMLs were plated on bovine pdFN and cultured from days 1 to 7 with DMSO or lapatinib prior to analysis of cell lysates for FN or αSMA. Results expressed as percent inhibition by lapatinib compared to DMSO only (n = 3; P ≤ 0.001 for both myofibroblast markers). Note that the avian-specific anti-FN antibody used for Western blotting does not recognize bovine pdFN. For comparison, data are shown for DCDMLs plated under standard conditions (e.g., on laminin [LM]) and treated with DMSO only. (F) DCDMLs were cultured from days 1 to 7 with no additions (ctrl), TGFβ, or 10 nM HB-EGF, TGFα, or NRG1. None of the ErbB ligands increased the expression of FN or αSMA relative to untreated controls in four of four experiments. Similar results were obtained with 1 nM of each ErbB ligand (n = 3).
Figure 1.
 
TGFβ-induced upregulation of EMyT in lens DCDMLs requires ErbB signaling. (A–C) DCDML cultures of primary lens epithelial cells preincubated for 1 hour with DMSO (vehicle control), the ErbB kinase inhibitor lapatinib or erlotinib, or the specific TGFβ receptor inhibitor SB-431542 (SB4) as indicated were cultured from days 1 to 7 in the presence of TGFβ. Controls were cultured in DMSO only. (A) Cells were then processed for immunofluorescence detection of αSMA and AQP0. Hoechst staining of nuclei is also shown. Typical of four experiments. (B, C) Western blotting of whole-cell lysates of DCDMLs cultured in either 100 µL/well M199/BOTS medium (B) or 200 µL/well M199 medium (C) for FN or αSMA. (D) Results from experiments shown in B and C were quantitated as fold inhibition relative to DMSO + TGFβ controls in the same experiment. For all conditions, P = 0.000. (E) DCDMLs were plated on bovine pdFN and cultured from days 1 to 7 with DMSO or lapatinib prior to analysis of cell lysates for FN or αSMA. Results expressed as percent inhibition by lapatinib compared to DMSO only (n = 3; P ≤ 0.001 for both myofibroblast markers). Note that the avian-specific anti-FN antibody used for Western blotting does not recognize bovine pdFN. For comparison, data are shown for DCDMLs plated under standard conditions (e.g., on laminin [LM]) and treated with DMSO only. (F) DCDMLs were cultured from days 1 to 7 with no additions (ctrl), TGFβ, or 10 nM HB-EGF, TGFα, or NRG1. None of the ErbB ligands increased the expression of FN or αSMA relative to untreated controls in four of four experiments. Similar results were obtained with 1 nM of each ErbB ligand (n = 3).
Figure 2.
 
ErbB inhibition does not block TGFβ-induced lens fiber cell differentiation or Smad3 signaling. (A, B) DCDMLs preincubated for 1 hour with DMSO, lapatinib, or erlotinib were cultured with or without TGFβ for 6 days (A) or 90 minutes (B) prior to analysis of the fiber cell markers δ-crystallin, CP115, and CP49 (A) or activation of Smad3 (B) by either metabolic labeling (δ-crystallin)40,44 or Western blot (CP115, CP49; pSmad3). (C) DCDMLs were transfected with the SBE4-Luc reporter construct on day 1 of culture and then incubated on day 2 for 1 hour with DMSO, lapatinib, erlotinib, or the TGFβR inhibitor SB-431542. The cells were then cultured for an additional 48 hours with no additions (0) or TGFβ prior to Western blot analysis of luciferase expression. (D) Results for lapatinib and erlotinib from experiments shown in A to C were quantitated as fold inhibition relative to TGFβ + DMSO controls in the same experiment. In no case did ErbB inhibitors significantly (P < 0.05) reduce the ability of TGFβ to upregulate expression of the indicated protein.
Figure 2.
 
ErbB inhibition does not block TGFβ-induced lens fiber cell differentiation or Smad3 signaling. (A, B) DCDMLs preincubated for 1 hour with DMSO, lapatinib, or erlotinib were cultured with or without TGFβ for 6 days (A) or 90 minutes (B) prior to analysis of the fiber cell markers δ-crystallin, CP115, and CP49 (A) or activation of Smad3 (B) by either metabolic labeling (δ-crystallin)40,44 or Western blot (CP115, CP49; pSmad3). (C) DCDMLs were transfected with the SBE4-Luc reporter construct on day 1 of culture and then incubated on day 2 for 1 hour with DMSO, lapatinib, erlotinib, or the TGFβR inhibitor SB-431542. The cells were then cultured for an additional 48 hours with no additions (0) or TGFβ prior to Western blot analysis of luciferase expression. (D) Results for lapatinib and erlotinib from experiments shown in A to C were quantitated as fold inhibition relative to TGFβ + DMSO controls in the same experiment. In no case did ErbB inhibitors significantly (P < 0.05) reduce the ability of TGFβ to upregulate expression of the indicated protein.
Cell Lines
Human kidney HEK 293 cells were from the American Type Culture Collection (ATCC; Manassas, VA, USA). The N/N1003A rabbit41 and the FHL-124 human42 lens epithelial cell lines were kindly provided by Dr. John Reddan (Oakland University) and cultured in MEM-Earles with glutamine and gentamycin sulfate, supplemented with either 8% normal rabbit serum (N/N1003A cells) or 10% heat-inactivated fetal calf serum and Na pyruvate (FHL-124 cells). 
Plasmids and Transient Transfection
One day after plating, DCDML cultures were transfected in M199 medium without BOTS or antibiotics using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). following the manufacturer's suggested protocol. Control experiments confirmed that the efficiency of transient transfection of DCDMLs is consistently ∼70%.43 The SBE4-Luc reporter construct was provided by Bert Vogelstein (Johns Hopkins University; Addgene plasmid #16495). pcDNA3 plasmids encoding the following ErbB species were obtained from Addgene (Watertown, MA, USA): ErbB2-EGFP (#39321), originally from Martin Offterdinger; EGFR-GFP (#32751) from Alexander Sorkin; and ErbB4 (#29527) from Yardena Samuels. 
Immunofluorescence Microscopy
DCDMLs grown on glass coverslips were fixed in 2% paraformaldehyde in PBS and processed as previously described.40,44 Images were captured using a Leica (Teaneck, NJ, USA) DM LD photomicrography system and Scion (Frederick, Maryland, USA) Image 1.60 software. 
Cell Surface Biotinylation
DCDMLs were chilled on ice before gently rinsing four times with cold PBS wash buffer (PBS with 1 mM MgCl2 and 0.5 mM CaCl2). Cells were incubated with freshly prepared 0.25 mg/mL EZ-Link Sulfo-NHS-SS-Biotin (Thermoscientific #21331, Thermo Fisher Scientific, Waltham, MA, USA) in PBS wash buffer for 15 minutes on ice with constant rocking. The reaction was quenched with five rinses of cold PBS wash buffer supplemented with 0.1 M glycine (pH 7.6). Cells were lysed in 5 mM Tris-HCl, 5 mM EDTA, 5 mM EGTA, 0.6% SDS supplemented with 25 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mg/mL leupeptin, 0.25 mg/mL soybean trypsin inhibitor, and 20 µM Na orthovanadate (pH 8.0) and then boiled for 3 minutes. After removal of 5% of the lysate for quantitation of β-actin and/or α-tubulin, the remainder was diluted with immunoprecipitation buffer plus 1.2% Triton X-100 to achieve a final 5:1 ratio of Triton to SDS. Biotinylated proteins were incubated with streptavidin agarose beads with rotation overnight at 4°C. Beads were washed with immunoprecipitation buffer as previously described,45 after which biotinylated proteins were eluted by boiling for 3 minutes in SDS-PAGE loading buffer containing 2% β-mercaptoethanol prior to analysis by Western blot. 
Detection of ErbB Ligand Bioactivity in Conditioned Medium
Medium was collected from DCDMLs cultured for 2 days with or without TGFβ and immediately centrifuged at 3000 rpm in a microfuge (0.8 rcf) for 5 minutes to remove any cell debris. DCDML and mock (no cell) conditioned medium were concentrated fivefold using Amicon (St Louis, MO, USA) UltraCel-0.5 3K spin filters prior to incubation with HEK cell recipients at 4°C for 15 minutes. 
Chemical Crosslinking
DCDMLs were incubated with 10 nM HB-EGF for 15 minutes at 4°C, rinsed three times with cold PBS, and then incubated for 30 minutes on ice with freshly made 1 mM dithiobis-(sulfosuccinimdylpropionate) (DTSSP) (Thermoscientific #21578) for 30 minutes on ice with constant rocking. The reaction was quenched by rinsing three times with chilled 20 mM Tris with 100 mM NaCl, pH 7.5.46 Cultures were then subjected to cell surface biotinylation and analyzed as described above, except that β-mercaptoethanol was omitted from the SDS-PAGE loading buffer. 
Immunoblot Analysis
Whole-cell lysates were solubilized directly into SDS-PAGE sample buffer and boiled. Equal volumes of total cell lysate were transferred to polyvinylidene fluoride membranes and the blots probed with primary antibodies. Immunoreactive proteins were detected using secondary antibodies conjugated to either IRDye800 (Rockland Immunochemicals, Pottstown, PA, USA) or Alexa Fluor 680 (Molecular Probes, Eugene, OR, USA) and directly quantified using the LI-COR Biosciences Odyssey (Lincoln, NE) infrared imaging system and associated software. The level of each protein was normalized to the level of β-actin or α-tubulin in the same sample. GAPDH could not be used as a loading control because its signal on Western blots became saturated at the protein loads required for accurate quantitation of our proteins of interest. Data are graphed as the mean ± standard deviation obtained in the number of experiments indicated in each figure. Data were analyzed for significance using the two-tailed paired Student's t-test. 
Results
ErbB Signaling Is Specifically Required for TGFβ-Induced Myofibroblast Differentiation of DCDML Lens Epithelial Cells
We performed an immunofluorescence screen of clinically investigated small-molecule kinase inhibitors47 to identify novel potential drugs for PCO and to shed light on the signaling pathways that underlie this disorder. Expression of α smooth muscle actin (αSMA) in stress fibers was used as a marker for EMyT.48 Differentiation of lens epithelial cells into lens fiber cells was assessed by staining for aquaporin 0 (AQP0), a protein uniquely enriched in lens fiber cells that in culture accumulates in mound-shaped lentoid bodies composed of enlarged lens fiber–like cells.1,21 We found that multiple structurally and mechanistically distinct ErbB kinase inhibitors inhibited TGFβ-induced EMyT in DCDMLs without reducing the expression of AQP0 or the formation of lentoid bodies (Fig. 1A and Supplementary Fig. S1). Other tested drugs either did not detectably influence the ability of TGFβ to upregulate myofibroblast differentiation at nontoxic concentrations (e.g., CYC202, GW786034, JAK inhibitor 1; MLN8054) or had the converse effect in that they enhanced myofibroblast formation and reduced lens fiber cell differentiation (e.g., the mTOR inhibitor rapamycin).21 
Quantitative Western blotting confirmed that the ErbB inhibitors lapatinib and erlotinib blocked expression of αSMA and of the mesenchymal marker fibronectin (FN) in response to TGFβ (Fig. 1B). (As observed in other primary lens epithelial cells, DCDMLs express detectable levels of αSMA in the absence of exogenously added TGFβ.21) ErbB inhibitors also decreased myofibroblast differentiation when cells were cultured under conditions that favor formation of myofibroblasts over lens fibers (i.e., in 200-µL/96-well M199 medium, instead of in 100 µL M199 with the BOTS supplement) (Musil L, IOVS 2016;57:ARVO E-Abstract 1374) (Fig. 1C). 
Lens epithelial cells constitutively express latent TGFβ. We have reported that this TGFβ becomes activated when DCDMLs are cultured in the presence of plasma-derived fibronectin (pdFN) at levels lens cells would be expected to encounter during cataract surgery. The resulting chronic increase in TGFβ signaling is sufficient to induce EMyT.22 We found that formation of myofibroblasts in response to plating on pdFN was blocked by lapatinib (Fig. 1E). ErbB signaling is therefore required for myofibroblast differentiation of lens epithelial cells induced by either exogenous or endogenous sources of TGFβ. 
Additional experiments demonstrated that culturing DCDMLs with the ErbB ligands TGFα, HB-EGF, or NRG1 in the absence of TGFβ failed to induce the expression of either αSMA or FN (Fig. 1F); data showing that DCDMLs respond to these ligands are presented in Figure 3. EGF was not tested due to the inability of mammalian forms to efficiently activate avian ErbB1.49,50 Thus, ErbB signaling is necessary, but not sufficient, to induce myofibroblast differentiation of lens epithelial cells. 
Figure 3.
 
DCDMLs are responsive to ErbB1 and/or ErbB4 ligands. DCDMLs were incubated for 5 minutes with the ErbB ligands HB-EGF (1 nM), TGFα (1 nM), or NRG1 (10 nM), with or without a 1-hour pretreatment with lapatinib (lap). Whole-cell lysates were analyzed for activated forms of ERK (pERK) or AKT (pAKT). Results quantitated as fold increase relative to no growth factor controls (0) in which medium was removed and replaced for 5 minutes (P = 0.000). For comparison, additional cultures were exposed to the ErbB-unrelated ERK and AKT agonist insulin-like growth factor 1 (3 nM). Note that avian cells express the ERK2, but not the ERK1, isoform.
Figure 3.
 
DCDMLs are responsive to ErbB1 and/or ErbB4 ligands. DCDMLs were incubated for 5 minutes with the ErbB ligands HB-EGF (1 nM), TGFα (1 nM), or NRG1 (10 nM), with or without a 1-hour pretreatment with lapatinib (lap). Whole-cell lysates were analyzed for activated forms of ERK (pERK) or AKT (pAKT). Results quantitated as fold increase relative to no growth factor controls (0) in which medium was removed and replaced for 5 minutes (P = 0.000). For comparison, additional cultures were exposed to the ErbB-unrelated ERK and AKT agonist insulin-like growth factor 1 (3 nM). Note that avian cells express the ERK2, but not the ERK1, isoform.
In keeping with our findings with AQP0 (Fig. 1A), lapatinib or erlotinib did not inhibit the expression of other established lens fiber cell differentiation markers in response to TGFβ, including δ-crystallin, CP115 (filensin, also known as BFSP1), and CP49 (phakinin, also known as BFSP2) (Fig. 2A). Moreover, neither inhibitor affected activation (C-terminal phosphorylation of S423/S425) of the canonical TGFβ signal transducer Smad3 when measured 1.5 hours after addition of TGFβ (Fig. 2B). Lapatinib or erlotinib also did not interfere with expression at 48 hours of a Smad3 transcriptional reporter (SBE4-luc) that contains four repeats of an eight-base palindromic Smad4-binding element driving a luciferase reporter51 (Fig. 2C). Taken together, these findings demonstrate that ErbB inhibitors do not act as global repressors of TGFβ signaling but instead have a specific inhibitory effect on EMyT. 
Expression of ErbB1, ErbB2, and ErbB4 in Lens Epithelial Cells
Existent lens cDNA and RNA sequence databases vary in their evidence for ErbB and ErbB ligand transcripts and provide no information on protein expression or function. Incubation of DCDMLs at 37°C for 5 minutes with TGFα (a ligand that binds to ErbB1 but not to any other ErbB family member), NRG1 (which binds to ErbB4 but not ErbB1), or HB-EGF (which binds to both ErbB1 and ErbB4)52,53 resulted in the rapid activation of the downstream ErbB effectors ERK and AKT in a manner inhibited by lapatinib (Fig. 3), demonstrating functional expression of both ligand-binding ErbB kinases in these cells (ErbB2 does not bind ligand but can form heterodimers with ErbB1 or ErbB4). 
We then used antibodies specific for ErbB1, ErbB2, or ErbB4 to directly examine ErbB protein expression. Each reagent recognized a Mr = ∼170 kD band in Western blots generated from DCDML whole-cell lysates, in keeping with the similar electrophoretic mobilities of these family members in chicken (Fig. 4A).54 Although ErbB1 has been reported in both primary rabbit and human lens epithelial cells,31,55,56 to our knowledge, protein expression of ErbB2 and ErbB4 in mammalian lens has not been reported. We found that both kinases as well as ErbB1 are present in lens central epithelial tissue surgically removed from adult rabbits during mock cataract surgery as assessed by Western blotting (Supplementary Fig. S2A). ErbB1 and ErbB2, but not ErbB4, were detected in two immortalized lens epithelium–derived cell lines, human FHL124 cells and rabbit N/N1003A cells (Supplementary Fig. S2B). 
Figure 4.
 
Effect of TGFβ and/or FGF on expression of ErbB1, 2, and 4 in DCDMLs. (A, B) DCDMLs plated under standard conditions (e.g., low density on laminin) were cultured from days 1 to 7 with no additions (0), TGFβ, FGF2, or TGFβ plus FGF2 prior to analysis of ErbBs from either whole-cell lysates (A) or after isolation of the plasma membrane pool by cell surface biotinylation (B). Surface expression in cells cultured with TGFβ is graphed relative to no TGFβ controls (P = 0.000 for all). (C) DCDMLs plated at low density on pdFN were cultured for 6 days with either DMSO or the TGFβR inhibitor SB-431542 to block endogenous TGFβ signaling. Whole-cell lysates were analyzed for the indicated ErbB or αSMA. (D) DCDMLs were plated at higher density on either laminin (LM) or pdFN and then cultured from days 1 to 7 with no additions, TGFβ, DMSO, or SB-431542 prior to whole-cell lysate analysis of ErbB1 and αSMA. In A, C, and D, the arrow denotes the position of ErbB1; the lower band detected in some experiments is a nonspecific, cytosolic species in that it is not recognized by the same antibody in strepavidin-precipitated samples from cell surface biotinylated DCDMLs (B) and is not detected in cell lysates by the anti-ErbB1 rat monoclonal antibody 20.3.6 (Fig. 7B). (E) Expression of the indicated protein in cell lysates from experiments shown in A, C, and D was graphed relative to no growth factor or inhibitor controls plated on the same substrate in the same experiment, all normalized to tubulin. For all, P ≤ 0.01, except bars labeled with either an asterisk (P = 0.032) or NS (P = 0.162).
Figure 4.
 
Effect of TGFβ and/or FGF on expression of ErbB1, 2, and 4 in DCDMLs. (A, B) DCDMLs plated under standard conditions (e.g., low density on laminin) were cultured from days 1 to 7 with no additions (0), TGFβ, FGF2, or TGFβ plus FGF2 prior to analysis of ErbBs from either whole-cell lysates (A) or after isolation of the plasma membrane pool by cell surface biotinylation (B). Surface expression in cells cultured with TGFβ is graphed relative to no TGFβ controls (P = 0.000 for all). (C) DCDMLs plated at low density on pdFN were cultured for 6 days with either DMSO or the TGFβR inhibitor SB-431542 to block endogenous TGFβ signaling. Whole-cell lysates were analyzed for the indicated ErbB or αSMA. (D) DCDMLs were plated at higher density on either laminin (LM) or pdFN and then cultured from days 1 to 7 with no additions, TGFβ, DMSO, or SB-431542 prior to whole-cell lysate analysis of ErbB1 and αSMA. In A, C, and D, the arrow denotes the position of ErbB1; the lower band detected in some experiments is a nonspecific, cytosolic species in that it is not recognized by the same antibody in strepavidin-precipitated samples from cell surface biotinylated DCDMLs (B) and is not detected in cell lysates by the anti-ErbB1 rat monoclonal antibody 20.3.6 (Fig. 7B). (E) Expression of the indicated protein in cell lysates from experiments shown in A, C, and D was graphed relative to no growth factor or inhibitor controls plated on the same substrate in the same experiment, all normalized to tubulin. For all, P ≤ 0.01, except bars labeled with either an asterisk (P = 0.032) or NS (P = 0.162).
Effect of TGFβ on Expression of ErbB1, ErbB2, and ErbB4
Next, we cultured DCDMLs for 6 days with 4 ng/mL TGFβ, 7 to 10 ng/mL FGF2 (which induces lens fiber but not myofibroblast formation),21 or a combination of the two. Western blot analysis of whole-cell lysates demonstrated that each ErbB responded to the panel of growth factors in a unique manner, supporting the specificity of the antibodies employed for their respective isoforms (Fig. 4A). To examine only the plasma membrane, ligand-responsive pools of ErbBs, we used cell surface biotinylation. As in whole-cell lysates, TGFβ increased the level of ErbB1 but decreased ErbB2 and ErbB4 on the plasma membrane (Fig. 4B). 
We also examined ErbB expression in DCDMLs grown on pdFN to induce the activation of endogenous TGFβ.22 Coculture with the highly selective TGFβ type 1 receptor (ALK5) kinase inhibitor SB-43154257 reduced the level of ErbB1 while upregulating ErbB2 and ErbB4 (Fig. 4C), demonstrating the same pattern of dependence on TGFβ signaling as was observed in cultures plated on laminin and treated with exogenous TGFβ. 
In all of the heretofore presented experiments, DCDMLs were subconfluent until at least day 6 of culture to model the low number of lens epithelial cells that survive cataract surgery. Cells plated at a higher density at which they achieved confluence by day 3 responded to TGFβ added on day 1 by promoting fiber cell differentiation, with very low expression of myofibroblast markers21 (Musil L, IOVS 2016;57:ARVO E-Abstract 1374). In higher density plated cells, TGFβ signaling reduced instead of increased expression of ErbB1 in that (1) in such cells plated on laminin, addition of exogenous TGFβ decreased ErbB1 levels, and (2) in cells plated densely on pdFN, which increased endogenous TGFβ signaling (Fig. 1E), the TGFβR inhibitor SB-431542 increased ErbB1 levels (Fig. 4D). The finding that ErbB1 was upregulated in cells densely plated on pdFN when EMyT was blocked by SB-431542 indicates that enhanced expression of ErbB1 is not simply a downstream consequence of myofibroblast differentiation. 
Lens Epithelial Cells Express Soluble ErbB Ligands
Many cell types that express a functional ErbB also produce one or more ErbB ligands. The resulting paracrine and/or autocrine signaling could be especially important to epithelial cells in the avascular lens, given the reported lack of ErbB ligands in the aqueous humor.5861 ErbB1 and/or ErbB4 have been reported to bind to more than 10 distinct polypeptide growth factors.26,37 Instead of pursuing the many individual ErbB ligand candidates, we developed an assay to detect overall ErbB-stimulating activity in DCDML-conditioned medium. HEK293 cells endogenously express ErbB1, ErbB2, and ErbB4 (Supplementary Fig. S2) and respond to low levels of exogenous ligand.62 HEKs were incubated either with recombinant HB-EGF or with (serum-free) DCDML-conditioned medium at 4°C, a temperature that allows ligand-induced tyrosine autophosphorylation of ErbBs,63,64 but is not permissive for their transactivation by other effectors. We then used the well-characterized 4G10 antiphosphotyrosine antibody to probe Western blots for activated ErbBs, as previously described by others.65 As expected, the 4G10 reagent detected a ∼170-kD band when HEK cells were incubated at 4°C in the presence of exogenous HB-EGF in a concentration-dependent manner (Fig. 5A). This species was not detected if cells had been pretreated at 37°C with lapatinib, confirming that it represents bona fide autophosphorylated ErbB. Conditioned medium from DCDMLs cultured for 2 days in the absence of TGFβ markedly stimulated lapatinib-inhibitable ErbB autophosphorylation in HEKs (Fig. 5B), in keeping with previous reports of ErbB ligand expression in lens epithelial cells.35,66 Culturing DCDMLs with TGFβ for 48 hours (a period too short to induce myofibroblast formation in DCDMLs)21,67 further increased the ErbB-activating activity released into the medium by 1.4-fold (±0.23; P = 0.019; n = 5) (Fig. 5B). Mock DCDML-conditioned medium, generated by incubating culture medium for 2 days with or without TGFβ in the absence of lens cells, did not induce the phosphotyrosine ErbB band in HEKs (Fig. 5B). In another series of experiments, we assessed the ability of DCDML-conditioned medium to activate ERK at 37°C in DCDML recipients in a lapatinib-inhibitable manner (Supplementary Fig. S3). Although the semiquantitative nature of these assays precluded a precise assessment of the magnitude of the effect, both methods indicate that culturing DCDMLs in the presence of TGFβ increased the ability of the conditioned medium to activate ErbBs in recipient cells to a statistically significant extent. Upregulation of functional ErbB ligand by TGFβ has previously been reported in other cell types.68 
Figure 5.
 
ErbB-stimulating activity in DCDML-conditioned medium. Untransfected HEK 293 cells pretreated for 1 hour with either DMSO or lapatinib (lap) were incubated at 4°C for 15 minutes with the medium indicated, prior to analysis of whole-cell lysates with the pan-phosphotyrosine antibody 4G10. All results shown are from the same blot of a single experiment, reprobed with antibodies against total ErbB1 to confirm equal loading. (A) HEKs were incubated at 4°C with fresh medium supplemented with 5-0.5 ng/mL HB-EGF. (B) HEKs were incubated at 4°C with medium conditioned for 2 days by DCDMLs (CM) cultured in either the absence (−) or presence (+) of TGFβ. Mock-conditioned medium (mock) was generated by incubating medium with or without TGFβ for 2 days in the absence of cells. All media were concentrated 5-fold prior to addition to HEK cell recipients.
Figure 5.
 
ErbB-stimulating activity in DCDML-conditioned medium. Untransfected HEK 293 cells pretreated for 1 hour with either DMSO or lapatinib (lap) were incubated at 4°C for 15 minutes with the medium indicated, prior to analysis of whole-cell lysates with the pan-phosphotyrosine antibody 4G10. All results shown are from the same blot of a single experiment, reprobed with antibodies against total ErbB1 to confirm equal loading. (A) HEKs were incubated at 4°C with fresh medium supplemented with 5-0.5 ng/mL HB-EGF. (B) HEKs were incubated at 4°C with medium conditioned for 2 days by DCDMLs (CM) cultured in either the absence (−) or presence (+) of TGFβ. Mock-conditioned medium (mock) was generated by incubating medium with or without TGFβ for 2 days in the absence of cells. All media were concentrated 5-fold prior to addition to HEK cell recipients.
ErbB Activity in DCDMLs in the Absence of TGFβ
If DCDMLs constitutively release ErbB ligands into the medium and express the ligand-binding ErbB1 and ErbB4 kinases, do DCDMLs have detectable levels of active ErbBs on the cell surface? In order to simultaneously assess all possible ErbB kinases, we subjected DCDMLs to cell surface biotinylation, collected labeled plasma membrane proteins with streptavidin beads, and probed Western blots with the 4G10 antiphosphotyrosine antibody. As in HEK cells (Fig. 5), Western blots of DCDMLs exposed to 10 nM HB-EGF for 15 minutes at 4°C showed a prominent cell surface, phosphotyrosine-positive band with the appropriate electrophoretic mobility for ErbBs that was not present if cells had been pretreated with lapatinib (Fig. 6). Active (e.g., phosphotyrosine-positive, lapatinib-inhibitable) cell surface ErbBs were also detected, albeit much less strongly, in DMSO-only treated DCDMLs (Fig. 6). 
Figure 6.
 
Basal ErbB activity in DCDMLs. DCDMLs pretreated with either DMSO or lapatinib (lap) were incubated at 4°C for 15 minutes in either the absence or presence of 10 nM HB-EGF as indicated prior to cell surface biotinylation. Strepavidin precipitates of biotin-labeled plasma membrane proteins were run on SDS-PAGE and blotted for phosphotyrosine, followed by reprobing with antibodies against total ErbB1 to confirm the position of ErbBs. Equal portions of whole-cell lysate were analyzed for β-actin to ensure equal loading. Typical of six independent experiments.
Figure 6.
 
Basal ErbB activity in DCDMLs. DCDMLs pretreated with either DMSO or lapatinib (lap) were incubated at 4°C for 15 minutes in either the absence or presence of 10 nM HB-EGF as indicated prior to cell surface biotinylation. Strepavidin precipitates of biotin-labeled plasma membrane proteins were run on SDS-PAGE and blotted for phosphotyrosine, followed by reprobing with antibodies against total ErbB1 to confirm the position of ErbBs. Equal portions of whole-cell lysate were analyzed for β-actin to ensure equal loading. Typical of six independent experiments.
To examine the functional state of ErbB1, we first took advantage of the fact that ErbB kinase inhibitors such as lapatinib can gradually increase the level of previously active ErbB1 by blocking its ligand-mediated downregulation.6971 We found that a 24-hour incubation with lapatinib consistently enhanced the total and cell surface amount of ErbB1 in DCDMLs (Fig. 7A). In contrast, this treatment did not increase the level of ErbB4, the ligand-mediated degradation of which occurs by a mechanism distinct from that utilized by ErbB1.72 
Figure 7.
 
ErbB1 is active in DCDMLs. (A) DCDMLs were cultured for 24 hours in the presence of DMSO or lapatinib (lap) prior to analysis of ErbB1 or ErbB4 from either whole-cell lysates (total) or after isolation of the plasma membrane pool by cell surface biotinylation (cell surface). The level of ErbB1 and ErbB4 recovered from lapatinib-treated cells is graphed relative to DMSO-only controls in the same experiment. (B) DCDMLs were pretreated for 1 hour at 37°C with DMSO or lapatinib (lap) and incubated for 5 minutes at 37°C in the presence or absence of 10 nM HB-EGF prior to analysis of whole-cell lysates with a rabbit antibody specific for the Y1068 autophosphorylated, activated form of ErbB1. The blot was reprobed with the rat anti-ErbB1 20.3.6 antibody to detect total ErbB1. Arrow in A denotes the position of ErbB1; the lower band is a nonspecific, cytosolic species not recognized by the rat 20.3.6 monoclonal.
Figure 7.
 
ErbB1 is active in DCDMLs. (A) DCDMLs were cultured for 24 hours in the presence of DMSO or lapatinib (lap) prior to analysis of ErbB1 or ErbB4 from either whole-cell lysates (total) or after isolation of the plasma membrane pool by cell surface biotinylation (cell surface). The level of ErbB1 and ErbB4 recovered from lapatinib-treated cells is graphed relative to DMSO-only controls in the same experiment. (B) DCDMLs were pretreated for 1 hour at 37°C with DMSO or lapatinib (lap) and incubated for 5 minutes at 37°C in the presence or absence of 10 nM HB-EGF prior to analysis of whole-cell lysates with a rabbit antibody specific for the Y1068 autophosphorylated, activated form of ErbB1. The blot was reprobed with the rat anti-ErbB1 20.3.6 antibody to detect total ErbB1. Arrow in A denotes the position of ErbB1; the lower band is a nonspecific, cytosolic species not recognized by the rat 20.3.6 monoclonal.
Next, we conducted Western blot experiments with a rabbit antibody specific for the Y1068 autophosphorylated form of ErbB1, a modification closely linked to receptor activation and function (Fig. 7B).26,73,74 As expected, pY1068 ErbB1 levels were rapidly increased after addition of exogenous HB-EGF ligand in a manner inhibited by a 1-hour pretreatment with lapatinib. This antibody also recognized a ∼170-kD species in whole-cell lysates of DMSO-only DCDMLs that was reduced by 91% (±6.3; n = 5; P = 0.00) if the cells had been pretreated with lapatinib, further demonstrating that ErbB1 was basally active in these cells (Fig. 7B). 
TGFβ Promotes Homodimerization of ErbB1
In order to mediate ligand-induced signal transduction, ErbBs must form either homo- or heterodimers. ErbB2 is considered the preferred dimerization partner, in part because it is constitutively conformationally competent to interact with other ErbBs.26,7577 Because culturing DCDMLs for 6 days with TGFβ increases the total (and cell surface) levels of ErbB1 but reduces expression of ErbB2 and ErbB4 (Figs. 4A, 4B), TGFβ would be expected to decrease the proportion of ErbB1 that can form ErbB1/ErbB2 or ErbB1/ErbB4 heterodimers and increase the fraction of ErbB1/ErbB1 homodimers. To test this prediction, we exploited the fact that ErbB1 homodimers are downregulated and degraded in response to ligands such as HB-EGF much more efficiently than any ErbB1 heterodimer, which has a greater tendency to recycle to the cell surface.78,79 DCDMLs cultured on laminin for 6 days with or without TGFβ were subsequently incubated for 4 hours at 37°C with a high concentration (10 nM; 100 ng/mL) of HB-EGF. In cells not exposed to TGFβ, this treatment resulted in the loss of <25% of the total cellular pools of ErbB1, ErbB2, and ErbB4 (Fig. 8A). In cells in the same experiment cultured for 6 days with TGFβ, the fraction of ErbB2 and ErbB4 subsequently downregulated in response to HB-EGF was minimally affected. In contrast, the loss of total ErbB1 induced by HB-EGF was much greater (∼84%) than in TGFβ-minus cells. Downregulation of ErbB1 was blocked if cells were pretreated with lapatinib prior to addition of ligand (Fig. 8A graph). ErbB1/ErbB1 homodimer formation also appeared to be promoted when DCDMLs were grown on pdFN to stimulate the activation of endogenous TGFβ (Fig. 8A). Cell surface biotinylation confirmed a major (>90%) decrease in the level of ErbB1, but not of ErbB2, on the plasma membrane of TGFβ-cultured cells in response to a 4-hour treatment with 10 nM HB-EGF (Fig. 8B). We repeated these experiments using NRG1, a ligand that binds to ErbB4 but not to ErbB1. A 4-hour treatment with 10 nM NRG1 reduced the total cellular levels of ErbB4 by ∼50% in DCDMLs cultured in either the absence or presence of TGFβ (Fig. 8C). NRG1 also induced a small (∼15%) reduction in the total level of ErbB1 in cells grown in the absence of TGFβ, indicating that a fraction of ErbB4 had formed heterodimers with ErbB1. In contrast, NRG1 had no statistically significant effect on ErbB1 levels in cells cultured with TGFβ (Fig. 8C). 
Figure 8.
 
ErbB1 behaves like a heterodimer in DCDMLs cultured without TGFβ but like a homodimer in plus TGFβ cells. (A–C) Lens cells plated on laminin (LM) or on pdFN were cultured from days 1 to 7 with or without TGFβ as indicated. The cells were then incubated at 37°C for 4 hours in the absence or presence of high levels (10 nM) of HB-EGF (A, B) or NRG1 (C) prior to analysis of ErbBs from either whole-cell lysates (A, C) or after isolation of the plasma membrane pool by cell surface biotinylation (B). Levels of ErbB graphed relative to no-ligand controls in the same experiment. (A) Increased expression of ErbB1 induced by either addition of exogenous TGFβ or plating on pdFN enhances the loss of total cellular ErbB1 in response to HBEGF. The graph also shows that a 1-hour pretreatment with lapatinib (+lap) blocks downregulation of ErbB1 after exposure to HB-EGF. NS, P ≥ 0.06. (B) Cell surface biotinylation confirms the loss of ErbB1 from the surface of TGFβ-cultured, HB-EGF–treated cells. (C) Culturing DCDMLs with TGFβ renders ErbB1 insensitive to downregulation by the ErbB4 ligand NRG1.
Figure 8.
 
ErbB1 behaves like a heterodimer in DCDMLs cultured without TGFβ but like a homodimer in plus TGFβ cells. (A–C) Lens cells plated on laminin (LM) or on pdFN were cultured from days 1 to 7 with or without TGFβ as indicated. The cells were then incubated at 37°C for 4 hours in the absence or presence of high levels (10 nM) of HB-EGF (A, B) or NRG1 (C) prior to analysis of ErbBs from either whole-cell lysates (A, C) or after isolation of the plasma membrane pool by cell surface biotinylation (B). Levels of ErbB graphed relative to no-ligand controls in the same experiment. (A) Increased expression of ErbB1 induced by either addition of exogenous TGFβ or plating on pdFN enhances the loss of total cellular ErbB1 in response to HBEGF. The graph also shows that a 1-hour pretreatment with lapatinib (+lap) blocks downregulation of ErbB1 after exposure to HB-EGF. NS, P ≥ 0.06. (B) Cell surface biotinylation confirms the loss of ErbB1 from the surface of TGFβ-cultured, HB-EGF–treated cells. (C) Culturing DCDMLs with TGFβ renders ErbB1 insensitive to downregulation by the ErbB4 ligand NRG1.
Taken together, the findings shown in Figure 8 indicate that TGFβ increases the capacity of DCDMLs to form ligand-induced ErbB1/ErbB1 homodimers and reduces ErbB1 heterodimers. Chemical cross-linking experiments provided further evidence for the preferential formation of ErbB1 homodimers over ErbB1/ErbB2 heterodimers in TGFβ-cultured cells (Supplementary Fig. S4). 
ErbB1 Signaling Is Preserved in TGFβ-Cultured Lens Cells
We next asked if the increase in soluble ErbB ligand production (Fig. 5) and shift to downregulation-prone ErbB1 homodimers (Fig. 8) induced by TGFβ decreased the activity of ErbB on the cell surface. The experiment shown in Figure 6 was repeated on DCDMLs cultured for 6 days either with or without TGFβ. As shown in Figure 9A, the level of tyrosine-phosphorylated cell surface ErbB recovered was similar under both conditions, suggesting that total ErbB function had been maintained. Culturing DCDMLs with TGFβ also did not significantly alter the amount of active ErbB1 on the plasma membrane as assessed with phospho-Y1068 ErbB1-specific antibodies. Notably, a 4°C treatment with the ErbB1-specific ligand TGFα resulted in ∼4.6-fold higher levels of 4G10-positive ErbB and 5.2-fold higher levels of autophosphorylated pY1068 ErbB1 in TGFβ-cultured cells relative to no TGFβ (+ TGFα) controls, demonstrating that the ErbB1 induced by TGFβ is ligand responsive (Fig. 9B). Thus, ErbB, particularly ErbB1, function is maintained throughout the process of EMyT and appears to be limited to basal levels by the amount of endogenous ErbB ligand. 
Figure 9.
 
Effect of TGFβ on ErbB function. (A) TGFβ does not change the level of endogenously active ErbBs in DCDMLs. DCDMLs were cultured for 6 days in the absence or presence of TGFβ. Cells were then cell surface biotinylated prior to analysis of plasma membrane proteins using antibodies against phosphotyrosine (4G10; phospho-Y) or specific for the Y1068 autophosphorylated form of ErbB1 as indicated. Blots were reprobed with antibodies against total ErbB1. Data graphed as fold versus no TGFβ control cultures in the same experiment. (B) ErbB1 induced by TGFβ is activatable by exogenous ligand. DCDMLs cultured for 6 days with or without TGFβ were incubated at 4°C for 15 minutes in either the absence or presence of 10 nM TGFα as indicated. Whole-cell lysates were then analyzed by Western blot with antiphosphotyrosine or anti-pY1068 ErbB1 antibodies, followed by reprobing for total ErbB1. Data graphed as fold versus no TGFβ, + TGFα cultures in the same experiment.
Figure 9.
 
Effect of TGFβ on ErbB function. (A) TGFβ does not change the level of endogenously active ErbBs in DCDMLs. DCDMLs were cultured for 6 days in the absence or presence of TGFβ. Cells were then cell surface biotinylated prior to analysis of plasma membrane proteins using antibodies against phosphotyrosine (4G10; phospho-Y) or specific for the Y1068 autophosphorylated form of ErbB1 as indicated. Blots were reprobed with antibodies against total ErbB1. Data graphed as fold versus no TGFβ control cultures in the same experiment. (B) ErbB1 induced by TGFβ is activatable by exogenous ligand. DCDMLs cultured for 6 days with or without TGFβ were incubated at 4°C for 15 minutes in either the absence or presence of 10 nM TGFα as indicated. Whole-cell lysates were then analyzed by Western blot with antiphosphotyrosine or anti-pY1068 ErbB1 antibodies, followed by reprobing for total ErbB1. Data graphed as fold versus no TGFβ, + TGFα cultures in the same experiment.
Relative Roles of ErbB1, ErbB2, and ErbB4 in Lens Cell Fibrosis
In addition to upregulating ErbB1, TGFβ induces a striking decrease in the total and cell surface levels of ErbB4 (Fig. 4). In the kidney, reduced expression of ErbB4 has been associated with increased fibrosis after unilateral ureteral obstruction,80 whereas deletion of ErbB1 had the opposite effect.81 Based on this precedent, we considered the possibility that induction of myofibroblast differentiation in lens cells by TGFβ might require the observed decrease in ErbB4 levels. We therefore examined the effect of overexpressed ErbB4 in DCDMLs on the fibrotic marker αSMA in the absence or presence of TGFβ. Control experiments demonstrated that transient transfection of a plasmid encoding human ErbB4 in DCDMLs increased the level of anti-ErbB4 immunoreactivity by 60 to 242 times on day 7 of culture and also greatly enhanced the level of tyrosine phosphorylated (e.g., anti–pY-reactive; lapatinib inhibitable) ErbBs, indicating that the exogenous ErbB4 was signaling competent (Supplementary Fig. S5). Overexpression of ErbB4 did not, however, block the ability of TGFβ to upregulate αSMA, FN, or the fiber cell marker CP49 (Fig. 10A). Similar results were obtained when DCDMLs were transiently transfected with a plasmid encoding ErbB1-GFP or ErbB2-GFP. 
Figure 10.
 
Effect of transient transfection of ErbB1, ErbB2, or ErbB4 in DCDMLs on αSMA. (A) Forced overexpression of ErbB 1, 2, or 4 does not prevent TGFβ from upregulating αSMA, FN, or CP49. DCDMLs were transiently transfected with plasmids encoding ErbB1-GFP, ErbB2-GFP, or ErbB4 on day 1 and cultured for 6 more days either with or without TGFβ prior to analysis of whole-cell lysates for the indicated protein. Controls were transfected with a plasmid encoding an irrelevant integral membrane protein (E208K Cx32).67 (B) Overexpression of ErbB1 selectively enhances αSMA in the absence of TGFβ. DCDMLs transfected with control or ErbB1-GFP (R1), ErbB2-GFP (R2), or ErbB4 (R4) plasmid were cultured for 6 days without TGFβ prior to Western blot assessment of αSMA and tubulin. (C) The level of αSMA in the presence of each exogenously expressed ErbB was graphed relative to the level of αSMA in control transfectants, all without TGFβ (n = 4). Overexpression of ErbB over endogenous ErbB protein achieved in the experiments quantitated was 75-206X (ErbB4), 61-358X (ErbB2), and 71-205X (ErbB1).
Figure 10.
 
Effect of transient transfection of ErbB1, ErbB2, or ErbB4 in DCDMLs on αSMA. (A) Forced overexpression of ErbB 1, 2, or 4 does not prevent TGFβ from upregulating αSMA, FN, or CP49. DCDMLs were transiently transfected with plasmids encoding ErbB1-GFP, ErbB2-GFP, or ErbB4 on day 1 and cultured for 6 more days either with or without TGFβ prior to analysis of whole-cell lysates for the indicated protein. Controls were transfected with a plasmid encoding an irrelevant integral membrane protein (E208K Cx32).67 (B) Overexpression of ErbB1 selectively enhances αSMA in the absence of TGFβ. DCDMLs transfected with control or ErbB1-GFP (R1), ErbB2-GFP (R2), or ErbB4 (R4) plasmid were cultured for 6 days without TGFβ prior to Western blot assessment of αSMA and tubulin. (C) The level of αSMA in the presence of each exogenously expressed ErbB was graphed relative to the level of αSMA in control transfectants, all without TGFβ (n = 4). Overexpression of ErbB over endogenous ErbB protein achieved in the experiments quantitated was 75-206X (ErbB4), 61-358X (ErbB2), and 71-205X (ErbB1).
Interestingly, overexpression of ErbB1 consistently increased the level of αSMA, but not of FN or CP49, in DCDMLs cultured in the absence of TGFβ (Figs. 10A, 10B). Upregulation of αSMA in ErbB1 transfectants was blocked by coculture with lapatinib or SB-431542, indicating that exogenous ErbB1 is able to cooperate with endogenous TGFβ signaling to enhance at least some aspects of EMyT (Supplementary Fig. S6). In contrast, overexpression of ErbB2 or ErbB4 did not significantly alter αSMA levels in the absence of added TGFβ (Figs. 10A–C; Supplementary Fig. S6). Together, these findings support a selective role for ErbB1 in promoting lens epithelial cell fibrosis. 
ErbB Inhibitor-Based Strategies for Prevention of Fibrotic PCO
Ideally, a one-time dosing at the time of cataract surgery would be the preferred means to administer an anti-PCO therapeutic. It has been reported that a single, brief treatment with a high concentration of the ErbB inhibitor erlotinib has a long-term effect on the fate of cancer cells.82 We found that a 1-hour incubation of DCDMLs with 40 µM lapatinib (but not with 4 µM lapatinib; not shown) followed by drug removal reduced active ErbB1 to undetectable levels when assessed 6 days later when cells were cultured in either the absence or the presence of TGFβ (Fig. 11A; n = 6). This treatment also blocked TGFβ-induced EMyT (Fig. 11B). Importantly, expression of the lens fiber cell differentiation markers CP115 and CP49 was not reduced, ruling out a nonspecific toxic effect on cell viability. 
Figure 11.
 
A single 1-hour, high-dose treatment with lapatinib blocks ErbB1 activity (A) and TGFβ-induced EMyT (B) for 6 days. DCDMLs were incubated on day 1 for 1 hour with 40 µM lapatinib or DMSO vehicle only. Medium was removed and the cells were cultured without drug for 6 days in the absence or presence of TGFβ. (A) Whole-cell lysates were analyzed for endogenous active ErbB using rabbit anti-pY1068 ErbB1 antibodies (n = 6). (B) Whole-cell lysates were analyzed for the EMyT markers FN and αSMA and for the fiber cell differentiation markers CP115 and CP49. Results quantitated as fold inhibition relative to controls treated with DMSO and then TGFβ in the same experiment. *P = 0.024. **P = 0.000.
Figure 11.
 
A single 1-hour, high-dose treatment with lapatinib blocks ErbB1 activity (A) and TGFβ-induced EMyT (B) for 6 days. DCDMLs were incubated on day 1 for 1 hour with 40 µM lapatinib or DMSO vehicle only. Medium was removed and the cells were cultured without drug for 6 days in the absence or presence of TGFβ. (A) Whole-cell lysates were analyzed for endogenous active ErbB using rabbit anti-pY1068 ErbB1 antibodies (n = 6). (B) Whole-cell lysates were analyzed for the EMyT markers FN and αSMA and for the fiber cell differentiation markers CP115 and CP49. Results quantitated as fold inhibition relative to controls treated with DMSO and then TGFβ in the same experiment. *P = 0.024. **P = 0.000.
We have previously reported that lens cell EMyT is also specifically blocked by a 1-hour 10-µM treatment with the small-molecule multikinase inhibitor rebastinib, most likely due to long-term blockade of more than one kinase in the p38 MAP kinase cascade.21 As expected from in vitro studies,83,84 rebastinib does not hinder ligand-induced stimulation of ErbBs, including ErbB1, in DCDMLs (Fig. 12A). Conversely, lapatinib (unlike rebastinib; Supplementary Fig. S7) has no effect on activation of p38 by stimuli as diverse as TGFβ, FGF, and anisomycin (Fig. 12B). It has been well established that treatments that interfere with multiple processes can be more efficacious than monotherapies for cancer and other complex diseases.8587 Because they block distinct kinases that both contribute to lens cell EMyT, we considered the possibility that lapatinib and rebastinib could act cooperatively when combined. Consistent with this concept, a 1-hour treatment with 4 µM lapatinib and 0.5 µM rebastinib on day 1 reduced TGFβ-induced myofibroblast differentiation on day 7 to a greater extent than either drug alone (Fig. 12C). In contrast, there was no significant inhibitory or additive effect of lapatinib plus rebastinib on expression of CP115 or CP49, consistent with the lack of involvement of ErbB (Fig. 2) or p3821 signaling in fiber cell differentiation downstream of TGFβ. These findings raise the possibility that ErbB inhibitors, alone or in combination with a mechanistically distinct drug such as rebastinib, could be administered once at the time of cataract surgery as a prophylactic treatment for fibrotic PCO. 
Figure 12.
 
A 1-hour treatment with lapatinib combines with a non-ErbB-targeted therapeutic to inhibit lens cell fibrosis. (A) Rebastinib does not hinder ligand-induced stimulation of ErbBs in DCDMLs. DCDMLs were preincubated with DMSO or 0.5 µM rebastinib (reb) for 4 hours prior to a 15-minute, 4°C treatment with either HB-EGF or TGFα. Autoactivation of ErbBs was assessed by Western blot of whole-cell lysates using mouse antibodies against either phosphotyrosine (to measure active ErbB1, ErbB2, and ErbB4) or pY1068 ErbB1 (to specifically assess active ErbB1). No inhibition by rebastinib was detected in five of five experiments (P = 0.000). (B) Lapatinib does not target activation of p38. DCDMLs were preincubated for 2 hours with DMSO or 4 µM lapatinib (lap) prior to a 90-minute incubation with 4 ng/mL TGFβ, 10 ng/mL FGF-2, or 3 µg/mL anisomycin. Whole-cell lysates were analyzed for active (pT180/pY182) p38 and total p38 by Western blotting. Typical of four experiments. (C) A single 1-hour treatment with 4 µM lapatinib combined with rebastinib blocks TGFβ-induced EMyT for 6 days. DCDMLs were treated for 1 hour with 0.5 µM rebastinib, 4 µM lapatinib, or both, after which the drug-containing medium was removed. Cells were then cultured for 6 days with TGFβ. Controls were pretreated with DMSO only and cultured with or without TGFβ. Western blots of whole-cell lysates were probed with antibodies against the indicated protein and results graphed as fold expression relative to DMSO, then TGFβ-treated controls. Asterisks indicate P ≤ 0.010 inhibition compared to control (n = 4).
Figure 12.
 
A 1-hour treatment with lapatinib combines with a non-ErbB-targeted therapeutic to inhibit lens cell fibrosis. (A) Rebastinib does not hinder ligand-induced stimulation of ErbBs in DCDMLs. DCDMLs were preincubated with DMSO or 0.5 µM rebastinib (reb) for 4 hours prior to a 15-minute, 4°C treatment with either HB-EGF or TGFα. Autoactivation of ErbBs was assessed by Western blot of whole-cell lysates using mouse antibodies against either phosphotyrosine (to measure active ErbB1, ErbB2, and ErbB4) or pY1068 ErbB1 (to specifically assess active ErbB1). No inhibition by rebastinib was detected in five of five experiments (P = 0.000). (B) Lapatinib does not target activation of p38. DCDMLs were preincubated for 2 hours with DMSO or 4 µM lapatinib (lap) prior to a 90-minute incubation with 4 ng/mL TGFβ, 10 ng/mL FGF-2, or 3 µg/mL anisomycin. Whole-cell lysates were analyzed for active (pT180/pY182) p38 and total p38 by Western blotting. Typical of four experiments. (C) A single 1-hour treatment with 4 µM lapatinib combined with rebastinib blocks TGFβ-induced EMyT for 6 days. DCDMLs were treated for 1 hour with 0.5 µM rebastinib, 4 µM lapatinib, or both, after which the drug-containing medium was removed. Cells were then cultured for 6 days with TGFβ. Controls were pretreated with DMSO only and cultured with or without TGFβ. Western blots of whole-cell lysates were probed with antibodies against the indicated protein and results graphed as fold expression relative to DMSO, then TGFβ-treated controls. Asterisks indicate P ≤ 0.010 inhibition compared to control (n = 4).
Discussion
PCO remains an intractable and costly clinical problem.8,15,88 Attempts to eliminate this condition using toxic chemicals have failed, largely due either to an inability to kill all lens epithelial cells or to unacceptably deleterious effects on other ocular tissues.8991 Moreover, total eradication of all residual lens cells could lead to “dead bag syndrome,” in which the IOL becomes displaced from the visual axis.92,93 
The emergence of small-molecule tyrosine kinase inhibitors for the treatment of human diseases94,95 led us to consider if such more specific therapeutics could be used against PCO. In this study, we have investigated how a target of such inhibitors—namely, ErbBs—participates in PCO-associated processes. Specifically, we used our DCDML system to address four fundamental issues: (1) which ErbB kinases are functionally expressed in lens cells, (2) whether lens cells produce biologically active ErbB ligands, (3) how TGFβ affects ErbB signaling, and (4) if anti-ErbB cancer therapies could be repurposed to combat fibrotic PCO. Combined with previously mentioned studies in human capsular bags,3133 an in vivo rat PCO model,34 and primary explants of postnatal rat lens epithelium,35 our data support a key role for ErbBs, particularly ErbB1, in PCO-associated fibrosis in vertebrates. 
ErbB Pathways in Lens Cells in the Absence and Presence of TGFβ
Our data show that under basal (e.g., no added growth factor) conditions, primary lens cells express ErbB1, ErbB2, and ErbB4 protein on the plasma membrane (Fig. 4). They also secrete into the medium one or more ErbB ligands (Fig. 5) that are most likely responsible for the tyrosine phosphorylated ErbB species detected on the cell surface (Fig. 6). These species include ErbB1 activated on the autophosphorylation site Y1068 (Fig. 7B). Based on our results (Fig. 8) and on the role of ErbB2 as the preferred ErbB dimerization partner,26,77 basal ErbB signaling is likely to be predominantly mediated by ErbB1/ErbB2 heterodimers. This species transduces particularly strong ligand-stimulated signaling due in part to its ability to activate the both the ErbB1 and ErbB2 kinases in response to the binding of a single molecule of (ErbB1) ligand and its resistance to degradation relative to ErbB1 homodimers.79,96 Our finding that a 24-hour exposure to lapatinib increases total and plasma membrane pools of ErbB1 in DCDMLs (Fig. 7A) suggests, however, that the level of endogenous ErbB ligand under basal conditions is sufficient to induce a partial downregulation of ErbB1. 
When DCDMLs are cultured with exogenous TGFβ, TGFβ signaling is enhanced, as has been observed after cataract surgery in human patients.18 TGFβ increases the production of bioactive, soluble ErbB ligand in DCDMLs (Fig. 5) and decreases the total and cell surface levels of ErbB2 and ErbB4 (Fig. 4). Because ErbB1/ErbB1 homodimers are particularly susceptible to ligand-induced destruction,78,79 and because TGFβ and/or ErbB ligands can induce the expression of ErbB inhibitors97 (several of which have been reported in lens cells),98101 this combination would be expected to result in a loss of ErbB function in the absence of other changes. Our data suggest that downregulation of ErbB activity in the presence of TGFβ may be prevented by the observed TGFβ-induced increase in the level of ErbB1, which serves to preserve the level of ErbB signaling required to sustain myofibroblast differentiation. 
Results shown in Figure 10A indicate that DCDMLs that exogenously overexpress ErbB2 or ErbB4 are still capable of undergoing TGFβ-induced EMyT, ruling out the possibility that reduced expression of either species is required for this process. The question thus arises as to why TGFβ selectively increases the level of ErbB1 instead of ErbB2 or ErbB4. An attractive possibility arises from the well-established concept that different ErbB dimers mediate distinct functions, due in part to isoform-specific binding of ligands, recruitment of intracellular signaling adaptors, and differences in the extent to which ErbBs are internalized, recycled, or degraded in response to specific ligands.25,102,103 It could be that ErbB1, perhaps especially in homodimers, promotes myofibroblast differentiation, proliferation, and/or survival in a manner that other ErbB species cannot. Our finding that the FHL124 lens cell line does not express ErbB4 (Supplementary Fig. S2) but is reported to undergo some aspects of TGFβ-induced EMT42 suggests that ErbB4 may not be essential for this process. Unfortunately, available small-molecule inhibitors and dominant-negative cDNA constructs block the activity of multiple ErbB family members in intact cells due in part to heterodimerization,104,105 and attaining efficient gene knockdown in DCDMLs under conditions that do not nonspecifically perturb their developmental fate remains challenging. We have therefore addressed the roles of ErbB1, ErbB2, and ErbB4 in DCDMLs by individually increasing their expression by transient transfection. Using this approach, we found that exogenous expression of ErbB1, but not of ErbB2 or ErbB4, is sufficient to increase the level of αSMA in the absence of added TGFβ (Fig. 10). This finding is consistent with a specific profibrotic role for ErbB1, as has been reported in kidney.80,81 Although beyond the scope of the current study, the signaling pathway(s) activated by ErbB1 that are essential for myofibroblast generation are the subject of ongoing investigations. Our current data support an essential role for ERK downstream of ErbB1 autophosphorylation in this process. 
Therapeutic Implications
It has been reported that within 1 month after cataract surgery, the square posterior edge of the IOL “shrink wraps” to the lens capsule. This forms a so-called capsular bend that physically blocks the further movement of lens cells to the posterior capsule, thereby stopping the progression of PCO.10,106108 Consequently, interventions that interfere with critical signaling events during the initial postoperative period may lead to a long-term reduction in fibrotic PCO. Our finding that a single 1-hour exposure to lapatinib inhibits EMyT in lens cells for at least 1 week (Fig. 11) raises the possibility that such a treatment at the time of cataract surgery could block fibrosis during the critical first few postoperative weeks. Equally importantly, no ocular toxicity has been reported in humans after systemic administration of lapatinib despite its likely ability to enter the eye based on its permeability to the blood–brain barrier.109 This is in contrast to the less specific84,110 ErbB inhibitors erlotinib and gefitinib.111,112 Lapatinib could be administered as a drug-eluting IOL or in viscoelastic solution, or by injection into the vitreous body to specifically gain access to the postsurgery population of epithelial cells at the posterior of the lens capsule that cause clinically deleterious fibrotic PCO. Both gefitinib and erlotinib have been loaded into, and eluted from, human IOLs at doses that slow lens cells spread in in vitro systems.33,113 Our finding that the antifibrotic effect of suboptimal concentrations of lapatinib can be enhanced by coadministration of the unrelated human therapeutic rebastinib (Fig. 12) suggests that PCO may be vulnerable to combination therapy. 
In addition to small-molecule kinase inhibitors, agents that block either the shedding114 or receptor binding115,116 of specific ErbB ligands have been considered as human therapeutics. Applying such an approach to PCO awaits the identification of the relevant growth factors; given the well-known ability of ErbB1 ligands to a cross-induce each other,117119 it is possible that TGFβ promotes the production of multiple species. A more promising strategy is based on our finding that ErbB1/B2 heterodimers and then ErbB1 homodimers are likely to mediate the effects of TGFβ on myofibroblast differentiation. Antibodies that specifically bind to, and block the function of, ErbB1 and ErbB2 hetero- and homodimers are approved by the US Food and Drug Administration for oncological use.27,120,121 Such antibodies, especially anti-ErbB1 agents, may protect lens cells against PCO if administered locally at the time of cataract surgery. Given that the receptor tyrosine kinase MET may be profibrotic in lens cells,122 recently developed bispecific antibodies that simultaneously inhibit ErbB1 and MET123 may also be of value. Unfortunately, the species specificity of available anti-ErbB therapeutic antibodies precludes their testing in small animal models such as DCDMLs. Our finding that lapatinib increases the amount of ErbB1 on the cell surface (Fig. 7A) suggests that it could be used to maximize the binding of function-blocking antibodies. Combined use of the two mechanistically distinct anti-ErbB therapies may therefore lead to a synergistic abrogation of fibrotic PCO. 
Acknowledgments
The authors thank Nick Mamalis, MD, and Liliana Werner, MD, PhD (Intermountain Ocular Research Lab in the John A. Moran Eye Center, University of Utah) for providing normal rabbit lens central epithelial tissue. They also thank Mike Hayman (Stonybrook University) for his generous gift of the anti-ErbB1 20.3.6 monoclonal antibody. 
Supported by R01 EY028558 from the National Eye Institute of the National Institutes of Health. 
Disclosure: J.K. VanSlyke, None; B.A. Boswell, None; L.S. Musil, None 
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Figure 1.
 
TGFβ-induced upregulation of EMyT in lens DCDMLs requires ErbB signaling. (A–C) DCDML cultures of primary lens epithelial cells preincubated for 1 hour with DMSO (vehicle control), the ErbB kinase inhibitor lapatinib or erlotinib, or the specific TGFβ receptor inhibitor SB-431542 (SB4) as indicated were cultured from days 1 to 7 in the presence of TGFβ. Controls were cultured in DMSO only. (A) Cells were then processed for immunofluorescence detection of αSMA and AQP0. Hoechst staining of nuclei is also shown. Typical of four experiments. (B, C) Western blotting of whole-cell lysates of DCDMLs cultured in either 100 µL/well M199/BOTS medium (B) or 200 µL/well M199 medium (C) for FN or αSMA. (D) Results from experiments shown in B and C were quantitated as fold inhibition relative to DMSO + TGFβ controls in the same experiment. For all conditions, P = 0.000. (E) DCDMLs were plated on bovine pdFN and cultured from days 1 to 7 with DMSO or lapatinib prior to analysis of cell lysates for FN or αSMA. Results expressed as percent inhibition by lapatinib compared to DMSO only (n = 3; P ≤ 0.001 for both myofibroblast markers). Note that the avian-specific anti-FN antibody used for Western blotting does not recognize bovine pdFN. For comparison, data are shown for DCDMLs plated under standard conditions (e.g., on laminin [LM]) and treated with DMSO only. (F) DCDMLs were cultured from days 1 to 7 with no additions (ctrl), TGFβ, or 10 nM HB-EGF, TGFα, or NRG1. None of the ErbB ligands increased the expression of FN or αSMA relative to untreated controls in four of four experiments. Similar results were obtained with 1 nM of each ErbB ligand (n = 3).
Figure 1.
 
TGFβ-induced upregulation of EMyT in lens DCDMLs requires ErbB signaling. (A–C) DCDML cultures of primary lens epithelial cells preincubated for 1 hour with DMSO (vehicle control), the ErbB kinase inhibitor lapatinib or erlotinib, or the specific TGFβ receptor inhibitor SB-431542 (SB4) as indicated were cultured from days 1 to 7 in the presence of TGFβ. Controls were cultured in DMSO only. (A) Cells were then processed for immunofluorescence detection of αSMA and AQP0. Hoechst staining of nuclei is also shown. Typical of four experiments. (B, C) Western blotting of whole-cell lysates of DCDMLs cultured in either 100 µL/well M199/BOTS medium (B) or 200 µL/well M199 medium (C) for FN or αSMA. (D) Results from experiments shown in B and C were quantitated as fold inhibition relative to DMSO + TGFβ controls in the same experiment. For all conditions, P = 0.000. (E) DCDMLs were plated on bovine pdFN and cultured from days 1 to 7 with DMSO or lapatinib prior to analysis of cell lysates for FN or αSMA. Results expressed as percent inhibition by lapatinib compared to DMSO only (n = 3; P ≤ 0.001 for both myofibroblast markers). Note that the avian-specific anti-FN antibody used for Western blotting does not recognize bovine pdFN. For comparison, data are shown for DCDMLs plated under standard conditions (e.g., on laminin [LM]) and treated with DMSO only. (F) DCDMLs were cultured from days 1 to 7 with no additions (ctrl), TGFβ, or 10 nM HB-EGF, TGFα, or NRG1. None of the ErbB ligands increased the expression of FN or αSMA relative to untreated controls in four of four experiments. Similar results were obtained with 1 nM of each ErbB ligand (n = 3).
Figure 2.
 
ErbB inhibition does not block TGFβ-induced lens fiber cell differentiation or Smad3 signaling. (A, B) DCDMLs preincubated for 1 hour with DMSO, lapatinib, or erlotinib were cultured with or without TGFβ for 6 days (A) or 90 minutes (B) prior to analysis of the fiber cell markers δ-crystallin, CP115, and CP49 (A) or activation of Smad3 (B) by either metabolic labeling (δ-crystallin)40,44 or Western blot (CP115, CP49; pSmad3). (C) DCDMLs were transfected with the SBE4-Luc reporter construct on day 1 of culture and then incubated on day 2 for 1 hour with DMSO, lapatinib, erlotinib, or the TGFβR inhibitor SB-431542. The cells were then cultured for an additional 48 hours with no additions (0) or TGFβ prior to Western blot analysis of luciferase expression. (D) Results for lapatinib and erlotinib from experiments shown in A to C were quantitated as fold inhibition relative to TGFβ + DMSO controls in the same experiment. In no case did ErbB inhibitors significantly (P < 0.05) reduce the ability of TGFβ to upregulate expression of the indicated protein.
Figure 2.
 
ErbB inhibition does not block TGFβ-induced lens fiber cell differentiation or Smad3 signaling. (A, B) DCDMLs preincubated for 1 hour with DMSO, lapatinib, or erlotinib were cultured with or without TGFβ for 6 days (A) or 90 minutes (B) prior to analysis of the fiber cell markers δ-crystallin, CP115, and CP49 (A) or activation of Smad3 (B) by either metabolic labeling (δ-crystallin)40,44 or Western blot (CP115, CP49; pSmad3). (C) DCDMLs were transfected with the SBE4-Luc reporter construct on day 1 of culture and then incubated on day 2 for 1 hour with DMSO, lapatinib, erlotinib, or the TGFβR inhibitor SB-431542. The cells were then cultured for an additional 48 hours with no additions (0) or TGFβ prior to Western blot analysis of luciferase expression. (D) Results for lapatinib and erlotinib from experiments shown in A to C were quantitated as fold inhibition relative to TGFβ + DMSO controls in the same experiment. In no case did ErbB inhibitors significantly (P < 0.05) reduce the ability of TGFβ to upregulate expression of the indicated protein.
Figure 3.
 
DCDMLs are responsive to ErbB1 and/or ErbB4 ligands. DCDMLs were incubated for 5 minutes with the ErbB ligands HB-EGF (1 nM), TGFα (1 nM), or NRG1 (10 nM), with or without a 1-hour pretreatment with lapatinib (lap). Whole-cell lysates were analyzed for activated forms of ERK (pERK) or AKT (pAKT). Results quantitated as fold increase relative to no growth factor controls (0) in which medium was removed and replaced for 5 minutes (P = 0.000). For comparison, additional cultures were exposed to the ErbB-unrelated ERK and AKT agonist insulin-like growth factor 1 (3 nM). Note that avian cells express the ERK2, but not the ERK1, isoform.
Figure 3.
 
DCDMLs are responsive to ErbB1 and/or ErbB4 ligands. DCDMLs were incubated for 5 minutes with the ErbB ligands HB-EGF (1 nM), TGFα (1 nM), or NRG1 (10 nM), with or without a 1-hour pretreatment with lapatinib (lap). Whole-cell lysates were analyzed for activated forms of ERK (pERK) or AKT (pAKT). Results quantitated as fold increase relative to no growth factor controls (0) in which medium was removed and replaced for 5 minutes (P = 0.000). For comparison, additional cultures were exposed to the ErbB-unrelated ERK and AKT agonist insulin-like growth factor 1 (3 nM). Note that avian cells express the ERK2, but not the ERK1, isoform.
Figure 4.
 
Effect of TGFβ and/or FGF on expression of ErbB1, 2, and 4 in DCDMLs. (A, B) DCDMLs plated under standard conditions (e.g., low density on laminin) were cultured from days 1 to 7 with no additions (0), TGFβ, FGF2, or TGFβ plus FGF2 prior to analysis of ErbBs from either whole-cell lysates (A) or after isolation of the plasma membrane pool by cell surface biotinylation (B). Surface expression in cells cultured with TGFβ is graphed relative to no TGFβ controls (P = 0.000 for all). (C) DCDMLs plated at low density on pdFN were cultured for 6 days with either DMSO or the TGFβR inhibitor SB-431542 to block endogenous TGFβ signaling. Whole-cell lysates were analyzed for the indicated ErbB or αSMA. (D) DCDMLs were plated at higher density on either laminin (LM) or pdFN and then cultured from days 1 to 7 with no additions, TGFβ, DMSO, or SB-431542 prior to whole-cell lysate analysis of ErbB1 and αSMA. In A, C, and D, the arrow denotes the position of ErbB1; the lower band detected in some experiments is a nonspecific, cytosolic species in that it is not recognized by the same antibody in strepavidin-precipitated samples from cell surface biotinylated DCDMLs (B) and is not detected in cell lysates by the anti-ErbB1 rat monoclonal antibody 20.3.6 (Fig. 7B). (E) Expression of the indicated protein in cell lysates from experiments shown in A, C, and D was graphed relative to no growth factor or inhibitor controls plated on the same substrate in the same experiment, all normalized to tubulin. For all, P ≤ 0.01, except bars labeled with either an asterisk (P = 0.032) or NS (P = 0.162).
Figure 4.
 
Effect of TGFβ and/or FGF on expression of ErbB1, 2, and 4 in DCDMLs. (A, B) DCDMLs plated under standard conditions (e.g., low density on laminin) were cultured from days 1 to 7 with no additions (0), TGFβ, FGF2, or TGFβ plus FGF2 prior to analysis of ErbBs from either whole-cell lysates (A) or after isolation of the plasma membrane pool by cell surface biotinylation (B). Surface expression in cells cultured with TGFβ is graphed relative to no TGFβ controls (P = 0.000 for all). (C) DCDMLs plated at low density on pdFN were cultured for 6 days with either DMSO or the TGFβR inhibitor SB-431542 to block endogenous TGFβ signaling. Whole-cell lysates were analyzed for the indicated ErbB or αSMA. (D) DCDMLs were plated at higher density on either laminin (LM) or pdFN and then cultured from days 1 to 7 with no additions, TGFβ, DMSO, or SB-431542 prior to whole-cell lysate analysis of ErbB1 and αSMA. In A, C, and D, the arrow denotes the position of ErbB1; the lower band detected in some experiments is a nonspecific, cytosolic species in that it is not recognized by the same antibody in strepavidin-precipitated samples from cell surface biotinylated DCDMLs (B) and is not detected in cell lysates by the anti-ErbB1 rat monoclonal antibody 20.3.6 (Fig. 7B). (E) Expression of the indicated protein in cell lysates from experiments shown in A, C, and D was graphed relative to no growth factor or inhibitor controls plated on the same substrate in the same experiment, all normalized to tubulin. For all, P ≤ 0.01, except bars labeled with either an asterisk (P = 0.032) or NS (P = 0.162).
Figure 5.
 
ErbB-stimulating activity in DCDML-conditioned medium. Untransfected HEK 293 cells pretreated for 1 hour with either DMSO or lapatinib (lap) were incubated at 4°C for 15 minutes with the medium indicated, prior to analysis of whole-cell lysates with the pan-phosphotyrosine antibody 4G10. All results shown are from the same blot of a single experiment, reprobed with antibodies against total ErbB1 to confirm equal loading. (A) HEKs were incubated at 4°C with fresh medium supplemented with 5-0.5 ng/mL HB-EGF. (B) HEKs were incubated at 4°C with medium conditioned for 2 days by DCDMLs (CM) cultured in either the absence (−) or presence (+) of TGFβ. Mock-conditioned medium (mock) was generated by incubating medium with or without TGFβ for 2 days in the absence of cells. All media were concentrated 5-fold prior to addition to HEK cell recipients.
Figure 5.
 
ErbB-stimulating activity in DCDML-conditioned medium. Untransfected HEK 293 cells pretreated for 1 hour with either DMSO or lapatinib (lap) were incubated at 4°C for 15 minutes with the medium indicated, prior to analysis of whole-cell lysates with the pan-phosphotyrosine antibody 4G10. All results shown are from the same blot of a single experiment, reprobed with antibodies against total ErbB1 to confirm equal loading. (A) HEKs were incubated at 4°C with fresh medium supplemented with 5-0.5 ng/mL HB-EGF. (B) HEKs were incubated at 4°C with medium conditioned for 2 days by DCDMLs (CM) cultured in either the absence (−) or presence (+) of TGFβ. Mock-conditioned medium (mock) was generated by incubating medium with or without TGFβ for 2 days in the absence of cells. All media were concentrated 5-fold prior to addition to HEK cell recipients.
Figure 6.
 
Basal ErbB activity in DCDMLs. DCDMLs pretreated with either DMSO or lapatinib (lap) were incubated at 4°C for 15 minutes in either the absence or presence of 10 nM HB-EGF as indicated prior to cell surface biotinylation. Strepavidin precipitates of biotin-labeled plasma membrane proteins were run on SDS-PAGE and blotted for phosphotyrosine, followed by reprobing with antibodies against total ErbB1 to confirm the position of ErbBs. Equal portions of whole-cell lysate were analyzed for β-actin to ensure equal loading. Typical of six independent experiments.
Figure 6.
 
Basal ErbB activity in DCDMLs. DCDMLs pretreated with either DMSO or lapatinib (lap) were incubated at 4°C for 15 minutes in either the absence or presence of 10 nM HB-EGF as indicated prior to cell surface biotinylation. Strepavidin precipitates of biotin-labeled plasma membrane proteins were run on SDS-PAGE and blotted for phosphotyrosine, followed by reprobing with antibodies against total ErbB1 to confirm the position of ErbBs. Equal portions of whole-cell lysate were analyzed for β-actin to ensure equal loading. Typical of six independent experiments.
Figure 7.
 
ErbB1 is active in DCDMLs. (A) DCDMLs were cultured for 24 hours in the presence of DMSO or lapatinib (lap) prior to analysis of ErbB1 or ErbB4 from either whole-cell lysates (total) or after isolation of the plasma membrane pool by cell surface biotinylation (cell surface). The level of ErbB1 and ErbB4 recovered from lapatinib-treated cells is graphed relative to DMSO-only controls in the same experiment. (B) DCDMLs were pretreated for 1 hour at 37°C with DMSO or lapatinib (lap) and incubated for 5 minutes at 37°C in the presence or absence of 10 nM HB-EGF prior to analysis of whole-cell lysates with a rabbit antibody specific for the Y1068 autophosphorylated, activated form of ErbB1. The blot was reprobed with the rat anti-ErbB1 20.3.6 antibody to detect total ErbB1. Arrow in A denotes the position of ErbB1; the lower band is a nonspecific, cytosolic species not recognized by the rat 20.3.6 monoclonal.
Figure 7.
 
ErbB1 is active in DCDMLs. (A) DCDMLs were cultured for 24 hours in the presence of DMSO or lapatinib (lap) prior to analysis of ErbB1 or ErbB4 from either whole-cell lysates (total) or after isolation of the plasma membrane pool by cell surface biotinylation (cell surface). The level of ErbB1 and ErbB4 recovered from lapatinib-treated cells is graphed relative to DMSO-only controls in the same experiment. (B) DCDMLs were pretreated for 1 hour at 37°C with DMSO or lapatinib (lap) and incubated for 5 minutes at 37°C in the presence or absence of 10 nM HB-EGF prior to analysis of whole-cell lysates with a rabbit antibody specific for the Y1068 autophosphorylated, activated form of ErbB1. The blot was reprobed with the rat anti-ErbB1 20.3.6 antibody to detect total ErbB1. Arrow in A denotes the position of ErbB1; the lower band is a nonspecific, cytosolic species not recognized by the rat 20.3.6 monoclonal.
Figure 8.
 
ErbB1 behaves like a heterodimer in DCDMLs cultured without TGFβ but like a homodimer in plus TGFβ cells. (A–C) Lens cells plated on laminin (LM) or on pdFN were cultured from days 1 to 7 with or without TGFβ as indicated. The cells were then incubated at 37°C for 4 hours in the absence or presence of high levels (10 nM) of HB-EGF (A, B) or NRG1 (C) prior to analysis of ErbBs from either whole-cell lysates (A, C) or after isolation of the plasma membrane pool by cell surface biotinylation (B). Levels of ErbB graphed relative to no-ligand controls in the same experiment. (A) Increased expression of ErbB1 induced by either addition of exogenous TGFβ or plating on pdFN enhances the loss of total cellular ErbB1 in response to HBEGF. The graph also shows that a 1-hour pretreatment with lapatinib (+lap) blocks downregulation of ErbB1 after exposure to HB-EGF. NS, P ≥ 0.06. (B) Cell surface biotinylation confirms the loss of ErbB1 from the surface of TGFβ-cultured, HB-EGF–treated cells. (C) Culturing DCDMLs with TGFβ renders ErbB1 insensitive to downregulation by the ErbB4 ligand NRG1.
Figure 8.
 
ErbB1 behaves like a heterodimer in DCDMLs cultured without TGFβ but like a homodimer in plus TGFβ cells. (A–C) Lens cells plated on laminin (LM) or on pdFN were cultured from days 1 to 7 with or without TGFβ as indicated. The cells were then incubated at 37°C for 4 hours in the absence or presence of high levels (10 nM) of HB-EGF (A, B) or NRG1 (C) prior to analysis of ErbBs from either whole-cell lysates (A, C) or after isolation of the plasma membrane pool by cell surface biotinylation (B). Levels of ErbB graphed relative to no-ligand controls in the same experiment. (A) Increased expression of ErbB1 induced by either addition of exogenous TGFβ or plating on pdFN enhances the loss of total cellular ErbB1 in response to HBEGF. The graph also shows that a 1-hour pretreatment with lapatinib (+lap) blocks downregulation of ErbB1 after exposure to HB-EGF. NS, P ≥ 0.06. (B) Cell surface biotinylation confirms the loss of ErbB1 from the surface of TGFβ-cultured, HB-EGF–treated cells. (C) Culturing DCDMLs with TGFβ renders ErbB1 insensitive to downregulation by the ErbB4 ligand NRG1.
Figure 9.
 
Effect of TGFβ on ErbB function. (A) TGFβ does not change the level of endogenously active ErbBs in DCDMLs. DCDMLs were cultured for 6 days in the absence or presence of TGFβ. Cells were then cell surface biotinylated prior to analysis of plasma membrane proteins using antibodies against phosphotyrosine (4G10; phospho-Y) or specific for the Y1068 autophosphorylated form of ErbB1 as indicated. Blots were reprobed with antibodies against total ErbB1. Data graphed as fold versus no TGFβ control cultures in the same experiment. (B) ErbB1 induced by TGFβ is activatable by exogenous ligand. DCDMLs cultured for 6 days with or without TGFβ were incubated at 4°C for 15 minutes in either the absence or presence of 10 nM TGFα as indicated. Whole-cell lysates were then analyzed by Western blot with antiphosphotyrosine or anti-pY1068 ErbB1 antibodies, followed by reprobing for total ErbB1. Data graphed as fold versus no TGFβ, + TGFα cultures in the same experiment.
Figure 9.
 
Effect of TGFβ on ErbB function. (A) TGFβ does not change the level of endogenously active ErbBs in DCDMLs. DCDMLs were cultured for 6 days in the absence or presence of TGFβ. Cells were then cell surface biotinylated prior to analysis of plasma membrane proteins using antibodies against phosphotyrosine (4G10; phospho-Y) or specific for the Y1068 autophosphorylated form of ErbB1 as indicated. Blots were reprobed with antibodies against total ErbB1. Data graphed as fold versus no TGFβ control cultures in the same experiment. (B) ErbB1 induced by TGFβ is activatable by exogenous ligand. DCDMLs cultured for 6 days with or without TGFβ were incubated at 4°C for 15 minutes in either the absence or presence of 10 nM TGFα as indicated. Whole-cell lysates were then analyzed by Western blot with antiphosphotyrosine or anti-pY1068 ErbB1 antibodies, followed by reprobing for total ErbB1. Data graphed as fold versus no TGFβ, + TGFα cultures in the same experiment.
Figure 10.
 
Effect of transient transfection of ErbB1, ErbB2, or ErbB4 in DCDMLs on αSMA. (A) Forced overexpression of ErbB 1, 2, or 4 does not prevent TGFβ from upregulating αSMA, FN, or CP49. DCDMLs were transiently transfected with plasmids encoding ErbB1-GFP, ErbB2-GFP, or ErbB4 on day 1 and cultured for 6 more days either with or without TGFβ prior to analysis of whole-cell lysates for the indicated protein. Controls were transfected with a plasmid encoding an irrelevant integral membrane protein (E208K Cx32).67 (B) Overexpression of ErbB1 selectively enhances αSMA in the absence of TGFβ. DCDMLs transfected with control or ErbB1-GFP (R1), ErbB2-GFP (R2), or ErbB4 (R4) plasmid were cultured for 6 days without TGFβ prior to Western blot assessment of αSMA and tubulin. (C) The level of αSMA in the presence of each exogenously expressed ErbB was graphed relative to the level of αSMA in control transfectants, all without TGFβ (n = 4). Overexpression of ErbB over endogenous ErbB protein achieved in the experiments quantitated was 75-206X (ErbB4), 61-358X (ErbB2), and 71-205X (ErbB1).
Figure 10.
 
Effect of transient transfection of ErbB1, ErbB2, or ErbB4 in DCDMLs on αSMA. (A) Forced overexpression of ErbB 1, 2, or 4 does not prevent TGFβ from upregulating αSMA, FN, or CP49. DCDMLs were transiently transfected with plasmids encoding ErbB1-GFP, ErbB2-GFP, or ErbB4 on day 1 and cultured for 6 more days either with or without TGFβ prior to analysis of whole-cell lysates for the indicated protein. Controls were transfected with a plasmid encoding an irrelevant integral membrane protein (E208K Cx32).67 (B) Overexpression of ErbB1 selectively enhances αSMA in the absence of TGFβ. DCDMLs transfected with control or ErbB1-GFP (R1), ErbB2-GFP (R2), or ErbB4 (R4) plasmid were cultured for 6 days without TGFβ prior to Western blot assessment of αSMA and tubulin. (C) The level of αSMA in the presence of each exogenously expressed ErbB was graphed relative to the level of αSMA in control transfectants, all without TGFβ (n = 4). Overexpression of ErbB over endogenous ErbB protein achieved in the experiments quantitated was 75-206X (ErbB4), 61-358X (ErbB2), and 71-205X (ErbB1).
Figure 11.
 
A single 1-hour, high-dose treatment with lapatinib blocks ErbB1 activity (A) and TGFβ-induced EMyT (B) for 6 days. DCDMLs were incubated on day 1 for 1 hour with 40 µM lapatinib or DMSO vehicle only. Medium was removed and the cells were cultured without drug for 6 days in the absence or presence of TGFβ. (A) Whole-cell lysates were analyzed for endogenous active ErbB using rabbit anti-pY1068 ErbB1 antibodies (n = 6). (B) Whole-cell lysates were analyzed for the EMyT markers FN and αSMA and for the fiber cell differentiation markers CP115 and CP49. Results quantitated as fold inhibition relative to controls treated with DMSO and then TGFβ in the same experiment. *P = 0.024. **P = 0.000.
Figure 11.
 
A single 1-hour, high-dose treatment with lapatinib blocks ErbB1 activity (A) and TGFβ-induced EMyT (B) for 6 days. DCDMLs were incubated on day 1 for 1 hour with 40 µM lapatinib or DMSO vehicle only. Medium was removed and the cells were cultured without drug for 6 days in the absence or presence of TGFβ. (A) Whole-cell lysates were analyzed for endogenous active ErbB using rabbit anti-pY1068 ErbB1 antibodies (n = 6). (B) Whole-cell lysates were analyzed for the EMyT markers FN and αSMA and for the fiber cell differentiation markers CP115 and CP49. Results quantitated as fold inhibition relative to controls treated with DMSO and then TGFβ in the same experiment. *P = 0.024. **P = 0.000.
Figure 12.
 
A 1-hour treatment with lapatinib combines with a non-ErbB-targeted therapeutic to inhibit lens cell fibrosis. (A) Rebastinib does not hinder ligand-induced stimulation of ErbBs in DCDMLs. DCDMLs were preincubated with DMSO or 0.5 µM rebastinib (reb) for 4 hours prior to a 15-minute, 4°C treatment with either HB-EGF or TGFα. Autoactivation of ErbBs was assessed by Western blot of whole-cell lysates using mouse antibodies against either phosphotyrosine (to measure active ErbB1, ErbB2, and ErbB4) or pY1068 ErbB1 (to specifically assess active ErbB1). No inhibition by rebastinib was detected in five of five experiments (P = 0.000). (B) Lapatinib does not target activation of p38. DCDMLs were preincubated for 2 hours with DMSO or 4 µM lapatinib (lap) prior to a 90-minute incubation with 4 ng/mL TGFβ, 10 ng/mL FGF-2, or 3 µg/mL anisomycin. Whole-cell lysates were analyzed for active (pT180/pY182) p38 and total p38 by Western blotting. Typical of four experiments. (C) A single 1-hour treatment with 4 µM lapatinib combined with rebastinib blocks TGFβ-induced EMyT for 6 days. DCDMLs were treated for 1 hour with 0.5 µM rebastinib, 4 µM lapatinib, or both, after which the drug-containing medium was removed. Cells were then cultured for 6 days with TGFβ. Controls were pretreated with DMSO only and cultured with or without TGFβ. Western blots of whole-cell lysates were probed with antibodies against the indicated protein and results graphed as fold expression relative to DMSO, then TGFβ-treated controls. Asterisks indicate P ≤ 0.010 inhibition compared to control (n = 4).
Figure 12.
 
A 1-hour treatment with lapatinib combines with a non-ErbB-targeted therapeutic to inhibit lens cell fibrosis. (A) Rebastinib does not hinder ligand-induced stimulation of ErbBs in DCDMLs. DCDMLs were preincubated with DMSO or 0.5 µM rebastinib (reb) for 4 hours prior to a 15-minute, 4°C treatment with either HB-EGF or TGFα. Autoactivation of ErbBs was assessed by Western blot of whole-cell lysates using mouse antibodies against either phosphotyrosine (to measure active ErbB1, ErbB2, and ErbB4) or pY1068 ErbB1 (to specifically assess active ErbB1). No inhibition by rebastinib was detected in five of five experiments (P = 0.000). (B) Lapatinib does not target activation of p38. DCDMLs were preincubated for 2 hours with DMSO or 4 µM lapatinib (lap) prior to a 90-minute incubation with 4 ng/mL TGFβ, 10 ng/mL FGF-2, or 3 µg/mL anisomycin. Whole-cell lysates were analyzed for active (pT180/pY182) p38 and total p38 by Western blotting. Typical of four experiments. (C) A single 1-hour treatment with 4 µM lapatinib combined with rebastinib blocks TGFβ-induced EMyT for 6 days. DCDMLs were treated for 1 hour with 0.5 µM rebastinib, 4 µM lapatinib, or both, after which the drug-containing medium was removed. Cells were then cultured for 6 days with TGFβ. Controls were pretreated with DMSO only and cultured with or without TGFβ. Western blots of whole-cell lysates were probed with antibodies against the indicated protein and results graphed as fold expression relative to DMSO, then TGFβ-treated controls. Asterisks indicate P ≤ 0.010 inhibition compared to control (n = 4).
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