Differentiation of Chick Lens Epithelial Cells: Involvement of the Epidermal Growth Factor Receptor and Endogenous Ligand

Abstract

purpose. To characterize the constitutively activated epidermal growth factor receptor in a lens epithelial cell population experiencing initial stages of lens fiber formation, the chick lens annular pad.

methods. Phosphotyrosine levels of the receptor were examined with western blot analysis and immunoprecipitation after ligand stimulation. Endogenous receptor ligands were immunologically identified in whole cell lysates of freshly isolated cells. The expression of lens fiber–specific differentiation marker proteins was examined with western blot analysis and enzyme-linked immunosorbent assay (ELISA) in short-term primary cultures of annular pad cells exposed to ligand.

results. The major phosphotyrosine-containing protein in annular pad cells comigrated with the epidermal growth factor receptor and increased its phosphotyrosine content after epidermal growth factor treatment. Both time- and dose-dependent responses were noted. The constitutive activation of the receptor was determined in the presence of phosphatase inhibitors. Endogenous transforming growth factor-α, but not epidermal growth factor, was detected in freshly isolated cells. Transforming growth factor-α (TGF-α) treatment produced greater increases in receptor phosphotyrosine levels than equimolar levels of epidermal growth factor. Finally, TGF-α treatment induced increased expression of the beaded filament protein filensin when compared with control cells. Filensin expression was increased further when cells were costimulated with TGF-α and cAMP analogs.

conclusions. At least in the postnatal lens, endogenous TGF-α may affect overall growth patterns by modulating differentiation-specific protein expression. Furthermore, signaling pathways elicited by TGF-α and cAMP analogs converge to cooperatively enhance lens fiber differentiation.

The control of lens growth throughout the life of an organism essentially involves two closely related processes. First, the production of cells (i.e., cell division) must be temporally and spatially regulated because the lens continues to grow in size by retaining every cell that has gone into its formation. Second, the terminal differentiation of lens fibers, which form the bulk of any lens in all but the earliest of developmental stages, must also be similarly regulated to produce a highly ordered and transparent refractive apparatus. Numerous receptor-mediated events are known to influence each process. Growth factors like the fibroblast growth factors (FGF), insulin-like growth factor-1, platelet-derived growth factor, and epidermal growth factor (EGF) promote normal lens growth patterns ^{1 2} as well as the expression of morphologic and biochemical characteristics of lens fiber differentiation. ^{3 4 5} Pathways involving adrenergic receptors have also been shown to influence crucial aspects of lens growth and differentiation. ^{6 7 8} Cooperative and synergistic effects of growth factors indicate that inter-related or intersecting signaling networks must also be developmentally/temporally integrated during normal lens growth. ^{2 3 4 5} Additional levels of regulatory mechanisms affecting lens development can be implied by reports showing the endogenous presence of both protein and message for a variety of growth regulatory substances. ^{9 10 11 12 13} Alterations in the continuous processes of cell division and fiber cell differentiation due to genetic mutations or biochemical alterations in the ocular environment may lead, in some cases, to the appearance of cataract. 

Both major aspects of lens growth can be affected by the same ligands. Although a strong and convincing case has been made for FGF concentrations being of prime importance in the regulation of cell division and differentiation, ¹⁴ other growth factors may have similar effects. ^{1 2 15 16 17} A consensus on what mechanisms are of prime importance in controlling lens growth may be difficult to arrive at due to the various models used (e.g., organ culture, explant culture, passaged cells), genuine species differences, or some degree of receptor-mediated signaling redundancy. Alternatively, the multiple processes that must be successfully integrated during normal fiber development may each require a unique set of temporally or spatially integrated signaling cues. We have chosen to study how lens fiber terminal differentiation is regulated in the juvenile chicken lens. This species presents an epithelial specialization known as the annular pad, which is composed of post-mitotic cells committed to and undergoing initial stages of lens fiber formation. ¹⁸ At least with regard to lens fiber formation, this model may provide additional mechanistic insights when compared with paradigms using central epithelial cells, extensively passaged/immortalized cultured cells, or pluripotent embryonic epithelial populations. 

In this report, we begin the characterization of epidermal growth factor receptors (EGFRs) in annular pad cells. We show that the EGFR is the major phosphotyrosine (PY) containing protein in cells experiencing initial stages of fiber development and that receptor activity may be influenced by endogenous ligands. In addition, receptor stimulation results in the increased expression of differentiated characteristics and augments cell signaling pathways previously implicated in sustaining lens fiber terminal differentiation. These data support the hypothesis that EGFRs, aside from influencing mitotic activity, may contribute to overall lens growth patterns after birth by increasing the expression of differentiation-specific proteins. 

Methods

Materials

Unless otherwise indicated, all reagents were from Sigma Chemical (St. Louis, MO). Cell culture supplies were from Celox (St. Paul, MN). Tissue culture plastic ware was from Corning Glass Works (Corning, NY). Antibodies were from the following sources: mouse monoclonal (clone PY20) anti-PY, rabbit polyclonal anti-recombinant human EGF, goat polyclonal anti-human transforming growth factor-α (Santa Cruz Biotechnology, Santa Cruz, CA); sheep polyclonal anti-human epidermal growth factor receptor (Upstate Biotechnology, Lake Placid, NY); and the previously characterized rabbit polyclonal anti-chicken filensin. ¹⁹ A431 cell lysates were from Upstate Biotechnology. Mouse EGF was purchased from Collaborative Biomedical/Becton Dickinson (Bedford, MA), and human recombinant transforming growth factor-α (TGF-α) was from Sigma Chemical. 

Cell Isolation and Treatment

All experiments were performed under the Guide for the Care and Use of Laboratory Animals, National Institutes of Health Publication No. 85-23 (revised 1985). Annular pad cells from freshly killed juvenile chickens (2–3 months of age) were isolated as previously described and placed in Medium 199 (M199) supplemented with 1 μCg/ml of pepstatin A, leupeptin, aprotinin, 1,10-phenanthroline, and benzamidine. ⁷ After a gentle trituration, the cells were layered onto a discontinuous gradient composed of 10% to 20% to 30% sucrose made up in M199. Cells were allowed to settle for 10 minutes before collection. Cells that had sedimented through the gradient or collected at the 20% to 30% and 10% to 20% interfaces were retrieved and combined. This procedure predominantly yields aggregates of 10 to 200 cells, whereas individual cells and cellular debris do not enter the 10% sucrose layer. Cell aggregates were rinsed several times with M199 supplemented with protease inhibitors and phosphatase inhibitors (2 mM sodium fluoride and 1 mM sodium orthovanadate). Aliquots were placed in microfuge tubes and stimulated with the indicated compounds for the indicated periods of time at 37°C in a humidified 95% air/5% CO₂ atmosphere. Cells were rapidly sedimented and dissolved in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer supplemented with phosphatase inhibitors. Protein concentrations of samples prepared for SDS–PAGE were determined as previously described ²⁰ so that equal amounts of protein could be subsequently compared. For the detection of endogenous growth factors, column aliquots were immediately dissolved in SDS–PAGE sample buffer. 

Immunoprecipitation

In some experiments, aliquots of treated cells were rinsed with ice-cold phosphate-buffered saline (PBS) supplemented with phosphatase inhibitors and lysed in cold 25 mM Tris–HCl (pH 7.5), 100 mM NaCl, and 1% NP-40 supplemented with protease inhibitors. All procedures were carried out at 4°C. Samples were tumbled end-over-end for 1 hour before microfuging at 13,000g for 15 minutes. Supernatants were transferred to fresh tubes, and 10 μg/ml of anti-EGFR was added. After overnight tumbling, a slurry of protein A–Sepharose beads was added to the lysate (50 μl protein A/ml lysate) and incubated for 3 hours. Beads were sedimented by microfuging at 10g for 2 minutes. Supernatants were removed, and the pellet was washed once with low salt HTNG (50 mM Tris–HCl [pH7.5], 150 mM NaCl, 0.1% NP-40, 10% glycerol), twice with high salt HTNG (same as low salt HTNG but with 500 mM NaCl), and once more with low salt HTNG. Twenty microliters of 0.25 M Tris–HCl (pH 6.8), 8% SDS, 40% glycerol, 0.1 mg/ml bromophenol blue, and 1% β-mercaptoethanol was added to each sample. Samples were boiled for 4 minutes, beads were spun down, and the entire supernatant loaded onto a polyacrylamide gel. 

SDS–PAGE and Western Blot Analysis

Samples were electrophoresed on 7%, 10%, or 15% polyacrylamide gels using the discontinuous buffer system of Laemmli. ²¹ After separation, proteins were electrophoretically transferred to Immobilon-P membranes (Millipore, Bedford, MA) according to the method described by Towbin et al. ²² Antigen visualization was accomplished with ECL using horseradish peroxide–conjugated secondary antibodies according to the manufacturer’s specifications (Amersham, Arlington Heights, IL). With immunoprecipitates, after visualization of PY, blots were stripped by incubating the blots for 30 minutes at 50°C in 62.5 mM Tris–HCl (pH 6.7), 100 mM β-mercaptoethanol, and 2% SDS and then reprobed with anti-EGFR antibodies. 

Primary Cell Culture

Annular pad cells were isolated as above and immediately cultured in 6-well plates coated overnight with a 1% Matrigel solution (Collaborative Biomedical/Becton Dickinson), as previously described. ²³ Cultures were treated immediately after plating and every 24 hours thereafter for a total of 3 days. On the fourth day, cultures were terminated by replacing the media with ice-cold PBS supplemented with protease inhibitors, scraping the cells with a rubber policeman, transferring the cells to a microfuge tube, rinsing several times with buffer, and dissolving the cells in SDS–PAGE sample buffer. 

ELISA Quantification

For quantitative measurements, primary cultures were established and grown in 5% fetal bovine serum (FBS) for 3 to 5 days. Single cell suspensions were obtained after trypsinization, and 75,000 cells were placed into the wells of a 24-well plate coated as above. We have found that trypsinizing and plating short-term primary cultures gives consistently more reliable results than trypsinizing and plating freshly isolated annular pad cells (data not shown). Cells were maintained in serum-free media and treated as indicated immediately after plating and every 24 hours thereafter for a total of 3 days. Each treatment was examined in quadruplicate. Cultures were terminated by removing the media and adding 0.5% Triton X-100 in PBS to the wells. The solution was allowed to completely dry down at 50°C for 2 days. The remaining antibody incubation procedures were performed at 37°C. The wells were blocked for 1 hour with 3% gelatin in Tris-buffered saline containing Tween 20 (TTBS: 0.1 M Tris–HCl [pH 7.4], 0.15 M NaCl, 0.05% Tween 20). The plate was rinsed three times with TTBS, and the wells were then reacted with a 1:250 dilution of anti-filensin primary antibody in 1% gelatin/TTBS for 1 hour. The wells were again rinsed three times with TTBS before the addition of a 1:400 dilution of secondary antibody (goat anti-rabbit IgG, horseradish peroxidase–conjugated) in 1% gelatin/TTBS for 1 hour. The wells were rinsed again and reacted for 10 minutes with 2,2′-azino-bis(3 ethylbenzothiazoline-6-sulfonic acid) (ABTS) developing solution (100 μl ABTS, 100 μl 1% H₂O₂, 10 ml 0.5 M sodium citrate buffer; Zymed Laboratories, San Francisco, CA) at room temperature. The plate was then read with a Bio-Tek EL 311SX microplate reader (Bio-Tek Instruments, Winooski, VT) at 405 nm to yield individual optical density values for each well. Data were analyzed using unpaired Student’s one-tailed t-tests. Nonspecific color development from empty wells was subtracted from all measurements. A level of P < 0.05 was accepted as statistically significant. 

Results

Characteristics of the EGFR in Annular Pad Cells

The major PY-containing protein in freshly isolated annular pad cells comigrates with the EGFR receptor, which is overexpressed in A431 cells (Fig. 1) . After EGF stimulation, the PY content of the putative EGFR was increased significantly along with several other unidentified protein species. Both dose- and time-dependent increases in the annular pad EGFR PY levels were also observed (Fig. 2) . Significant changes in the phosphotyrosine level of the annular pad EGFR were elicited within 5 minutes of stimulation and by as little as 10 ng/ml of EGF. In addition, the tyrosine kinase activity of the EGFR could be greatly reduced with the tyrosine kinase inhibitor genistein (Fig. 3) . The apparent high intrinsic activity of the annular pad EGFR, as evidenced by its PY content in freshly isolated cells, was further examined by exposing cells to increasing levels of phosphatase inhibitors. This produced a dose-dependent increase in PY content of the EGFR in nonstimulated cells. Identical experiments in which cells were stimulated with EGF produced a further elevation of the EGFR PY content (Fig. 4) . These experiments indicate that the annular pad EGFRs may have been undergoing ligand binding at the time of isolation, resulting in their apparent constitutive activity. Experiments conducted with freshly isolated superficial cortical fiber cells identified a PY-containing band that comigrated with the A431 EGFR. However, stimulation with EGF failed to affect the PY content of the putative lens fiber EGFR (data not shown). 

One way to account for the constitutive activity of annular pad EGFRs would be through the presence of endogenous ligands. This possibility was examined by immunostaining whole cell lysates of freshly isolated annular pad cells for the presence of EGF and TGF-α, well characterized ligands for the EGFR. In addition to reacting with an exogenous source of TGF-α, the antibodies used in this study also identified a comigrating low abundance protein in annular pad cells (Fig. 5) . Complementary studies using antibodies that recognize authentic EGF failed to identify a cross-reacting species in annular pad cells. 

The sensitivity of annular pad EGFRs to stimulation by EGF or TGF-α was examined with immunoprecipitation (Fig. 6) . When aliquots of freshly isolated annular pad cells were stimulated with equimolar concentrations of EGF or TGF-α followed by immunoprecipitation of the EGFR, TGF-α produced a greater increase in PY content than EGF. To confirm that equal amounts of EGFR were being compared, the blot was stripped and then reprobed with the original EGFR antibody. 

EGFR Stimulation and Annular Pad Differentiation

Because annular pad cells are in the early stages of lens fiber terminal differentiation, ¹⁸ we wondered whether the high constitutive activity of the EGFR might be influencing some aspect(s) of normal fiber cell development. To examine this possibility, we treated primary cultures of freshly isolated annular pad cells maintained in serum-free media with TGF-α, the potential endogenous EGFR ligand, and 8-bromoadenosine 3′:5-cyclic monophosphate (8bcAMP), a previously identified factor in promoting the differentiation of chick lens annular pad cells. ^{7 8 23} After 3 days of culture, both treatments caused the increased accumulation of filensin, a novel intermediate filament family member whose expression is restricted to differentiating lens fiber cells (Fig. 7) . When combined, the two treatments resulted in an even greater effect on the expression of filensin. Densitometric analysis of Figure 7 indicated that TGF-α or 8bcAMP induced a 1.5- to 1.6-fold increase in filensin immunoreactivity, and combining the treatments yielded a 1.9-fold increase. Complementary statistically significant levels of filensin accumulation were obtained with passaged cells similarly treated but analyzed with the ELISA methodology (Fig. 7 , inset). 

Discussion

The present results indicate that functional EGFRs in the juvenile chicken lens annular pad influence protein expression during early stages of fiber terminal differentiation. We were able to accomplish this by using an abundant, routinely accessible cell population composed of post-mitotic epithelial cells committed to and undergoing initial stages of lens fiber formation. The functional nature of the annular pad EGFR was determined by observing rapid, dose-dependent increases in their PY content and the ability of genistein, a tyrosine kinase inhibitor, to reduce receptor autophosphorylation in response to ligand binding. The apparent constitutive activity of annular pad EGFRs may be due to the presence of endogenous TGF-α, an EGFR ligand with a greater capacity than EGF to stimulate receptor tyrosine autophosphorylation. Exogenous TGF-α was also shown to positively influence the increased expression of fiber cell–specific proteins during short-term primary culture. Finally, stimulation of annular pad EGFRs cooperatively increases differentiation-specific protein accumulation elicited by previously characterized cAMP-mediated mechanisms. Whether EGFR-generated signals augment the cAMP response or operate through a separate signaling pathway remains to be determined. 

Is There a Role for the EGFR during Lens Growth and Development?

A role for the EGFR and its relevant ligands in normal lens development has not been established with certainty. Available evidence indicates that embryonic lens development does not involve the EGFR, whereas postnatal and adult lens growth does seem to be influenced by the EGFR in several species including humans. 

The EGFR could not be detected using in situ hybridization in embryonic lenses from mice engineered to express altered components of this signaling system. ^{24 25} With knockouts of receptor function, lens abnormalities can most easily be attributed to mechanical trauma resulting from the thin fibrotic corneas that develop and open eyelids at birth. ^{24 26} This leads to the prolapse and adherence of the lens to the cornea. Mouse mutants with ablated tyrosine kinase activity of the EGFR or engineered for TGF-α deficiency also show gross lens abnormalities or no lens at all. ^{27 28} In these cases, it is fairly certain that the abnormalities do not arise as a differentiation defect but rather are due to failure of the lens to separate from the cornea, failure of an anterior chamber to form, or extrusion of the lens through the underdeveloped cornea. With lenticular TGF-α overexpressors, ^{25 29} the perioptic mesenchyme proliferates and migrates abnormally to surround the lens, most likely in response to the high levels of TGF-α being released locally into the eye globe. Until this point, the lens develops normally. It is felt that the abnormal presence of the perioptic mesenchyme effectively deprives the developing lens of important developmental factors originating in the surrounding ocular tissues. ^{30 31}  

Although clearly not implicated during embryonic lens development, the EGFR may have additional significant roles during postnatal and adult life, the time when most lens growth occurs. Similar to the mouse, the embryonic chicken lens does not apparently express the EGFR as assessed by reverse transcription–polymerase chain reaction (RT–PCR). ³² However, the current data and additional studies with cultured cells and freshly isolated tissues from postnatal and adult lenses provide strong evidence for an important role for the EGFR in maintaining lens growth patterns. The presence of EGFRs in cultured lens epithelial cells from several species was indicated through the use of conventional receptor binding assays. ^{33 34} More recently, RT–PCR has been used to amplify the message for the EGFR in cultures of rabbit and human epithelial cells and in freshly isolated rabbit epithelial cells. ³⁵ The effects of receptor occupancy on lens epithelial cell behavior have been dependent on experimental conditions and the lineage of the cell type examined. In general, EGF has been shown to be a potent mitogen for normally amitotic central epithelial cells in organ culture or in cultures derived from central epithelial cells. ^{17 33} Similar proliferative responses to TGF-α have also been noted in cultured central epithelial cells. ³⁶ However, cultures initiated from more peripheral regions of the anterior epithelium (which may be more annular pad–like with regard to fiber cell commitment) did not proliferate in response to EGF. ³⁴ This indicates that central and the most peripheral lens epithelial cells may respond differentially to EGF or TGF-α treatment. In preliminary experiments, we have not observed any significant proliferative response during the time course of our experiments in response to either EGF or TGF-α (data not shown). To date, a role for EGF/TGF-α in regulating the cell division responsible for continuous lens growth remains to be determined. EGF has also been implicated, along with several other growth factors, in promoting the appearance of lens fiber–like structures called lentoids in cultured human epithelial cells. ^{15 16} However in these studies, lentoid formation occurred only after vigorous proliferation and therefore may be a secondary response to cellular crowding. Our results clearly show that EGFR occupancy may directly and significantly increase the accumulation of differentiation-specific cytoskeletal proteins in short-term cell culture in the absence of any significant cell proliferation. 

Cooperativity and Synergism during EGFR-Mediated Processes

Accumulating evidence suggests that whatever function(s) the EGFR mediates during normal lens growth and development, additional signaling systems are required that cooperate with or synergistically enhance the effects of EGFR-ligand interactions. Although normal rat lens growth and transparency optimally require the simultaneous pulsatile application of insulin plus platelet-derived growth factor or EGF during organ culture, it is highly probable that these treatments support both coordinated cell division and lens fiber differentiation. ² Similarly, in rat and human lens cells, DNA synthesis depends on costimulation with EGF and insulin. ^{37 38} Analogous situations also occur in the rat that influence the regulation of cell division and fiber differentiation in response to FGF and insulin-like growth factor-1 or insulin. ^{3 4 5} These later studies also suggest that post-receptor signaling via protein kinase C is an integral part of the FGF cellular response. ⁵ Our data indicate that protein kinase A could also be involved in augmenting differentiation as evidenced by the effects of cAMP analogs. However, it is not clear whether cyclic nucleotide production is affected through EGFR stimulation or whether previously characterized β-adrenergic receptors, possibly responding to cyclical levels of aqueous humor catecholamines, can fulfill this role. ^{39 40 41 42 43}  

Endogenous Control of EGFR Actions

The ligand for presentation to lens EGFRs could be derived from several sources. EGF applied topically on the corneal surface is rapidly taken up by and retained within lens cells. ⁴⁴ Therefore, EGF detected within the aqueous humor could readily be an exogenous source for lens stimulation (see ^{Ref. 45} and references within). In those instances where EGF has been found in the aqueous, its cellular origin could not be determined. An alternative endogenous source for EGF has also been proposed in human and rabbit lenses. ^{34 46} In these studies, lenticular EGF was found in regions that include the most peripheral epithelial cells plus superficial cortical fibers, which again supports the hypothesis that EGFRs may also influence certain aspects of fiber cell differentiation. Interestingly, EGF levels were often found to be significantly elevated in relation to cataract formation. ⁴⁶ Our data indicate that endogenous TGF-α, which is structurally related to EGF and elicits greater EGFR tyrosine phosphorylation, is the likely source for EGFR stimulation in the chick lens. Similar observations localizing TGF-α in rabbit epithelial and superficial cortical fibers have been made with immunohistochemistry. ³⁶ Because TGF-α typically influences neighboring cells in a paracrine or juxtacrine fashion, ⁴⁷ it is therefore a prime candidate for the endogenous regulation of gene expression during epithelial differentiation into lens fibers, at least in the chicken. In this regard, autocrine stimulation affecting lens cell behaviors must also be considered. Highly localized effects of endogenous stores of TGF-α would also account for the apparent constitutive activity of the EGFR within the annular pad, a cell population committed to and experiencing initial stages of fiber cell differentiation. 

In summary, we have shown that the EGFR may have a meaningful role in the endogenous control of lens fiber formation with regard to upregulating the expression of differentiation-specific proteins during postnatal lens growth. This was shown by determining that EGFRs in a cell population undergoing initial stages of fiber formation are normally activated and that this activation may be in response to endogenous stores of ligand. Furthermore, eliciting differentiated characteristics through EGFR stimulation is augmented by signaling molecules possibly generated by other pathways. This supports the view that lens growth, in general, and fiber differentiation, in particular, require the sustained integration of multifactorial processes. Furthermore, because the EGFR may not be present in the embryonic lens ^{25 32} but is widely distributed in adult lens tissues from several species, our data also indicate that mechanisms affecting lens growth and differentiation are differentially regulated during development and ageing. Whether the EGFR directly affects gene expression or improves overall cellular metabolism conducive to improved growth patterns remains to be determined. 

 Supported by Grants EY09346 (MEI) and EY04068 (Core Grant for Vision Research).

 Submitted for publication April 6, 1999; revised August 10, 1999; accepted August 27, 1999.

 Commercial relationships policy: N.

 

Figure 1.

 

The EGFR is the major PY-containing protein in freshly isolated annular pad cells. Aliquots of annular pad cells isolated on a discontinuous sucrose gradient were incubated in the absence (Control) or presence (EGF) of 100 ng/ml EGF for 30 minutes. The resulting lysates were stained with anti-PY antibodies (1:1000 dilution) for western blot analysis. The major PY-containing protein in annular pad cells comigrated with the EGFR overexpressed in A431 cells (A431) and increased its PY content in the presence of EGF.

Figure 2.

 

Dose- (A) and time-dependent (B) increases of the annular pad EGFR PY content in response to EGF treatment. (A) Aliquots of annular pad cells isolated on a discontinuous sucrose gradient were incubated in the presence of the indicated concentrations of EGF for 30 minutes. The resulting lysates were stained with anti-PY antibodies (1:1000 dilution), and results of western blot analysis show a dose-dependent increase in the PY content of the EGFR. (B) Aliquots of annular pad cells were incubated with 100 ng/ml EGF for the indicated periods of time before processing for western blot analysis. Anti-PY antibodies were able to detect increases in the annular pad EGFR with 5 minutes of stimulation. Samples of the EGFR in A431 cells are shown for comparative purposes.

Figure 3.

 

Genistein reduces EGF-mediated increases in annular pad EGFR PY levels. Results of western blot analysis show staining with anti-PY antibodies (1:1000 dilution) of annular pad cell aliquots incubated in the absence (C) or presence (EGF) of 100 ng/ml EGF for 30 minutes. Inclusion of 10μ M genistein (EGF + genistein) during incubation with EGF reduced increases in PY content of the annular pad EGFR. A431, sample of A431 cells.

Figure 4.

 

Constitutive activity of the annular pad EGFR. Results of western blot analysis show staining with anti-PY antibodies (1:1000 dilution) of annular pad cell aliquots incubated in the absence (−EGF) or presence (+EGF) of 100 ng/ml EGF for 30 minutes. Incubations were performed in the indicated micromolar concentrations of the phosphatase inhibitor sodium orthovanadate (VO₄ ⁼). Note that annular pad EGFR PY levels increased in response to elevated orthovanadate concentrations even in the absence of EGF treatment. Phosphotyrosine levels were increased further still in the presence of EGF. A431, sample of A431 cells.

Figure 5.

 

Identification of endogenous TGF-α in annular pad cells. (A) Coomassie blue stained 15% PAGE of: MW, high molecular weight standards; CLAP, 150 μg of chick lens annular pad whole cell lysate; TGF-α, 100 ng of authentic TGF-α; and EGF, 100 ng of authentic EGF. (B) Results of western blot analysis show replicate gel pictured in (A) stained with TGF-α antibodies (1:1000 dilution of primary antisera). Note positive staining of a minor component in CLAP sample lysate, which comigrates with authentic TGF-α. (C) Results of western blot analysis show replicate gel pictured in (A) stained with EGF antibodies (1:500 dilution of primary antisera). Although this antisera recognized authentic EGF, no such cross-reactivity was noted in the CLAP sample.

Figure 6.

 

Increased PY levels of the annular pad EGFR in response to TGF-α stimulation. Aliquots of annular pad cells were incubated in the absence (Control) or the presence of 100 ng/ml EGF or TGF-α for 30 minutes before immunoprecipitation with anti-EGFR antibodies. (A) Results of western blot analysis of immunoprecipitates stained with anti-PY antibodies (1:1000 dilution). TGF-α produced a greater increase in the annular pad EGFR phosphotyrosine content than an equimolar concentration of EGF. (B) Western blot pictured in upper panel was stripped and reprobed with the EGFR antibodies (1:1000 dilution) used for immunoprecipitation. A431, sample of A431 cells.

Figure 7.

 

TGF-α stimulates differentiation of annular pad cells. Primary cultures of annular pad cells were grown for 3 days in unsupplemented media (Control) or media supplemented with 100 ng/ml TGF-α, 100 μM 8-bromoadenosine 3′:5-cyclic monophosphate (8bcAMP), or both reagents (TGF-α + 8bcAMP). Left: Coomassie blue–stained 10% SDS–PAGE of equivalent protein loads of cells grown under the various conditions. Right: Western blot of boxed area indicated in left panel stained with anti-filensin antibodies (1:3000 dilution of primary antiserum). Relative densitometric quantification appears below each respective treatment. Note that combining treatments produced a further increase in differentiation when compared with either treatment alone. Inset: ELISA quantification of filensin levels in passaged cells. Note that TGF-α or 8bcAMP each produced a statistically significant increase in filensin levels, and combining the treatments resulted in a further significant increase. The average filensin level (as a percentage of control) is indicated within each bar of the histogram.