Free
Glaucoma  |   March 2014
Pharmacological Regulation of SPARC by Lovastatin in Human Trabecular Meshwork Cells
Author Notes
  • Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts 
  • Correspondence: Douglas J. Rhee, Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, 243 Charles Street, Boston, MA 02114; DougRhee@aol.com
Investigative Ophthalmology & Visual Science March 2014, Vol.55, 1657-1665. doi:10.1167/iovs.13-12712
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Guadalupe Villarreal, Jr, Ayan Chatterjee, Sarah S. Oh, Dong-Jin Oh, Douglas J. Rhee; Pharmacological Regulation of SPARC by Lovastatin in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(3):1657-1665. doi: 10.1167/iovs.13-12712.

      Download citation file:


      © 2016 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

Purpose.: Statins have been shown to increase aqueous outflow facility. The matricellular protein SPARC (secreted protein acidic and rich in cysteine) is a critical mediator of aqueous outflow and intraocular pressure (IOP). Here, we examine the effects of lovastatin on SPARC expression in trabecular meshwork (TM) cells, exploring the molecular mechanisms involved.

Methods.: Primary cultured human TM cells were incubated for 24, 48, and 72 hours with 10 μM lovastatin. In separate cultures, media was supplemented with either farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) for the duration of the 72-hour time point experiment. Trabecular meshwork cells were also pretreated for 24 hours with lovastatin followed by 24-hour stimulation with 3 ng/mL TGF-β2. Cell lysates and media were harvested and relative mRNA and protein level changes were determined. Krüppel-like factor 4 (KLF4) localization in normal human anterior segments was examined by immunofluorescence. Adenovirus expressing human KLF4 was used and relative changes in SPARC mRNA and protein levels were assessed.

Results.: Incubating TM cells with lovastatin suppressed SPARC mRNA and protein levels. This effect was reversed upon media supplementation with GGPP but not FPP. Pretreating cells with lovastatin inhibited TGF-β2 induction of SPARC. The KLF4 transcription factor was expressed throughout the TM and the inner and outer walls of Schlemm's canal. Lovastatin treatment upregulated KLF4 mRNA and protein levels. Overexpression of KLF4 downregulated SPARC expression.

Conclusions.: Collectively, our data identify lovastatin as an important pharmacological suppressor of SPARC expression in TM cells, and provide further insight into the molecular mechanisms mediating statin enhancement of aqueous outflow facility.

Introduction
Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness worldwide. 1 Among persons of African American or Latino descent in the United States, POAG represents the leading cause of preventable blindness. 13 Progressive optic nerve damage and the resultant visual field loss in POAG are believed to be due to an elevated intraocular pressure (IOP) beyond the structural and/or vascular capabilities of the optic nerve to withstand. The elevated IOP of POAG is caused by impaired aqueous drainage through the trabecular meshwork (TM). 4  
Between 80% and 90% of aqueous outflow occurs through the TM (“conventional” pathway), with the remaining 10% to 20% exiting through the ciliary body face (“uveoscleral” pathway). 5 The juxtacanicular (JCT) region, which includes the inner wall of Schlemm's canal and underlying JCT TM, is the anatomic location of highest outflow resistance. 6,7 Outflow facility studies using human anterior segment perfusion systems with either glycosaminoglycan degradation enzymes, matrix metalloproteinases, or inhibition of glycosaminoglycan sulfation, strongly indicate that the balance between ECM synthesis and degradation regulates aqueous outflow. 810  
Matricellular proteins are nonstructural secreted glycoproteins that facilitate cellular control over their surrounding ECM. 11 Secreted protein acidic and rich in cysteine (SPARC) is the prototypical matricellular protein and has been associated with increased tissue fibrosis and aberrant tissue remodeling, processes that perturb ECM homeostasis and may have a role in glaucoma pathogenesis. 12 SPARC is expressed throughout the TM with prominent levels within the JCT region. 13 Quantitative proteomic studies have demonstrated that SPARC is the most highly upregulated protein in human TM cells following TGF-β2 treatment. 14 TGF-β2 is elevated up to 3-fold in the aqueous humor of patients with POAG. 1518 In perfused cadaveric human anterior segments and live rats, TGF-β2 increases IOP and causes alterations of JCT ECM. 19,20 We have previously shown that SPARC-null mice have a 15% to 20% lower IOP, with more uniform outflow and decreased collagen fibril diameter compared with control wild-type mice. 21,22 We have further demonstrated that SPARC overexpression elevates IOP with a corresponding qualitative increase in the JCT ECM of perfused cadaveric human anterior segments. 23 Together, these data suggest that SPARC may function as an important node in the regulation of outflow facility and IOP, and have provided an impetus for the identification of novel pharmacological molecules and/or signaling pathways that modulate SPARC expression. 
HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA) reductase inhibitors, or statins, are a widely prescribed class of cholesterol-lowering medications that have been shown to exert beneficial effects independent of their lipid-lowering properties, so-called “pleiotropic” effects. 24 Growing clinical data have suggested that statins may be protective against the development and progression of open-angle glaucoma in specific patient populations. 2529 Studies using organ-cultured porcine anterior segments have demonstrated that statins increase aqueous outflow facility. 30 Statins have also been shown to suppress ECM synthesis in various nonocular tissues. 3133 The Krüppel-like factor 4 (KLF4) transcription factor is induced by statins and functions as an important mediator of statin-related vasoprotection. 34,35 Recent work in other ocular and nonocular tissues has identified SPARC as a downstream target of KLF4. 36,37 We hypothesized that statins may suppress SPARC expression in TM cells via the induction of KLF4. 
Materials and Methods
Trabecular Meshwork Cell Culture
Primary human trabecular meshwork (TM) cells were isolated, in accordance with the tenets of the Declaration of Helsinki, and cultured as previously described. 38 Independent primary human TM cell lines were generated from donors ranging in age from 35 to 70 years and no known history of ocular disease. Cell cultures were maintained, unless otherwise stated, in Dulbecco's modified Eagle's medium (Life Technologies, Grand Island, NY) supplemented with 20% fetal bovine serum, 1% L-glutamine (2 mM), and gentamicin (0.1 mg/mL) at 37°C in a 10% CO2 atmosphere. Trabecular meshwork cells from passage 4 to 5 cultures were used. 
RNA Isolation and Real-Time Gene Expression Assays
Total RNA was isolated using the RNAqueous-4PCR kit (Life Technologies) as described by the manufacturer. Following isolation, RNA was treated with a commercial kit (RNase-free DNase I; Life Technologies) to remove DNA contaminants. RNA concentration was assessed via spectrophotometry. Total RNA (500 ng) was then reverse transcribed using the M-MLV reverse transcription kit (Promega, Madison, WI). Quantitative real-time PCR for SPARC (Hs00234160_m1), KLF4 (Hs00358836_m1), and internal control GAPDH (Hs02758991_g1) was performed using predesigned gene expression assays as described by the manufacturer's instructions (TaqMan; Life Technologies). Levels of SPARC and KLF4 were normalized to GAPDH using the formula 2−ΔCt. The relative fold change in expression between control and treatment groups was then assessed using the 2−ΔΔCt method. 
Lovastatin Time Course Experiments
Trabecular meshwork endothelial cells at 90% to 100% confluency (equal between control and treatment groups) were cultured in serum-free media for 19 hours, and subsequently incubated for 24, 48, or 72 hours in serum-free media containing 10 μM activated lovastatin (Calbiochem #438186, San Diego, CA) or vehicle (dimethyl sulfoxide 1% vol/vol). Cell culture media containing lovastatin or vehicle was replaced every 24 hours for the 48- and 72-hour time points. For isoprenoid supplementation experiments, primary human TM cells were treated with 10 μM lovastatin or DMSO vehicle with concurrent addition of either 10 μM farnesyl pyrophosphate (FPP), 10 μM geranylgeranyl pyrophosphate (GGPP), or vehicle for 72 hours (Sigma-Aldrich, St. Louis, MO). 
Adenoviral-Mediated Infection Experiments
Primary human TM endothelial cells at 70% to 90% confluency were infected in 2% FBS media with either Ad-KLF4 (multiplicity of infection [MOI] = 50; SignaGen Laboratories, Rockville, MD) or Ad-Null control virus (MOI = 50; SignaGen Laboratories). Multiplicity of infection is defined as the ratio of infectious viral units to cells. 39 After 18 hours, an equal volume of 10% FBS media was added to each well and cells were incubated for an additional 24 hours. Cells were then washed once with PBS and incubated for 24 hours in serum-free media containing either 3 ng/mL activated, recombinant human TGF-β2 (R&D Systems, Minneapolis, MN), or 4 mM HCl solution containing 0.1% human serum albumin as the vehicle. 
TGF-β2 Stimulatory Experiments
Trabecular meshwork cells at 90% to 100% confluency (equal between control and treatment groups) were cultured in serum-free media for 19 hours, and subsequently incubated for 24 hours in serum-free media containing either 10 μM lovastatin or vehicle (dimethyl sulfoxide 1% vol/vol). Cells were then treated for an additional 24 hours with either 3 ng/mL activated, recombinant human TGF-β2 or 4 mM HCl solution containing 0.1% human serum albumin as vehicle. 
Immunoblot Analysis
For SPARC detection, conditioned media from TM cell cultures was harvested and centrifuged at 2300g for 10 minutes at 4°C. The supernatant was then concentrated (Amicon Ultra-4 Filter Unit, 10 kDa; Millipore Corp., Milford, MA), and protein content quantified (DC Protein Assay; Bio-Rad Laboratories, Inc., Hercules, CA). For KLF4 protein detection, TM cells were lysed for 3 minutes on ice with cold 1× RIPA buffer containing 0.5% aprotinin, 0.1% EDTA, 1% EGTA, 0.5% phenylmethanesulfonyl fluoride, and 0.01% Leupeptin. Samples were then centrifuged at 18000g for 15 minutes at 4°C and supernatant protein content was isolated and quantified. In all experiments, equal amounts of protein were treated with 6× reducing buffer and boiled for 5 minutes. Samples were then electrophoresed in 10% SDS-polyacrylamide gels, and proteins were transferred to nitrocellulose membranes (0.45-μm pore size; Invitrogen, Carlsbad, CA). Membranes were blocked for 1 hour at room temperature in a 1:1 mixture of 1× TBS-T (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% Tween-20) and blocking buffer (Rockland, Inc., Gilbertsville, PA), followed by overnight incubation at 4°C with the indicated primary antibody at 1:10,000 for SPARC (Hematologic Technologies, Inc., Essex Junction, VT); 1:1000 for KLF4 (Cell Signaling Technology, Inc., Danvers, MA); and 1:1000 for GAPDH (Trevigen, Inc., Gaithersburg, MD). Following overnight incubation with the primary antibody, the membranes were washed three times with 1× TBS-T and incubated for 1 hour at room temperature with dye-conjugated affinity purified 680 anti-mouse or 800 anti-rabbit IgG antibodies, respectively (IRDye; 1:10,000 dilution; Rockland, Inc.). The membranes were then washed three times with 1× TBS-T and scanned, and integrated band intensities were calculated using an infrared imaging system (Odyssey; LI-COR Biosciences, Lincoln, NE). For evaluation of KLF4 expression in normal human TM tissue, samples were isolated from donors aged 42 and 47 years and processed as above, with β-actin loading control at 1:1000 dilution (Cell Signaling Technology, Inc.). 
Immunofluorescence
Human donor eyes (aged 44 and 69 years) were immersion-fixed in 10% neutral buffered formalin, sequentially dehydrated in ethanol solutions (75%, 85%, 95%, 100%), and embedded in paraffin. Six micron sections were mounted on poly-L-lysine-coated glass slides and baked at 60°C for 2 hours. Slides were deparaffinized in xylene, sequentially rehydrated in ethanol solutions, and washed three times for 10 minutes in phosphate-buffered saline with 0.1% Tween-20 (PBS-T). Following 1 hour incubation in 10% goat serum, tissues were permeabilized for 5 minutes in 0.2% Triton-100 in 1× PBS and again washed three times in PBS-T. The sections were then incubated at 4°C overnight either in primary KLF4 monoclonal antibody (Thermo Scientific, Billerica, MA) diluted 1:200 in PBS or in PBS alone. Slides were washed three times in PBS-T prior to incubation in 1:200 goat anti-mouse 594 AlexaFluor secondary IgG (Invitrogen) and then washed three more times following incubation. Nuclei were stained in DAPI antifade reagent (SlowFade Gold; Invitrogen). Labeled tissues were imaged and analyzed by fluorescent light microscopy using a Zeiss Observer3.1. 
Statistics
Statistical analysis was performed using graphing and statistics software (GraphPad Prism 6; GraphPad, La Jolla, CA). A two-tailed Student's t-test was used for comparing differences between two groups. Comparisons between multiple groups were made using one-way ANOVA followed by Tukey's HSD post hoc test for multiple comparisons. Differences were considered statistically significant for P < 0.05. 
Results
Lovastatin Suppresses SPARC Expression in a Time-Dependent Manner
To determine whether statins regulate SPARC expression in human TM cells, SPARC mRNA and protein levels were analyzed after 24, 48, and 72 hours of incubation with lovastatin. Real-time quantitative PCR analysis of SPARC mRNA levels revealed a statistically significant reduction at 24 and 72 hours (Fig. 1A). Immunoblot analysis of the TM-conditioned media demonstrated significant decreases in SPARC protein levels at 48 and 72 hours (Fig. 1B). Cells treated with lovastatin appeared elongated by 24 hours and remained so through 72 hours (data not shown), which is consistent with changes to TM cellular morphology observed and well-documented in previous reports. 30,40  
Figure 1
 
Lovastatin suppresses SPARC expression. (A) SPARC mRNA levels (n = 4). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 8 for 24 hours, n = 5 for 48 hours and 72 hours) from conditioned media of primary human TM cells cultured for 24, 48, and 72 hours with either 10 μM lovastatin or DMSO vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus its time-corresponding vehicle control; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 1
 
Lovastatin suppresses SPARC expression. (A) SPARC mRNA levels (n = 4). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 8 for 24 hours, n = 5 for 48 hours and 72 hours) from conditioned media of primary human TM cells cultured for 24, 48, and 72 hours with either 10 μM lovastatin or DMSO vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus its time-corresponding vehicle control; n refers to the number of independent experiments performed using n different primary human TM cell strains).
GGPP Supplementation Reverses Lovastatin-Induced SPARC Suppression
In addition to reducing cholesterol biosynthesis, lovastatin has also been shown to suppress the synthesis of intermediaries such as the isoprenoids FPP and GGPP. 41,42 Isoprenoid supplementation has been shown to reverse lovastatin-induced cytoskeletal changes in cultured porcine and human TM cells, suggesting their involvement in regulating downstream RhoA GTPase control over cellular tone and ECM deposition. 30,40,43 We examined whether lovastatin-induced SPARC suppression could be reversed with concurrent addition of either 10 μM FPP or GGPP for 72 hours. Immunoblot analysis of the TM-conditioned media demonstrated full recovery in SPARC protein levels at 72 hours when GGPP, but not FPP, had been supplemented in the media (Fig. 2). 
Figure 2
 
Geranylgeranyl pyrophosphate, but not FPP, supplementation reverses lovastatin-induced suppression of SPARC protein levels. SPARC levels (n = 6) were assessed in the conditioned media of primary human TM cells incubated for 72 hours with 10 μM lovastatin or DMSO vehicle with concurrent addition of either 10 μM FPP, 10 μM GGPP, or vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus control; #P < 0.05 versus lovastatin + vehicle; δP < 0.05 versus lovastatin + FPP; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 2
 
Geranylgeranyl pyrophosphate, but not FPP, supplementation reverses lovastatin-induced suppression of SPARC protein levels. SPARC levels (n = 6) were assessed in the conditioned media of primary human TM cells incubated for 72 hours with 10 μM lovastatin or DMSO vehicle with concurrent addition of either 10 μM FPP, 10 μM GGPP, or vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus control; #P < 0.05 versus lovastatin + vehicle; δP < 0.05 versus lovastatin + FPP; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Lovastatin Inhibits SPARC Induction Under TGF-β2 Stimulatory Conditions
TGF-β2 is an important regulator of ECM synthesis and deposition in the TM. 19,20,44,45 In TM cells, SPARC is the most highly upregulated protein by TGF-β2. 14 We next investigated whether lovastatin could suppress SPARC induction under TGF-β2 stimulatory conditions. Analysis of SPARC mRNA and protein levels revealed a significant suppression in TGF-β2–mediated induction of SPARC (Figs. 3A, 3B). 
Figure 3
 
Lovastatin inhibits TGF-β2 induction of SPARC. (A) SPARC mRNA expression (n = 5). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 6) from conditioned media of primary human TM cells pretreated for 24 hours with 10 μM lovastatin or DMSO vehicle followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2, or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.05; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 3
 
Lovastatin inhibits TGF-β2 induction of SPARC. (A) SPARC mRNA expression (n = 5). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 6) from conditioned media of primary human TM cells pretreated for 24 hours with 10 μM lovastatin or DMSO vehicle followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2, or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.05; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Evaluation of KLF4 Expression in Human TM
SPARC has been identified as an important downstream target of KLF4 in ocular and nonocular tissues. 36,37 KLF4 expression in the human trabecular meshwork is unknown. To characterize its expression, immunoblot analysis and immunofluorescent staining of KLF4 in normal cadaveric human TM tissue was performed. Immunoblot analysis of human TM tissue demonstrated KLF4 expression (Fig. 4A). Immunofluorescent staining identified KLF4 expression throughout the TM as well as the inner and outer walls of Schlemm's canal (Fig. 4B). 
Figure 4
 
KLF4 expression in human TM. (A) Representative immunoblot showing detection of KLF4 in normal cadaveric human TM tissue from a 42-year-old donor. (B) Immunolocalization of KLF4 in human trabecular meshwork from an 84-year-old donor. Representative images demonstrate KLF4 expression (red) throughout the TM as well as inner and outer walls of Schlemm's canal. Cell nuclei stained with DAPI (blue). AC, anterior chamber; SC, Schlemm's canal. Scale bar: 50 μm.
Figure 4
 
KLF4 expression in human TM. (A) Representative immunoblot showing detection of KLF4 in normal cadaveric human TM tissue from a 42-year-old donor. (B) Immunolocalization of KLF4 in human trabecular meshwork from an 84-year-old donor. Representative images demonstrate KLF4 expression (red) throughout the TM as well as inner and outer walls of Schlemm's canal. Cell nuclei stained with DAPI (blue). AC, anterior chamber; SC, Schlemm's canal. Scale bar: 50 μm.
Lovastatin Treatment Upregulates KLF4 Expression
To determine whether lovastatin has an effect on KLF4 expression in human TM cells, mRNA and protein levels were quantified 24, 48, and 72 hours after initiation of lovastatin treatment. KLF4 mRNA and protein levels were significantly increased at each time point measured (Figs. 5A, 5B). 
Figure 5
 
Lovastatin induces expression of KLF4. (A) KLF4 mRNA levels (n = 5). (B) Representative immunoblot and densitometric analysis of KLF4 protein expression (n = 4) from cell lysates of primary human TM cells incubated for 24, 48, and 72 hours with either 10 μM lovastatin or DMSO vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus its time-corresponding vehicle control; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 5
 
Lovastatin induces expression of KLF4. (A) KLF4 mRNA levels (n = 5). (B) Representative immunoblot and densitometric analysis of KLF4 protein expression (n = 4) from cell lysates of primary human TM cells incubated for 24, 48, and 72 hours with either 10 μM lovastatin or DMSO vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus its time-corresponding vehicle control; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Effect of TGF-β2 Stimulation on KLF4 Expression
We next assessed whether TGF-β2 stimulation affects KLF4 expression under basal and lovastatin conditions. KLF4 mRNA levels were not significantly affected by TGF-β2 under both basal and lovastatin conditions (Fig. 6A). While TGF-β2 did not affect KLF4 protein levels under lovastatin conditions, a small, statistically significant 2.4-fold increase in KLF4 protein expression was seen under basal conditions (Fig. 6B). 
Figure 6
 
Effect of TGF-β2 stimulation on KLF4 expression. (A) KLF4 mRNA levels (n = 6). (B) Representative immunoblot and densitometric analysis of KLF4 protein levels (n = 4) from cell lysates of primary human TM cells pretreated for 24 hours with 10 μM lovastatin or DMSO vehicle followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2, or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.01; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 6
 
Effect of TGF-β2 stimulation on KLF4 expression. (A) KLF4 mRNA levels (n = 6). (B) Representative immunoblot and densitometric analysis of KLF4 protein levels (n = 4) from cell lysates of primary human TM cells pretreated for 24 hours with 10 μM lovastatin or DMSO vehicle followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2, or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.01; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Overexpression of KLF4 Suppresses SPARC Under Basal and TGF-β2 Stimulatory Conditions
To assess the effect of KLF4 on SPARC expression, human TM cells were infected with control adenovirus or virus expressing human KLF4. Overexpression of KLF4 led to an increase in SPARC mRNA levels under basal conditions but decrease under TGF-β2 stimulation (Fig. 7A). By contrast, overexpression of KLF4 significantly suppressed SPARC protein levels under both basal and TGF-β2 stimulatory conditions (Fig. 7B). 
Figure 7
 
Overexpression of KLF4 suppresses SPARC levels under basal and TGF-β2 stimulatory conditions. (A) SPARC mRNA expression (n = 4). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 4) from conditioned media of primary human TM cells infected with either Ad-Null or Ad-KLF4 adenovirus followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2 or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.05; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 7
 
Overexpression of KLF4 suppresses SPARC levels under basal and TGF-β2 stimulatory conditions. (A) SPARC mRNA expression (n = 4). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 4) from conditioned media of primary human TM cells infected with either Ad-Null or Ad-KLF4 adenovirus followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2 or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.05; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Discussion
Growing clinical evidence has suggested a protective role for statins in the development and progression of open-angle glaucoma in specific patient populations. 2529 Studies of organ cultured porcine eye anterior segments have shown that statins increase aqueous outflow facility. 30 Matricellular proteins, and in particular SPARC, have emerged as important modulators of outflow facility and IOP. 2123 Here, we identify lovastatin as a pharmacologic suppressor of SPARC expression in TM cells and provide evidence suggesting that these effects may be mediated via the upregulation of transcription factor KLF4 (Fig. 8). 
Figure 8
 
Working model for the regulation of SPARC by lovastatin.
Figure 8
 
Working model for the regulation of SPARC by lovastatin.
In the present study, we demonstrated that lovastatin suppressed SPARC levels in a time-dependent pattern in cultured TM cells. The reversibility of lovastatin-induced SPARC suppression upon supplementation with equimolar GGPP—but not FPP—suggests that GGPP is an important intermediary in this process. Additionally, the pretreatment of cells with lovastatin significantly inhibited TGF-β2–mediated induction of SPARC. Transforming growth factor-β2 has been implicated in the pathogenesis of POAG, with multiple studies demonstrating elevated levels of TGF-β2 in the aqueous humor of POAG patients. 1518 Importantly, TGF-β2 has been shown to increase ECM accumulation in the TM and aqueous outflow resistance. 19,20,44,46 Current work from our laboratory strongly suggests that SPARC is critical for TGF-β2–mediated ECM accumulation and ocular hypertension in mice (Swaminathan SS, Oh D-J, Kang MH, Shepard AR, Pang I-H, unpublished observations, 2013). Pharmacological modulation of SPARC expression may serve as an important therapeutic avenue for the reestablishment of a functional aqueous drainage system through the TM. 
In other ocular and nonocular tissues, the transcription factor KLF4 regulates SPARC expression. 36,37 Immunofluorescent analysis of KLF4 in cadaveric human anterior segments revealed expression throughout the TM as well as inner and outer walls of Schlemm's canal, suggesting that KLF4 is biologically relevant to transcriptional signaling in the conventional outflow pathway. Incubation of TM cells with lovastatin resulted in a significant induction in KLF4 mRNA and protein expression. The mechanisms mediating KLF4 upregulation by lovastatin in TM cells remain unknown. However, in vascular endothelial cells, the MEK5/ERK5/MEF2 pathway is an important regulator of KLF4 induction. 34,35 Further clarification of the role of this signaling pathway in TM cells is needed. 
Overexpression of KLF4 reduced SPARC protein levels under basal and TGF-β2 stimulatory conditions, suggesting that KLF4 may be an attractive target for therapeutic modulation of SPARC. Interestingly, under basal conditions, KLF4 overexpression upregulated SPARC mRNA levels by 1.3-fold, while suppressing SPARC protein levels by 3.3-fold. These results suggest that posttranscriptional mechanisms may play an important role in KLF4-mediated regulation of SPARC. MicroRNAs are small, single-stranded noncoding RNAs that modulate posttranscriptional gene expression. KLF4 has been shown to regulate the expression of various microRNAs. 4749 Further investigation is necessary to assess whether microRNA modulation by KLF4 functions as an important posttranscriptional regulatory mechanism for SPARC expression. Under TGF-β2 stimulatory conditions, KLF4 overexpression suppressed both SPARC mRNA and protein levels. Activation of the SMAD3 transcription factor is critical for TGF-β2–mediated induction of SPARC. 50 In nonocular tissues, KLF4 can directly interact with SMAD3 and inhibit its ability to bind target gene promoters. 51 Additional studies would be necessary to determine whether KLF4 binds and inhibits SMAD3 activity in TM cells. 
Rho GTPase functions as an important mediator of ECM induction by TGF-β2. 52 In TM cells, inhibition of Rho GTPase suppresses TGF-β2-mediated ECM production. 52 Statins have been shown to indirectly inhibit Rho GTPase signaling via the prevention of isoprenoid intermediate synthesis. 53,54 In nonocular tissues, RhoA inhibits KLF4 DNA binding. 55 Thus, indirect inhibition of RhoA by statins may allow for enhanced DNA binding of KLF4 and transcriptional suppression of SPARC. It is possible that Rho signaling may have a role in KLF4 regulation of SPARC in TM cells. 
Collectively, the results presented here identify lovastatin as a novel pharmacological modulator of SPARC expression and provide further insight into the molecular mechanisms mediating the effects of statins on outflow facility. 
Acknowledgments
Supported by a Howard Hughes Medical Institute Research Training Fellowship for Medical Students (AC), National Eye Institute Grant EY 019654-01 (DJR), and MEEI Vision-Core Grant EY 014104. 
Disclosure: G. Villarreal Jr, None; A. Chatterjee, None; S.S. Oh, None; D.-J. Oh, None; D.J. Rhee, Alcon (F, C), Aquesys (F, C), Merck (F, C), Aerie (C), Alcon (C), Allergan (C), Johnson & Johnson (C), Santen (C) 
References
Allingham RR Liu Y Rhee DJ. The genetics of primary open-angle glaucoma: a review. Exp Eye Res . 2009; 88: 837–844. [CrossRef] [PubMed]
Hyman L Wu SY Connell AM Prevalence and causes of visual impairment in the Barbados Eye Study. Ophthalmology . 2001; 108: 1751–1756. [CrossRef] [PubMed]
Rodriguez J Sanchez R Munoz B Causes of blindness and visual impairment in a population-based sample of U.S. Hispanics. Ophthalmology . 2002; 109: 737–743. [CrossRef] [PubMed]
Larsson LI Rettig ES Brubaker RF. Aqueous flow in open-angle glaucoma. Arch Ophthalmol . 1995; 113: 283–286. [CrossRef] [PubMed]
Pederson JE Gaasterland DE MacLellan HM. Uveoscleral aqueous outflow in the rhesus monkey: importance of uveal reabsorption. Invest Ophthalmol Vis Sci . 1977; 16: 1008–1017. [PubMed]
Bill A Phillips CI. Uveoscleral drainage of aqueous humour in human eyes. Exp Eye Res . 1971; 12: 275–281. [CrossRef] [PubMed]
Seiler T Wollensak J. The resistance of the trabecular meshwork to aqueous humor outflow. Graefes Arch Clin Exp Ophthalmol . 1985; 223: 88–91. [CrossRef] [PubMed]
Gum GG Samuelson DA Gelatt KN. Effect of hyaluronidase on aqueous outflow resistance in normotensive and glaucomatous eyes of dogs. Am J Vet Res . 1992; 53: 767–770. [PubMed]
Bradley JM Vranka J Colvis CM Effect of matrix metalloproteinases activity on outflow in perfused human organ culture. Invest Ophthalmol Vis Sci . 1998; 39: 2649–2658. [PubMed]
Keller KE Bradley JM Kelley MJ Acott TS. Effects of modifiers of glycosaminoglycan biosynthesis on outflow facility in perfusion culture. Invest Ophthalmol Vis Sci . 2008; 49: 2495–2505. [CrossRef] [PubMed]
Chiodoni C Colombo MP Sangaletti S. Matricellular proteins: from homeostasis to inflammation, cancer, and metastasis. Cancer Metastasis Rev . 2010; 29: 295–307. [CrossRef] [PubMed]
Rhee DJ Haddadin RI Kang MH Oh DJ. Matricellular proteins in the trabecular meshwork. Exp Eye Res . 2009; 88: 694–703. [CrossRef] [PubMed]
Rhee DJ Fariss RN Brekken R Sage EH Russell P. The matricellular protein SPARC is expressed in human trabecular meshwork. Exp Eye Res . 2003; 77: 601–607. [CrossRef] [PubMed]
Bollinger KE Crabb JS Yuan X Putliwala T Clark AF Crabb JW. Quantitative proteomics: TGFβ2 signaling in trabecular meshwork cells. Invest Ophthalmol Vis Sci . 2011; 52: 8287–8294. [CrossRef] [PubMed]
Ochiai Y Ochiai H. Higher concentration of transforming growth factor-beta in aqueous humor of glaucomatous eyes and diabetic eyes. Jpn J Ophthalmol . 2002; 46: 249–253. [CrossRef] [PubMed]
Ozcan AA Ozdemir N Canataroglu A. The aqueous levels of TGF-beta2 in patients with glaucoma. Int Ophthalmol . 2004; 25: 19–22. [CrossRef] [PubMed]
Picht G Welge-Luessen U Grehn F Lutjen-Drecoll E. Transforming growth factor beta 2 levels in the aqueous humor in different types of glaucoma and the relation to filtering bleb development. Graefes Arch Clin Exp Ophthalmol . 2001; 239: 199–207. [CrossRef] [PubMed]
Tripathi RC Li J Chan WF Tripathi BJ. Aqueous humor in glaucomatous eyes contains an increased level of TGF-beta 2. Exp Eye Res . 1994; 59: 723–727. [CrossRef] [PubMed]
Shepard AR Millar JC Pang IH Jacobson N Wang WH Clark AF. Adenoviral gene transfer of active human transforming growth factor-{beta}2 elevates intraocular pressure and reduces outflow facility in rodent eyes. Invest Ophthalmol Vis Sci . 2010; 51: 2067–2076. [CrossRef] [PubMed]
Gottanka J Chan D Eichhorn M Lutjen-Drecoll E Ethier CR. Effects of TGF-beta2 in perfused human eyes. Invest Ophthalmol Vis Sci . 2004; 45: 153–158. [CrossRef] [PubMed]
Haddadin RI Oh DJ Kang MH SPARC-null mice exhibit lower intraocular pressures. Invest Ophthalmol Vis Sci . 2009; 50: 3771–3777. [CrossRef] [PubMed]
Swaminathan SS Oh DJ Kang MH Secreted protein, acidic and rich in cysteine (SPARC)-null mice exhibit more uniform outflow. Invest Ophthalmol Vis Sci . 2013; 54: 2035–2047. [CrossRef] [PubMed]
Oh DJ Kang MH Ooi YH Choi KR Sage EH Rhee DJ. Overexpression of SPARC in human trabecular meshwork increases intraocular pressure and alters extracellular matrix. Invest Ophthalmol Vis Sci . 2013; 54: 3309–3319. [CrossRef] [PubMed]
Wang CY Liu PY Liao JK. Pleiotropic effects of statin therapy: molecular mechanisms and clinical results. Trends Mol Med . 2008; 14: 37–44. [CrossRef] [PubMed]
Stein JD Newman-Casey PA Talwar N Nan B Richards JE Musch DC. The relationship between statin use and open-angle glaucoma. Ophthalmology . 2012; 119: 2074–2081. [CrossRef] [PubMed]
Marcus MW Muskens RP Ramdas WD Cholesterol-lowering drugs and incident open-angle glaucoma: a population-based cohort study. PLoS One . 2012; 7: e29724. [CrossRef] [PubMed]
McGwin G Jr McNeal S Owsley C Girkin C Epstein D Lee PP. Statins and other cholesterol-lowering medications and the presence of glaucoma. Arch Ophthalmol . 2004; 122: 822–826. [CrossRef] [PubMed]
Leung DY Li FC Kwong YY Tham CC Chi SC Lam DS. Simvastatin and disease stabilization in normal tension glaucoma: a cohort study. Ophthalmology . 2010; 117: 471–476. [CrossRef] [PubMed]
De Castro DK Punjabi OS Bostrom AG Effect of statin drugs and aspirin on progression in open-angle glaucoma suspects using confocal scanning laser ophthalmoscopy. Clin Experiment Ophthalmol . 2007; 35: 506–513. [CrossRef] [PubMed]
Song J Deng PF Stinnett SS Epstein DL Rao PV. Effects of cholesterol-lowering statins on the aqueous humor outflow pathway. Invest Ophthalmol Vis Sci . 2005; 46: 2424–2432. [CrossRef] [PubMed]
Xu H Zeng L Peng H HMG-CoA reductase inhibitor simvastatin mitigates VEGF-induced “inside-out” signaling to extracellular matrix by preventing RhoA activation. Am J Physiol Renal Physiol . 2006; 291: F995–F1004. [CrossRef] [PubMed]
Schaafsma D Dueck G Ghavami S The mevalonate cascade as a target to suppress extracellular matrix synthesis by human airway smooth muscle. Am J Respir Cell Mol Biol . 2011; 44: 394–403. [CrossRef] [PubMed]
Li YB Yin JJ Wang HJ Wang J Tian H Yang M. Effect of simvastatin on expression of transforming growth factor-β and collagen type IV in rat mesangial cells. Pharmacology . 2011; 88: 188–192. [CrossRef] [PubMed]
Ohnesorge N Viemann D Schmidt N Erk5 activation elicits a vasoprotective endothelial phenotype via induction of Kruppel-like factor 4 (KLF4). J Biol Chem . 2010; 285: 26199–26210. [CrossRef] [PubMed]
Villarreal G Jr Zhang Y Larman HB Gracia-Sancho J Koo A Garcia-Cardena G. Defining the regulation of KLF4 expression and its downstream transcriptional targets in vascular endothelial cells. Biochem Biophys Res Commun . 2010; 391: 984–989. [CrossRef] [PubMed]
Zhou Y Hofstetter WL He Y KLF4 inhibition of lung cancer cell invasion by suppression of SPARC expression. Cancer Biol Ther . 2010; 9: 507–513. [CrossRef] [PubMed]
Swamynathan SK Davis J Piatigorsky J. Identification of candidate Klf4 target genes reveals the molecular basis of the diverse regulatory roles of Klf4 in the mouse cornea. Invest Ophthalmol Vis Sci . 2008; 49: 3360–3370. [CrossRef] [PubMed]
Stamer DW Roberts BC Epstein DL Allingham RR. Isolation of primary open-angle glaucomatous trabecular meshwork cells from whole eye tissue. Curr Eye Res . 2000; 20: 347–350. [CrossRef] [PubMed]
Gutierrez S Yvon M Thebaud G Monsion B Michalakis Y Blanc S. Dynamics of the multiplicity of cellular infection in a plant virus. PLoS Pathog . 2010; 6: e1001113. [CrossRef] [PubMed]
Von Zee CL Richards MP Bu P Perlman JI Stubbs EB Jr. Increased RhoA and RhoB protein accumulation in cultured human trabecular meshwork cells by lovastatin. Invest Ophthalmol Vis Sci . 2009; 50: 2816–2823. [CrossRef] [PubMed]
Goldstein JL Brown MS. Regulation of the mevalonate pathway. Nature . 1990; 343: 425–430. [CrossRef] [PubMed]
Fenton RG Kung HF Longo DL Smith MR. Regulation of intracellular actin polymerization by prenylated cellular proteins. J Cell Biol . 1992; 117: 347–356. [CrossRef] [PubMed]
Von Zee CL Stubbs EB Jr. Geranylgeranylation facilitates proteasomal degradation of rho G-proteins in human trabecular meshwork cells. Invest Ophthalmol Vis Sci . 2011; 52: 1676–1683. [CrossRef] [PubMed]
Fleenor DL Shepard AR Hellberg PE Jacobson N Pang IH Clark AF. TGFbeta2-induced changes in human trabecular meshwork: implications for intraocular pressure. Invest Ophthalmol Vis Sci . 2006; 47: 226–234. [CrossRef] [PubMed]
Zode GS Sethi A Brun-Zinkernagel AM Chang IF Clark AF Wordinger RJ. Transforming growth factor-β2 increases extracellular matrix proteins in optic nerve head cells via activation of the Smad signaling pathway. Mol Vis . 2011; 17: 1745–1758. [PubMed]
Zhao X Ramsey KE Stephan DA Gene Russell P. and protein expression changes in human trabecular meshwork cells treated with transforming growth factor-beta. Invest Ophthalmol Vis Sci . 2004; 45: 4023–4034. [CrossRef] [PubMed]
Lin CC Liu LZ Addison JB Wonderlin WF Ivanov AV Ruppert JMA. KLF4-miRNA-206 autoregulatory feedback loop can promote or inhibit protein translation depending upon cell context. Mol Cell Biol . 2011; 31: 2513–2527. [CrossRef] [PubMed]
Kim H Lee G Ganat Y miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells. Cell Stem Cell . 2011; 8: 695–706. [CrossRef] [PubMed]
Sun SG Zheng B Han M miR-146a and Krüppel-like factor 4 form a feedback loop to participate in vascular smooth muscle cell proliferation. EMBO Rep . 2011; 12: 56–62. [CrossRef] [PubMed]
Kang MH Oh DJ Kang JH Rhee DJ. Regulation of SPARC by transforming growth factor β2 in human trabecular meshwork. Invest Ophthalmol Vis Sci . 2013; 54: 2523–2532. [CrossRef] [PubMed]
Hu B Wu Z Liu T Ullenbruch MR Jin H Phan SH. Gut-enriched Krüppel-like factor interaction with Smad3 inhibits myofibroblast differentiation. Am J Respir Cell Mol Biol . 2007; 36: 78–84. [CrossRef] [PubMed]
Pattabiraman PP Rao PV. Mechanistic basis of rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells. Am J Physiol Cell Physiol . 2010; 298: C749–C763. [CrossRef] [PubMed]
Liao JK Laufs U. Pleiotropic effects of statins. Annu Rev Pharmacol Toxicol . 2005; 45: 89–118. [CrossRef] [PubMed]
Stubbs EB Jr, Von Zee CL. Prenylation of rho G-proteins: a novel mechanism regulating gene expression and protein stability in human trabecular meshwork cells. Mol Neurobiol . 2012; 46: 28–40. [CrossRef] [PubMed]
Zeng Y Zhuang S Gloddek J Tseng CC Boss GR Pilz RB. Regulation of cGMP-dependent protein kinase expression by rho and Kruppel-like transcription factor-4. J Biol Chem . 2006; 281: 16951–16961. [CrossRef] [PubMed]
Footnotes
 GV Jr and AC contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Figure 1
 
Lovastatin suppresses SPARC expression. (A) SPARC mRNA levels (n = 4). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 8 for 24 hours, n = 5 for 48 hours and 72 hours) from conditioned media of primary human TM cells cultured for 24, 48, and 72 hours with either 10 μM lovastatin or DMSO vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus its time-corresponding vehicle control; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 1
 
Lovastatin suppresses SPARC expression. (A) SPARC mRNA levels (n = 4). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 8 for 24 hours, n = 5 for 48 hours and 72 hours) from conditioned media of primary human TM cells cultured for 24, 48, and 72 hours with either 10 μM lovastatin or DMSO vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus its time-corresponding vehicle control; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 2
 
Geranylgeranyl pyrophosphate, but not FPP, supplementation reverses lovastatin-induced suppression of SPARC protein levels. SPARC levels (n = 6) were assessed in the conditioned media of primary human TM cells incubated for 72 hours with 10 μM lovastatin or DMSO vehicle with concurrent addition of either 10 μM FPP, 10 μM GGPP, or vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus control; #P < 0.05 versus lovastatin + vehicle; δP < 0.05 versus lovastatin + FPP; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 2
 
Geranylgeranyl pyrophosphate, but not FPP, supplementation reverses lovastatin-induced suppression of SPARC protein levels. SPARC levels (n = 6) were assessed in the conditioned media of primary human TM cells incubated for 72 hours with 10 μM lovastatin or DMSO vehicle with concurrent addition of either 10 μM FPP, 10 μM GGPP, or vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus control; #P < 0.05 versus lovastatin + vehicle; δP < 0.05 versus lovastatin + FPP; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 3
 
Lovastatin inhibits TGF-β2 induction of SPARC. (A) SPARC mRNA expression (n = 5). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 6) from conditioned media of primary human TM cells pretreated for 24 hours with 10 μM lovastatin or DMSO vehicle followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2, or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.05; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 3
 
Lovastatin inhibits TGF-β2 induction of SPARC. (A) SPARC mRNA expression (n = 5). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 6) from conditioned media of primary human TM cells pretreated for 24 hours with 10 μM lovastatin or DMSO vehicle followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2, or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.05; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 4
 
KLF4 expression in human TM. (A) Representative immunoblot showing detection of KLF4 in normal cadaveric human TM tissue from a 42-year-old donor. (B) Immunolocalization of KLF4 in human trabecular meshwork from an 84-year-old donor. Representative images demonstrate KLF4 expression (red) throughout the TM as well as inner and outer walls of Schlemm's canal. Cell nuclei stained with DAPI (blue). AC, anterior chamber; SC, Schlemm's canal. Scale bar: 50 μm.
Figure 4
 
KLF4 expression in human TM. (A) Representative immunoblot showing detection of KLF4 in normal cadaveric human TM tissue from a 42-year-old donor. (B) Immunolocalization of KLF4 in human trabecular meshwork from an 84-year-old donor. Representative images demonstrate KLF4 expression (red) throughout the TM as well as inner and outer walls of Schlemm's canal. Cell nuclei stained with DAPI (blue). AC, anterior chamber; SC, Schlemm's canal. Scale bar: 50 μm.
Figure 5
 
Lovastatin induces expression of KLF4. (A) KLF4 mRNA levels (n = 5). (B) Representative immunoblot and densitometric analysis of KLF4 protein expression (n = 4) from cell lysates of primary human TM cells incubated for 24, 48, and 72 hours with either 10 μM lovastatin or DMSO vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus its time-corresponding vehicle control; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 5
 
Lovastatin induces expression of KLF4. (A) KLF4 mRNA levels (n = 5). (B) Representative immunoblot and densitometric analysis of KLF4 protein expression (n = 4) from cell lysates of primary human TM cells incubated for 24, 48, and 72 hours with either 10 μM lovastatin or DMSO vehicle. All data are expressed as the mean ± SEM (*P < 0.05 versus its time-corresponding vehicle control; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 6
 
Effect of TGF-β2 stimulation on KLF4 expression. (A) KLF4 mRNA levels (n = 6). (B) Representative immunoblot and densitometric analysis of KLF4 protein levels (n = 4) from cell lysates of primary human TM cells pretreated for 24 hours with 10 μM lovastatin or DMSO vehicle followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2, or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.01; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 6
 
Effect of TGF-β2 stimulation on KLF4 expression. (A) KLF4 mRNA levels (n = 6). (B) Representative immunoblot and densitometric analysis of KLF4 protein levels (n = 4) from cell lysates of primary human TM cells pretreated for 24 hours with 10 μM lovastatin or DMSO vehicle followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2, or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.01; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 7
 
Overexpression of KLF4 suppresses SPARC levels under basal and TGF-β2 stimulatory conditions. (A) SPARC mRNA expression (n = 4). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 4) from conditioned media of primary human TM cells infected with either Ad-Null or Ad-KLF4 adenovirus followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2 or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.05; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 7
 
Overexpression of KLF4 suppresses SPARC levels under basal and TGF-β2 stimulatory conditions. (A) SPARC mRNA expression (n = 4). (B) Representative immunoblot and densitometric analysis of SPARC protein levels (n = 4) from conditioned media of primary human TM cells infected with either Ad-Null or Ad-KLF4 adenovirus followed by 24-hour stimulation with either 3 ng/mL activated, recombinant human TGF-β2 or HCl vehicle. All data are expressed as the mean ± SEM (*P < 0.05; n refers to the number of independent experiments performed using n different primary human TM cell strains).
Figure 8
 
Working model for the regulation of SPARC by lovastatin.
Figure 8
 
Working model for the regulation of SPARC by lovastatin.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×