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1. Transforming Growth Factor-β Promotes Rho G-protein Mediated Synthesis and Secretion of Endothelin-1 in Human Trabecular Meshwork Cells Cynthia L. Von Zee1,2A, Kelly A. Langert1,2B, Evan B. Stubbs, Jr.1,2A 1Research Service (151), Edward Hines, Jr. VA Hospital, Hines, IL AOphthalmology, BProgramof Neuroscience, 2Loyola University Chicago, Maywood, IL Introduction Results Primary open-angle glaucoma (POAG) is one of the most common causes of blindness worldwide, affecting approximately 1.86% or nearly 2 million individuals 40 years and older in the US.A poorly-understood risk factor for the initiation and progression of POAG is high intraocular pressure (IOP).In healthy eyes, normal IOP is sustained through balanced production and outflow of aqueous humor (AH). In adults, the majority (>50%) of AH exits the eye through a conventional outflow pathway involving the trabecular meshwork (TM).Resistance to AH outflow through the TM is mediated, in part, through Rho G-protein mediated enhancement of TM cell actin cytoskeleton contractile tone. Analysis of AH samples from POAG patients reveal marked increases in the content of endothelin-1 (ET-1), a potent vasoconstrictor that promotes Rho G-protein mediated TM cell contraction. Levels of transforming growth factor (TGF)-β2, a key regulator of extracellular matrix structure within the TM, are similarly aberrantly increased in AH of POAG patients. Whereas the significance of these independent clinical observations remains unclear, emerging evidence strongly supports a pathologic association between either ET-1 or TGF-β2 with elevated IOP in the pathogenesis of POAG. Mechanisms responsible for regulating endogenous synthesis and secretion of ET-1 and TGF-β2 within the eye are currently unknown. In other cell types, application of TGF-β2 has been shown to significantly enhance both prepro (pp) ET-1 gene transcription as well as secretion of mature ET-1 peptide. Interestingly, a role for Rho G-protein activation in TGF-β2 mediated upregulation of ppET-1 mRNA has been proposed.In this study, we determined the effect of TGF-β2 signaling on expression and secretion of ET-1 by human TM cells. TGF-β2 increases ppET-1 mRNA content TGF-β2 increases mature ET-1 peptide secretion TGF-β2 increases Rho G-protein mediated filamentous actin stress fiber organization A. B. A. B. C. NTM5 GTM3 NTM5 GTM3 Primary NTM5 C. D. D. E. GTM3 GTM3 GTM3 GTM3 Vehicle TGF-β2 GTM3 Vehicle TGF-β2 ET-1 content present in culture medium was quantified by ELISA. Data shown are the mean ± SD (N=3-9) from 1-3 separate experiments each performed in triplicate. Statistical significance between groups is shown. (A-C) *p < 0.01;**p < 0.0001, Student’s t-test.(D) *p < 0.05; **p < 0.01 compared with 0 ng/ml, one-way ANOVA with Dunnett’s post-hoc analysis. (E) *p < 0.01; **p < 0.001 compared with Baseline, one-way ANOVA with Bonferroni’s post-hoc analysis. Levels of ppET-1 mRNA were quantified by qRT-PCR. Data shown are the GAPDH-normalized fold changes from 2-3 separate experiments (N=6-9) each performed in triplicate and expressed as mean ± SD. Statistical significance between groups is shown. (A, B) *p < 0.01, unpaired Student’s t-test.(C) *p < 0.01 compared with 0 ng/ml, one-way ANOVA with Dunnett’s post-hoc analysis. (D) *p < 0.01 compared with Baseline, one-way ANOVA with Bonferroni’s post-hoc analysis. TGF-β2 increases ppET-1 mRNA content through TGFβRI/ALK-5, not ETA or ETB TGF-β2 increases ET-1 secretion through TGFβRI/ALK-5, not ETA or ETB B. A. B. A. GTM3 GTM3 GTM3 GTM3 Methods Cell Culture: Primary human TM cells were harvested from discarded human corneoscleral rims and cultured to confluence as approved by the Hines V.A. and Loyola University Chicago institutional review boards. SV40-transformed human TM cell lines derived from patients without or with glaucoma (NTM5 and GTM3, respectively) were a generous gift from Alcon Laboratories. All cell cultures were maintained at 37ºC under an atmosphere of 5% CO2/95% air. Treatment of Human TM Cells: Prior to treatment, sub-confluent human TM cells were cultured x 24h in serum-free media containing antibiotics. Recombinant human TGF-β2 (Cell Signaling Technology) was reconstituted as a stock solution in 4 mM HCl containing 0.1% bovine serum albumin (BSA) for 30 min at 23ºC prior to use. Serum-starved cultures were then treated (0-24h) with vehicle (200 nM HCl) or activated TGF-β2 (0-20ng/ml) in fresh serum-free medium containing antibiotics. Real-Time RT-PCR: Total RNA was extracted from human TM cells using TRIzol reagent, and 5 µg was reverse-transcribed using Super Script III First Strand Synthesis system (Life Technologies) as described previously (Von Zee et al., 2009; Stubbs and Von Zee, 2012). Human-specific ppET-1 or GAPDH cDNA sequences were amplified by real-time PCR on a Mini-Opticon PCR detection system. For each sample, the specificity of the real-time reaction product was determined by melting curve analysis. The endogenous expression of GAPDH was unaltered by drug treatments; therefore, relative fold-changes in gene expression were normalized to GAPDH. Endothelin-1 ELISA: Levels of ET-1 in cell culture supernatants were assessed using a commercially-available ELISA kit (R&D Systems, Minneapolis, MN) according to package instructions. Filamentous Actin Staining: Human TM cells grown to subconfluenceon chambered coverslips were treated as described above, fixed (buffered 4% paraformaldehyde), and filamentous actin was stained with AlexaFluor488-conjugated phalloidin. Stained cells were visualized by confocal microscopy. Statistical Analysis:Results are expressed as mean ± SD of duplicate or triplicate cultures, repeated at least one additional time unless otherwise specified. Parametric data were analyzed by Student’s t-test or by one-way ANOVA followed by either a Dunnett’s or Bonferroni’s multiple comparison post-hoc analysis. In all cases, p < 0.05 was considered statistically significant. TGF-β2+ Lovastatin TGF-β2 + GGTI-298 Levels of ppET-1 mRNA were quantified by qRT-PCR. Data shown are the GAPDH-normalized fold changes (N=3-6) from 1-2 separate experiments each performed in triplicate and expressed as mean ± SD. *p < 0.01, one-way ANOVA with Dunnett’s post-hoc analysis. Data shown are confocal photomicrographs of stained cells representative of 7-20 separate cultures. Nuclei are counterstained with DAPI. Bar, 20 µm. ET-1 content present in culture medium was quantified by ELISA. Data shown are the mean ± SD (N=3) from single experiments performed in triplicate. Statistical significance between groups is shown. *p < 0.01, one-way ANOVA with Dunnett’s post-hoc analysis. Inhibition of geranylgeranylation attenuates TGF-β2 mediated ET-1 secretion Lovastatin attenuates TGF-β2 mediated increases in ppET-1 Summary/Conclusion B. B. A. A. TGF-β2 induces ET-1 mRNA expression and enhances ET-1 peptide secretion, in part, through a non-canonical mechanism facilitated by the Rho G-protein subfamily (RhoA/B/C) TGF-β2 markedly promotes Rho G-protein mediated filamentous actin stress fiber organization Elevated levels of TGF-β2 present in AH of POAG patients may promote elevated IOP, in part, by eliciting Rho G-protein mediated aberrant ET-1 synthesis and secretion, and concurrent actin stress fiber organization, in human TM cells. GTM3 GTM3 Primary GTM3 Levels of ppET-1 mRNA content were quantified by qRT-PCR. Data shown are the GAPDH-normalized fold changes from two separate experiments (N=4-6) each performed in (A) triplicate or (B) duplicate and expressed as mean ± SD. Statistical significance between groups is shown. *, p < 0.05; **, p < 0.01 compared with control, one-way ANOVA with Bonferroni’s post-hoc analysis. ET-1 content present in culture medium was quantified by ELISA. Data shown are the mean ± SD (N=3-6) from 1-2 experiments performed in triplicate. Statistical significance between groups is shown. *p < 0.001 compared with control, one-way ANOVA with Bonferroni’s post-hoc analysis. Inhibition of Rho G-protein signaling attenuates TGF-β2 mediated increases in ppET-1 mRNA content Inhibition of Rho G-protein signaling attenuates TGF-β2-mediated increases in secreted ET-1 content B. A. Acknowledgments Primary GTM3 The authors would like to acknowledge Dr. Beatrice Y.J.T. Yueand Mr. Xiang Shen (University of Illinois Chicago) for assistance with procuring and culture of human TM cells. Supported, in part, by grants from the Department of Veterans Affairs (C3638R & B3756-F (EBS), C7506M (CVZ), Pre-Doctoral Associated Health Rehabilitation Research Fellowship (CVZ & KAL)), the Illinois Society for the Prevention of Blindness, the Midwest Eye Banks, and the Richard A. Perritt Charitable Foundation. Content of ppET-1 mRNA in GTM3 cells was quantified by qRT-PCR. Data shown are representative GAPDH-normalized fold changes from 2 separate experiments performed in triplicate (N = 6), expressed as the mean ± SD percent of Control. Statistical significance to respective control groups is shown (Student’s t-test). ET-1 content present in culture medium was quantified by ELISA. Data shown are the mean ± SD (N=3) from single experiments performed in triplicate. Statistical significance between groups is shown. *p < 0.001 compared with control, one-way ANOVA with Bonferroni’s post-hoc analysis.