MicroRNAs have emerged in recent years as critical modulators of posttranscriptional gene expression. Aberrant changes in microRNA profiles have been shown to have profound pathophysiological consequences, including most notably tissue fibrosis.33
Using a combination of gain- and loss-of-function experiments, we have identified the miR-29 family as an important regulator of ECM synthesis in the TM.
Analysis of miR-29 family expression in cultured TM cells revealed the presence of all three paralogs. Interestingly, the levels of miR-29a were found to be significantly greater than those of miR-29b and miR-29c, suggesting that endogenous miR-29a may mediate a more expansive role in global gene regulation than miR-29b and miR-29c. Loss-of-function studies using whole-genome microarrays will be necessary to evaluate the global importance of each miR-29 paralog.
Previous studies have shown that TGF-β2 promotes ECM synthesis in the TM.20,25,26
TM cells incubated with TGF-β2 for 24, 48, and 72 hours displayed a significant induction in miR-29a expression at 48 hours and 72 hours. The delayed induction of miR-29a may represent a negative feedback or homeostatic response by the TM to curtail TGF-β2–mediated ECM deposition. Several reports have provided evidence for the existence of an ECM homeostatic mechanism in the TM.51,52
In contrast to miR-29a, miR-29b expression was significantly reduced by TGF-β2 at 24, 48, and 72 hours, suggesting that its suppression may be important in facilitating ECM deposition by TGF-β2. Although no significant changes were observed in miR-29c expression after TGF-β2 treatment, the overexpression of miR-29c suppressed the synthesis of several ECM components by TGF-β2, suggesting an important role for miR-29c in the regulation of TGF-β2–mediated ECM production.
TGF-β2 signaling promotes the deposition of various ECM components in the TM. Our laboratory and others have recently revealed that TGF-β2 induces SPARC expression in the TM (Kang M, et al. IOVS
2010;51:ARVO E-Abstract 6100; Bollinger KE, et al. IOVS
2010;51:ARVO E-Abstract 3207). Matricellular proteins, and in particular SPARC, are associated with increased fibrosis and aberrant tissue remodeling, processes implicated as major contributors to glaucoma pathogenesis. Mice bearing a homozygous deficiency in SPARC exhibit a 15% to 20% lower IOP than do control wild-type mice.53
Despite these findings, the regulatory mechanisms governing the TGF-β2–mediated synthesis of ECM in the TM remain incompletely understood. The SMAD3 transcription factor functions as an important downstream mediator of TGF-β2 signaling.54
Our data demonstrate that SMAD3 inhibition modulates miR-29b expression differently under basal and TGF-β2 stimulatory conditions. This effect may be attributed to differing gene expression environments, resulting in the altered recruitment of regulatory complexes. A working model for the role of miR-29 in TGF-β2–mediated signaling is shown in . Although we have identified SMAD3 as an important modulator of miR-29b expression, recent studies have suggested the involvement of additional pathways in the control of miR-29b levels by TGF-β2. One such candidate is the p38 mitogen-activated protein kinase signaling pathway. p38 is activated by TGF-β2 and is important in its production of ECM proteins.54
A recent report55
has demonstrated the involvement of p38 in the regulation of miR-29b expression. Another possible factor involved in the regulation of miR-29b is SMAD7, an inhibitory SMAD that has been shown to antagonize TGF-β2–mediated signaling in the TM.56
Future studies will be necessary to assess the role of both p38 and SMAD7 in the regulation of miR-29b by TGF-β2 in the TM.
Figure 6. Working model for the role of the miR-29 family in TGF-β2–mediated signaling. TGF-β2 stimulation activates SMAD3 signaling, which is necessary for both miR-29b expression and ECM induction by TGF-β2. Concurrently, TGF-β2 (more ...)
The miR-29 family is transcribed from two bicistronic clusters: miR-29a and miR-29b-1 are coexpressed from one cluster, whereas miR-29c and miR-29b-2 are coexpressed from another.33
The exclusive effect of SMAD3 inhibition on miR-29b expression after treatment for 24 hours with TGF-β2 or its vehicle suggests that SMAD3 modulates miR-29b expression by a posttranscriptional mechanism. Noncanonical functions for SMAD3 have been identified.47
Specifically, SMAD3 has been reported to facilitate the posttranscriptional processing of microRNAs by its association with the DROSHA microprocessor complex.46,47
Dynamic modulation of ECM turnover is critical for IOP homeostasis. Combined data from our gain- and loss-of-function experiments reveal the importance of the miR-29 family in the regulation of ECM synthesis in the TM. In particular, the miR-29 family suppressed various ECM proteins in the context of both basal and TGF-β2 stimulation, including SPARC, collagen I, collagen IV, and laminin. Consistent with these observations, a recent study41
demonstrated, by microarray and real-time quantitative PCR analyses, that miR-29b overexpression suppresses basal levels of multiple ECM components in the TM, including SPARC, COL1A1, COL1A2, COL4A1, and LAMC1. Transgenic mice containing alterations in SPARC, collagen I, and collagen IV gene expression have been shown to develop significant changes in IOP.53,57,58
Future studies examining the consequence of alterations in miR-29 family expression on IOP in ex vivo and in vivo glaucoma models will be important.
Multiple reports have demonstrated the presence of elevated levels of TGF-β2 in the aqueous humor of POAG patients.21–24
The molecular mechanisms governing TGF-β2 signaling are complex and multifactorial.59
Although our data show that miR-29a levels are upregulated and miR-29b levels are downregulated by TGF-β2 stimulation, the relative contributions of these effects to the overall synthesis of the ECM remain unknown. As we have demonstrated, the relative expression of these paralogs varies significantly, making it unlikely that their effects would cancel. Additional studies using ex vivo and in vivo glaucoma models will be necessary to characterize the relative expression of the miR-29 paralogs in the TM and to assess their functional roles with regard to TGF-β2 signaling and ECM synthesis.
Collectively, the results presented here identify an important role for the miR-29 family in the coordinated regulation of ECM gene networks in the TM. Our findings lend further insight into the complex regulatory mechanisms governing ECM homeostasis and TGF-β2 signaling in the TM and may serve in the development of novel therapeutics for glaucoma.