A major risk factor for the development of POAG is elevated IOP (Kass et al. 2002
, AGIS. 2000
). Elevated IOP is primarily due to increased AH outflow resistance within the juxtacanalicular region (JCT) of the TM. This resistance is associated with local morphologic and biochemical changes in the JCT (Rohen. 1983
). In the normal JCT there are adequate homeostatic control mechanisms with respect to ECM protein deposition, turnover and modification. However in POAG, distinct structural changes occur in the JCT including the accumulation of fibrillar proteins (Lutjen-Drecoll. 2005
, Rohen. 1983
). These changes are thought to lead to decreased outflow facility and increased IOP. The accumulation of ECM proteins may be the result of abnormal regulation of ECM protein turnover by MMPs and PAI-1 (Bradley et al. 1998
, Fleenor et al. 2006
, Fuchshofer et al. 2003
, Pang et al. 2003
). It is also possible that elevated expression of cross-linking enzymes within the TM may lead to ECM proteins that are resistant to enzymatic digestion by MMPs. Growth factors present in the TM and AH are known to regulate both MMPs and cross-linking enzymes in TM cells. Thus growth factors are considered important regulators of ECM homeostasis and altered growth factor expression may result in deleterious consequences to the TM and/or regulation of AH outflow pathways.
TGF-β2 influences many aspects of cellular behavior including proliferation, differentiation, migration, and ECM synthesis and breakdown (Zhu and Burgess. 2001
)(Verrecchia and Mauviel. 2002
). With respect to glaucoma, a number of studies have reported elevated levels of TGF-β2 in the AH of glaucoma patients compared to age-matched normal eyes (Inatani et al. 2001
, Ochiai and Ochiai. 2002
, Ozcan et al. 2004
, Picht et al. 2001
, Tripathi et al. 1994
). Experimentally, perfusion of TGF-β2 in ex-vivo anterior segment organ culture models causes an accumulation of fibrillar material in the TM (Gottanka et al. 2004
). Fleenor et al. (2006)
, also reported that perfusion of TGF-β2 in ex-vivo anterior segment organ culture models elevated IOP in a time dependent manner and also increased fibronectin and PAI-I levels in the elutes while decreasing outflow facility (Fleenor et al. 2006
). Intraocular injection of bioactive TGF-β2 has been reported to induce ocular hypertension in the rat and mouse and to reduce AH outflow facility in the mouse (Shepard et al., 2010). Thus, it is reasonable to suspect TGF-β2 may have a direct role in deposition and accumulation of ECM proteins in the TM of glaucoma patients.
TGM2 is a cross-linking enzyme that may contribute to the increase resistance of aqueous humor outflow. We speculate that cross-linking of proteins by TGM2 would stiffen the trabecular meshwork. In fact, recent evidence by atomic force microscopy have shown that the GTM tissue is significantly stiffer compared to NTM tissues (Last et al., 2011
). Welge-Lüssen and co-workers (2000)
first reported the presence and induction of TGM2 by TGF-β1 and β2 in cultured HTM. Our laboratory previously reported that TGM2 protein levels and enzyme activities were elevated in GTM cells and tissues (Tovar-Vidales et al., 2008
). Despite the fact that TGF-β1 and β2 induces TGM2 and TGM2 is elevated in glaucomatous TM cells, the exact role or molecular mechanism for TGM2 in the pathogenesis of glaucoma is not clear. Therefore, the purpose of this study was three-fold: (a) to examine the induction of TGM2 by TGF-β2, (b) to identify if the canonical TGF-β2 signaling pathway is utilized to regulate TGM2 in cultured TM cells and (c), to determine if CTGF acts as a downstream mediator for TGF-β2 - regulation of TGM2 in human TM cells.
Within the glaucomatous eye, TGF-β2 has been reported to be as high as 2.7ng/ml compared to 1.48ng/ml in normal eyes (Tripathi et al. 1994
). For our purposes we performed a dose response experiment using TGF-β2 at concentrations of 0.5ng/ml, 1.25ng/ml, 2.5ng/ml, 5.0ng/ml, and 10.0ng/ml. Interestingly, our data did not show a dose dependent increase of TGM2 with TGF-β2. We observed an induction of TGM2 by TGF-β2 from 1.25 to 10.0 ng/ml concentrations when compared to untreated controls. Thus even a very low concentration of TGF-β2 (1.25ng/ml) is capable of stimulating TGM2 expression in TM cells.
However the presence of TGM2 protein does not always correlate with enzyme activity. Therefore we examined the induction of TGM2 enzyme activity by TGF-β2 in cultured TM cells. Enzyme activity was measured by the incorporation of biotin-cadaverine, a pseudo-substrate for TGM2 (Tovar-Vidales et al. 2008
). Our results demonstrated significantly increased enzymatic activity of TGM2 in cultured TM cells following exogenous treatment by TGF-β2. These results demonstrate that TGM2 is induced by TGF-β2 in primary TM cells and is biologically active.
We had previously reported that TGM2 protein levels and enzyme activities were elevated in GTM cells and tissues (Tovar-Vidales et al., 2008
). Previous reports had indicated that TGF-β2 levels were also elevated in the aqueous humor of glaucomatous patients (Inatani et al. 2001
, Picht et al. 2001
, Tripathi et al. 1994
). However previous reports had not examined endogenous TGF-β2 protein levels in glaucomatous TM cells nor did they co-localize TGF-β2 and TGM2 in glaucomatous TM tissues. Our results demonstrated significantly increased protein levels of TGF-β2. , Although, the molecular weight of the TGF-β2 protein band was detected at 60 kDa, we presume the antibody is detecting the latent form of TGF-β2, thus there is greater expression of the TGF-β2 latent form in glaucomatous TM cells when compared to normal TM cells. Interestingly, Tritschler et al., (2009)
demonstrated a similar molecular weight of the latent form of TGF-β1 and TGF-β2 in the conditioned media of human malignant glioma cells. (Tritschler et al. 2009
). Secondly, to verify that co-localization of TGF-β2 with TGM2 was increased in glaucoma, normal TM and glaucomatous TM tissues were evaluated with immunohistochemistry. Our immunohistochemical results indicated increased expression and increased co-localization for both TGF-β2 and TGM2 in glaucoma TM tissues in comparison to normal TM tissues. This is of interest since the endothelium of Schlemm’s canal is considered to be the site of aqueous humor outflow resistance (Tamm and Fuchshofer. 2007
). The increase co-localization for both TGF-β2 and TGM2 are interesting since there is evidence that TGM2 can increase the conversion of latent TGF-β to its biologically active form, and then TGF-β can further increase TGM2 providing a positive feedback mechanism in glaucomatous TM (Kojima et al., 1993
, Kojima S et al., 1995
, Nunes et al., 1997
). These results suggest a direct role of TGM2 induction by TGF-β2 in the TM of human glaucomatous eyes.
The canonical signaling pathway utilized by TGF-β2 involves ligand binding to a receptor complex and subsequent downstream signaling via receptor Smad proteins (Massague. 1998
, Massague and Chen. 2000
, Massague. 2000
). Specific Smad proteins for TGF-β2 include Smad2 and Smad3 (Massague. 1998
, Massague and Chen. 2000
, Massague. 2000
). However several reports indicate that Smad3 is mainly utilized for fibrotic responses to TGF-β2 (Flanders. 2004
, Roberts et al. 2003
). As a first step to evaluate TGF-β2 regulation of TGM2, we wanted to determine if isolated TM cells express Smad3 and respond to exogenous TGF-β2 via Smad3 phosphorylation in a time dependent manner. Our data demonstrated that TM cells express endogenous phosphorylated Smad3 (p-Smad3) and respond to exogenous TGF-β2 with increased protein levels of p-Smad3. The endogenous presence of p-Smad-3 indicates an autocrine loop is present in TM cells. We also showed that SIS3, a inhibitor of the phosphorylation of Smad3, down-regulated TGM2 protein levels in TM cells. In addition, silencing of Smad3 gene expression via siRNA also decreased TGM2 protein expression. Our immunocytochemistry results indicated that upon TGF-β2 treatment, there is more Smad3 in the nucleus and there is none to little staining in the Smad3 siRNA. Treatment with Smad3 siRNA also reduced TGM2 protein levels in TM cells. These results indicate that human TM cells can respond to exogenous TGF-β2 via phosphorylation of Smad3 and that blocking Smad 3 protein phosphorylation or reducing the Smad3 protein levels, prevents TGF-β2 upregulation of TGM2.
Connective tissue growth factor (CTGF) is highly expressed in the TM (Tomarev et al. 2003
) and has been shown to be an important downstream mediator of fibrotic changes induced by TGF-β2. (Leask and Abraham. 2004
). Specifically, Junglas et al., (2009)
demonstrated that CTGF is a critical mediator of the effects of TGF-β2 on the synthesis of ECM proteins in TM cells (Junglas et al. 2009
). Fuchshofer et al., (2007)
, previously demonstrated that TGF-β2 induces CTGF in TM cell cultures (Fuchshofer et al. 2007
). Our current results verify and are in agreement with their study. We also determined if the induction of TGM2 by TGF-β2 was mediated by CTGF in TM cell strains. We first demonstrated reduction of endogenous CTGF protein levels in TM cells following treatment with CTGF siRNA. While this reduction in CTGF blocked TGF-β2 upregulation of FN, there was no reduction of TGM2 protein levels in TM cells. Interestingly, using optic nerve head astrocytes (ONHA) , Fuchshofer and coworkers (2005)
, demonstrated that TGF-β2 did induce TGM2 mRNA expression and protein levels and that this action was mediated via CTGF (Fuchshofer et al. 2005
). Priglinger has reported that TGM2 is present in PVR membranes and the level is related to the state of differentiation of retinal pigmented epithelial (RPE) cells and TGF-β2 stimulation. They further showed that the TGF-β2-mediated increase of TGM2 was also independent of CTGF. Thus, ONA regulation of TGM2 is CTGF dependent and in RPE and TM cells it is not dependent on CTGF. Therefore, it appears that the regulation of endpoints may be cell context dependent.
In conclusion, our results demonstrated that cultured glaucomatous TM cells and tissues from human donors have elevated levels of TGF-β2. We have shown that TM cells express TGM2 and expression and enzyme activity are stimulated by TGF-β2. In addition, we have demonstrated that the canonical pathway is stimulated by TGF-β2 in TM cells. We also showed Smad3 siRNA and SIS3 down-regulated TGM2 protein levels. Lastly we demonstrated that TGF-β2 regulation of TGM2 in human TM cells is independent of CTGF action.