TGF-β2 induces transdifferentiation and fibrosis in human lens epithelial cells via regulating gremlin and CTGF
Introduction
Posterior capsule opacification (PCO) is the most common postoperative complication after extracapsular cataract extraction or phacoemulsification surgery, it is mainly caused by the transdifferentiation, proliferation, migration, and collagen-production of the residual lens epithelial cells (LECs) in the capsule.
A wide range of cytokines and growth factors are involved in the development of PCO [1]. Among them, TGF-β2 and CTGF have been shown to be critical. Both TGF-β2 and CTGF exert biological functions by binding to their respective receptors and induce cell growth and differentiation, extracellular matrix (ECM) synthesis, and tissue fibrosis [2]. A recent report from Lee and Joo has shown that both TGF-β2 and CTGF enhance the synthesis of epithelial mesenchymal transition (EMT)-specific proteins α-SMA and various ECM proteins including Fn, COL-I, collagen type IV, but reduce the expression of E-cadherin and other intrinsic proteins of LECs [3]. As α-SMA, Fn and COL-I are primary constituents of ECM and crucial for EMT induction, and E-cadherin plays an important role in the maintenance of morphology and structural integrity of normal epithelial cells, the role of TGF-β and CTGF in transdifferentiation and fibrosis of intraocular LECs, trabecular meshwork (TM) cells and retinal pigment epithelial cells (RPE) has been considered to be the main causes for many of the pathological processes in the eye [4], [5].
Gremlin, a member of the DAN family protein, is one of the major endogenous antagonists of bone morphogenetic protein (BMP) which plays an important role in many organ development [6]. BMP has a few isoforms such as BMP2, BMP4 and BMP7, they selectively regulate the proliferation and differentiation of many kinds of tissue cells such as osteocytes and tumor cells [7], [8]. Gremlin can effectively inhibit BMP activity by binding to BMP extra- and intra-cellularly. Binding of gremlin to intracellular BMP prevents the secretion of mature BMP [9], therefore it in essence regulates the embryonic development, growth, and cell differentiation [10]. Some recent reports have demonstrated that gremlin induces ECM synthesis in TM cells and it further increases the expression of Fn protein when added together with BMP4 and TGF-β, whereas addition of BMP4 and TGF-β2 without gremlin fails to do so [11], [12]. The hypothesis is that BMP can inhibit the profibrotic and transdifferentiation effect of TGF-β, while gremlin can bind to BMP and abolish its inhibitory effect on TGF-β, thus enhancing the profibrotic effect of TGF-β [13]. Although the effects of TGF-β2 and CTGF on the EMT and ECM synthesis of LECs have been reported, it remains to be determined whether gremlin plays a role in the EMT and ECM synthesis of LECs, and what are underlying mechanisms of its function.
The signaling pathway of TGF-β2 is complex and it involves the reciprocal interactions among different signal transduction pathways [14], [15] such as the canonical Smad signaling pathway. TGF-β2 binds to a type II receptor, which phosphorylates a type I receptor. The type I receptor then phosphorylates receptor-regulated Smad2 and Smad3 which can bind the coSmad Smad4. CoSmad complexes accumulate in the nucleus where they act as transcription factors and participate in the regulation of target gene expression. Some recent studies have demonstrated that Smad signaling pathway plays an important role in assisting TGF-β2 on EMT and ECM production in HLECs [14], and that gremlin can activate Smad signaling pathway too [11]. However, it is not clear whether CTGF and/or gremlin are regulated by the TGF-β/Smad signaling pathway and blockade of Smad signaling pathway can effectively inhibited gremlin induced expression of α-SMA, Fn, COL-I, in HLECs.
In this study we first induced in vitro cultured HLECs with various concentrations of TGF-β2, CTGF, and gremlin, respectively, to examine the expression of TGF-β2, CTGF and gremlin-induced EMT-associated proteins and ECM synthesis, as well as the activation of Smad signaling pathway. Following that we investigated the induction of CTGF and gremlin expressions, in HLECs by TGF-β2 and whether the effect of TGF-β2 on the expression of EMT-associated proteins, ECM synthesis, and activation of Smad in HLECs can be blocked by specifically silencing CTGF and gremlin. Data obtained from this study will provide experimental basis for better understanding the functional relationship among TGF-β2, CTGF and gremlin, and the potential underlying mechanisms for PCO.
Section snippets
Culture and treatment of HLECs
HLEC line SRA01/04 was purchased from ATCC (Manassas, VA, USA). 1 × 106 cells within 20 passages were seeded into culture flask with DMEM containing 10% fetal bovine serum (FBS). The culture medium was replaced with serum-free DMEM when the cells approached 70% confluence and cells were cultured for 24 h. The cells in the experimental group were then treated with 3 ml of serum-free medium containing TGF-β2, CTGF, or gremlin at various concentrations for a further 24 h before cells were harvested for
The effect of TGF-β2, CTGF and gremlin on the mRNA and protein expressions of α-SMA, Fn, COL-I, and E-cadherin in HLECs
TGF-β2, CTGF, and gremlin are believed to be involved in the process of fibrosis in different cells and tissues, and TGF-β2 can induce the expression of CTGF and gremlin in some cells [2], [16]. Our results confirmed these findings in HLECs. After 24 h treatment of HLECs with TGF-β2, the mRNA (Fig. 1A, ∗P < 0.05; ∗∗P < 0.001) and protein (Fig. 1D) levels of CTGF, gremlin, α-SMA, Fn, and COL-I were significantly up-regulated in a dose-dependent manner. In contrast, E-cadherin expression was
Discussion
Cell transdifferentiation, proliferation, migration, and collagen synthesis in the capsule after cataract surgery are the main reasons of PCO. A variety of regulatory factors have been implicated in this pathological process. Among them, TGF-β plays a key role in the development of the disease. CTGF is functionally related to TGF-β as the downstream factor of TGF-β and is involved in the pathogenesis of LECs and vascular smooth muscle cells related diseases [17], [18], while gremlin enhances
Acknowledgments
This study was supported by the fund from the National Natural Science Foundation of China (No. 81070721) and International Exchange Program of Shaanxi Province (No. 2012KW-31). We thank institute of Neurobiology Xi’an Jiaotong University College of Medicine for assisting in the study.
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