Angiogenic and Fibrogenic Dual-effect of Gremlin1 on Proliferative Diabetic Retinopathy
Xinjing WuBing QinRuiwen ChengRu ZhouXingxing WangZhengyu ZhangXiying MaoZhan XieMingkang ChenLin JiangPing XieJiangdong JiWeiwei ZhangSongtao YuanZizhong HuQinghuai Liu
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Ocular angiogenic diseases, such as proliferative diabetic retinopathy (PDR), are often characterized by pathological new vessels and fibrosis formation.Anti-vascular endothelial growth factor (VEGF) therapy, despite of its efficiency to inhibit new vessels, has limitations, including drug resistance and retinal fibrosis.Here, we identified that Gremlin1, a novel angiogenesis and fibrosis inducer, was secreted from Müller glial cells, and its expression increased in the vitreous fluid from patients with PDR.Mechanistically, Gremlin1 triggered angiogenesis by promoting endothelial-mesenchymal transition via the EGFR/RhoA/ROCK pathway.In addition, Gremlin1 activated microglia to present profibrotic and fibrogenic properties.Further, anti-Gremlin1 antibody inhibited ocular angiogenesis and microglia fibrosis in mouse models.Collectively, Gremlin1 could be a potential therapeutic target in the treatment of ocular angiogenic diseases.Previous studies have demonstrated that expression of either constitutively‐active (CA) or dominant negative (DN) RhoA reduced tight junction barrier function in cultured MDCK monolayers (Jou et al. 1998). However, the mechanisms by which RhoA disruption causes barrier loss have not been defined. The aims of this study were to determine whether RhoA regulates barrier function in mammary epithelia and to identify the molecular events responsible. V14 and N19 (CA and DN, respectively) RhoA were expressed in EpH4 mammary epithelial cells. RhoA (CA or DN) expression in confluent monolayers caused a dose‐dependent loss of transepithelial electrical resistance. Neither trafficking of ZO‐1 and occludin to the tight junction nor cell viability were affected by CA or DN RhoA expression, but western blots showed that ERK and Akt were phosphorylated (consistent with activation) after either CA or DN RhoA expression. Transcript content of several claudins was altered after CA or DN RhoA expression. Most notably, claudin‐2 mRNA and protein expression were increased following either CA or DN RhoA expression. These data demonstrate that RhoA regulates the barrier in mammary epithelia and suggest that increased claudin‐2 expression contributes to RhoA‐mediated tight junction regulation. Supported by Charite Universitaetsmedizin Berlin.
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How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, RhoA can promote focal adhesion (FA) growth or disassembly, but how RhoA activity mediates these opposite outcomes is not clear. Here, we develop a photoswitchable RhoA guanine nucleotide exchange factor (GEF), psRhoGEF, to precisely control endogenous RhoA activity. Using this optical tool, we discover that peak FA disassembly selectively occurs upon activation of RhoA to submaximal levels. We also find that Src activation at FAs selectively occurs upon submaximal RhoA activation, identifying Src as an amplitude-dependent RhoA effector. Finally, a pharmacological Src inhibitor reverses the direction of the FA response to RhoA activation from disassembly to growth, demonstrating that Src functions to suppress FA growth upon RhoA activation. Thus, rheostatic control of RhoA activation by psRhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.
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Al-Shabrawey M, Mussell R, Kahook K, Tawfik A, Eladl M, Sarthy V, Nussbaum J, El-Marakby A, Park SY, Gurel Z, Sheibani N, Maddipati KR. Increased expression and activity of 12-lipoxygenase in oxygen-induced ischemic retinopathy and proliferative diabetic retinopathy: implications in retinal neovascularization. Diabetes 2011;60:614–624
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In this study, we addressed the potential relationship between prominin-1 (prom1) and vascular endothelial growth factor (VEGFA) in diabetes-induced retinopathy. In total, we examined 28 retinas from 14 rats with streptozotocin-induced diabetes and 30 retinas from 15 untreated control rats. ELISA was used to measure the level of prom1 and VEGFA in retinal tissue homogenates. Immunohistochemical techniques were used with antibodies directed against prom1, VEGFA, and CASP-3. After 180 days of diabetes induction, we performed light and electron microscopy studies on rat eyes to evaluate histopathological changes and to estimate the de novo metric “Diabetic Retinopathy Histopathological Index” (DRHI). These changes were then correlated to the tissue and immunoexpression levels of prom1 and VEGFA. The data showed a significant upregulation of the tissue levels and optical densities (ODs) of VEGFA and prom1 immunoreactivity in diabetic retinas compared with controls. Both the tissue levels and OD values of prom1 and VEGFA correlated significantly with each other and to the diabetic structural changes as calculated by DRHI. Taken together, these data provide new insight into the potential role of prom1 and VEGFA in the development of diabetic retinopathy.
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The pro-oncogenic role of RhoA has been well identified in other cancers, but rarely in cervical cancer (CC), one of the main causes of cancer-related death in women. In the present study, we identified the overexpression of RhoA and its downstream effectors, ROCK-1 and ROCK-II, in CC specimens using western blotting. Then, we determined the effect of RhoA on the proliferation and migration of Hela cells, one of CC cell lines, by upregulating or downregulating the RhoA expression in Hela cells. We found that there was an overexpression of RhoA, ROCK-I/II in CC, which was associated with the progression of CC. And we confirmed that RhoA promoted the proliferation and migration of CC cells. In conclusion, we found a positive correlation among RhoA with the progression of CC by in vivo and in vitro evidences. A high RhoA expression in CC may predict a high metastatic potential of CC.
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The small GTPase RhoA controls many important cellular processes through its ability to activate multiple downstream effector pathways. Most RhoA effectors contain a Rho-binding domain (RBD), and interaction between active RhoA and the RBD typically induces a conformational change in effectors that stimulates their recruitment or activity. Isolated GTPase binding domains fused to GST have been widely used in so-called pulldown assays to measure the activation state of other GTPases in cell lysates. Similarly, GST fusions containing the RBD of the RhoA effector Rhotekin have been widely adopted as a standardized tool for the measurement of RhoA activation. RBDs have also been used to generate fluorescent reporter constructs to localize sites of GTPase activation in intact cells. In this report, we demonstrate that not all forms of active RhoA are capable of interacting with the Rhotekin RBD. A constitutively active RhoA-G14V mutant, which interacted with the RBDs of ROCK2 and mDIA1, was unable to bind the Rhotekin RBD as evidenced by both conventional GST pulldown assay and our newly established BRET assay. Furthermore, active RhoA induced by different stimuli in cells also displayed binding preference for its diverse effectors. Our data demonstrate that RhoA may undergo effector-specific activation for differential regulation of its downstream pathways, and that RhoA activation should not be defined solely by its interaction with Rhotekin.
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rhoA p21, a ras p21-like small GTP-binding protein, purified from bovine aortic smooth muscle is similarly modified at its C-terminal region as described for ras p21s. In this study, I examined the role of the post-translational modifications of the C-terminal region of rhoA p21 by comparing bovine rhoA p21 with bacterially-produced rhoA p21 because the bacterial protein was not modified. Bovine rhoA p21 bound to plasma membranes, but bacterial rhoA p21 did not. Both the stimulatory and inhibitory GDP/GTP exchange proteins for bovine rhoA p21 were inactive for bacterial rhoA p21. On the other hand, the GTPase activating protein for bovine rhoA p21 was also active for bacterial rhoA p21. These results indicate that the post-translational modifications of the C-terminal region of bovine rhoA p21, which are absent in bacterial rhoA p21, are essential for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins but not with the GTPase activating protein.
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