TAT-mediated PRDX6 protein transduction protects against eye lens epithelial cell death and delays lens opacity

2008 ◽  
Vol 294 (3) ◽  
pp. C842-C855 ◽  
Author(s):  
Eri Kubo ◽  
Nigar Fatma ◽  
Yoshio Akagi ◽  
David R. Beier ◽  
Sanjay P. Singh ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Lens Epithelial Cell ◽  
Biologically Active ◽  
Lens Epithelial Cells ◽  
Protein Levels ◽  
Endogenous Antioxidant ◽  
Tgf Β1

A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-β1 and of α-smooth muscle actin and βig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-β1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS.

Overexpression of matrix metalloproteinase-2 mediates phenotypic transformation of lens epithelial cells

2001 ◽  
Vol 358 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Young SEOMUN ◽  
Jeong-a KIM ◽  
Eunjoo H. LEE ◽  
Choun-Ki JOO
Keyword(s):  
Epithelial Cells ◽  
Matrix Metalloproteinase ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Transforming Growth Factor Β ◽  
Causative Factor ◽  
Matrix Metalloproteinase 2 ◽  
Lens Epithelial Cells ◽  
Cell Phenotype ◽  
Tgf Β1

Transforming growth factor-β (TGF-β) is known to be a causative factor in pathological fibrosis and the metastasis of cancer cells, through effects on molecules of the extracellular matrix (ECM). We evaluated the influence of TGF-β1 on the gene expression of matrix metalloproteinase-2 (MMP-2) in lens epithelial cells (LECs). The results showed that TGF-β1 induced the expression of mRNA for MMP-2 in LECs. Subsequently, in order to examine the role of MMP-2, we overexpressed MMP-2 in LECs by stable transfection. The MMP-2-overexpressing LECs showed typical indicators of a myofibroblast-like cell phenotype, such as multiple layers of cells, elongated morphology, and expression of α-smooth muscle actin. We also showed that an MMP inhibitor blocked the TGF-β1-induced morphological change in LECs. These results demonstrate that MMP-2 plays a role in the transformation of LECs, which has implications for the pathological fibrosis of these cells.

Transforming growth factor-β2-mediated mesenchymal transition in lens epithelial cells is repressed in the absence of RAGE

2021 ◽  
Vol 478 (12) ◽  
pp. 2285-2296
Author(s):  
Mi-Hyun Nam ◽  
Mina B. Pantcheva ◽  
Johanna Rankenberg ◽  
Ram H. Nagaraj
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Knockout Mice ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Lens Epithelial Cells ◽  
Wild Type ◽  
Mesenchymal Transition ◽  
Smad Signaling ◽  
Transforming Growth Factor Β2

Transforming growth factor-β2 (TGFβ2)-mediated epithelial to mesenchymal transition (EMT) in lens epithelial cells (LECs) has been implicated in fibrosis associated with secondary cataracts. In this study, we investigated whether the receptor for advanced glycation end products (RAGE) plays a role in TGFβ2-mediated EMT in LECs. Unlike in the LECs from wild-type mice, TGFβ2 failed to elicit an EMT response in LECs from RAGE knockout mice. The lack of RAGE also diminished TGFβ2-mediated Smad signaling. In addition, treatment with TGFβ2 increased IL-6 levels in LECs from wild-type mice but not in those from RAGE knockout mice. Treatment of human LECs with the RAGE inhibitor FPS-ZM1 reduced TGFβ2-mediated Smad signaling and the EMT response. Unlike that in wild-type lenses, the removal of fiber cell tissue in RAGE knockout lenses did not result in elevated levels of α-smooth muscle actin (α-SMA), fibronectin (FN), and integrin β1 in capsule-adherent LECs. Taken together, these results suggest that TGFβ2 signaling is intricately linked to RAGE. Targeting RAGE could be explored as a therapeutic strategy against secondary cataracts.

MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells

2008 ◽  
Vol 294 (5) ◽  
pp. F1116-F1128 ◽  
Author(s):  
Gerard Elberg ◽  
Lijuan Chen ◽  
Dorit Elberg ◽  
Michael D. Chan ◽  
Charlotte J. Logan ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Epithelial Cells ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Primary Cultures ◽  
Muscle Actin ◽  
Mesenchymal Transition ◽  
Endogenous Expression ◽  
Tgf Β1 ◽  
Stimulation Of

Transforming growth factor-β1 (TGF-β1) is known to induce epithelial-mesenchymal transition in the kidney, a process involved in tubulointerstitial fibrosis. We hypothesized that a coactivator of the serum response factor (SRF), megakaryoblastic leukemia factor-1 (MKL1), stimulates α-smooth muscle actin (α-SMA) transcription in primary cultures of renal tubular epithelial cells (RTC), which convert into myofibroblasts on treatment with TGF-β1. Herein, we study the effect of MKL1 expression on α-SMA in these cells. We demonstrate that TGF-β1 stimulation of α-SMA transcription is mediated through CC(A/T)6-rich GG elements known to bind to SRF. These elements also mediate the MKL1 effect that dramatically activates α-SMA transcription in serum-free media. MKL1 fused to green fluorescent protein localizes to the nucleus and induces α-SMA expression regardless of treatment with TGF-β1. Using proteasome inhibitors, we also demonstrate that the proteolytic ubiquitin pathway regulates MKL1 expression. These data indicate that MKL1 overexpression is sufficient to induce α-SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF-β1 stimulation of α-SMA expression. Therefore, MKL1 is also absolutely required for TGF-β1 stimulation of α-SMA expression. Western blot and immunofluorescence analysis show that overexpressed and endogenous MKL1 are located in the nucleus in non-stimulated RTC. Chromatin immunoprecipitation assay demonstrates that TGF-β1 induces binding of endogenous SRF and MKL1 to the α-SMA promoter in chromatin. Since MKL1 constitutes a potent factor regulating α-SMA expression, modulation of endogenous MKL1 expression or activity may have a profound effect on myofibroblast formation and function in the kidney.

Release of biologically active TGF-β1 by alveolar epithelial cells results in pulmonary fibrosis

2003 ◽  
Vol 285 (3) ◽  
pp. L527-L539 ◽  
Author(s):  
Ying Dong Xu ◽  
Jiesong Hua ◽  
Alice Mui ◽  
Robert O'Connor ◽  
Gary Grotendorst ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Pulmonary Fibrosis ◽  
Transforming Growth Factor ◽  
Inflammatory Cells ◽  
Alveolar Epithelial Cells ◽  
Biologically Active ◽  
Aberrant Expression ◽  
Alveolar Epithelial ◽  
Fibrotic Lung Disease ◽  
Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal fibrotic lung disease. Transforming growth factor (TGF)-β1 is present in a biologically active conformation in the epithelial cells lining lesions with advanced IPF. To determine the role of aberrant expression of biologically active TGF-β1 by alveolar epithelial cells (AECs), the AECs of explanted normal rat lungs were transfected with the TGF-β1 gene using the retrovirus pMX-L-s223,225-TGF-β1. In situ hybridization using a digoxigenin-labeled cDNA of the puromycin resistance gene contained in the pMX demonstrated that pMX-L-s233,225-TGF-β1 was selectively transfected into AECs of the explants. Conditioned media overlying explants obtained 7 days after being treated with pMX-L-s223,225-TGF-β1 contained 14.5 ± 3.15 pg/ml of active TGF-β1. With the use of Masson's trichrome staining of explant sections obtained 14 days after transfection, there were lesions similar to those in IPF, characterized by type II AEC hyperplasia, interstitial thickening, extensive increase in interstitial and subepithelial collagen, an increase in the number of fibroblasts, and areas resembling fibroblast buds. Collagens I, III, IV, and V and fibronectin were increased in explants treated with pMX-L-s223,225-TGF-β1. The findings in the current study suggest that IPF may be a disorder of epithelial cells and not inflammatory cells.

scholarly journals Platelet-Rich Plasma and Bone Marrow-Derived Mesenchymal Stromal Cells Prevent TGF-β1-Induced Myofibroblast Generation but Are Not Synergistic when Combined: Morphological in vitro Analysis

2018 ◽  
Vol 206 (6) ◽  
pp. 283-295 ◽  
Author(s):  
Flaminia Chellini ◽  
Alessia Tani ◽  
Larissa Vallone ◽  
Daniele Nosi ◽  
Paola Pavan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transforming Growth Factor ◽  
Synergistic Effects ◽  
Platelet Rich Plasma ◽  
Therapeutic Option ◽  
Smooth Muscle Actin ◽  
Combined Treatment ◽  
Biologically Active ◽  
Tgf Β1

The persistence of activated myofibroblasts is a hallmark of fibrosis of many organs. Thus, the modulation of the generation/functionality of these cells may represent a strategical anti-fibrotic therapeutic option. Bone marrow-derived mesenchymal stromal cell (MSC)-based therapy has shown promising clues, but some criticisms still limit the clinical use of these cells, including the need to avoid xenogeneic compound contamination for ex vivo cell amplification and the identification of appropriate growth factors acting as a pre-conditioning agent and/or cell delivery vehicle during transplantation, thus enabling the improvement of cell survival in the host tissue microenvironment. Many studies have demonstrated the ability of platelet-rich plasma (PRP), a source of many biologically active molecules, to positively influence MSC proliferation, survival, and functionality, as well as its anti-fibrotic potential. Here we investigated the effects of PRP, murine and human bone marrow-derived MSCs, and of the combined treatment PRP/MSCs on in vitro differentiation of murine NIH/3T3 and human HDFα fibroblasts to myofibroblasts induced by transforming growth factor (TGF)-β1, a well-known pro-fibrotic agent. The myofibroblastic phenotype was evaluated morphologically (cell shape and actin cytoskeleton assembly) and immunocytochemically (vinculin-rich focal adhesion clustering, α-smooth muscle actin and type-1 collagen expression). We found that PRP and MSCs, both as single treatments and in combination, were able to prevent the TGF-β1-induced fibroblast-myofibroblast transition. Unexpectedly, the combination PRP/MSCs had no synergistic effects. In conclusion, within the limitations related to an in vitro experimentation, our study may contribute to providing an experimental background for supporting the anti-fibrotic potential of the combination PRP/MSCs which, once translated “from bench to bedside,” could potentially offer advantages over the single treatments.

Tissue-type plasminogen activator deficiency attenuates peritoneal fibrosis in mice

2009 ◽  
Vol 297 (6) ◽  
pp. F1510-F1517 ◽  
Author(s):  
Kei Kurata ◽  
Shoichi Maruyama ◽  
Sawako Kato ◽  
Waichi Sato ◽  
Jun-ichiro Yamamoto ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Basement Membranes ◽  
Tissue Type ◽  
Vascular Density ◽  
Peritoneal Fibrosis ◽  
Plasmin Activity ◽  
Protein Levels ◽  
Tgf Β1

Peritoneal fibrosis (PF) is an important complication of peritoneal dialysis therapy. The present study was performed to examine the mechanisms of PF in view of the plasminogen activator (PA)/plasmin/matrix metalloproteinase (MMP) cascade. PF was induced in tissue-type PA (tPA) deficient mice and wild-type mice by intraperitoneal injection of chlorhexidine gluconate. Mice were killed on day 21, and tissue samples were taken. Histopathological studies were performed. Plasmin activity, gelatinases activity, and the levels of tPA, transforming growth factor-β1 (TGF-β1), and MMP-2 mRNA were determined. Protein levels of MMP-3, tissue inhibitor of metalloproteinases (TIMP)-1, -2, and -3, phospho-Smad3, membrane-type 1 (MT1)-MMP, and MT3-MMP were also studied. On day 21, tPA +/+ mice showed severe PF, whereas tPA −/− mice showed milder change. Submesothelial basement membranes were dissolved in tPA +/+ mice while they were relatively preserved in tPA −/− mice. The levels of macrophage infiltration, staining for α-smooth muscle actin (α-SMA) and collagen type III, and vascular density were all significantly lower in tPA −/− mice than in tPA +/+ mice. The levels of plasmin activity, pro- and active MMP-2, mRNA expression of tPA and TGF-β1, and phospho-Smad3 protein were also lower in tPA −/− mice. No difference was observed between the two groups concerning the protein levels of MMP-3, TIMP-1, TIMP-2, TIMP-3, MT1-MMP, or MT3-MMP. These results indicate that the presence of tPA enhances inflammation, angiogenesis, and fibrogenesis in the peritoneum of the PF model mice. Activation of the PA/plasmin/MMP cascade may play a pivotal role in the pathogenesis of PF.

scholarly journals Matrix-bound AGEs enhance TGFβ2-mediated mesenchymal transition of lens epithelial cells via the noncanonical pathway: implications for secondary cataract formation

2018 ◽  
Vol 475 (8) ◽  
pp. 1427-1440 ◽  
Author(s):  
Mi-Hyun Nam ◽  
Ram H. Nagaraj
Keyword(s):  
Epithelial Cells ◽  
P38 Mapk ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Lens Epithelial Cells ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Noncanonical Pathway ◽  
Αb Crystallin

Advanced glycation end products (AGEs) are post-translational modifications formed from the reaction of reactive carbonyl compounds with amino groups in proteins. Our laboratory has previously shown that AGEs in extracellular matrix (ECM) proteins promote TGFβ2 (transforming growth factor-beta 2)-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs), which could play a role in fibrosis associated with posterior capsule opacification. We have also shown that αB-crystallin plays an important role in TGFβ2-mediated EMT of LECs. Here, we investigated the signaling mechanisms by which ECM-AGEs enhance TGFβ2-mediated EMT in LECs. We found that in LECs cultured on AGE-modified basement protein extract (AGE-BME), TGFβ2 treatment up-regulated the mesenchymal markers α-SMA (α-smooth muscle actin) and αB-crystallin and down-regulated the epithelial marker E-cadherin more than LECs cultured on unmodified BME and treated with TGFβ2. Using a Multiplex Assay, we found that AGE-BME significantly up-regulated the noncanonical pathway by promoting phosphorylation of ERK (extracellular signal-regulated kinases), AKT, and p38 MAPK (mitogen-activated protein kinases) during TGFβ2-mediated EMT. This EMT response was strongly suppressed by inhibition of AKT and p38 MAPK phosphorylation. The AKT inhibitor LY294002 also suppressed TGFβ2-induced up-regulation of nuclear Snail and reduced phosphorylation of GSK3β. Inhibition of Snail expression suppressed TGFβ2-mediated α-SMA expression. αB-Crystallin was up-regulated in an AKT-dependent manner during AGE-BME/TGFβ2-mediated EMT in LECs. The absence of αB-crystallin in LECs suppressed TGFβ2-induced GSK3β phosphorylation, resulting in lower Snail levels. Taken together, these results show that ECM-AGEs enhance the TGFβ2-mediated EMT response through activation of the AKT/Snail pathway, in which αB-crystallin plays an important role as a linker between the TGFβ2 and AGE-mediated signaling pathways.

scholarly journals The E3 Ligase PIAS1 Regulates p53 Sumoylation to Control Stress-Induced Apoptosis of Lens Epithelial Cells Through the Proapoptotic Regulator Bax

2021 ◽  
Vol 9 ◽  
Author(s):  
Qian Nie ◽  
Huimin Chen ◽  
Ming Zou ◽  
Ling Wang ◽  
Min Hou ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
E3 Ligase ◽  
Positive Control ◽  
Lens Epithelial Cells ◽  
Post Translational Modifications ◽  
Transcription Activity ◽  
Protein Levels ◽  
Induced Apoptosis ◽  
Transcription Factor P53

Protein sumoylation is one of the most important post-translational modifications regulating many biological processes (Flotho A & Melchior F. 2013. Ann Rev. Biochem. 82:357–85). Our previous studies have shown that sumoylation plays a fundamental role in regulating lens differentiation (Yan et al., 2010. PNAS, 107(49):21034-9.; Gong et al., 2014. PNAS. 111(15):5574–9). Whether sumoylation is implicated in lens pathogenesis remains elusive. Here, we present evidence to show that the protein inhibitor of activated STAT-1 (PIAS1), a E3 ligase for sumoylation, is implicated in regulating stress-induced lens pathogenesis. During oxidative stress-induced cataractogenesis, expression of PIAS1 is significantly altered at both mRNA and protein levels. Upregulation and overexpression of exogenous PIAS1 significantly enhances stress-induced apoptosis. In contrast, silence of PIAS1 with CRISPR/Cas9 technology attenuates stress-induced apoptosis. Mechanistically, different from other cells, PIAS1 has little effect to activate JNK but upregulates Bax, a major proapoptotic regulator. Moreover, Bax upregulation is derived from the enhanced transcription activity of the upstream transcription factor, p53. As revealed previously in other cells by different laboratories, our data also demonstrate that PIAS1 promotes SUMO1 conjugation of p53 at K386 residue in lens epithelial cells and thus enhances p53 transcription activity to promote Bax upregulation. Silence of Bax expression largely abrogates PIAS1-mediated enhancement of stress-induced apoptosis. Thus, our results demonstrated that PIAS1 promotes oxidative stress-induced apoptosis through positive control of p53, which specifically upregulates expression of the downstream proapoptotic regulator Bax. As a result, PIAS1-promoted apoptosis induced by oxidative stress is implicated in lens pathogenesis.

Increased expression of the transforming growth factor β–inducible gene HIC-5 in systemic sclerosis skin and fibroblasts: a novel antifibrotic therapeutic target

Rheumatology ◽  
2020 ◽  
Vol 59 (10) ◽  
pp. 3092-3098 ◽  
Author(s):  
Sonsoles Piera-Velazquez ◽  
Jolanta Fertala ◽  
Gonzalo Huaman-Vargas ◽  
Natalia Louneva ◽  
Sergio A Jiménez
Keyword(s):  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Normal Skin ◽  
Transforming Growth Factor Β ◽  
Dermal Fibroblasts ◽  
Skin Fibroblasts ◽  
Tissue Fibrosis ◽  
Protein Levels ◽  
Antifibrotic Therapy ◽  
Tgf Β1

Abstract Objective SSc is a systemic fibrotic disease affecting skin, numerous internal organs and the microvasculature. The molecular pathogenesis of SSc tissue fibrosis has not been fully elucidated, although TGF-β1 plays a crucial role. The Hic-5 protein encoded by the TGF-β1-inducible HIC-5 gene participates in numerous TGF-β-mediated pathways, however, the role of Hic-5 in SSc fibrosis has not been investigated. The aim of this study was to examine HIC-5 involvement in SSc tissue fibrosis. Methods Affected skin from three patients with diffuse SSc and dermal fibroblasts cultured from affected and non-affected SSc skin were examined for HIC-5 and COL1A1 gene expression. Real-time PCR, IF microscopy, western blotting and small interfering RNA–mediated HIC-5 were performed. Results HIC-5 and COL1A1 transcripts and Hic-5, type 1 collagen (COL1) and α-smooth muscle actin (α-SMA) protein levels were increased in clinically affected SSc skin compared with normal skin and in cultured dermal fibroblasts from affected SSc skin compared with non-affected skin fibroblasts from the same patients. HIC-5 knockdown caused a marked reduction of COL1 production in SSc dermal fibroblasts. Conclusion HIC-5 expression is increased in affected SSc skin compared with skin from normal individuals. Affected SSc skin fibroblasts display increased HIC-5 and COL1A1 expression compared with non-affected skin fibroblasts from the same patients. Hic-5 protein was significantly increased in cultured SSc dermal fibroblasts. HIC-5 mRNA knockdown in SSc fibroblasts caused >50% reduction of COL1 production. Although these are preliminary results owing to the small number of skin samples studied, they indicate that Hic-5 plays a role in the profibrotic activation of SSc dermal fibroblasts and may represent a novel molecular target for antifibrotic therapy in SSc.

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