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      High Glucose Decreases Matrix Metalloproteinase-2 Activity in Rat Mesangial Cells via Transforming Growth Factor-β 1

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          Abstract

          Diabetic nephropathy is characterized by accumulation of mesangial matrix. Glucose-induced inhibition of matrix-degrading enzymes such as collagenases is believed to contribute to matrix accumulation. We have previously demonstrated that 72 kDa type IV collagenase activity is decreased in the rat mesangial cells cultured in high glucose media [ Diabetes 1995;44:929–935]. The present studies were designed to investigate if the cytokine transforming growth factor-β<sub>1</sub> (TGF-β<sub>1</sub>) mediates this effect of glucose. Type IV collagenases degrade type IV collagen as well as gelatin (denatured collagen) and are thus also called gelatinases. They belong to the family of matrix metalloproteinases (MMPs); MMP activity is controlled by tissue inhibitors of metalloproteinases (TIMPs). The activity of 72 kDa type IV collagenase, also known as matrix metalloproteinase-2 (MMP-2), was assessed using three methods: (1) fluoresceinated gelatin degradation assay to detect free enzyme activity (activity which is present in excess of TIMP-inhibited activity); (2) zymography to measure total (free + TIMP-bound) enzyme activity; (3) ELISA using specific antibodies to measure MMP-2 levels. TGF-β<sub>1</sub> and TIMP-2 levels were also determined by ELISA. Incubation of primary cultures of rat mesangial cells for 5 days in 30 vs. 5 m M glucose resulted in a 3-fold increase in production of total TGF-β<sub>1</sub>, a significant decrease in MMP-2 activity and immunoreactive MMP-2 levels, and an increase in TIMP-2 levels. Addition of exogenous TGF-β<sub>1</sub> to mesangial cells incubated in 5 m M glucose replicated the high glucose effect by producing a significant decrease in MMP-2 levels with a concurrent increase in TIMP-2 levels. Furthermore, glucose-induced inhibition of MMP-2 activity was completely blocked by neutralization of TGF-β<sub>1</sub> with anti-TGF-β<sub>1</sub> antibody. We conclude that the decrease in MMP-2 activity induced by glucose loading is mediated via TGF-β<sub>1</sub>.

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          Most cited references 2

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          Sequential effects of high glucose on mesangial cell transforming growth factor-beta 1 and fibronectin synthesis.

           David J. Oh,  J. Yu,  E Ha (1998)
          Transforming growth factor (TGF)-beta is recognized as the final common mediator of the principal lesions of diabetic nephropathy such as renal hypertrophy and mesangial expansion. To gain better understanding of the temporal relationships between high glucose (HG) and mesangial cell (MC) TGF-beta 1 synthesis and between TGF-beta 1 and extracellular matrix (ECM) synthesis, the present study examined early and sequential effects of HG on TGF-beta 1 and fibronectin (FN) mRNA expression and protein synthesis. Confluent primary rat MC was stimulated with 5.6 (control) or 30 (high) mM glucose after synchronizing the growth by incubation with serum-free media for 48 hours. Mesangial cell TGF-beta 1 mRNA expression increased significantly in six hours and continued to increase until 48 hours in response to HG. The level of TGF-beta 1 mRNA was 1.5-fold higher than that of control glucose at six hours and 1.8-fold at 48 hours. TGF-beta activity in heat-activated conditioned media under HG increased 1.5- and 1.6-fold at 24 and 48 hours, respectively, compared to control glucose. FN mRNA increased significantly at 24 and 48 hours and 1.4-fold that of control glucose at both time points. FN protein also increased 1.5-fold that of control glucose at 48 hours. Anti-TGF-beta antibody completely abolished HG-induced FN synthesis. The present finding demonstrate that HG stimulates TGF-beta 1 very early and prior to FN production and that HG-induced FN production is mediated by TGF-beta. This finding is consistent with the view that TGF-beta mediates increased ECM accumulation by MC under high glucose conditions.
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            Transcriptional activation of transforming growth factor-beta1 in mesangial cell culture by high glucose concentration.

            Transforming growth factor-beta (TGF-beta) is an important hypertrophic and prosclerotic cytokine in the pathogenesis of diabetic nephropathy. The mechanisms of regulation of the TGF-beta system by high ambient glucose in kidney cells are incompletely defined. This study examined the mechanisms of regulation of TGF-beta1 expression by high glucose in murine mesangial cells (MMCs) in culture. MMCs were cultured in either normal (100 mg/dl) or high (450 mg/dl) D-glucose concentration. Total TGF-beta1 protein secretion and bioactivity, mRNA expression and stability, and gene transcription rate were measured; promoter-reporter chloramphenicol acetyltransferase (CAT) assays and electrophoretic mobility shift assay (EMSA) were performed to investigate the presence of putative glucose-response elements. Raising the ambient D-glucose concentration for 72 hours increased TGF-beta1 bioactivity in cell culture medium by 47% and total TGF-beta1 secretion by approximately 90%. Northern analysis demonstrated that the steady-state TGF-beta1 mRNA level was increased nearly twofold after 48 hours of growth in high glucose. This increase was not due to increased stability, as the half-life of the message was approximately five hours in both normal and high glucose conditions. Transcriptional activity of the TGF-beta1 gene (nuclear run-on assay) was increased by 73% in cells grown in high glucose for 24 hours. Transiently transfected MMCs with CAT constructs containing varying lengths of the murine TGF-beta1 promoter demonstrated that high glucose selectively increased the expression of only one of the constructs, pA835. Sequence inspection revealed the presence of a putative glucose responsive element, CACGTG, within this construct. High glucose in MMC culture for 24 hours increased nuclear protein binding to a probe containing this element when analyzed using EMSA. High glucose stimulates total TGF-beta1 protein production and bioactivity as well as the steady-state level of TGF-beta1 mRNA. The latter effect is due primarily to stimulation of gene transcription rate rather than message stability. Transcriptional activation by high glucose may involve a region in the TGF-beta1 promoter containing a putative glucose-response element.
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              Author and article information

              Journal
              EXN
              Nephron Exp Nephrol
              10.1159/issn.1660-2129
              Cardiorenal Medicine
              S. Karger AG
              1660-2129
              2001
              2001
              27 June 2001
              : 9
              : 4
              : 249-257
              Affiliations
              aDepartment of Medicine, Veterans Affairs Hospital, Hines, Ill., and Loyola University Stritch School of Medicine, Maywood, Ill.; bDepartment of Medicine, Veterans Affairs Hospital, Westside, Chicago, Ill., and cHektoen Institute for Medical Research, Chicago, Ill., USA
              Article
              52619 Exp Nephrol 2001;9:249–257
              10.1159/000052619
              11423724
              © 2001 S. Karger AG, Basel

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              Page count
              Figures: 5, References: 32, Pages: 9
              Product
              Self URI (application/pdf): https://www.karger.com/Article/Pdf/52619
              Categories
              Original Paper

              Cardiovascular Medicine, Nephrology

              Matrix, Diabetes, Cytokine, Gelatinase A, Collagenase

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