Expression of connective tissue growth factor in human renal fibrosis

370

302

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor ␤1 (TGF-␤1), and addition of TGF-␤1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-␤1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGF␤1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGF␤1-driven matrix production within a diabetic milieu.

Section: Enhanced Ctgf Expression In Renal Cortex and Isolated Glomersupporting

confidence: 54%

Suppression Subtractive Hybridization Identifies High Glucose Levels as a Stimulus for Expression of Connective Tissue Growth Factor and Other Genes in Human Mesangial Cells

Murphy¹,

Godson²,

Cannon³

et al. 1999

370

302

“…This has been suggested to be a mechanism behind the nephropathy that leads to renal failure in diabetic patients. Similarly, it is possible that overexpression of CTGF due to WT1 mutation accounts for the renal fibrosis and ultimate failure that is observed in patients with DDS (50).…”

Section: Discussionmentioning

confidence: 99%

Identification of Connective Tissue Growth Factor as a Target of WT1 Transcriptional Regulation

Stanhope-Baker¹,

Williams

2000

The Wilms tumor suppressor WT1 has transcriptionactivating and -suppressing capabilities. WT1-responsive promoters have been described; however, in large part, it remains unclear which potential downstream genes are physiologically relevant and mediate the function of WT1 in tumorigenesis and development. To identify genes regulated by WT1 in vivo, we used a dominantnegative version of WT1 to modulate WT1 activity in a Wilms tumor cell line. Screening oligonucleotide arrays with RNA from these cells uncovered a number of genes whose expression was altered by abrogation of WT1 function. Several of the genes encode members of the CCN family of growth regulators. The promoter of one of these genes, connective tissue growth factor (CTGF), is suppressed by WT1 both in its endogenous location and in reporter constructs. WT1 regulation of CTGF expression is not mediated by previously identified WT1 recognition elements and may therefore involve a novel mechanism. Our results indicate that CTGF is a bona fide target of WT1 transcriptional suppression and likely plays a role in Wilms tumorigenesis and associated disease syndromes.Wilms tumor (pediatric nephroblastoma) is one of the most common solid tumors found in children. A subset (5-10%) of Wilms tumors is caused by mutations in the tumor suppressor gene, WT1 (reviewed in Refs. 1 and 2). In addition to its role in tumorigenesis, WT1 is also required for normal kidney and urogenital development. WT1 is expressed in a temporally and spatially restricted pattern in the developing kidney and urogenital structures, and WT1 knockout mice die before birth with both kidney and urogenital development blocked at an early stage (3). Based upon these findings, it has been suggested that WT1 is required for the expression of signaling molecules and receptors involved in the reciprocal inductive events of early kidney differentiation (reviewed in Ref. Although it is clear that WT1 plays an important role in both development and tumorigenesis, the mechanisms through which it functions in these processes remain poorly understood. WT1 has been implicated in such diverse pathways as RNA splicing, DNA replication, and apoptosis (reviewed in Refs. 2 and 4). The most well characterized function of WT1, however, is that of a transcription factor. The amino terminus of WT1 is rich in proline and glutamine, a feature characteristic of transactivation domains, and the carboxyl terminus contains four C2H2 zinc finger DNA-binding motifs and a nuclear localization signal sequence. In vivo, there are four major isoforms of WT1 generated by alternative splicing at two sites. Splicing of exon 5 removes 17 amino acids from the middle of the protein, and a second alternative splicing event removes three amino acids (KTS) from between the third and fourth zinc fingers of the protein. The four isoforms of WT1 are present in a constant ratio that is conserved among species, suggesting that they have non-overlapping functions.WT1 binds to DNA via its zinc finger motifs, but this binding is isoform-depen...

“…CTGF mRNA levels are up-regulated in skin wound healing models (16), and subcutaneous injection of CTGF into newborn mice produces large granulation tissue formation, fibroplasia, and increased extracellular matrix deposition (22). Overexpression of CTGF is also a feature of a number of fibrotic and fibroproliferative disorders, including localized scleroderma, keloid tissue (23), the fibrous stroma of mammary tumors (46), the fibrotic areas of atherosclerotic lesions (26), renal and pulmonary fibrosis (24,25), and inflammatory bowel disease (47). The mechanism by which CTGF expression is up-regulated during tissue repair and the development of tissue fibrosis is still unknown, although there is good in vitro and in vivo evidence that TGF-␤ plays an important role.…”

Section: Thrombin Exerts Its Stimulatory Effects On Ctgf Expression Vmentioning

confidence: 99%

“…CTGF mRNA levels are strongly up-regulated in skin wound healing models in vivo (16), and subcutaneous injection of CTGF into newborn mice results in increased connective tissue deposition (22). CTGF is also thought to be involved in the development of tissue fibrosis, based on the observation that CTGF expression is increased in skin and internal organ fibrosis (23)(24)(25) and the fibrotic areas of atherosclerotic lesions (26). There is also growing evidence that CTGF may be the downstream autocrine mediator responsible for mediating some of the cellular effects of TGF-␤ 1 , the most fibrogenic mediator characterized to date.…”

mentioning

confidence: 99%

Thrombin Is a Potent Inducer of Connective Tissue Growth Factor Production via Proteolytic Activation of Protease-activated Receptor-1

Chambers

Leoni

Blanc‐Brude

et al. 2000

223

170

The coagulation protease thrombin plays a critical role in hemostasis and exerts pro-inflammatory and profibrotic effects via proteolytic activation of the major thrombin receptor, protease-activated receptor-1 (PAR-1). Connective tissue growth factor (CTGF) is a novel fibroblast mitogen and also promotes extracellular matrix protein production. It is selectively induced by transforming growth factor ␤ (TGF-␤) and is thought to be the autocrine agent responsible for mediating its pro-fibrotic effects. CTGF is up-regulated during tissue repair and in fibrotic conditions associated with activation of the coagulation cascade. We therefore hypothesized that coagulation proteases promote the production of CTGF by cells at sites of tissue injury. To begin to address this hypothesis, we assessed the effect of coagulation proteases on fibroblast CTGF expression in vitro, and we show that thrombin, at physiological concentrations, up-regulated CTGF mRNA levels 5-fold relative to base line (p < 0.01) in fetal fibroblasts and 7-fold in primary adult fibroblasts (p < 0.01). These effects were cycloheximide-insensitive and were not blocked with a pan-specific TGF-␤-neutralizing antibody. They were further paralleled by a concomitant increase in CTGF protein production and could be mimicked with selective PAR-1 agonists. In addition, fibroblasts derived from PAR-1 knockout mice were unresponsive to thrombin but responded normally to TGF-␤ 1 . Finally, factor Xa, which is responsible for activating prothrombin during blood coagulation, exerted similar stimulatory effects. We propose that coagulation proteases and PAR-1 may play a role in promoting connective tissue formation during normal tissue repair and the development of fibrosis by up-regulating fibroblast CTGF expression.