Thrombin stimulates production of transforming growth factor-beta by cultured human mesangial cells

Abstract

Fibrin formation within the glomeruli occurs in various forms of human and experimental glomerulonephritis and it may play an important role in progressive glomerular injury. Transforming growth factor-beta (TGF-beta) has been shown to participate in the glomerular accumulation of extracellular matrix in glomerulonephritis. We investigated whether thrombin, an important coagulation factor, could modulate the production of TGF-beta by cultured human mesangial cells (HMC). TGF-beta levels in the culture supernatants were measured by ELISA using a specific antibody. The TGF-beta concentration was significantly increased by incubation of HMC with thrombin in a time-dependent manner. The stimulating effect of thrombin on TGF-beta was inhibited by addition of hirudin (a natural thrombin inhibitor) and argatroban (a synthetic thrombin inhibitor). In addition DFP-inactivated thrombin, which has no enzymatic activity, did not stimulate TGF-beta production. A protein kinase C inhibitor (H7) and a tyrosine kinase inhibitor (herbimycin A) also inhibited thrombin induced TGF-beta production. These findings suggested that thrombin may modulate the synthesis of TGF-beta via protein kinase C- and tyrosine kinase-dependent mechanisms in cultured HMC. Thus thrombin may participate in the accumulation of extracellular matrix in glomeruli through the augmentation of TGF-beta production.

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

“…MLEC were lysed after 16 h, and luciferase activity was measured as an index of PAI-1 promoter activity as described under "Experimental Thrombin elicits a number of its cellular responses, including its pro-inflammatory and pro-fibrotic effects, via the induction or release of secondary mediators. Of particular relevance to the present study, thrombin has been reported to promote TGF-␤ 1 secretion by human mesangial cells (34) and its release from the pericellular matrix of cultured fibroblasts (35), although it was produced in a latent form and over long incubation periods in both of these reports. In the present study, there was good reason to believe that thrombin was exerting its stimulatory effects on CTGF expression independently of TGF-␤ production or release, based on the observations that CTGF mRNA levels were increased very early and independently of de novo protein synthesis in response to thrombin, whereas TGF-␤ 1 , at concentrations that could not be generated by fibroblasts in our culture conditions, did not affect fibroblast CTGF mRNA levels until much later.…”

“…We also performed experiments to rule out more definitively the possibility that thrombin may be acting via the induction or the release of TGF-␤, as this has been previously reported for thrombin in other cell types (34,35). We first assessed whether the stimulatory effects of thrombin on CTGF mRNA levels could be blocked with pan-specific TGF-␤ neutralizing antibodies.…”

Roles of Coagulation Pathway and Factor Xa in the Progression of Diabetic Nephropathy in db/db Mice

“…Factor Xa is not only a key factor in coagulation cascades, but also a potent mitogen for endothelial cells. 34) Furthermore, thrombin stimulates TGF-b production in cultured mesangial cells, 7) and factor Xa upregulates the synthesis of TGF-b in cultured tu- Immunohistochemical findings of CD31, an endothelial marker, are shown. CD31 positive capillaries were detected in the glomerulus of the mϩ/mϩ group (A), and they were abundantly observed in the control db/db group (B).…”

“…6) Regarding its relationship with coagulation, thrombin stimulates TGF-b production in mesangial cells in vitro. 7) On the other hand, plasminogen-activator-inhibitor type 1 (PAI-1) is an inhibitor of the fibrinolytic system and is known to increase ECM components through the inhibition of plasmin. 8) In diabetes mellitus, impaired fibrinolytic activity may lead to vascular abnormalities and fibrosis, and patients with greater urinary albumin excretion exhibit a significantly higher ratio of PAI-1/tissue-type plasminogen activator in the circulation.…”

Mizoribine Suppresses Proliferation of Rat Glomerular Epithelial Cells in Culture and Inhibits Increase of Monocyte Chemoattractant Protein-1 and Macrophage Inflammatory Protein-2 Stimulated by Thrombin

“…16) We have also reported that thrombin mediated various factors. [17][18][19][20][21][22] In the previous study, we showed that thrombin increased the production of MCP-1 and MIP-2 by GEC and enhanced mRNA of these cytokines. 23) It is well known that neutrophils are involved in acute phase of inflammation and following macrophages infiltrate.…”