| Objective: (1) To observe the expression and regulation of connective tissue growth factor(CTGF) by transforming growth factor?1(TGF?1),angiotensinâ…¡(Angâ…¡) and platelet derived growth factor (PDGF) respectively on cultured mouse podocytes.(2) To study the signaling pathway by which TGF?1 up-regulates the expression of CTGF on podocytes.Method: (1) Cultured immortalized mouse podcytes were stimulated by TGF?1, Angâ…¡ and PDGF respectively. Western Blotting and RT-PCR were performed to detect the levels of CTGF protein and mRNA after treatment. Podocytes without stimulation were used as control. (2) Cytochemical immunofluorescence was performed to observe the location of CTGF expression on podocytes stimulated by TGF?1.(3) Cultured podocytes were incubated with different doses of TGF?1 for 12 hours, and with 1ng/ml of TGF?1 for various lengths of time. Smad2 and ERK1/2 activation were tested by Western Blotting. (4) Cultured podocytes were divided into four groups: the control group, TGF?1 stimulated group, pretreated by certain blocker plus TGF?1 stimulated group, and blocker treated only group. The activation of Smad2 and ERK1/2, along with CTGF protein levels were evaluated by Western blotting.Result: (1) TGF?1 up-regulated the protein level of CTGF, while PDGF and Angâ…¡ did not have as much regulating effects as TGF?1 did.(2) The mRNA level of CTGF was also up-regulated by TGF?1.(3) Both Smad2 and ERK1/2 signaling were activated by TGF?1 on podocytes.(4) Application of Staurosporine, inhibitor of Serine and Threonine kinases, and PD98059, inhibitor of MEK1/2 activation, abrogated Smad2 and ERK 1/2 activation respectively. (5) Blockade of Smad2 activation reduced the level of CTGF protein up-regulated by TGF?1, while blocking ERK1/2 did not. Conclusion: The up-regulation of CTGF by TGF?1 was mediated probably by Smad2 activation-dependent, while ERK1/2 activation- independent pathway, the latter may be involved in other biological effects of TGF?1 on podocytes.
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