| Budget Amount *help |
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Our data show that TGF-beta1 can induce apoptosis of bronchiolar epithelial cells (SAEC) through the caspase-3 activation and the downregulation of the p21expression. In addition, a low concentration of TGF-beta1 that could not induce apoptosis alone enhanced Fas-mediated apoptosis of SAEC. We found the p53-independent downregulation of p21 expression by TGF-beta1, even at the low concentration. Detached apoptotic cells exhibited significantly reduced levels of p21 expression, whereas attached living cells exhibited elevated levels. Thus, for SAEC, induction of p21 appears to be correlated with resistance to TGF-beta1-mediated apoptosis. It is also possible that in the presence of TGF-betal, p21 protects SAEC from apoptosis. To confirm this protective effect, we infected SAEC with AdV-p21. Ectopic expression of p21 protected SAEC from TGF-beta1-mediated apoptosis and reduced caspase-3 activity. Interestingly, some investigators have demonstrated that activation of caspase-3 is regulate
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d by p21, and procaspase-3-p21 complex formation is an essential system for cell survival. Therefore, the downregulation of p21 by TGF-beta1 may enhance Fas-mediated apoptosis through caspase-3 activation. We previously demonstrated that the concentrations of soluble FasL (sFasL) in brochoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF) and hypersensitivity pneumonia (HP) were significantly higher than those in BALF from normal volunteers. Here, we demonstrated that BALF from patients with IPF but not HP could induce apoptosis of SAEC, despite the fact that BALF from both IPF and HP contained similar concentrations of sFasL. Anti-Fas neutralizing antibody could significantly inhibit SAEC apoptosis induced by BALF from patients with IPF. However, much higher concentrations of recombinant FasL were required for the induction of apoptosis in vitro. Anti-TGF-beta neutralizing antibody inhibited apoptosis of SAEC induced by BALF administration. Thus, TGF-beta1 may act synergistically as a cofactor for the development of a full apoptotic response to sFasL in BALF from patients with IPF. This may be one explanation for the difference in prognosis between IPF and HP.
Finally, to confirm the direct link of TGF-beta1 to apoptotic effects in a physiologically relevant system in vivo, we designed the Fas-mediated lung injury model of mice. The intratracheal instillation of 50 g anti-Fas antibody killed 90% of mice within 24 hours. In this model, TGF-beta1 enhanced lung epithelial cell apoptosis and inflammation induced by low dose of anti-Fas antibody, and increased the mortality of mice. Western blot analysis using extracts from lung homogenates demonstrated that the cleaved caspase-3 was significantly increased from 12 to 18h after the treatment of anti-Fas antibody with TGF-beta1. Thus, TGF-beta1 augments Fas-mediated apoptosis on lung epithelium via the caspase-3 activation in vivo. As seen in the vitro system, TGF-beta1 alone also induced the caspase-3 activation and apoptosis on lung epithelial cells. We conclude that TGF-beta1 is a potent inducer of apoptosis through the caspase-3 activation and the downregulation of p21 and is also an enhancer of Fas-mediated apoptosis of lung epithelial cells. This novel function of TGF-b1 in apoptosis of lung epithelial cells should be considered in the treatment of pulmonary fibrosis, and could be a new treatment strategy. Less
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