Understanding the functions of vimentin filaments in collagen expression and targeting vimentin filaments for the treatment of fibrosis.

摘要

In the first part of the dissertation, we report that vimentin filaments associate with type I collagen mRNAs in a 5'SL and LARP6 dependent manner. This association is needed for stabilization of type I collagen mRNAs. Our conclusion was based on the following lines of evidence. First, RNA immunoprecipitation and cellular fractionation experiments showed that collagen &agr;1(I) and &agr;2(I) mRNAs exhibit specific interaction with vimentin intermediate filaments. This was substantiated by RNA-FISH experiments which showed that collagen I mRNAs colocalize with vimentin filaments. Second, we showed that the binding of collagen mRNAs to vimentin intermediate filaments is dependent on the 5' stem-loop. Collagen mRNAs from mouse embryonic fibroblasts carrying a knock-in mutation in the 5'SL failed to interact with vimentin intermediate filaments. Third, interaction of collagen mRNAs with collagen mRNAs was mediated by the RNA binding protein, LARP6, which specifically binds the 5'SL of type I collagen mRNAs. siRNA knock down of LARP6 abrogated the interaction of type I collagen mRNAs with vimentin intermediate filaments. We also found that LARP6 interacts and colocalizes with vimentin intermediate filaments. Mapping of the domain of LARP6 needed for interaction with vimentin revealed that the La domain of LARP6 is necessary and sufficient to interact with viemntin. Fourth, disruption of vimentin filaments using the vimentin disrupting drug β,β'-imminodipropionitrile or by expressing a dominant negative intermediate filament markedly reduced production of type I collagen. The reduction in collagen synthesis was due to decreased stability of collagen mRNAs. Last, but not least, we observed a marked reduction in collagen synthesis from vimentin -/- mouse fibroblasts. This reduced collagen production was also due to reduced stability of type I collagen mRNAs in vimentin deficient fibroblasts. This was consistent with the impaired wound healing phenotype displayed by vimentin deficient mice further verifying the important role of vimentin filaments in collagen synthesis. We concluded that vimentin intermediate filaments play a key role in the development of tissue fibrosis by stabilizing type I collagen mRNAs. This finding served as a basis for targeting vimentin in the investigation of a novel anti-fibrotic therapy.;In the second part of the project, we examined the antifibrotic effects of Withaferin-A. The intermediate filament vimentin is the primary target of Withaferin-A. In light of our finding of the role of vimentin in stabilizing collagen mRNAs, we hypothesized that Withaferin A may reduce collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. We thus aimed to determine if Withafrein-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms by which WF-A exerts its anti-fibrotic effects. We found that, in tissue culture, Withaferin-A suppresses collagen expression, both at transcriptional and post-transcriptional level, by inhibiting the TGF-β signaling pathway and by disrupting vimentin filaments, respectively. Withaferin-A disrupted vimentin filaments and caused degradation of vimentin in fibroblasts. The toxicity of Withaferin-A is not due to disruption of vimentin filaments as the disruption occurs at concentrations lower than the toxic range. WF-A can potently inhibit the expression of type I collagen in human and rodent fibroblasts. Withaferin-A increases the rate of decay of &agr;1(I) and &agr;2(I) collagen mRNAs. The effect of WF-A on half-life of collagen mRNAs is dependent on the presence of vimentin. In addition to destabilizing collagen mRNAs by disrupting vimentin filaments, WF-A also interferes with TGF-β induced transcription of collagen genes. WF-A inhibits both the TGF-β induced as well as the culture induced phosphorylation of Smad3. Withaferin-A inhibits in vitro activation of hepatic stellate cells (HSCs) and decreases collagen production by HSCs. In vivo, WF-A inhibits isoproterenol-induced myocardial fibrosis and results in downregulation type I and type III collagen as well as &agr;-smooth muscle actin. Our findings provide a strong evidence base for the further exploration of Withaferin-A as a therapeutic drug against fibroproliferative diseases, including but not limited to cardiac interstitial fibrosis. (Abstract shortened by UMI.).