MECHANISMS OF TGF-BETA &SMAD SIGNALING IN ADIPOGENESIS

University Of California San Francisco, San Francisco CA

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Abstract

DESCRIPTION (adapted from the application) TGF-beta regulates extracellular matrix synthesis, cell growth and differentiation. Aberrant or uncontrolled signaling by TGF-beta has been implicated in many disease states. TGF-beta has also long been known to inhibit adipogenesis, but the mechanism has remained uncharacterized. Adipogenesis is regulated via activation of the transcription factors PPARgamma,ADD-1, andC/EBP's alpha, Beta, and delta, whereas Smad proteins are known to mediate TGF-beta signaling. Little is known about how TGF-beta/Smads affect adipocyte transcription factors. This candidate has shown that endogenous TGF-beta receptor signaling inhibits differentiation of cultured preadipocytes, and that Smad2 and Smad3 mediate this inhibition whereas the inhibitory Smads 6 and 7 were also found to be key regulators of differentiation. This application builds upon these findings and proposes to characterize the mechanisms by which TGF-beta and Smad signaling regulate adipocyte differentiation. Specific Aim 1 is to determine the mechanism(s) by which Smads regulate adipocyte transcription factors. The physical interaction between Smads and adipocyte transcription factors will be characterized using GST-adsorption assays and coimmunoprecipitations, while functional interactions will be tested in transcriptional cooperation assays and gel shift/supershift assays, using promoters for adipocyte differentiation markers and defined DNA sequences that bind adipocyte transcription factors. We will also examine adipocyte transcription factors for TGF-beta-induced modifications at the protein level. Specific Aim 2 is to define the mechanism by which Smad6/7 regulate adipogenesis. Endogenous Smad6 or 7 function will be blocked by stably overexpressing antisense Smad6/7 RNAs or mutant versions of the proteins. Also, activated or dominant negative versions of other TGF-beta family member receptors or effector Smads will be overexpressed. The consequences of these manipulations on growth and differentiation will be assessed. Specific Aim 3 is to verify the relevance of our findings about the role of autocrine TGF-beta and Smad signaling in adipocyte differentiation in vivo. We will characterize the capacity of our F442A cell lines, which express the dominant-negative type II TGF-beta receptor or different TGF-beta Smads, to develop fat pads when injected into nude mice. We will also use transgenic mice to assess the effect of expressing dominant-negative type Il TGF-beta or Smad7 from a fat-specific promoter transgenic mice, on adipose tissue development.