Collagen Triple Helix Repeat Containing protein 1 (Cthrd) is a gene product with novel biochemical activities and its ability to reduce collagen deposition by inhibition of Smad 2/3 activation could have major clinical applications. Both overexpression and knockout of Cthrd result in various levels of lethality, while the surviving animals are characteristic of vascular defects. These observations suggest that the characterization of Cthrd will be instructive in understanding vascular development, repair and fibrosis. We hypothesize that Cthrd expression in vascular cells regulate transforming growth factor-p (TGF-B) responsiveness, thereby impacting TGF-P target genes, including collagens, and regulation or activation of Cthrd is strictly controlled in these cells. The proposed experiments specifically address the following hypotheses: 1) Removal of the Cthrd propeptide is essential for the inhibition of TGF-B signaling via Smad 2/3 signaling and 2) Cthrd directly or indirectly inhibits TGF-B signaling by blocking signal transduction via TGF- 3 receptors. Plasmin is capable of cleaving Cthrd in two locations and the activity of these fragments will be assessed in vitro using a collagen deposition assay in smooth muscle cells and luciferase reporter assay using PAC1 cells stably transfected with the TGF-B sensitive (CAGA) 12-Luc reporter. The generation of transgenic mouse lines overexpressing each of these fragments will be instrumental in addressing the effect of these processed Cthrd fragments during development. Harvesting of various embryonic time points and histology will allow us to examine the phenotypic consequences of overexpression of cleaved forms of Cthrd. The potential mechanism of collagen regulation is hypothesized to be a novel interaction of Cthrd with the TGF-B signaling pathway.
Specific aim two will begin to examine the location of Cthrd inhibition of TGF-B signaling. The effect of Cthrd (both full length, and the plasmin generated fragments), on levels of Smad 2/3 activation in cell lines stably transected with a series of constitutively active TGF-B receptors will allow us to address this question. Using various luciferase reporter constructs we also seek to determine whether Cthrd is specific to inhibition of signaling via Smad 2 or 3. Determining Cthrd activation (or inactivation) and addressing the mechanism of TGF-B inhibition are key aspects to translating this work into clinically relevant applications. Diseases involving fibrosis involve nearly every major organ and without effective treatment, often result in mortality. The development of therapies involving regulation of Cthrd has the potential to impact society and the biochemical characterization of this gene is the first step towards achieving these goals.
|Leclair, Renee J; Wang, Qiaozeng; Benson, Meredith A et al. (2008) Intracellular localization of Cthrc1 characterizes differentiated smooth muscle. Arterioscler Thromb Vasc Biol 28:1332-8|