Cthrc1 Is a Novel Inhibitor of Transforming Growth Factor-β Signaling and Neointimal Lesion Formation

Abstract

Abstract-We identified collagen triple helix repeat containing-1 (Cthrc1) as a novel gene expressed in the adventitia and neointima on arterial injury and found that it functionally increases cell migration while reducing collagen deposition.To address the in vivo role of Cthrc1, we generated transgenic mouse lines that constitutively overexpress Cthrc1. An intercross of 2 transgenic lines produced offspring with brittle bones caused by a reduction in collagenous bone matrix. Hemizygous Cthrc1 transgenic mice developed normally but neointimal lesion formation and adventitial collagen deposition in response to carotid artery ligation were significantly reduced compared with wild-type littermates. In 75% of Cthrc1 transgenic mice, cartilaginous metaplasia of medial smooth muscle cells was observed as assessed by Alcian blue staining and expression of the chondrocyte marker collagen type II. Transforming growth factor-␤ signaling was reduced in smooth muscle cells of Cthrc1 transgenic arteries, as demonstrated by reduced phospho-Smad2/3 immunoreactivity, whereas Smad signaling related to bone morphogenetic proteins was unaffected. Similarly, primary smooth muscle cells and PAC1 smooth muscle cells overexpressing Cthrc1 had reduced levels of phospho-Smad2/3 as well as procollagen. Furthermore, Cthrc1 inhibited transforming growth factor-␤-sensitive reporter constructs in smooth muscle but not endothelial cells. These data indicate that Cthrc1 is a cell-type-specific inhibitor of transforming growth factor-␤, which in turn impacts collagen type I and III deposition, neointimal formation, and dedifferentiation of smooth muscle cells. Key Words: intimal hyperplasia Ⅲ fibrosis Ⅲ bone morphogenetic protein Ⅲ TGF-␤ Ⅲ SMC differentiation W e originally identified collagen triple helix repeat containing-1 (Cthrc1) in a screen for differentially expressed genes in normal versus balloon-injured arteries. 1 In the vasculature, Cthrc1 expression is limited to injured tissue, where it is induced abundantly in adventitial fibroblasts and neointimal smooth muscle cells (SMCs). Expression of Cthrc1 in the injured artery is transient, with Cthrc1 levels becoming undetectable 4 weeks following injury. Thus Cthrc1 expression coincides and colocalizes with adventitial fibrosis, which is responsible for constrictive remodeling following arterial injury and contributes to the failure of angioplasty procedures. 2 Cthrc1 was also found in the matrix of calcifying human atherosclerotic plaques and in mineralized bone of skeletal tissues. 3 The injury response of tissues involves the activated fibroblast termed myofibroblasts based on its characteristic expression of the SMC marker smooth muscle ␣-actin (␣-SMA). 4 As part of the general wound healing process, these myofibroblasts exhibit proliferation, migration, production of collagen types I and III, and other extracellular matrix (ECM) molecules. 5 Cthrc1 enhances migration of SMCs and fibroblasts in a scratch wound assay. 1 We also demonstrated that Cthrc1 reduces collagen type I mR...

Intracellular Localization of Cthrc1 Characterizes Differentiated Smooth Muscle

“…Furthermore, we recently reported that the TGF-␤ signaling pathway is highly active in SMCs during neointima formation as demonstrated by the presence of pSmad2/3 in intimal SMCs, whereas pSmad2/3 was not detectable in uninjured medial SMCs. 1 This is in contrast to adventitial fibroblasts and endothelial cells of normal vessels, which displayed constitutive TGF-␤ signaling as detected by the presence of prominent nuclear pSmad2/3.…”

“…1 Immunoblotting of SMCs in vitro, however, revealed various lower molecular weight immunoreactive Cthrc1 species. 1 As many proteins, including TGF-␤ family members, require proteolytic processing for the generation of active molecules, questions about the identity and function of the lower molecular weight Cthrc1 fragments were raised. This prompted us to generate antisera specific for the N terminus of unprocessed Cthrc1.…”

“…1 TGF-␤ signaling can be decreased by overexpressing Cthrc1, which results in a reduction of phosphorylated Smad 2/3 (pSmad2/3) levels and decreased intimal lesion formation. 1 We therefore hypothesized that increased levels of Cthrc1 in the vasculature function to inhibit dedifferentiation of SMCs.…”

“…1 TGF-␤ signaling can be decreased by overexpressing Cthrc1, which results in a reduction of phosphorylated Smad 2/3 (pSmad2/3) levels and decreased intimal lesion formation. 1 We therefore hypothesized that increased levels of Cthrc1 in the vasculature function to inhibit dedifferentiation of SMCs. 1 TGF-␤ has been implicated in transdifferentiation of fibroblasts to myofibroblasts 3,4 by promoting expression of the SMC marker gene smooth muscle ␣-actin (SMA).…”

“…1 We therefore hypothesized that increased levels of Cthrc1 in the vasculature function to inhibit dedifferentiation of SMCs. 1 TGF-␤ has been implicated in transdifferentiation of fibroblasts to myofibroblasts 3,4 by promoting expression of the SMC marker gene smooth muscle ␣-actin (SMA). 2 Despite the involvement of TGF-␤ in positive regulation of smooth muscle marker gene expression, we found that in vivo TGF-␤ stimulates SMC proliferation, 3 which is linked to dedifferentiation.…”