Cthrc1, a Novel Circulating Hormone Regulating Metabolism J. Patrizia Stohn, Nicole G. Perreault, Qiaozeng Wang, Lucy Liaw, Volkhard Lindner* Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, United States of America Abstract Background: We discovered the gene Collagen Triple Helix Repeat Containing 1 (Cthrc1) and reported its developmental expression and induction in adventitial cells of injured arteries and dermal cells of skin wounds. The role of Cthrc1 in normal adult tissues has not yet been determined. Methodology/Principal Findings: We generated mutant mice with a novel Cthrc1 allele by homologues recombination. Cthrc1 mice appeared developmentally normal. On the C57BL/6J background, livers from Cthrc1 mice accumulated vast quantities of lipid, leading to extensive macrovesicular steatosis. Glycogen levels in skeletal muscle and liver of Cthrc1 mice on the 129S6/SvEv background were significantly increased. However, Cthrc1 expression is not detectable in these tissues in wild-type mice, suggesting that the lipid and glycogen storage phenotype may be a secondary effect due to loss of Cthrc1 production at a distant site. To investigate potential hormonal functions of Cthrc1, tissues from adult mice and pigs were examined for Cthrc1 expression by immunohistochemistry with monoclonal anti-Cthrc1 antibodies. In pigs, Cthrc1 was detected around chromophobe cells of the anterior pituitary, and storage of Cthrc1 was observed in colloid-filled follicles and the pituitary cleft. Pituitary follicles have been observed in numerous vertebrates including humans but none of the known pituitary hormones have hitherto been detected in them. In C57BL/6J mice, however, Cthrc1 was predominantly expressed in the paraventricular and supraoptic nucleus of the hypothalamus but not in the posterior pituitary. In human plasma, we detected Cthrc1 in pg/ml quantities and studies with 125I-labeled Cthrc1 revealed a half-life of 2.5 hours in circulation. The highest level of Cthrc1 binding was observed in the liver. Conclusions: Cthrc1 has characteristics of a circulating hormone generated from the anterior pituitary, hypothalamus and bone. Hormonal functions of Cthrc1 include regulation of lipid storage and cellular glycogen levels with potentially broad implications for cell metabolism and physiology. Citation: Stohn JP, Perreault NG, Wang Q, Liaw L, Lindner V (2012) Cthrc1, a Novel Circulating Hormone Regulating Metabolism. PLoS ONE 7(10): e47142. doi:10.1371/journal.pone.0047142 Editor: Raul M. Luque, University of Cordoba, Spain Received June 12, 2012; Accepted September 11, 2012; Published October 8, 2012 Copyright: 2012 Stohn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by National Institutes of Health grants HL69182 (V.L.) and P20 RR-15555 from the National Center for Research Resources. The Histopathology Core Facility is supported by P20RR018789/P20GM103465 (D.Wojchowski, PI) and P30RR030927/P30GM103392 (R.Friesel, PI). The Mouse Transgenic and Imaging Core is supported by P30RR030927/P30GM103392 (R.Friesel, PI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lindnv@mmc.org Introduction We originally discovered collagen triple helix repeat containing 1 (Cthrc1) in a screen for novel sequences induced in rat carotid arteries upon balloon catheter injury [1]. The response to this injury results in constrictive remodeling with reduction in lumen size and fibrosis of the adventitia. Cthrc1 was not expressed in normal vessels, but was induced in adventitial cells in remodeling arteries. In addition, Cthrc1 expression was observed in dermal fibroblasts during skin wound healing [1]. Targeted replacement of the first exon of the Cthrc1 gene by a LacZ reporter gene in mice was reported to demonstrate expression of Cthrc1 in inner ear hair cells [2]. This study described abnormalities in inner ear development when Cthrc1 mice were crossed with mice carrying one mutant allele of Vangl2, but these abnormalities were only observed when the compound mutants were on a mixed 129/SvEv-C57BL/6 genetic background and not when the mutants were crossed with outbred CD-1 mice. In connection with in vitro data derived from co- cultures of transfected HEK293T, the authors concluded that Cthrc1 is involved in non-canonical Wnt signaling as part of the planar cell polarity pathway [2]. A separate mutant Cthrc1 mouse with deletion of exon 2 was reported to have reduced bone mass [3]. Expression analyses at the RNA level using in situ hybridization have identified the sites of Cthrc1 expression during embryonic development. In addition, our studies have also shown that Cthrc1 is expressed by the activated fibroblast of remodeling tissues following injury [4]. Whether Cthrc1 protein is constitutively expressed in any tissues of normal adult animals has so far remained unclear largely because reliable antibodies suitable for detection of Cthrc1 at the cellular level were not available. The pituitary gland is the master endocrine gland, with the anterior pituitary expressing and secreting a variety of hormones and the posterior pituitary releasing oxytocin as well as vasopressin expressed by neurosecretory cells of the hypothalamus. Colloid- PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e47142