Hormonal Functions of Cthrc1 Figure 11. Isolated cells in the rat pituitary express Cthrc1. (A) Cthrc1 immunohistochemistry on pituitary glands from three month old male Sprague Dawley rats identified Cthrc1 expression by isolated cells. Cytoplasmic immunoreactivity is clearly detectable in cells adjacent to extracellular accumulations of Cthrc1 (arrows), suggesting Cthrc1 synthesis by these cells. (B) Pre-absorption of antibody with peptide antigen completely eliminates staining on an adjacent section. Scale bar = 50 mm. doi:10.1371/journal.pone.0047142.g011 monoclonal antibodies that can detect native Cthrc1 by ELISA do not cross-react with mouse Cthrc1 and in addition, relatively large amounts of plasma were necessary to detect Cthrc1 in human plasma. Therefore, we have not been able to demonstrate the presence of Cthrc1 in mouse plasma. In the absence of a quantitative assay, we can only estimate the Cthrc1 levels detected in the plasma sample. Based on experience with the antibodies and the levels of Cthrc1 expressed by transduced CHO- K1 cells we estimate the levels of Cthrc1 in the plasma sample analyzed here to be below 100 pg/ml, which would be several orders of magnitude lower than those of adiponectin (typically several mg/ml) [14]. The current study also sheds light on the identity of colloid-filled follicles and the anterior pituitary as a source of Cthrc1. In guinea pigs, the first few colloid follicles of the anterior pituitary are detected at the age of 6 months with an average of just over 4 mm in size [6]. They increase in size and number with age and are found in many vertebrates including humans [5,6]. Focusing on the pig pituitary, here we identify follicles as well as the pituitary cleft separating the anterior lobe from the pars intermedia as storage sites for Cthrc1. However, not all accumulations of Cthrc1 in the pituitary were encapsulated by folliculostellate cells. Staining of adjacent sections with hematoxylin and eosin suggests that Cthrc1 originates from chromophobe cells (Fig. 7), which are thought to represent acidophil and basophilic cells that recently released their secretory vesicles. Our data indicate that chromo- phobe cells may be the primary source of Cthrc1 in the pituitary. We saw no expression of Cthrc1 in the pituitary of young adult mice and this raises the question whether the pituitary becomes References 1. Pyagay P, Heroult M, Wang Q, Lehnert W, Belden J, et al. (2005) Collagen triple helix repeat containing 1, a novel secreted protein in injured and diseased arteries, inhibits collagen expression and promotes cell migration. Circ Res 96: 261–268. 2. Yamamoto S, Nishimura O, Misaki K, Nishita M, Minami Y, et al. (2008) Cthrc1 selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex. Dev Cell 15: 23–36. 3. Kimura H, Kwan KM, Zhang Z, Deng JM, Darnay BG, et al. (2008) Cthrc1 is a positive regulator of osteoblastic bone formation. PLoS ONE 3: e3174. 4. LeClair RJ, Durmus T, Wang Q, Pyagay P, Terzic A, et al. (2007) Cthrc1 is a novel inhibitor of transforming growth factor-beta signaling and neointimal lesion formation. Circ Res 100: 826–833. a more significant provider of Cthrc1 with age when tissue remodeling is limited. Alternatively, the origin of Cthrc1 could differ depending on the species and with the pig physiology being more similar to the human physiology, we expect our findings consistently seen in the pig to be more relevant to humans. To further address species-dependent expression of Cthrc1, pituitary glands from three month old male Sprague Dawley rats were examined and isolated foci of Cthrc1 expression by cells surrounding Cthrc1 accumulations were found (Fig. 11). Cthrc1 is expressed in many mesenchyme-derived cells during growth and tissue remodeling especially the skeletal system, where it continues to be expressed in adult bone (Fig. 6O), a tissue that constantly undergoes remodeling. With bone mass accounting for a sub- stantial amount of total body mass, it is likely that osteocytes and osteoblasts contribute substantially to circulating Cthrc1 levels. Interestingly, osteocalcin, another hormone derived from osteo- blastic cells has recently been shown to regulate glucose metabolism and fat mass [15]. Acknowledgments The authors thank Armie Mangoba, Katrina Abramo, Anne Harrington and Victoria DeMambro for expert technical assistance and James Schwob for critical input in experimental design. Author Contributions Conceived and designed the experiments: JPS LL VL. Performed the experiments: JPS NGP QW LL VL. Analyzed the data: JPS NGP QW LL VL. Wrote the paper: JPS LL VL. 5. Ogawa S, Couch EF, Kubo M, Sakai T, Inoue K (1996) Histochemical study of follicles in the senescent porcine pituitary gland. Arch Histol Cytol 59: 467–478. 6. Kameda Y (1991) Occurrence of colloid-containing follicles in the pars distalis of pituitary glands from aging guinea pigs. Cell Tissue Res 263: 115–124. 7. Rosen CJ, Ackert-Bicknell CL, Adamo ML, Shultz KL, Rubin J, et al. (2004) Congenic mice with low serum IGF-I have increased body fat, reduced bone mineral density, and an altered osteoblast differentiation program. Bone 35: 1046–1058. 8. Ciocca DR, Gonzalez CB (1978) The pituitary cleft of the rat: an electron microscopic study. Tissue Cell 10: 725–733. 9. Wong GW, Krawczyk SA, Kitidis-Mitrokostas C, Ge G, Spooner E, et al. (2009) Identification and characterization of CTRP9, a novel secreted glycoprotein, PLOS ONE | www.plosone.org 11 October 2012 | Volume 7 | Issue 10 | e47142