(1986) J

(1986) J. I procollagen processing and deposition into the extracellular matrix. The reduced collagen matrix deposition is partly a consequence of reduced fibronectin matrix formation in the CRT-deficient cells. Together, these data show that CRT complexes with collagen in cells and that CRT plays critical roles at multiple stages of collagen expression Rabbit Polyclonal to STEA3 and processing. These data identify CRT as an important regulator of collagen and suggest that intracellular CRT signaling plays an important role in tissue remodeling and fibrosis. model (22). CRT is an important regulator of Ca2+ homeostasis within the ER (23, 24). Total CRT expression is up-regulated by many forms of cellular stress, including amino acid deprivation, depletion of Ca2+ stores, oxidative stress, and hypoxia (25,C29). Despite its lack of a transmembrane domain, CRT is on the surface of many cell types, including fibroblasts, endothelial cells, and apoptotic cells. From the cell surface, CRT signals multiple cellular processes, including apoptotic clearance of cells, focal adhesion turnover, proliferation, migration, and anoikis resistance (4, 13, 20, 30,C33). Many of these responses to cell surface CRT are mediated by CRT binding to LRP1 (low density lipoprotein receptor-related protein 1) (13, 33,C35). Recent studies from our laboratory show that engagement of the CRT-LRP1 complex on the cell surface by thrombospondin-1 stimulates fibrillar collagen expression and (housekeeping gene) were assayed using TaqMan gene expression assay primers designed and optimized by Applied Biosystems. primer ID is Mm01165187_m1; primer ID FICZ is Mm00802331_m1; primer ID is Mm01335418_m1; and primer ID is Mm00469845_m1. levels were normalized to levels. Results are expressed as the mean S.D. of 3C8 samples (indicated in figure legend) assayed in triplicate. Soluble Collagen Assays Wild type and CRT?/? MEFs were cultured for 48 h in DMEM supplemented with FICZ 10% FBS and 2 mm l-glutamine (Glutamax, Invitrogen). Cells were then switched to DMEM with 0.5% FBS, and cells were cultured for another 72 h. Cells were dosed daily with treatments in DMEM with 0.5% FBS. Mouse L fibroblasts were cultured under the same conditions, except that these cells were plated on wells coated with fibronectin and cultured in the presence of 100 g/ml G418. Conditioned medium was collected in the presence of protease inhibitor mixture (Sigma) and centrifuged at 15,000 for 5 min to remove cell debris. For measurement of soluble collagens, the SircolTM assay was used (Biocolor, Ireland). Two hundred microliters of medium were added to 1 ml of SircolTM reagent containing Sirius Red in picric acid. Conditioned medium and SircolTM reagent were rotated for 30 min at room temperature and then pelleted at 12,000 for 15 min at room temperature. Excess dye was removed from the tube, and the pellet was reconstituted in the provided alkali reagent. Absorbance was read at 540 nm using a plate reader. A standard curve was made using rat tail collagen (provided by manufacturer). The concentration of soluble collagen in the conditioned media was determined from FICZ the standard curve. Results were normalized to cell number determined by counting of trypsinized cells using a hemocytometer. Protein Concentration Assay Cells were cultured for 24 h in DMEM with 10% FBS and 2 mm l-glutamine. Cells were then switched to serum and phenol-red free DMEM with 2 mm FICZ l-glutamine for 24 h. Conditioned medium was.