Type I collagen is the most abundant vertebrate protein. Its abnormal biosynthesis contributes to fibrosis, cancer, osteoporosis, skeletal dysplasias and other disorders. It is a heterotrimer of two alpha-1 and one alpha-2 chains, but homotrimers of alpha-1 chains are produced in fetal tissues and some disorders. We discovered that these homotrimers are resistant to cleavage by all matrix metalloproteinases (MMPs) and characterized the mechanism of this resistance. We observed homotrimer synthesis by cancer cells (20-40% of type I collagen in culture and even more in vivo) but not by normal cells or fibroblasts recruited into tumors. More rigid matrix made of the homotrimers supported faster proliferation and migration of cancer cells. MMP-resistant homotrimer fibers laid down by these cells may serve as tracks for outward cell migration and tumor growth. The homotrimers may thus present an appealing diagnostic and therapeutic target in cancer. Mutations in type I collagen typically cause osteogenesis imperfecta (OI), Ehlers-Danlos syndrome (EDS) or a combination of OI and EDS. Most OI mutations are substitutions of an obligatory glycine in the repeating Gly-X-Y triplets of the collagen triple helix. Disruption of the triple helix folding and structure by these mutations is involved in the disease, but no relationship between different substitutions and OI severity has been found so far. We established that the effect of Gly substitutions on the overall collagen stability depends on their location within different regions of the triple helix but not on the identity of the substituting residues. These regions appear to align with regions important for collagen folding, fibril assembly and ligand binding as well as some of the observed regional variations in OI phenotypes. In an ongoing study, we continue analysis of the role of these regions in OI severity. Our current studies suggest that the primary cause of bone pathology in OI is osteoblast malfunction. It was believed that OI results primarily from collagen mutations and deficiency/malfunction in the extracellular matrix. However, recent discoveries revealed OI-like bone pathologies caused by mutations in: (a) Endoplasmic Reticulum (ER) chaperones involved in procollagen folding; (b) proteins important for maturation and function of osteoblasts but not directly involved in collagen biosynthesis; and (c) proteins that are involved in osteoblast signaling and interaction with other cells and organs. Collagen deficiency/malfunction might be a modulating factor rather than the primary cause of the disease. We believe that collagen mutations might be prevalent in OI because of osteoblast malfunction associated with excessive cell stress response to abnormal collagen precursor (procollagen) folding, trafficking and secretion. Similarly, misfolding of normal procollagen in aging osteoblasts might contribute to bone pathology in age-related osteoporosis. Testing of these ideas might open up new approaches to pharmacological treatment of OI and osteoporosis through cell stress targeting. In particular, we are examining procollagen folding and cell stress response to procollagen misfolding in dermal fibroblasts from OI patients with Gly substitutions as well as in fibroblasts and osteoblasts from mouse models of Gly substitutions. Recently, we developed a novel assay for procollagen folding, trafficking and secretion based on metabolic labeling with azidohomoalanine, a noncanonical amino acid that replaces methionine in newly synthesized proteins. This approach is more versatile, efficient and economical than radioisotope labeling and it also has fewer unintended consequences for cell differentiation and function. Our experiments revealed procollagen folding delays in OI cells as well as retention and accumulation of partially unfolded or misfolded mutant procollagen in the ER. We observed an unusual cell stress response to this accumulation. The cells do not activate the conventional unfolded protein response signaling. Instead, they downregulate procollagen synthesis and activate pathways reminiscent but not identical to those previously described in an ER overload response to aggregation of misfolded proteins. We are currently examining molecular mechanisms of these cell stress responses and potential ways of their modulation. Our cell culture studies emphasized the importance of examining the cell stress response of fibroblasts and osteoblasts in vivo as well. In addition to utilizing the Brtl mouse model developed earlier at NICHD, we generated and characterized a second, G610C mouse model with a different Gly substitution. Our study of the latter mice revealed similarities as well as important differences in osteoblast cell stress response and malfunction in vivo compared to cell culture. Over the last three years, we identified macroautophagy as a key step in degradation of misfolded procollagen molecules and as an important adaptation mechanism of osteoblasts to such misfolding. Autophagy enhancement in cultured osteoblasts restored deposition of normal collagen matrix. Autophagy enhancement in mice by low protein diet improved bone material properties, but this diet suppressed overall animal growth. Bone marrow stromal cells (BMSCs) from low protein diet animals exhibited significantly improved osteoblast differentiation in culture at the same conditions as BMSCs from normal protein diet animals, suggesting epigenetic changes caused by the diet. To further test the role of autophagy and therapeutic potential of targeting autophagy, we modified the G610C mouse to enable conditional autophagy suppression or enhancement in osteoblasts in vivo by manipulation of the expression of an essential autophagy gene, Atg5. Our preliminary observations indicate that conditional knockout of Atg5 in mature osteoblasts dramatically increases the severity of OI in G610C mice without producing similar bone abnormalities in wild type controls. We are currently testing whether conditional overexpression of Atg5 in mature osteoblast can reduce the severity of OI, as suggested by our low protein diet studies. By exploiting the G610C mouse model, we have also found that misfolded procollagen (unlike most other misfolded proteins) is targeted to degradation in autophagosomes after rather than before it is exported from the ER. ER markers do not appear to be present inside procollagen-containing autophagosomes. Even HSP47 is not found there, although it binds to procollagen in the ER and accompanies procollagen to an intermediate ER-Golgi compartment or cis-Golgi,. These observations suggest very unusual procollagen trafficking and autophagy in osteoblasts, which is currently under investigation in our laboratory. Abnormal differentiation and function of osteoblasts also plays an important role in bone tumors. In collaboration with Dr. Stratakis, we are investigating bone pathology associated with osteoblast malfunction is caudal vertebrae tumors in mice with deficiencies in different catalytic and regulatory subunits of protein kinase A, which is a crucial enzyme for cAMP signaling. In these tumors, we found accelerated bone matrix formation and deficient mineralization reminiscent of the McCune-Albright syndrome as well as very unusual collagen matrix organization and bone structures, which appear to be associated with improper maturation and/or function of osteoblasts. We are currently characterizing the latter abnormalities and the origin of novel bone structures formed in these tumors. We hope that further studies of these animals will not only shed new light on the role of cAMP signaling in osteoblasts but also promote better general understanding of normal and pathological bone formation mechanisms.
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