Type I collagen is the most abundant vertebrate protein. Its abnormal biosynthesis contributes to fibrosis, cancer, osteoporosis, skeletal dysplasias and other disorders. Normally, type I collagen is a heterotrimer of two alpha-1 and one alpha-2 chains. However, homotrimers of three alpha-1 chains are produced in fetal and some fibrotic tissues as well as in rare genetic alpha-2 chain deficiencies. We discovered that these homotrimers are resistant to cleavage by all major collagenolytic matrix metalloproteinases (MMPs), including MMP-1,2,8,13, and 14 and characterized the mechanism of this resistance. We observed homotrimer synthesis by a variety of 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. We are now trying to understand the mechanism of their synthesis and develop approaches to selective targeting of this synthesis and the molecules themselves. 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 clearly 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 mapping of these regions and analysis of their association with OI phenotype variations. It has long been believed that bone pathology in OI results primarily from collagen deficiency and/or collagen malfunction in the extracellular matrix. However, recent discoveries are inconsistent with this idea. First, OI-like bone pathologies are also caused by deficiencies in other proteins, including: (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 affect osteoblast function from a distance, e.g., by altering serotonin synthesis in duodenum. Second, normal bone homeostasis requires not only osteoblast synchronization with osteoclasts but also osteoblast coordination with other cells and organs. Our studies suggest that the primary cause of bone pathology in OI is osteoblast malfunction. Collagen mutations might be prevalent in OI because of their autosomal dominant inheritance and osteoblast malfunction associated with excessive cell stress response to abnormal collagen precursor (procollagen) folding, trafficking and secretion. Collagen deficiency and/or malfunction is likely a modulating factor rather than the primary cause of the disease, potentially explaining why other connective tissues are usually less affected by collagen mutations than bones. Furthermore, procollagen misfolding in aging osteoblasts might contribute to bone pathology in common, age-related osteoporosis. Experimental testing of these ideas might open up new approaches to pharmacological treatment of OI and osteoporosis through cell stress targeting in osteoblasts. In particular, we are examining procollagen folding and cell stress response to procollagen misfolding in dermal fibroblasts from several OI patients with Gly substitutions as well as in fibroblasts and osteoblasts from mouse models of Gly substitutions. We have just completed development of 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. We have found not only that this approach is more versatile, efficient and economical than radioisotope labeling but that it also has fewer (if any) unintended consequences for cell differentiation and function. Our experiments revealed qualitatively similar 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 signaling pathways reminiscent of those previously described in serpinopathies as 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 assisted Dr. McBride (U Maryland) in generating a second model with a different Gly substitution, which mimics the mutation in a large group of patients from the Old Order Amish community in Pennsylvania. Our study of the latter mice revealed important differences in osteoblast cell stress response and malfunction in vivo compared to cell culture. Over the last two 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 mice by low protein diet resulted in a noticeable improvement in bone material properties (reduced hypermineralization) but suppressed overall bone and animal growth. Amazingly, bone marrow stromal cells (BMSCs) from animals kept on a low protein diet exhibited significantly improved osteoblast differentiation in culture at the same conditions as BMSCs from animals on normal protein diet, suggesting possible epigenetic changes caused by the diet. Better understanding of the latter changes not only might reveal mechanisms of osteoblast adaptation but also might be exploited for designing treatment protocols that incorporate intermittent low protein diet. More detailed analysis of mechanisms of misfolded procollagen autophagy and potential therapeutic targets in this pathway is currently under way. 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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