Type I collagen is the most abundant protein in higher vertebrates. Its mutations typically result in Osteogenesis Imperfecta (OI), Ehlers-Danlos syndrome (EDS) or a combination of OI and EDS. Most known pathogenic mutations are substitutions of an obligatory glycine in the repeating Gly-X-Y triplets of the collagen triple helix. It is generally accepted that disruption of the triple helix folding and structure by these mutations is somehow involved in the disease, but no clear relationship between different substitutions and variations in the disease severity associated with them has been found so far. We have 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 of different substitutions, we are continuing mapping of these regions and analysis of their association with OI phenotype variations. Since mutations affecting type I collagen triple helix structure are the most common genetic defects in severe OI, it has long been believed that abnormal collagen biosynthesis and function are the main causes of OI bone pathology. However, recent discoveries by several research teams, including our group, suggest otherwise. First, OI-like bone pathologies have also been found to be 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 (e.g., PERK, osteopotentia and osterix);and (c) proteins that affect osteoblast function from a distance, e.g., by altering serotonin synthesis in duodenum. Second, it has been demonstrated that normal bone homeostasis requires not only osteoblast synchronization with osteoclasts but also osteoblast coordination with other cells and organs. As a part of these studies, we characterized collagen biosynthesis defects in a new recessive form of OI caused by the lack of cyclophilin B, a general ER peptidylprolyl isomerase and an essential procollagen chaperone. We also investigated procollagen folding defects caused by several substitutions of obligatory Gly residues in the collagen triple helix in the classical autosomal dominant OI. Based on the findings discussed above, we proposed that the primary cause of bone pathology in OI is osteoblast malfunction rather than collagen malfunction. Collagen mutations are prevalent in OI simply because of their autosomal dominant inheritance and osteoblast malfunction associated with excessive ER stress response to procollagen misfolding. 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. Experimental testing of this hypothesis, which may open up new approaches to pharmacological OI treatment through ER stress targeting in osteoblasts, is currently under way. In addition to OI and EDS patients, we continued analysis of murine OI models with Gly substitutions. We extended our studies of bone structure and mineralization pathology in mice with a Cys substitution for Gly-610 in the alpha-2 chain and in mice with a Cys substitution for Gly-349 in the alpha-1 chain of type I collagen. We also continued to study effects of intrauterine transplantation of bone marrow stromal cells on the OI progression and bone quality in the Gly-349 OI model. Murine models seem to support the idea that abnormal bone quality in OI is associated primarily with osteoblast malfunction, e.g., normal donor osteoblasts in transplanted mice likely improve the bone quality by normalizing the coordination of the osteoblast pool with osteoclasts and other cells. We also investigated bone pathology is caudal vertebrae of mice with bone tumors caused by defects in protein kinase A, a crucial protein in cAMP signaling. We found that these tumors caused periosteal deposition of immature cortical bone, in which collagen matrix and mineral organization were intermediate between the ones expected in woven and lamellar bones. Accelerated matrix formation and deficient mineralization were reminiscent of the McCuneAlbright syndrome, caused by deficient cAMP signaling associated with defects in the guanine binding protein Gs-alpha. One of our most significant advances in the past several years was the characterization of a collagenase-resistant isoform of type I collagen and its potential role in cancer, fibrosis, and other disorders. The normal isoform of type I collagen is a heterotrimer of two alpha-1 and one alpha-2 chains. However, homotrimers of three alpha-1 chains were found in some carcinomas, fibrotic tissues, and rare forms of OI and EDS associated with alpha-2 chain deficiency. Our studies of the homotrimeric collagen from an EDS patient revealed its resistance to cleavage by all major collagenolytic matrix metalloproteinases (MMPs), including MMP-1,2,8,13, and 14. A more detailed investigation showed this resistance to be related to an increased stability of the homotrimer triple helix at the primary MMP cleavage site, inhibiting unwinding of the helix at this site (necessary for the cleavage). MMP overexpression is a hallmark of invasive cancers;cleavage of stromal type I collagen fibers by cancer cells and recruited fibroblasts is an essential step in clearing an invasion path for the cancer. Therefore, we hypothesized that synthesis of MMP-resistant collagen isoform may support cancer cell proliferation and tumor invasion;the MMP-resistant fibers laid down by these cells may serve as tracks, supporting outward cell migration and tumor growth. Our measurements confirmed the synthesis of a significant fraction of the homotrimers by cancer cells (20-40% in culture and even larger in vivo) but no homotrimer synthesis by normal mesenchymal cells or fibroblasts recruited into tumors. We found that more rigid homotrimeric type I collagen matrix supported faster proliferation and migration of cancer cells. Since the homotrimers do not appear to be produced in normal tissues, they may present a novel, appealing diagnostic and therapeutic target. Presently, we are investigating how the homotrimer vs. heterotrimer synthesis is regulated in different cells and testing different molecular probes for selective targeting of the homotrimers. In addition to carcinomas, type I collagen homotrimers were reported in liver fibrosis and other fibrotic disorders. In the past two years, we established that their resistance to MMP is indeed involved in glomerular sclerosis in two different murine models. We obtained initial evidence suggesting that it may also be a factor in the murine model of cAMP deficiency discussed above. These and other studies suggest that the homotrimers are produced by undifferentiated, dedifferentiated, or transformed cells but not by normal or activated collagen-producing mesenchymal cells. Presently, we are investigating the molecular mechanism which prevents the homotrimer formation in the latter cells.
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