Collagen-related diseases

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Type I collagen is the most abundant human protein that forms the structural scaffold of bone, skin and other tissues. Normal type I collagen is a heterotrimer of two alpha-1 and one alpha-2 chains. Homotrimers of alpha-1 chains can also be produced in fetal tissues and some disorders. We discovered that alpha-1 homotrimers are resistant to cleavage by all collagenases and characterized the mechanism of this resistance. In tumors, the homotrimers are synthesized by cancer but not normal cells. More rigid matrix made of the homotrimers supports faster proliferation and migration of cancer cells. Collagenase-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. The most prominent human development pathologies associated with type I collagen mutations are fragility of bones in OI and laxity and fragility of skin, tendons, and ligaments in EDS. Over 80% of severe OI cases are caused by substitutions of glycine (Gly) required in every third position for maintaining the triple helical structure of collagen. By altering the triple helix folding and structure, Gly substitutions cause malfunction of bone producing cells (osteoblasts) and alter formation and function of the collagen scaffold of bone. Over the years, our studies revealed that osteoblast malfunction is a major pathogenic factor in Gly substitutions. The cause of this malfunction is cell stress resulting from accumulation of misfolded collagen precursor (procollagen) in osteoblast Endoplasmic Reticulum (ER). We characterized some of the key features of this cell stress response and identified potential therapeutic targets for its alleviation. To understand and target osteoblast cell stress, we created and characterized a novel G610C mouse model of OI, which mimics a Gly610 to Cys substitution in the alpha-2 chain found in a large group of patients. We found that misfolding of mutant procollagen in G610C mice does not activate unfolded protein response (UPR) pathways of canonical ER stress. From single-cell RNA sequencing, we recently discovered, e.g., increased expression of cell stress response genes Atf5 and Hspa9 instead of their homologues Atf4 and Hspa5 activated by UPR. We have confirmed this noncanonical cell stress response by in-situ RNA hybridization, identified several candidate pathways involved in it and are currently examining the underlying molecular mechanisms. We also found that removal of excess misfolded mutant procollagen from the ER and its delivery to lysosomes for degradation (autophagy) is an important adaptation mechanism to cell stress in osteoblasts. We therefore created additional G610C mouse models, in which autophagy can be suppressed or enhanced by altering expression of Atg5 gene, which is required for an autophagy pathway known as macro-autophagy. We observed more severe bone pathology upon reduced Atg5 expression, yet we discovered that osteoblasts recycle misfolded procollagen primarily by another pathway of ER exit site (ERES) micro-autophagy (described below), for which Atg5 is an enhancer rather than a required gene. Importantly, we observed perinatal lethality of some G610C animals due to deficient embryonic lung development, apparently associated with lung fibroblast malfunction. Given that lung malfunction is a common complication and cause of death in OI patients, we are examining how fibroblast malfunction causes lung pathology and how the fibroblast function can be normalized by pharmacological and dietary approaches. In addition to studies of cells stress associated with procollagen misfolding in mouse models of OI, we are also pursuing more fundamental cell biology studies of procollagen biosynthesis by osteoblasts and fibroblasts. The goal of these studies is better understanding of underlying molecular mechanisms, many of which are still unknown. For instance, observations made by us and others suggest that deficient procollagen trafficking and autophagy might be involved in a variety of pathologies spanning the entire lifespan, from skeletal dysplasia in early development to osteoporosis in aging. Better understanding of the trafficking and autophagy mechanisms might therefore reveal new therapeutic targets and approaches. To study procollagen trafficking and autophagy, we developed novel fluorescent constructs of procollagen for live cell imaging and correlative light and electron microscopy. Contrary to published models, we observed that transport vesicles carrying procollagen from the ER to Golgi have no COPII coat and no procollagen chaperone HSP47. Our study revealed that export of normally folded procollagen from ERES is dependent on COPI coat machinery. Procollagen is delivered to cis-Golgi from ERES by COPI-dependent rapidly moving transport vesicles or through membrane ERES-cis-Golgi tunnels. Misfolded procollagen molecules are recognized at ERES and rerouted from this secretory pathway to autophagy. ERESs containing misfolded molecules are modified by autophagic machinery and directly engulfed by lysosomes in a non-canonical process of ERES micro-autophagy. We are currently investigating the mechanism of the lysosomal recruitment to ERES, which is an appealing target for therapeutic applications. We are also investigating whether ERES micro-autophagy is a more general protein quality control mechanism, which might be utilized by cells for many proteins and not just procollagen. To facilitate these studies, we are utilizing CRISPR/CAS gene editing technology for creating novel cell lines, in which endogenous procollagen is fluorescently tagged and can be manipulated by Flp-recombinase mediated cassette exchange to introduce mutations and change the fluorescent tags. Abnormal differentiation and function of collagen-producing cells also plays an important role in fibrosis and tumor formation. In collaboration with Dr. Stratakis, we investigated 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 characterized the latter abnormalities and the origin of novel bone structures formed in these tumors. In collaboration with Dr. Leppert, we described abnormal composition of collagen deposited in uterine fibromas, which could be involved in the dysregulation of uterine fibroblasts underlying this pathology. In addition, we are collaborating with NIH and extramural researchers on studies of collagen-related pathology in a variety of human patients and animal models. For instance, we assisted Dr. Marini in discovering several novel forms of OI and characterizing underlying pathology. In collaboration with Dr. Byers, we investigated OI caused by arginine substitutions in type I collagen, demonstrating procollagen misfolding and accumulation in the ER similar to Gly substitutions. We assisted Dr. Bonnemann in characterization of a complex connective tissue disorder involving pathology of multiple tissues, which is caused by deficient function of prolyl 4 hydroxylase 1, an enzyme primarily responsible for hydroxylation of proline in type I collagen. We are collaborating with Dr. Otsuru on studies of growth plate pathology and growth deficiency in the G610C mouse model of OI. We are also collaborating with Dr. Forlino on characterization of type I collagen processing in zebra fish models of OI.