Type I collagen is the most abundant protein in vertebrates. Its mutations typically result in 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 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, 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 from abnormal collagen biosynthesis and function. However, recent discoveries by several research teams, including our group, are inconsistent with this idea. 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. Based on our studies of OI mutations in collagen and other proteins, we argue that the primary cause of bone pathology in OI is osteoblast rather than collagen malfunction. Collagen mutations might be 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. Furthermore, ER stress response to procollagen misfolding in aging osteoblasts in the absence of any mutations might contribute to bone pathology is common, age-related osteoporosis. Experimental testing of these ideas, which may open up new approaches to pharmacological treatment of OI and osteoporosis through ER stress targeting in osteoblasts, is currently under way. During the last several months, we focused on examining procollagen folding and ER stress response to procollagen misfolding in dermal fibroblasts from several OI patients with Gly substitutions. Immunofluorescence imaging revealed clear retention of partially unfolded or misfolded procollagen in the ER of affected cells compared to normal control fibroblasts, while analysis of gene expression showed an unusual ER stress response to this accumulation. We observed no activation of the conventional unfolded protein response signaling. Instead, in some of the cells we found dramatic downregulation of procollagen transcription and in some activation of signaling pathways previously described in serpinopathies as an ER overload response to aggregation of misfolded proteins. We are currently examining molecular mechanisms of these ER stress responses and potential ways of their modulation. To gain better understanding of osteoblast malfunction in skeletal disorders and develop novel approaches to treatment, in addition to cells and tissues from human patients, we utilize murine models. Our studies of mice with a Cys substitution for Gly-610 in the alpha-2 chain and mice with a Cys substitution for Gly-349 in the alpha-1 chain of type I collagen further support the idea of osteoblast malfunction as the primary cause of bone pathology in OI. We are now trying to translate this understanding into developing treatments that could reduce OI severity by normalizing osteoblast function. We are investigating ER stress response of mouse cells to procollagen misfolding in vitro and in vivo as well as testing transplantation of bone marrow stromal cells, dietary changes, and pharmacological approaches. Another important murine model of 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. Among our other important 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). We observed synthesis of a significant fraction of the homotrimers by a variety of cancer cells (20-40% in culture and even larger in vivo) but no homotrimer synthesis by normal mesenchymal cells or fibroblasts recruited into tumors. More rigid homotrimeric type I collagen matrix supported faster proliferation and migration of cancer cells. MMP-resistant homotrimer fibers laid down by these cells may serve as tracks, supporting outward cell migration and tumor growth. The homotrimers may thus present an appealing diagnostic and therapeutic target in cancer. In addition to carcinomas, type I homotrimers are produced in some but not all fibrotic disorders;e.g., we established their involvement in glomerular sclerosis in two different murine models but we did not find them in uterine fibroids or scleroderma skin lesions. Our studies suggest that they might also be involved in tumors in the murine model of cAMP deficiency discussed above. More surprisingly, we recently observed their synthesis by calvarial osteoblasts from one of the murine models of OI discussed above. Based on these and other studies, we hypothesize that the homotrimers are produced by undifferentiated, abnormally differentiated, or transformed cells but not by normal or activated collagen-producing mesenchymal cells. We are now trying to understand the molecular mechanism regulating their synthesis in different cells.

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Makareeva, Elena; Sun, Guoli; Mirigian, Lynn S et al. (2018) Substitutions for arginine at position 780 in triple helical domain of the ?1(I) chain alter folding of the type I procollagen molecule and cause osteogenesis imperfecta. PLoS One 13:e0200264
Saloustros, Emmanouil; Liu, Sisi; Mertz, Edward L et al. (2017) Celecoxib treatment of fibrous dysplasia (FD) in a human FD cell line and FD-like lesions in mice with protein kinase A (PKA) defects. Mol Cell Endocrinol 439:165-174
Zou, Yaqun; Donkervoort, Sandra; Salo, Antti M et al. (2017) P4HA1 mutations cause a unique congenital disorder of connective tissue involving tendon, bone, muscle and the eye. Hum Mol Genet 26:2207-2217
Gioia, Roberta; Tonelli, Francesca; Ceppi, Ilaria et al. (2017) The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta. Hum Mol Genet :
Gistelinck, C; Gioia, R; Gagliardi, A et al. (2016) Zebrafish Collagen Type I: Molecular and Biochemical Characterization of the Major Structural Protein in Bone and Skin. Sci Rep 6:21540
Mertz, E L; Makareeva, E; Mirigian, L S et al. (2016) Makings of a brittle bone: Unexpected lessons from a low protein diet study of a mouse OI model. Matrix Biol 52-54:29-42
Cabral, Wayne A; Ishikawa, Masaki; Garten, Matthias et al. (2016) Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta. PLoS Genet 12:e1006156
Lindert, Uschi; Cabral, Wayne A; Ausavarat, Surasawadee et al. (2016) MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta. Nat Commun 7:11920
Mirigian, Lynn S; Makareeva, Elena; Mertz, Edward L et al. (2016) Osteoblast Malfunction Caused by Cell Stress Response to Procollagen Misfolding in ?2(I)-G610C Mouse Model of Osteogenesis Imperfecta. J Bone Miner Res 31:1608-1616
Liu, Sisi; Saloustros, Emmanouil; Mertz, Edward L et al. (2015) Haploinsufficiency for either one of the type-II regulatory subunits of protein kinase A improves the bone phenotype of Prkar1a+/- mice. Hum Mol Genet :

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