Type I collagen is the most abundant protein in vertebrates. Its abnormalities contribute to pathology in a variety of diseases, including fibrosis, cancer, osteoporosis, and skeletal dysplasias. We characterized an isoform of type I collagen, which is resistant to cleavage by all major collagenolytic matrix metalloproteinases (MMPs), including MMP-1,2,8,13, and 14. The normal isoform of type I collagen is a heterotrimer of two alpha-1 and one alpha-2 chains. However, the collagenase-resistant homotrimers of three alpha-1 chains are produced in most carcinomas, some fibrotic tissues, and rare forms of osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS) associated with alpha-2 chain deficiency. 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. We are now trying to understand the molecular mechanism regulating their synthesis in different cells and develop approaches to selective targeting of this synthesis and the molecules themselves. Type I collagen 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 primarily from collagen deficiency and/or malfunction in the extracellular matrix. 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,WNT1 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 believe 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 ER stress response to collagen precursor (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. In particular, we are examining procollagen folding and ER 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. Our experiments have revealed qualitatively similar procollagen folding delays in all of these cells as well as retention and accumulation of partially unfolded or misfolded mutant procollagen in the ER. Gene expression analysis has shown an unusual ER stress response to this accumulation. The cells do not activate the conventional unfolded protein response signaling. Instead, they downregulate procollagen synthesis and/or activate 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. Our cell culture studies emphasized the importance of examining the ER 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 much more severe osteoblast ER stress and malfunction in vivo compared to cell culture. Based on our observations indicating an important role of autophagy in degradation of misfolded procollagen molecules and adaptation of osteoblasts to ER stress, we designed a dietary treatment strategy for the animals. We are presently examining the effectiveness of this approach in the first group of treated animals. Our preliminary data indicate a significant improvement in osteoblast function in animals on a low protein diet supplemented with essential and aromatic amino acids. However, many additional experiments remain to be completed before we arrive at definite conclusions. 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.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Zip Code
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
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
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
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 α2(I)-G610C Mouse Model of Osteogenesis Imperfecta. J Bone Miner Res 31:1608-16
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 :
Cabral, Wayne A; Perdivara, Irina; Weis, MaryAnn et al. (2014) Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta. PLoS Genet 10:e1004465
Barnes, Aileen M; Duncan, Geraldine; Weis, Maryann et al. (2013) Kuskokwim syndrome, a recessive congenital contracture disorder, extends the phenotype of FKBP10 mutations. Hum Mutat 34:1279-88
Keupp, Katharina; Beleggia, Filippo; Kayserili, Hülya et al. (2013) Mutations in WNT1 cause different forms of bone fragility. Am J Hum Genet 92:565-74
Valli, M; Barnes, A M; Gallanti, A et al. (2012) Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix. Clin Genet 82:453-9

Showing the most recent 10 out of 21 publications