Type I collagen is the most abundant vertebrate protein. Its abnormal biosynthesis contributes to fibrosis, cancer, osteoporosis, skeletal dysplasias and other disorders. It is a heterotrimer of two alpha-1 and one alpha-2 chains, but homotrimers of alpha-1 chains are produced in fetal tissues and some disorders. We discovered that these homotrimers are resistant to cleavage by all matrix metalloproteinases (MMPs) and characterized the mechanism of this resistance. We observed homotrimer synthesis by 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. 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 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 analysis of the role of these regions in OI severity. Our current studies suggest that the two main causes of bone pathology in OI are (a) insufficient synthesis of the collagen matrix of bone, particularly in mild type I OI and (b) malfunction of bone producing cells (osteoblasts), particularly in more severe forms of OI. It has been demonstrated by others that the primary cause of insufficient bone matrix synthesis in mild OI is null-allele mutations that prevent synthesis of type I collagen chains from the corresponding allele. Our findings indicate that the primary cause of osteoblast malfunction is accumulation of misfolded type I procollagen (collagen precursor) in the osteoblast Endoplasmic Reticulum (ER). We believe that the prevalence of Gly substitutions in severe OI can be explained by major effects of disruptions in the Gly-X-Y sequence on procollagen folding and resulting ER accumulation of misfolded molecules. Our recent studies directly demonstrated such accumulation of misfolded procollagen and its detrimental effects on osteoblast differentiation and function. We created and characterized a novel G610C mouse OI model, which mimics a Gly610 to Cys substitution in the alpha-2 chain found in a large group of patients. We developed a novel assay for procollagen folding, trafficking and secretion based on metabolic pulse-chase labeling with azidohomoalanine, a noncanonical amino acid that replaces methionine in newly synthesized proteins. To image the accumulation and handling of misfolded procollagen by live osteoblasts, we generated novel fluorescent constructs of procollagen chains. By combining the pulse-chase labeling with live cell imaging, we discovered a novel mechanism for the quality control of procollagen folding in the ER and redirection of misfolded molecules for degradation in lysosomes via autophagy. We demonstrated that suppression of autophagy by reducing or completely eliminating expression of an essential autophagy gene, Atg5 causes increased accumulation of misfolded procollagen and increased severity of bone pathology in G610C animals. We also found that stimulation of autophagy improves differentiation and function of G610C osteoblasts as well as the quality of bone produced by the cells. Our studies revealed that misfolded procollagen accumulation causes an unconventional cell stress response in osteoblasts, which does not follow canonical unfolded protein response pathways. We identified some of the key markers of this cell stress response. We are currently investigating its relationship with abnormal differentiation and function of osteoblasts. One goal of these studies is novel therapeutic approaches to OI treatment, e.g., we are currently developing autophagy enhancement strategies for improving osteoblast function and consequently reducing the severity of bone pathology. Another goal is better general understanding of misfolded procollagen handling by osteoblasts. Changes in the ER environment with aging and sickness might cause accumulation of misfolded procollagen and resulting osteoblast malfunction, contributing to common forms of osteoporosis. Testing of these ideas might therefore open up new approaches to osteoporosis treatment in the general population. 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. In addition, we are assisting NIH and extramural researchers in describing and understanding procollagen folding and handling by cells in other connective tissue disorders and animal models. In particular, we assisted Dr. Marini and other clinical researchers in discovering several novel forms of OI and characterizing defects of procollagen folding and handling by cells in these disorders. We collaborated with Dr. Forlino on characterization of normal and abnormal type I collagen processing in a zebra fish model of OI. In collaboration with Dr, Byers, we investigated OI caused by substitutions of Y-position Arg in the Gly-X-Y triplets. We demonstrated that such substitutions cause procollagen misfolding and accumulation in the ER similar to Gly substitutions. Y-Arg is essential for increasing the stability of procollagen triple helix and for binding an important collagen chaperone HSP47 in the ER. In sharp contrast, X-position Arg is not essential for proper procollagen folding in the cell. Consequently, X-Arg mutations do not cause bone pathology and affect other tissues, likely due to aberrant interactions of the mutant molecules in the extracellular matrix. We are currently assisting 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.
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