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. Bone pathology in OI is caused by (a) insufficient synthesis and abnormal function of extracellular collagen matrix of bone and (b) malfunction of bone producing cells (osteoblasts). Our studies revealed that osteoblast malfunction is a major and likely dominant pathogenic factor in many cases of OI associated with Gly substitutions. The primary cause of this malfunction is accumulation of misfolded type I procollagen (collagen precursor) in the osteoblast Endoplasmic Reticulum (ER) that causes cell stress. We demonstrated that Gly substitutions cause 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 believe that the prevalence of Gly substitutions in severe OI can be explained by their major effects on procollagen folding and by cell stress resulting from ER accumulation of misfolded molecules. To understand and target osteoblast cell stress and resulting malfunction, 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 found that removal of excess misfolded mutant procollagen from the ER and its delivery to lysosomes for degradation is an important adaptation mechanism to cell stress in osteoblasts. The process of cellular cargo isolation and delivery to lysosomes for degradation is referred to as autophagy. We demonstrated that suppression of autophagy by reducing or eliminating expression of an essential autophagy gene, Atg5 caused increased accumulation of misfolded procollagen and increased severity of bone pathology in G610C animals. Stimulation of autophagy improved differentiation and function of G610C osteoblasts as well as the quality of bone produced by the cells. 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. To understand misfolded procollagen handling by osteoblasts, we recently developed novel fluorescent constructs of procollagen chains and imaged their trafficking in live cells. We discovered that misfolded procollagen molecules are recognized and rerouted from normal secretory pathway to autophagy via a novel quality control mechanism at ER exit sites (ERES). ERES that contain misfolded molecules are prevented from maturation into transport vesicles, which normally deliver procollagen from the ER to Golgi. Instead, they are ubiquitinated, resulting in recruitment of autophagy machinery and insertion of autophagosome membrane proteins into ERES membranes. The latter proteins trigger recruitment of lysosomes that engulf ERES, disconnect ERES from the ER and degrade ERES content in a non-canonical micro-ERES-phagy process reminiscent of micro-autophagy described for other cargo. We are currently investigating the mechanism of the lysosomal recruitment, which is an appealing target for micro-ERES-phagy enhancement that might be utilized in therapeutic applications. At the same time, we are investigating whether micro-ERES-phagy is a more general protein quality control mechanism, which might be utilized by cells for many proteins and not just procollagen. We are also examining whether this process might be involved in rerouting of proteins from the secretory pathway to lysosomal degradation not only in the case of protein misfolding but under starvation conditions as well. 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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