Collagen-related diseases

胶原蛋白相关疾病

• 批准号: 7968474
• 负责人: Sergey Leikin
• 金额: $ 63.19万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968474
• 关键词: Affect; Animals; Arginine; Biomechanics; Biopsy; Bone Marrow; Bone Matrix; Cancer Model; Carcinoma; Cell Culture Techniques; Cell Proliferation; Cells; Clinical; Collagen; Collagen Fiber; Collagen Fibril; Collagen Type I; Connective Tissue; Cysteine; Data; Defect; Degenerative polyarthritis; Development; Diagnostic; Disease; Disease Progression; Ehlers-Danlos Syndrome; Engraftment; Family; Fiber; Fibroblasts; Fibrosis; Functional disorder; Glycine; Goals; Individual; Interstitial Collagenase; Investigation; Knock-in Mouse; Ligand Binding; Literature; Liver Fibrosis; Location; Malignant Neoplasms; Maps; Matrix Metalloproteinases; Measurement; Mesenchymal; Modeling; Molecular; Mus; Mutation; Normal tissue morphology; Osteoblasts; Osteogenesis Imperfecta; Osteoporosis; Pathology; Patients; Phenotype; Physiologic calcification; Play; Positioning Attribute; Property; Protein Isoforms; Proteins; Recruitment Activity; Reporting; Resistance; Role; Rupture; Scleroderma; Severity of illness; Site; Skin; Stromal Cells; Structure; Symptoms; Technology; Testing; Time; Tissues; Triplet Multiple Birth; Tumor Cell Invasion; Undifferentiated; Uterine Fibroids; Variant; Vertebrates; base; bone; cancer cell; cell motility; cell transformation; collagenase; disulfide bond; experience; glomerulosclerosis; in utero transplantation; in vivo; interest; mature animal; member; migration; mineralization; molecular pathology; mutant; novel; overexpression; prevent; scaffold; skeletal abnormality; spectroscopic imaging; stem cell therapy; therapeutic target; triple helix; tumor; tumor growth; tumor xenograft; type I collagen alpha 1

Type I collagen is the most abundant protein in higher vertebrates. It forms fibrous scaffolds of bone, skin, vasculature, and other tissues. Mutations in type I collagen typically result in Osteogenesis Imperfecta (OI), Ehlers-Danlos syndrome (EDS) or a combination of OI and EDS. Most known pathogenic mutations are substitutions of an obligatory glycine in the repeating Gly-X-Y triplets of the collagen triple helix. It is generally accepted that disruption of the triple helix folding and structure by these mutations is somehow involved in the disease, but no clear relationship between different substitutions and rather dramatic variations in the disease severity associated with them 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 fibril assembly and ligand binding as well as with some (but not all) of the observed regional variations in OI phenotypes. In an ongoing study of different substitutions, we are continuing mapping of these regions and analysis of their association with OI phenotype variations. Substitutions at the X or Y position in the triple helix, mostly of cysteine for arginine, were recently identified in individuals with OI, osteopenia, arterial rupture, EDS, and Caffey disease. Since these non-glycine substitutions were not considered to be detrimental for the triple helix folding and structure, their molecular pathophysiology remained completely unclear. In 2007, we reported a change in the triple helix stability caused by a Cys substitution for an Y position Arg-888 in several members of the same family affected with a combination of OI and EDS symptoms. During the past year, we described a similar change in the triple helix stability in unrelated patients, in which Arg-780 in the alpha-1 chain was substituted with Cys or Leu. This study confirmed the role of Y-position Arg residues in the triple helix stability proposed by several authors. It suggested that the OI phenotype of patients with Y-Arg substitutions might be caused by the destabilizing effect of an Y-Arg loss rather than by the gain of a Cys residue not normally present in the triple helix. At the same time, analysis of our and literature data indicated that the Cys gain might be responsible for the arterial rupture and EDS phenotypes. We observed formation of aberrant disulfide bonds between the Cys residues in molecules with two mutant alpha-1 chains. These bonds resulted in kinking, register shift, and abnormal N-propeptide cleavage from the triple helix, the latter effect known to be involved in EDS. In addition to characterization of molecular defects in OI and EDS patients, we continued studies of murine OI models with knocked-in Gly substitutions. In particular, we utilized Raman microspectroscopy to characterize defects in organic matrix composition and bone mineralization in mice with a Cys substitution for Gly-610 in the alpha-2 chain and in mice with a Cys substitution for Gly-349 in the alpha-1 chain of type I collagen. These studies revealed significantly reduced collagen content and hypermineralization of the matrix, likely contributing to the bone brittleness in these animals observed in biomechanical measurements. In the Gly-349-Cys mice, we investigated the effect of intrauterine transplantation of bone marrow stromal cells on the disease progression. One of the major challenges in the stem cell therapy is low engraftment of donor cells. However, our study revealed that the donor cells comprising just 2% of osteoblasts were responsible for synthesis of about 20% of bone matrix and dramatic improvement in the matrix composition and mineralization, eliminating 30% perinathal lethality associated with this mutation in mice and nearly normalizing biomechanical properties of bones in adult animals. One of our most significant advances in the past two 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 surprising resistance to cleavage by the most common interstitial collagenase (MMP-1). While pursuing this observation, we discovered that the homotrimers were at least 5-10 more resistant to all major collagenolytic matrix metalloproteinases (MMPs), including MMP-1,2,8,13, and 14. A more detailed investigation revealed 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). Overexpression of MMPs is a hallmark of invasive cancers; cleavage of stromal type I collagen fibers by cancer cells and recruited fibroblasts is an essential step in clearing an invasion path for the cancer. Therefore, we hypothesized that synthesis of MMP-resistant collagen isoform may support cancer cell proliferation and tumor invasion; the MMP-resistant fibers laid down by these cells may serve as tracks, supporting outward cell migration and tumor growth. Our measurements in cell culture and xenograft tumor models confirmed the synthesis of a significant fraction of the homotrimers by cancer cells (20-40% in culture and even larger in vivo) but no homotrimer synthesis by normal mesenchymal cells or fibroblasts recruited into tumors. Furthermore, we found that more rigid homotrimeric type I collagen matrix supported faster proliferation and migration of cancer cells. Presently, we are investigating how the homotrimer vs. heterotrimer synthesis is regulated in different cells and developing approaches to selective targeting of the homotrimers. Since the homotrimers do not appear to be produced in normal tissues, they may present a novel, appealing diagnostic and therapeutic target. In addition to carcinomas, type I collagen homotrimers were reported in liver fibrosis and other fibrotic disorders. Since collagen fiber degradation by MMPs is essential for preventing fibrosis, it was reasonable to assume that MMP-resistant isoform of type I collagen would be involved in the disease and would be a common factor in different forms of fibrosis. In the past year, we found that the homotrimers were indeed involved in glomerular sclerosis in at least one murine model. However, further testing revealed no homotrimers in tissue biopsies from scleroderma or uterine fibroma patients. Based on these observations, our experience gained in the studies of cancer models, and literature reports, we now hypothesize that the homotrimers are not produced by normal or activated mesenchymal cells but only by non-mesenchymal, undifferentiated, dedifferentiated, or transformed cells. While we still believe that the homotrimers may play an important role in some forms of fibrosis, they are not likely to be involved in fibrosis within mesenchymal tissues.

I型胶原蛋白是高等脊椎动物中含量最多的蛋白质。它形成骨、皮肤、脉管系统和其他组织的纤维支架。I型胶原蛋白的突变通常会导致成骨不全症（OI）、Ehlers-Danlos综合征（EDS）或成骨不全症和EDS的合并。大多数已知的致病性突变是在胶原蛋白三螺旋的重复Gly-X-Y三联体中置换一个强制性甘氨酸。人们普遍认为，这些突变对三螺旋折叠和结构的破坏在某种程度上与疾病有关，但到目前为止，还没有发现不同的替代与与之相关的疾病严重程度的显著变化之间的明确关系。我们已经确定，Gly取代对整体胶原稳定性的影响取决于它们在三螺旋的不同区域内的位置，而不是取代残基的身份。这些区域似乎与胶原纤维组装和配体结合的重要区域一致，也与一些（但不是全部）观察到的成骨不全症表型的区域差异一致。在一项正在进行的不同替代研究中，我们正在继续绘制这些区域的图谱，并分析它们与OI表型变异的关系。