OI caused by collagen type I mutations

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ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94

158 Type I collagen glomerulopathy: oim (COL1A2 deficient) mice C.L. Phillips a, A.C. Brodeur a, A.M. Roberts-Pilgrim a, A.L. Walker a, J.H. Miner b, C.L. Franklin a b

University of Missouri, Columbia, MO 65212, United States Washington University, St Louis, MO 63110, United States

A novel type I collagen glomerulopathy was identified in oim mice (COL1A2 deficient), which synthesize exclusively homotrimeric type I collagen, [α1(I)3]. Though type I collagen exists as a heterotrimer [α1(I)2α2(I)], the homotrimeric form is present in small amounts in skin, embryologic tissues and healing wounds. Its role is uncertain. The homotrimer accumulates in the renal mesangium of oim mice. Under normal physiologic conditions type I collagen is not present in glomeruli; its accumulation is pathologic. In the following, we investigate the pathological progression and mechanistic significance of the type I collagen glomerulopathy. We evaluated kidneys from homozygous (oim), heterozygous (oim/+), and wildtype (WT) mice at various ages, 1 day-3 months. Picrosirius red staining of formalin-fixed kidneys demonstrated that oim and oim/+mice develop glomerular collagen deposition in a gene dose effect, resulting in albuminuria in severely affected mice. The glomerulopathy initiates postnatally within the 1st week of life, appearing to follow glomerular maturation. Electron microscopy demonstrated fibrillar collagen deposition in the mesangium and subendothelial space between the fenestrated endothelium and the basement membrane. In areas of severe deposition, podocyte foot process effacement was seen. Quantitative RT-PCR using whole kidney demonstrated a two-fold increase in steady-state proα1(I)collagen mRNA in 1-month oim mice relative to WT mice. This suggests that the glomerular collagen deposition seen in the type I collagen glomerulopathy is, in part, due to pretranslational mechanisms. doi:10.1016/j.matbio.2006.08.181

159 The protective function of lubricin at the cartilage surface Y. Cui, M. Warman Department of Genetics, Case Western Reserve University, Cleveland, OH 44106, United States The human autosomal recessive disorder camptodactylyarthropathy-coxa vara-pericarditis syndrome (CACP) results from loss-of-function mutations in lubricin (encoded by the gene PRG4). The lubricin (Prg4) knock-out mouse recapitulates features of human CACP, such as synovial hyperplasia

and precocious joint failure. To investigate when lubricin is required to protect joints we created a transgenic mouse in which lubricin expression can be induced conditionally using doxycycline. We bred the tet-inducible lubricin transgene and a ubiquitously expressed reverse tet-transactivator (rtTA) transgene onto the lubricin-null background and began feeding pregnant dams doxycyline at embryonic day 0.5, 9.5, or at postnatal day 1. Their offspring continued to receive doxycycline until 1 month of age, at which time they were sacrificed and their knee joints examined. Preliminary results indicate that inducing lubricin expression in the lubricin-null background reduces protein deposition at the cartilage surface that would otherwise occur in lubricin-null mice. Protein deposition was reduced even in mice that had their lubricin expression induced postnatally. We have also created a BAC transgenic mouse that expresses rtTA under the control of Prg4 regulatory sequences. This latter mouse expresses rtTA in superficial zone chondrocytes and cells at the meniscal surface. These transgenic mouse strains will enable us to address fundamental questions regarding lubricin biology and, more broadly, the cell biology at the cartilage surface. doi:10.1016/j.matbio.2006.08.182

160 EDS/OI caused by collagen type I mutations F. Malfait a, S. Symoens a , Y. Vander Haeghen b, J. Naeyaert b, N. Goemans c, E. Holmberg d, M. Petersen e, P. Coucke a, A. De¨Paepe a a

Centre for Medical Genetics, Ghent University Hospital, Ghent, Belgium b Department of Dermatology, Ghent University Hospital, Ghent, Belgium c Department of Pediatrics, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium d Department of Clinical Genetics, Sahlgrenska University Hospital/East, S-41685 Gothenburg, Sweden e Department of Genetics, Institute of Child Health, Aghia Sophia Children's Hospital, Athens, Greece Glycine substitutions and exon-skipping mutations in COL1A1 and COL1A2, encoding type I procollagen, cause OI, a connective tissue disorder characterized by bone fragility. A specific class of mutations, resulting in skipping of COL1A1 or COL1A2-exon 6, and loss of the procollagenI-N proteinase cleavage site results in EDS arthrochalasis type, characterized by joint hypermobility and congenital hip dislocation. We present a series of patients with glycine substitutions or exon-skipping defects in the N-terminal region of the helical domain of the α1(I) or α2(I) collagen chain. In addition to mild OI they present characteristics of EDS, with joint hypermobility, skin hyperextensibility and

ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94

easy bruising. Two of the patients experienced vascular rupture. We show that all mutations interfere with removal of the procollagen-I-N propeptide, even though the N-proteinase cleavage site remains intact. Incorporation of these uncleaved pN-collagen chains into the mature collagen fibrils results in restricted lateral growth of these fibrils, as evidenced by decreased dermal fibril diameters in vivo. While these helical mutations are directly responsible for the OI-features, they are, by interference of N-propeptide removal, indirectly responsible for the EDS features. doi:10.1016/j.matbio.2006.08.183

161 Conditional expression of collagen II mutants in SW1353 cells A. Steplewski, V. Hintze, R.J.Brittingham, K. Holmes, A. Fertala Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA 19107, United States Mutations in collagen II are associated with chondrodysplasias. It is expected that pathological effects of collagen mutations can be counterbalanced by gene or cell therapy approaches. Due to a number of limitations, which include problems with delivery of therapeutic agents into patients, transgenic mice harboring collagen II mutations, and native chondrocytes molecular targets that have to be reached in order to change diseased phenotype have not been defined. We developed an experimental model that will allow us to test gene and cell therapy approaches aimed at alleviating the effects of the presence of mutant collagen II molecules. We designed DNA constructs for R75C, R519C, R789C, and G853E collagen II mutants found in chondrodysplasia patients under a Tet-responsive promoter. These constructs were designed to be expressed in the SW1353 chondrocytic cell line, which forms cartilage-like constructs in 3D cultures. We tested the behavior of these cells and the morphology of cartilage-like constructs formed in conditions allowing expression of recombinant collagen II. Subsequently, the behavior of cells and the morphological changes were monitored in these 3D constructs after switching off expression of exogenous collagen II. We demonstrated great utility of this system for studies on the effects of blocking expression of mutant collagen II on repair of damaged cartilaginous tissues. Moreover, we demonstrated a great utility of this system for defining minimal therapeutic targets that have to be reached to change a diseased phenotype caused by collagen II mutants. doi:10.1016/j.matbio.2006.08.184

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162 ADAMTS10 interacts with fibrillin in Weill-Marchesani syndrome W.E. Kutz a, L.W. Wang a, D.R. Keene b, L.Y. Sakai b, S.S. Apte a a

Department of Biomedical Engineering, Cleveland Clinic, Cleveland, OH 44195, United States b Shriners Hospital for Children, Portland, OR 97239, United States Mutations in ADAMTS10 or fibrillin-1 cause the recessive and dominant forms of Weill-Marchesani syndrome (WMS), respectively. WMS is a rare connective tissue disorder characterized by short stature, brachydactyly, ectopia lentis, and aortic stenosis. In most respects, WMS is the opposite of Marfan syndrome (MFS), a more common connective tissue disorder caused by dominant mutations in fibrillin-1. Given this genetic evidence, we hypothesize that ADAMTS10 and fibrillin-1 proteins interact to regulate extracellular networks in connective tissues. Adamts10 mRNA, like fibrillin-1, was widely expressed in tissues relevant to WMS and MFS, such as aorta, lung, skin and the skeleton. Using blot overlay assays and BIAcore, we found that ADAMTS10 binds to the N-and C-terminal halves of fibrillin-1. Binding was specific to ADAMTS10, as ADAMTS1, 2, 4, 5, and 13 did not bind to fibrillin-1. In tissues, light and immuno-EM showed specific immunolocalization of ADAMTS10 along fibrillin microfibrils. Cell-based proteolysis assays suggested furin-activated ADAMTS10 is capable of cleaving the C-terminal portion of fibrillin-1. Lastly, immunocytochemistry of WMS patient cells, as well as MEFS from TS10−/− mice showed disorganized and sometimes absent fibrillin1 matrices. These results suggest an intimate relationship between ADAMTS10 and fibrillin-1, with fibrillin-1 likely being a substrate for ADAMTS10. The absence of well-defined fibrillin-1 microfibrils in cells lacking ADAMTS10 points toward a role for ADAMTS10 in fibrillin-1 assembly or modification doi:10.1016/j.matbio.2006.08.185

163 Skeletal phenotype of mice overexpressing mutant human COMP K. Posey a, Y. Yang b, E.S. Bales a, A. Veerisetty a, R. Haynes c, S.K. Sharan b, J.T. Hecht a,c a

Department of Pediatrics, University of Texas Medical School at Houston, Houston, TX 77030, USA b Genetics of Cancer Susceptibility, National Cancer Institute, Frederick, MD 21702, United States c Shriners Hospital for Children, Houston, TX 77030, USA Cartilage oligomeric matrix protein (COMP) is an extracellular matrix protein found primarily in cartilage and