Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
PLoS One ; 4(12): e8481, 2009 Dec 29.
Article in English | MEDLINE | ID: mdl-20041163

ABSTRACT

BACKGROUND: Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development. METHODOLOGY/PRINCIPAL FINDINGS: We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically. CONCLUSIONS/SIGNIFICANCE: Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype.


Subject(s)
Body Patterning/genetics , Calcification, Physiologic/physiology , Fibrillar Collagens/genetics , Notochord/embryology , Spine/embryology , Zebrafish Proteins/genetics , Zebrafish/embryology , Animals , Body Patterning/drug effects , Calcification, Physiologic/drug effects , Cartilage/drug effects , Cartilage/metabolism , Cartilage/pathology , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/ultrastructure , Fibrillar Collagens/metabolism , Gene Expression Regulation, Developmental/drug effects , Humans , Notochord/abnormalities , Notochord/drug effects , Notochord/ultrastructure , Oligonucleotides, Antisense/pharmacology , Osteogenesis/drug effects , Osteogenesis/genetics , Phylogeny , Spine/abnormalities , Spine/drug effects , Spine/pathology , Zebrafish/genetics , Zebrafish Proteins/metabolism
2.
Hum Mutat ; 29(12): 1435-42, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18566967

ABSTRACT

Autosomal dominant osteogenesis imperfecta (OI) is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Recently, dysregulation of hydroxylation of a single proline residue at position 986 of both the triple-helical domains of type I collagen alpha1(I) and type II collagen alpha1(II) chains has been implicated in the pathogenesis of recessive forms of OI. Two proteins, cartilage-associated protein (CRTAP) and prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene) form a complex that performs the hydroxylation and brings the prolyl cis-trans isomerase cyclophilin-B (CYPB) to the unfolded collagen. In our screen of 78 subjects diagnosed with OI type II or III, we identified three probands with mutations in CRTAP and 16 with mutations in LEPRE1. The latter group includes a mutation in patients from the Irish Traveller population, a genetically isolated community with increased incidence of OI. The clinical features resulting from CRTAP or LEPRE1 loss of function mutations were difficult to distinguish at birth. Infants in both groups had multiple fractures, decreased bone modeling (affecting especially the femurs), and extremely low bone mineral density. Interestingly, "popcorn" epiphyses may reflect underlying cartilaginous and bone dysplasia in this form of OI. These results expand the range of CRTAP/LEPRE1 mutations that result in recessive OI and emphasize the importance of distinguishing recurrence of severe OI of recessive inheritance from those that result from parental germline mosaicism for COL1A1 or COL1A2 mutations.


Subject(s)
Extracellular Matrix Proteins/genetics , Membrane Glycoproteins/genetics , Osteogenesis Imperfecta/genetics , Proteoglycans/genetics , Collagen/metabolism , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Consanguinity , Cyclophilins/genetics , DNA Mutational Analysis , Humans , Infant, Newborn , Molecular Chaperones , Osteogenesis Imperfecta/diagnosis , Osteogenesis Imperfecta/physiopathology , Prenatal Diagnosis , Prolyl Hydroxylases
3.
J Biol Chem ; 283(23): 16061-7, 2008 Jun 06.
Article in English | MEDLINE | ID: mdl-18375391

ABSTRACT

Type I procollagen is a heterotrimer composed of two proalpha1(I) chains and one proalpha2(I) chain, encoded by the COL1A1 and COL1A2 genes, respectively. Mutations in these genes usually lead to dominantly inherited forms of osteogenesis imperfecta (OI) by altering the triple helical domains, but a few affect sequences in the proalpha1(I) C-terminal propeptide (C-propeptide), and one, which has a phenotype only in homozygotes, alters the proalpha2(I) C-propeptide. Here we describe four dominant mutations in the COL1A2 gene that alter sequences of the proalpha2(I) C-propeptide in individuals with clinical features of a milder form of the disease, OI type IV. Three of the four appear to interfere with disulfide bonds that stabilize the C-propeptide conformation and its interaction with other chains in the trimer. Cultured cells synthesized proalpha2(I) chains that were slow to assemble with proalpha1(I) chains to form heterotrimers and that were retained intracellularly. Some alterations led to the uncharacteristic formation of proalpha1(I) homotrimers. These findings show that the C-propeptide of proalpha2(I), like that of the proalpha1(I) C-propeptide, is essential for efficient assembly of type I procollagen heterotrimers. The milder OI phenotypes likely reflect a diminished amount of normal type I procollagen, small populations of overmodified heterotrimers, and proalpha1(I) homotrimers that are compatible with normal skeletal growth.


Subject(s)
Bone Development/genetics , Collagen Type I/metabolism , Collagen/metabolism , Mutation , Osteogenesis Imperfecta/metabolism , Adult , Child , Collagen/genetics , Collagen Type I/genetics , Collagen Type I, alpha 1 Chain , Female , Humans , Male , Osteogenesis Imperfecta/genetics , Osteogenesis Imperfecta/pathology , Pedigree , Protein Structure, Quaternary/genetics
4.
Bone ; 41(4): 535-42, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17693149

ABSTRACT

COL27A1 is a member of the collagen fibrillar gene family and is expressed in cartilaginous tissues including the anlage of endochondral bone. To begin to understand its role in skeletogenesis, the temporospatial distributions of its RNA message and protein product, type XXVII collagen, were determined in developing human skeletal tissues. Laser capture microdissection and quantitative reverse-transcription polymerase chain reaction demonstrated that gene expression occurred throughout the growth plate and that it was higher in the resting and proliferative zones than in hypertrophic cartilage. Immunohistochemical analyses showed that type XXVII collagen was most evident in hypertrophic cartilage at the primary ossification center and at the growth plate and that it accumulated in the pericellular matrix. Synthesis of type XXVII collagen overlapped partly with that of type X collagen, a marker of chondrocyte hypertrophy, preceded the transition of cartilage to bone, and was associated with cartilage calcification. Immunogold electron microscopy of extracted ECM components from mouse growth plate showed that type XXVII collagen was a component of long non-banded fibrous structures, filamentous networks, and thin banded fibrils. The timing and location of synthesis suggest that type XXVII collagen plays a role during the calcification of cartilage and the transition of cartilage to bone.


Subject(s)
Bone and Bones/cytology , Bone and Bones/metabolism , Cartilage/cytology , Cartilage/metabolism , Cell Differentiation , Fibrillar Collagens/metabolism , Skeleton , Animals , Fibrillar Collagens/genetics , Humans , Mice , Microscopy, Immunoelectron , RNA, Messenger/genetics
5.
Cell ; 127(2): 291-304, 2006 Oct 20.
Article in English | MEDLINE | ID: mdl-17055431

ABSTRACT

Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.


Subject(s)
Extracellular Matrix Proteins/metabolism , Mutation , Osteogenesis Imperfecta/genetics , Procollagen-Proline Dioxygenase/metabolism , Proteins/metabolism , Amino Acid Sequence , Animals , Bone Diseases, Metabolic/genetics , Bone Diseases, Metabolic/metabolism , Bone Diseases, Metabolic/pathology , Bone and Bones/embryology , Bone and Bones/metabolism , Bone and Bones/pathology , Cells, Cultured , DNA Mutational Analysis , Extracellular Matrix Proteins/genetics , Fibrillar Collagens/metabolism , Fibroblasts/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Chaperones , Molecular Sequence Data , Osteochondrodysplasias/genetics , Osteochondrodysplasias/metabolism , Osteochondrodysplasias/pathology , Osteogenesis Imperfecta/metabolism , Proteins/genetics , RNA, Messenger/metabolism , Time Factors
6.
J Biol Chem ; 281(50): 38507-18, 2006 Dec 15.
Article in English | MEDLINE | ID: mdl-17018525

ABSTRACT

The function of the NH(2)-terminal propeptide of type I procollagen (N-propeptide) is poorly understood. We now show that a recombinant trimeric N-propeptide interacts with transforming growth factor-beta1 and BMP2 and exhibits functional effects in stably transfected cells. The synthesis of N-propeptide by COS-7 cells results in an increase in phosphorylation of Akt and Smad3 and is associated with a marked reduction in type I procollagen synthesis and impairment in adhesion. In C2C12 cells, N-propeptide inhibits the osteoblastic differentiation induced by BMP2. Our data suggest that these effects are mediated by the interaction of N-propeptide with an intracellular receptor in the secretory pathway, because they are not observed when recombinant N-propeptide is added to the culture medium of either COS-7 or C2C12 cells. Both the binding of N-propeptide to cytokines and its functional properties are entirely dependent on the exon 2-encoded globular domain, and a mutation that substitutes a serine for a highly conserved cysteine in exon 2 abolishes its function. Our findings suggest that N-propeptide performs an important feedback regulatory function and provides a rationale for the prominence of a homotrimeric form of type I procollagen (alpha1 trimer) during vertebrate development.


Subject(s)
Collagen Type I/physiology , Animals , Cell Adhesion , Cell Line , Collagen Type I/chemistry , Collagen Type I/genetics , Mice , Mutation , Osteogenesis Imperfecta/genetics , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Smad2 Protein/metabolism
7.
Matrix Biol ; 24(3): 177-84, 2005 May.
Article in English | MEDLINE | ID: mdl-15922909

ABSTRACT

The most recently discovered collagen gene, COL27A1, codes for type XXVII collagen. The COL27A1 gene is strongly expressed in developing cartilage and weakly expressed in many other tissue types. The present study was undertaken to identify transcriptional regulatory mechanisms that govern the expression of COL27A1 in cartilage, and in particular to determine whether SOX9, a key regulator of chondrogenesis, could activate COL27A1. The first intron of COL27A1 was examined to identify sites with homology to the Sox consensus sequence (A)/(T)(A)/(T)CAA(A)/(T)G. Three 50-bp regions that contained paired Sox sites arranged in opposite orientation to each other and separated by 3 or 4 bp were targeted for further analysis. The elements were tested by transient transfection of reporter plasmids, and two of the three elements showed enhancer activity in chondrocytic cells. The same two elements bound SOX9 in electrophoretic mobility shift assays (EMSA). They were not transcriptionally active in fibroblasts, but cotransfection with a SOX9 expression plasmid resulted in activation. The independent mutation of either Sox site in a pair prevented SOX9 binding to the enhancers in EMSA experiments, indicating that SOX9 binds these enhancers only as a dimer. Mutation of either site in a pair also abolished enhancer activity in chondrocytes, indicating that dimeric binding of SOX9 is required for transcriptional activation of the two new enhancers. In summary, these results suggest that SOX9 may play an important role in the transcriptional activation of the newest collagen gene, COL27A1.


Subject(s)
Enhancer Elements, Genetic/physiology , Fibrillar Collagens/genetics , High Mobility Group Proteins/physiology , Transcription Factors/physiology , Animals , Base Sequence , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel , Fibroblasts/metabolism , Gene Expression Regulation , Humans , Introns , Molecular Sequence Data , Mutation , Rats , SOX9 Transcription Factor
8.
Hum Mutat ; 25(4): 348-52, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15776436

ABSTRACT

Marfan Syndrome (MFS) is an autosomal dominant disorder caused by mutations in the fibrillin-1 gene (FBN1). Several calves, all sired by a phenotypically normal bull, were found to exhibit the major clinical and pathological characteristics of human MFS (aortic dissection, joint laxity, lens dislocation), and were recognized as potential models of the human disease. In this study, Fbn1 cDNA from affected animals was sequenced and a heterozygous c.3598G > A transition was detected in exon 29, which predicted the substitution of an evolutionarily conserved glutamic acid by lysine at position 1200 (p.E1200K). This residue is part of a calcium-binding epidermal growth factor-like (cbEGF-like) module, a domain that is frequently altered in human MFS. Analysis of genomic DNA from the original bull's sperm showed that less than 20% of the sperm harbored the mutation, consistent with the presence of germline mosaicism. This study validates the use of these animals as models of human MFS. These cows will be valuable for investigations into the molecular pathogenesis of MFS, and may lead to better therapeutic testing and evaluation of human Marfan patients.


Subject(s)
Calcium/chemistry , Epidermal Growth Factor/chemistry , Marfan Syndrome/genetics , Marfan Syndrome/metabolism , Microfilament Proteins/genetics , Amino Acid Sequence , Animals , Cattle , DNA, Complementary/metabolism , Disease Models, Animal , Fibrillin-1 , Fibrillins , Germ-Line Mutation , Humans , Molecular Sequence Data , Protein Structure, Tertiary
9.
Science ; 303(5661): 1198-201, 2004 Feb 20.
Article in English | MEDLINE | ID: mdl-14976317

ABSTRACT

Adult stem cells offer the potential to treat many diseases through a combination of ex vivo genetic manipulation and autologous transplantation. Mesenchymal stem cells (MSCs, also referred to as marrow stromal cells) are adult stem cells that can be isolated as proliferating, adherent cells from bones. MSCs can differentiate into multiple cell types present in several tissues, including bone, fat, cartilage, and muscle, making them ideal candidates for a variety of cell-based therapies. Here, we have used adeno-associated virus vectors to disrupt dominant-negative mutant COL1A1 collagen genes in MSCs from individuals with the brittle bone disorder osteogenesis imperfecta, demonstrating successful gene targeting in adult human stem cells.


Subject(s)
Collagen Type I/genetics , Gene Targeting , Mesenchymal Stem Cells/physiology , Osteogenesis Imperfecta/genetics , Osteogenesis Imperfecta/therapy , Alleles , Animals , Bone Marrow Cells/physiology , Cell Differentiation , Cells, Cultured , Collagen Type I/chemistry , Collagen Type I/metabolism , Dependovirus/genetics , Genetic Therapy , Genetic Vectors , Humans , Kanamycin Kinase/genetics , Male , Mice , Osteogenesis , Point Mutation , Recombination, Genetic , Stem Cell Transplantation
10.
Matrix Biol ; 22(1): 3-14, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12714037

ABSTRACT

The fibrillar collagens provide structural scaffolding and strength to the extracellular matrices of connective tissues. We identified a partial sequence of a new fibrillar collagen gene in the NCBI databases and completed the sequence with bioinformatic approaches and 5' RACE. This gene, designated COL27A1, is approximately 156 kbp long and has 61 exons located on chromosome 9q32-33. The homologous mouse gene is located on chromosome 4. The gene encodes amino- and carboxyl-terminal propeptides similar to those in the 'minor' fibrillar collagens. The triple-helical domain is, however, shorter and contains 994 amino acids with two imperfections of the Gly-Xaa-Yaa repeat pattern. There were three sites of alternative RNA splicing, only one of which led to the intact mRNA that encodes this full-length collagen proalpha chain. Phylogenetic analyses indicated that COL27A1 forms a clade with COL24A1 that is distinct from the two known lineages of fibrillar collagens. Expression analyses of the mouse col27a1 gene demonstrated high expression in cartilage, the eye and ear, but also in lung and colon. It is likely that the major protein product of COL27A1, proalpha1(XXVII), is a component of the extracellular matrices of cartilage and these other tissues. Study of this collagen should yield insights into normal chondrogenesis, and provide clues to the pathogenesis of some chondrodysplasias and disorders of other tissues in which this gene is expressed.


Subject(s)
Fibrillar Collagens/chemistry , Fibrillar Collagens/genetics , Alternative Splicing/genetics , Amino Acid Motifs , Amino Acid Sequence , Animals , Base Sequence , Evolution, Molecular , Fibrillar Collagens/metabolism , Gene Expression Profiling , Humans , Mice , Molecular Sequence Data , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Nucleic Acid
SELECTION OF CITATIONS
SEARCH DETAIL
...