RESUMO
Collagen V is a regulatory fibril-forming collagen that forms heterotypic fibrils with collagen I. Deletion of collagen V in the mouse is associated with a lack of fibril assembly in the embryonic mesenchyme, with a resultant lethal phenotype. The current work elucidates the regulatory roles of collagen V during development and growth of tissues. A conditional mouse model with a mutation in Col5a1 was developed using a Cre-loxP approach. Col5a1 was ablated in Col5a1(flox/flox) mice using a cornea stroma-specific Kera-Cre driver mouse to produce a bitransgenic Col5a1(Δst/Δst) line that is null for collagen V. This permits analyses of the corneal stroma, a widely used model for studies of collagen V. The collagen-V-knockout stroma demonstrated severe dysfunctional regulation of fibrillogenesis. Fibril diameters were significantly increased, with an abnormal, heterogeneous distribution; fibril structure was abnormal, fibril number was decreased and lamellae were disorganized with decreased stroma thickness. The phenotype was more severe in the anterior versus posterior stroma. Opacity was demonstrated throughout the Col5a1(Δst/Δst) stroma, with significantly increased haze intensity compared with control mice. These data indicate central regulatory roles for collagen V in fibril and matrix assembly during tissue development, with dysfunctional regulation resulting in a functional loss of transparency.
Assuntos
Colágeno Tipo V/metabolismo , Substância Própria/patologia , Regulação da Expressão Gênica no Desenvolvimento , Alelos , Animais , Colágeno Tipo V/genética , Opacidade da Córnea/patologia , Substância Própria/metabolismo , Substância Própria/ultraestrutura , Modelos Animais de Doenças , Feminino , Deleção de Genes , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , FenótipoRESUMO
Collagens V and XI comprise a single regulatory type of fibril-forming collagen with multiple isoforms. Both co-assemble with collagen I or II to form heterotypic fibrils and have been implicated in regulation of fibril assembly. The objective of this study was to determine the roles of collagens V and XI in the regulation of tendon fibrillogenesis. Flexor digitorum longus tendons from a haplo-insufficient collagen V mouse model of classic Ehlers Danlos syndrome (EDS) had decreased biomechanical stiffness compared with controls consistent with joint laxity in EDS patients. However, fibril structure was relatively normal, an unexpected finding given the altered fibrils observed in dermis and cornea from this model. This suggested roles for other related molecules, i.e. collagen XI, and compound Col5a1(+/-),Col11a1(+/-) tendons had altered fibril structures, supporting a role for collagen XI. To further evaluate this, transcript expression was analyzed in wild type tendons. During development (E18-P10) both collagen V and XI were comparably expressed; however, collagen V predominated in mature (P30) tendons. The collagens had a similar expression pattern. Tendons with altered collagen V and/or XI expression (Col5a1(+/-); Col11a1(+/-); Col5a1(+/-),Col11a1(+/-); Col11a1(-/-); Col5a1(+/-),Col11a1(-/-)) were analyzed at E18. All genotypes demonstrated a reduced fibril number and altered structure. This phenotype was more severe with a reduction in collagen XI. However, the absence of collagen XI with a reduction in collagen V was associated with the most severe fibril phenotype. The data demonstrate coordinate roles for collagens V and XI in the regulation of fibril nucleation and assembly during tendon development.
Assuntos
Colágeno Tipo V/metabolismo , Colágeno Tipo XI/metabolismo , Síndrome de Ehlers-Danlos/metabolismo , Tendões/crescimento & desenvolvimento , Tendões/metabolismo , Animais , Colágeno Tipo V/genética , Colágeno Tipo V/ultraestrutura , Colágeno Tipo XI/genética , Colágeno Tipo XI/ultraestrutura , Modelos Animais de Doenças , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/patologia , Humanos , Camundongos , Camundongos KnockoutRESUMO
Genetic mutations in minor fibrillar collagen types Va1 (ColVa1) and XIa1 (ColXI) have been identified in connective tissue disorders including Ehlers-Danlos syndrome and chondrodysplasias. ColVa1+/- and ColXIa1-/- mutant mice recapitulate these human disorders and show aberrations in collagen fiber organization in connective tissue of the skin, cornea, cartilage, and tendon. In the heart, fibrous networks of collagen fibers form throughout the ventricular myocardium and heart valves, and alterations in collagen fiber homeostasis are apparent in many forms of cardiac disease associated with myocardial dysfunction and valvular insufficiency. There is increasing evidence for cardiac dysfunction in connective tissue disorders, but the mechanisms have not been addressed. ColVa1+/- and ColXIa1-/- mutant mice were used to identify roles for ColVa1 and ColXIa1 in ventricular myocardial morphogenesis and heart valve development. These affected cardiac structures show a compensatory increase in type I collagen deposition, similar to that previously described in valvular and cardiomyopathic disease. Morphological cardiac defects associated with changes in collagen fiber homeostasis identified in ColVa1+/- and ColXIa1-/- mice provide an insight into previously unappreciated forms of cardiac dysfunction associated with connective tissue disorders.
Assuntos
Colágeno Tipo IX/metabolismo , Colágeno Tipo V/metabolismo , Coração Fetal/embriologia , Coração Fetal/metabolismo , Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Animais , Sequência de Bases , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Colágeno Tipo IX/deficiência , Colágeno Tipo IX/genética , Colágeno Tipo V/deficiência , Colágeno Tipo V/genética , Primers do DNA/genética , Modelos Animais de Doenças , Síndrome de Ehlers-Danlos/embriologia , Síndrome de Ehlers-Danlos/genética , Exostose Múltipla Hereditária/embriologia , Exostose Múltipla Hereditária/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , GravidezRESUMO
The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proalpha1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS.
Assuntos
Colágeno Tipo V/genética , Colágeno Tipo V/fisiologia , Síndrome de Ehlers-Danlos/genética , Alelos , Animais , Fenômenos Biomecânicos , Colágeno/química , Colágeno/metabolismo , Derme/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Heterozigoto , Camundongos , Camundongos Transgênicos , Modelos Biológicos , MutaçãoRESUMO
Hammerhead ribozymes are small catalytic RNA molecules that can be targeted to any RNA molecule containing a putative cleavage site. We developed a vector (pCOLZ) that uses the COL1A1 promoter to drive expression of a self-cleaving multimeric ribozyme (M8Rz547) and its monomeric counterpart (Rz547). The ribozymes were stably coexpressed in MC3T3-E1 osteoblasts expressing a truncated COL1A1 target transcript. The multimeric ribozyme exhibited self-cleavage to derivative fragments, including monomers. Increased expression of ribozymes was found in cells expressing the multimeric ribozyme. A modest reduction of truncated target transcript and protein was seen in cells expressing the ribozyme monomer, while nearly complete ablation of target transcript and protein occurred in cells expressing the ribozyme multimer. A reversion to a more normal collagen phenotype, measured as an increase in fibril diameter and restored fibrillar architecture, and a decreased rate of collagen turnover were seen in cells expressing the ribozyme multimer.
Assuntos
Colágeno Tipo I/metabolismo , RNA Catalítico/fisiologia , Animais , Sequência de Bases , Bovinos , Linhagem Celular , Colágeno Tipo I/genética , Colágeno Tipo I/ultraestrutura , Cadeia alfa 1 do Colágeno Tipo I , Camundongos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutação , Osteoblastos/metabolismo , Regiões Promotoras GenéticasRESUMO
Vertebrate collagen fibrils are heterotypically composed of a quantitatively major and minor fibril collagen. In non-cartilaginous tissues, type I collagen accounts for the majority of the collagen mass, and collagen type V, the functions of which are poorly understood, is a minor component. Type V collagen has been implicated in the regulation of fibril diameter, and we reported recently preliminary evidence that type V collagen is required for collagen fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113-124). The purpose of this study was to define the roles of type V collagen in the regulation of collagen fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-alpha1(V) chains were created by homologous recombination. The col5a1-/- animals die in early embryogenesis, at approximately embryonic day 10. The type V collagen-deficient mice demonstrate a virtual lack of collagen fibril formation. In contrast, the col5a1+/- animals are viable. The reduced type V collagen content is associated with a 50% reduction in fibril number and dermal collagen content. In addition, relatively normal, cylindrical fibrils are assembled with a second population of large, structurally abnormal collagen fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V collagen in the regulation of fibrillogenesis. The complete dependence of fibril formation on type V collagen is indicative of the critical role of the latter in early fibril initiation. In addition, this fibril collagen is important in the determination of fibril structure and matrix organization.
Assuntos
Colágeno Tipo V/fisiologia , Animais , Western Blotting , Técnicas de Cultura de Células , Colágeno/química , Colágeno/metabolismo , Colágeno Tipo V/química , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Embrião de Mamíferos/metabolismo , Éxons , Matriz Extracelular/metabolismo , Vetores Genéticos , Genótipo , Hibridização In Situ , Camundongos , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Modelos Genéticos , Pele/metabolismo , Fatores de TempoRESUMO
To examine mechanisms by which reduced type V collagen causes weakened connective tissues in the Ehlers-Danlos syndrome (EDS), we examined matrix deposition and collagen fibril morphology in long-term dermal fibroblast cultures. EDS cells with COL5A1 haplo-insufficiency deposited less than one-half of hydroxyproline as collagen compared to control fibroblasts, though total collagen synthesis rates are near-normal because type V collagen represents a small fraction of collagen synthesized. Cells from patients with osteogenesis imperfecta (OI) and haplo-insufficiency for proalpha1(I) chains of type I collagen also incorporated about one-half the collagen as controls, but this amount was proportional to their reduced rates of total collagen synthesis. Collagen fibril diameter was inversely proportional to type V/type I collagen ratios (EDS > control > OI). However, a reduction of type V collagen, in the EDS derived cells, was associated with the assembly of significantly fewer fibrils compared to control and OI cells. These data indicate that in cell culture, the quantity of collagen fibrils deposited in matrix is highly sensitive to reduction in type V collagen, far out of proportion to type V collagen's contribution to collagen mass.