RESUMEN
Osteogenesis imperfecta (OI) type V is caused by an autosomal dominant mutation in the IFITM5 gene, also known as BRIL. The c.-14C>T mutation in the 5'UTR of BRIL creates a novel translational start site adding 5 residues (MALEP) in frame with the natural coding of BRIL. A neomorphic function has been proposed for the MALEP-BRIL but the mechanisms at play are still unknown. In order to further understand the effects of MALEP-BRIL in vivo, we generated a knockin (KI) mouse model having the exact genetic -14C>T replica of patients with OI type V. Live KI descendants were never obtained from 2 male mosaic founders. Skeletal staining with alizarin red/alcian blue and µCT imaging of KI embryos revealed striking skeletal anomalies such as hypomineralized skull, short and bent long bones, and frail and wavy ribs. Histology and histochemical labeling revealed that midshaft of long bones was filled with hypertrophic chondrocytes, lacked a defined primary ossification center with the absence of defined cortices. Gene expression monitoring at E15.5 and E17.5 showed no change in Osx but decreased Bril itself as well as other differentiated osteoblast markers (Ibsp, Bglap, Sost). However, upregulation of Ptgs2 and Nr4a3 suggested that a pro-inflammatory reaction was activated. Primary osteoblasts from KI calvaria showed delayed differentiation and mineralization, with decreased abundance of BRIL. However, the upregulation AdipoQ and Fabp4 in young cultures indicated a possible switch in fate towards adipogenesis. Altogether our data suggest that the low level expression of MALEP-BRIL in Osx+ mesenchymal progenitors blunted their further differentiation into mature osteoblasts, which may have resulted in part from an inflammatory response.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas de la Membrana/genética , Osteoblastos/patología , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/patología , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas , Diferenciación Celular/genética , Edición Génica/métodos , Técnicas de Sustitución del Gen , Inflamación/genética , Inflamación/patología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Mutación , Osteoblastos/metabolismo , Osteogénesis/genéticaRESUMEN
BACKGROUND: In vitro studies suggest that the multiple functions of decorin are related to both its core protein and its dermatan sulfate chain. To determine the contribution of the dermatan sulfate chain to the functional properties of decorin in vivo, a mutant mouse whose decorin lacked a dermatan sulfate chain was generated. RESULTS: Homozygous mice expressing only the decorin core protein developed and grew in a similar manner to wild type mice. In both embryonic and postnatal mice, all connective tissues studied, including cartilage, skin and cornea, appeared to be normal upon histological examination, and their collagen fibrils were of normal diameter and organization. In addition, abdominal skin wounds healed in an identical manner in the mutant and wild type mice. CONCLUSIONS: The absence of a dermatan sulfate chain on decorin does not appear to overtly influence its functional properties in vivo.
Asunto(s)
Decorina/metabolismo , Dermatán Sulfato/metabolismo , Desarrollo Embrionario , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Secuencia de Bases , Cartílago/patología , Cartílago/ultraestructura , Decorina/química , Decorina/genética , Técnicas de Sustitución del Gen , Homocigoto , Ratones Endogámicos C57BL , Cicatrización de HeridasRESUMEN
STUDY DESIGN: Histologic analysis of spine development in cartilage-specific knockout mice. OBJECTIVE: To evaluate the role hyaluronan produced by hyaluronan synthase-2 (Has2) in spine development. SUMMARY OF BACKGROUND DATA: The Has2 gene is responsible for most hyaluronan production throughout the body, including the skeleton. However, it is not possible to study the involvement of hyaluronan in skeletal development using constitutive Has2 knockout mice, as the embryonic mice die early before skeletal development has occurred. This problem can be overcome by the use of cartilage-specific knockout mice. METHODS: Mice possessing floxed Has2 genes were crossed with mice expressing Cre recombinase under control of the type II collagen promoter to generate cartilage-specific Has2 knockout mice. Spine development was studied by histology. RESULTS: Knockout mice died near birth and displayed severe abnormality in skeletal development. The spine showed defects in vertebral body size and the formation of the intervertebral discs. There was no evidence for the formation of an organized primary center of ossification within the vertebrae, and the appearance and organization of the hypertrophic chondrocytes was abnormal. Although no organized endochondral ossification appeared to be taking place, there was excessive bone formation at the center of the vertebrae. There was also a generalized increased cellularity of the vertebral cartilage and a corresponding decrease in the abundance of extracellular matrix. The nucleus pulposus of the intervertebral discs were less flattened than in the control mice and possessed an increased amount of large vacuolated cells. Remnants of the notochord could also be seen between adjacent discs. CONCLUSION: Hyaluronan production by Has2 is essential for normal vertebral and intervertebral disc development within the spine, and the absence of this synthase impairs the organization of both soft and hard tissue elements.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Glucuronosiltransferasa/genética , Ácido Hialurónico/fisiología , Columna Vertebral/metabolismo , Agrecanos/biosíntesis , Animales , Huesos/anomalías , Huesos/metabolismo , Cartílago/metabolismo , Colágeno Tipo II/genética , Matriz Extracelular/metabolismo , Femenino , Glucuronosiltransferasa/metabolismo , Hialuronano Sintasas , Ácido Hialurónico/biosíntesis , Inmunohistoquímica , Integrasas/genética , Integrasas/metabolismo , Disco Intervertebral/embriología , Disco Intervertebral/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Columna Vertebral/embriología , Factores de TiempoRESUMEN
In long bone development, the evolution of the cartilaginous anlagen into a secondary ossification center is initiated by the formation of canals. The excavation to create the canals is achieved through lysis of the two major cartilage components, aggrecan, and the type II collagen (COL2) fibril. The present study examines the lysis of the fibril. Because it is known that matrix metalloproteinases (MMPs) cleave COL2 in vitro at the Gly(775)-Leu(776) bond, it has been reasoned that, if such cleavage is detected in relation to the canals, it can be concluded that a collagenase is involved. Furthermore, because MMPs undergo change in domain structure with activation resulting in propeptide domain loss then, if such a loss is revealed in relation to the cleavage of COL2, this MMP is likely involved. The collective findings reveal that COL2 is attacked at the afore-described susceptible peptide bond at the surface of cartilage canals and, that MMP-13 cleaves it. Developmental Dynamics 238:1547-1563, 2009. (c) 2009 Wiley-Liss, Inc.
Asunto(s)
Colágeno Tipo II/metabolismo , Colagenasas/metabolismo , Epífisis , Epítopos , Agrecanos/metabolismo , Animales , Anticuerpos/metabolismo , Colágeno Tipo II/genética , Activación Enzimática , Epífisis/anatomía & histología , Epífisis/embriología , Isoenzimas/genética , Isoenzimas/metabolismo , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Precursores de Proteínas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.