RESUMO
Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y-Neo Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y-Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS.
Assuntos
Cartilagem/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Osteocondrodisplasias/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Cartilagem/crescimento & desenvolvimento , Cartilagem/ultraestrutura , Moléculas de Adesão Celular/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Cromatografia Líquida , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/patologia , Ontologia Genética , Glicosaminoglicanos/metabolismo , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/genética , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Osteoartrite/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteocondrodisplasias/genética , Espectrometria de Massas em TandemRESUMO
Perlecan/Hspg2, a large monomeric heparan sulfate proteoglycan, is found in the basement membrane and extracellular matrix, where it acts as a matrix scaffold, growth factor depot, and tissue barrier. Perlecan deficiency leads to skeletal dysplasia in Schwartz-Jampel Syndrome (SJS) and is a risk factor for osteoporosis. In the SJS-mimicking murine model (Hypo), inferior cortical bone quality and impaired mechanotransduction in osteocytes were reported. This study focused on trabecular bone, where perlecan deficiency was hypothesized to result in structural deficit and altered response to disuse and re-loading. We compared the Hypo versus WT trabecular bone in both axial and appendicular skeletons of 8-38-week-old male mice, and observed severe trabecular deficit in Hypo mice, approximately 50% reduction of Tb.BV/TV regardless of skeletal site and animal age. Defects in endochondral ossification (e.g., accelerated mineralization), increases in osteoclast activity, and altered differentiation of bone progenitor cells in marrow contributed to the Hypo phenotype. The Hypo trabecular bone deteriorated further under three-week hindlimb suspension as did the WT. Re-ambulation partially recovered the lost trabecular bone in Hypo, but not in WT mice. The novel finding that low-impact loading could counter detrimental disuse effects in the perlecan-deficient skeleton suggests a strategy to maintain skeletal health in SJS patients.
Assuntos
Osso Esponjoso/patologia , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/genética , Osteócitos/citologia , Animais , Fêmur/patologia , Células-Tronco Hematopoéticas/citologia , Proteoglicanas de Heparan Sulfato/fisiologia , Cifose , Masculino , Mecanotransdução Celular , Metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteogênese , Fenótipo , Fatores de Risco , Estresse Mecânico , Caminhada , Microtomografia por Raio-XRESUMO
Islet amyloid is a pathologic feature of type 2 diabetes (T2D) that is associated with ß-cell loss and dysfunction. These amyloid deposits form via aggregation of the ß-cell secretory product islet amyloid polypeptide (IAPP) and contain other molecules including the heparan sulfate proteoglycan perlecan. Perlecan has been shown to bind amyloidogenic human IAPP (hIAPP) via its heparan sulfate glycosaminoglycan (HS GAG) chains and to enhance hIAPP aggregation in vitro. We postulated that reducing the HS GAG content of perlecan would also decrease islet amyloid deposition in vivo. hIAPP transgenic mice were crossed with Hspg2Δ3/Δ3 mice harboring a perlecan mutation that prevents HS GAG attachment (hIAPP;Hspg2Δ3/Δ3), and male offspring from this cross were fed a high fat diet for 12 months to induce islet amyloid deposition. At the end of the study body weight, islet amyloid area, ß-cell area, glucose tolerance and insulin secretion were analyzed. hIAPP;Hspg2Δ3/Δ3 mice exhibited significantly less islet amyloid deposition and greater ß-cell area compared to hIAPP mice expressing wild type perlecan. hIAPP;Hspg2Δ3/Δ3 mice also gained significantly less weight than other genotypes. When adjusted for differences in body weight using multiple linear regression modeling, we found no differences in islet amyloid deposition or ß-cell area between hIAPP transgenic and hIAPP;Hspg2Δ3/Δ3 mice. We conclude that loss of perlecan exon 3 reduces islet amyloid deposition in vivo through indirect effects on body weight and possibly also through direct effects on hIAPP aggregation. Both of these mechanisms may promote maintenance of glucose homeostasis in the setting of T2D.
Assuntos
Peso Corporal , Proteoglicanas de Heparan Sulfato/deficiência , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Animais , Contagem de Células , Humanos , Camundongos , Camundongos TransgênicosRESUMO
We investigated the role of glycosaminoglycans (GAGs) in the regulation of endothelial nitric oxide synthase (eNOS) activity in wild-type CHO-K1 cells and in xylosyltransferase-deficient CHO-745 cells. GAGs inhibit the integrin/FAK/PI3K/AKT signaling pathway in CHO-K1 cells, decreasing the phosphorylation of eNOS at Ser1177. Furthermore, in CHO-K1 cells, eNOS and PKCα are localized at sphingolipid- and cholesterol-rich domains in the plasma membrane called caveolae. At caveolae, PKCα activation stimulates the phosphorylation of eNOS on Thr495, resulting in further inhibition of NO production in these cells. In our data, CHO-745 cells generate approximately 12-fold more NO than CHO-K1 cells. Increased NO production in CHO-745 cells promotes higher rates of protein S-nitrosylation and protein tyrosine nitration. Regarding reactive oxygen species (ROS) production, CHO-745 cells show lower basal levels of superoxide (O2- ) than CHO-K1 cells. In addition, CHO-745 cells express higher levels of GPx, Trx1, and catalase than CHO-K1 cells, suggesting that CHO-745 cells are in a constitutive nitrosative/oxidative stress condition. Accordingly, we showed that CHO-745 cells are more sensitive to oxidant-induced cell death than CHO-K1 cells. The high concentration of NO and reactive oxygen species generated by CHO-745 cells can induce simultaneous mitochondrial biogenesis and antioxidant gene expression. These observations led us to propose that GAGs are part of a regulatory mechanism that participates in eNOS activation and consequently regulates nitrosative/oxidative stress in CHO cells.
Assuntos
Proteoglicanas de Heparan Sulfato/deficiência , Espaço Intracelular/metabolismo , Óxido Nítrico/biossíntese , Regulação para Cima , Animais , Células CHO , Cricetinae , Cricetulus , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Oligopeptídeos/metabolismo , Biogênese de Organelas , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Frações Subcelulares/metabolismoRESUMO
The pericellular matrix (PCM) is a component of the extracellular matrix that is found immediately surrounding individual chondrocytes in developing and adult cartilage, and is rich in the proteoglycan perlecan. Mutations in perlecan are the basis of several developmental disorders, which are thought to arise from disruptions in the mechanical stability of the PCM. We tested the hypothesis that defects in PCM organization will reduce the stiffness of chondrocytes in developing cartilage by combining a murine model of Schwartz-Jampel syndrome, in which perlecan is knocked down, with our novel atomic force microscopy technique that can measure the stiffness of living cells and surrounding matrix in embryonic and postnatal tissues in situ. Perlecan knockdown altered matrix organization and significantly decreased the stiffness of both chondrocytes and interstitial matrix as a function of age and genotype. Our results demonstrate that the knockdown of a spatially restricted matrix molecule can have a profound influence on cell and tissue stiffness, implicating a role for outside-in mechanical signals from the PCM in regulating the intracellular mechanisms required for the overall development of cartilage.
Assuntos
Cartilagem/fisiopatologia , Proteínas da Matriz Extracelular/deficiência , Proteoglicanas de Heparan Sulfato/deficiência , Animais , Fenômenos Biomecânicos , Cartilagem/crescimento & desenvolvimento , Cartilagem/patologia , Condrócitos/patologia , Condrócitos/fisiologia , Modelos Animais de Doenças , Matriz Extracelular/patologia , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Microscopia de Força Atômica , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Osteocondrodisplasias/fisiopatologia , GravidezRESUMO
The autophagy-lysosome system is essential for muscle protein synthesis and degradation equilibrium, and its dysfunction has been linked to various muscle disorders. It has been reported that a diverse collection of extracellular matrix constituents, including decorin, collagen VI, laminin α2, endorepellin, and endostatin, can modulate autophagic signaling pathways. However, the association between autophagy and perlecan in muscle homeostasis remains unclear. The mechanical unloading of perlecan-deficient soleus muscles resulted in significantly decreased wet weights and cross-section fiber area compared with those of control mice. We found that perlecan deficiency in slow-twitch soleus muscles enhanced autophagic activity. This was accompanied by a decrease in autophagic substrates, such as p62, and an increase in LC3II levels. Furthermore, perlecan deficiency caused a reduction in the phosphorylation levels of p70S6k and Akt and increased the phosphorylation of AMPKα. Our findings suggested that perlecan inhibits the autophagic process through the activation of the mTORC1 pathway. This autophagic response may be a novel target for enhancing the efficacy of skeletal muscle atrophy treatment.
Assuntos
Autofagia/genética , Proteoglicanas de Heparan Sulfato/genética , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteoglicanas de Heparan Sulfato/deficiência , Homeostase/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fibras Musculares de Contração Rápida/patologia , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , TenotomiaRESUMO
Perlecan/HSPG2 (Pln) is a large heparan sulfate proteoglycan abundant in the extracellular matrix of cartilage and the lacunocanalicular space of adult bones. Although Pln function during cartilage development is critical, evidenced by deficiency disorders including Schwartz-Jampel Syndrome and dyssegmental dysplasia Silverman-Handmaker type, little is known about its function in development of bone shape and quality. The purpose of this study was to understand the contribution of Pln to bone geometric and mechanical properties. We used hypomorph mutant mice that secrete negligible amount of Pln into skeletal tissues and analyzed their adult bone properties using micro-computed tomography and three-point-bending tests. Bone shortening and widening in Pln mutants was observed and could be attributed to loss of growth plate organization and accelerated osteogenesis that was reflected by elevated cortical thickness at older ages. This effect was more pronounced in Pln mutant females, indicating a sex-specific effect of Pln deficiency on bone geometry. Additionally, mutant females, and to a lesser extent mutant males, increased their elastic modulus and bone mineral densities to counteract changes in bone shape, but at the expense of increased brittleness. In summary, Pln deficiency alters cartilage matrix patterning and, as we now show, coordinately influences bone formation and calcification.
Assuntos
Desenvolvimento Ósseo/fisiologia , Proteoglicanas de Heparan Sulfato/deficiência , Osteogênese/fisiologia , Envelhecimento , Animais , Osso e Ossos , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Confocal , Microtomografia por Raio-XRESUMO
Perlecan is a heparan sulfate proteoglycan assembled into the vascular basement membranes (BMs) during vasculogenesis. In the present study we have investigated vessel formation in mice, teratomas and embryoid bodies (EBs) in the absence of perlecan. We found that perlecan was dispensable for blood vessel formation and maturation until embryonic day (E) 12.5. At later stages of development 40% of mutant embryos showed dilated microvessels in brain and skin, which ruptured and led to severe bleedings. Surprisingly, teratomas derived from perlecan-null ES cells showed efficient contribution of perlecan-deficient endothelial cells to an apparently normal tumor vasculature. However, in perlecan-deficient EBs the area occupied by an endothelial network and the number of vessel branches were significantly diminished. Addition of FGF-2 but not VEGF(165) rescued the in vitro deficiency of the mutant ES cells. Furthermore, in the absence of perlecan in the EB matrix lower levels of FGFs are bound, stored and available for cell surface presentation. Altogether these findings suggest that perlecan supports the maintenance of brain and skin subendothelial BMs and promotes vasculo- and angiogenesis by modulating FGF-2 function.
Assuntos
Proteoglicanas de Heparan Sulfato/fisiologia , Microvasos/embriologia , Microvasos/fisiologia , Neovascularização Fisiológica , Animais , Membrana Basal/irrigação sanguínea , Membrana Basal/embriologia , Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Corpos Embrioides/citologia , Corpos Embrioides/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Feminino , Fator 2 de Crescimento de Fibroblastos/fisiologia , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/ultraestrutura , Neovascularização Patológica , Gravidez , Pele/irrigação sanguínea , Pele/embriologia , Teratoma/irrigação sanguíneaRESUMO
Congenital peripheral nerve hyperexcitability (PNH) is usually associated with impaired function of voltage-gated K(+) channels (VGKCs) in neuromyotonia and demyelination in peripheral neuropathies. Schwartz-Jampel syndrome (SJS) is a form of PNH that is due to hypomorphic mutations of perlecan, the major proteoglycan of basement membranes. Schwann cell basement membrane and its cell receptors are critical for the myelination and organization of the nodes of Ranvier. We therefore studied a mouse model of SJS to determine whether a role for perlecan in these functions could account for PNH when perlecan is lacking. We revealed a role for perlecan in the longitudinal elongation and organization of myelinating Schwann cells because perlecan-deficient mice had shorter internodes, more numerous Schmidt-Lanterman incisures, and increased amounts of internodal fast VGKCs. Perlecan-deficient mice did not display demyelination events along the nerve trunk but developed dysmyelination of the preterminal segment associated with denervation processes at the neuromuscular junction. Investigating the excitability properties of the peripheral nerve suggested a persistent axonal depolarization during nerve firing in vitro, most likely due to defective K(+) homeostasis, and excluded the nerve trunk as the original site for PNH. Altogether, our data shed light on perlecan function by revealing critical roles in Schwann cell physiology and suggest that PNH in SJS originates distally from synergistic actions of peripheral nerve and neuromuscular junction changes.
Assuntos
Axônios/fisiologia , Proteoglicanas de Heparan Sulfato/fisiologia , Osteocondrodisplasias/patologia , Células de Schwann/fisiologia , Potenciais de Ação/fisiologia , Envelhecimento/fisiologia , Animais , Membrana Basal/metabolismo , Doenças Desmielinizantes/etiologia , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/genética , Canal de Potássio Kv1.1/biossíntese , Camundongos , Camundongos Mutantes , Microscopia Eletrônica , Mutação , Bainha de Mielina/fisiologia , Bainha de Mielina/ultraestrutura , Junção Neuromuscular/fisiopatologia , Osteocondrodisplasias/complicações , Osteocondrodisplasias/fisiopatologia , Nós Neurofibrosos/metabolismo , Nós Neurofibrosos/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células de Schwann/metabolismo , Nervo Isquiático/fisiopatologia , Nervo Isquiático/ultraestruturaRESUMO
Multiple Osteochondromas (MO; previously known as multiple hereditary exostosis) is an autosomal dominant genetic condition that is characterized by the formation of cartilaginous bone tumours (osteochondromas) at multiple sites in the skeleton, secondary bursa formation and impingement of nerves, tendons and vessels, bone curving, and short stature. MO is also known to be associated with arthritis, general pain, scarring and occasional malignant transformation of osteochondroma into secondary peripheral chondrosarcoma. MO patients present additional complains but the relevance of those in relation to the syndromal background needs validation. Mutations in two enzymes that are required during heparan sulphate synthesis (EXT1 or EXT2) are known to cause MO. Previously, we have used zebrafish which harbour mutations in ext2 as a model for MO and shown that ext2â»/â» fish have skeletal defects that resemble those seen in osteochondromas. Here we analyse dental defects present in ext2â»/â» fish. Histological analysis reveals that ext2â»/â» fish have very severe defects associated with the formation and the morphology of teeth. At 5 days post fertilization 100% of ext2â»/â» fish have a single tooth at the end of the 5(th) pharyngeal arch, whereas wild-type fish develop three teeth, located in the middle of the pharyngeal arch. ext2â»/â» teeth have abnormal morphology (they were shorter and thicker than in the WT) and patchy ossification at the tooth base. Deformities such as split crowns and enamel lesions were found in 20% of ext2âº/â» adults. The tooth morphology in ext2â»/â» was partially rescued by FGF8 administered locally (bead implants). Our findings from zebrafish model were validated in a dental survey that was conducted with assistance of the MHE Research Foundation. The presence of the malformed and/or displaced teeth with abnormal enamel was declared by half of the respondents indicating that MO might indeed be also associated with dental problems.
Assuntos
Exostose Múltipla Hereditária/patologia , Proteoglicanas de Heparan Sulfato/deficiência , Doenças Dentárias/patologia , Peixe-Zebra/metabolismo , Adulto , Envelhecimento/patologia , Animais , Biomarcadores/metabolismo , Exostose Múltipla Hereditária/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Larva , Mutação/genética , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/metabolismo , Fenótipo , Transdução de Sinais , Dente/crescimento & desenvolvimento , Dente/metabolismo , Dente/patologia , Doenças Dentárias/genética , Peixe-Zebra/genéticaRESUMO
Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder in which the aggregation and deposition of amyloid-ß (Aß) peptides in the brain are central to its pathogenesis. In healthy brains, Aß is effectively metabolized with little accumulation. Cellular uptake and subsequent degradation of Aß is one of the major pathways for its clearance in the brain. Increasing evidence has demonstrated significant roles for the low-density lipoprotein receptor-related protein 1 (LRP1) in the metabolism of Aß in neurons, glia cells, and along the brain vasculatures. Heparan sulfate proteoglycan (HSPG) has also been implicated in several pathogenic features of AD, including its colocalization with amyloid plaques. Here, we demonstrate that HSPG and LRP1 cooperatively mediate cellular Aß uptake. Fluorescence-activated cell sorter and confocal microscopy revealed that knockdown of LRP1 suppresses Aß uptake, whereas overexpression of LRP1 enhances this process in neuronal cells. Heparin, which antagonizes HSPG, significantly inhibited cellular Aß uptake. Importantly, treatment with heparin or heparinase blocked LRP1-mediated cellular uptake of Aß. We further showed that HSPG is more important for the binding of Aß to the cell surface than LRP1. The critical roles of HSPG in cellular Aß binding and uptake were confirmed in Chinese hamster ovary cells genetically deficient in HSPG. We also showed that heparin and a neutralizing antibody to LRP1 suppressed Aß uptake in primary neurons. Our findings demonstrate that LRP1 and HSPG function in a cooperative manner to mediate cellular Aß uptake and define a major pathway through which Aß gains entry to neuronal cells.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Fibroblastos/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de LDL/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Transporte Biológico , Western Blotting , Células CHO , Linhagem Celular , Células Cultivadas , Cricetinae , Cricetulus , Embrião de Mamíferos , Endocitose/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Proteoglicanas de Heparan Sulfato/antagonistas & inibidores , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/genética , Heparina/farmacologia , Hipotálamo/citologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Microscopia Confocal , Neurônios/efeitos dos fármacos , Gravidez , RNA Interferente Pequeno , Receptores de LDL/genética , Transfecção , Proteínas Supressoras de Tumor/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Schwartz-Jampel syndrome (SJS) is an autosomal-recessive condition characterized by muscle stiffness and chondrodysplasia. It is due to loss-of-function hypomorphic mutations in the HSPG2 gene that encodes for perlecan, a proteoglycan secreted into the basement membrane. The origin of muscle stiffness in SJS is debated. To resolve this issue, we performed an electrophysiological investigation of an SJS mouse model with a missense mutation in the HSPG2 gene. Compound muscle action potential amplitudes, distal motor latencies, repetitive nerve stimulation tests, and sensory nerve conduction velocities of SJS mice were normal. On electromyography (EMG), neuromyotonic discharges, that is, bursts of motor unit action potentials firing at high rates (120-300 HZ), were constantly observed in SJS mice in all muscles, except in the diaphragm. Neuromyotonic discharges were not influenced by general anesthesia and disappeared with curare administration. They persisted after complete motor nerve section, terminating only with Wallerian degeneration. These results demonstrate that perlecan deficiency in SJS provokes a neuromyotonic syndrome. The findings further suggest a distal axonal localization of the generator of neuromyotonic discharges. SJS should now be considered as an inherited disorder with peripheral nerve hyperexcitability.
Assuntos
Fibras Musculares Esqueléticas/fisiologia , Osteocondrodisplasias/patologia , Osteocondrodisplasias/fisiopatologia , Nervos Periféricos/fisiopatologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Fenômenos Biofísicos , Curare/farmacologia , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Eletromiografia/métodos , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/genética , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , Condução Nervosa/efeitos dos fármacos , Condução Nervosa/genética , Condução Nervosa/fisiologia , Fármacos Neuromusculares não Despolarizantes/farmacologia , Osteocondrodisplasias/genética , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologiaRESUMO
Schwartz-Jampel syndrome (SJS) is a recessive neuromyotonia with chondrodysplasia. It results from hypomorphic mutations of the gene encoding perlecan, leading to a decrease in the levels of this heparan sulphate proteoglycan in basement membranes (BMs). It has been suggested that SJS neuromyotonia may result from endplate acetylcholinesterase (AChE) deficiency, but this hypothesis has never been investigated in vivo due to the lack of an animal model for neuromyotonia. We used homologous recombination to generate a knock-in mouse strain with one missense substitution, corresponding to a human familial SJS mutation (p.C1532Y), in the perlecan gene. We derived two lines, one with the p.C1532Y substitution alone and one with p.C1532Y and the selectable marker Neo, to down-regulate perlecan gene activity and to test for a dosage effect of perlecan in mammals. These two lines mimicked SJS neuromyotonia with spontaneous activity on electromyogramm (EMG). An inverse correlation between disease severity and perlecan secretion in the BMs was observed at the macroscopic and microscopic levels, consistent with a dosage effect. Endplate AChE levels were low in both lines, due to synaptic perlecan deficiency rather than major myofibre or neuromuscular junction disorganization. Studies of muscle contractile properties showed muscle fatigability at low frequencies of nerve stimulation and suggested that partial endplate AChE deficiency might contribute to SJS muscle stiffness by potentiating muscle force. However, physiological endplate AChE deficiency was not associated with spontaneous activity at rest on EMG in the diaphragm, suggesting that additional changes are required to generate such activity characteristic of SJS.
Assuntos
Acetilcolinesterase/deficiência , Acetilcolinesterase/genética , Síndrome de Isaacs/enzimologia , Síndrome de Isaacs/genética , Placa Motora/enzimologia , Osteocondrodisplasias/enzimologia , Osteocondrodisplasias/genética , Alelos , Animais , Modelos Animais de Doenças , Eletromiografia , Feminino , Dosagem de Genes , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/genética , Humanos , Síndrome de Isaacs/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Placa Motora/fisiopatologia , Contração Muscular/genética , Contração Muscular/fisiologia , Mutação de Sentido Incorreto , Osteocondrodisplasias/fisiopatologia , FenótipoRESUMO
Schwartz-Jampel syndrome (SJS) is a rare autosomal recessive disorder characterized by the association of myotonia with chondrodysplasia. A positional cloning strategy allowed the localization and the identification of perlecan as the disease causing gene. Another human recessive disorder, the Dyssegmental Dysplasia, Silverman-Handmaker type (DDSH), is caused by functional null mutations of the perlecan gene. A gene-dosage effect seems to account for the correlation between the phenotype and the mutations within the gene: SJS would be associated with hypomorph mutations of the perlecan gene and DDSH would be due to the absence of functional perlecan. Perlecan is the major heparan sulfate proteoglycan of extracellular matrix and basement membranes that displays various functions. Based on these functions, several hypotheses are evoked to explain chondrodysplasia and the unusual myotonia in SJS. Mouse models are invaluable tools to explore these hypotheses. Since knock-out mice develop a phenotype similar to DDSH, we have developed a SJS mouse model by reproducing the hypomorph effect of SJS perlecan mutation in this species. The characterization of this mouse model will help to understand the pathophysiological mechanism leading to this multisystemic human disorder.
Assuntos
Proteoglicanas de Heparan Sulfato/deficiência , Osteocondrodisplasias/genética , Animais , Membrana Basal/metabolismo , Cartilagem/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/fisiologia , Humanos , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/fisiopatologiaRESUMO
OBJECTIVE: Low-molecular-weight heparin (LMWH) exerts antitumor activity in clinical trials. The K5 polysaccharide from Escherichia coli has the same structure as the heparin precursor. Chemical and enzymatic modifications of K5 polysaccharide lead to the production of biotechnological heparin-like compounds. We investigated the fibroblast growth factor-2 (FGF2) antagonist and antiangiogenic activity of a series of LMW N,O-sulfated K5 derivatives. METHODS AND RESULTS: Surface plasmon resonance analysis showed that LMW-K5 derivatives bind FGF2, thus inhibiting its interaction with heparin immobilized to a BIAcore sensor chip. Interaction of FGF2 with tyrosine-kinase receptors (FGFRs), heparan sulfate proteoglycans (HSPGs), and alpha(v)beta3 integrin is required for biological response in endothelial cells. Similar to LMWH, LMW-K5 derivatives abrogate the formation of HSPG/FGF2/FGFR ternary complexes by preventing FGF2-mediated attachment of FGFR1-overexpressing cells to HSPG-bearing cells and inhibit FGF2-mediated endothelial cell proliferation. However, LMW-K5 derivatives, but not LMWH, also inhibit FGF2/alpha(v)beta3 integrin interaction and consequent FGF2-mediated endothelial cell sprouting in vitro and angiogenesis in vivo in the chick embryo chorioallantoic membrane. CONCLUSIONS: LMW N,O-sulfated K5 derivatives affect both HSPG/FGF2/FGFR and FGF2/alpha(v)beta3 interactions and are endowed with FGF2 antagonist and antiangiogenic activity. These compounds may provide the basis for the design of novel LMW heparin-like angiostatic compounds.
Assuntos
Inibidores da Angiogênese/biossíntese , Escherichia coli/química , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Engenharia Genética/métodos , Heparina de Baixo Peso Molecular/biossíntese , Polissacarídeos Bacterianos/biossíntese , Inibidores da Angiogênese/genética , Animais , Cápsulas Bacterianas , Células CHO/química , Células CHO/metabolismo , Bovinos , Adesão Celular/fisiologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Cricetinae , Cricetulus , Células Endoteliais/química , Células Endoteliais/metabolismo , Escherichia coli/genética , Fator 2 de Crescimento de Fibroblastos/análogos & derivados , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/análogos & derivados , Fatores de Crescimento de Fibroblastos/metabolismo , Proteoglicanas de Heparan Sulfato/análogos & derivados , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina de Baixo Peso Molecular/síntese química , Heparina de Baixo Peso Molecular/genética , Integrina alfaVbeta3/metabolismo , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Polissacarídeos Bacterianos/genéticaAssuntos
Proteoglicanas de Heparan Sulfato/fisiologia , Camundongos Knockout , Junção Neuromuscular/fisiologia , Osteocondrodisplasias/genética , Acetilcolina/fisiologia , Acetilcolinesterase/deficiência , Acetilcolinesterase/fisiologia , Animais , Condrogênese/genética , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/genética , Humanos , Camundongos , Morfogênese/genética , Mutação , Doenças Neuromusculares/genética , Junção Neuromuscular/embriologiaRESUMO
Perlecan, a modular proteoglycan carrying primary heparan sulfate (HS) side chains, is a major component of blood vessel basement membranes. It sequesters growth factors such as fibroblast growth factor 2 (FGF-2) and regulates the ligand-receptor interactions on the cell surface, and thus it has been implicated in the control of angiogenesis. Both stimulatory and inhibitory effects of perlecan on FGF-2 signaling have been reported. To understand the in vivo function of HS carried by perlecan, the perlecan gene heparan sulfate proteoglycan 2 (Hspg2) was mutated in mouse by gene targeting. The HS at the NH(2) terminus of perlecan was removed while the core protein remained intact. Perlecan HS-deficient (Hspg2(Delta3/Delta3)) mice survived embryonic development and were apparently healthy as adults. However, mutant mice exhibited significantly delayed wound healing, retarded FGF-2-induced tumor growth, and defective angiogenesis. In the mouse corneal angiogenesis model, FGF-2-induced neovascularization was significantly impaired in Hspg2(Delta3/Delta3) mutant mice. Our results suggest that HS in perlecan positively regulates the angiogenesis in vivo.
Assuntos
Proteoglicanas de Heparan Sulfato/deficiência , Neoplasias Experimentais/irrigação sanguínea , Neovascularização Fisiológica/fisiologia , Cicatrização/fisiologia , Animais , Membrana Basal/patologia , Vasos Sanguíneos/patologia , Divisão Celular/fisiologia , Córnea/irrigação sanguínea , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/fisiologia , Proteoglicanas de Heparan Sulfato/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neovascularização Patológica/patologia , TransfecçãoRESUMO
We were interested in the elucidation of the interaction between the heparan sulfate proteoglycan, perlecan, and PTEN in the regulation of vascular smooth muscle cell (SMC) growth. We verified serum-stimulated DNA synthesis, and Akt and FAK phosphorylation were significantly reduced in SMCs overexpressing wild-type PTEN. Our previous studies showed perlecan is a potent inhibitor of serum-stimulated SMC growth. We report in the present study, compared with SMCs plated on fibronectin, serum-stimulated SMCs plated on perlecan exhibited increased PTEN activity, decreased FAK and Akt activities, and high levels of p27, consistent with SMC growth arrest. Adenoviral-mediated overexpression of constitutively active Akt reversed perlecan-induced SMC growth arrest while morpholino antisense-mediated loss of endogenous PTEN resulted in increased growth and phosphorylation of FAK and Akt of SMCs on perlecan. Immunohistochemical and Western analyses of balloon-injured rat carotid artery tissues showed a transient increase in phosphoPTEN (inactive) after injury, correlating to high rates of neointimal cell replication; phosphoPTEN was largely limited to actively replicating SMCs. Similarly, in the developing rat aorta, we found increased PTEN activity associated with increased perlecan deposition and decreased SMC replication rates. However, significantly decreased PTEN activity was detected in aortas of perlecan-deficient mouse embryos, consistent with SMC hyperplasia observed in these animals, compared with E17.5 heterozygous controls that produce abundant amounts of perlecan at this developmental time point. Our data show PTEN is a potent endogenously produced inhibitor of SMC growth and increased PTEN activity mediates perlecan-induced suppression of SMC proliferation.
Assuntos
Proteoglicanas de Heparan Sulfato/fisiologia , Músculo Liso Vascular/citologia , Proteínas Serina-Treonina Quinases , Animais , Aorta Torácica/citologia , Aorta Torácica/embriologia , Membrana Basal/fisiologia , Lesões das Artérias Carótidas/patologia , Cateterismo/efeitos adversos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura Livres de Soro , Replicação do DNA/efeitos dos fármacos , Fibronectinas/farmacologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Glicosaminoglicanos/fisiologia , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/farmacologia , Heparitina Sulfato/fisiologia , Masculino , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/fisiologiaRESUMO
The role of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) in modulation of vascular cell proliferation is believed to be mediated, in part, by its ability to regulate the activity of certain growth factors through direct binding. In this study, we demonstrate that SPARC does not bind to basic fibroblast growth factor (bFGF/FGF-2) or interfere with complex formation between FGF-2 and its high-affinity FGF receptor-1 (FGFR1), yet both native SPARC and a peptide derived from the C-terminal high-affinity Ca(2+)-binding region of protein significantly inhibit ligand-induced autophosphorylation of FGFR1 (>80%), activation of mitogen-activated protein kinases (MAPKs) (>75%), and DNA synthesis in human microvascular endothelial cells (HMVEC) stimulated by FGF-2 (>80%). We also report that in the presence of FGF-2, a factor which otherwise stimulates myoblast proliferation and the repression of terminal differentiation, both native SPARC and the Ca(2+)-binding SPARC peptide significantly promote (>60%) the differentiation of the MM14 murine myoblast cell line that expresses FGFR1 almost exclusively. Moreover, using heparan sulfate proteoglycan (HSPG)-deficient myeloid cells and porcine aortic endothelial cells (PAECs) expressing chimeric FGFR1, we show that antagonism of FGFR1-mediated DNA synthesis and MAPK activation by SPARC does not require the presence of cell-surface, low-affinity FGF-2 receptors, but can be mediated by an intracellular mechanism that is independent of an interaction with the extracellular ligand-binding domain of FGFR1. We also report that the inhibitory effect of SPARC on DNA synthesis and MAPK activation in endothelial cells is mediated in part (>50%) by activation of protein kinase A (PKA), a known regulator of Raf-MAPK pathway. SPARC thus modulates the mitogenic effect of FGF-2 downstream from FGFR1 by selective regulation of the MAPK signaling cascade.
Assuntos
Diferenciação Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Osteonectina/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Aorta/metabolismo , Cálcio/metabolismo , Divisão Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , DNA/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteoglicanas de Heparan Sulfato/deficiência , Humanos , Ligantes , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fosforilação , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Proteínas Recombinantes/metabolismo , Transdução de Sinais , SuínosRESUMO
BACKGROUND: Chondrodystrophic myotonia or Schwartz-Jampel syndrome is a rare genetic disorder characterized by myotonia and skeletal dysplasia. It may be progressive in nature. Recently, the gene responsible for Schwartz-Jampel syndrome has been found and the defective protein it encodes leads to abnormal cartilage development and anomalous neuromuscular activity. CASE PRESENTATION: We report the clinical findings and the management of an 8-year-old boy with this disorder. The molecular findings confirm that the patient is a compound heterozygote with a different splicing mutation in each Perlecan allele. This resulted in a significant reduction in the production of the encoded normal protein. CONCLUSION: We discuss the multi-disciplinary management of Schwartz-Jampel syndrome that will facilitate optimal care and timely intervention of patients with this disorder.