RESUMO
Nephrogenic systemic fibrosis (NSF) is a devastating condition associated with gadolinium (Gd3+)-based contrast agents (GBCAs) in patients with kidney disease. The release of toxic Gd3+ from GBCAs likely plays a major role in NSF pathophysiology. The cause and etiology of Gd3+ release from GBCAs is unknown. Increased Acidic Serine Aspartate Rich MEPE-associated peptides (ASARM peptides) induce bone mineralization abnormalities and contribute to renal phosphate-handling defects in inherited hypophosphatemic rickets and tumor-induced osteomalacia. The proteolytic cleavage of related bone matrix proteins with ASARM motifs results in release of ASARM peptide into bone and circulation. ASARM peptides are acidic, reactive, phosphorylated inhibitors of mineralization that bind Ca2+ and hydroxyapatite. Since the ionic radius of Gd3+ is close to that of Ca2+, we hypothesized that ASARM peptides increase the risk of NSF by inducing release of Gd3+ from GBCAs. Here, we show 1) ASARM peptides bind and induce release of Gd3+ from GBCAs in vitro and in vivo; 2) A bioengineered peptide (SPR4) stabilizes the Gd3+-GBCA complex by specifically binding to ASARM peptide in vitro and in vivo; and 3) SPR4 peptide infusion prevents GBCA-induced NSF-like pathology in a murine model with increased ASARM peptide (Hyp mouse). We conclude ASARM peptides may play a role in NSF and SPR4 peptide is a candidate adjuvant for preventing or reducing risk of disease.
Assuntos
Meios de Contraste , Proteínas da Matriz Extracelular/metabolismo , Gadolínio DTPA , Glicoproteínas/metabolismo , Rim/metabolismo , Meglumina/análogos & derivados , Dermopatia Fibrosante Nefrogênica/prevenção & controle , Compostos Organometálicos , Endopeptidase Neutra Reguladora de Fosfato PHEX/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosfoproteínas/metabolismo , Animais , Citoproteção , Modelos Animais de Doenças , Estabilidade de Medicamentos , Raquitismo Hipofosfatêmico Familiar/complicações , Raquitismo Hipofosfatêmico Familiar/genética , Raquitismo Hipofosfatêmico Familiar/metabolismo , Fator de Crescimento de Fibroblastos 23 , Rim/diagnóstico por imagem , Rim/patologia , Imageamento por Ressonância Magnética , Masculino , Camundongos Endogâmicos C57BL , Dermopatia Fibrosante Nefrogênica/induzido quimicamente , Dermopatia Fibrosante Nefrogênica/diagnóstico , Dermopatia Fibrosante Nefrogênica/genética , Dermopatia Fibrosante Nefrogênica/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Microtomografia por Raio-XRESUMO
More than 300 million years ago, vertebrates emerged from the vast oceans to conquer gravity and the dry land. With this transition, new adaptations occurred that included ingenious changes in reproduction, waste secretion, and bone physiology. One new innovation, the egg shell, contained an ancestral protein (ovocleidin-116) that likely first appeared with the dinosaurs and was preserved through the theropod lineage in modern birds and reptiles. Ovocleidin-116 is an avian homolog of matrix extracellular phosphoglycoprotein (MEPE) and belongs to a group of proteins called short integrin-binding ligand-interacting glycoproteins (SIBLINGs). These proteins are all localized to a defined region on chromosome 5q in mice and chromosome 4q in humans. A unifying feature of SIBLING proteins is an acidic serine aspartate-rich MEPE-associated motif (ASARM). Recent research has shown that the ASARM motif and the released ASARM peptide have regulatory roles in mineralization (bone and teeth), phosphate regulation, vascularization, soft-tissue calcification, osteoclastogenesis, mechanotransduction, and fat energy metabolism. The MEPE ASARM motif and peptide are physiological substrates for PHEX, a zinc metalloendopeptidase. Defects in PHEX are responsible for X-linked hypophosphatemic rickets (HYP). There is evidence that PHEX interacts with another ASARM motif containing SIBLING protein, dentin matrix protein-1 (DMP1). DMP1 mutations cause bone and renal defects that are identical with the defects caused by a loss of PHEX function. This results in autosomal recessive hypophosphatemic rickets (ARHR). In both HYP and ARHR, increased FGF23 expression plays a major role in the disease and in autosomal dominant hypophosphatemic rickets (ADHR), FGF23 half-life is increased by activating mutations. ASARM peptide administration in vitro and in vivo also induces increased FGF23 expression. FGF23 is a member of the fibroblast growth factor (FGF) family of cytokines, which surfaced 500 million years ago with the boney fish (i.e., teleosts) that do not contain SIBLING proteins. In terrestrial vertebrates, FGF23, like SIBLING proteins, is expressed in the osteocyte. The boney fish, however, are an-osteocytic, so a physiological bone-renal link with FGF23 and the SIBLINGs was cemented when life ventured from the oceans to the land during the Triassic period, approximately 300 million years ago. This link has been revealed by recent research that indicates a competitive displacement of a PHEX-DMP1 interaction by an ASARM peptide that leads to increased FGF23 expression. This review discusses the new discoveries that reveal a novel PHEX, DMP1, MEPE, ASARM peptide, and FGF23 bone-renal pathway. This pathway impacts not only bone formation, bone-renal mineralization, and renal phosphate homeostasis but also energy metabolism. The study of this new pathway is relevant for developing therapies for several diseases: bone-teeth mineral loss disorders, renal osteodystrophy, chronic kidney disease and bone mineralization disorders (CKD-MBD), end-stage renal diseases, ectopic arterial-calcification, cardiovascular disease renal calcification, diabetes, and obesity.
Assuntos
Doenças Ósseas Metabólicas/metabolismo , Osso e Ossos/metabolismo , Metabolismo Energético/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Rim/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Fosfoproteínas/metabolismo , Animais , Desmineralização Patológica Óssea/metabolismo , Desmineralização Patológica Óssea/terapia , Doenças Ósseas Metabólicas/terapia , Calcificação Fisiológica/fisiologia , Distúrbio Mineral e Ósseo na Doença Renal Crônica/metabolismo , Distúrbio Mineral e Ósseo na Doença Renal Crônica/terapia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/terapia , Proteínas do Ovo/metabolismo , Feminino , Fator de Crescimento de Fibroblastos 23 , Humanos , Hipofosfatemia/metabolismo , Hipofosfatemia/terapia , Falência Renal Crônica/metabolismo , Falência Renal Crônica/terapia , Masculino , Mecanotransdução Celular/fisiologia , Camundongos , Camundongos Transgênicos , Obesidade/metabolismo , Obesidade/terapia , Osteócitos/metabolismo , Osteomalacia/metabolismo , Osteomalacia/terapia , Raquitismo/metabolismo , Raquitismo/terapiaRESUMO
The eggshell is an ancient innovation that helped the vertebrates' transition from the oceans and gain dominion over the land. Coincident with this conquest, several new eggshell and noncollagenous bone-matrix proteins (NCPs) emerged. The protein ovocleidin-116 is one of these proteins with an ancestry stretching back to the Triassic. Ovocleidin-116 is an avian homolog of Matrix Extracellular Phosphoglycoprotein (MEPE) and belongs to a group of proteins called Small Integrin-Binding Ligand Interacting Glycoproteins (SIBLINGs). The genes for these NCPs are all clustered on chromosome 5q in mice and chromosome 4q in humans. A unifying feature of the SIBLING proteins is an Acidic Serine Aspartate-Rich MEPE (ASARM)-associated motif. The ASARM motif and the released ASARM peptide play roles in mineralization, bone turnover, mechanotransduction, phosphate regulation and energy metabolism. ASARM peptides and motifs are physiological substrates for phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX), a Zn metalloendopeptidase. Defects in PHEX are responsible for X-linked hypophosphatemic rickets. PHEX interacts with another ASARM motif containing SIBLING protein, Dentin Matrix Protein-1 (DMP1). DMP1 mutations cause bone-renal defects that are identical with the defects caused by loss of PHEX function. This results in autosomal recessive hypophosphatemic rickets (ARHR). In both X-linked hypophosphatemic rickets and ARHR, increased fibroblast growth factor 23 (FGF23) expression occurs, and activating mutations in FGF23 cause autosomal dominant hypophosphatemic rickets (ADHR). ASARM peptide administration in vitro and in vivo also induces increased FGF23 expression. This review will discuss the evidence for a new integrative pathway involved in bone formation, bone-renal mineralization, renal phosphate homeostasis and energy metabolism in disease and health.
Assuntos
Proteínas do Ovo/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Animais , Proteínas do Ovo/química , Proteínas do Ovo/classificação , Metabolismo Energético , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Homeostase , Humanos , Osteogênese , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Fosfatos/metabolismoRESUMO
Spinal cord injury (SCI) is a complex syndrome that has profound effects on patient well-being, including the development of medically-resistant chronic pain. The mechanisms underlying SCI pain have been the subject of thorough investigation but remain poorly understood. While the majority of the research has focused on changes occurring within and surrounding the site of injury in the spinal cord, there is now a consensus that alterations within the peripheral nervous system, namely sensitization of nociceptors, contribute to the development and maintenance of chronic SCI pain. Using an ex vivo skin/nerve/DRG/spinal cord preparation to characterize afferent response properties following SCI, we found that SCI increased mechanical and thermal responding, as well as the incidence of spontaneous activity (SA) and afterdischarge (AD), in below-level C-fiber nociceptors 24 hr following injury relative to naïve controls. Interestingly, the distribution of nociceptors that exhibit SA and AD are not identical, and the development of SA was observed more frequently in nociceptors with low heat thresholds, while AD was found more frequently in nociceptors with high heat thresholds. We also found that SCI resulted in hindpaw edema and elevated cutaneous calcitonin gene-related peptide (CGRP) concentration that were not observed in naïve mice. These results suggest that SCI causes a rapidly developing nociceptor sensitization and peripheral inflammation that may contribute to the early emergence and persistence of chronic SCI pain.
RESUMO
Increased acidic serine aspartate-rich MEPE-associated motif (ASARM) peptides cause mineralization defects in X-linked hypophosphatemic rickets mice (HYP) and "directly" inhibit renal phosphate uptake in vitro. However, ASARM peptides also bind to phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and are a physiological substrate for this bone-expressed, phosphate-regulating enzyme. We therefore tested the hypothesis that circulating ASARM peptides also "indirectly" contribute to a bone-renal PHEX-dependent hypophosphatemia in normal mice. Male mice (n = 5; 12 wk) were fed for 8 wk with a normal phosphorus and vitamin D(3) diet (1% P(i) diet) or a reduced phosphorus and vitamin D(3) diet (0.1% P(i) diet). For the final 4 wk, transplantation of mini-osmotic pumps supplied a continuous infusion of either ASARM peptide (5 mg·day(-1)·kg(-1)) or vehicle. HYP, autosomal recessive hypophosphatemic rickets (ARHR), and normal mice (no pumps or ASARM infusion; 0.4% P(i) diet) were used in a separate experiment designed to measure and compare circulating ASARM peptides in disease and health. ASARM treatment decreased serum phosphate concentration and renal phosphate cotransporter (NPT2A) mRNA with the 1% P(i) diet. This was accompanied by a twofold increase in serum ASARM and 1,25-dihydroxy vitamin D(3) [1,25 (OH)(2)D(3)] levels without changes in parathyroid hormone. For both diets, ASARM-treated mice showed significant increases in serum fibroblast growth factor 23 (FGF23; +50%) and reduced serum osteocalcin (-30%) and osteopontin (-25%). Circulating ASARM peptides showed a significant inverse correlation with serum P(i) and a significant positive correlation with fractional excretion of phosphate. We conclude that constitutive overexpression of ASARM peptides plays a "component" PHEX-independent part in the HYP and ARHR hypophosphatemia. In contrast, with wild-type mice, ASARM peptides likely play a bone PHEX-dependent role in renal phosphate regulation and FGF23 expression. They may also coordinate FGF23 expression by competitively modulating PHEX/DMP1 interactions and thus bone-renal mineral regulation.
Assuntos
Ácido Aspártico , Proteínas da Matriz Extracelular/fisiologia , Glicoproteínas/fisiologia , Rim/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/fisiologia , Peptídeos/fisiologia , Fosfatos/sangue , Fosfoproteínas/fisiologia , Serina , Sequência de Aminoácidos , Animais , Proteínas da Matriz Extracelular/metabolismo , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Hipofosfatemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Dados de Sequência Molecular , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismoRESUMO
Matrix extracellular phosphoglycoprotein/osteoblast factor 45 (MEPE/OF45) was cloned in 2000 with functions related to bone metabolism. We identified MEPE/OF45 for the first time as a new co-factor of CHK1 in mammalian cells to protect cells from DNA damage induced killing. We demonstrate here that MEPE/OF45 directly interacts with CHK1. Knocking down MEPE/OF45 decreases CHK1 levels and sensitizes the cells to DNA damage inducers such as ionizing radiation (IR) or camptothicin (CPT)-induced killing. Over-expressing wild-type MEPE/OF45, but not the mutant MEPE/OF45 (depleted the key domain to interact with CHK1) increases CHK1 levels in the cells and increases the resistance of the cells to IR or CPT. MEPE/OF45, interacting with CHK1, increases CHK1 half-life and decreases CHK1 degradation through the ubiquitine-mediated pathway. In addition, the interaction of MEPE/OF45 with CHK1 decreases CHK1 levels in the ubiquitin E3 ligases (Cul1 and Cul4A) complex, which suggests that MEPE/OF45 competes with the ubiquitin E3 ligases binding to CHK1 and thus decreases CHK1 from ubiquitin-mediated proteolysis. These findings reveal an important role of MEPE/OF45 in protecting cells from DNA damage induced killing through stabilizing CHK1, which would provide MEPE/OF45 as a new target for sensitizing tumor cells to radiotherapy or chemotherapy.
Assuntos
Dano ao DNA , Proteínas da Matriz Extracelular/fisiologia , Glicoproteínas/fisiologia , Fosfoproteínas/fisiologia , Proteínas Quinases/metabolismo , Animais , Linhagem Celular Transformada , Quinase 1 do Ponto de Checagem , Citoproteção , Estabilidade Enzimática , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Fosfoproteínas/metabolismo , Ratos , Ubiquitinas/metabolismoRESUMO
Matrix extracellular phosphoglycoprotein (MEPE) is an inhibitor of mineralization in situ and in cell cultures where altered expression is associated with oncogenic osteomalacia and hypophosphatemic rickets. The purpose of this study was to determine whether the intact protein or the peptide(s) originating from this protein was responsible for the inhibition. The ability of the intact protein and the acidic, serine- and aspartate-rich MEPE-associated motif (ASARM) peptide to promote or inhibit de novo hydroxyapatite formation and growth of hydroxyapatite seed crystals, in both phosphorylated and dephosphorylated forms, was assessed at room temperature in a dynamic gel diffusion system at 3.5 and 5 days. The most effective nucleator concentration was also examined when associated with fibrillar type I collagen. The phosphorylated intact protein was an effective promoter of mineralization in the gelatin gel diffusion system, while the ASARM peptide was an effective inhibitor. When dephosphorylated both the intact protein and the ASARM peptide had no effect on mineralization. Associated with collagen fibrils, some of the effect of the intact protein was lost. This study demonstrates the importance of posttranslational modification for the site-specific activity of MEPE and its ASARM peptide.
Assuntos
Osso e Ossos/metabolismo , Calcificação Fisiológica/fisiologia , Durapatita/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/fisiopatologia , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Cristalização , Proteínas da Matriz Extracelular/farmacologia , Géis/química , Glicoproteínas/farmacologia , Humanos , Osteomalacia/genética , Osteomalacia/metabolismo , Osteomalacia/fisiopatologia , Fragmentos de Peptídeos/farmacologia , Fosfoproteínas/farmacologia , Fosforilação/efeitos dos fármacos , Modificação Traducional de Proteínas/efeitos dos fármacos , Modificação Traducional de Proteínas/fisiologiaRESUMO
Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) and DMP1 (dentin matrix protein 1) result in X-linked hypophosphatemic rickets (HYP) and autosomal-recessive hypophosphatemic-rickets (ARHR), respectively. Specific binding of PHEX to matrix extracellular phosphoglycoprotein (MEPE) regulates the release of small protease-resistant MEPE peptides [acidic serine- and aspartate-rich MEPE-associated motif (ASARM) peptides]. ASARM peptides are potent inhibitors of mineralization (minhibins) that also occur in DMP1 [MEPE-related small integrin-binding ligand, N-linked glycoprotein (SIBLING) protein]. It is not known whether these peptides are directly responsible for the mineralization defect. We therefore used a bone marrow stromal cell (BMSC) coculture model, ASARM peptides, anti-ASARM antibodies, and a small synthetic PHEX peptide (SPR4; 4.2 kDa) to examine this. Surface plasmon resonance (SPR) and two-dimensional (1)H/(15)N nuclear magnetic resonance demonstrated specific binding of SPR4 peptide to ASARM peptide. When cultured individually for 21 d, HYP BMSCs displayed reduced mineralization compared with wild type (WT) (-87%, P < 0.05). When cocultured, both HYP and WT cells failed to mineralize. However, cocultures (HYP and WT) or monocultures of HYP BMSCs treated with SPR4 peptide or anti-ASARM neutralizing antibodies mineralized normally. WT BMSCs treated with ASARM peptide also failed to mineralize properly without SPR4 peptide or anti-ASARM neutralizing antibodies. ASARM peptide treatment decreased PHEX mRNA and protein (-80%, P < 0.05) and SPR4 peptide cotreatment reversed this by binding ASARM peptide. SPR4 peptide also reversed ASARM peptide-mediated changes in expression of key osteoclast and osteoblast differentiation genes. Western blots of HYP calvariae and BMSCs revealed massive degradation of both MEPE and DMP1 protein compared with the WT. We conclude that degradation of MEPE and DMP-1 and release of ASARM peptides are chiefly responsible for the HYP mineralization defect and changes in osteoblast-osteoclast differentiation.
Assuntos
Calcificação Fisiológica , Proteínas da Matriz Extracelular/metabolismo , Raquitismo Hipofosfatêmico Familiar/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X , Glicoproteínas/metabolismo , Fosfoproteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Dados de Sequência Molecular , Osteoclastos/fisiologia , Endopeptidase Neutra Reguladora de Fosfato PHEX/química , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Inactivating PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome) mutations cause X-linked hypophosphatemia in humans and mice (Hyp) through overproduction of fibroblast growth factor 23 (FGF23) a phosphaturic factor, by osteocytes. Matrix extracellular phosphoglycoprotein (MEPE) is also elevated in Hyp and other hypophosphatemic disorders. In addition, the administration of an ASARM (acidic serine-aspartate rich MEPE-associated motif) peptide derived from MEPE causes phosphaturia and inhibits bone mineralization in mice, suggesting that MEPE also plays a role in phosphate homeostasis. Since recent studies found that MEPE binds specifically to PHEX in vitro, we tested the effect of recombinant-MEPE and its ASARM peptide on PHEX enzyme activity in vitro and FGF23 expression in bone marrow stromal cell cultures ex vivo. We found that both recombinant MEPE and synthetic phosphorylated ASARM peptide (ASARM-PO(4)) inhibit PHEX enzyme activities in an in vitro fluorescent-quenched PHEX enzyme activity assay. The ASARM-PO(4) peptide inhibits PHEX enzyme activity in a dose-dependent manner with a K(i) of 128 nM and V(max-i) of 100%. Recombinant MEPE also inhibits PHEX activity (K(i) = 2 nM and V(max-i) = 26%). Long-term bone marrow stromal cell cultures supplemented with 10 microM ASARM-PO(4) peptide resulted in significant elevation of FGF23 transcripts and inhibition of mineralization. These findings suggest that MEPE inhibits mineralization and PHEX activity and leads to increased FGF23 production. The resulting coordination of mineralization and release of a phosphaturic factor by MEPE may serve a physiological role in regulating systemic phosphate homeostasis to meet the needs for bone mineralization.
Assuntos
Células da Medula Óssea/metabolismo , Proteínas da Matriz Extracelular/farmacologia , Raquitismo Hipofosfatêmico Familiar/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X , Glicoproteínas/farmacologia , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Fosfoproteínas/farmacologia , Animais , Células da Medula Óssea/patologia , Células Cultivadas , Ciclofilina A/genética , Relação Dose-Resposta a Droga , Raquitismo Hipofosfatêmico Familiar/patologia , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/análise , Fatores de Crescimento de Fibroblastos/genética , Homeostase , Camundongos , Camundongos Knockout , Endopeptidase Neutra Reguladora de Fosfato PHEX/antagonistas & inibidores , Fosfatos/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Increased expression of several osteoblastic proteases and MEPE (a bone matrix protein) occurs in X-linked hypophosphatemic rickets (hyp). This is associated with an increased release of a protease-resistant MEPE peptide (ASARM peptide), a potent inhibitor of mineralization. Cathepsin B cleaves MEPE releasing ASARM peptide and hyp osteoblast/osteocyte cells hypersecrete cathepsin D, an activator of cathepsin B. Our aims were to determine whether cathepsin inhibitors correct the mineralization defect in vivo and whether hyp-bone ASARM peptide levels are reduced after protease treatment. Normal littermates and hyp mice (n = 6) were injected intraperitoneally once a day for 4 weeks with pepstatin, CAO74 or vehicle. Animals were then sacrificed and bones plus serum removed for comprehensive analysis. All hyp mice groups (treated and untreated) remained hypophosphatemic with serum 1,25 vitamin D3 inappropriately normal. Serum PTH was significantly elevated in all hyp mice groups relative to normal mice (P = 0.0017). Untreated hyp mice had six-fold elevated levels of serum alkaline-phosphatase and two-fold elevated levels of ASARM peptides relative to normal mice (P < 0.001). In contrast, serum alkaline phosphatase and serum ASARM peptides were significantly reduced (normalized) in hyp mice treated with CA074 or pepstatin. Serum FGF23 levels remained high in all hyp animal groups (P < 0.0001). Hyp mice treated with protease inhibitors showed dramatic reductions in unmineralized osteoid (femurs) compared to control hyp mice (Goldner staining). Also, hyp animals treated with protease inhibitors showed marked and significant improvements in growth plate width (42%), osteoid thickness (40%) and cortical area (40%) (P < 0.002). The mineralization apposition rate, bone formation rate and mineralization surface were normalized by protease-treatment. High-resolution pQCT mineral histomorphometry measurements and uCT also confirmed a marked mineralization improvement. Finally, the growth plate and cortical bone of hyp femurs contained a massive accumulation of osteoblast-derived ASARM peptide(s) that was reduced in hyp animals treated with CA074 or pepstatin. This study confirms in vivo administration of cathepsin inhibitors improves bone mineralization in hyp mice. This may be due to a protease inhibitor mediated decrease in proteolytic degradation of the extracellular matrix and a reduced release of ASARM peptides (potent mineralization inhibitors).
Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Pepstatinas/farmacologia , Inibidores de Proteases/farmacologia , Animais , Catepsina B/análise , Catepsina B/antagonistas & inibidores , Catepsina D/análise , Catepsina D/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fêmur/efeitos dos fármacos , Fêmur/patologia , Fator de Crescimento de Fibroblastos 23 , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Modelos Biológicos , Osteoblastos/metabolismo , Pepstatinas/administração & dosagem , Inibidores de Proteases/administração & dosagem , Tomografia Computadorizada por Raios XRESUMO
Matrix Extracellular Phospho-glycoprotEin (MEPE) and proteases are elevated and PHEX is defective in HYP. PHEX prevents proteolysis of MEPE and release of a protease-resistant MEPE-ASARM peptide, an inhibitor of mineralization (minhibin). Thus, in HYP, mutated PHEX may contribute to increased ASARM peptide release. Moreover, binding of MEPE by PHEX may regulate this process in normal subjects. The nature of the PHEX-MEPE nonproteolytic interaction(s) (direct or indirect) is/are unknown. Our aims were to determine (1) whether PHEX binds specifically to MEPE, (2) whether the binding involves the ASARM motif region, and (3) whether free ASARM peptide affects mineralization in vivo in mice. Protein interactions between MEPE and recombinant soluble PHEX (secPHEX) were measured using surface plasmon resonance (SPR). Briefly, secPHEX, MEPE, and control protein (IgG) were immobilized on a Biacore CM5 sensor chip, and SPR experiments were performed on a Biacore 3000 high-performance research system. Pure secPHEX was then injected at different concentrations, and interactions with immobilized proteins were measured. To determine MEPE sequences interacting with secPHEX, the inhibitory effects of MEPE-ASARM peptides (phosphorylated and nonphosphorylated), control peptides, and MEPE midregion RGD peptides on secPHEX binding to chip-immobilized MEPE were measured. ASARM peptide and etidronate-mediated mineralization inhibition in vivo and in vitro were determined by quenched calcein fluorescence in hind limbs and calvariae in mice and by histological Sanderson stain. A specific, dose-dependent and Zn-dependent protein interaction between secPHEX and immobilized MEPE occurs (EC50 of 553 nM). Synthetic MEPE PO4-ASARM peptide inhibits the PHEX-MEPE interaction (K(D(app)) = 15 uM and B(max/inhib) = 68%). In contrast, control and MEPE-RGD peptides had no effect. Subcutaneous administration of ASARM peptide resulted in marked quenching of fluorescence in calvariae and hind limbs relative to vehicle controls indicating impaired mineralization. Similar results were obtained with etidronate. Sanderson-stained calvariae also indicated a marked increase in unmineralized osteoid with ASARM peptide and etidronate groups. We conclude that PHEX and MEPE form a nonproteolytic protein interaction via the MEPE carboxy-terminal ASARM motif, and the ASARM peptide inhibits mineralization in vivo. The binding of MEPE and ASARM peptide by PHEX may explain why loss of functional osteoblast-expressed PHEX results in defective mineralization in HYP.
Assuntos
Calcificação Fisiológica , Proteínas da Matriz Extracelular/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Glicoproteínas/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Raquitismo/fisiopatologia , Sequência de Aminoácidos , Ácido Etidrônico/farmacologia , Fluorescência , Humanos , Dados de Sequência Molecular , Endopeptidase Neutra Reguladora de Fosfato PHEX , Ressonância de Plasmônio de SuperfícieRESUMO
CONTEXT: Mice with null mutations in matrix extracellular phosphoglycoprotein (MEPE) have increased bone mass, increased trabecular density and abnormal cancellous bone (MN-mice). These defects worsen with age and MEPE overexpression induces opposite effects. Also, genome wide association studies show that MEPE plays a major role in bone mass. We hypothesized that the conserved C-terminal MEPE ASARM-motif is chiefly responsible for regulating bone mass and trabecular structure. DESIGN: To test our theory we overexpressed C-terminal ASARM-peptide in MN-mice using the Col1α1 promoter (MNAt-mice). We then compared the bone and renal phenotypes of the MNAt-mouse with the MN-mouse and the X-linked hypophosphatemic rickets mouse (HYP). The HYP mouse overexpresses ASARM-peptides and is defective for the PHEX gene. RESULTS: The MN-mouse developed increased bone mass, bone strength and trabecular abnormalities that worsened markedly with age. Defects in bone formation were chiefly responsible with suppressed sclerostin and increased active ß-catenin. Increased uric acid levels also suggested that abnormalities in purine-metabolism and a reduced fractional excretion of uric acid signaled additional renal transport changes. The MN mouse developed a worsening hyperphosphatemia and reduced FGF23 with age. An increase in the fractional excretion of phosphate (FEP) despite the hyperphosphatemia confirms an imbalance in kidney-intestinal phosphate regulation. Also, the MN mice showed an increased creatinine clearance suggesting hyperfiltration. A reversal of the MN bone-renal phenotype changes occurred with the MNAt mice including the apparent hyperfiltration. The MNAt mice also developed localized hypomineralization, hypophosphatemia and increased FGF23. CONCLUSIONS: The C-terminal ASARM-motif plays a major role in regulating bone-mass and cancellous structure as mice age. In healthy mice, the processing and release of free ASARM-peptide are chiefly responsible for preserving normal bone and renal function. Free ASARM-peptide also affects renal mineral phosphate handling by influencing FGF23 expression. These findings have implications for understanding age-dependent osteoporosis, unraveling drug-targets and developing treatments.
Assuntos
Osso e Ossos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Rim/metabolismo , Osteoporose/metabolismo , Fosfoproteínas/metabolismo , Absorciometria de Fóton , Animais , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Fator de Crescimento de Fibroblastos 23 , Glicoproteínas/genética , Immunoblotting , Imuno-Histoquímica , Testes de Função Renal , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoporose/genética , Fosfoproteínas/genética , Reação em Cadeia da PolimeraseRESUMO
CONTEXT: ASARM-peptides are substrates and ligands for PHEX, the gene responsible for X-linked hypophosphatemic rickets (HYP). PHEX binds to the DMP1-ASARM-motif to form a trimeric-complex with α5ß3-integrin on the osteocyte surface and this suppresses FGF23 expression. ASARM-peptide disruption of this complex increases FGF23 expression. We used a 4.2kDa peptide (SPR4) that binds to ASARM-peptide and ASARM-motif to study DMP1-PHEX interactions and to assess SPR4 for treating inherited hypophosphatemic rickets. DESIGN: Subcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle into wild-type mice (WT) and HYP-mice for 4 weeks. RESULTS: Asymmetrically distributed mineralization defects occurred with WT-SPR4 femurs. Specifically, SPR4 induced negative effects on trabecular bone and increased bone volume and mineralization in cortical-bone. Markedly increased sclerostin and reduced active ß-catenin occurred with HYP mice. SPR4-infusion suppressed sclerostin and increased active ß-catenin in WT and HYP mice and improved HYP-mice trabecular mineralization defects but not cortical mineralization defects. CONCLUSIONS: SPR4-peptide has bimodal activity and acts by: (1) preventing DMP1 binding to PHEX and (2) sequestering an inhibitor of DMP1-PHEX binding, ASARM-peptide. In PHEX defective HYP-mice the second pathway predominates. Although SPR4-peptide improved trabecular calcification defects, decreased sclerostin and increased active ß-catenin it did not correct HYP-mice cortical mineralization defects on a normal phosphate diet. Thus, for inherited hypophosphatemic rickets patients on a normal phosphate diet, SPR4-peptide is not a useful therapeutic.
Assuntos
Osso e Ossos/metabolismo , Raquitismo Hipofosfatêmico Familiar/fisiopatologia , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Glicoproteínas/metabolismo , Peptídeos/química , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Animais , Dieta , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Raquitismo Hipofosfatêmico Familiar/genética , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular , Rim/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Fosfatos/química , Ligação Proteica , RNA Mensageiro/metabolismo , Microtomografia por Raio-XRESUMO
MEPE (Matrix Extracellular PhosphoglycoprotEin) expression is markedly elevated in X-linked-hypophosphatemic-rickets (HYP) and tumor-induced osteomalacia (TIO). In normal individuals, circulating serum-levels of MEPE are tightly correlated with serum-phosphorus, parathyroid hormone (PTH) and bone mineral density (BMD). Also, MEPE derived, C-terminal ASARM-peptides are candidate minhibins and/or phosphatonins. Our aims were to determine: 1. whether MEPE-ASARM-peptide(s) are abnormally elevated in HYP/hyp serum, and, 2. whether the ASARM-peptide(s) accumulate in hyp mice kidney renal-tubules. Using a specific competitive ELISA we measured a five fold increase (P=0.007) of serum ASARM-peptide(s) in human HYP patients (normal subjects 3.25 microM n=9; S.E.M.=0.51 and HYP-patients 15.74 microM, n=9; S.E.M.=3.32). A 6.23 fold increase (P=0.008) was measured in hyp male mice compared with their normal male siblings (normal-siblings, 3.73 muM, S.E.M.=0.57, n=3; and hyp-mice 23.4 microM, n=3, S.E.M.=4.01). Renal immuno-histological screening also revealed a dramatic increase of ASARM-peptides in regions anatomically consistent with the proximal convoluted tubules. This study demonstrates for the first time that markedly elevated serum levels of protease-resistant ASARM-peptide(s) occur in HYP/hyp and they accumulate in murine hyp kidneys. These peptides are thus likely responsible for the phosphaturia and defective mineralization in HYP/hyp and TIO.
Assuntos
Proteínas da Matriz Extracelular/sangue , Glicoproteínas/sangue , Hipofosfatemia Familiar/sangue , Fosfoproteínas/sangue , Animais , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas da Matriz Extracelular/análise , Feminino , Glicoproteínas/análise , Humanos , Imuno-Histoquímica/métodos , Túbulos Renais/química , Masculino , Camundongos , Fosfatos/urina , Fosfoproteínas/análiseRESUMO
CONTEXT: PHEX or DMP1 mutations cause hypophosphatemic-rickets and altered energy metabolism. PHEX binds to DMP1-ASARM-motif to form a complex with α5ß3 integrin that suppresses FGF23 expression. ASARM-peptides increase FGF23 by disrupting the PHEX-DMP1-Integrin complex. We used a 4.2 kDa peptide (SPR4) that binds to ASARM-peptide/motif to study the DMP1-PHEX interaction and to assess SPR4 for the treatment of energy metabolism defects in HYP and potentially other bone-mineral disorders. DESIGN: Subcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle (VE) into wild-type mice (WT) and HYP-mice (PHEX mutation) for 4 weeks. RESULTS: SPR4 partially corrected HYP mice hypophosphatemia and increased serum 1.25(OH)2D3. Serum FGF23 remained high and PTH was unaffected. WT-SPR4 mice developed hypophosphatemia and hypercalcemia with increased PTH, FGF23 and 1.25(OH)2D3. SPR4 increased GAPDH HYP-bone expression 60× and corrected HYP-mice hyperglycemia and hypoinsulinemia. HYP-VE serum uric-acid (UA) levels were reduced and SPR4 infusion suppressed UA levels in WT-mice but not HYP-mice. SPR4 altered leptin, adiponectin, and sympathetic-tone and increased the fat mass/weight ratio for HYP and WT mice. Expression of perlipin-2 a gene involved in obesity was reduced in HYP-VE and WT-SPR4 mice but increased in HYP-SPR4 mice. Also, increased expression of two genes that inhibit insulin-signaling, ENPP1 and ESP, occurred with HYP-VE mice. In contrast, SPR4 reduced expression of both ENPP1 and ESP in WT mice and suppressed ENPP1 in HYP mice. Increased expression of FAM20C and sclerostin occurred with HYP-VE mice. SPR4 suppressed expression of FAM20C and sclerostin in HYP and WT mice. CONCLUSIONS: ASARM peptides and motifs are physiological substrates for PHEX and modulate osteocyte PHEX-DMP1-α5ß3-integrin interactions and thereby FGF23 expression. These interactions also provide a nexus that regulates bone and energy metabolism. SPR4 suppression of sclerostin and/or sequestration of ASARM-peptides improves energy metabolism and may have utility for treating familial rickets, osteoporosis, obesity and diabetes.
Assuntos
Raquitismo Hipofosfatêmico Familiar/tratamento farmacológico , Endopeptidase Neutra Reguladora de Fosfato PHEX/química , Peptídeos/uso terapêutico , Animais , Metabolismo Energético/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Raquitismo Hipofosfatêmico Familiar/metabolismo , Fator de Crescimento de Fibroblastos 23 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/químicaRESUMO
Increased matrix extracellular phosphoglycoprotein (MEPE) expression occurs in several phosphate and bone-mineral metabolic disorders. To resolve whether MEPE plays a role, we created a murine model overexpressing MEPE protein (MEPE tgn) in bone. MEPE tgn mice displayed a growth and mineralization defect with altered bone-renal vascularization that persisted to adulthood. The growth mineralization defect was due to a decrease in bone remodeling, and MEPE tgn mice were resistant to diet-induced renal calcification. MEPE protein-derived urinary ASARM peptides and reduced urinary Ca X PO4 product mediated the suppressed renal calcification. Osteoblastic cells displayed reduced activity but normal differentiation. Osteoclastic precursors were unable to differentiate in the presence of osteoblasts. In the kidney, NPT2a up-regulation induced an increase in phosphate renal reabsorption, leading to hyperphosphatemia. We conclude MEPE and MEPE-phosphate-regulating gene with homologies to endopeptidases on the X chromosome (MEPE-PHEX) interactions are components to an age-diet-dependent pathway that regulates bone turnover and mineralization and suppresses renal calcification. This novel pathway also modulates bone-renal vascularization and bone turnover.
Assuntos
Proteínas da Matriz Extracelular/fisiologia , Glicoproteínas/fisiologia , Fosfoproteínas/fisiologia , Envelhecimento , Aldosterona/urina , Fenômenos Fisiológicos da Nutrição Animal , Animais , Desenvolvimento Ósseo/efeitos dos fármacos , Osso e Ossos/irrigação sanguínea , Calcitriol/sangue , Hiperfosfatemia/fisiopatologia , Hipocalcemia/fisiopatologia , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Rim/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica/genética , Osteopontina/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/fisiologia , Hormônio Paratireóideo/sangue , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
The last 350 years since the publication of the first medical monograph on rickets (old English term wrickken) (Glisson et al., 1651) have seen spectacular advances in our understanding of mineral-homeostasis. Seminal and exciting discoveries have revealed the roles of PTH, vitamin D, and calcitonin in regulating calcium and phosphate, and maintaining healthy teeth and skeleton. However, it is clear that the PTH/Vitamin D axis does not account for the entire picture, and a new bone-renal metabolic milieu has emerged, implicating a novel set of matrix proteins, hormones, and Zn-metallopeptidases. The primary defects in X-linked hypophosphatemic rickets (HYP) and autosomal-dominant hypophosphatemic rickets (ADHR) are now identified as inactivating mutations in a Zn-metalloendopeptidase (PHEX) and activating mutations in fibroblast-growth-factor-23 (FGF23), respectively. In oncogenic hypophosphatemic osteomalacia (OHO), several tumor-expressed proteins (MEPE, FGF23, and FRP-4) have emerged as candidate mediators of the bone-renal pathophysiology. This has stimulated the proposal of a global model that takes into account the remarkable similarities between the inherited diseases (HYP and ADHR) and the tumor-acquired disease OHO. In HYP, loss of PHEX function is proposed to result in an increase in uncleaved full-length FGF23 and/or inappropriate processing of MEPE. In ADHR, a mutation in FGF23 results in resistance to proteolysis by PHEX or other proteases and an increase in half-life of full-length phosphaturic FGF23. In OHO, over-expression of FGF23 and/or MEPE is proposed to result in abnormal renal-phosphate handling and mineralization. Although this model is attractive, many questions remain unanswered, suggesting a more complex picture. The following review will present a global hypothesis that attempts to explain the experimental and clinical observations in HYP, ADHR, and OHO, plus diverse mouse models that include the MEPE null mutant, HYP-PHEX transgenic mouse, and MEPE-PHEX double-null-mutant.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Hipofosfatemia Familiar/genética , Osteomalacia/genética , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Animais , Calcificação Fisiológica/genética , Proteínas da Matriz Extracelular/genética , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Glicoproteínas/genética , Humanos , Hipofosfatemia Familiar/metabolismo , Camundongos , Camundongos Mutantes , Mutação , Osteomalacia/etiologia , Osteomalacia/metabolismo , Osteossarcoma/complicações , Endopeptidase Neutra Reguladora de Fosfato PHEX , Fosfatos/metabolismo , Fosfoproteínas/genética , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
X-linked hypophosphatemia (XLH) and the Hyp-mouse disease homolog are caused by inactivating mutations of Phex which results in the local accumulation of an unknown autocrine/paracrine factor in bone that inhibits mineralization of extracellular matrix. In these studies, we evaluated whether the matrix phosphoglycoprotein MEPE, which is increased in calvaria from Hyp mice, is a substrate for Phex. Using recombinant full-length Phex (rPhexWT) produced in Sf9 cells, we failed to observe Phex-dependent hydrolysis of recombinant human MEPE (rMEPE). Rather, we found that rPhex-WT inhibited cleavage of rMEPE by endogenous cathepsin-like enzyme activity present in Sf9 membrane. Sf9 membranes as well as purified cathepsin B cleaved MEPE into two major fragments of approximately 50 and approximately 42kDa. rPhexWT protein in Sf9 membrane fractions, co-incubation of rPhexWT and cathepsin B, and pre-treatment of Sf9 membranes with leupeptin prevented the hydrolysis of MEPE in vitro. The C-terminal domain of Phex was required for inhibition of MEPE cleavage, since the C-terminal deletion mutant rPhex (1-433) [rPhex3(')M] failed to inhibit Sf9-dependent metabolism of MEPE. Phex-dependent inhibition of MEPE degradation, however, did not require Phex enzymatic activity, since EDTA, an inhibitor of rPhex, failed to block rPhexWT inhibition of MEPE cleavage by Sf9 membranes. Since we were unable to identify interactions of Phex with MEPE or actions of Phex to metabolize cathepsin B, Phex may be acting to interfere with the actions of other enzymes that degrade extracellular matrix proteins. Although the molecular mechanism and biological relevance of non-enzymatic actions of Phex need to be established, these findings indicate that MEPE may be involved in the pathogenesis defective mineralization due to Phex deficiency in XLH and the Hyp-mouse.
Assuntos
Proteínas da Matriz Extracelular , Glicoproteínas/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Animais , Catepsina B/metabolismo , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Células Cultivadas , Insetos/citologia , Camundongos , Endopeptidase Neutra Reguladora de Fosfato PHEX , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Hephaestin was implicated in mammalian iron homeostasis following its identification as the defective gene in murine sex-linked anaemia. It is a member of the family of copper oxidases that includes mammalian ceruloplasmin, factors V and VIII, yeast fet3 and fet5 and bacterial ascorbate oxidase. Hephaestin is different from ceruloplasmin, a soluble ferroxidase, in having a membrane-spanning region towards the C-terminus. Here we report the gene structure, spanning approximately 100 kb, of the human homologue of mouse hephaestin. The sequence was assembled from the cDNA clones and the chromosome X genomic sequence data available at the Sanger Centre. It has an open reading frame that encodes a protein of 1158 residues, 85% identical with the murine homologue. A model of the N-terminal ecto-domain has been built based on the known three-dimensional structure of human ceruloplasmin. The overall tertiary structure for the hephaestin and the putative residues involved in binding copper and iron appear to be highly conserved between these proteins, which suggests they share the same fold and a conserved function.