RESUMO
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
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 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
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
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
Polycystic kidney disease (PKD) is a lethal disorder characterized by progressive expansion of renal cysts. Genetic mutations associated with PKD are thought to disrupt intracellular Ca2+ regulation, leading to abnormal proliferation of tubule epithelial cells. cAMP stimulates the B-Raf/MEK/extracellular signal-regulated kinase (B-Raf/MEK/ERK) pathway and accelerates the proliferation of cells that are cultured from PKD cysts. By contrast, cAMP inhibits the proliferation of cells from normal human kidneys (NHK) and M-1 mouse collecting duct cells. Previously, it was found that a sustained reduction of intracellular Ca2+ levels in NHK and M-1 cells that were treated with Ca2+ entry blockers allowed cAMP activation of the B-Raf/MEK/ERK pathway, switching the cells to a cAMP-growth stimulated phenotype. In this study, primary cultures of cyst epithelial cells from autosomal dominant (ADPKD) and recessive (ARPKD) PKD kidneys were used to determine whether controlled addition of Ca2+ could reverse the aberrant mitogenic response to cAMP. Steady-state intracellular Ca2+ levels were found to be 20 nM lower in cyst-derived ADPKD cells (57 +/- 2 nM) compared with NHK cells (77 +/- 2 nM). Treatment of ADPKD cells or ARPKD cells with either Bay K8644, a Ca2+ channel activator, or A23187, a Ca2+ ionophore, caused sustained increases in intracellular Ca2+ levels and completely reversed the mitogenic response to cAMP. Elevation of intracellular Ca2+ levels in ADPKD cells increased Akt activity and blocked cAMP-dependent B-Raf and ERK activation. Thus, increases in [Ca2+]i are able to restore the normal anti-mitogenic response to cAMP in cells that are derived from two genetically distinct forms of PKD.
Assuntos
Cálcio/metabolismo , Doenças Renais Policísticas/patologia , Proliferação de Células , Células Cultivadas , AMP Cíclico/farmacologia , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Rim/metabolismo , MAP Quinase Quinase Quinases/fisiologia , Fenótipo , Doenças Renais Policísticas/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Recessivo/metabolismo , Proteínas Proto-Oncogênicas B-raf/fisiologia , Transdução de SinaisRESUMO
Transepithelial salt and fluid secretion mediated by cAMP in initial inner medullary collecting ducts (IMCDi) may be important for making final adjustments to urine composition. We examined in primary cultures of human IMCDi cells the effects of adrenergic receptor (AR) agonists and antagonists on intracellular cAMP levels, short-circuit current (I(SC)), and fluid secretion. Epinephrine (1 microM), norepinephrine (1 microM), and isoproterenol (10 nM) individually increased intracellular cAMP levels 57-, 2-, and 25-fold, respectively, and stimulated I(SC) 3.3-, 2.9-, and 3.4-fold, respectively. beta-AR activation increased net fluid secretion by cultured human IMCDi cell monolayers from 0.09 +/- 0.04 to 0.26 +/- 0.05 microl x h(-1) x cm(-2) and freshly isolated rat IMCDi from 0.02 +/- 0.01 to 0.09 +/- 0.02 nl x h(-1) x mm(-1). In monolayers, these effects were eliminated by blocking beta2-AR, but not beta1-AR. Activation of alpha2-AR with guanabenz inhibited isoproterenol-induced I(SC) by 37% in human IMCDi monolayers and fluid secretion by 91% in rat IMCDi. Immunohistochemistry of human medullary tissue sections revealed greater expression of beta2-AR than beta1-AR; beta2-AR was localized to the basolateral membranes of human IMCDi. Immunoblots identified alpha2A-AR and alpha2B-AR in cultured human IMCDi cell monolayers. We conclude that 1) catecholamines stimulate cAMP-dependent anion and fluid secretion by IMCDi cells primarily through beta2-AR activation and 2) alpha2-AR activation attenuates cAMP-dependent anion secretion.