RESUMO
Calcium (Ca2+) signaling is the first messenger signal exhibited by osteocytes. The present study aimed to better understand the link between Ca2+ concentration, and the levels of bone mineralization regulator proteins [phosphateregulating neutral endopeptidase on chromosome X (PHEX), matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1)] and the levels of oxidative stress in osteocytes. The viability of MLOY4 cells was determined using the live/dead assay following treatment with various Ca2+ concentrations (1.8, 6, 12, 18, 24 and 50 mM) for different durations (15 and 60 min, and 24 h). Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and NADPH oxidase (NOX) enzymes were analyzed using a colorimetric method. Apoptosis was detected by caspase3 analysis. Furthermore, the protein expression levels of PHEX, MEPE and DMP1 were analyzed using immunoblotting, and oxidative stress was examined using the total antioxidant and total oxidant status (TOS) assay. Notably, after 15 min, there were more live cells than dead cells; however, after 60 min, the number of dead cells was increased following treatment with 24 and 50 mM Ca2+. After 24 h, there were more dead cells than live cells following treatment with 50 mM Ca2+. After 24 h of Ca2+ treatment, the highest protein expression levels of PHEX, MEPE and DMP1 were measured in cells treated with 24 mM Ca2+. In addition, as Ca2+ concentration increased, the TOS and the oxidative stress index values were also increased. In conclusion, these results suggested that 24 mM Ca2+ may trigger bone mineralization proteins, such as PHEX, MEPE and DMP1, and could be considered an applicable dosage for the treatment of bone damage in the future.
Assuntos
Osteócitos , Endopeptidase Neutra Reguladora de Fosfato PHEX , Osteócitos/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Cálcio/metabolismo , Caspase 3/metabolismo , Catalase/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Neprilisina/metabolismo , Antioxidantes/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Glicoproteínas/metabolismo , Fosfatos/metabolismo , Glutationa/metabolismo , NADPH Oxidases/metabolismo , Oxidantes/metabolismo , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: X-linked hypophosphatemic rickets (XLH) is a heterogeneous genetic phosphate wasting disorder that occupies the majority of inheritable hypophosphatemic rickets (HR). XLH is caused by loss-of-function mutations in the phosphate-regulating endopeptidase gene (PHEX) located on the X chromosome. METHOD: In this study, we performed whole-exome sequencing (WES) on the proband to identify the causative gene. The mutations were analyzed by predictive online software, such as PolyPhen-2. Plasmids containing the wild-type (WT) and mutant cDNA of the candidate gene were transfected into HEK293, then, the expression, cellular localization, and glycosylation state of the candidate proteins were detected by western blot, immunostaining, and endoglycosidase H digestion. The expression and concentration of related factor were measured by RT-PCR and ELISA. RESULTS: We identified a novel missense mutation c.2179T>C in the PHEX that results in the substitution of p.Phe727Leu (F727L). This mutation was predicted to be disease-causing by all four predictive online software. In vitro studies demonstrated that the F727L substitution hindered the intracellular trafficking of the mutant PHEX, with ~59% of mutant PHEX protein retained in the endoplasmic reticulum (ER) and only ~16% of the mutant protein localized on the cell surface. Endoglycosidase H digestion assay showed that the mutant F727L PHEX protein was not fully glycosylated. The concentration of intact FGF23 in hFOB1.19 cell culture medium collected from the mutant PHEX group was the highest (62.9 pg/ml) compared to the WT group (32.1 pg/ml) and control group (23.5 pg/ml). CONCLUSION: Our results confirmed that the mutant PHEX protein was lowly glycosylated and retarded within the ER, the intact FGF23 level in cell culture media caused by the mutant PHEX protein was significantly elevated compared to that of the WT group, which may explain why the single base mutation in the PHEX led to XLH syndrome in this family.
Assuntos
Raquitismo Hipofosfatêmico Familiar/genética , Mutação de Sentido Incorreto , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Adulto , Idoso , Retículo Endoplasmático/metabolismo , Raquitismo Hipofosfatêmico Familiar/patologia , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Glicosilação , Células HEK293 , Humanos , Masculino , Endopeptidase Neutra Reguladora de Fosfato PHEX/química , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Linhagem , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Transporte ProteicoRESUMO
Hypophosphatemic rickets is caused by renal phosphate wasting that is most commonly due to X-linked dominant mutations in PHEX. PHEX mutations cause hypophosphatemia indirectly, through the increased expression of fibroblast growth factor 23 (FGF23) by osteocytes. FGF23 decreases renal phosphate reabsorption and thereby increases phosphate excretion. The lack of phosphate leads to a mineralization defect at the level of growth plates (rickets), bone tissue (osteomalacia), and teeth, where the defect facilitates the formation of abscesses. The bone tissue immediately adjacent to osteocytes often remains unmineralized ("periosteocytic lesions"), highlighting the osteocyte defect in this disorder. Common clinical features of XLH include deformities of the lower extremities, short stature, enthesopathies, dental abscesses, as well as skull abnormalities such as craniosynostosis and Chiari I malformation. For the past four decades, XLH has been treated by oral phosphate supplementation and calcitriol, which improves rickets and osteomalacia and the dental manifestations, but often does not resolve all aspects of the mineralization defects. A newer treatment approach using inactivating FGF23 antibodies leads to more stable control of serum inorganic phosphorus levels and seems to heal rickets more reliably. However, the long-term benefits of FGF23 antibody treatment remain to be elucidated.
Assuntos
Raquitismo Hipofosfatêmico Familiar/patologia , Fatores de Crescimento de Fibroblastos/metabolismo , Osteomalacia/patologia , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Fosfatos/metabolismo , Absorciometria de Fóton , Desenvolvimento Ósseo/efeitos dos fármacos , Desenvolvimento Ósseo/genética , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/genética , Calcitriol/administração & dosagem , Raquitismo Hipofosfatêmico Familiar/diagnóstico , Raquitismo Hipofosfatêmico Familiar/tratamento farmacológico , Raquitismo Hipofosfatêmico Familiar/genética , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Humanos , Osteócitos/metabolismo , Osteomalacia/diagnóstico , Osteomalacia/tratamento farmacológico , Osteomalacia/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Comunicação Parácrina/genética , Fosfatos/administração & dosagem , Fosfatos/sangue , Reabsorção Renal/efeitos dos fármacos , Reabsorção Renal/genética , Dente/crescimento & desenvolvimento , Dente/patologia , Resultado do TratamentoRESUMO
Peroxisomes are essential for lipid metabolism and disruption of liver peroxisomal function results in neonatal death. Little is known about how peroxisomal content and activity respond to changes in the lipid environment in human skeletal muscle (HSkM). AIMS: We hypothesized and tested that increased peroxisomal gene/protein expression and functionality occur in HSkM as an adaptive response to lipid oversupply. MATERIALS AND METHODS: HSkM biopsies, derived from a total of sixty-two subjects, were collected for 1) examining correlations between peroxisomal proteins and intramyocellular lipid content (IMLC) as well as between peroxisomal functionality and IMLC, 2) assessing peroxisomal gene expression in response to acute- or 7-day high fat meal (HFM), and in human tissue derived primary myotubes for 3) treating with high fatty acids to induce peroxisomal adaptions. IMLC were measured by both biochemical analyses and fluorescent staining. Peroxisomal membrane protein PMP70 and biogenesis gene (PEX) expression were assessed using western blotting and realtime qRT-PCR respectively. 1-14C radiolabeled lignocerate and palmitate oxidation assays were performed for peroxisomal and mitochondrial functionality respectively. RESULTS: 1) Under fasting conditions, HSkM tissue demonstrated a significant correlation (Pâ¯âªâ¯0.05) between IMCL and the peroxisomal biogenesis factor 19 (PEX19) protein as well as between lipid content and palmitate and lignocerate complete oxidation. 2) Similarly, post-HFM, additional PEX genes (Pex19, PEX11A, and PEX5) were significantly (Pâ¯âªâ¯0.05) upregulated. 3) Increments in PMP70, carnitine octanoyl transferase (CrOT), PGC-1α, and ERRα mRNA were observed post-fatty acid incubation in HSkM cells. PMP70 protein was significantly (Pâ¯âªâ¯0.05) elevated 48-h post lipid treatment. CONCLUSIONS: These results are the first to associate IMLC with peroxisomal gene/protein expression and function in HSkM suggesting an adaptive role for peroxisomes in lipid metabolism in this tissue.
Assuntos
Dieta Hiperlipídica , Expressão Gênica/fisiologia , Músculo Esquelético/metabolismo , Peroxissomos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Biópsia , Ácidos Graxos/metabolismo , Feminino , Humanos , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Peroxissomos/genética , Cultura Primária de Células , Adulto JovemRESUMO
The type I transmembrane protein αKlotho (Klotho) serves as a coreceptor for the phosphaturic hormone fibroblast growth factor 23 (FGF23) in kidney, while a truncated form of Klotho (soluble Klotho, sKL) is thought to exhibit multiple activities, including acting as a hormone, but whose mode(s) of action in different organ systems remains to be fully elucidated. FGF23 is expressed primarily in osteoblasts/osteocytes and aberrantly high levels in the circulation acting via signaling through an FGF receptor (FGFR)-Klotho coreceptor complex cause renal phosphate wasting and osteomalacia. We assessed the effects of exogenously added sKL on osteoblasts and bone using Klotho-deficient (kl/kl) mice and cell and organ cultures. sKL induced FGF23 signaling in bone and exacerbated the hypomineralization without exacerbating the hyperphosphatemia, hypercalcemia and hypervitaminosis D in kl/kl mice. The same effects were seen in rodent bone models in vitro, in which we also detected formation of a sKL complex with FGF23-FGFR and decreased Phex (gene responsible for X-linked hypophosphatemic rickets (XLH)/osteomalacia) expression. Further, sKL-FGF23-dependent hypomineralization in vitro was rescued by soluble PHEX. These data suggest that exogenously added sKL directly participates in FGF23 signaling in bone and that PHEX is a downstream effector of the sKL-FGF23-FGFR axis in bone.
Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Glucuronidase/genética , Glucuronidase/farmacologia , Osteomalacia/genética , Raquitismo/genética , Animais , Animais Recém-Nascidos , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/fisiologia , Células Cultivadas , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Klotho , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteócitos/efeitos dos fármacos , Osteócitos/fisiologia , Osteomalacia/sangue , Osteomalacia/induzido quimicamente , Osteomalacia/patologia , Endopeptidase Neutra Reguladora de Fosfato PHEX/efeitos dos fármacos , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Gravidez , Isoformas de Proteínas/farmacologia , Ratos , Ratos Wistar , Raquitismo/sangue , Raquitismo/induzido quimicamente , Raquitismo/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , SolubilidadeRESUMO
Skeletal development and mineralization are essential processes driven by the coordinated action of neural signals, circulating molecules and local factors. Our previous studies revealed that the novel neuropeptide Pth4, synthesized by hypothalamic cells, was involved in bone metabolism via phosphate regulation in adult zebrafish. Here, we investigate the role of pth4 during skeletal development using single-cell resolution, two-photon laser ablation of Pth4:eGFP-expressing cells and confocal imaging in vivo. Using a stable transgenic Pth4:eGFP zebrafish line, we identify Pth4:eGFP-expressing cells as post-mitotic neurons. After targeted ablation of eGFP-expressing cells in the hypothalamus, the experimental larvae exhibited impaired mineralization of the craniofacial bones whereas cartilage development was normal. In addition to a decrease in pth4 transcript levels, we noted altered expression of phex and entpd5, genes associated with phosphate homeostasis and mineralization, as well as a delay in the expression of osteoblast differentiation markers such as sp7 and sparc. Taken together, these results suggest that Pth4-expressing hypothalamic neurons participate in the regulation of bone metabolism, possibly through regulating phosphate balance during zebrafish development.
Assuntos
Calcificação Fisiológica/genética , Calcinose/genética , Hipotálamo/metabolismo , Neurônios/metabolismo , Osteoblastos/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteínas de Xenopus/genética , Animais , Animais Geneticamente Modificados , Densidade Óssea , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Calcinose/patologia , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipotálamo/crescimento & desenvolvimento , Hipotálamo/lesões , Larva , Terapia a Laser , Neurônios/patologia , Osteoblastos/patologia , Osteogênese/genética , Osteonectina/genética , Osteonectina/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Fosfatos/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Transdução de Sinais , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Mesenchymal stem cells (MSCs) hold tremendous potential as a targeted cell-based delivery platform for inflammatory and cancer therapy. Genetic manipulation of MSCs, however, is challenging, and therefore, most studies using MSCs as therapeutic cell carriers have utilized viral vectors to transduce the cells. Here, we demonstrate, for the first time, an alternative approach for the efficient transfection of MSCs; therapeutic ultrasound (TUS). Using TUS with low intensities and moderate frequencies, MSCs were transfected with a pDNA encoding for PEX, a protein that inhibits tumor angiogenesis, and studied as a cell vehicle for in vivo tumor therapy. TUS application did not alter the MSCs' stemness or their homing capabilities, and the transfected MSCs transcribed biologically active PEX. Additionally, in a mouse model, 70% inhibition of prostate tumor growth was achieved following a single I.V. administration of MSCs that were TUS-transfected with pPEX. Further, the repeated I.V. administration of TUS-pPEX transfected-MSCs enhanced tumor inhibition up to 84%. Altogether, these results provide a proof of concept that TUS-transfected MSCs can be effectively used as a cell-based delivery approach for the prospective treatment of cancer.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Terapia de Alvo Molecular/métodos , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Neoplasias da Próstata/terapia , Transfecção/métodos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Nus , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ratos , Sonicação/métodos , Transfecção/instrumentação , Transgenes , Transplante Heterólogo , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Proteolytic enzymes mediate the activation or inactivation of many physiologic and pathologic processes. The PHEX gene (Phosphate-regulating gene with homologies to endopeptidase on the X chromosome) encodes a metallopeptidase, which is mutated in patients with a prevalent form (1:20,000) of inherited rickets-X-linked hypophosphatemia (XLH). XLH shows growth retardation, hypophosphatemia, osteomalacia, and defective renal phosphate reabsorption and metabolism of vitamin D. Most PHEX studies have focused on bone, and recently we identified osteopontin (OPN) as the first protein substrate for PHEX, demonstrating in the murine model of XLH (Hyp mice) an increase in OPN that contributes to the osteomalacia. Besides its role in bone mineralization, OPN is expressed in many tissues, and therein has different functions. In tumor biology, OPN is known to be associated with metastasis. Here, we extend our PHEX-OPN studies to investigate PHEX expression in a squamous cell carcinoma (SCC) cell line and its possible involvement in modulating OPN function. Real-time PCR showed PHEX-OPN co-expression in SCC cells, with sequencing of the 22 exons showing no mutation of the PHEX gene. Although recombinant PHEX hydrolyze SCC-OPN fragments, unlike in bone cells, SCC-PHEX protein was not predominantly at the plasma membrane. Enzymatic activity assays, FACs and immunoblotting analyses demonstrated that membrane PHEX is degraded by cysteine proteases and the decreased PHEX activity could contribute to inappropriate OPN regulation. These results highlight for the first time PHEX in tumor biology.
Assuntos
Carcinoma de Células Escamosas/patologia , Regulação Neoplásica da Expressão Gênica , Osteopontina/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Proteólise , Membrana Celular/metabolismo , Cisteína Proteases/metabolismo , Ativação Enzimática , Humanos , Osteopontina/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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
Bone sialoprotein (BSP) belongs to the "small integrin-binding ligand N-linked glycoprotein" (SIBLING) family, whose members interact with bone cells and bone mineral. BSP is strongly expressed in bone and we previously showed that BSP knockout (BSP-/-) mice have a higher bone mass than wild type (BSP+/+) littermates, with lower bone remodelling. Because baseline bone formation activity is constitutively lower in BSP-/- mice, we studied the impact of the absence of BSP on in vitro osteogenesis in mouse calvaria cell (MCC) cultures. MCC BSP-/- cultures exhibit fewer fibroblast (CFU-F), preosteoblast (CFU-ALP) and osteoblast colonies (bone nodules) than wild type, indicative of a lower number of osteoprogenitors. No mineralized colonies were observed in BSP-/- cultures, along with little/no expression of either osteogenic markers or SIBLING proteins MEPE or DMP1. Osteopontin (OPN) is the only SIBLING expressed in standard density BSP-/- culture, at higher levels than in wild type in early culture times. At higher plating density, the effects of the absence of BSP were partly rescued, with resumed expression of osteoblast markers and cognate SIBLING proteins, and mineralization of the mutant cultures. OPN expression and amount are further increased in high density BSP-/- cultures, while PHEX and CatB expression are differentiatlly regulated in a manner that may favor mineralization. Altogether, we found that BSP regulates mouse calvaria osteoblast cell clonogenicity, differentiation and activity in vitro in a cell density dependent manner, consistent with the effective skeletogenesis but the low levels of bone formation observed in vivo. The BSP knockout bone microenvironment may alter the proliferation/cell fate of early osteoprogenitors.
Assuntos
Osteogênese , Osteopontina/genética , Crânio/citologia , Animais , Apoptose , Células da Medula Óssea/citologia , Catepsina B/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Feminino , Masculino , Camundongos , Camundongos Knockout , Osteopontina/deficiência , Osteopontina/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Crânio/metabolismoRESUMO
Fibroblast growth factor-23 (FGF23), produced by osteocytes, is the key physiological regulator of phosphate homeostasis. Sepsis patients often experience transient hypophosphataemia, suggesting the regulation of FGF23 levels by pro-inflammatory factors. Here, we used the osteocyte-like cell line IDG-SW3 to investigate the effect of pro-inflammatory stimuli on FGF23 production. In differentiated IDG-SW3 cultures, basal Fgf23 mRNA was dose-dependently up-regulated by pro-inflammatory cytokines TNF, IL-1ß and TWEAK, and bacterial LPS. Similar effects were observed in human bone samples. TNF- and IL-1ß-induced Fgf23 expression was NF-κB-dependent. Conversely, mRNA encoding negative regulators of FGF23, Phex, Dmp1 and Enpp1, were suppressed by TNF, IL-1ß, TWEAK and LPS, independent of NF-κß signalling. Galnt3, the protein product of which protects intact FGF23 protein from furin/furin-like proprotein convertase cleavage, increased in response to these treatments. C-terminal FGF23 and intact FGF23 protein levels also increased, the latter only in the presence of Furin inhibitors, suggesting that enzymatic cleavage exerts critical control of active FGF23 secretion by osteocytes. Our results demonstrate in principle that pro-inflammatory stimuli are capable of increasing osteocyte secretion of FGF23, which may contribute to hypophosphataemia during sepsis and possibly other inflammatory conditions.
Assuntos
Osso e Ossos/metabolismo , Diferenciação Celular , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Osteócitos/metabolismo , Osso e Ossos/patologia , Linhagem Celular , Citocina TWEAK , Proteínas da Matriz Extracelular/metabolismo , Fator de Crescimento de Fibroblastos 23 , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/toxicidade , Lipopolissacarídeos/toxicidade , Osteócitos/patologia , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Fosfoproteínas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Fatores de Necrose Tumoral/toxicidadeRESUMO
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
Familial hypophosphatemic rickets (HR), the most common inherited form of rickets, is a group of inherited renal phosphate wasting disorders characterized by growth retardation, rickets with bone deformities, osteomalacia, poor dental development, and hypophosphatemia. The purpose of this study was to identify the genetic defect responsible for familial HR in a four-generation Chinese Han pedigree by exome sequencing and Sanger sequencing. Clinical features include skeletal deformities, teeth abnormalities, hearing impairments and variable serum phosphate level in patients of this family. A novel deletion mutation, c.1553delT (p.F518Sfs*4), was identified in the X-linked phosphate regulating endopeptidase homolog gene (PHEX). The mutation is predicted to result in prematurely truncated and loss-of-function PHEX protein. Our data suggest that exome sequencing is a powerful tool to discover mutation(s) in HR, a disorder with genetic and clinical heterogeneity. The findings may also provide new insights into the cause and diagnosis of HR, and have implications for genetic counseling and clinical management.
Assuntos
Exoma/genética , Raquitismo Hipofosfatêmico Familiar/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Adolescente , Adulto , Criança , China , Feminino , Aconselhamento Genético , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Análise de SequênciaRESUMO
Developing sensory neurons require neurotrophic support for survival, neurite outgrowth and myelination. The low-density lipoprotein receptor-related protein-1 (LRP1) transactivates Trk receptors and thereby functions as a putative neurotrophin. Herein, we show that LRP1 is abundantly expressed in developing dorsal root ganglia (DRG) and that LRP1-dependent cell signaling supports survival, neurite extension and receptivity to Schwann cells even in the absence of neurotrophins. Cultured embryonic DRG neurons (E15) were treated with previously characterized LRP1 ligands, LRP1-receptor binding domain of α2-macroglobulin (RBD), hemopexin domain of MMP-9 (PEX) or controls (GST) for two weeks. These structurally diverse LRP1 ligands significantly activated and sustained extracellular signal-regulated kinases (ERK1/2) 5-fold (p<0.05), increased expression of growth-associated protein-43(GAP43) 15-fold (P<0.01), and increased neurite outgrowth 20-fold (P<0.01). Primary sensory neurons treated with LRP1 ligands survived > 2 weeks in vitro, to an extent equaling NGF, a finding associated with canonical signaling mechanisms and blockade of caspase-3 cleavage. LRP1 ligand-induced survival and sprouting were blocked by co-incubation with the LRP1 antagonist, receptor associated protein (RAP), whereas RAP had no effect on NGF-induced activity. Site directed mutagenesis of the LRP1 ligand, RBD, in which Lys(1370) and Lys(1374) are converted to alanine to preclude LRP1 binding, were ineffective in promoting cell signaling, survival or inducing neurite extension in primary sensory neurons, confirming LRP1 specificity. Furthermore, LRP1-induced neurite sprouting was mediated by Src-family kinase (SFK) activation, suggesting transactivation of Trk receptors. Co-cultures of primary embryonic neurons and Schwann cells showed that LRP1 agonists promoted axonal receptivity to myelination to Schwann cells. Collectively, these findings identify LRP1 as a novel and perhaps essential trophic molecule for sensory neuronal survival and development.
Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Proteína GAP-43/metabolismo , Gânglios Espinais/metabolismo , Ligantes , Sistema de Sinalização das MAP Quinases/fisiologia , Fatores de Crescimento Neural/metabolismo , Neuritos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Células PC12 , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor trkA/metabolismo , Células de Schwann , alfa-Macroglobulinas/metabolismo , Quinases da Família src/metabolismoRESUMO
Bone is mineralized when hydroxyapatite crystals derived from calcium ions and inorganic phosphate (Pi) grow along collagen fibrils in the extracellular matrix. Mineralization is initiated by nucleation of those crystals. Mature osteoblasts secrete matrix vesicles into osteoid, which contain growing hydroxyapatite crystal seeds. After rupture of the lipid bilayer of those vesicles, crystals continue to grow as a mineralized nodule and adhere to collagen fibrils. It remains controversial whether nucleation occurs mainly in matrix vesicles or also extra-vesicularly around collagen fibrils. Mineralization is inhibited by pyrophosphate (PPi) and by SIBLING family proteins, which carry an acidic serine- and aspartate-rich motif (ASARM) and include osteopontin, dentin matrix protein 1 and MEPE. Intracellular and extracellular activity of these factors is regulated by the PPi-generating ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1) , the PPi-transporter progressive ankylosis (ANK) protein, the PPi-degrading/Pi-generating ectoenzyme alkaline phosphatase (ALPL, TNAP) , and PHEX endopeptidase. Gain- or loss-of-function mutations in genes encoding these proteins are associated with mineralization disorders such as ectopic calcification and other pathologies.
Assuntos
Calcificação Fisiológica/fisiologia , Raquitismo/metabolismo , Animais , Difosfatos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Transdução de SinaisRESUMO
Calcium, in combination with vitamin D, is an effective treatment for osteoporosis. Since bone mineralisation occurs concurrently with osteoblast to osteocyte transition, we hypothesised that calcium would stimulate this process. The effect of calcium (1.8-11.8mM) was tested on human primary osteoblast (NHBC) differentiation in vitro. Cultures were assayed for cell-associated mineral and gene expression associated with osteoblast differentiation and mineralisation. Treatment with calcium resulted in a striking dose- and time-dependent increase in cell-associated mineralisation. Calcium appeared to promote osteoblast to osteocyte differentiation, as indicated by increased expression of osteocalcin (OCN), E11, dentin matrix protein 1 (DMP1) and SOST mRNA. The expression of the osteoclast inhibitor, osteoprotegerin, was dramatically enhanced by calcium. Calcium also increased the ratio of PHEX mRNA expression relative to that of MEPE, suggesting a mechanism for the pro-anabolic effect. Consistent with this, calcium-dependent mineralisation was reversed in the presence of MEPE-ASARM peptides. This study suggests that calcium promotes osteoblast to osteocyte transition and concurrent matrix mineralisation, at least in part through the PHEX-MEPE axis.
Assuntos
Cálcio/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , RNA Mensageiro/genética , Proteínas Adaptadoras de Transdução de Sinal , Biomarcadores/metabolismo , Proteínas Morfogenéticas Ósseas/agonistas , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Relação Dose-Resposta a Droga , Proteínas da Matriz Extracelular/agonistas , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Marcadores Genéticos/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/agonistas , Osteocalcina/genética , Osteocalcina/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Osteoprotegerina/agonistas , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Fosfoproteínas/agonistas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Cultura Primária de Células , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
X-linked hypophosphatemia (XLH/HYP)-with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscesses-is caused by mutations in the zinc-metallopeptidase PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization-inhibiting, acidic serine- and aspartate-rich motif (ASARM)-containing peptides, which are proteolytically derived from the mineral-binding matrix proteins of the SIBLING family (small, integrin-binding ligand N-linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motif-osteopontin (OPN) and bone sialoprotein (BSP)-as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full-length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an â¼35 kDa OPN fragment that was not present in wild-type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild-type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. In conclusion, these results identify full-length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization-inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP.
Assuntos
Osso e Ossos/metabolismo , Raquitismo Hipofosfatêmico Familiar/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X , Osteopontina/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Osteopontina/química , ProteóliseRESUMO
Dentin matrix protein-1 (DMP1) or phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) inactivation results in elevation of the phosphaturic hormone fibroblast growth factor (FGF)-23, leading to hypophosphatemia, aberrant vitamin D metabolism, and rickets/osteomalacia. Compound mutant Phex-deficient Hyp and Dmp1(ko) mice exhibit nonadditive phenotypes, suggesting that DMP1 and PHEX may have interdependent effects to regulate FGF23 and bone mineralization. To determine the relative importance of DMP1 and PHEX in regulating FGF23 and mineralization, we tested whether the transgenic expression of full-length [Dmp1(Tg(full-length))] or C-terminal Dmp1 [Dmp1(Tg(57kDa))] could rescue the phenotype of Hyp mice. We found that Dmp1(ko) and Hyp mice have similar phenotypes characterized by decreased cortical bone mineral density (-35% vs. wild type, P < 0.05) and increased serum FGF23 levels (~12-fold vs. wild type, P < 0.05). This was significantly corrected by the overexpression of either the full-length or the C-terminal transgene in Dmp1(ko) mice. However, neither of the transgenes rescued the Hyp mice phenotype. Hyp/Dmp1(Tg(full-length)) and Hyp mice were similar, but Hyp/Dmp1(Tg(57 kDa)) mice exhibited worsening of osteomalacia (-20% cortical bone mineral density) in association with increased serum FGF23 levels (+2-fold) compared with Hyp mice. Bone FGF23 mRNA expression was decreased and a 2-fold increase in the ratio of the full-length/degraded circulating FGF23 was observed, indicating that degradation of FGF23 was impaired in Hyp/Dmp1(Tg(57 kDa)) mice. The paradoxical effects of the C-terminal Dmp1 transgene were observed in Hyp/Dmp1(Tg(57 kDa)) but not in Dmp1(Tg(57 kDa)) mice expressing a functional PHEX. These findings indicate a functional interaction between PHEX and DMP1 to regulate bone mineralization and circulating FGF23 levels and for the first time demonstrate effects of the C-terminal DMP1 to regulate FGF23 degradation.
Assuntos
Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Raquitismo Hipofosfatêmico Familiar/patologia , Fatores de Crescimento de Fibroblastos/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Peso Corporal , Calcificação Fisiológica , Raquitismo Hipofosfatêmico Familiar/sangue , Raquitismo Hipofosfatêmico Familiar/diagnóstico por imagem , Raquitismo Hipofosfatêmico Familiar/urina , Fêmur/diagnóstico por imagem , Fêmur/patologia , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Modelos Biológicos , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , 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
PHosphate-regulating gene with homology to Endopeptidase on the X chromosome (PHEX) has been identified as the gene mutated in X-linked hypophosphatemia (XLH) syndrome, the most prevalent form of rickets in humans. The predominant expression of PHEX in bones and teeth, and the defective mineralization of these tissues in XLH patients indicate that PHEX is an important regulator of mineralization. Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are known to regulate the expression of numerous genes in osteoblastic cells through activation of the protein kinase A pathway, including repression of PHEX. PTH also activates the transcriptional repressor E4BP4 through the same pathway, suggesting that PTH or PTHrP-mediated repression of PHEX expression could involve E4BP4. To evaluate this possibility, we treated UMR-106 osteoblastic cells with PTHrP(1-34), and used RT-PCR and immunoblotting to analyze PHEX and E4BP4 expression. E4BP4 mRNA and protein levels were rapidly increased in cells treated with PTHrP(1-34), with a concomitant decrease in PHEX expression. This downregulation of PHEX could be reproduced by overexpression of E4BP4. Moreover, PTHrP(1-34)-mediated PHEX repression was blocked when cells were transfected with a siRNA targeting E4BP4 mRNA. Finally, DNA pull-down and luciferase assays showed that two E4BP4 response elements located in PHEX promoter were functional. These results underline the important role of E4BP4 in osteoblastic cells and further define the repression mechanism of PHEX gene by PTHrP(1-34).