Caspase-1 and the inflammasome promote polycystic kidney disease progression.

We and others have previously shown that the presence of renal innate immune cells can promote polycystic kidney disease (PKD) progression. In this study, we examined the influence of the inflammasome, a key part of the innate immune system, on PKD. The inflammasome is a system of molecular sensors, receptors, and scaffolds that responds to stimuli like cellular damage or microbes by activating Caspase-1, and generating critical mediators of the inflammatory milieu, including IL-1ß and IL-18. We provide evidence that the inflammasome is primed in PKD, as multiple inflammasome sensors were upregulated in cystic kidneys from human ADPKD patients, as well as in kidneys from both orthologous (PKD1 RC/RC or RC/RC) and non-orthologous (jck) mouse models of PKD. Further, we demonstrate that the inflammasome is activated in female RC/RC mice kidneys, and this activation occurs in renal leukocytes, primarily in CD11c+ cells. Knock-out of Casp1, the gene encoding Caspase-1, in the RC/RC mice significantly restrained cystic disease progression in female mice, implying sex-specific differences in the renal immune environment. RNAseq analysis implicated the promotion of MYC/YAP pathways as a mechanism underlying the pro-cystic effects of the Caspase-1/inflammasome in females. Finally, treatment of RC/RC mice with hydroxychloroquine, a widely used immunomodulatory drug that has been shown to inhibit the inflammasome, protected renal function specifically in females and restrained cyst enlargement in both male and female RC/RC mice. Collectively, these results provide evidence for the first time that the activated Caspase-1/inflammasome promotes cyst expansion and disease progression in PKD, particularly in females. Moreover, the data suggest that this innate immune pathway may be a relevant target for therapy in PKD.

Correction of Vascular Calcification and Hyperphosphatemia in CKD Rats Treated with ASARM Peptide.

Background: Abnormalities in calcium, phosphorus, PTH, vitamin D metabolism, bone, and vascular calcification occur in chronic kidney disease mineral bone disorder (CKD-MBD). Calciphylaxis, involving painful, ulcerative skin lesions, is also a major problem associated with CKD-MBD. There are no quality medical interventions to address these clinical issues. Bone ASARM peptides are strong inhibitors of mineralization and induce hypophosphatemia by inhibiting phosphate uptake from the gut. We hypothesize treatment of CKD-MBD rats with ASARM peptides will reverse hyperphosphatemia, reduce soft-tissue calcification, and prevent calciphylaxis. Methods: To test our hypothesis, we assessed the effects of synthetic ASARM peptide in rats that had undergone a subtotal 5/6th nephrectomy (56NEPHREX), a rodent model of CKD-MBD. All rats were fed a high phosphate diet (2% Pi) to worsen mineral metabolism defects. Changes in serum potassium, phosphate, BUN, creatinine, PTH, FGF23, and calcium were assessed in response to 28 days of ASARM peptide infusion. Also, changes in bone quality, soft-tissue calcification, and expression of gut Npt2b (Slc34a2) were studied following ASARM peptide treatment. Results: Rats that had undergone 56NEPHREX treated with ASARM peptide showed major improvements in hyperphosphatemia, blood urea nitrogen (BUN), and bone quality compared with vehicle controls. Also, ASARM-infused 56NEPHREX rats displayed improved renal, brain, and cardiovascular calcification. Notably, ASARM peptide infusion prevented the genesis of subdermal medial blood vessel calcification and calciphylaxis-like lesions in 56NEPHREX rats compared with vehicle controls. Conclusions: ASARM peptide infusion corrects hyperphosphatemia and improves vascular calcification, renal calcification, brain calcification, bone quality, renal function, and skin mineralization abnormalities in 56NEPHREX rats. These findings confirm our hypothesis and support the utility of ASARM peptide treatment in patients with CKD-MBD.

Critical Role of Osteopontin in Maintaining Urinary Phosphate Solubility in CKD.

Background: Nephron loss dramatically increases tubular phosphate to concentrations that exceed supersaturation. Osteopontin (OPN) is a matricellular protein that enhances mineral solubility in solution; however, the role of OPN in maintaining urinary phosphate solubility in CKD remains undefined. Methods: Here, we examined (1) the expression patterns and timing of kidney/urine OPN changes in CKD mice, (2) if tubular injury is necessary for kidney OPN expression in CKD, (3) how OPN deletion alters kidney mineral deposition in CKD mice, (4) how neutralization of the mineral-binding (ASARM) motif of OPN alters kidney mineral deposition in phosphaturic mice, and (5) the in vitro effect of phosphate-based nanocrystals on tubular epithelial cell OPN expression. Results: Tubular OPN expression was dramatically increased in all studied CKD murine models. Kidney OPN gene expression and urinary OPN/Cr ratios increased before changes in traditional biochemical markers of kidney function. Moreover, a reduction of nephron numbers alone (by unilateral nephrectomy) was sufficient to induce OPN expression in residual nephrons and induction of CKD in OPN-null mice fed excess phosphate resulted in severe nephrocalcinosis. Neutralization of the ASARM motif of OPN in phosphaturic mice resulted in severe nephrocalcinosis that mimicked OPN-null CKD mice. Lastly, in vitro experiments revealed calcium-phosphate nanocrystals to induce OPN expression by tubular epithelial cells directly. Conclusions: Kidney OPN expression increases in early CKD and serves a critical role in maintaining tubular mineral solubility when tubular phosphate concentrations are exceedingly high, as in late-stage CKD. Calcium-phosphate nanocrystals may be a proximal stimulus for tubular OPN production.

Comprehensive phenotyping of cutaneous afferents reveals early-onset alterations in nociceptor response properties, release of CGRP, and hindpaw edema following spinal cord injury.

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.

Do ASARM peptides play a role in nephrogenic systemic fibrosis?

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.

Age dependent regulation of bone-mass and renal function by the MEPE ASARM-motif.

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.

A unified model for bone-renal mineral and energy metabolism.

The beginning of the millennium saw the discovery of a new bone-matrix protein, Matrix Extracellular PhosphoglycoprotEin (MEPE) and an associated C-terminal motif called ASARM. This motif and other distinguishing features occur in a group of proteins called SIBLINGs. These proteins include dentin matrix protein 1 (DMP1), osteopontin, dentin-sialophosphoprotein (DSPP), statherin, bone sialoprotein (BSP) and MEPE. MEPE, DMP1 and ASARM-motifs regulate expression of a phosphate regulating cytokine FGF23. Further, a trimeric interaction between phosphate regulating endopeptidase homolog X-linked (PHEX), DMP1, and α5ß3-integrin that occurs on the plasma-membrane of the osteocyte mediates FGF23 regulation (FAP pathway). ASARM-peptides competitively inhibit the trimeric complex and increase FGF23. A second pathway involves specialized structures, matrix vesicles pathway (MVP). This review will discuss the FAP and MVP pathways and present a unified model for mineral and energy metabolism.

SPR4-peptide alters bone metabolism of normal and HYP mice.

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.

PHEX mimetic (SPR4-peptide) corrects and improves HYP and wild type mice energy-metabolism.

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.

MEPE-derived ASARM peptide inhibits odontogenic differentiation of dental pulp stem cells and impairs mineralization in tooth models of X-linked hypophosphatemia.

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide - a substrate for PHEX and a strong inhibitor of mineralization - derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.

The chicken or the egg: PHEX, FGF23 and SIBLINGs unscrambled.

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.

Regulation of bone-renal mineral and energy metabolism: the PHEX, FGF23, DMP1, MEPE ASARM pathway.

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.

Sclerostin is a locally acting regulator of late-osteoblast/preosteocyte differentiation and regulates mineralization through a MEPE-ASARM-dependent mechanism.

The identity of the cell type responsive to sclerostin, a negative regulator of bone mass, is unknown. Since sclerostin is expressed in vivo by mineral-embedded osteocytes, we tested the hypothesis that sclerostin would regulate the behavior of cells actively involved in mineralization in adult bone, the preosteocyte. Differentiating cultures of human primary osteoblasts exposed to recombinant human sclerostin (rhSCL) for 35 days displayed dose- and time-dependent inhibition of in vitro mineralization, with late cultures being most responsive in terms of mineralization and gene expression. Treatment of advanced (day 35) cultures with rhSCL markedly increased the expression of the preosteocyte marker E11 and decreased the expression of mature markers DMP1 and SOST. Concomitantly, matrix extracellular phosphoglycoprotein (MEPE) expression was increased by rhSCL at both the mRNA and protein levels, whereas PHEX was decreased, implying regulation through the MEPE-ASARM axis. We confirmed that mineralization by human osteoblasts is exquisitely sensitive to the triphosphorylated ASARM-PO4 peptide. Immunostaining revealed that rhSCL increased the endogenous levels of MEPE-ASARM. Importantly, antibody-mediated neutralization of endogenous MEPE-ASARM antagonized the effect of rhSCL on mineralization, as did the PHEX synthetic peptide SPR4. Finally, we found elevated Sost mRNA expression in the long bones of HYP mice, suggesting that sclerostin may drive the increased MEPE-ASARM levels and mineralization defect in this genotype. Our results suggest that sclerostin acts through regulation of the PHEX/MEPE axis at the preosteocyte stage and serves as a master regulator of physiologic bone mineralization, consistent with its localization in vivo and its established role in the inhibition of bone formation.

ASARM peptides: PHEX-dependent and -independent regulation of serum phosphate.

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.

Abnormal presence of the matrix extracellular phosphoglycoprotein-derived acidic serine- and aspartate-rich motif peptide in human hypophosphatemic dentin.

Severe dental troubles are associated with X-linked hypophosphatemic rickets and are mainly related to impaired dentin mineralization. In dentin matrix, matrix extracellular phosphoglycoprotein (MEPE) may be protected from proteolysis by a specific interaction with PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome). The objective of our work was to determine whether PHEX impairment induces MEPE cleavage in dentin and the subsequent release of the C-terminal acidic serine- and aspartate-rich motif (ASARM) peptide, which is known to inhibit mineralization. By Western blot analysis, we explored dentin extracts from seven hypophosphatemic patients with mutations of the PHEX gene. A proteomic approach combining immunoprecipitation, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry and matrix-assisted laser desorption ionization-time of flight analysis of the samples completed this exploration. This study shows a 4.1-kDa peptide containing the MEPE-derived ASARM peptide in hypophosphatemic samples. The presence of ASARM was less marked in patients treated with 1-hydroxylated vitamin D and phosphate during growth. Moreover, recombinant ASARM implanted in a rat pulp injury model disturbed the formation of the reparative dentin bridge. These results suggest that abnormal MEPE cleavage occurs when PHEX activity is deficient in humans, the ASARM peptide may be involved in the mineralization defects and the PHEX-MEPE interaction may be indirect, as ensuring a better phosphate and vitamin D environment to the mineralizing dentin prevents MEPE cleavage.

MEPE's diverse effects on mineralization.

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.

MEPE/OF45 protects cells from DNA damage induced killing via stabilizing CHK1.

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.

Matrix extracellular phosphoglycoprotein (MEPE) is a new bone renal hormone and vascularization modulator.

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.

Dentin noncollagenous matrix proteins in familial hypophosphatemic rickets.

Familial hypophosphatemic rickets is transmitted in most cases as an X-linked dominant trait and results from the mutation of the PHEX gene predominantly expressed in osteoblast and odontoblast. Patients with rickets have been reported to display important dentin defects. Our purpose was to explore the structure, composition and distribution of noncollagenous proteins (NCPs) of hypophosphatemic dentin. We collected teeth from 10 hypophosphatemic patients whose mineralization occurred either in a hypophosphatemic environment or in a corrected phosphate and vitamin environment. Teeth were examined by scanning electron microscopy, immunohistochemistry and Western blot analysis. An abnormal distribution (accumulation in interglobular spaces) and cleavage of the NCPs and particularly of matrix extracellular phosphoglycoprotein were observed in deciduous dentin. In contrast, it was close to normal in permanent dentin mineralized under corrected conditions. In conclusion, dentin mineralization in a corrected phosphate and vitamin D environment compensates the adverse effect of PHEX mutation.

Aberrant Phex function in osteoblasts and osteocytes alone underlies murine X-linked hypophosphatemia.

Patients with X-linked hypophosphatemia (XLH) and the hyp-mouse, a model of XLH characterized by a deletion in the Phex gene, manifest hypophosphatemia, renal phosphate wasting, and rickets/osteomalacia. Cloning of the PHEX/Phex gene and mutations in affected patients and hyp-mice established that alterations in PHEX/Phex expression underlie XLH. Although PHEX/Phex expression occurs primarily in osteoblast lineage cells, transgenic Phex expression in hyp-mouse osteoblasts fails to rescue the phenotype, suggesting that Phex expression at other sites underlies XLH. To establish whether abnormal Phex in osteoblasts and/or osteocytes alone generates the HYP phenotype, we created mice with a global Phex knockout (Cre-PhexDeltaflox/y mice) and conditional osteocalcin-promoted (OC-promoted) Phex inactivation in osteoblasts and osteocytes (OC-Cre-PhexDeltaflox/y). Serum phosphorus levels in Cre-PhexDeltaflox/y, OC-Cre-PhexDeltaflox/y, and hyp-mice were lower than those in normal mice. Kidney cell membrane phosphate transport in Cre-PhexDeltaflox/y, OC-Cre-PhexDeltaflox/y, and hyp-mice was likewise reduced compared with that in normal mice. Abnormal renal phosphate transport in Cre-PhexDeltaflox/y and OC-Cre-PhexDeltaflox/y mice was associated with increased bone production and serum FGF-23 levels and decreased kidney membrane type IIa sodium phosphate cotransporter protein, as was the case in hyp-mice. In addition, Cre-PhexDeltaflox/y, OC-Cre-PhexDeltaflox/y, and hyp-mice manifested comparable osteomalacia. These data provide evidence that aberrant Phex function in osteoblasts and/or osteocytes alone is sufficient to underlie the hyp-mouse phenotype.