Inactivation of a novel FGF23 regulator, FAM20C, leads to hypophosphatemic rickets in mice.

Family with sequence similarity 20,-member C (FAM20C) is highly expressed in the mineralized tissues of mammals. Genetic studies showed that the loss-of-function mutations in FAM20C were associated with human lethal osteosclerotic bone dysplasia (Raine Syndrome), implying an inhibitory role of this molecule in bone formation. However, in vitro gain- and loss-of-function studies suggested that FAM20C promotes the differentiation and mineralization of mouse mesenchymal cells and odontoblasts. Recently, we generated Fam20c conditional knockout (cKO) mice in which Fam20c was globally inactivated (by crossbreeding with Sox2-Cre mice) or inactivated specifically in the mineralized tissues (by crossbreeding with 3.6 kb Col 1a1-Cre mice). Fam20c transgenic mice were also generated and crossbred with Fam20c cKO mice to introduce the transgene in the knockout background. In vitro gain- and loss-of-function were examined by adding recombinant FAM20C to MC3T3-E1 cells and by lentiviral shRNA-mediated knockdown of FAM20C in human and mouse osteogenic cell lines. Surprisingly, both the global and mineralized tissue-specific cKO mice developed hypophosphatemic rickets (but not osteosclerosis), along with a significant downregulation of osteoblast differentiation markers and a dramatic elevation of fibroblast growth factor 23 (FGF23) in the serum and bone. The mice expressing the Fam20c transgene in the wild-type background showed no abnormalities, while the expression of the Fam20c transgene fully rescued the skeletal defects in the cKO mice. Recombinant FAM20C promoted the differentiation and mineralization of MC3T3-E1 cells. Knockdown of FAM20C led to a remarkable downregulation of DMP1, along with a significant upregulation of FGF23 in both human and mouse osteogenic cell lines. These results indicate that FAM20C is a bone formation "promoter" but not an "inhibitor" in mouse osteogenesis. We conclude that FAM20C may regulate osteogenesis through its direct role in facilitating osteoblast differentiation and its systemic regulation of phosphate homeostasis via the mediation of FGF23.

Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism.

The osteocyte, a terminally differentiated cell comprising 90%-95% of all bone cells, may have multiple functions, including acting as a mechanosensor in bone (re)modeling. Dentin matrix protein 1 (encoded by DMP1) is highly expressed in osteocytes and, when deleted in mice, results in a hypomineralized bone phenotype. We investigated the potential for this gene not only to direct skeletal mineralization but also to regulate phosphate (P(i)) homeostasis. Both Dmp1-null mice and individuals with a newly identified disorder, autosomal recessive hypophosphatemic rickets, manifest rickets and osteomalacia with isolated renal phosphate-wasting associated with elevated fibroblast growth factor 23 (FGF23) levels and normocalciuria. Mutational analyses showed that autosomal recessive hypophosphatemic rickets family carried a mutation affecting the DMP1 start codon, and a second family carried a 7-bp deletion disrupting the highly conserved DMP1 C terminus. Mechanistic studies using Dmp1-null mice demonstrated that absence of DMP1 results in defective osteocyte maturation and increased FGF23 expression, leading to pathological changes in bone mineralization. Our findings suggest a bone-renal axis that is central to guiding proper mineral metabolism.

The rescue of dentin matrix protein 1 (DMP1)-deficient tooth defects by the transgenic expression of dentin sialophosphoprotein (DSPP) indicates that DSPP is a downstream effector molecule of DMP1 in dentinogenesis.

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are essential for the formation of dentin. Previous in vitro studies have indicated that DMP1 might regulate the expression of DSPP during dentinogenesis. To examine whether DMP1 controls dentinogenesis through the regulation of DSPP in vivo, we cross-bred transgenic mice expressing normal DSPP driven by a 3.6-kb rat Col1a1 promoter with Dmp1 KO mice to generate mice expressing the DSPP transgene in the Dmp1 KO genetic background (referred to as "Dmp1 KO/DSPP Tg mice"). We used morphological, histological, and biochemical techniques to characterize the dentin and alveolar bone of Dmp1 KO/DSPP Tg mice compared with Dmp1 KO and wild-type mice. Our analyses showed that the expression of endogenous DSPP was remarkably reduced in the Dmp1 KO mice. Furthermore, the transgenic expression of DSPP rescued the tooth and alveolar bone defects of the Dmp1 KO mice. In addition, our in vitro analyses showed that DMP1 and its 57-kDa C-terminal fragment significantly up-regulated the Dspp promoter activities in a mesenchymal cell line. In contrast, the expression of DMP1 was not altered in the Dspp KO mice. These results provide strong evidence that DSPP is a downstream effector molecule that mediates the roles of DMP1 in dentinogenesis.

Big endothelin changes the cellular miRNA environment in TMOb osteoblasts and increases mineralization.

INTRODUCTION: Endothelin (ET1) promotes the growth of osteoblastic breast and prostate cancer metastases. Conversion of big ET1 to mature ET1, catalyzed primarily by endothelin converting enzyme 1 (ECE1), is necessary for ET1's biological activity. We previously identified the Ece1, locus as a positional candidate gene for a pleiotropic quantitative trait locus affecting femoral size, shape, mineralization, and biomechanical performance. METHODS: We exposed TMOb osteoblasts continuously to 25 ng/ml big ET1. Cells were grown for 6 days in growth medium and then switched to mineralization medium for an additional 15 days with or without big ET1, by which time the TMOb cells form mineralized nodules. We quantified mineralization by alizarin red staining and analyzed levels of miRNAs known to affect osteogenesis. Micro RNA 126-3p was identified by search as a potential regulator of sclerostin (SOST) translation. RESULTS: TMOb cells exposed to big ET1 showed greater mineralization than control cells. Big ET1 repressed miRNAs targeting transcripts of osteogenic proteins. Big ET1 increased expression of miRNAs that target transcripts of proteins that inhibit osteogenesis. Big ET1 increased expression of 126-3p 121-fold versus control. To begin to assess the effect of big ET1 on SOST production we analyzed both SOST transcription and protein production with and without the presence of big ET1 demonstrating that transcription and translation were uncoupled. CONCLUSION: Our data show that big ET1 signaling promotes mineralization. Moreover, the results suggest that big ET1's osteogenic effects are potentially mediated through changes in miRNA expression, a previously unrecognized big ET1 osteogenic mechanism.

Overexpression of Dmp1 fails to rescue the bone and dentin defects in Fam20C knockout mice.

FAM20C is a kinase phosphorylating the small-integrin-binding ligand, N-linked glycoproteins (SIBLINGs), a group of extracellular matrix proteins that are essential for bone and dentin formation. Previously, we showed that Sox2-Cre;Fam20Cfl/fl mice had bone and dentin defects, along with hypophosphatemia and significant downregulation of dentin matrix protein 1 (DMP1). While the assumed phosphorylation failure of the SIBLINGs is likely associated with the defects in the Fam20C-deficient mice, it remains unclear if the downregulation of Dmp1 contributes to these phenotypes. In this study, we crossed 3.6 kb Col1-Dmp1 transgenic mice with 3.6 kb Col1-Cre;Fam20Cfl/fl mice to overexpress Dmp1 in the mineralized tissues of Fam20C conditional knockout (cKO) mice. X-ray, micro-computed tomography, serum biochemistry and histology analyses showed that expressing the Dmp1 transgene failed to rescue the bone and dentin defects, as well as the serum levels of FGF23 and phosphate in the Fam20C-cKO mice. These results indicated that the downregulation of Dmp1 may not directly associate with, or significantly contribute to the bone and dentin defects in the Fam20C-cKO mice.

FAM20C plays an essential role in the formation of murine teeth.

FAM20C is highly expressed in bone and tooth. Previously, we showed that Fam20C conditional knock-out (KO) mice manifest hypophosphatemic rickets, which highlights the crucial roles of this molecule in promoting bone formation and mediating phosphate homeostasis. In this study, we characterized the dentin, enamel, and cementum of Sox2-Cre-mediated Fam20C KO mice. The KO mice exhibited small malformed teeth, severe enamel defects, very thin dentin, less cementum than normal, and overall hypomineralization in the dental mineralized tissues. In situ hybridization and immunohistochemistry analyses revealed remarkable down-regulation of dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein in odontoblasts, along with a sharply reduced expression of ameloblastin and amelotin in ameloblasts. Collectively, these data indicate that FAM20C is essential to the differentiation and mineralization of dental tissues through the regulation of molecules critical to the differentiation of tooth-formative cells.

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.

Therapeutics for enhancement of spinal fusion: A mini review.

OBJECTIVE: With the advances in biological technologies over the past 20 years, a number of new therapies to promote bone healing have been introduced. Particularly in the spinal surgery field, more unprecedented biological therapeutics become available to enhance spinal fusion success rate along with advanced instrumentation approaches. Yet surgeons may not have been well informed about their safety and efficacy profiles in order to improve clinical practices. Therefore there is a need to summarize the evidence and bring the latest progress to surgeons for better clinical services for patients. METHODS: We comprehensively reviewed the literatures in regard to the biological therapeutics for enhancement of spinal fusion published in the last two decades. RESULTS: Autograft bone is still the gold standard for bone grafting in spinal fusion surgery due to its good osteoconductive, osteoinductive, and osteogenic abilities. Accumulating evidence suggests that adding rhBMPs in combination with autograft effectively promotes the fusion rate and improves surgical outcomes. However, the stimulating effect on spinal fusion of other growth factors, including PDGF, VEGF, TGF-beta, and FGF, is not convincing, while Nell-1 and activin A exhibited preliminary efficacy. In terms of systemic therapeutic approaches, the osteoporosis drug Teriparatide has played a positive role in promoting bone healing after spinal surgery, while new medications such as denosumab and sclerostin antibodies still need further validation. Currently, other treatment, such as controlled-release formulations and carriers, are being studied for better releasing profile and the administration convenience of the active ingredients. CONCLUSION: As the world's population continues to grow older, the number of spinal fusion cases grows substantially due to increased surgical needs for spinal degenerative disease (SDD). Critical advancements in biological therapeutics that promote spinal fusion have brought better clinical outcomes to patients lately. With the accumulation of higher-level evidence, the safety and efficacy of present and emerging products are becoming more evident. These emerging therapeutics will shift the landscape of perioperative therapy for the enhancement of spinal fusion.

Endothelin signaling regulates mineralization and posttranscriptionally regulates SOST in TMOb cells via miR 126-3p.

Previously, our laboratory identified ECE-1, encoding endothelin-converting enzyme-1 (ECE-1), as a positional candidate for a pleiotropic quantitative trait locus affecting femoral size, shape, and biomechanical performance. We hypothesized that endothelin-1 (ET-1) signaling promotes osteogenesis. Exposure of immortalized mouse osteoblast (TMOb) cells to big ET-1 increased mineralization. Following big ET-1 treatment, we measured the secretion of insulin-like-growth factor-1 (IGF1), dickkopf-homolog-1 protein 1 (DKK1), and sclerostin (SOST). In each case, big ET-1 signaling changed secretion in a manner that favored increased osteogenic activity. Treatment with ECE-1, endothelin receptor A (EDNRA), or WNT receptor antagonists inhibited the big ET-1-mediated increase in mineralization. In the presence of big ET-1, message levels of Runx2, Igf1, Dkk1, and Sost are uncoupled from protein production, suggesting posttranscriptional regulation. To evaluate the role of big ET-1 in normal bone physiology, we inhibited EDNRA signaling during mineralization in the absence of exogenous ET-1. EDNRA blockade reduced mineralization, decreased IGF1, and increased DKK1 and SOST secretion, responses opposite to those induced by exogenous big ET-1. Pharmacological and siRNA knockdown to inhibit ECE-1 reduced mineralization and IGF1 secretion with decreasing DKK1 and decreasing or stable SOST secretion, suggesting a further, unknown role of ECE-1 in osteoblast maturation. Previously we identified miR 126-3p as a potential ET-1-responsive regulator of SOST in murine cells. Overexpression of miR126-3p increased mineralization in TMOb cells and decreased SOST secretion. Osteoblasts express the ET-1 signaling pathway and ET-1 signaling is necessary for normal osteoblast differentiation and mineralization, acting through regulation of miRs that target osteogenic molecules.

A novel auditory ossicles membrane and the development of conductive hearing loss in Dmp1-null mice.

Genetic mouse models are widely used for understanding human diseases but we know much less about the anatomical structure of the auditory ossicles in the mouse than we do about human ossicles. Furthermore, current studies have mainly focused on disease conditions such as osteomalacia and rickets in patients with hypophosphatemia rickets, although the reason that these patients develop late-onset hearing loss is unknown. In this study, we first analyzed Dmp1 lac Z knock-in auditory ossicles (in which the blue reporter is used to trace DMP1 expression in osteocytes) using X-gal staining and discovered a novel bony membrane surrounding the mouse malleus. This finding was further confirmed by 3-D micro-CT, X-ray, and alizarin red stained images. We speculate that this unique structure amplifies and facilitates sound wave transmissions in two ways: increasing the contact surface between the eardrum and malleus and accelerating the sound transmission due to its mineral content. Next, we documented a progressive deterioration in the Dmp1-null auditory ossicle structures using multiple imaging techniques. The auditory brainstem response test demonstrated a conductive hearing loss in the adult Dmp1-null mice. This finding may help to explain in part why patients with DMP1 mutations develop late-onset hearing loss, and supports the critical role of DMP1 in maintaining the integrity of the auditory ossicles and its bony membrane.

Sclerostin antibody (Scl-Ab) improves osteomalacia phenotype in dentin matrix protein 1(Dmp1) knockout mice with little impact on serum levels of phosphorus and FGF23.

Unlike treatments for most rickets, the treatment using 1,25-(OH)2 vitamin D3 has little efficacy on patients with hypophosphatemic rickets, a set of rare genetic diseases. Thus, understanding the local cause for osteomalacia in hypophosphatemic rickets and developing an effective treatment to restore mineralization in this rare disease has been a longstanding goal in medicine. Here, we used Dmp1 knockout (KO) mice (whose mutations led to the same type of autosomal recessive hypophosphatemic rickets in humans) as the model in which the monoclonal antibody of sclerostin (Scl-Ab) was tested in two age groups for 8weeks: the prevention group (starting at age 4weeks) and the treatment group (starting at age 12weeks). Applications of Scl-Ab greatly improved the osteomalacia phenotype (>15%) and the biomechanical properties (3-point bending, ~60%) in the treated long-bone group. Our studies not only showed improvement of the osteomalacia in the alveolar bone, which has the highest bone metabolism rate, as well as the long bone phenotypes in treated mice. All these improvements attributed to the use of Scl-Ab are independent of the change in serum levels of phosphorus and FGF23, since Scl-Ab had little efficacy on those parameters. Finally, we propose a model to explain how Scl-Ab can improve the Dmp1 KO osteomalacia phenotype, in which the sclerostin level is already low.

Dmp1 Null Mice Develop a Unique Osteoarthritis-like Phenotype.

Patients with hypophosphatemia rickets (including DMP1 mutations) develop severe osteoarthritis (OA), although the mechanism is largely unknown. In this study, we first identified the expression of DMP1 in hypertrophic chondrocytes using immunohistochemistry (IHC) and X-gal analysis of Dmp1-knockout-lacZ-knockin heterozygous mice. Next, we characterized the OA-like phenotype in Dmp1 null mice from 7-week-old to one-year-old using multiple techniques, including X-ray, micro-CT, H&E staining, Goldner staining, scanning electronic microscopy, IHC assays, etc. We found a classical OA-like phenotype in Dmp1 null mice such as articular cartilage degradation, osteophyte formation, and subchondral osteosclerosis. These Dmp1 null mice also developed unique pathological changes, including a biphasic change in their articular cartilage from the initial expansion of hypertrophic chondrocytes at the age of 1-month to a quick diminished articular cartilage layer at the age of 3-months. Further, these null mice displayed severe enlarged knees and poorly formed bone with an expanded osteoid area. To address whether DMP1 plays a direct role in the articular cartilage, we deleted Dmp1 specifically in hypertrophic chondrocytes by crossing the Dmp1-loxP mice with Col X Cre mice. Interestingly, these conditional knockout mice didn't display notable defects in either the articular cartilage or the growth plate. Because of the hypophosphatemia remained in the entire life span of the Dmp1 null mice, we also investigated whether a high phosphate diet would improve the OA-like phenotype. A 8-week treatment of a high phosphate diet significantly rescued the OA-like defect in Dmp1 null mice, supporting the critical role of phosphate homeostasis in maintaining the healthy joint morphology and function. Taken together, this study demonstrates a unique OA-like phenotype in Dmp1 null mice, but a lack of the direct impact of DMP1 on chondrogenesis. Instead, the regulation of phosphate homeostasis by DMP1 via the axis of "FGF23-renal phosphorus reabsorption" is vital for maintaining a healthy joint.

The in vivo role of DMP-1 and serum phosphate on bone mineral composition.

Human DMP1 mutations or Dmp1-null (KO) mice display hypophosphatemia rickets, suggesting a causative role of low phosphate (P) in development of osteomalacia. To address the direct contribution of P to the in vivo bone mineralization we analyzed the properties of femurs obtained from Dmp1 null mice and wild type (WT) mice under a normal or high phosphorous (HiP) diet using combined assays, including histological examination, micro computed tomography (µCT), X-ray absorption near edge structure (XANES) spectroscopy and Raman spectroscopy. Histology and XANES indicate that WT mice have phosphate coordinated with Ca in the form of hydroxyapatite and tricalcium phosphate, while the KO mice have poorly coordinated soluble phosphates in their structure in both the normal and HiP diets. Raman spectroscopy and XANES indicate a higher carbonate/phosphate ratio and a low mineral/matrix ratio in the osteoid clusters in the KO femurs, which was only partially improved by HiP diets. Thus, we conclude that the hypophosphatemia induced osteomalacia phenotype in Dmp1 KO mice is contributed by at least two factors: the low Pi level and the DMP1 local function in mineralization.

Insights into Dahl salt-sensitive hypertension revealed by temporal patterns of renal medullary gene expression.

Dahl salt-sensitive SS and consomic, salt-resistant SS-13(BN)/Mcw rats possess a highly similar genetic background but exhibit substantial differences in blood pressure salt sensitivity. We used cDNA microarrays to examine sequential changes of mRNA expression of approximately 2,000 currently known rat genes in the renal medulla (a tissue critical for long-term blood pressure regulation) in SS and SS-13(BN)/Mcw rats in response to a high-salt diet (16 h, 3 days, or 2 wk). Differentially expressed genes in each between-group comparison were identified based on a threshold determined experimentally using a reference distribution that was constructed by comparing rats within the same group. A difference analysis of 54 microarrays identified 50 genes that exhibited the most distinct temporal patterns of expression between SS and SS-13(BN)/Mcw rats over the entire time course. Thirty of these genes could be linked to the regulation of arterial blood pressure or renal injury based on their known involvement in functional pathways such as renal tubular transport, metabolism of vasoactive substances, extracellular matrix formation, and apoptosis. Importantly, the majority of the 30 genes exhibited temporal expression patterns that would be expected to lower arterial pressure and reduce renal injury in SS-13(BN)/Mcw compared with SS rats. The phenotypic impact of the other 20 genes was less clear. These 50 genes are widely distributed on chromosome 13 and several other chromosomes. This suggested that primary genetic defects, although important, are unlikely to be solely responsible for the full manifestation of this type of hypertension and associated injury phenotypes. In summary, the results of this study identified a number of pathways potentially important for the amelioration of hypertension and renal injury in SS-13(BN)/Mcw rats, and these results generated a series of testable hypotheses related to the role of the renal medulla in the complex mechanism of salt-sensitive hypertension.

Renal medullary genes in salt-sensitive hypertension: a chromosomal substitution and cDNA microarray study.

Substitution of chromosome 13 from Brown Norway BN/SsNHsd/Mcw (BN/Mcw) rats into the Dahl salt-sensitive SS/JrHsd/Mcw (SS/Mcw) rats resulted in substantial reduction of blood pressure salt sensitivity in this consomic rat strain designated SSBN13. In the present study, we attempted to identify genes associated with salt-sensitive hypertension by utilizing a custom, known-gene cDNA microarray to compare the mRNA expression profiles in the renal medulla (a tissue playing a pivotal role in long-term blood pressure regulation) of SS/Mcw and SSBN13 rats on either low-salt (0.4% NaCl) or high-salt (4% NaCl, 2 wk) diets. To increase the reliability of microarray data, we designed a four-way comparison experiment incorporating several levels of replication and developed a conservative yet robust data analysis method. Using this approach, from the 1,751 genes examined (representing more than 80% of all currently known rat genes), we identified 80 as being differentially expressed in at least 1 of the 4 comparisons. Substantial agreements were found between the microarray results and the results predicted on the basis of the four-way comparison as well as the results of Northern blots of 20 randomly selected genes. Analysis of the four-way comparison further indicated that approximately 75% of the 80 differentially expressed genes were likely related to salt-sensitive hypertension. Many of these genes had not previously been recognized to be important in hypertension, whereas several genes/pathways known to be involved in hypertension were confirmed. These results should provide an informative source for designing future functional studies in salt-sensitive hypertension.

Evidence for abnormal translational regulation of renal 25-hydroxyvitamin D-1alpha-hydroxylase activity in the hyp-mouse.

Hyp-mice exhibit abnormal regulation of 25-hydroxyvitamin D [25(OH)D]-1alpha-hydroxylase activity. Previous observations suggest such aberrant modulation is posttranscriptional. To investigate this possibility further, we examined whether hyp-mice manifest abnormal translation of 25(OH)D-1alpha-hydroxylase mRNA. We compared phosphate, parathyroid, and calcitonin effects on renal 25(OH)D-1alpha-hydroxylase protein as well as mRNA and enzyme activity in normal and hyp-mice. We assayed protein by Western blots, mRNA by real-time RT-PCR, and enzyme activity by measuring 1,25-dihydroxyvitamin D production. Although phosphate-depleted mice exhibited enhanced enzyme function, with significantly increased mRNA and protein expression, hyp-mice comparably increased mRNA but failed to augment enzyme activity, concordant with an inability to increase protein expression. PTH stimulation increased mRNA and protein expression as well as enzyme activity in normal mice but in hyp-mice, despite effecting mRNA enhancement, did not increment enzyme function or protein. The inability of hypophosphatemia and PTH to increase 25(OH)D-1alpha-hydroxylase activity and protein expression in hyp-mice was not universal because calcitonin stimulation was normal, suggesting proximal convoluted tubule localization of the defect. These data, in accord with absent undue enhancement of protein expression in hyp-mice treated with protease inhibitors, establish that abberrant regulation of vitamin D metabolism results from abnormal translational activity.

Osteocyte regulation of phosphate homeostasis and bone mineralization underlies the pathophysiology of the heritable disorders of rickets and osteomalacia.

Although recent studies have established that osteocytes function as secretory cells that regulate phosphate metabolism, the biomolecular mechanism(s) underlying these effects remain incompletely defined. However, investigations focusing on the pathogenesis of X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), and autosomal recessive hypophosphatemic rickets (ARHR), heritable disorders characterized by abnormal renal phosphate wasting and bone mineralization, have clearly implicated FGF23 as a central factor in osteocytes underlying renal phosphate wasting, documented new molecular pathways regulating FGF23 production, and revealed complementary abnormalities in osteocytes that regulate bone mineralization. The seminal observations leading to these discoveries were the following: 1) mutations in FGF23 cause ADHR by limiting cleavage of the bioactive intact molecule, at a subtilisin-like protein convertase (SPC) site, resulting in increased circulating FGF23 levels and hypophosphatemia; 2) mutations in DMP1 cause ARHR, not only by increasing serum FGF23, albeit by enhanced production and not limited cleavage, but also by limiting production of the active DMP1 component, the C-terminal fragment, resulting in dysregulated production of DKK1 and ß-catenin, which contributes to impaired bone mineralization; and 3) mutations in PHEX cause XLH both by altering FGF23 proteolysis and production and causing dysregulated production of DKK1 and ß-catenin, similar to abnormalities in ADHR and ARHR, but secondary to different central pathophysiological events. These discoveries indicate that ADHR, XLH, and ARHR represent three related heritable hypophosphatemic diseases that arise from mutations in, or dysregulation of, a single common gene product, FGF23 and, in ARHR and XLH, complimentary DMP1 and PHEX directed events that contribute to abnormal bone mineralization.

Hexa-D-arginine treatment increases 7B2•PC2 activity in hyp-mouse osteoblasts and rescues the HYP phenotype.

Inactivating mutations of the "phosphate regulating gene with homologies to endopeptidases on the X chromosome" (PHEX/Phex) underlie disease in patients with X-linked hypophosphatemia (XLH) and the hyp-mouse, a murine homologue of the human disorder. Although increased serum fibroblast growth factor 23 (FGF-23) underlies the HYP phenotype, the mechanism(s) by which PHEX mutations inhibit FGF-23 degradation and/or enhance production remains unknown. Here we show that treatment of wild-type mice with the proprotein convertase (PC) inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (Dec), increases serum FGF-23 and produces the HYP phenotype. Because PC2 is uniquely colocalized with PHEX in osteoblasts/bone, we examined if PC2 regulates PHEX-dependent FGF-23 cleavage and production. Transfection of murine osteoblasts with PC2 and its chaperone protein 7B2 cleaved FGF-23, whereas Signe1 (7B2) RNA interference (RNAi) transfection, which limited 7B2 protein production, decreased FGF-23 degradation and increased Fgf-23 mRNA and protein. The mechanism by which decreased 7B2•PC2 activity influences Fgf-23 mRNA was linked to reduced conversion of the precursor to bone morphogenetic protein 1 (proBMP1) to active BMP1, which resulted in limited cleavage of dentin matrix acidic phosphoprotein 1 (DMP1), and consequent increased Fgf-23 mRNA. The significance of decreased 7B2•PC2 activity in XLH was confirmed by studies of hyp-mouse bone, which revealed significantly decreased Sgne1 (7B2) mRNA and 7B2 protein, and limited cleavage of proPC2 to active PC2. The expected downstream effects of these changes included decreased FGF-23 cleavage and increased FGF-23 synthesis, secondary to decreased BMP1-mediated degradation of DMP1. Subsequent Hexa-D-Arginine treatment of hyp-mice enhanced bone 7B2•PC2 activity, normalized FGF-23 degradation and production, and rescued the HYP phenotype. These data suggest that decreased PHEX-dependent 7B2•PC2 activity is central to the pathogenesis of XLH.

Protective roles of DMP1 in high phosphate homeostasis.

PURPOSE: Dmp1 (dentin matrix protein1) null mice (Dmp1(-/-)) display hypophosphatemic rickets with a sharp increase in fibroblast growth factor 23 (FGF23). Disruption of Klotho (the obligatory co-receptor of FGF23) results in hyperphosphatemia with ectopic calcifications formed in blood vessels and kidneys. To determine the role of DMP1 in both a hyperphosphatemic environment and within the ectopic calcifications, we created Dmp1/Klotho compound deficient (Dmp1(-/-)kl/kl) mice. PROCEDURES: A combination of TUNEL, immunohistochemistry, TRAP, von Kossa, micro CT, bone histomorphometry, serum biochemistry and Scanning Electron Microscopy techniques were used to analyze the changes in blood vessels, kidney and bone for wild type control, Dmp1(-/-), Klotho deficient (kl/kl) and Dmp1(-/-)kl/kl animals. FINDINGS: Interestingly, Dmp1(-/-)kl/kl mice show a dramatic improvement of rickets and an identical serum biochemical phenotype to kl/kl mice (extremely high FGF23, hyperphosphatemia and reduced parathyroid hormone (PTH) levels). Unexpectedly, Dmp1(-/-)kl/kl mice presented elevated levels of apoptosis in osteocytes, endothelial and vascular smooth muscle cells in small and large blood vessels, and within the kidney as well as dramatic increase in ectopic calcification in all these tissues, as compared to kl/kl. CONCLUSION: These findings suggest that DMP1 has an anti-apoptotic role in hyperphosphatemia. Discovering this novel protective role of DMP1 may have clinical relevance in protecting the cells from apoptosis in high-phosphate environments as observed in chronic kidney disease (CKD).

The biological function of DMP-1 in osteocyte maturation is mediated by its 57-kDa C-terminal fragment.

Dentin matrix protein 1 (DMP-1) is a key molecule in controlling osteocyte formation and phosphate homeostasis. Based on observations that full-length DMP-1 is not found in bone, but only cleaved fragments of 37 and 57 kDa are present, and in view of the finding that mutations in the 57-kDa fragment result in disease, we hypothesized that the 57-kDa C-terminal fragment is the functional domain of DMP-1. To test this hypothesis, a 3.6-kb type I collagen promoter was used to express this 57-kDa C-terminal fragment for comparison with full-length DMP-1 in Dmp1 null osteoblasts/osteocytes. Not only did expression of the full-length DMP-1 in bone cells fully rescue the skeletal abnormalities of Dmp1 null mice, but the 57-kDa fragment also had similar results. This included rescue of growth plate defects, osteomalacia, abnormal osteocyte maturation, and the abnormal osteocyte lacunocanalicular system. In addition, the abnormal fibroblast growth factor 23 (FGF-23) expression in osteocytes, elevated circulating FGF-23 levels, and hypophosphatemia were rescued. These results show that the 57-kDa C-terminal fragment is the functional domain of DMP-1 that controls osteocyte maturation and phosphate metabolism.