Degradation of MEPE, DMP1, and Release of SIBLING ASARM-Peptides (Minhibins): ASARM-Peptide(s) Are Directly Responsible for Defective Mineralization in HYP

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) and DMP1 (dentin matrix protein 1) result in X-linked hypophosphatemic rickets (HYP) and autosomal-recessive hypophosphatemic-rickets (ARHR), respectively. Specific binding of PHEX to matrix extracellular phosphoglycoprotein (MEPE) regulates the release of small protease-resistant MEPE peptides [acidic serine- and aspartate-rich MEPE-associated motif (ASARM) peptides]. ASARM peptides are potent inhibitors of mineralization (minhibins) that also occur in DMP1 [MEPE-related small integrin-binding ligand, N-linked glycoprotein (SIBLING) protein]. It is not known whether these peptides are directly responsible for the mineralization defect. We therefore used a bone marrow stromal cell (BMSC) coculture model, ASARM peptides, anti-ASARM antibodies, and a small synthetic PHEX peptide (SPR4; 4.2 kDa) to examine this. Surface plasmon resonance (SPR) and two-dimensional 1H/15N nuclear magnetic resonance demonstrated specific binding of SPR4 peptide to ASARM peptide. When cultured individually for 21 d, HYP BMSCs displayed reduced mineralization compared with wild type (WT) (−87%, P < 0.05). When cocultured, both HYP and WT cells failed to mineralize. However, cocultures (HYP and WT) or monocultures of HYP BMSCs treated with SPR4 peptide or anti-ASARM neutralizing antibodies mineralized normally. WT BMSCs treated with ASARM peptide also failed to mineralize properly without SPR4 peptide or anti-ASARM neutralizing antibodies. ASARM peptide treatment decreased PHEX mRNA and protein (−80%, P < 0.05) and SPR4 peptide cotreatment reversed this by binding ASARM peptide. SPR4 peptide also reversed ASARM peptide-mediated changes in expression of key osteoclast and osteoblast differentiation genes. Western blots of HYP calvariae and BMSCs revealed massive degradation of both MEPE and DMP1 protein compared with the WT. We conclude that degradation of MEPE and DMP-1 and release of ASARM peptides are chiefly responsible for the HYP mineralization defect and changes in osteoblast-osteoclast differentiation.We acknowledge the very kind gift of pure sPHEX by Dr. Philippe Crine (Department of Biochemistry, University of Montreal, and Enobia Pharma). Also, we acknowledge the anti-DMP1 antibodies generously donated by Dr. Larry Fisher, National Institute of Dental and Craniofacial Research, Bethesda, MD. Address all correspondence and requests for reprints to: Peter S. N. Rowe, Department of Internal Medicine, Division of Nephrology and Hypertension, The Kidney Institute, MS 3018, 3901 Rainbow Boulevard, Kansas City, Kansas 66160. E-mail: [email protected]. We acknowledge the generous financial support from the National Institutes of Health to P.S.N.R. (RO-1 AR51598-01; National Institute of Arthritis and Musculoskeletal Diseases). Also, the SPR experiments were performed in the UTHSCSA Center for Macromolecular Interactions, which is supported by grants from the National Cancer Institute (CA54174) and UTHSCSA Executive Research Committee Research fund

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

PHEX or DMP1 mutations cause hypophosphatemic-rickets and altered energy metabolism. PHEX binds to DMP1-ASARM-motif to form a complex with α5β3 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.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.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.ASARM peptides and motifs are physiological substrates for PHEX and modulate osteocyte PHEX-DMP1-α5β3-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

SPR4-peptide induces a dramatic increase in fat mass/weight in HYP and WT mice.

<p>Dual Energy X-ray Absorptiometry (DEXA) measurements using a Lunar PIXImus system were carried out as described previously and discussed in methods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone.0097326-David2" target="_blank">[140]</a>. Measurements are shown for mice prior to pump implantation and after sacrifice 28 days later. The temporal percentage change measurements are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-g007" target="_blank">Figure 7</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t005" target="_blank">Table 5</a>. (<b>A</b>) <b><u>Percentage Fat Mass (% FAT Mass)</u></b>. HYPVE %-Fat-Mass was significantly less than WTVE %-Fat-Mass at all time-points. SPR4 peptide treatment significantly increased time-dependent gain in %-Fat-Mass for HYP mice (HYPSPR4) but not WT mice (WTSPR4) relative to respective vehicle groups. Following 2-way ANOVA analysis, phenotypic variation (including SPR4-treatment) was highly significant accounting for 31.86% of the total variance (F = 46.72, DF_n = 3, Df_d = 32 and <i>P<0.0001</i>). Also, time-changes were highly significant accounting for 53.69% of the total variance (F = 236.22, DF_n = 1, Df_d = 32 and <i>P<0.0001</i>). The phenotype/time <i><u>interaction</u></i> was also significant accounting for 7.18% of the total variance (F = 10.52, DF_n = 3, Df_d = 32 and <i>P<0.0001</i>). (<b>B</b>) <b><u>Total Weight (gm).</u></b> HYPVE-mice weight was significantly less than WTVE-mice weight at all time-points. SPR4 peptide treatment significantly decreased time-dependent gain in weight for both HYP mice (HYPSPR4) and WT mice (WTSPR4) relative to respective vehicle groups. Following 2-way ANOVA analysis, phenotypic variation (including SPR4-treatment) was highly significant accounting for 64.65% of the total variance (F = 40.6, DF_n = 3, Df_d = 32 and <i>P<0.0001</i>). Also, time-changes were highly significant accounting for 13.64% of the total variance (F = 25.69, DF_n = 1, Df_d = 32 and <i>P<0.0001</i>). The phenotype/time <i><u>interaction</u></i> was significant accounting for 4.73% of the total variance (F = 2.97, DF_n = 3, Df_d = 32 and <i>P = 0.0463</i>). (<b>C</b>) <b><u>Ratio of Fat mass/Weight (% Ratio).</u></b> No significant differences in fat-mass/weight ratios were observed between groups at 0 weeks (baseline, prior to pump implantation). In contrast, SPR4 peptide treatment significantly increased time-dependent gain in fat-Mass/weight ratio for both HYP mice (HYP-SPR4) and WT mice (WTSPR4) relative to respective vehicle groups. The gain in HYP-SPR4 fat-Mass/weight ratio was more marked and significantly greater than the WT-SPR4 mice. Following 2-way ANOVA analysis, phenotypic variation (including SPR4-treatment) was highly significant accounting for 30.60% of the total variance (F = 9.86, DF_n = 3, Df_d = 32 and <i>P<0.0001</i>). Also, time-changes were highly significant accounting for 17.14% of the total variance (F = 16.58, DF_n = 1, Df_d = 32 and <i>P = 0.0003</i>). The phenotype/time <i><u>interaction</u></i> was significant accounting for 19.17% of the total variance (F = 6.18, DF_n = 3, Df_d = 32 and <i>P = 0.002</i>). <b><i><u>Index</u></i></b><u>: </u><b>WTVE</b> =  wild type mice infused with vehicle (0.9% physiological saline); <b>HYPVE</b>  =  X-linked hypophosphatemic rickets mice infused with vehicle (0.9% physiological saline); <b>WTSPR4</b>  =  wild type mice infused SPR4-peptide; <b>HYPSPR4</b>  =  X-linked hypophosphatemic rickets mice infused SPR4-peptide.</p

Table of primary antibodies used in the study:

<p>Table of primary antibodies used in the study:</p

Percentage difference serum chemistry comparisons between wild type (WT) and HYP mice and mice infused with vehicle or SPR4-peptide.

<p>For absolute measurements in tabulated form see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t001" target="_blank"><b>Table 1</b></a>. Mice were sacrificed on day 28 and sera prepared from 16 hour fasted mice housed in metabolic cages. Values are means of percentage difference and are significant (* = P<0.05) unless indicated by NS (unpaired t test, confidence interval  = 95%; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t002" target="_blank">Table 2</a> for absolute numbers). Column headings represent: <b>WT</b>  =  wild type mice; <b>HYP</b>  =  X-linked hypophosphatemic rickets mice; <b>SPR4</b>  =  infused SPR4-peptide; <b>Vehicle</b>  =  Saline infused; <b>NS</b>  =  not significant; <b>ND</b>  =  not done; * = P<0.05. Histogram bars to the left of zero on the axis indicate down regulation and to the right up regulation.</p

Table of primers used for quantitative RT-PCR (qRT-PCR).

<p>Table of primers used for quantitative RT-PCR (qRT-PCR).</p

Temporal changes in (Weight, fat mass and fat mass/weight ratio) as measured by Dual Energy X-ray Absorptiometry (DEXA) for wild type and HYP mice infused with vehicle or SPR4-peptide for 28 days (See <b>Figure 6</b> for static changes).

<p>The mean percentage change over the 28 days for each metric was calculated and the percentage differences between the groups plotted as a histogram (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t005" target="_blank"><b>Table 5</b></a>). Values are mean percentage differences and are significant (* = P<0.05) unless indicated by NS (unpaired t test confidence interval  = 95%). Column headings represent; <b>WT</b>  =  wild type mice, <b>HYP</b>  =  X-linked hypophosphatemic rickets mice, <b>SPR4</b>  =  infused SPR4-peptide and <b>Vehicle</b>  =  Saline infused. Histogram bars to the left of zero on the y-axis indicate down regulation and to the right up regulation. DEXA measurements using a Lunar PIXImus system were carried out as described previously and discussed in methods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone.0097326-David2" target="_blank">[140]</a>. <b><i><u>Index</u></i></b><u>: </u><b>Weight (Wt)</b>  =  percentage difference in weight change over 4 weeks; <b>Fat-Mass</b>  =  percentage difference in total fat-mass change over 4 weeks; <b>Fat/Wt Ratio</b>  =  percentage difference in total fat-mass/weight change over 4 weeks.</p

Immunohistochemistry of kidney sections confirm changes in protein expression for Na dependent phosphate co-transporter (NPT2A; Slc34a1).

<p>NPT2a protein-expression (purple-stain) in renal cortex sections is markedly decreased in HYP mice (compare photos 1 and 3). SPR4 peptide suppresses NPT2a expression in WT mice (compare photos 1 and 2) but increases NPT2a expression in HYP mice (compare photos 3 and 4). Staining is localized to proximal convoluted tubules with little glomerular staining. Magnifications are 20× and are from representative sections (matched regions).</p

Bone (femur) gene expression (mRNA) comparisons as measured by quantitative RT/PCR (qRT-PCR) for wild type (WT) and HYP mice infused with vehicle or SPR4-peptide for 28 days.

<p>Mice were sacrificed on day 28 and femurs collected for RNA purification as described in methods. Column headings represent; WT  =  wild type mice, HYP  =  X-linked hypophosphatemic rickets mice, SPR4  =  infused SPR4-peptide and Vehicle  =  Saline infused. For gene analysis mRNA was prepared from bone marrow stromal cell “<i>depleted</i>” femurs as detailed in methods. For qRT-PCR gene analysis fold differences in expression calculated by the Pfaffl method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone.0097326-Pfaffl1" target="_blank">[163]</a> were statistically analyzed for significance using the One Sample t-test and the Wilcoxon Signed rank-test with theoretical means set to 1. Results are significant (* = p<0.05) unless indicated by NS (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-t004" target="_blank"><b>Table 4</b></a> for detailed statistics). <b><i><u>Index</u></i></b><u>: </u><b>FAM20C</b>  =  Family with sequence similarity 20, member C Kinase also known as DMP4; <b>ENPP1</b>  =  Ectonucleotide Pyrophosphatase Phosphodiesterase 1; <b>ESP</b>  =  Osteotesticular protein tyrosine (OST-PTP); <b>Plin-2</b>  =  Perlipin-2; phosphatase; <b>Cyclophilin</b>  =  peptidylprolyl isomerase A (cyclophilin A); <b>BGLAP</b>  =  Osteocalcin or Bone Gamma-Carboxyglutamate (gla) protein; <b>PHEX</b>  =  Phosphate-regulating gene with Homologies to Endopeptidases on the X chromosome; <b>GAPDH</b>  =  Glyceraldehyde 3-phosphate dehydrogenase; <b>VEGF</b>  =  Vascular Endothelial Growth factor; <b>DMP1</b>  =  Dentin Matrix Protein 1; <b>SOST</b>  =  Sclerostin; <b>MEPE</b>  =  Matrix Extracellular Phosphoglycoprotein with ASARM -motif; <b>FGF23</b>  =  Fibroblast Growth Factor 23; <b>NS</b>  =  not significant; <b>NA</b>  =  not applicable, PHEX mutated in HYP; * = P<0.05. Histogram bars to the left of zero on the axis indicate down regulation and to the right up regulation.</p

Urine chemistry results and comparisons for wild type and HYP mice infused with vehicle or SPR4-peptide.

<p>Mice were sacrificed on day 28 and sera prepared from 16 hour fasted mice housed in metabolic cages. Column headings represent; WT  =  wild type mice, HYP  =  X-linked hypophosphatemic rickets mice, SPR4  =  infused SPR4-peptide and Vehicle  =  Saline infused. Values are means with ± SEM (N = 6). Unpaired t tests with confidence intervals of 95% were used for statistical analysis. Unpaired t tests with confidence intervals of 95% were used for statistical analysis (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097326#pone-0097326-g002" target="_blank"><b>Figure 2</b></a>). Superscript letters (a, b or c) added to the calculated serum values indicate; a =  significantly different to WT vehicle (WT-VE) infused mice p<0.008, b =  significantly different to HYP vehicle (HYP-VE) infused mice p<0.002 and c =  significantly different to HYP SPR4-peptide (HYP-SPR4) infused mice p<0.02. ND =  Not done. The percentage changes between <i><u>WT-VE versus WT-SPR4</u></i>, <i><u>HYP-VE versus HYP-SPR4</u></i> and <i><u>WT-VE versus HYP-VE</u></i> are shown in columns 3, 6 and 7 as indicated in the headings. In these columns (3, 6 and 7), the numbers that are significantly different (p<0.01) are denoted by a superscript asterisk (*). <b><i><u>Index</u></i></b><u>:</u><b>FEP(%)</b>  =  Fractional Excretion of Phosphate, <b>Fe Ca (%)</b>  =  Fractional Excretion of Calcium.</p