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Review
. 2012;22(1):61-86.
doi: 10.1615/critreveukargeneexpr.v22.i1.50.

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

Peter S N Rowe  1
Affiliations
Review

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

Peter S N Rowe. Crit Rev Eukaryot Gene Expr. 2012.

Abstract

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.

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Figures

FIGURE 1
FIGURE 1
Scheme showing the chromosomal locations of MEPE and SIBLING proteins, (Rowe et al 2000). All the SIBLINGs including MEPE map to the long arm of chromosome 4 (4q12) between markers D4S1534 and D4S3381 in humans and chromosome 5 in mice. Codes are translated as follows: (1) BMP3, bone morphogenetic protein 3; (2) DSPP, dentin sialo phospho protein; (3) OPN, osteopontin; (4) DMP-1, dentin matrix protein 1; (5) ANX, annexin; (6) BSP, integrin binding sialo protein/bone sialoprotein II; (7) BMPR1B, bone morphogenetic receptor protein 1B; 8, MEPE, matrix extracellular phosphoglycoprotein; (9) statherin, salivary statherin; (10) ENAM, enamelin. Human ASARM sequence is shown above the scheme with conserved casein kinase serine-phosphorylation sites. See also Figure 6 and 7 for illustrations of the casein kinase ASARM-conservation across species for DMP1 and MEPE.
FIGURE 2
FIGURE 2
Scheme illustrating the sequence alignments and positions of the ASARM-peptide in MEPE, DMP1, DSPP, osteopontin (OPN), and statherin. The sequence similarity analysis was carried out using “sim” and Lalnview mathematical and software tools.– In each computation the gap open penalty was set to 12, and the gap extension penalty was 4. Comparison matrix was set to BLOSUM62 with similarity a configured score of 70%. The highlighted and colored blocks shown on each protein scheme represent sequence percentage homologies that are color indexed on the similarity scale at the top of the figure. Notably, in MEPE versus DSPP (B) there are several repeated ASARM homologous blocks that extend across the COOH terminal dentin phosphoprotein (DPP) region of DSPP. This region also contains a single integrin binding RGD motif. The MEPE and DMP1 ASARM motif sequence (DDSSESSDSGSSSESDGD) is shown in Figures 6 and 7. For other SIBLING ASARM sequences, see Rowe et al. 2000 and other recent publications.,,
FIGURE 3
FIGURE 3
Signal peptide analysis prediction using signal peptide-NN software confirming that all SIBLING proteins have strong signal-peptide motifs and are therefore secreted extracellular matrix proteins. Analysis was conducted using the SignalP 4.0 software and server at http://www.cbs.dtu.dk/services/SignalP/. Three scores (C, S, and Y) are provided as shown in the scheme. The red line shows the C-score or “cleavage site” score. The C-score is calculated for each sequence position. In all Siblings (MEPE, DMP1, DSPP, OPN, BSP, and statherin) a highly significant cleavage score (>0.8) was reported for the exact same sequence position. Y-max is a derivative of the C-score combined with the S-score resulting in a better cleavage site prediction than the raw C-score alone (blue peak). This is due to the fact that multiple high-peaking C-scores can be found in one sequence, where only one is the true cleavage site. The cleavage site is assigned from the Y-score where the slope of the S-score is steep and a significant C-score is found. The S-mean is the average of the S-score, ranging from the N-terminal amino acid to the amino acid assigned with the highest Y-max score, thus the S-mean score is calculated for the length of the predicted signal peptide. The calculated S-mean score for all SIBLINGs indicates strongly that all the SIBLINGs are secretory proteins.
FIGURE 4
FIGURE 4
Secondary structure prediction for MEPE as calculated using GCG peptide structure software, (see also Accelrys computer platform software details at http://accelrys.com/products/). The primary amino acidbackbone is shown as a central line with curves indicating regions of predicted turn. Regions of hydrophilicity and hydrophobicity are represented as ellipsoids (red) and diamonds (blue) respectively. The RGD motif is highlighted with a pentagon. The N-glycosylation sites are represented as blue ellipsoids on stalks (C-terminus) and an alpha helix is indicated by undulating regions on the primary backbone. The signal peptide is indicated by a checkered box and coincides with a hydrophobic region at the N-terminus. The COOH-terminal ASARM motif is highlighted and the PHEX binding region is indicated.
FIGURE 5
FIGURE 5
The MEPE RGD integrin binding region (dentonin or AC100) is highly conserved across species as illustrated in the clustalW alignment. There is complete conservation of a sequence (FSGDG) N-terminal to the RGD region and the consensus sequence RGDNDISPFSGDGQ is highly conserved. Species sequences for MEPE were searched for and downloaded from the “Ensembl” project resource at http://www.ensembl.org. The dentonin/AC100 peptide is a strong stimulator of bone/teeth formation in vitro and in vivo.,,–,– This contrasts with the ASARM peptide, an inhibitor of mineralization in vitro and in vivo (minhibin).–, ,–,,,,
FIGURE 6
FIGURE 6
DMP1 ASARM region (COOH residues 464 to 478) shows strong homology to MEPE ASARM peptide (across species) and the free ASARM peptide likely competes for PHEX binding. Flanking this region is a sequence also conserved in DMP1 (minfostin motif) and mutations here result in ARHR., Since MEPE and osteopontin ASARM peptides bind specifically to PHEX,–,,, we would propose the DMP1 ASARM motif also interacts with PHEX protein. Thus, free ASARM peptides likely compete with PHEX for DMP1 binding as described in Figure 9. The ASARM region (DMP1 and MEPE) consists of serine residues interspersed with acidic aspartate (D) and glutamic acid (E) residues. The serine residues are phosphorylated and the SXSSSE(S/D) conserved sequence of DMP1 and ASARM contains identical consensus casein-kinase II phosphorylation sites (see also Figure 7). Of relevance, a highly conserved GD sequence occurs in both MEPE-ASARM peptide and DMP1-ASARM motif as highlighted in the alignment. The GD sequence resides in the DMP1 minfostin region and likely plays an important role in PHEX binding. A DMP1 frame-shift mutation in this minfostin region that alters the COOH-terminal sequence at position 498 following residues LTVDA (with loss of the GD domain and an increase of 13 residues) results in ARHR., The species alignments (top to bottom) for DMP1 are human, chimpanzee, gibbon, macaque, cat, elephant, tarsier, cow, dolphin, tenrec, kangaroo, rabbit, squirrel, mouse, rat, hedgehog, and vicuna. The species alignments for MEPE (top to bottom) are human, chimpanzee, orangutan, gorilla, gibbon, macaque, rhesus monkey, marmoset, cow, tree shrew, sloth, alpaca, rock hyrax, tarsier, elephant, rabbit, squirrel, ground squirrel, cat, dog, dolphin, pig, microbat, hedgehog, kangaroo, rat and mouse. The above species sequences for MEPE and DMP1 were searched for and downloaded from the “Ensembl” project resource at http://www.ensembl.org.
FIGURE 7
FIGURE 7
DMP1 ASARM region (COOH residues 464 to 478) shows strong homology to MEPE ASARM peptide (across species) and the free ASARM-peptide likely competes for PHEX binding. The ASARM region in both DMP1 and MEPE also contain casein kinase II serine phosphorylation sites as depicted in the scheme. These phosphoserine sites are highly conserved between DMP1 and MEPE and across species. The clustal W alignment compares DMP1 and MEPE and two representative species (human and rabbit).
FIGURE 8
FIGURE 8
Cleavage sites for cysteine proteases (cathepsin K and B) that are N-terminal and in close proximity to the MEPE ASARM motif are highly conserved as shown in the alignment. The ASARM motif and peptide sequence is resistant to proteases (except for PHEX). The ASARM peptide is the only known physiological substrate for PHEX. Species sequences for MEPE were searched for and downloaded from the “Ensembl” project resource at http://www.ensembl.org.
FIGURE 9
FIGURE 9
A: ASARM displacement of the PHEX-DMP1-integrin complex regulates FGF23 expression (see text for detailed discussion of the scheme). The highlighted and encircled letters in the diagram link to the discussion in subsection-V1 titled “ASARM displacement of PHEX-DMP1-integrin complex regulates FGF23”. B: ASARM-peptide regulation of bone mineral and renal phosphate regulation through FGF23 and 1,25 Vit D3 (see text for detailed discussion of the scheme). The highlighted and encircled letters in the diagram link to the discussion in subsection titled: “V(2) ASARM & bone-renal PO4 regulation through FGF23 & 1,25 VitD3”. C: The ASARM pathway and the processing of BMP1 and DMP1 by SPC2 convertase and its co-activator 7B2 (see text for detailed discussion of the scheme). The highlighted and encircled letters in the diagram link to the discussion in subsection titled: “V(3) The ASARM pathway: processing of BMP1 & DMP1 by SPC2 & 7B2”.
FIGURE 9
FIGURE 9
A: ASARM displacement of the PHEX-DMP1-integrin complex regulates FGF23 expression (see text for detailed discussion of the scheme). The highlighted and encircled letters in the diagram link to the discussion in subsection-V1 titled “ASARM displacement of PHEX-DMP1-integrin complex regulates FGF23”. B: ASARM-peptide regulation of bone mineral and renal phosphate regulation through FGF23 and 1,25 Vit D3 (see text for detailed discussion of the scheme). The highlighted and encircled letters in the diagram link to the discussion in subsection titled: “V(2) ASARM & bone-renal PO4 regulation through FGF23 & 1,25 VitD3”. C: The ASARM pathway and the processing of BMP1 and DMP1 by SPC2 convertase and its co-activator 7B2 (see text for detailed discussion of the scheme). The highlighted and encircled letters in the diagram link to the discussion in subsection titled: “V(3) The ASARM pathway: processing of BMP1 & DMP1 by SPC2 & 7B2”.
FIGURE 9
FIGURE 9
A: ASARM displacement of the PHEX-DMP1-integrin complex regulates FGF23 expression (see text for detailed discussion of the scheme). The highlighted and encircled letters in the diagram link to the discussion in subsection-V1 titled “ASARM displacement of PHEX-DMP1-integrin complex regulates FGF23”. B: ASARM-peptide regulation of bone mineral and renal phosphate regulation through FGF23 and 1,25 Vit D3 (see text for detailed discussion of the scheme). The highlighted and encircled letters in the diagram link to the discussion in subsection titled: “V(2) ASARM & bone-renal PO4 regulation through FGF23 & 1,25 VitD3”. C: The ASARM pathway and the processing of BMP1 and DMP1 by SPC2 convertase and its co-activator 7B2 (see text for detailed discussion of the scheme). The highlighted and encircled letters in the diagram link to the discussion in subsection titled: “V(3) The ASARM pathway: processing of BMP1 & DMP1 by SPC2 & 7B2”.
FIGURE 10
FIGURE 10
Competitive displacement of DMP1 by ASARM peptide modulates PHEX–DMP1-mediated FGF23 expression (see central osteoblast in green). Specifically, DMP1 and PHEX interact and signal a down regulation of FGF23 as discussed in the text. ASARM disrupts this binding (PHEX + DMP1), resulting in an up-regulation of FGF23 signaling as discussed in Figure 9. This in turn leads to major changes in bone mineralization, bone turnover, vitamin D metabolism with hypophosphatemia. Recent evidence indicates that this also impacts fat energy metabolism pathways as depicted in the scheme and discussed in the text. Profound changes in fat mass, weight, glucose metabolism, insulin sensitivity, leptin levels, serotonin levels, sympathetic tone, aldosterone levels, vascularization, and sympathetic tone, occur in mice with defects in PHEX, DMP1, FGF23, MEPE, and ASARM expression. ,,,–,,,,– Index: SNS, sympathetic nervous system; Adrβ2, Osteoblast β2 adrenergic receptors (responsive to epinephrine/norepinephrine); Gla-OCN, γ-carboxylated osteocalcin (inactive form); Unc-OCN, γ-de-carboxylated osteocalcin (active form); Esp, Gene for OST-PTP (tyrosine phosphatase) that phosphorylates and inactivates the insulin receptor. This directly/indirectly results in reduced active osteocalcin (reduced γ-decarboxylation); Tcirg1, Osteoclast proton (H+) pump. Increases resorption lacuna acidity (acidic ASARM-peptides likely contribute) and thereby increases active osteocalcin (Unc-OCN) by acidic γ-decarboxylation; ASARM, Acidic Serine Aspartate Rich MEPE associated peptide or motif.
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