Regulation of Bone Mineralization by Inorganic Peptide Factors Negri A.L. Postgraduate Department of Osteology, Metabolic Research Institute, Universidad del Salvador, Buenos Aires

ABSTRACT

Orthotopic mineralization begins with the production of matrix vesicles that are produced by polarized budding of the surface of condrocytes, osteoblasts and odontoblasts. This occurs in two steps: The first one is the formation of hydroxyapatite crystals within the matrix vesicles, followed by the propagation of the hydroxyapatite crystals through the membrane vesicle into the extra cellular matrix. In the regulation of orthotopic mineralization, apart from tissue-specific cells, a great number of enzymes, inorganic and peptide factors participate, that have complex interactions among them. Inorganic pyrophosphate (PPi) antagonizes the ability of phosphate (Pi) to crystallize with calcium and to form hydroxyapatite, thus suppressing its propagation. For the normal mineralization to continue, an adjusted balance of the extra cellular Pi and PPi levels is needed. Three molecules have been identified that have a central role in the regulation of extra cellular PPi levels: tissue non-specific alkaline phosphatase (TNAP), which hydrolyzes PPi, the nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), which generates PPi from triphosphate nucleosides, and the multiple-steps transmembrane ANK, which transfers PPi from the intracellular to the extracellular compartment. There are, in turn, two SIBLING called DMP1 and MEPE that regulate mineralization. The expression of DMP1 by the osteocyte is dramatically induced in response to mechanical loading increasing bone mineralization. MEPE protein contains a protease resistant motif called ASARM, which is believed to be the candidate for the mineralization inhibitor (minhibin). is another mineralization inhibitor in its phosphorylated form and its secretion is markedly reduced in knockout mice for NPP1. Present data seem to support the hypothesis that these molecules could be the translators of bone strain and participate in the regulation of mineralization of the perilacunar osteocytic space.

Rev Argent endocrinol Metab 48: x-x, 2011

No financial conflicts of interest exist.

Key Words: mineralization, regulation, pyrophosphate, MEPE, DMP1, osteocyte.

INTRODUCTION Biomineralization is the process by which minerals are deposited inside or outside a wide variety of organisms(1). In vertebrate tissues, mineral is deposited as hidroxyapatite, one of the forms of calcium phosphate. Physiological mineralization occurs in hard tissues such as bones, with this process being highly regulated by tissue-specific cells(2). On the contrary, heterotopic calcification or soft tissue calcification may occur in the articular cartilage, in cardiovascular tissues such as vessels and in the kidney(3), etc. In the regulation of orthotopic mineralization, apart from tissue-specific cells, a great number of enzymes, inorganic and peptide factors, participate, that have complex interactions among them and which are the reason for this review.

Matrix vesicles in the process of bone mineralization

Matrix vesicles are vesicular elements whose diameter ranges from 50 to 200 nm which are produced by polarized budding of the surface of condrocytes, osteoblasts and odontoblasts(2). The necessary signs for the release of these matrix vesicles are not accurately known, although it is presumed that intracellular calcium and extracellular phosphate concentrations could be significant factors.

The lipid composition of matrix vesicles is different from that of the cell membrane from where they originate. These matrix vesicles are rich in several phospholipids, mainly phosphatidylserine, a lipid with high calcium affinity(4). Matrix vesicles are also rich in A2 (II), A5 (V) and A6 (VI) and in CaATasa, D9K, carbonic anhydrase, collagen X, alkaline phosphatase, pyrophosphatase phosphodiesterase 1 (NPP1), Na/Pi III cotransporter and PHOSPHO1(2,3,5) (Graph 1).

(References: Hidroxiapatita=Hydroxyapatite; Vesícula de matriz=Matrix vesicle; Transportador Na/Pi= Na/Pi transporter; Annexina=) Graph 1. Matrix vesicle. Pi: Phosphorus; PPi Pyrophosphate; TNAP: Tissue non- specific alkaline phosphatase: NPP1; nucleotide pyrophosphatase phosphodiesterase 1; PCho: phosphatidylcholine; PEA: phosphatidylethanolamine.

(References: Hidroxiapatita=Hydroxyapatite; Vesícula de matriz=Matrix vesicle; Osteoblasto: osteoblast; cristal de hidroxiapatita: hydroxiapatite crystal) Graph 2. interaction between matrix vesicles and osteoblasts. Pi: phosphorus; PPi pyrophosphate; TNAP: tissue-nonspecific alkaline phosphatase; NPP1: Nucleotide pyrophosphatases/phosphodiesterases; OPN: osteopontin; MEPE: matrix extracellular phosphoglycoprotein

Mineralization Steps Mineralization occurs in two (2) steps. The first one is the formation of hydroxyapatite crystals within the matrix vesicles, followed by the propagation of the hydroxyapatite crystals through the membrane vesicle into the extra cellular matrix. First step: calcium-binding phospholipids such as phosphatidylserine, calcium binding proteins such as calbindin D9K and bone sialoprotein promote calcium accumulation inside matrix vesicles(2,6). Annexins in matrix vesicles form calcium channels that incorporate calcium into vesicles(5,7) Inorganic phosphate (Pi) is provided by Type III Na/Pi co-transporter found both in the membrane of cells originating vesicles and in matrix vesicles(8,9). Cytosolic phosphotase PHOSPHO1 also produces phosphates through the hydrolysis of phosphocholine and phosphoethanolamine derived from membrane phospholipids by phospholipase C(10,11). When the accumulation of calcium and phosphorus exceeds the solubility point for calcium phosphate (CaPO4), the latter is deposited as hydroxyapatite in vesicles. Second step: The hydroxyapatite crystal generated in the first step penetrates the membrane wall of the matrix vesicle and is extended into the matrix extracellular space. The extracellular fluid has enough calcium and inorganic phosphorus to support the continuous formation of new hydroxyapatite crystals. Hydroxyapatite is propagated around matrix vesicles thus filling the space between the collagen fibrils of the matrix(12,13). The propagation of hydroxyapatite crystals mainly depends on the relation between Pi and inorganic pyrophosphate (PPi), since the latter inhibits hydroxyapatite formation.

Biomineralization Regulators Inorganic regulators: phyrophosphate/phosphate relation

Exposure of the hydroxyapatite crystals to the extracellular milieu further enables (13,15) growth and proliferation of the crystals . Inorganic pyrophosphate (PPi) antagonizes the ability of Pi to crystallize with calcium to form hydroxyapatite and thereby suppresses hydroxyapatite crystal propagation. For normal mineral deposition to proceed, a tight balance is required between the levels of extracellular Pi and PPi.

Three molecules have been identified as central regulators of extracellular PPi and Pi (16,20) levels, ie, tissue-nonspecific alkaline phosphatase (TNAP), which hydrolyzes PPi, nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), which generates PPi from nucleoside triphosphates(21,23) and the multiple-pass transmembrane protein ANK, (24,25) which mediates intracellular to extracellular channeling of PPi. NPP1 and TNAP but not ANK localize to matrix vesicles.

TNAP is an important promoter of mineralization because it catalyzes the hydrolysis of PPi thereby decreasing the concentrations of this calcification inhibitor, while concomitantly increasing the levels of Pi. Mice in which the TNAP has been inactivated (Akp2−/−) mimic the most severe form of hypophosphatasia, a disease characterized by rickets, osteomalacia, spontaneous bone fractures, and increased PPi levels (26,27). Akp2−/− skeletal preparations show poor mineralization in the parietal bones, scapulae, vertebral bones, and ribs(28-30). NPP1 serves as a physiological (21-23,31) inhibitor of calcification, at least in part by generating PPi . In human infants, severe NPP1 deficiency states were recently linked to a syndrome of spontaneous infantile arterial and periarticular calcification(32,33). NPP1 knockout mice (Enpp1−/−) also known as tiptoe walking (ttw/ttw) mice, spontaneously develop progressive ankylosing intervertebral and peripheral joint hyperostosis and articular cartilage calcification(34-37). Despite the different manner in which NPP1 and ANK supply PPi to bone matrix, a similar phenotype is associated with a naturally occurring truncation mutation of the C- terminal cytosolic domain of ANK that appears to attenuate PPi channeling in ank/ank mutant mice(24,25,38). Therefore, mice deficient in NPP1 (Enpp1−/−), or defective in the PPi, channeling function of ANK (ank/ank), have decreased levels of extracellular PPi and are hypermineralized. The TNAP-, NPP1-, and ANK-deficient mice all have altered levels of PPi and thus, these mice are valuable tools to further understand the function of PPi in the process of bone mineralization. In the understanding of the biomineralization process it is not only important how osteoblasts and chondrocytes make and dispose of PPi, but what effects PPi has on bone forming cells, in particular osteoblast . Crossbreeding alkaline phosphatase knockout mice −/− −/− (Akp2 ) to NPP1 knockout mice (Enpp1 ) rescues the PPi levels of the single- deficient animals, resulting in a correction of the mineralization defect(20). Given the similarity in function between ANK and NPP1 and also between the phenotype of deficient mice, crossbreeding Akp2−/− mice to ank/ank mice produces a partial −/− normalization of the mineralization phenotypes and PPi levels. Examination of Enpp1 and ank/ank mice revealed that Enpp1−/− mice have a more severe hypermineralized phenotype than ank/ank mice and that NPP1 but not ANK localizes to matrix vesicles, thus suggesting that failure of ANK deficiency to correct hypomineralization in Akp2−/− mice reflects the lack of ANK activity in the matrix vesicle compartment.

Peptide regulators of mineralization: ASARM-peptides Hypothesis

Hyp mice have a disease similar to human X-linked hypophosphatemia (XLH) rickets. Both Hyp mouse and XLH are caused by mutations of the phosphate-regulating gene PHEX with homologies to endopeptidases on the X , a member of the endothelin-converting enzyme family, located in the external part of the cell membrane(39). Both Hyp mice and XLH patients have a defect in bone mineralization. The hypothesis is that Hyp mice produce an unknown secreted factor called Minhibin which inhibits the mineralization of the extracellular matrix. Minhibin is a theoretical substrate for PHEX and would be predicted to accumulate in bone in patients with XLH or the Hyp mouse homologue of this disease(40). There is another disease with a phenotype similar to that of XLH: autosomal recessive hypophosphatemic rickets (ARHR). Like XLH, this disease presents rickets/osteomalacia, hypophosphatemia and abnormal regulation of calcitriol, due to high levels of bone phophatonine, FGF23. ARHR is caused by inactivating mutations of DMP1 (Dental Matrix Protein 1), an extracellular matrix small integrin-binding ligand N-linked glycoprotein (SIBLING protein)(41). Both diseases (XLH and ARHR) are associated with elevated FGF23 production by osteocytes what might indicate the presence of autocrine/paracrine pathways in bone that coordinate the mineralization process with renal phosphorus reabsorption. There is another SIBLING protein called MEPE (Matrix Extracellular Phosphogycoprotein). MEPE contains a protease-resistant peptide motif called ASARM (Acidic serine-aspartate-rich-motif) which is believed to be a candidate for elusive minhibin(42). The idea that MEPE is a source of minhibin is derived from the observations that inactivation of PHEX in Hyp mice is associated with increased proteolytic activity that releases ASARM peptides that accumulate in the extracellular matrix to inhibit the mineralization process(43). In addition, this ASARM peptide appears to be degraded by PHEX ectopeptidase, which further contributes to its accumulation in the absence of PHEX(44). Other studies demonstrate that anti-ASARM antibodies or soluble Phex-derived peptides sequestrate ASARM and correct the defective mineralization of Hyp-derived osteoblasts and bone marrow stromal cells in vitro(45). The relevance of MEPE to bone is also supported by mapping of a bone mineral density loci in humans to 4q21.1, a region where the MEPE gene is located in humans(46) and the small age-dependent increase in bone density in MEPE null mice(47). Finally, MEPE overexpression in mice, under the control of the Col1a1 promoter, leads to increased MEPE and ASARM levels in bone and defective mineralization. Recent studies have shown the importance of posttranslational modification for site- specific activity of MEPE and ASARM peptides. Intact phosphorylated protein was an effective promoter of mineralization, while ASARM peptide was an effective inhibitor. When both the intact protein and the ASARM peptide were dephosphorylated, they did not have any effect on mineralization(48).

Regulation of peptide factors by pyrophosphate and phosphorus Osteopontin (OPN) was another mineralization inhibitor protein which was increased in −/− Akp2 , and decreased in ank/ank mice. PPi and OPN levels were normalized in [Akp2−/−; Enpp1−/−] and [Akp2−/−; ank/ank] double knockout mice, at both the mRNA (49) level and in serum . Wild-type osteoblasts treated with PPi showed an increase in OPN, and a decrease in Enpp1 and Ank expression. Thus TNAP, NPP1, and ANK −/− coordinately regulate PPi and OPN levels. The hypomineralization observed in Akp2 mice arises from the combined inhibitory effects of PPi and OPN. In contrast, NPP1 or ANK deficiencies cause a decrease in the PPi and OPN pools that leads to hypermineralization. We do not know how the PPi regulates the osteopontin gene and if there are cell receptors for PPi.

The expression of several that codify proteins that regulate mineralization specific to bone cells, such as DMP1, osteopontin and matrix-gla-protein, are stimulated by phosphorus at transcriptional level, while TNAP expression is suppressed(50-52). Cellular uptake of phosphorus by specialized transporters appears to be required since addition of phosphonophormic acid (forscarnet), an inhibitor of sodium-phosphate co-transporters, blocks these effects of phosphorus(52-54). Downstream effects resulting in TNAP suppression may require signaling the bone morphogenetic protein, as noticed in mouse marrow stromal cell line ST-2(55). In contrast, in ATDC5 cells, phosphate signaling depends on the p42/p44- MAPK (mitogen activated protein kinase) / ERK (extracellular signal-regulated kinase) pathway(56). Similarly, the induction of osteopontin and matrix-gla-protein requires the activation of p42/p44-MAPK/ERK pathway and may be blocked by UO126, an inhibitor specific to MAPK/ERK kinase(57,58).

Regulation of mineralization in the osteocyte lacuna-canalicular space

SIBLING, DMP1, MEPE and osteopontin proteins, as previously mentioned, have a major role in the regulation of bone mineralization. DMP1 and MEPE are expressed by the osteoblasts (in late stages) and osteocytes located in osteocyte lacunar walls and canaliculi. In contrast with DMP1, MEPE appears to be a negative regulator of mineralization since null mouse for MEPE, has an increase in bone mineral density(59). ASARM peptides, resulting from MEPE cleavage, may directly block mineralization both “in vitro”(60) and “in vivo”(61). Current data seem to support the hypothesis that these molecules may be the transducers of bone “strain” and participate in the regulation of the perilacunar space mineralization. The proposed model would be as follows: the ASARM fragments derived from MEPE would reduce the local mineral content in the perilacunar area while DMP1 would be a positive regulator of mineralization in the same site(62-64).

Null mouse for DMP1 provides a good model to understand how these genes and their products are related to the mineralization process. DMP1 is a serine-rich acidic protein, which has numerous potential sites of phosphorylation that promote mineral formation in perilacunar areas(65,66). DMP1 is susceptible to proteolytic cleavage resulting in the generation of specific N-terminal and C-terminal fragments. In spite of that, the biological functions of the intact molecule versus fragments are still not clear. Although it has been established in these mice that DMP1 is not essential to mineralization, in early development [7], mice show significant defects in osteocyte morphology, bone remodeling and mineralization during postnatal development when the skeleton supports mechanical loading(63-65,66). These studies have confirmed that the loss of DMP1 results in deep changes both in osteocyte structure and in total bone mineralization(67-71) and agree with previous studies in adult wild animals, where DMP1 expression was dramatically induced, in response to the mechanical loading induced by bone remodeling(72,73). Taken as a whole, the resulting data suggest that SIBLING proteins regulate mineralization in the interface of bone surfaces covering the lacuna- canalicular space. It has been further hypothesized that the degree of mineralization dictates the flexibility of the osteocyte, thereby altering how it responds to mechanical induction and how it transmits signals that regulate active remodeling on the bone surface. Sclerostin is a protein that regulates bone formation and is expressed “in vivo” in the osteocytes submerged in the bone mineral. Recently, we have evaluated the hypothesis that sclerostin might regulate the behavior of cells actively involved in mature bone mineralization, the pre-osteocytes(74). Primary cultures of human osteoblasts exposed to recombinant human sclerostin (rhSCL) for 35 days, showed a 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 cultures with rhSCL markedly increased the expression of the pre-osteocyte marker E11 and decreased the expression of mature markers, DMP1 and SOST. Concomitantly, MEPE expression was increased by rhSCL at both the mRNA and protein levels, while PHEX was decreased, implying regulation through the MEPE- ASARM axis. These authors confirmed that mineralization by human osteoblasts is exquisitely sensitive to the tri-phosphorylated ASARM peptide. Immunostaining revealed that rhSCL increased the endogenous levels of MEPE-ASARM. Antibody- mediated neutralization of endogenous MEPE-ASARM antagonized the effect of rhSCL on mineralization, as did the PHEX synthetic peptide, SPR4. Finally, they found elevated Sost mRNA expression in long bones of HYP mice, suggesting that sclerostin may drive the increased MEPE-ASARM levels and mineralization defect in this genotype. Therefore, these results suggest that sclerostin acts through regulation of the PHEX/MEPE axis at the pre-osteocyte stage and serves as a master regulator of physiological bone mineralization, consistent with its localization in vivo and its established role in the inhibition of bone formation.

REFERENCES