Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice.

Acid transport is required for bone synthesis by osteoblasts. The osteoblast basolateral surface extrudes acid by Na+/H+ exchange, but apical proton uptake is undefined. We found high expression of the Cl-/H+ exchanger ClC3 at the bone apical surface. In mammals ClC3 functions in intracellular vesicular chloride transport, but when we found Cl- dependency of H+ transport in osteoblast membranes, we queried whether ClC3 Cl-/H+ exchange functions in bone formation. We used ClC3 knockout animals, and closely-related ClC5 knockout animals: In vitro studies suggested that both ClC3 and ClC5 might support bone formation. Genotypes were confirmed by total exon sequences. Expression of ClC3, and to a lesser extent of ClC5, at osteoblast apical membranes was demonstrated by fluorescent antibody labeling and electron microscopy with nanometer gold labeling. Animals with ClC3 or ClC5 knockouts were viable. In ClC3 or ClC5 knockouts, bone formation decreased ~40 % by calcein and xylenol orange labeling in vivo. In very sensitive micro-computed tomography, ClC5 knockout reduced bone relative to wild type, consistent with effects of ClC3 knockout, but varied with specific histological parameters. Regrettably, ClC5-ClC3 double knockouts are not viable, suggesting that ClC3 or ClC5 activity are essential to life. We conclude that ClC3 has a direct role in bone formation with overlapping but probably slightly smaller effects of ClC5. The mechanism in mineral formation might include ClC H+ uptake, in contrast to ClC3 and ClC5 function in cell vesicles or other organs.

The ecto-nucleotide pyrophosphatase/phosphodiesterase 2 promotes early osteoblast differentiation and mineralization in stromal stem cells.

The phosphodiesterase enzymes mediate calcium-phosphate deposition in various tissues, although which enzymes are active in bone mineralization is unclear. Using gene array analysis, we found that a member of ecto-nucleotide pyrophosphatase/phosphodiesterase family, ENPP2, was strongly down-regulated with age in stromal stem cells that produce osteoblasts and make bone. This is in keeping with reduced bone formation in older animals. Thus, we hypothesized that ENPP2 is, at least in part, an early mediator of bone formation and thus may reflect reduced bone formation with age. Since ENPP2 has not previously been shown to have a role in osteoblast differentiation, we studied its effect on bone differentiation from stromal stem cells, verified by flow cytometry for stem cell antigens. In these remarkably uniform osteoblast precursors, we did transfection with ENPP2 DsiRNA, scrambled DsiRNA, or no transfection to make cells with normal or greatly reduced ENPP2 and analyzed osteoblast differentiation and mineralization. Osteoblast differentiation down-regulation was shown by alizarin red binding, silver staining, and alkaline phosphatase activity. Differences were confirmed by real-time PCR for alkaline phosphatase (ALPL), osteocalcin (BGLAP), and ENPP2 and by Western Blot for Enpp2. These were decreased, ∼50%, in osteoblasts transfected with ENPP2 DsiRNA compared with cells transfected with a scrambled DsiRNA or not transfected (control) cells. This finding is the first evidence for the role of ENPP2 in osteoblast differentiation and mineralization.NEW & NOTEWORTHY We report the discovery that the ecto-nucleotide pyrophosphatase/phosphodiesterase, ENPP2, is an important regulator of early differentiation of bone-forming osteoblasts.

Epithelial-like transport of mineral distinguishes bone formation from other connective tissues.

We review unique properties of bone formation including current understanding of mechanisms of bone mineral transport. We focus on formation only; mechanism of bone degradation is a separate topic not considered. Bone matrix is compared to other connective tissues composed mainly of the same proteins, but without the specialized mechanism for continuous transport and deposition of mineral. Indeed other connective tissues add mechanisms to prevent mineral formation. We start with the epithelial-like surfaces that mediate transport of phosphate to be incorporated into hydroxyapatite in bone, or in its ancestral tissue, the tooth. These include several phosphate producing or phosphate transport-related proteins with special expression in large quantities in bone, particularly in the bone-surface osteoblasts. In all connective tissues including bone, the proteins that constitute the protein matrix are mainly type I collagen and γ-carboxylate-containing small proteins in similar molar quantities to collagen. Specialized proteins that regulate connective tissue structure and formation are surprisingly similar in mineralized and non-mineralized tissues. While serum calcium and phosphate are adequate to precipitate mineral, specialized mechanisms normally prevent mineral formation except in bone, where continuous transport and deposition of mineral occurs.

Creatine energy substrate increases bone density in the Pahenu2 classical PKU mouse in the context of phenylalanine restriction.

Pathophysiology of osteopenia in phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) is poorly characterized. The Pahenu2 mouse is universally osteopenic where dietary phenylalanine (Phe) management with amino acid defined chow does not improve bone density. We previously demonstrated Pahenu2 osteopenia owes to a skeletal stem cell (SSC) developmental deficit mediated by energy dysregulation and oxidative stress. This investigation demonstrates complexity of Pahenu2 SSC energy dysregulation. Creatine use by bone tissue is recognized. In vitro Pahenu2 SSCs in osteoblast differentiation respond to creatine with increased in situ alkaline phosphatase activity and increased intracellular ATP content. Animal studies applied a 60-day creatine regimen to Pahenu2 and control cohorts. Control cohorts include unaffected littermates (wt/wt), Pahenu2 receiving no intervention, and dietary Phe restricted Pahenu2. Experimental cohorts (Phe unrestricted Pahenu2, Phe restricted Pahenu2) were provided 1% creatine ad libitum in water. After 60 days, microcomputed tomography assessed bone metrics. Equivalent osteopenia occurs in Phe-restricted and untreated Pahenu2 control cohorts. In Phe unrestricted Pahenu2, creatine was without effect as bone density remained equivalent to Pahenu2 control cohorts. Alternatively, Phe-restricted Pahenu2 receiving creatine present increased bone density. We hypothesize small molecule dysregulation in untreated Pahenu2 disallows creatine utilization; therefore, osteopenia persisted. Dietary Phe restriction enables creatine utilization to enhance SSC osteoblast differentiation and improve in vivo bone density. PKU intervention singularly focused on Phe reduction enables residual disease including osteopenia and neurologic elements. Intervention concurrently addressing Phe homeostasis and energy dysregulation will improve disease elements refractory to standard of care Phe reduction mono-therapy.

Age-related decline in bone mineral transport and bone matrix proteins in osteoblasts from stromal stem cells.

We studied osteoblast bone mineral transport and matrix proteins as a function of age. In isolated bone marrow cells from long bones of young (3 or 4 mo) and old (18 or 19 mo) mice, age correlated with reduced mRNA of mineral transport proteins: alkaline phosphatase (ALP), ankylosis (ANK), the Cl-/H+ exchanger ClC3, and matrix proteins collagen 1 (Col1) and osteocalcin (BGLAP). Some proteins, including the neutral phosphate transporter2 (NPT2), were not reduced. These are predominately osteoblast proteins, but in mixed cell populations. Remarkably, in osteoblasts differentiated from preparations of stromal stem cells (SSCs) made from bone marrow cells in young and old mice, differentiated in vitro on perforated polyethylene terephthalate membranes, mRNA confirmed decreased expression with age for most transport-related and bone matrix proteins. Additional mRNAs in osteoblasts in vitro included ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), unchanged, and ENPP2, reduced with age. Decrease with age in ALP activity and protein by Western blot was also significant. Transport protein findings correlated with micro-computed tomography of lumbar vertebra, showing that trabecular bone of old mice is osteopenic relative to young mice, consistent with other studies. Pathway analysis of osteoblasts differentiated in vitro showed that cells from old animals had reduced Erk1/2 phosphorylation and decreased suppressor of mothers against decapentaplegic 2 (Smad2) mRNA, consistent with TGFß pathway, and reduced ß-catenin mRNA, consistent with WNT pathway regulation. Our results show that decline in bone density with age reflects selective changes, resulting effectively in a phenotype modification. Reduction of matrix and mineral transport protein expression with age is regulated by multiple signaling pathways.NEW & NOTEWORTHY This work for the first time showed that specific enzymes in bone mineral transport, and matrix synthesis proteins, in the epithelial-like bone-forming cell layer are downregulated with aging. Results were compared using cells extracted from long bones of young and old mice, or in essentially uniform osteoblasts differentiated from stromal stem cells in vitro. The age effect showed memory in the stromal stem cells, a remarkable finding.

The calcium channel Orai1 is required for osteoblast development: Studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1.

The calcium-selective ion channel Orai1 has a complex role in bone homeostasis, with defects in both bone production and resorption detected in Orai1 germline knock-out mice. To determine whether Orai1 has a direct, cell-intrinsic role in osteoblast differentiation and function, we bred Orai1 flox/flox (Orai1fl/fl) mice with Runx2-cre mice to eliminate its expression in osteoprogenitor cells. Interestingly, Orai1 was expressed in a mosaic pattern in Orai1fl/fl-Runx2-cre bone. Specifically, antibody labeling for Orai1 in vertebral sections was uniform in wild type animals, but patchy regions in Orai1fl/fl-Runx2-cre bone revealed Orai1 loss while in other areas expression persisted. Nevertheless, by micro-CT, bones from Orai1fl/fl-Runx2-cre mice showed reduced bone mass overall, with impaired bone formation identified by dynamic histomorphometry. Cortical surfaces of Orai1fl/fl-Runx2-cre vertebrae however exhibited patchy defects. In cell culture, Orai1-negative osteoblasts showed profound reductions in store-operated Ca2+ entry, exhibited greatly decreased alkaline phosphatase activity, and had markedly impaired substrate mineralization. We conclude that defective bone formation observed in the absence of Orai1 reflects an intrinsic role for Orai1 in differentiating osteoblasts.

Glutamine energy substrate anaplerosis increases bone density in the Pahenu2 classical PKU mouse in the absence of phenylalanine restriction.

Osteopenia is an under-investigated clinical presentation of phenylalanine hydroxylase (PAH)-deficient phenylketonuria (PKU). While osteopenia is not fully penetrant in human PKU, the Pahenu2 mouse is universally osteopenic and ideal to study the phenotype. We determined Pahenu2 mesenchymal stem cells (MSCs) are developmentally impaired in the osteoblast lineage. Moreover, we determined energy dysregulation and oxidative stress contribute to the osteoblast developmental deficit. The MSC preferred substrate glutamine (Gln) was applied to enhance energy homeostasis. In vitro Pahenu2 MSCs, in the context of 1200 µM Phe, respond to Gln with increased in situ alkaline phosphatase activity indicating augmented osteoblast differentiation. Oximetry applied to Pahenu2 MSCs in osteoblast differentiation show Gln energy substrate increases oxygen consumption, specifically maximum respiration and respiratory reserve. For 60 days post-weaning, Pahenu2 animals received either no intervention (standard lab chow), amino acid defined chow maintaining plasma Phe at ~200 µM, or standard lab chow where ad libitum water was a 2% Gln solution. Bone density was assessed by microcomputed tomography and bone growth assessed by dye labeling. Bone density and dye labeling in Phe-restricted Pahenu2 was indistinguishable from untreated Pahenu2. Gln energy substrate provided to Pahenu2, in the context of uncontrolled hyperphenylalaninemia, present increased bone density and dye labeling. These data provide further evidence that Pahenu2 MSCs experience a secondary energy deficit that is responsive both in vitro and in vivo to Gln energy substrate and independent of hyperphenylalaninemia. Energy support may have effect to treat human PKU osteopenia and elements of PKU neurologic disease resistant to standard of care systemic Phe reduction. Glutamine energy substrate anaplerosis increased Pahenu2 bone density and improved in vitro MSC function in the context of hyperphenylalaninemia in the classical PKU range.

Growth and mineralization of osteoblasts from mesenchymal stem cells on microporous membranes: Epithelial-like growth with transmembrane resistance and pH gradient.

Osteoblasts in vivo form an epithelial-like layer with tight junctions between cells. Bone formation involves mineral transport into the matrix and acid transport to balance pH levels. To study the importance of the pH gradient in vitro, we used Transwell inserts composed of polyethylene terephthalate (PET) membranes with 0.4 µm pores at a density of (2 ± 0.4) x 106 pores per cm2. Mesenchymal stem cells (MSCs) prepared from murine bone marrow were used to investigate alternative conditions whereby osteoblast differentiation would better emulate in vivo bone development. MSCs were characterized by flow cytometry with more than 90% CD44 and 75% Sca-1 labeling. Mineralization was validated with paracellular alkaline phosphatase activity, collagen birefringence, and mineral deposition confirming MSCs identity. We demonstrate that MSCs cultured and differentiated on PET inserts form an epithelial-like layer while mineralizing. Measurement of the transepithelial resistance was ∼1400 Ω•cm2 at three weeks of differentiation. The pH value of the media above and under the cells were measured while cells were in proliferation and differentiation. In mineralizing cells, a difference of 0.145 pH unit was observed between the medium above and under the cells indicating a transepithelial gradient. A significant difference in pH units was observed between the medium above and below the cells in proliferation compared to differentiation. Data on pH below membranes were confirmed by pH-dependent SNARF1 fluorescence. Control cells in proliferative medium did not form an epithelial-like layer, displayed low transepithelial resistance, and there was no significant pH gradient. By transmission electron microscopy, membrane attached osteoblasts in vitro had abundant mitochondria consistent with active transport that occurs in vivo by surface osteoblasts. In keeping with osteoblastic differentiation, scanning electron microscopy identified deposition of extracellular collagen surrounded by hydroxyapatite. This in vitro model is a major advancement in modeling bone in vivo for understanding of osteoblast bone matrix production.

A New View of Bone Loss in Phenylketonuria.

Osteopenia is common in phenylalanine hydroxylase deficient phenylketonuria (PKU). PKU is managed by limiting dietary phenylalanine. Osteopenia in PKU might reflect a therapeutic diet, with reduced bone forming materials. However, osteopenia occurs in patients who never received dietary therapy or following short-term therapy. Humans and animal studies find no correlation between bone loss, plasma hyperphenylalaninemia, bone formation, and resorption markers. Work in the Pahenu2 mouse recently showed a mesenchymal stem cell (MSC) developmental defect in the osteoblast pathway. Specifically, Pahenu2 MSCs are affected by energy dysregulation and oxidative stress. In PKU, MSCs oximetry and respirometry show mitochondrial respiratory-chain complex 1 deficit and over-representation of superoxide, producing reactive oxygen species affecting mitochondrial function. Similar mechanisms are involved in aging bone and other rare defects including alkaptonuria and homocysteinemia. Novel interventions to support energy and reduce oxidative stress may restore bone formation PKU patients, and in metabolic diseases with related mechanisms.

The function of the calcium channel Orai1 in osteoclast development.

To determine the intrinsic role of Orai1 in osteoclast development, Orai1-floxed mice were bred with LysMcre mice to delete Orai1 from the myeloid lineage. PCR, in situ labelling and Western analysis showed Orai1 deletion in myeloid-lineage cells, including osteoclasts, as expected. Surprisingly, bone resorption was maintained in vivo, despite loss of multinucleated osteoclasts; instead, a large number of mononuclear cells bearing tartrate resistant acid phosphatase were observed on cell surfaces. An in vitro resorption assay confirmed that RANKL-treated Orai1 null cells, also TRAP-positive but mononuclear, degraded matrix, albeit at a reduced rate compared to wild type osteoclasts. This shows that mononuclear osteoclasts can degrade bone, albeit less efficiently. Further unexpected findings included that Orai1fl/fl -LysMcre vertebrae showed slightly reduced bone density in 16-week-old mice, despite Orai1 deletion only in myeloid cells; however, this mild difference resolved with age. In summary, in vitro analysis showed a severe defect in osteoclast multinucleation in Orai1 negative mononuclear cells, consistent with prior studies using less targeted strategies, but with evidence of resorption in vivo and unexpected secondary effects on bone formation leaving bone mass largely unaffected.

Mesenchymal stem cell energy deficit and oxidative stress contribute to osteopenia in the Pahenu2 classical PKU mouse.

Osteopenia occurs in a subset of phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) patients. While osteopenia is not fully penetrant in patients, the Pahenu2 classical PKU mouse is universally osteopenic, making it an ideal model of the phenotype. Pahenu2 Phe management, with a Phe-fee amino acid defined diet, does not improve bone density as histomorphometry metrics remain indistinguishable from untreated animals. Previously, we demonstrated Pahenu2 mesenchymal stem cells (MSCs) display impaired osteoblast differentiation. Oxidative stress is recognized in PKU patients and PKU animal models. Pahenu2 MSCs experience oxidative stress determined by intracellular superoxide over-representation. The deleterious impact of oxidative stress on mitochondria is recognized. Oximetry applied to Pahenu2 MSCs identified mitochondrial stress by increased basal respiration with concurrently reduced maximal respiration and respiratory reserve. Proton leak secondary to mitochondrial complex 1 dysfunction is a recognized superoxide source. Respirometry applied to Pahenu2 MSCs, in the course of osteoblast differentiation, identified a partial complex 1 deficit. Pahenu2 MSCs treated with the antioxidant resveratrol demonstrated increased mitochondrial mass by MitoTracker green labeling. In hyperphenylalaninemic conditions, resveratrol increased in situ alkaline phosphatase activity suggesting partial recovery of Pahenu2 MSCs osteoblast differentiation. Up-regulation of oxidative energy production is required for osteoblasts differentiation. Our data suggests impaired Pahenu2 MSC developmental competence involves an energy deficit. We posit energy support and oxidative stress reduction will enable Pahenu2 MSC differentiation in the osteoblast lineage to subsequently increase bone density.

Phylogeny and chemistry of biological mineral transport.

Three physiologically mineralizing tissues - teeth, cartilage and bone - have critical common elements and important evolutionary relationships. Phylogenetically the most ancient densely mineralized tissue is teeth. In jawless fishes without skeletons, tooth formation included epithelial transport of phosphates, a process echoed later in bone physiology. Cartilage and mineralized cartilage are skeletal elements separate from bone, but with metabolic features common to bone. Cartilage mineralization is coordinated with high expression of tissue nonspecific alkaline phosphatase and PHOSPHO1 to harvest available phosphate esters and support mineralization of collagen secreted locally. Mineralization in true bone results from stochastic nucleation of hydroxyapatite crystals within the cross-linked collagen fibrils. Mineral accumulation in dense collagen is, at least in major part, mediated by amorphous aggregates - often called Posner clusters - of calcium and phosphate that are small enough to diffuse into collagen fibrils. Mineral accumulation in membrane vesicles is widely suggested, but does not correlate with a definitive stage of mineralization. Conversely mineral deposition at non-physiologic sites where calcium and phosphate are adequate has been shown to be regulated in large part by pyrophosphate. All of these elements are present in vertebrate bone metabolism. A key biological element of bone formation is an epithelial-like cellular organization which allows control of phosphate, calcium and pH during mineralization.

Correction: The high-density lipoprotein receptor Scarb1 is required for normal bone differentiation in vivo and in vitro.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Generation of an immunodeficient mouse model of tcirg1-deficient autosomal recessive osteopetrosis.

BACKGROUND: Autosomal recessive osteopetrosis is a rare skeletal disorder with increased bone density due to a failure in osteoclast bone resorption. In most cases, the defect is cell-autonomous, and >50% of patients bear mutations in the TCIRG1 gene, encoding for a subunit of the vacuolar proton pump essential for osteoclast resorptive activity. The only cure is hematopoietic stem cell transplantation, which corrects the bone pathology by allowing the formation of donor-derived functional osteoclasts. Therapeutic approaches using patient-derived cells corrected ex vivo through viral transduction or gene editing can be considered, but to date functional rescue cannot be demonstrated in vivo because a relevant animal model for xenotransplant is missing. METHODS: We generated a new mouse model, which we named NSG oc/oc, presenting severe autosomal recessive osteopetrosis owing to the Tcirg1 oc mutation, and profound immunodeficiency caused by the NSG background. We performed neonatal murine bone marrow transplantation and xenotransplantation with human CD34+ cells. RESULTS: We demonstrated that neonatal murine bone marrow transplantation rescued NSG oc/oc mice, in line with previous findings in the oc/oc parental strain and with evidence from clinical practice in humans. Importantly, we also demonstrated human cell chimerism in the bone marrow of NSG oc/oc mice transplanted with human CD34+ cells. The severity and rapid progression of the disease in the mouse model prevented amelioration of the bone pathology; nevertheless, we cannot completely exclude that minor early modifications of the bone tissue might have occurred. CONCLUSION: Our work paves the way to generating an improved xenograft model for in vivo evaluation of functional rescue of patient-derived corrected cells. Further refinement of the newly generated mouse model will allow capitalizing on it for an optimized exploitation in the path to novel cell therapies.

Cellular and extracellular matrix of bone, with principles of synthesis and dependency of mineral deposition on cell membrane transport.

Bone differs from other connective tissues; it is isolated by a layer of osteoblasts that are connected by tight and gap junctions. This allows bone to create dense lamellar type I collagen, control pH, mineral deposition, and regulate water content forming a compact and strong structure. New woven bone formed after degradation of mineralized cartilage is rapidly degraded and resynthesized to impart structural order for local bone strength. Ossification is regulated by thickness of bone units and by patterning via bone morphogenetic receptors including activin, other bone morphogenetic protein receptors, transforming growth factor-ß receptors, all part of a receptor superfamily. This superfamily interacts with receptors for additional signals in bone differentiation. Important features of the osteoblast environment were established using recent tools including osteoblast differentiation in vitro. Osteoblasts deposit matrix protein, over 90% type I collagen, in lamellae with orientation alternating parallel or orthogonal to the main stress axis of the bone. Into this organic matrix, mineral is deposited as hydroxyapatite. Mineral matrix matures from amorphous to crystalline hydroxyapatite. This process includes at least two-phase changes of the calcium-phosphate mineral as well as intermediates involving tropocollagen fibrils to form the bone composite. Beginning with initiation of mineral deposition, there is uncertainty regarding cardinal processes, but the driving force is not merely exceeding the calcium-phosphate solubility product. It occurs behind a epithelial-like layer of osteoblasts, which generate phosphate and remove protons liberated during calcium-phosphate salt deposition. The forming bone matrix is discontinuous from the general extracellular fluid. Required adjustment of ionic concentrations and water removal from bone matrix are important details remaining to be addressed.

The high-density lipoprotein receptor Scarb1 is required for normal bone differentiation in vivo and in vitro.

We examined bone formation and turnover in high-density lipoprotein (HDL) receptor, scavenger receptor type I (Scarb1), knockout animals relative to wild-type (WT) controls. Scarb1-/- animals have elevated serum adrenocorticotropic hormone (ACTH) due to the role of Scarb1 in glucocorticoid production, which might cause increased bone mass. However, this was not observed: Scarb1-/- mice, with ACTH, over 1000 pg/ml relative to wild-type ACTH ~ 25 pg/ml, bone of the knockout animals was osteopenic relative to the wild type at 16 weeks, including bone volume/total volume and trabecular thickness. Other serum parameters of WT and Scarb1-/- animals in cortisol or calcium were unaffected, although Scarb1-/- animals had significantly elevated PTH and decreased phosphate. Osteoblast and osteoclast-related mRNAs extracted from bone were greatly decreased at 8 or 16 weeks. Importantly, in normal ACTH, osteogenic differentiation in vitro from mesenchymal stem cells showed reduced alkaline phosphatase and mineralization. In Scarb1-/- cells relative to WT, mRNAs for RunX2, alkaline phosphatase, type I collagen, and osteocalcin were reduced 40-90%, all p < 0.01, indicating a role of Scarb1 in osteoblast differentiation independent of ACTH. Additionally, in vitro osteoblast differentiation at variable ACTH in WT cells confirmed ACTH increasing bone differentiation, mineralization, alkaline phosphatase, and osteocalcin mRNA at 0-10 nM ACTH, but reduced bone differentiation at 100-1000 nM ACTH. Overall Scarb1-/- animals show inhibited bone formation with age. This may be a mixed effect on direct bone formation and of very high ACTH. Further, this work shows that both ACTH concentration and the HDL receptor Scarb1 play important independent roles in osteoblast differentiation.

A bone mineralization defect in the Pahenu2 model of classical phenylketonuria involves compromised mesenchymal stem cell differentiation.

Osteopenia is observed in some patients affected by phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU). Bone density studies, in diverse PKU patient cohorts, have demonstrated bone disease is neither fully penetrant nor uniform in bone density loss. Biochemical assessment has generated a muddled perspective regarding mechanisms of the PKU bone phenotype where the participation of hyperphenylalaninemia remains unresolved. Osteopenia is realized in the Pahenu2 mouse model of classical PKU; although, characterization is incomplete. We characterized the Pahenu2 bone phenotype and assessed the effect of hyperphenylalaninemia on bone differentiation. Employing Pahenu2 and control animals, cytology, static and dynamic histomorphometry, and biochemistry were applied to further characterize the bone phenotype. These investigations demonstrate Pahenu2 bone density is decreased 33% relative to C57BL/6; bone volume/total volume was similarly decreased; trabecular thickness was unchanged while increased trabecular spacing was observed. Dynamic histomorphometry demonstrated a 25% decrease in mineral apposition. Biochemically, control and PKU animals have similar plasma cortisol, adrenocorticotropic hormone, and 25-hydroxyvitamin D. PKU animals show moderately increased plasma parathyroid hormone while plasma calcium and phosphate are reduced. These data are consistent with a mineralization defect. The effect of hyperphenylalaninemia on bone maturation was assessed in vitro employing bone-derived mesenchymal stem cells (MSCs) and their differentiation into bone. Using standard culture conditions, PAH deficient MSCs differentiate into bone as assessed by in situ alkaline phosphatase activity and mineral staining. However, PAH deficient MSCs cultured in 1200 µM PHE (metric defining classical PKU) show significantly reduced mineralization. These data are the first biological evidence demonstrating a negative impact of hyperphenylalaninemia upon bone maturation. In PAH deficient MSCs, expression of Col1A1 and Rankl are suppressed by hyperphenylalaninemia consistent with reduced bone formation and bone turnover. Osteopenia is intrinsic to PKU pathology in untreated Pahenu2 animals and our data suggests PHE toxicity participates by inhibiting mineralization in the course of MSC bone differentiation.

Sphingosine-1-Phosphate Modulates the Effect of Estrogen in Human Osteoblasts.

Production of sphingosine-1-phosphate (S1P) is linked to 17ß-estradiol (E2) activity in many estrogen-responsive cells; in bone development, the role of S1P is unclear. We studied effects of S1P on proliferation and differentiation of human osteoblasts (hOB). Ten nM E2, 1 µM S1P, or 1 µM of the S1P receptor 1 (S1PR1) agonist SEW2871 increased hOB proliferation at 24 hours. S1PR 1, 2, and 3 mRNAs are expressed by hOB but not S1PR4 or S1PR5. Expression of S1PR2 was increased at 7 and 14 days of differentiation, in correspondence with osteoblast-related mRNAs. Expression of S1PR1 was increased by E2 or S1P in proliferating hOB, whereas S1PR2 mRNA was unaffected in proliferating cells; S1PR3 was not affected by E2 or S1P. Inhibiting sphingosine kinase (SPHK) activity with sphingosine kinase inhibitor (Ski) greatly reduced the E2 proliferative effect. Both E2 and S1P increased SPHK mRNA at 24 hours in hOB. S1P promoted osteoblast proliferation via activating MAP kinase activity. Either E2 or S1P increased S1P synthesis in a fluorescent S1P assay. Interaction of E2 and S1P signaling was indicated by upregulation of E2 receptor mRNA after S1P treatment. E2 and S1P also promoted alkaline phosphatase expression. During osteoblast differentiation, S1P increased bone-specific mRNAs, similarly to the effects of E2. However, E2 and S1P showed differences in the activation of some osteoblast pathways. Pathway analysis by gene expression arrays was consistent with regulation of pathways of osteoblast differentiation; collagen and cell adhesion proteins centered on Rho/Rac small GTPase signaling and Map kinase or signal transducer and activator of transcription (Stat) intermediates. Transcriptional activation also included significant increases in superoxide dismutase 1 and 2 transcription by either S1P or E2. We demonstrate that the SPHK system is a co-mediator for osteoblast proliferation and differentiation, which is mainly, but not entirely, complementary to E2, whose effects are mediated by S1PR1 and S1PR2.

Support of bone mineral deposition by regulation of pH.

Osteoblasts secrete collagen and isolate bone matrix from extracellular space. In the matrix, alkaline phosphatase generates phosphate that combines with calcium to form mineral, liberating 8 H+ per 10 Ca+2 deposited. However, pH-dependent hydroxyapatite deposition on bone collagen had not been shown. We studied the dependency of hydroxyapatite deposition on type I collagen on pH and phosphate by surface plasmon resonance in 0-5 mM phosphate at pH 6.8-7.4. Mineral deposition saturated at <1 mM Ca2+ but was sensitive to phosphate. Mineral deposition was reversible, consistent with amorphous precipitation; stable deposition requiring EDTA removal appeared with time. At pH 6.8, little hydroxyapatite deposited on collagen; mineral accumulation increased 10-fold at pH 7.4. Previously, we showed high expression Na+/H+ exchanger (NHE) and ClC transporters in osteoblasts. We hypothesized that, in combination, these move protons across osteoblasts to the general extracellular space. We made osteoblast membrane vesicles by nitrogen cavitation and used acridine orange quenching to characterize proton transport. We found H+ transport dependent on gradients of chloride or sodium, consistent with apical osteoblast ClC family Cl-,H+ antiporters and basolateral osteoblast NHE family Na+/H+ exchangers. Little, if any, active H+ transport, supported by ATP, occurred. Major transporters include cariporide-sensitive NHE1 in basolateral membranes and ClC3 and ClC5 in apical osteoblast membranes. The mineralization inhibitor levamisole reduced bone formation and expression of alkaline phosphatase, NHE1, and ClC5. We conclude that mineral deposition in bone collagen is pH-dependent, in keeping with H+ removal by Cl-,H+ antiporters and Na+/H+-exchangers. Periodic orientation hydroxyapatite is organized on type I collagen-coiled coils.

Adrenocorticotropic hormone and 1,25-dihydroxyvitamin D3 enhance human osteogenesis in vitro by synergistically accelerating the expression of bone-specific genes.

To improve definition of the physical and hormonal support of bone formation, we studied differentiation of human osteoblasts in vitro at varying combinations of ACTH, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D), and extracellular calcium, with and without added cortisol. Bone mineralization, alkaline phosphatase activity, and osteoblast-specific markers RunX2, osterix, and collagen I increased with 10 pM ACTH, 10 nM 1,25(OH)2D, or at 2 mM calcium with important synergistic activity of combinations of any of these stimuli. Signals induced by ACTH at 10-30 min included cAMP, TGF-ß, and Erk1/2 phosphorylation. Affymetrix gene expression analysis showed that 2 h treatment of ACTH or 1,25(OH)2D increased the expression of bone regulating and structural mRNAs, including collagen I, biglycan, the vitamin D receptor, and TGF-ß. Accelerating expression of these bone-specific genes was confirmed by quantitative PCR. Expression of 1,25(OH)2D 1α-hydroxylase (1α-hydroxylase) increased with 1,25(OH)2D, ACTH, and extracellular calcium from 0.5 to 2 mM. Unlike renal 1α-hydroxylase, in osteoblasts, 1α-hydroxylase activity is independent of parathyroid hormone. In keeping with calcium responsivity, calcium-sensing receptor RNA and protein increased with 10 nM ACTH or 1,25(OH)2D. Inclusion of 200 nM cortisol or 10 nM ACTH in differentiation media blunted osteoblasts alkaline phosphatase response to 1,25(OH)2D and calcium. Our results point to the importance of ACTH in bone maintenance and that extra skeletal (renal) 1,25(OH)2D is required for bone mineralization despite 1α-hydroxylase expression by osteoblasts.