ALPL Expression Research Articles

The effect of Lycii Radicis Cortex extract on bone formation in vitro and in vivo.

Osteoporosis is a common skeletal disease caused by decreased bone mass; it enhances the risk of bone fracture. This study aimed to discover novel herbal extract(s) for the treatment of osteoporosis. We screened 64 ethanol extracts of edible plants native to Korea for their ability to increase the cellular proliferation and differentiation of two osteoblastic cell lines: C3H10T1/2 and MC3T3-E1. We selected a Lycii Radicis Cortex (LRC), Lycium Chinese root bark as the primary candidate. Treatment with LRC extract showed enhanced alkaline phosphatase activity and increased expression of bone metabolic markers Alpl, Runx2, and Bglap genes in both osteoblastic cell lines. There was no effect on the osteoclastic differentiation of primary-cultured monocytes from the mouse bone marrows. Furthermore, the study examined the effect of LRC extract in vivo in ovariectomizd (OVX) mice for 8 weeks and 16 weeks, respectively. Bone mineral density (BMD) was significantly higher in LRC extract-administered group than in the non-LRC-administered OVX control group. The results indicated that LRC extract prevented the OVX-induced BMD loss in mice via promoting the differentiation of osteoblast linage cells. These results suggest that LRC extract may be a good natural herbal medicine candidate for the treatment of osteoporosis.

Galectin-3 Inhibits Osteoblast Differentiation through Notch Signaling

Patients with bone cancer metastasis suffer from unbearable pain and bone fractures due to bone remodeling. This is caused by tumor cells that disturb the bone microenvironment. Here, we have investigated the role of tumor-secreted sugar-binding protein, i.e., galectin-3, on osteoblast differentiation and report that it downregulates the expression of osteoblast differentiation markers, e.g., RUNX2, SP7, ALPL, COL1A1, IBSP, and BGLAP, of treated human fetal osteoblast (hFOB) cells. Co-culturing of hFOB cells with human breast cancer BT-549 and prostate cancer LNCaP cells harboring galectin-3 has resulted in inhibition of osteoblast differentiation by the secreted galectin-3 into culture medium. The inhibitory effect of galectin-3 was found to be through its binding to Notch1 in a sugar-dependent manner that has led to accelerated Notch1 cleavage and activation of Notch signaling. Taken together, our findings show that soluble galectin-3 in the bone microenvironment niche regulates bone remodeling through Notch signaling, suggesting a novel bone metastasis therapeutic target.

Effects of egg yolk-derived peptide on osteogenic gene expression and MAPK activation.

The present study investigated the effects of egg yolk-derived peptide (YPEP) on osteogenic activities and MAPK-regulation of osteogenic gene expressions. The effects of YPEP on cell proliferation, alkaline phosphatase activity, collagen synthesis, and mineralization were measured in human osteoblastic MG-63 cells. Activation of MAPKs and downstream transcription factors such as extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2), p38, ELK1, and cJUN were examined using western blot analysis. YPEP dose-dependently increased MG-63 cell proliferation, ALP activity, collagen synthesis, and calcium deposition. YPEP activated ERK1/2, p38, and ELK1 phosphorylation whereas JNK and cJUN were not affected by YPEP. The COL1A1 (collagen, type I, alpha 1), ALPL (alkaline phosphatase), and SPP1 (secreted phosphoprotein 1, osteopontin) gene expressions were increased while BGLAP (osteocalcin) was not affected by YPEP. The ERK1/2 inhibitor (PD98509) blocked the YPEP-induced COL1A1 and ALPL gene expressions as well as ELK1 phosphorylation. The p38 inhibitor (SB203580) blocked YPEP-induced COL1A1 and ALPL gene expressions. SPP1 gene expression was not affected by these MAPK inhibitors. In conclusion, YPEP treatment stimulates the osteogenic differentiation via the MAPK/ELK1 signaling pathway. These results could provide a mechanistic explanation for the bone-strengthening effects of YPEP.

Zebrafish (Danio rerio) in calcium-poor water mobilise calcium and phosphorus from scales

Summary Calcium is of vital importance in vertebrates and plasma levels are tightly regulated. Terrestrial vertebrates depend for their calcium uptake exclusively on the diet, while fish have an essentially infinite directly accessible source of calcium in the surrounding water. The need to store calcium seems thus less evident in fish. It was the goal of the present study to investigate the possible role of the scale compartment of zebrafish (Danio rerio) as an accessible pool for calcium and phosphorus. We restricted the calcium availability to zebrafish both in water and diet. Following 12 days restriction, we found that scalar contents of calcium and phosphorus had declined, while magnesium was unaffected, indicating resorption of calcium hydroxyapatite. Osteoclast activity on scales of calcium-restricted fish increased, as we conclude from enlarged demineralized areas on scales, increased TRAcP activity staining, and higher ctsk gene expression. Osteoblasts respond to calcium limitation by increasing expression of col1a2 and alpl. In the presence of water and dietary calcium, removal of scales from one flank of the fish does not affect scalar mineral contents, scale demineralization and osteoclastic TRAcP activity; expression of sp7, alpl and ctsk are up-regulated. Apparently, a high demand for calcium in zebrafish is preferentially complemented with exogenous calcium, while calcium is recruited from the scales when environmental calcium is limited. We conclude that the scales represent an important deposit of available calcium and phosphorus from which these minerals can be recruited in periods of high demand. This finding contributes to a niche for scales as model in bone research.

The effect of hormone replacement therapy on the expression of the alkaline phosphatase gene (ALPL) within the mucosal epithelium of the cheek and in peripheral blood lymphocytes

Abstract In adult life, proper bone metabolism requires efficient regulation of bone formation and resorption processes. Bone turnover markers allow for assessing the rate of bone formation and resorption processes. In menopausal period, female patients experience gradual reduction in blood estradiol levels. The deficit of estrogens leads to enhanced osteoclastogenesis and bone resorption. Alkaline phosphatase (ALP) is a membranebound enzyme that stimulates the osteoblast activity and bone mineralization. It is synthesized by osteoblasts and incorporated into the newly formed bone tissue. The produced enzyme stimulates the osteoblast activity and bone mineralization. The goal of this study is to determine the effect of hormone replacement therapy in postmenopausal women on the expression of alkaline phosphatase gene (ALPL) within the mucosal epithelium of the cheek and in peripheral blood lymphocytes. The studies show that hormone replacement therapy has no significant effect on the increase in ALPL expression within the mucosal epithelium of the cheek. Only in women having undergone ovarectomy (OV), the epithelial ALPL expression level was higher than in the remaining groups.

Brief Report: Importance of SOX8 for In Vitro Chondrogenic Differentiation of Human Mesenchymal Stromal Cells

The transcription factor SOX9 is believed to be the master regulator of chondrogenesis. SOX8 is another SOX group E transcription factor with a high degree of homology to SOX9. Here, we demonstrate that SOX8 mRNA levels decrease during in vitro dedifferentiation of human articular chondrocytes and increase during chondrogenic differentiation of mesenchymal stromal cells. Knockdown of SOX9 reduced the expression of SOX8, COL2A1, and a range of other chondrogenic molecules. SOX8 knockdown reduced the expression of a large number of overlapping chondrogenic molecules, but not SOX9. Neither siSOX9 nor siSOX8 altered expression of the hypertrophic marker gene COL10A1. siSOX9, but not siSOX8 led to upregulation of hypertrophy associated genes MMP13 and ALPL. Transfection of synthetic SOX5, 6, and 9 mRNA trio upregulated SOX8, COL2A1, and ACAN, but not COL10A1 mRNA. Replacement of synthetic SOX9 by SOX8 in the SOX trio showed similar but lower chondrogenic effect. We conclude that SOX8 expression is regulated by SOX9, and that both together with SOX5 and SOX6 are required as a SOX quartet for transcription of COL2A1 and a large number of other chondrogenic molecules. Neither SOX8 nor SOX9 affect COL10A1 expression, but SOX9 inhibits chondrocyte hypertrophy through inhibition of MMP13 and ALPL expression.

β-Arrestin-1 mediates thyrotropin-enhanced osteoblast differentiation.

Thyrotropin (TSH) activation of the TSH receptor (TSHR), a 7-transmembrane-spanning receptor (7TMR), may have osteoprotective properties by direct effects on bone. TSHR activation by TSH phosphorylates protein kinases AKT1, p38α, and ERK1/2 in some cells. We found TSH-induced phosphorylation of these kinases in 2 cell lines engineered to express TSHRs, human embryonic kidney HEK-TSHR cells and human osteoblastic U2OS-TSHR cells. In U2OS-TSHR cells, TSH up-regulated pAKT1 (7.1±0.5-fold), p38α (2.9±0.4-fold), and pERK1/2 (3.1±0.2-fold), whereas small molecule TSHR agonist C2 had no or little effect on pAKT1 (1.8±0.08-fold), p38α (1.2±0.09-fold), and pERK1/2 (1.6±0.19-fold). Furthermore, TSH increased expression of osteoblast marker genes ALPL (8.2±4.6-fold), RANKL (21±5.9-fold), and osteopontin (OPN; 17±5.3-fold), whereas C2 had little effect (ALPL, 1.7±0.5-fold; RANKL, 1.3±0.6-fold; and OPN, 2.2±0.7-fold). β-Arrestin-1 and -2 can mediate activatory signals by 7TMRs. TSH stimulated translocation of β-arrestin-1 and -2 to TSHR, whereas C2 failed to translocate either β-arrestin. Down-regulation of β-arrestin-1 by siRNA inhibited TSH-stimulated phosphorylation of ERK1/2, p38α, and AKT1, whereas down-regulation of β-arrestin-2 increased phosphorylation of AKT1 in both cell types and of ERK1/2 in HEK-TSHR cells. Knockdown of β-arrestin-1 inhibited TSH-stimulated up-regulation of mRNAs for OPN by 87 ± 1.7% and RANKL by 73 ± 2.4%, and OPN secretion by 74 ± 10%. We conclude that TSH enhances osteoblast differentiation in U2OS cells that is, in part, caused by activatory signals mediated by β-arrestin-1.

Sex and Genetic Factors Determine Osteoblastic Differentiation Potential of Murine Bone Marrow Stromal Cells

Sex and genetic factors determine skeletal mass, and we tested whether bone histomorphometric parameters were sexually dimorphic in femurs from 1 to 6 month old C57BL/6 mice. Trabecular bone volume declined more rapidly in female mice than in male littermates because of enhanced bone resorption. Although bone formation was not different between sexes, female mice exhibited a higher number of osteoblasts than male littermates, suggesting that osteoblasts from female mice may have a reduced ability to form bone. To determine the impact of sex on osteoblastogenesis, we investigated the potential for osteoblastic differentiation of bone marrow stromal cells from C57BL/6, Friend leukemia virus-B (FVB), C3H/HeJ and BALB/c mice of both sexes. Bone marrow stromal cells from female FVB, C57BL/6 and C3H/HeJ mice exhibited lower Alpl and Osteocalcin expression and alkaline phosphatase activity, and formed fewer mineralized nodules than cells from male littermates. Proliferative capacity was greater in cells from male than female C57BL/6, but not FVB, mice. Sorting of bone marrow stromal cells from mice expressing an α-Smooth muscle actin-green fluorescent protein transgene, revealed a higher yield of mesenchymal stem cells in cultures from male mice than in those from female littermates. Sex had a modest impact on osteoblastic differentiation of mesenchymal stem cells. To determine the influence of sex and genetic factors on osteoblast function, calvarial osteoblasts were harvested from C57BL/6, FVB, C3H/HeJ and BALB/c mice. Alpl expression and activity were lower in osteoblasts from C57BL/6 and C3H/HeJ, but not FVB or BALB/c, female mice than in cells from littermates. Sex had no effect on osteoclastogenesis of bone marrow cultures of C57BL/6 mice, but osteoblasts from female mice exhibited higher Rankl and lower Opg expression than cells from male littermates. In conclusion, osteoblastogenesis is sexually dimorphic and influenced by genetic factors.

Collagen hydrolysates increased osteogenic gene expressions via a MAPK signaling pathway in MG-63 human osteoblasts

The present study investigated the effects of CHs on osteogenic activities and MAPK-regulation on bone matrix gene expressions. The effects of CHs on cell proliferation, alkaline phosphatase (ALP) activity, collagen synthesis, and mineralization were measured in human osteoblastic MG-63 cells. Activation of MAPKs and downstream transcription factors such as extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2), p38, ELK1, and cJUN was examined using Western blot analysis. The expressions of osteogenic genes were measured by quantitative real-time PCR. CHs dose-dependently increased MG-63 cell proliferation, ALP activity, collagen synthesis, and calcium deposition. CHs activated ERK1/2, JNK1/2, p38, and ELK1 phosphorylation except cJUN. The COL1A1 (collagen, type I, alpha 1), ALPL (alkaline phosphatase), BGLAP (osteocalcin), and SPP1 (secreted phosphoprotein 1, osteopontin) gene expressions were increased by CH treatment. The ERK1/2 inhibitor (PD98509) blocked the CH-induced COL1A1 and ALPL gene expression, as well as ELK1 phosphorylation. The JNK1/2 inhibitor (SP600125) abolished CH-induced COL1A1 expression. The p38 inhibitor (SB203580) blocked CH-induced COL1A1 and SPP1 gene expression. In conclusion, CH treatment stimulates the osteogenic activities and increases bone matrix gene expressions via the MAPK/ELK1 signaling pathway. These results could provide a mechanistic explanation for the bone-strengthening effects of CHs.

Comparative study of biphasic calcium phosphate with beta-tricalcium phosphate in rat cranial defects—A molecular-biological and histological study

The aim of this study was to evaluate the in vivo biocompatibility of a biphasic calcium phosphate (BCP) bone graft substitute consisting of 60% hydroxyapatite and 40% β-tricalcium phosphate (β-TCP) in comparison to a pure β-TCP of identical shape and porosity. The materials were evaluated using an established rat cranial defect model in 24 animals. One bone defect with a diameter of 5mm was created per animal. The defects were filled with either BCP or β-TCP and left to heal for 4 weeks. Twelve samples (6 per material) were processed for histological evaluation and immunohistochemistry. The remaining 12 samples were processed for mRNA expression analysis. No signs of inflammation or adverse material reactions were detected. New bone formation in the former defect site did not differ between the two groups (BCP: 49.2%; β-TCP: 52.4%). Osteoblast-like and TRAP-positive osteoclast-like cells were found at the surface of the bone graft substitute granules. The β-TCP group showed significantly higher mRNA levels for the bone resorption marker Acp5 and osteogenic differentiation marker Runx2. The expression of IGF1, IGF2, VEGF, Phex, Alpl, Col1, Col2, Bglap and MMP8 did not differ between the groups. The in vivo biocompatibility of BCP is to a large part identical to those of TCP. Within the limitation of the animal model, the implantation study shows that BCP can be used as bone graft substitute, due to the fact that the material integrates into tissue, remains stable in the implantation bed and serves as an osteoconductive scaffold.

Involvement of MAPK signaling pathway in the osteogenic gene expressions of Cervi Pantotrichum Cornu in MG-63 human osteoblast-like cells

AimsThe purposes of this study were to determine whether Cervi Pantotrichum Cornu (CPC) has osteogenic activities in human osteoblastic MG-63 cells and to investigate the underlying molecular mechanism. Main methodsThe effects of CPC on alkaline phosphatase activity, collagen synthesis, and calcium deposits were measured. The COL1A1, ALPL, BGLAP, and SPP1 expressions were measured by real-time PCR. Phosphorylated MAP kinases (ERK1/2, JNK1/2, p38, ELK1, and cJUN) were studied by western blot analysis. The involvement of MAPK pathway in osteogenic gene expressions was determined by using each selective MAPK inhibitor (PD98059, SP600125, and SB203580). Key findingsCPC increased alkaline phosphatase activity, collagen synthesis, and calcium deposits. CPC activated ERK1/2, JNK1/2, p38, and ELK1 phosphorylation except cJUN. CPC increased the COL1A1, ALPL, BGLAP, and SPP1 gene expressions. The elevated COL1A1 and BGLAP expressions were inhibited by PD98059, SP600125 or SB203580. The elevated ALPL expression was blocked by SB203580. The elevated SPP1 expression was inhibited by SP600125 or SB203580. CPC increased COL1A1 and BGLAP expressions via ERK1/2, JNK1/2, and p38 MAPKs pathways and SPP1 expression via JNK1/2 and p38 pathways. p38 pathway is needed for ALPL expression. SignificanceThese results imply that MAPK signaling pathway is an indispensable factor for bone matrix genes expression of CPC in MG-63 human osteoblast-like cells.

Differentiation of osteoprogenitor cells is affected by trauma-haemorrhage

IntroductionIn multiple trauma patients an increased incidence of delayed healing and non-union has been observed. The exact mechanisms underlying this delayed fracture healing are still not fully understood. Material and Methods40 male C57BL/6N-mice underwent standardized midline laparotomy and pressure-controlled haemorrhage (TH) or implantation of catheters without blood loss (sham procedure). Animals were sacrificed 24h or 72h later. Osteoprogenitor cells derived from bone marrow were isolated and differentiated into osteoblasts for 20 days and osteoclasts for 7 days. Osteoblast mineralization and osteoclast numbers were determined, and gene expression of Alpl, Bglap, Opg, Rankl was measured in osteoblast cell culture, as well as gene expression of Rank, Ctsk and Nfatc1 was determined in osteoclast cell culture. Furthermore, plasma Opg, Rankl and TRAP were measured. ResultsMineralization capacity of osteoblasts was unchanged after TH, but Alpl gene expression after 23 days was significantly decreased compared to sham. Osteoclast number of group TH 8 days was significantly decreased compared to sham. Furthermore, gene expression of Ctsk and Nfatc1 were increased in group TH 10 days compared to group TH 8 days. Plasma Opg concentration was significantly elevated and Rankl concentrations were significantly declined in TH groups compared to sham groups after 24h and 72h. ConclusionTH results in a diminished osteoclast number after 8 days, whereas differentiation of osteoblasts seems to be unaffected. The reduction of osteoclast number seems to be mediated through the Rankl-Opg-signalling pathway. However, further studies in models including a fractured extremity with a longer observation period are needed to identify the relevance of the Rankl-Opg- pathway in delayed fracture healing after TH and to focus on possible therapeutic interventions.

Abstract 2991: Coordinate methylation of the alkaline phosphatase gene family in colon cancer. Implications for understanding the manifestation of the CpG island methylator phenotype (CIMP). .

Abstract The HDAC-inhibitor sodium-butyrate, promotes differentiation of colon cancer cells as evidenced by induction of alkaline phosphatase (ALP) enzyme activity. Screening of a panel of 34 colon cancer cell lines identified cell lines responsive (44%) and refractory (56%) to butyrate-induced differentiation. Refractory cell lines were significantly enriched for those harbouring the CpG island methylator phenotype (CIMP) (P=0.016). The ALP gene family comprises 4 members - ALPi, ALPL, ALPP and ALPPL2 - each of which was induced by butyrate in responsive cell lines, demonstrating co-ordinate regulation of this gene family in response to HDACi treatment. Deletion analyses of the ALPi promoter identified a KLF/Sp1 cis element essential for butyrate-induction of ALPi promoter activity. Remarkably, we observed that a CpG dinucleotide within this site was preferentially methylated in refractory cell lines. Furthermore, the majority of cell lines with methylation of this site in the ALPi promoter also harboured methylation of similar sites within the ALPL and ALPP promoters, indicating coordinate methylation of this gene family in refractory cell lines. Frequent methylation and reduced expression of ALPi and ALPL were also observed in primary human colon cancers. Targeted inactivation of the DNA methyltransferases Dnmt1 and Dnmt3b (HCT116-DKO) and co-treatment with decitabine restored butyrate-inducibility of ALP in the methylated HCT116 cell line. However, butyrate-induction of an exogenous ALPi promoter-reporter paralleled induction of endogenous ALPi mRNA across the cell lines, suggesting ALPi promoter methylation may not be the primary determinant of ALP inducibility. We therefore explored whether the differential induction of the ALP gene family in colon cancer cells was due to selective induction of a common transcriptional regulator. To address this, responsive and refractory cell lines were treated with butyrate for 72h and transcription factors selectively induced in responsive cell lines identified by microarray. By focussing on factors which bind KLF/Sp1 regulatory elements, we identified KLF5 as selectively induced in responsive cell lines. KLF5 was also preferentially induced by butyrate in HCT116 DKO cells. Importantly, knockdown of KLF5 markedly attenuated butyrate-induction of ALP mRNA expression and enzyme activity. These findings demonstrate that the ALP gene family is co-ordinately inducible by butyrate in select colon cancer cell lines, in a KLF5-dependent manner. Cells lines refractory to butyrate-induction of the ALP gene family were enriched for CIMP high lines, and displayed coordinate promoter methylation. We propose that the coordinate methylation of this gene family in CIMP high colon cancers is likely linked to their coordinate regulation. Citation Format: John Mariadason, Azadeh Carr, Georgia A. Corner, Sheren Al-Obaidi, Anderly Chueh, Fiona Chionh, Lars Tögel, Naseem Ahmed, Diego Arango, Daniel Buchanan, Joanne Young, Leonard H. Augenlicht, Madhu S. Malo, Richard Hodin, Oliver M. Sieber, Thomas Weber, Joongho Shin. Coordinate methylation of the alkaline phosphatase gene family in colon cancer. Implications for understanding the manifestation of the CpG island methylator phenotype (CIMP). . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2991. doi:10.1158/1538-7445.AM2013-2991

Adipose derived mesenchymal stem cells – Their osteogenicity and osteoblast in vitro mineralization on titanium granule carriers

PurposeAdipose derived mesenchymal stem cells (ADMSCs) may be osteogenic, may generate neoangiogenisis and may be progenitors for differentiated osteoblast mineralization. Titanium granules may be suitable as carriers for these cells. The aim was to demonstrate the osteogenic potential of ADMSCs and the effect of porous non-oxidized (Ti) and oxidized titanium (TiO2) granules as carriers for ADMSCs mineralization in vitro. Materials and methodsADMSCs were isolated, cultivated in osteoblast medium and evaluated for alkaline phosphatase (ALP) assay, RNA isolation, and ALP staining. Osteoblast in vitro mineralization cells without granules or seeded on Ti or TiO2 granules were evaluated for Alizarin Red assay and RNA isolation for later gene expressing. ResultsADMSCs express osteoblastic lineage genes, CBFA-1 and stain strongly for ALP. Mineralization was significantly higher for cells seeded on TiO2 than on Ti granules or pure cells. Expression of ALPL and RUNX2 was significantly higher for cells seeded on TiO2 granules and expression of COL1α1 for pure cells was significantly higher than for cells seeded on granules. ConclusionADMSCs have osteogenic potential. Mineralization was significantly high when cells were seeded on TiO2 granules. TiO2 granules may be used as carriers for adipose derived mesenchymal osteoblastic cells from laboratory bench to the patient.

Loss of equilibrative nucleoside transporter 1 in mice leads to progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis in humans

Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory spondyloarthropathy, characterized by ectopic calcification of spinal tissues. Symptoms include spine pain and stiffness, and in severe cases dysphagia and spinal cord compression. The etiology of DISH is unknown and there are no specific treatments. Recent studies have suggested a role for purine metabolism in the regulation of biomineralization. Equilibrative nucleoside transporter 1 (ENT1) transfers hydrophilic nucleosides, such as adenosine, across the plasma membrane. In mice lacking ENT1, we observed the development of calcified lesions resembling DISH. By 12 months of age, ENT1(-/-) mice exhibited signs of spine stiffness, hind limb dysfunction, and paralysis. Micro-computed tomography (µCT) revealed ectopic mineralization of paraspinal tissues in the cervical-thoracic region at 2 months of age, which extended to the lumbar and caudal regions with advancing age. Energy-dispersive X-ray microanalysis of lesions revealed a high content of calcium and phosphorus with a ratio similar to that of cortical bone. At 12 months of age, histological examination of ENT1(-/-) mice revealed large, irregular accumulations of eosinophilic material in paraspinal ligaments and entheses, intervertebral discs, and sternocostal articulations. There was no evidence of mineralization in appendicular joints or blood vessels, indicating specificity for the axial skeleton. Plasma adenosine levels were significantly greater in ENT1(-/-) mice than in wild-type, consistent with loss of ENT1--a primary adenosine uptake pathway. There was a significant reduction in the expression of Enpp1, Ank, and Alpl in intervertebral discs from ENT1(-/-) mice compared to wild-type mice. Elevated plasma levels of inorganic pyrophosphate in ENT1(-/-) mice indicated generalized disruption of pyrophosphate homeostasis. This is the first report of a role for ENT1 in regulating the calcification of soft tissues. Moreover, ENT1(-/-) mice may be a useful model for investigating pathogenesis and evaluating therapeutics for the prevention of mineralization in DISH and related disorders.

Candidate Human Primary Mesenchymal Stem/Progenitor Cells Are Highly Enriched in Linneg/CD45neg/CD271pos/PDGFRαneg Bone Marrow Cells

Abstract 3460Human bone marrow (BM) contains a rare population of non-hematopoietic mesenchymal stem cells (BM-MSC) which can differentiate toward skeletal lineages such as osteoblasts, adipocytes, chondrocytes and hematopoiesis-supporting stromal cells. In vivo, BM-MSC are essential constituents of the hematopoietic stem cell niche, thus playing an important role in supporting, maintaining and controlling hematopoiesis.We and others have previously shown that primary BM-MSC were exclusively found in the linneg/CD45neg/CD271pos cell fraction in human bone marrow, and we furthermore reported that expression of CD146 on BM-MSC correlated with in-situ localization (Tormin et al., Blood 2011,117[19]:5067–5077). Although BM-MSC were highly enriched in linneg/CD45neg/CD271pos cells as reflected by CFU-F frequencies of about 1 in 20, there was still a considerable fraction of non-colony forming cells present in this population. Therefore, the current study aimed to identify novel MSC markers that would allow for a more precise definition of the candidate stromal stem cell population in human bone marrow.Human bone marrow linneg/CD45neg cells were sorted based on CD271 expression and comparative gene expression profiling was performed using the Illumina Human HT-12 expression v4 BeadChip comprising 48,107 probes. In total, 215 genes were found to be significantly up-regulated in the linneg/CD45neg/CD271pos subset compared to linneg/CD45neg/CD271neg cells, whereas 97 genes were down-regulated. Twenty eight of the upregulated genes correlated to surface markers and expression of thirteen of them could be verified by FACS. Several of the surface markers identified by this approach, such as CD140b, CD10 and CD106 were previously described in the context of MSC isolation. However, the majority of them represented novel MSC markers including molecules such as CD151, CD81, IFNGR2, LEPR, TGFBR3, IL1R1, CD18, CD140a, and FGFR3.FACS analysis of these markers on linneg/CD45neg/CD271pos cells revealed two staining patterns, i.e. A) marker expression either correlated directly with CD271 expression, or B) the novel maker was only expressed on a fraction of linneg/CD45neg/CD271pos cells. CD151 and CD106 are examples for pattern A markers and, as expected, CFU-F frequencies in sorted linneg/CD45neg/CD271pos/CD151pos and linneg/CD45neg/CD271pos/CD106pos cells were comparable with linneg/CD45neg/CD271pos cells. Furthermore, proliferation and in-vitro/in-vivo differentiation capacities were comparable. On the other hand, using CD140a (platelet-derived growth factor receptor α, PDGFRα) - one of the pattern B markers - allowed to clearly identify a population of linneg/CD45neg/CD271pos/CD140aneg cells which were highly enriched for CFU-F (24.15 ± 4.51 CFU-Fs per 100 plated cells, n=6) compared to linneg/CD45neg/CD271pos/CD140apos cells (1.13 ± 0.65 CFU-Fs per 100 plated cells, n=6). The high CFU-F frequency in CD140aneg cells was furthermore confirmed in single cell sorting and limiting dilution experiments. Quantitative RT-PCR of sorted primary CD140neg MSC showed considerably higher expression of ALPL, PPARγ, and ACAN as well as Oct4, Sox2 and Nanog compared to CD140apos cells, and multicolor FACS analysis revealed that linneg/CD45neg/CD271pos/CD140aneg cells co-expressed typical primary MSC markers (CD90, CD105, CD140b, STRO-1), but not CD31 and CD34. Furthermore, linneg/CD45neg/CD271pos/CD140aneg cells (bulk and single cell sorted) gave rise to typical cultured MSC (expression of standard surface markers, in-vitro differentiation capacity). Moreover, linneg/CD45neg/CD271pos/CD140aneg -derived stromal cells formed bone, adipocytes and hematopoietic stroma when transplanted s.c. into NOD-SCID mice.Taken together, sorting of linneg/CD45neg/CD271pos cells based on CD140a (PDGFRα) expression enabled to isolate CFU-F with thus far unmet precision. Linneg/CD45neg/CD271pos/CD140aneg cells had typical BM-MSC properties, thus possibly representing a close to pure population of the candidate human primary mesenchymal stem/progenitor cells. These findings will enable to better characterize native BM-MSC and establish their physiological role in vivo. Disclosures:No relevant conflicts of interest to declare.

Systems Genetics of Metabolism: The Use of the BXD Murine Reference Panel for Multiscalar Integration of Traits

Metabolic homeostasis is achieved by complex molecular and cellular networks that differ significantly among individuals and are difficult to model with genetically engineered lines of mice optimized to study single gene function. Here, we systematically acquired metabolic phenotypes by using the EUMODIC EMPReSS protocols across a large panel of isogenic but diverse strains of mice (BXD type) to study the genetic control of metabolism. We generated and analyzed 140 classical phenotypes and deposited these in an open-access web service for systems genetics (www.genenetwork.org). Heritability, influence of sex, and genetic modifiers of traits were examined singly and jointly by using quantitative-trait locus (QTL) and expression QTL-mapping methods. Traits and networks were linked to loci encompassing both known variants and novel candidate genes, including alkaline phosphatase (ALPL), here linked to hypophosphatasia. The assembled and curated phenotypes provide key resources and exemplars that can be used to dissect complex metabolic traits and disorders.

Influence of BONITmatrix® and OSSA NOVA on the expression of bone specific genes

The aim of this study was to examine the effects of BONITmatrix(®) and OSSA NOVA bone graft materials on osteogenic differentiation. For this, mRNA expression of Alpl, Acp5, Bglap, Phex and Runx2 was analysed using quantitative RT-PCR in surgically created defects to the cranium of adult male rats. Cranial samples were collected after implantation of BONITmatrix(®) or OSSA NOVA scaffolds into these defects for 4 weeks and gene expressions of Alpl, Acp5, Bglap, Phex and Runx2 were measured by quantitative RT-PCR. Real-time PCR analyses showed significantly higher expressions of Acp5, Alpl and Bglap only in the group treated with BONITmatrix(®) compared to untreated controls. No changes in transcript expression of Phex and Runx2 were detectable between the analysed groups. Furthermore, in rats treated with OSSA NOVA the same transcript level of all tested genes was detected as shown in controls. In conclusion, it can be stated that only the granular dosage form of BONITmatrix(®) is able to stimulate osteoblast differentiation, whereas OSSA NOVA has no influence on the gene expression of osteoblasts.

SP7 Inhibits Osteoblast Differentiation at a Late Stage in Mice

RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation. RUNX2 induced Sp7 expression in Runx2 −/− calvarial cells. Adenoviral transfer of sh-Sp7 into primary osteoblasts reduced the expression of Alpl, Col1a1, and Bglap2 and mineralization, whereas that of Sp7 reduced Bglap2 expression and mineralization at a late stage of osteoblast differentiation. Sp7 transgenic mice under the control of 2.3 kb Col1a1 promoter showed osteopenia and woven-bone like structure in the cortical bone, which was thin and less mineralized, in a dose-dependent manner. Further, the number of processes in the osteoblasts and osteocytes was reduced. Although the osteoblast density was increased, the bone formation was reduced. The frequency of BrdU incorporation was increased in the osteoblastic cells, while the expression of Col1a1, Spp1, Ibsp, and Bglap2 was reduced. Further, the osteopenia in Sp7 or Runx2 transgenic mice was worsened in Sp7/Runx2 double transgenic mice and the expression of Col1a1 and Bglap2 was reduced. The expression of Sp7 and Runx2 was not increased in Runx2 and Sp7 transgenic mice, respectively. The expression of endogenous Sp7 was increased in Sp7 transgenic mice and Sp7-transduced cells; the introduction of Sp7 activated and sh-Sp7 inhibited Sp7 promoter; and ChIP assay showed the binding of endogenous SP7 in the proximal region of Sp7 promoter. These findings suggest that SP7 and RUNX2 inhibit osteoblast differentiation at a late stage in a manner independent of RUNX2 and SP7, respectively, and SP7 positively regulates its own promoter.

Epigenetic regulation of alkaline phosphatase in human cells of the osteoblastic lineage

Epigenetic mechanisms play an important role in the tissue-specific regulation of gene expression. This study analyzed the relationship between tissue non-specific alkaline phosphatase (ALPL) gene expression and the methylation of a CpG island located in its proximal region. Gene expression was analyzed by real time RT-qPCR in primary human osteoblasts (hOBs), the osteoblastic cell line MG-63, the mammary cell line MCF-7, and bone tissue. DNA methylation was analyzed by qMSP in those cells and also in lining osteoblasts and in osteocytes obtained from human bone samples by laser-assisted capture. hOBs expressed much more ALPL mRNA than MG-63 cells (7.3±3.2 vs. 0.2±0.1 arbitrary units, respectively). hOBs showed a very weak DNA methylation (<10%), whereas MG-63 had a higher degree of methylation (58±6%). Likewise, MCF-7 cells, which scarcely expressed ALPL, had a hypermethylated CpG island. Thus, the degree of methylation in the CpG island was inversely associated with the transcriptional levels of ALPL in the studied cells. Furthermore, treatment with the DNA demethylating agent AzadC induced a 30-fold increase in ALPL expression, in MG-63 cells, accompanied by a parallel increase in alkaline phosphatase activity. However, AzadC did not affect ALPL levels in the already hypomethylated hOBs. In addition, in microdissected osteocytes, which do not express alkaline phosphatase, the CpG island was highly methylated (>90%), whereas lining osteoblasts showed an intermediate degree of methylation (58±13%). These results suggest an important role of DNA methylation in the regulation of ALPL expression through the osteoblast-osteocyte transition.