The Interaction between Vitamin D and Extracellular Calcium on Osteogenic Differentiation
Dongqing Yang
B.Sc, M.Biotechnology
Thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
October, 2014
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Thesis abstract...... I
Declaration...... III
Acknowledgements...... IV
Publications and Presentations ...... VI
Chapter 1 Literature review: The interaction between vitamin D and
extracellular calcium in osteogenic differentiation...... 1
1.1 Osteoblast biology ...... 2
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1.2 Nutritional factors interacting with osteoblasts ...... 13
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1.3 Conclusions and proposal for study ...... 25
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Chapter 2 Materials and Methods...... 28
2.1 Materials ...... 28
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2.2 Nucleotide preparations ...... 29
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2.3 Isolation of osteoblasts ...... 32
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2.4 Histochemical staining ...... 38
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2.5 Osteoblast proliferation assay ...... 40
2.6 Osteoblast differentiation and mineralisation assay ...... 40
Chapter 3 Differential effects of 1,25-dihydroxyvitamin D on mineralisation and differentiation in two different types of osteoblast- like cultures...... 42
Abstract ...... 46
3.1 Introduction ...... 46
3.2 Materials and methods ...... 47
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3.3 Results ...... 49
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3.4 Discussion ...... 50
References ...... 50
Erratum ...... 51
Chapter 4 The regulation of osteogenic differentiation of calvaria- derived osteoblasts in response to 1,25D and calcium, and the influence of the level of VDR expression ...... 52
4.1 Introduction ...... 52
4.2 Experimental methods ...... 54
4.3 Results ...... 60
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4.4 Discussion ...... 81
4.5 Conclusion ...... 90 LY
Chapter 5 Vitamin D metabolites and extracellular calcium promote mineral deposition by a mature osteoblast cell line MLO-A5 ...... 91
Abstract ...... 95
5.1 Introduction ...... 96
5.2 Materials and methods ...... 97
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5.3 Results ...... 99
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5.4 Discussion ...... 102
Acknowledgements ...... 105
References ...... 106
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Chapter 6 Conclusion remarks...... 122
References ...... 129
Appendix I...... 151
Appendix II ...... 156
Appendix III...... 170
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,KHUHE\FHUWLI\WKDWWKHVWDWHPHQWRIFRQWULEXWLRQLVDFFXUDWH 6LJQ 'DWH +$0RUULV 3ODQQHGDQGVXSHUYLVHGUHVHDUFKZRUNKHOSHGZLWKGDWDLQWHUSUHWDWLRQDQGPDQXVFULSW SUHSDUDWLRQ6HQLRUDXWKRU ,KHUHE\FHUWLI\WKDWWKHVWDWHPHQWRIFRQWULEXWLRQLVDFFXUDWH 6LJQ 'DWH Journal of Steroid Biochemistry & Molecular Biology 136 (2013) 166–170 Contents lists available at SciVerse ScienceDirect Journal of Steroid Biochemistry and Molecular Biology journa l homepage: www.elsevier.com/locate/jsbmb Review Differential effects of 1,25-dihydroxyvitamin D on mineralisation and differentiation in two different types of osteoblast-like cultures a,b,c,∗ c b,d b,d a,b,d D. Yang , G.J. Atkins , A.G. Turner , P.H. Anderson , H.A. Morris a Discipline of Medicine, University of Adelaide, Adelaide, SA 5005, Australia b Endocrine Bone Research, Chemical Pathology, SA Pathology, Adelaide, SA 5000, Australia c Bone Cell Biology Group, Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, SA 5005, Australia d Musculoskeletal Biology Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia a r t i c l e i n f o a b s t r a c t Article history: In osteoblast cultures, 1,25-dihydroxyvitamin D (1,25D) has been shown to play either catabolic or Received 5 July 2012 anabolic roles on differentiation and mineralisation. We have employed osteoblast-like cells extracted Received in revised form 6 November 2012 from neonatal mouse calvariae and cells derived from juvenile mouse long bones to compare the bio- Accepted 28 November 2012 logical effects of 1,25D on differentiation and mineralisation in vitro. 1,25D exerts differential effects on osteoblast-like cells depending on their stage of maturation and possibly their skeletal origin. Keywords: This article is part of a Special Issue entitled ‘Vitamin D Workshop’. Osteogenesis © 2012 Elsevier Ltd. All rights reserved. 1,25-Dihydroxyvitamin D Osteoblast differentiation Mineralisation Contents 1. Introduction ...... 166 2. Materials and methods...... 167 2.1. Isolation of osteoblasts from juvenile mouse cortical bones ...... 167 2.2. Isolation of osteoblast from mouse neonatal calvarial bones ...... 167 2.3. Differentiation assays ...... 167 2.4. Statistics ...... 169 3. Results ...... 169 3.1. Characterisation of cells ...... 169 3.2. In vitro mineralisation and response to 1,25D ...... 170 3.3. 1,25D effects on gene expression ...... 170 4. Discussion ...... 170 References ...... 170 eral deposition. A proportion of these mature cells will become 1. Introduction embedded in the matrix, forming an osteoid- or pre-osteocyte, and finally differentiate into mature osteocytes embedded in mineral. Osteoblastogenesis is essential for bone formation requiring Understanding the process of osteoblast differentiation and miner- a sequential process of cell proliferation and maturation [1]. alisation is important for elucidating the cellular activities required Immature osteoblasts are capable of proliferating and express- for bone health and a number of in vitro models have been utilised ing genes required for synthesis of extracellular matrix compared [2]. to mature osteoblasts which exhibit activities required for min- Numerous studies have demonstrated an interaction between vitamin D activity and osteoblast differentiation with the expres- sion of a multitude of osteoblast genes directly regulated by ∗ Corresponding author at: Endocrine Bone Research, Chemical Pathology, SA 1,25-dihydroxyvitamin D(1,25D) [3]. However there remains con- Pathology, Box 14 Rundle Mall PO, Adelaide, SA 5000, Australia. Tel.: +61 882223514; siderable controversy regarding whether the overall activity of fax: +61 882223518. 1,25D is catabolic or anabolic for bone. 1,25D was reported to inhibit E-mail addresses: [email protected], [email protected] (D. Yang). proliferation and enhance differentiation of osteoblasts isolated 0960-0760/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jsbmb.2012.11.016 46 D. Yang et al. / Journal of Steroid Biochemistry & Molecular Biology 136 (2013) 166–170 167 Table 1 Primer sets used for real-time PCR for gene amplification. Gene Sequence (5 –3 ) Forward: TCCTGACCAAAAACCTCAAAGG TNAP Reverse: TGCTTCATGCAGAGCCTGC Forward: AGGGTGTGATGGTGGGAAT -Actin Reverse: GCTGGGGTGTTGAAGGTCT Forward: AGGCATAAAGGGTCATCGTG Col-IA1 Reverse: CGTTGAGTCCGTCTTTGCCA Forward: CCTGAAGGTGAATGACGCCAGA MEPE Reverse: TGCTCCTGTCTTCATTCGGCATT Forward: AGACCTAGCAGACACCATGA OCN Reverse: GAAGGCTTTGTCAGACTCAG Forward: GCGTCCTCTCTGCTTGAGG OSX Reverse: GGCTTCTTTGTGCCTCCTTTC Forward: TGAAGACACACTACCTGACTCCTG RANKL Reverse: CTGGCAGCATTGATGGTGAG Forward: CACAAGGACAGAGTCAGATTACAGAT RUNX2 Reverse: CGTGGTGGAGTGGATGGAT Forward: CTGAATGAAGAAGGCTCCGAT VDR Reverse: AAGCAGGACAATCTGGTCATCA from rat calvaria in the early 1990s [4]. The response of osteoblasts to 1,25D appears to depend on the stage of osteoblast matura- tion, with preferential induction of the catabolic factor, receptor activator of nuclear factor kappa B ligand (RANKL), in osteoblast cultures related to an immature phenotype rather than a mature phenotype [5]. In the osteoblastic cell line MC3T3-E1, 1,25D had pro-anabolic activity by enhancing the production of matrix vesi- cles and mineral deposition [6]. Osteogenesis, as measured by mineral deposition in vitro, is a critical readout of osteoblast differ- entiation and maturation. In vitro mineralisation by VDR-null mouse primary calvarial cells was enhanced compared to wild type cells [7], while mineral nodule formation by primary bone marrow cells was reduced by the ablation of vitamin D activity [8]. To address the controversy around the role of vitamin D activity on in vitro osteogenesis, we examined the effects of 1,25D on two types of Fig. 1. Calvarial cells express a gene profile consistent with an immature osteoblast phenotype compared to cortical cells. Ratios of Col-IA1, TNAP and OCN mRNA mouse primary osteoblast-like cells either extracted from neona- species to -actin mRNA from calvarial cells (closed bar) and cortical cells (open tal calvariae or derived from pre-pubertal mouse cortical bone. We bar) (mean ± SEM, n = 3; *p < 0.05; **p < 0.01). report here that 1,25D acts on these two types of osteoblast popu- lations distinctly, including effects on mineral deposition and gene expression. 2.2. Isolation of osteoblast from mouse neonatal calvarial bones Calvaria were dissected from 1 to 3 day old C57BL/6 mouse pups and carefully cleaned with a scalpel [10]. Multiple calvariae 2. Materials and methods were pooled and subjected to sequential enzymatic digestion using collagenase-I (1 mg/ml) and dispase (2 mg/ml) (Invitrogen, ◦ 2.1. Isolation of osteoblasts from juvenile mouse cortical bones Australia) for 10 min at 37 C in a shaking water bath. After this time, cells were collected and the digestion protocol repeated 6 Explant cultures of osteoblast-like cells were derived from times in total. Cells pooled from digests 2–6 were used as primary mouse long bones [9]. Femora and tibiae from 4-week old C57BL/6 cultures. mice were excised and all connected soft tissue removed with a scalpel. Bone marrow was removed by flushing with PBS 2.3. Differentiation assays through a syringe after removal of growth plate regions. Dia- 3 ∼ physeal cortical bone was then minced into 1 mm pieces Cells were seeded into 24-well plates at a density of 4 and digested by 2 mg/ml of collagenase-I (Invitrogen, Australia) 3 × 10 cells/well and cultured in growth media, comprising of ◦ ␣ ␣ at 37 C for 1 h. Cortical bone chips were incubated in - MEM (Invitrogen) with 10% FCS (Hyclone), antibiotics and l- ␣ Minimal Essential Medium ( MEM) (Invitrogen, Australia) with glutamine, for 3 days to reach 100% confluence. Cells were then 10% Foetal Calf Serum (FCS) (Hyclone, Australia), antibiotics cultured in differentiation media comprising growth media con- l and -glutamine. The osteoblast-like cells which grew from taining 50 mg/ml ascorbic acid (Sigma–Aldrich, St Louis, MO, USA) the bone chips were passaged to provide sufficient cells for and 10 mM -glycerol phosphate (Sigma). Fresh differentiation experimentation. medium was supplied to cultures every 72 h with either 1,25D 47 168 D. Yang et al. / Journal of Steroid Biochemistry & Molecular Biology 136 (2013) 166–170 Fig. 2. mRNA expression of RUNX2 (A) and OSX (B) by calvarial cells (left column) and cortical cells (right column) treated with vehicle (broken line) and 1 nM 1,25D (solid line) (mean ± SEM, n = 3). (1 nM, Wako, Japan) or vehicle (0.1% ethanol final concentration) hydroxide, and quantified by measuring absorption at 405 nm in added at each media change. In vitro mineralisation was visualised a spectrophotometer. by staining with Alizarin Red. The alizarin–calcium complex was Total RNA was extracted from the cell cultures using the tri- dissolved in acetic acid, neutralised to pH 4.2 with ammonium zol method (Invitrogen) and cDNA synthesised (superscript III) to &$/9$5,$/ 9(+,&/( ' &257,&$/ 0,1(5$/,6$7,21 QP 0,1(5$/,6$7,21 QP '$< '$< Fig. 3. Calvarial cells deposit less mineral, which is further reduced with 1,25D treatment, compared with cortical cells. In vitro mineralisation by calvarial cells (left) and ## cortical cells (right) treated with vehicle (broken line) and 1 nM 1,25D (solid line) (mean ± SEM, n = 3; effect of 1,25D treatment: **p < 0.01; effect of time: p < 0.01). 48 D. Yang et al. / Journal of Steroid Biochemistry & Molecular Biology 136 (2013) 166–170 169 Fig. 4. Differential effects of 1,25D on mRNA levels in calvarial and cortical cells. Ratios of VDR (A), RANKL (B) and MEPE (C) mRNA to -actin mRNA from calvarial cells (left column) and cortical cells (right column) treated with vehicle (broken line) and 1 nM 1,25D (solid line) (mean ± SEM, n = 3; MEPE mRNA levels were undetectable in cortical # cells (data not shown). Effect of 1,25D treatment: **p < 0.01; effect of time: p < 0.05). measure the expression of genes of interest using real-time PCR. 3. Results The primer sets for each gene are listed in Table 1. Each experi- ment was conducted in triplicate and repeated on three occasions. 3.1. Characterisation of cells Gene expression was normalised to that of the housekeeping gene,  -actin. At 100% confluence, the relative mRNA level of type-I collagen alpha 1 (Col-IA1) in calvarial cells was 3-fold higher than in cortical 2.4. Statistics cells (p < 0.05), whereas the expression of tissue non-specific alka- line phosphatase (TNAP) and osteocalcin (OCN) in cortical cells was All data were analysed by GraphPadPrism 5.04 (Graphpad Soft- 5-fold (p < 0.05) and 30-fold (p < 0.01) higher, respectively, than in ware, San Diego, USA). t-Tests were used to analyse data from single calvarial cells (Fig. 1). time point experiments. Two-way ANOVA, followed by Bonferroni Expression levels of early osteoblast transcriptional factors post-tests, were used to assess the effect of 1,25D treatment in RUNX2 and OSX were initially relatively high in cortical cells and long-term cultures. reduced with time, whereas levels in calvarial cultures remained 49 170 D. Yang et al. / Journal of Steroid Biochemistry & Molecular Biology 136 (2013) 166–170 unchanged. The expression of RUNX2 and OSX mRNA was unaf- Our findings suggest that the interpretation of data from in vitro fected by 1,25D (1 nM) treatment in both cell types (Fig. 2A and osteoblast models requires characterisation of the maturation stage B). of the cell preparations under study. Each culture type displays a particular differentiation potential and phenotypic profile. Fur- 3.2. In vitro mineralisation and response to 1,25D thermore 1,25D, an important regulator of osteoblast activity, also has distinct effects at different stages of osteoblast differentiation. The level of mineralisation in cortical cell cultures over 21 days Thus, interpretation of data related to the activities of 1,25D on bone was 6-fold greater than in calvarial cell cultures after 24 days. cells must also be made in the context of the maturation stage of Moreover, the mineralisation in cortical cell cultures was not sig- the osteoblasts. It is possible that at least some of the differential nificantly impacted by 1,25D treatment while mineral deposition response to 1,25D was due to the different skeletal sites from which in calvarial cell cultures was inhibited by 1,25D (p < 0.01), and by the cultures originated. It is interesting from this point of view that day 24 was reduced by 28% (p < 0.01) (Fig. 3). the skull is protected from the age-related bone loss observed in the axial skeleton; it will be of interest in future studies to com- 3.3. 1,25D effects on gene expression pare directly the effect of vitamin D on cranial and axial skeletal bone mass. The findings presented here suggest that the choice of In calvarial cell cultures, relative mRNA levels of matrix extra- osteoblast model is critical to the outcome of studies into osteoblast cellular phosphoglycoprotein (MEPE) showed a significant increase differentiation and their response to 1,25D. over time (p < 0.05) while VDR showed a trend to increase over time (p = 0.082). Relative RANKL mRNA levels did not change with References time (p = 0.23). VDR and RANKL mRNAs were further significantly [1] G.S. Stein, J.B. Lian, Molecular mechanisms mediating prolifera- induced by 1,25D treatment (p < 0.01) while MEPE mRNA was tion/differentiation interrelationships during progressive development unchanged (p = 0.13 at 24 days) (Fig. 4A–C). Notably, the relative of the osteoblast phenotype, Endocrine Reviews 14 (4) (1993) 424–442. level of RANKL mRNA expression in calvarial cultures was 1000-fold [2] V. Kartsogiannis, K.W. Ng, Cell lines and primary cell cultures in the study of bone cell biology, Molecular and Cellular Endocrinology 228 (1–2) (2004) higher than in cortical cells (Fig. 4B). 79–102. In cortical cell cultures, the mRNA levels of VDR and RANKL were [3] G.J. Atkins, D.M. Findlay, P.H. Anderson, H.A. Morris, Target genes: bone pro- detectable but unaffected by incubation with 1,25D (Fig. 4A and B). teins, in: D. Feldman, J.W. Pike, J.S. Adams (Eds.), Vitamin D, 3rd edition, Elsevier, The expression of MEPE was undetectable in cortical cultures (data San Diego, 2011. [4] T.A. Owen, et al., Pleiotropic effects of vitamin D on osteoblast gene expression not shown) in the presence or absence of 1,25D. are related to the proliferative and differentiated state of the bone cell pheno- type: dependency upon basal levels of gene expression, duration of exposure, and bone matrix competency in normal rat osteoblast cultures, Endocrinology 4. Discussion 128 (3) (1991) 1496–1504. [5] G.J. Atkins, et al., RANKL expression is related to the differentiation state Our results show that two different mouse primary osteoblast of human osteoblasts, Journal of Bone and Mineral Research 18 (6) (2003) 1088–1098. preparations used in this study had different properties prior to [6] V.J. Woeckel, et al., 1Alpha,25-(OH)2D3 acts in the early phase of osteoblast in vitro differentiation, suggesting that the calvarial preparation differentiation to enhance mineralization via accelerated production of represents a more immature osteoblast phenotype than the corti- mature matrix vesicles, Journal of Cellular Physiology 225 (2) (2010) cal preparation. Compared to calvarial cells, cortical cells expressed 593–600. [7] K. Sooy, Y. Sabbagh, M.B. Demay, Osteoblasts lacking the vitamin D receptor less Col-IA1 but more TNAP and OCN mRNA. At early stages of display enhanced osteogenic potential in vitro, Journal of Cellular Biochemistry differentiation, osteoblasts express more Col-IA1 mRNA synthesis- 94 (1) (2005) 81–87. ing extracellular matrix than in the post-matrix producing period, [8] D.K. Panda, et al., Inactivation of the 25-hydroxyvitamin D 1alpha-hydroxylase and vitamin D receptor demonstrates independent and interdependent effects when the expression of TNAP, an enzyme that degrades the potent of calcium and vitamin D on skeletal and mineral homeostasis, Journal of Bio- mineralisation inhibitor pyrophosphate, is increased enabling min- logical Chemistry 279 (16) (2004) 16754–16766. eralisation to proceed [11]. OCN is defined as a mature osteoblast [9] A. Bakker, J. Klein-Nulend, Osteoblast isolation from murine calvariae and long bones, Methods in Molecular Medicine 80 (2003) 19–28. marker [12,13]. The ability of cortical cells to deposit mineral in vitro [10] H. Zhou, et al., Osteoblasts directly control lineage commitment of mesenchy- was significantly enhanced compared to calvarial cells consistent mal progenitor cells through Wnt signaling, Journal of Biological Chemistry 283 with the difference in their apparent stage of maturation. More- (4) (2008) 1936–1945. [11] L.D. Quarles, et al., Distinct proliferative and differentiated stages of murine over, the ability of the pro-catabolic gene RANKL to be induced by MC3T3-E1 cells in culture: an in vitro model of osteoblast development, Journal 1,25D in calvarial cultures is also consistent with these cells repre- of Bone and Mineral Research 7 (6) (1992) 683–692. senting a more immature osteoblast model than the cortical cells [12] M. Morike, et al., Expression of osteoblastic markers in cultured human bone [5]. and fracture callus cells, Journal of Molecular Medicine (Berlin) 73 (11) (1995) 571–575. The osteoblast gene product MEPE, which increased with time [13] R. Yamamoto, et al., 1alpha,25-dihydroxyvitamin D3 acts predominately in the calvarial cell cultures, exerts a post-translational, inhibitory in mature osteoblasts under conditions of high extracellular phosphate to role on osteoblast mineralisation [14,15]. Furthermore when cul- increase fibroblast growth factor 23 production in vitro, Journal of Endocrinol- ogy 206 (3) (2010) 279–286. tures were treated with 1,25D, the levels of VDR and RANKL mRNAs [14] W.N. Addison, et al., MEPE-ASARM peptides control extracellular matrix min- were increased in calvarial cells but not in cortical cells. This pro- eralization by binding to hydroxyapatite: an inhibition regulated by PHEX catabolic, anti-anabolic gene expression profile in calvarial cultures cleavage of ASARM, Journal of Bone and Mineral Research 23 (10) (2008) 1638–1649. is consistent with the overall lower levels of mineral deposition by [15] G.J. Atkins, et al., Sclerostin is a locally acting regulator of late- these cells and the inhibitory effect of 1,25D compared with the 6- osteoblast/preosteocyte differentiation and regulates mineralization through fold greater level of mineral deposition and the absence of a 1,25D a MEPE-ASARM-dependent mechanism, Journal of Bone and Mineral Research 26 (7) (2011) 1425–1436. effect in cortical cells. 50 G Model SBMB 4225 No. of Pages 1 Journal of Steroid Biochemistry & Molecular Biology xxx (2014) exxx Contents lists available at ScienceDirect Journal of Steroid Biochemistry & Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb Erratum Erratum to “Differential effects of 1,25-dihydroxyvitamin D on mineralisation and differentiation in two different types of osteoblast-like cultures” [J. Steroid Biochem. Mol. Biol. 136 (2013) 166–170] a,b,c, c b,d b,d a,b,d D. Yang *, G.J. Atkins , A.G. Turner , P.H. Anderson , H.A. Morris a Discipline of Medicine, University of Adelaide, Adelaide, SA 5005, Australia b Endocrine Bone Research, Chemical Pathology, SA Pathology, Adelaide, SA 5000, Australia c Bone Cell Biology Group, Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, SA 5005, Australia d Musculoskeletal Biology research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia The authors regret that there was an error in this article describing cortical cells as primary cells isolated from juvenile mouse long bones. Our subsequent analysis has demonstrated unequivocally that the cortical cells used in this study were the well-characterised MLO- A5 cells as described by Kato et al., J. Bone Miner. Res. 16 (2001) 1622–1633. The MLO-A5 cell line was isolated as a single colony cultured from the long bones of juvenile mice expressing the osteocalcin promoter-driven T-antigen as a transgene (Kato et al., J. Bone Miner. Res. 12 (1997) 2014–2023). As such, this study should be considered a comparison between murine calvarium derived osteoblasts and a murine long bone derived osteoblast cell line. The authors do not wish to change the presentation or interpretation of the data related to this study. Section 2.1 should be replaced by the following paragraph: 2.1 MLO-A5 cells as a model of cortical bone cells The MLO-A5 cell line, utilised as a model of cortical postosteoblast/preosteocyte cells, was generously provided by Prof. Lynda Bonewald (University of Missouri–Kansas City, MO, USA) and cultured as previously described (Kato et al., J. Bone Miner. Res. 16 (2001) 1622–1633). DOI of original article: http://dx.doi.org/10.1016/j.jsbmb.2012.11.016 * Corresponding author. 0960-0760/$ – see front matter ã 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jsbmb.2014.07.006 Please cite this article in press as: D. 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igure 4.3.1 A-C Vitamin D receptor status in WT and OSVDR cells The mRNA ratios of mouse vitamin D receptor (mVDR) to E-Actin by WT (A) and OSVDR (B) cells and the mRNA ratio of human vitamin D receptor (hVDR) to E-Actin by OSVDR cells only (C), with vehicle (black line), acute 1,25D (1 nM) (red dots) and chronic 1,25D (1 nM) (blue line) treatments in standard osteogenic differentiation media (1.8 mM total calcium). Three independent experiments for gene expression analyses were carried out and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; * indicates the effects of 1,25D treatments matching with the color of treatment groups, actue 1,25D (red), or chronic 1,25D (blue); * p <0.05;**p< 0.01; *** p <0.001). 61 ' &<3$,1'8&7,21%<:7&(//6 $&7,1 P51$ E &<3$ '$<6 ( &<3$,1'8&7,21%<269'5&(//6 $&7,1 P51$ E &<3$ '$<6 VEHICLE ACUTE 1,25D CHRONIC 1,25D Figure 4.3.1 D and E Cyp24a1 induction in WT and OSVDR cells by 1,25D treatments The mRNA ratios of 25-hydroxyvitamin D 24-hydroxylase (Cyp24a1)toE-Actin by WT (D) and OSVDR (E) cells, with vehicle (black line), acute 1,25D (1 nM) (red dots) and chronic 1,25D (1 nM) (blue line) treatments in standard osteogenic differentiation media (1.8 mM total calcium). Three independent experiments for gene expression analyses were carried out and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; * indicates the effects of 1,25D treatments matching with the color of treatment groups, actue 1,25D (red), or chronic 1,25D (blue); * p <0.05;**p<0.01;***p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igure4.3.2A-HComparisonofgeneexpressionprofileofWTandVDRKOcells Relative gene expression by wild type (WT) (solid line) and vitamin D receptor knockout (VDRKO) (broken line) cells over the 24-day culture with standard osteogenicdifferentiationmediasupplied(1.8mMtotalcalcium).Threeindependent experiments for gene expression analyses were carried out and the error bars on graphs represent the standard errors of mean data collected from the three experiments(meanfSEM,n=3). A: Col1D1 (type-I collagen) B: Tnap (tissue nonspecific alkaline phosphatase) C: Ank (ankloysis protein) D: Enpp1 (ecto-nucleotide pyrophosphatase/phosphodiesterase-1) E: Ocn (osteocalcin) F: E11 (gp38) G: Phex (phosphate regulating endopeptidase homolog, X-linked) H: Mepe (matrix extracellular phosphoglycoprotein) * p<0.0 65 0 2 . , 581; E$&7,1 P51$ 23* E$&7,1 P51$ '03 E$&7,1 P51$ 6267 E$&7,1 P51$ 581; 6267 '03 23* '$<6 '$<6 '$<6 '$<6 3 :7 66 1 / 3 - 26; E$&7,1 P51$ 5$1./ 23* P51$ 5$1./ E$&7,1 P51$ )*) E$&7,1 P51$ 9'5.2 5$1./23* 5$1./ )*) '$<6 '$<6 '$<6 '$<6 26; Figure4.3.2I-PComparisonofgeneexpressionprofileofWTandVDRKOcells Relative gene expression by wild type (WT) (solid line) and vitamin D receptor knockout (VDRKO) (broken line) cells over the 24-day culture with standard osteogenicdifferentiationmediasupplied(1.8mMtotalcalcium).Threeindependent experiments for gene expression analyses were carried out and the error bars on graphs represent the standard errors of mean data collected from the three experiments(meanfSEM,n=3). , Sost VFOHURVWLQ - Fgf23 ILEUREODVWJURZWKIDFWRU . Dmp1 GHQWLQPDWUL[SURWHLQ / Rankl UHFHSWRUDFWLYDWRURI1)NDSSD%OLJDQG 0 Opg RVWHRSURWHJHULQ 1 Rankl/Opg 2 Runx2 UXQWUHODWHGWUDQVFULSWLRQIDFWRU 3 Osx RVWHUL[ S S 67 :7 269'5 $ % &2/D 71$3 $&7,1 P51$ E $&7,1 P51$ E D 71$3 &2/ '$<6 '$<6 & ' $1. (133 3 $&7,1 P51$ $&7,1 P51$ E E $1. (133 '$<6 '$<6 ( ) 2&1 ( $&7,1 P51$ $&7,1 P51$ E E 2&1 ( '$<6 '$<6 * + 3+(; 0(3( 3 $&7,1 P51$ $&7,1 P51$ E E 0(3( 3+(; '$<6 '$<6 68 Figure4.3.3A-HComparisonofgeneexpressionprofileofWTandOSVDRcells Relativegeneexpressionbywildtype(WT)(solidline)andover-expressedvitaminD receptor in osteocalcin expressing (OSVDR) (broken line) cells over the 24-day culturewithstandardosteogenicdifferentiationmediasupplied(1.8mMtotalcalcium). Three independent experiments for gene expression analyses were carried out and theerrorbarsongraphsrepresentthestandarderrorsofmeandatacollectedfromthe threeexperiments(meanfSEM,n=3). $ Col1D1 W\SH,FROODJHQ % Tnap WLVVXHQRQVSHFLILFDONDOLQHSKRVSKDWDVH & Ank DQNOR\VLV SURWHLQ ' Enpp1 HFWRQXFOHRWLGHS\URSKRVSKDWDVHSKRVSKRGLHVWHUDVH ( Ocn RVWHRFDOFLQ ) E11 JS * Phex SKRVSKDWHUHJXODWLQJHQGRSHSWLGDVH KRPRORJ;OLQNHG + Mepe PDWUL[H[WUDFHOOXODUSKRVSKRJO\FRSURWHLQ S 69 :7 269'5 , - 6267 )*) $&7,1 P51$ $&7,1 P51$ E E 6267 )*) '$<6 '$<6 . / '03 5$1./ $&7,1 P51$ E $&7,1 P51$ E 5$1./ '03 '$<6 '$<6 0 1 23* 5$1./23* $&7,1 P51$ E 23* 5$1./ 23* P51$ '$<6 '$<6 70 Figure4.3.3I-NComparisonofgeneexpressionprofileofWTandOSVDRcells Relativegeneexpressionbywildtype(WT)(solidline)andover-expressedvitaminD receptor in osteocalcin expressing (OSVDR) (broken line) cells over the 24-day culturewithstandardosteogenicdifferentiationmediasupplied(1.8mMtotalcalcium). Threeindependentexperimentsforgeneexpressionanalyseswerecarriedoutand theerrorbarsongraphsrepresentthestandarderrorsofmeandatacollectedfrom thethreeexperiments(meanfSEM,n=3). , Sost VFOHURVWLQ - Fgf23 ILEUREODVWJURZWKIDFWRU . 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(133 $&7,1 P51$ E $&7,1 P51$ E $1. (133 :7 269'5 :7 269'5 ( ) 2&1 ( $&7,1 P51$ $&7,1 P51$ E E S ( 2&1 :7 269'5 :7 269'5 * + 3+(; 0(3( S $&7,1 P51$ $&7,1 P51$ E E 3+(; 0(3( :7 269'5 :7 269'5 9(+,&/($&87( ' &+521,& ' 74 Figure4.3.4A-FOsteogenicdifferentiationrelatedgeneexpressionsunderacute andchronic1,25DtreatmentsbyDay-24WTandOSVDRcellcultures The mRNA ratios of osteogenic differentiation related genes, Col1D1 (A), Tnap (B), Ank(C),Enpp1(D),Ocn(E),E11(F),Phex(G),Mepe(H)toE-ActinbyDay-24ofWT and OSVDR cell cultures,with vehicle (blackbar),acute1,25D (1nM)(red bar)and chronic1,25D(1nM)(bluebar)treatmentsinstandardosteogenicdifferentiationmedia (1.8mMtotalcalcium).Threeindependentexperimentsforgeneexpressionanalyses werecarriedoutandtheerrorbarsongraphsrepresentthestandarderrorsofmean datacollectedfromthethreeexperiments.(meanfSEM,n=3;*indicatestheeffects of1,25Dtreatmentsmatchingwiththecoloroftreatmentgroups,actue1,25D(red),or chronic1,25D(blue);*p<0.05;**p<0.01;***p<0.001) 75 , - 6267 )*) $&7,1 P51$ $&7,1 P51$ E E 6267 )*) :7 269'5 :7 269'5 . / '03 5$1./ $&7,1 P51$ E $&7,1 P51$ E 5$1./ '03 :7 269'5 :7 269'5 1 0 5$1./23* 23* $&7,1 P51$ E 23* 5$1./ 23* P51$ :7 269'5 :7 269'5 9(+,&/($&87( ' &+521,& ' Figure 4.3.4 I-N Osteogenic differentiation related gene expressions under acute and chronic 1,25D treatments by Day-24 WT and OSVDR cell cultures The mRNA ratios of osteogenic differentiation related genes, Sost (I), Fgf23 (J), Dmp1 (K), Rankl (L), Opg (M) to E-Actin and Rankl/Opg (N), by Day-24 of WT and OSVDR cell cultures, with vehicle (black bar), acute 1,25D (1nM) (red bar) and chronic 1,25D (1nM) (blue bar) treatments in standard osteogenic differentiation media (1.8 mM total calcium). Three independent experiments for gene expression analyses were carried out and the error bars on graphs represent the standard errors of mean data collected from the three experiments. (meanfSEM, n = 3; * indicates the effects of 1,25D treatments matching with the color of treatment groups, actue 1,25D (red), or chronic 1,25D (blue); * p < 0.05; ** p< 0.01; *** p <0.001) 76 FKURQLF'WUHDWPHQW )LJ&'(*,- WKHP51$OHYHORI'PSZDV GHFUHDVHGE\DFXWH'WUHDWPHQWEXWQRWFKDQJHGE\FKURQLF' )LJ. 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ppendix II WT OSVDR VDRKO 30 20 -ACTIN mRNA E 1: 10 D COL1 0 03 9 15 24 03 9 15 24 03 9 15 24 DAYS DAYS DAYS 1.8 mM calcium 2.8 mM calcium 30 20 -ACTIN mRNA E 1: 10 D COL1 0 03 9 15 24 03 9 15 24 03 9 15 24 DAYS DAYS DAYS VEHICLE CHRONIC 1,25D Appendix II. A mRNA levels of Col1D1 The mRNA ratios of Col1D to E-Actin by WT (left), OSVDR (middle) and VDRKO (right) cells, with vehicle (solid line) and chronic 1,25D (1nM) (broken line) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3). 156 h RArto of ratios mRNA The B II. Appendix fma aacletdfo h he xeiet (mean experiments three errors the standard from the collected represent data graphs mean on of bars error the and conducted were analyses expression Mttlclim(oe)peetdi utr media. culture in 2.8 and presented (upper) (lower) mM calcium 1.8 with total treatments mM line) (broken (1nM) 1,25D chronic and line) (solid vehicle TNAP : E-ACTIN mRNA TNAP : E-ACTIN mRNA 0.03 0.06 0.09 0.03 0.06 0.09 0 0 3915 9 03 3915 9 03 RAlvl of levels mRNA DAYS DAYS Tnap WT to E -Actin Tnap 24 24 yW lf) SD mdl)adVRO(ih)cls with cells, (right) VDRKO and (middle) OSVDR (left), WT by VEHICLE 3915 9 03 391 24 15 9 03 . Mcalcium 2.8 mM calcium 1.8 mM 157 OSVDR DAYS DAYS he needn xeiet o gene for experiments independent Three HOI 1,25D CHRONIC f E,n=3 <.5 * p<0.001). *** p<0.05, * 3; = n SEM, *** * 24 3915 9 03 3915 9 03 VDRKO DAYS DAYS 24 24 h RArto of ratios mRNA The C II. Appendix . Mttlclim(oe)peetdi utr media. culture and in (upper) presented mM 1.8 (lower) calcium with treatments total line) mM (broken 2.8 (1nM) 1,25D chronic and line) (solid vehicle roso endt olce rmtetreeprmns(mean experiments three the standard from the collected represent data graphs mean on of bars errors error the and conducted were analyses expression gene ANK : E-ACTIN mRNA ANK : E-ACTIN mRNA 0.08 0.08 0.06 0.06 0.02 0.04 0.02 0.04 0 0 3915 9 03 3915 9 03 RAlvl of levels mRNA DAYS DAYS Ank WT to E -Actin ** Ank 24 24 yW lf) SD mdl)adVRO(ih)cls with cells, (right) VDRKO and (middle) OSVDR (left), WT by VEHICLE 3915 9 03 3915 9 03 . Mcalcium 2.8 mM calcium 1.8 mM 158 OSVDR DAYS DAYS HOI 1,25D CHRONIC he needn xeiet for experiments independent Three f 24 24 E,n=3 *p<0.01). ** 3; = n SEM, 3915 9 03 3915 9 03 VDRKO DAYS DAYS 24 24 fma aacletdfo h he xeiet (mean errors experiments three standard the the from represent collected graphs data mean on of bars error the and conducted were analyses expression Mttlclim(oe)peetdi utr media. culture 2.8 in and presented (upper) mM (lower) 1.8 calcium with total treatments line) mM (broken (1nM) 1,25D chronic and line) (solid vehicle h RArto of ratios mRNA The D II. Appendix 0.003 0.004 0.002 ENPP1 : -ACTIN mRNA ENPP10.001 : -ACTIN mRNA E 0.003 E 0.004 0.002 0.001 0 0 3915 9 03 3915 9 03 RAlvl of levels mRNA DAYS DAYS Enpp1 WT to E -Actin ** Enpp1 * 24 24 yW lf) SD mdl)adVRO(ih)cls with cells, (right) VDRKO and (middle) OSVDR (left), WT by 3915 9 03 VEHICLE 3915 9 03 159 . Mcalcium 2.8 mM calcium 1.8 mM OSVDR DAYS DAYS he needn xeiet o gene for experiments independent Three HOI 1,25D CHRONIC f E,n=3 <.5 *p<0.01). ** p<0.05, * 3; = n SEM, 24 24 3915 9 03 3915 9 03 VDRKO DAYS DAYS 24 24 Mttlclim(oe)peetdi utr media. culture in 2.8 and presented (upper) (lower) mM calcium 1.8 with total treatments mM line) (broken (1nM) 1,25D chronic and line) (solid vehicle h RArto of ratios mRNA The pedxI.E II. Appendix fma aacletdfo h he xeiet (mean experiments three errors the standard from the collected represent data graphs mean on of bars error the and conducted were analyses expression OCN : E-ACTIN mRNA OCN : E-ACTIN mRNA 8 8 6 2 4 0 6 2 4 0 3915 9 03 3915 9 03 RAlvl of levels mRNA DAYS DAYS WT Ocn to ** E -Actin 24 24 Ocn VEHICLE 3915 9 03 3915 9 03 yW lf) SD mdl)adVRO(ih)cls with cells, (right) VDRKO and (middle) OSVDR (left), WT by . Mcalcium 2.8 mM calcium 1.8 mM OSVDR DAYS 160 DAYS HOI 1,25D CHRONIC he needn xeiet o gene for experiments independent Three f E,n=3 <.5 *p<0.01). ** p<0.05, * 3; = n SEM, * 24 24 3915 9 03 3915 9 03 VDRKO DAYS DAYS 24 24 E11 : E-ACTIN mRNA E11 : E-ACTIN mRNA 0.15 0.15 0.05 0.10 0.05 0.10 n . Mttlclim(oe)peetdi utr media. culture in presented (upper) (lower) mM calcium 1.8 total with mM treatments 2.8 line) and (broken (1nM) 1,25D chronic and line) (solid vehicle with h RArto of ratios mRNA The F II. Appendix o eeepeso nlsswr odce n h ro aso rpsrpeetthe (mean experiments represent three graphs the from on collected bars data error mean of the errors and standard conducted were analyses expression gene for 0 0 3915 9 03 3915 9 03 RAlvl of levels mRNA DAYS DAYS WT E11 to E -Actin 24 24 E11 3915 9 03 VEHICLE 3915 9 03 yW lf) SD mdl)adVRO(ih)cells, (right) VDRKO and (middle) OSVDR (left), WT by 161 . Mcalcium 2.8 mM calcium 1.8 mM OSVDR DAYS DAYS HOI 1,25D CHRONIC 24 24 he needn experiments independent Three 3915 9 03 3915 9 03 f VDRKO E,n=3). = n SEM, DAYS DAYS 24 24 Mttlclim(oe)peetdi utr media. culture in 2.8 and presented (upper) (lower) mM calcium 1.8 with total treatments mM line) (broken (1nM) 1,25D chronic and line) (solid vehicle h RArto of ratios mRNA The G II. Appendix fma aacletdfo h he xeiet (mean experiments three errors the standard from the collected represent data graphs mean on of bars error the and conducted were analyses expression PHEX : E-ACTIN mRNA PHEX : E-ACTIN mRNA 0.06 0.06 0.02 0.04 0.02 0.04 0 0 3915 9 03 3915 9 03 RAlvl of levels mRNA DAYS DAYS Phex WT to E -Actin Phex 24 24 yW lf) SD mdl)adVRO(ih)cls with cells, (right) VDRKO and (middle) OSVDR (left), WT by VEHICLE 3915 9 03 3915 9 03 . Mcalcium 2.8 mM calcium 1.8 mM 162 OSVDR DAYS DAYS HOI 1,25D CHRONIC he needn xeiet o gene for experiments independent Three f E,n=3 <.5 * p<0.001). *** p<0.05, * 3; = n SEM, *** * 24 24 3915 9 03 3915 9 03 VDRKO DAYS DAYS 24 24 roso endt olce rmtetreeprmns(mean experiments three the standard from the collected represent data graphs mean on of bars errors error the and conducted were analyses expression gene ihvhce(oi ie n hoi ,5 1M boe ie ramnswt . M(upper) media. mM culture 1.8 in with presented (lower) treatments calcium line) total (broken mM 2.8 (1nM) and 1,25D chronic and line) (solid vehicle with h RArto of ratios mRNA The H II. Appendix MEPE : E-ACTIN mRNA MEPE : E-ACTIN mRNA 0.03 0.03 0.01 0.02 0.01 0.02 0.04 0.04 0 0 3915 9 03 3915 9 03 RAlvl of levels mRNA DAYS DAYS WT Mepe to E Mepe -Actin 24 24 VEHICLE 3915 9 03 3915 9 03 yW lf) SD mdl)adVRO(ih)cells, (right) VDRKO and (middle) OSVDR (left), WT by 163 . Mcalcium 2.8 mM calcium 1.8 mM OSVDR DAYS DAYS HOI 1,25D CHRONIC he needn xeiet for experiments independent Three * f 24 24 E,n=3 p<0.05). * 3; = n SEM, 3915 9 03 3915 9 03 VDRKO DAYS DAYS 24 24 pedxI.I II. Appendix Mttlclim(oe)peetdi utr media. culture 2.8 in and presented (upper) mM (lower) 1.8 calcium with total treatments mM line) (broken (1nM) 1,25D chronic and line) (solid vehicle fma aacletdfo h he xeiet (mean experiments errors three standard the the from represent collected data graphs mean on of bars error the and conducted were analyses expression h RArto of ratios mRNA The SOST : E-ACTIN mRNA 0.0025 0.0050 SOST : -ACTIN mRNA 0.0025 E 0.0050 0.0075 0.0075 0 0 3915 9 03 3915 9 03 RAlvl of levels mRNA DAYS Sost DAYS WT to E -Actin Sost * 24 24 yW lf) SD mdl)adVRO(ih)cls with cells, (right) VDRKO and (middle) OSVDR (left), WT by VEHICLE 3915 9 03 3915 9 03 164 . Mcalcium 2.8 mM calcium 1.8 mM OSVDR DAYS DAYS he needn xeiet o gene for experiments independent Three HOI 1,25D CHRONIC f E,n=3 <.5 * p<0.001). *** p<0.05, * 3; = n SEM, *** *** 24 24 3915 9 03 3915 9 03 VDRKO DAYS DAYS 24 24 h RArto of ratios mRNA The pedxI.J II. Appendix eil sldln)adcrnc12D(n)(rknln)tetet ih18m upr n . mM 2.8 media. and culture (upper) in mM presented 1.8 (lower) with calcium treatments total line) (broken (1nM) 1,25D chronic and line) (solid vehicle nlsswr odce n h ro aso rpsrpeettesadr roso endata mean of errors standard (mean the experiments represent three the graphs from on collected bars error the and conducted were analyses 0.003 0.004 0.002 FGF23 : -ACTIN mRNA FGF230.001 : -ACTIN mRNA E 0.003 E 0.004 0.002 0.001 0 0 3915 9 03 3915 9 03 RAlvl of levels mRNA Fgf23 DAYS DAYS WT to E Fgf23 -Actin ** ** 24 24 yW lf) SD mdl)adVRO(ih)cls with cells, (right) VDRKO and (middle) OSVDR (left), WT by VEHICLE 3915 9 03 391 24 15 9 03 f E,n=3 <.5 *p00,**p<0.001). *** p<0.01, ** p<0.05, * 3; = n SEM, he needn xeiet o eeexpression gene for experiments independent Three 165 . Mcalcium 2.8 mM calcium 1.8 mM OSVDR DAYS DAYS HOI 1,25D CHRONIC *** * 24 3915 9 03 3915 9 03 VDRKO DAYS DAYS 24 24 DM11 : E-ACTIN mRNA DMP1 : E-ACTIN mRNA 0.08 0.08 0.06 0.06 0.02 0.04 0.02 0.04 o eeepeso nlsswr odce n h ro aso rpsrpeetthe (mean represent experiments three graphs the on from collected bars data error mean of the errors and standard conducted were analyses expression gene for n . Mttlclim(oe)peetdi utr media. culture in presented (upper) mM (lower) calcium 1.8 with total mM treatments 2.8 line) and (broken (1nM) 1,25D chronic and line) (solid vehicle with h RArto of ratios mRNA The pedxI.K II. Appendix 0 0 3915 9 03 3915 9 03 RAlvl of levels mRNA DAYS DAYS WT Dmp1 to E 24 24 -Actin Dmp1 VEHICLE 3915 9 03 3915 9 03 yW lf) SD mdl)adVRO(ih)cells, (right) VDRKO and (middle) OSVDR (left), WT by . Mcalcium 2.8 mM calcium 1.8 mM 166 OSVDR DAYS DAYS HOI 1,25D CHRONIC he needn experiments independent Three 24 24 3915 9 03 3915 9 03 f VDRKO E,n=3). = n SEM, DAYS DAYS 24 24 WT OSVDR VDRKO 0.018 0.012 -ACTIN mRNA ** E 0.006 RANKL : 0 03 9 15 24 03 9 15 24 03 9 15 24 DAYS DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.018 0.012 -ACTIN mRNA E 0.006 RANKL : 0 03 9 15 24 03 9 15 24 03 9 15 24 DAYS DAYS DAYS VEHICLE CHRONIC 1,25D Appendix II. L mRNA levels of Rankl The mRNA ratios of Rankl to E-Actin by WT (left), OSVDR (middle) and VDRKO (right) cells, with vehicle (solid line) and chronic 1,25D (1nM) (broken line) treatments with 1.8 mM (upper) and 2.8 mM total calcum (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; ** p<0.01). 167 n . Mttlclim(oe)peetdi utr media. culture in (upper) presented mM (lower) 1.8 calcium with total treatments mM line) 2.8 (broken and (1nM) 1,25D chronic and line) (solid vehicle with h RArto of ratios mRNA The M II. Appendix o eeepeso nlsswr odce n h ro aso rpsrpeetthe represent graphs (mean experiments on three the bars from collected error data the mean of and errors standard conducted were analyses expression gene for OPG : E-ACTIN mRNA OPG : E-ACTIN mRNA 0.08 0.08 0.06 0.06 0.02 0.04 0.02 0.04 0 0 3915 9 03 3915 9 03 RAlvl of levels mRNA DAYS DAYS WT Opg to E -Actin Opg 24 24 VEHICLE yW lf) SD mdl)adVRO(ih)cells, (right) VDRKO and (middle) OSVDR (left), WT by 15 9 03 3915 9 03 . Mcalcium 2.8 mM calcium 1.8 mM 168 OSVDR DAYS DAYS HOI 1,25D CHRONIC he needn experiments independent Three 24 24 3915 9 03 3915 9 03 f E,n=3). = n SEM, VDRKO DAYS DAYS 24 24 RANKL : OPG mRNA RANKL : OPG mRNA eeepeso nlsswr odce n h ro aso rpsrpeetthe represent graphs (mean on experiments three bars the from error collected data the mean of and errors standard conducted were analyses expression gene h RArto of ratios mRNA The N II. Appendix . Mttlclim(oe)peetdi utr media. culture in and presented (upper) (lower) mM calcium 1.8 total with mM treatments 2.8 line) (broken (1nM) 1,25D chronic and line) (solid vehicle 0.5 1.5 0.5 1.5 1.0 1.0 2.0 2.0 0 0 3915 9 03 3915 9 03 RArto of ratios mRNA DAYS DAYS WT Rankl/Opg 24 24 Rankl/Opg yW lf) SD mdl)adVRO(ih)cls with cells, (right) VDRKO and (middle) OSVDR (left), WT by VEHICLE 3915 9 03 3915 9 03 . Mcalcium 2.8 mM calcium 1.8 mM 169 OSVDR DAYS DAYS HOI 1,25D CHRONIC he needn xeiet for experiments independent Three 24 24 3915 9 03 3915 9 03 f VDRKO E,n=3). = n SEM, DAYS DAYS 24 24 Appendix III WT OSVDR 40 20 -ACTIN mRNA E 1: D COL1 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 40 20 -ACTIN mRNA E 1: D COL1 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. A mRNA levels of Col1D1 The mRNA ratios of ColID to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3). 170 WT OSVDR 0.09 0.06 -ACTIN mRNA E 0.03 *** TNAP : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.09 0.06 -ACTIN mRNA E 0.03 ** TNAP : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. B mRNA levels of Tnap The mRNA ratios of Tnap to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; ** p<0.01, *** p<0.001). 171 WT OSVDR 0.08 0.06 0.04 * -ACTIN mRNA E 0.02 ANK : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.08 0.06 0.04 ** -ACTIN mRNA E 0.02 ANK : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. C mRNA levels of Ank The mRNA ratios of Ank to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; * p<0.05, ** p<0.01). 172 WT OSVDR 0.020 0.015 0.005 -ACTIN0.002 mRNA E 0.001 ENPP1 : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.020 0.015 0.005 -ACTIN0.002 mRNA E 0.001 ENPP1 : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. D mRNA levels of Enpp1 The mRNA ratios of Enpp1 to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcum (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 1 with OSVDR under acute 1,25D treated group and n = 3 with all other groups). 173 WT OSVDR 8 6 4 -ACTIN mRNA E 2 OCN : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 8 6 4 -ACTIN mRNA E 2 OCN : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. E mRNA levels of Ocn The mRNA ratios of Ocn to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3). 174 WT OSVDR 0.15 0.10 -ACTIN mRNA 0.05 E E11 : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.15 0.10 -ACTIN mRNA 0.05 E E11 : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. F mRNA levels of E11 The mRNA ratios of E11 to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3). 175 WT OSVDR 0.06 0.04 -ACTIN mRNA E 0.02 ** PHEX : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.06 0.04 -ACTIN mRNA E 0.02 * PHEX : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. G mRNA levels of Phex The mRNA ratios of Phex to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; * p<0.05, ** p<0.01). 176 WT OSVDR 0.04 0.03 0.02 -ACTIN mRNA E 0.01 * MEPE : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.04 0.03 0.02 -ACTIN mRNA E 0.01 ** MEPE : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. H mRNA levels of Mepe The mRNA ratios of Mepe to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; * p<0.05, ** p<0.01). 177 WT OSVDR 0.008 0.006 0.004 -ACTIN mRNA E ** 0.002 SOST : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.008 0.006 0.004 -ACTIN mRNA E 0.002 *** SOST : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. I mRNA levels of Sost The mRNA ratios of Sost to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; ** p<0.01, *** p<0.001). 178 WT OSVDR 0.0008 0.0006 0.0004 -ACTIN mRNA E 0.0002 FGF23 : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.0008 0.0006 0.0004 -ACTIN mRNA E 0.0002 FGF23 : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. J mRNA levels of Fgf23 The mRNA ratios of Fgf23 to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3). 179 WT OSVDR 0.08 0.06 0.04 -ACTIN mRNA E 0.02 * DMP1 : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.08 0.06 0.04 -ACTIN mRNA E 0.02 * DMP1 : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. K mRNA levels of Dmp1 The mRNA ratios of Dmp1 to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; * p<0.05). 180 WT OSVDR 0.04 0.03 0.02 -ACTIN mRNA *** E 0.01 ** RANKL : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.04 0.03 *** 0.02 -ACTIN mRNA E 0.01 * RANKL : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. L mRNA levels of Rankl The mRNA ratios of Rankl to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; * p<0.05, ** p<0.01, *** p<0.001). 181 WT OSVDR 0.08 0.06 0.04 -ACTIN mRNA E 0.02 * OPG : 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 0.08 0.06 0.04 -ACTIN mRNA E 0.02 OPG : 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. M mRNA levels of Opg The mRNA ratios of Opg to E-Actin by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; * p<0.05). 182 WT OSVDR 4 3 2 *** * 1 RANKL : OPG mRNA 0 03 9 15 24 03 9 15 24 DAYS DAYS 1.8 mM calcium 2.8 mM calcium 4 3 2 *** ** 1 RANKL : OPG mRNA 0 03 9 15 24 03 9 15 24 DAYS DAYS VEHICLE ACUTE 1,25D Appendix III. N mRNA ratios of Rankl/Opg The mRNA ratios of Rankl/Opg by WT (left) and OSVDR (right) cells, with vehicle (black lines) and acute 1,25D (1nM) (dots) treatments with 1.8 mM (upper) and 2.8 mM total calcium (lower) presented in culture media. Three independent experiments for gene expression analyses were conducted and the error bars on graphs represent the standard errors of mean data collected from the three experiments (meanfSEM, n = 3; * p<0.05, ** p<0.01, *** p<0.001). 183