The effect of PTH on IDG-SW3 cell morphology and motility.

<p>Images of GFP positive day 30 IDG-SW3 cells treated with PBS (<b>A</b>) or 50nM PTH (<b>B</b>) for 48 hours and captured by confocal microscopy. Scale bar = 25μm. (<b>C</b>) Mean velocity of mature IDG-SW3 cell motility in response to 50nM PTH over a 63 hour time course. Representative time lapse images can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125731#pone.0125731.s005" target="_blank">S1 Video</a> (n = 3 observation fields±SD, with each field containing 30–40 cells, *p<0.05) Changes in IDG-SW3 cell morphology induced by PTH, cAMP and forskolin. Effect on mature IDG-SW3 cell morphology induced by 48 hours treatment with dose response of (<b>D</b>) PTH, (<b>E</b>) 8-bromo-cAMP and (<b>F</b>) forskolin. Scale bar = 20μm.</p

L-type calcium channels mediate the effects of PTH on IDG-SW3 cell morphology and motility.

<p>(A, B) Real-time PCR analysis showing expression of the L and T-type channel subunits in IDG-SW3 cells cultured over a 29 day time course and treated with 50nM PTH for 24 hours at days 0, 7, 14, 21 and 28. Expression was normalized to <i>Actb</i> and is relative to day 1 control samples. (C) The L-type calcium channel blockers Nifedipine (250μM) and Diltiazem (100μM) inhibit the effect of 50nM PTH on mature IDG-SW3 cell morphology. (D) Mean velocity of mature IDG-SW3 cells treated with 50nM PTH alone or 50nM PTH with 100μM Diltiazem (DT) over a 46 hour time course (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125731#pone.0125731.s007" target="_blank">S3 Video</a>) (n = 3 observation fields±SD, with each field containing 30–40 cells, *p<0.05). Scale bar = 20μm.</p

Gene set enrichment analysis of PTH Responses in the IDG-SW3 Osteocyte Enriched Cell model and <i>Ex Vivo</i> Cortical Bone Osteocyte Model.

<p><b>(A)</b> 33,277 gene expression values were analyzed against the entire 1794 geneset of PTH responsive genes in the IDG-SW3 cell model. Highly significant enrichment (NES -1.44) was found in the genes in both models that were negatively responsive to PTH (365, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125731#pone.0125731.s003" target="_blank">S1 Table</a>). <b>(B)</b> The 574 genes that responded positively to PTH in the IDG-SW3 cell model, was analyzed against the 33,277 gene expression values in the <i>ex vivo</i> bone cortical osteocyte model, and a highly significant enrichment (NES = +1.98) was found in a set of genes that positively responded to PTH (131 geneset, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125731#pone.0125731.s004" target="_blank">S2 Table</a>).</p

The effect of PTH on osteoblast and osteocyte marker gene and protein expression in IDG-SW3 cells and primary osteocytes.

<p><b>(A)</b> Real-time PCR analysis of IDG-SW3 cells cultured over a 29 day time course and treated with 50nM PTH, or PBS control, for 24 hours at days 0, 7, 14, 21 and 28. Expression was normalized to <i>Actb</i> and is relative to day 1 control samples. <i>Kera</i> and <i>E11</i> were upregulated by PTH treatment, whereas <i>Dmp1</i>, <i>Phex</i>, <i>Mepe</i> and <i>Sost</i> expression was decreased, particularly at the later time points (n = 3±SD, *p<0.05). <b>(B)</b> Real-time PCR analysis of osteocyte-enriched bone fragments cultured in the presence of 50nM PTH, or PBS, for 24 hours. Expression was normalized to <i>Actb</i> and is relative to control samples. <i>Kera</i> and <i>E11</i> were upregulated by PTH treatment. <i>Dmp1</i>, <i>Phex</i>, <i>Mepe</i> and <i>Sost</i> expression was decreased in the primary osteocytes (n = 4±SD, *p<0.05). <b>(C)</b> IDG-SW3 cells were cultured over a 30 day time course and treated with 50nM PTH, or PBS, for 48 hours at days 0, 7, 14, 21 and 28. E11 and sclerostin expression was assessed by western blotting. <b>(D)</b> Quantitation of E11 protein expression with and without PTH treatment, relative to day 2 control samples. <b>(E)</b> GFP expression in mature (day 28) IDG-SW3 cells treated with 50nM PTH for 48 hours. Scale bar = 20μm. <b>(F)</b> Quantification of GFP in IDG-SW3 cells cultured over a 30 day time course and treated with 50nM PTH for 48 hours at days 0, 7, 14, 21 and 28 (n = 4±SD, *p<0.05).</p

Fold change values of motility-associated genes upregulated by PTH treatment in IDG-SW3 cells and corresponding values in <i>ex vivo</i> cortical bone osteocytes as determined by David analysis.

<p>The full geneset can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125731#pone.0125731.s004" target="_blank">S2 Table</a>.</p><p>Fold change values of motility-associated genes upregulated by PTH treatment in IDG-SW3 cells and corresponding values in <i>ex vivo</i> cortical bone osteocytes as determined by David analysis.</p

The calcium channel blockers Diltiazem and Nifedipine have no effect on PTH changes in gene expression except for Keratocan.

<p>(A) Real-time PCR analysis showing effects of Diltiazem on gene expression in mature IDG-SW3 cells. No effects on PTH changes in gene expression were observed (n = 3±SD, p<0.05 relative to control (a), PTH (b), DH (c) and PTH & DH (d)). (B) Effects of Nifedipine on gene expression. No effects of Nifedipine were observed on PTH induced gene expression except that Nifedipine inhibited PTH induction of <i>Kera</i>. (n = 3±SD, p<0.05 relative to control (a), PTH (b), NI (c) and PTH & NI (d)) Expression was normalized to <i>Actb</i>.</p

Lack of effect of inhibitors on PTH induced cell morphology.

<p>(A) The PKA inhibitor PKI 14–22 (50μM) does not block the effects of 50nM PTH on IDG-SW3 cell morphology. (B) Activation of the PKA pathway using 6-Bnz-cAMP does not reproduce the effect of PTH. (C) The Epac specific activator 8-CPT-2Me-cAMP (100μM) does not reproduce the effect of PTH on IDG-SW3 cell morphology. (D) The PKC activator PMA (100μM) does not affect IDG-SW3 cell morphology. (E) The broad spectrum PKC inhibitor Go6983 (10μM) blocked the effects of 50nM PTH on IDG-SW3 cell morphology. (F) The atypical PKC inhibitor PKC-zeta pseudosubstrate (PS) (100μM) failed to block the effect of 50nM PTH on IDG-SW3 cell morphology. (G) The Pi3K inhibitor LY294002 (25μM) blocked the effect of 50nM PTH on IDG-SW3 cell morphology, however the more potent and specific inhibitor Wortmannin (1μM) failed to do so (H). Scale bar = 20μm.</p

E11/gp38 is not involved in increased motility in response to PTH.

<p>(A). Primary osteocytes from E11-hypomorphic (fl/fl) mice do not express detectable E11 protein in the absence or presence of PTH, whereas E11 protein expression is induced in by PTH in E11-expressing cells. (B) PTH induces changes in cell morphology of both E11-expressing and E11-deficient primary osteocytes. Scale bar = 20μm.</p