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Loss of menin in osteoblast lineage affects osteocyte – osteoclast crosstalk causing osteoporosis

View Article: PubMed Central - PubMed

ABSTRACT

During osteoporosis bone formation by osteoblasts is reduced and/or bone resorption by osteoclasts is enhanced. Currently, only a few factors have been identified in the regulation of bone integrity by osteoblast-derived osteocytes. In this study, we show that specific disruption of menin, encoded by multiple endocrine neoplasia type 1 (Men1), in osteoblasts and osteocytes caused osteoporosis despite the preservation of osteoblast differentiation and the bone formation rate. Instead, an increase in osteoclast numbers and bone resorption was detected that persisted even when the deletion of Men1 was restricted to osteocytes. We demonstrate that isolated Men1-deficient osteocytes expressed numerous soluble mediators, such as C-X-C motif chemokine 10 (CXCL10), and that CXCL10-mediated osteoclastogenesis was reduced by CXCL10-neutralizing antibodies. Collectively, our data reveal a novel role for Men1 in osteocyte–osteoclast crosstalk by controlling osteoclastogenesis through the action of soluble factors. A role for Men1 in maintaining bone integrity and thereby preventing osteoporosis is proposed.

No MeSH data available.


Related in: MedlinePlus

Men1 deficiency in osteocytes leads to an enhanced osteoclastogenesis via increased Cxcl10 expression. (a–c) Osteoclastogenesis was visualized by TRAP staining of co-cultures of primary wild-type osteocytes and BMCs supplemented with conditioned medium from 4-OHT-pretreated Men1flox or Men1gtRosaCreERT2 osteocyte cultures (a). Number of multinucleated TRAP-positive cells (b) and their area (c) were determined (n=3). (d–f) Microarray analysis of Men1-deficient enriched osteocyte fractions. (d) Scatter plot of normalized expression levels of control (Men1flox, x axis) and Men1-deficient (Men1gtRosaCreERT2, y axis) enriched osteocyte fractions. Probe sets outside of the two diagonal lines represent 1.4-fold upregulated or 0.7-fold downregulated genes in Men1-deficient cells compared with Men1flox control cells. (e) Venn diagram of Men1-regulated genes annotated as ‘secreted' by DAVID. Numbers indicate genes highly expressed in Men1flox cells (left), no change (middle), or highly expressed in Men1gtRosaCreERT2 cells (right), respectively. (f) Heat map of normalized expression levels of control (WT, Men1flox) and Men1-deficient (KO, Men1gtRosaCreERT2) osteocytes. Secreted factors with high expression levels (>50% of the total probe signals) with fold change values (2expression levels (KO-WT)) of >1.4 (30 gene data sets) or <0.7 (81 gene data sets) were visualized in the heat map. (g) mRNA levels of Cxcl10 were determined from calvarial bone of 3-day-old Men1flox and Men1Dmp1Cre mice by QRT-PCR (n=4 or 5). (h) CXCL10 levels in the supernatant after 5 days of organ culturing of calvaria from neonatal Men1Dmp1Cre mice were determined by ELISA (n=3 or 4). (i) mRNA levels of Cxcl10 were determined by QRT-PCR in 4-OHT-pretreated enriched osteocyte fractions isolated from Men1flox and Men1gtRosaCreERT2 mice (n=3). (j and k) mRNA levels of Men1 (j) and Cxcl10 (k) were determined by QRT-PCR in Men1-overexpressing MLO-Y4 cells after treatment with control media or vitamin D3 (n=3). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean±S.E.M. Scale bar: 25 μm
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fig4: Men1 deficiency in osteocytes leads to an enhanced osteoclastogenesis via increased Cxcl10 expression. (a–c) Osteoclastogenesis was visualized by TRAP staining of co-cultures of primary wild-type osteocytes and BMCs supplemented with conditioned medium from 4-OHT-pretreated Men1flox or Men1gtRosaCreERT2 osteocyte cultures (a). Number of multinucleated TRAP-positive cells (b) and their area (c) were determined (n=3). (d–f) Microarray analysis of Men1-deficient enriched osteocyte fractions. (d) Scatter plot of normalized expression levels of control (Men1flox, x axis) and Men1-deficient (Men1gtRosaCreERT2, y axis) enriched osteocyte fractions. Probe sets outside of the two diagonal lines represent 1.4-fold upregulated or 0.7-fold downregulated genes in Men1-deficient cells compared with Men1flox control cells. (e) Venn diagram of Men1-regulated genes annotated as ‘secreted' by DAVID. Numbers indicate genes highly expressed in Men1flox cells (left), no change (middle), or highly expressed in Men1gtRosaCreERT2 cells (right), respectively. (f) Heat map of normalized expression levels of control (WT, Men1flox) and Men1-deficient (KO, Men1gtRosaCreERT2) osteocytes. Secreted factors with high expression levels (>50% of the total probe signals) with fold change values (2expression levels (KO-WT)) of >1.4 (30 gene data sets) or <0.7 (81 gene data sets) were visualized in the heat map. (g) mRNA levels of Cxcl10 were determined from calvarial bone of 3-day-old Men1flox and Men1Dmp1Cre mice by QRT-PCR (n=4 or 5). (h) CXCL10 levels in the supernatant after 5 days of organ culturing of calvaria from neonatal Men1Dmp1Cre mice were determined by ELISA (n=3 or 4). (i) mRNA levels of Cxcl10 were determined by QRT-PCR in 4-OHT-pretreated enriched osteocyte fractions isolated from Men1flox and Men1gtRosaCreERT2 mice (n=3). (j and k) mRNA levels of Men1 (j) and Cxcl10 (k) were determined by QRT-PCR in Men1-overexpressing MLO-Y4 cells after treatment with control media or vitamin D3 (n=3). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean±S.E.M. Scale bar: 25 μm

Mentions: We could not detect differences in Tnfsf11 (RANKL) and Tnfrsf11b (OPG) expression in the bone of Men1Dmp1Cre mice (Supplementary Figures S6a and b). We therefore tested whether other soluble factors derived from Men1-deficient osteocytes affected osteoclastogenesis. Transfer of conditioned medium from cultured Men1-deficient osteocyte-enriched fractions to wild-type osteocyte-enriched fractions/osteoclast co-cultures revealed a higher osteoclast formation than the transfer of medium from wild-type osteocyte-enriched fractions (Figures 4a–c). Furthermore, the conditioned medium from osteocyte-enriched fractions was added to wild-type osteoclast cultures. Again, the conditioned medium from Men1-deficient osteocyte-enriched fractions triggered higher osteoclast formation than the medium from control osteocyte-enriched fractions (Supplementary Figures S6c–e).


Loss of menin in osteoblast lineage affects osteocyte – osteoclast crosstalk causing osteoporosis
Men1 deficiency in osteocytes leads to an enhanced osteoclastogenesis via increased Cxcl10 expression. (a–c) Osteoclastogenesis was visualized by TRAP staining of co-cultures of primary wild-type osteocytes and BMCs supplemented with conditioned medium from 4-OHT-pretreated Men1flox or Men1gtRosaCreERT2 osteocyte cultures (a). Number of multinucleated TRAP-positive cells (b) and their area (c) were determined (n=3). (d–f) Microarray analysis of Men1-deficient enriched osteocyte fractions. (d) Scatter plot of normalized expression levels of control (Men1flox, x axis) and Men1-deficient (Men1gtRosaCreERT2, y axis) enriched osteocyte fractions. Probe sets outside of the two diagonal lines represent 1.4-fold upregulated or 0.7-fold downregulated genes in Men1-deficient cells compared with Men1flox control cells. (e) Venn diagram of Men1-regulated genes annotated as ‘secreted' by DAVID. Numbers indicate genes highly expressed in Men1flox cells (left), no change (middle), or highly expressed in Men1gtRosaCreERT2 cells (right), respectively. (f) Heat map of normalized expression levels of control (WT, Men1flox) and Men1-deficient (KO, Men1gtRosaCreERT2) osteocytes. Secreted factors with high expression levels (>50% of the total probe signals) with fold change values (2expression levels (KO-WT)) of >1.4 (30 gene data sets) or <0.7 (81 gene data sets) were visualized in the heat map. (g) mRNA levels of Cxcl10 were determined from calvarial bone of 3-day-old Men1flox and Men1Dmp1Cre mice by QRT-PCR (n=4 or 5). (h) CXCL10 levels in the supernatant after 5 days of organ culturing of calvaria from neonatal Men1Dmp1Cre mice were determined by ELISA (n=3 or 4). (i) mRNA levels of Cxcl10 were determined by QRT-PCR in 4-OHT-pretreated enriched osteocyte fractions isolated from Men1flox and Men1gtRosaCreERT2 mice (n=3). (j and k) mRNA levels of Men1 (j) and Cxcl10 (k) were determined by QRT-PCR in Men1-overexpressing MLO-Y4 cells after treatment with control media or vitamin D3 (n=3). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean±S.E.M. Scale bar: 25 μm
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fig4: Men1 deficiency in osteocytes leads to an enhanced osteoclastogenesis via increased Cxcl10 expression. (a–c) Osteoclastogenesis was visualized by TRAP staining of co-cultures of primary wild-type osteocytes and BMCs supplemented with conditioned medium from 4-OHT-pretreated Men1flox or Men1gtRosaCreERT2 osteocyte cultures (a). Number of multinucleated TRAP-positive cells (b) and their area (c) were determined (n=3). (d–f) Microarray analysis of Men1-deficient enriched osteocyte fractions. (d) Scatter plot of normalized expression levels of control (Men1flox, x axis) and Men1-deficient (Men1gtRosaCreERT2, y axis) enriched osteocyte fractions. Probe sets outside of the two diagonal lines represent 1.4-fold upregulated or 0.7-fold downregulated genes in Men1-deficient cells compared with Men1flox control cells. (e) Venn diagram of Men1-regulated genes annotated as ‘secreted' by DAVID. Numbers indicate genes highly expressed in Men1flox cells (left), no change (middle), or highly expressed in Men1gtRosaCreERT2 cells (right), respectively. (f) Heat map of normalized expression levels of control (WT, Men1flox) and Men1-deficient (KO, Men1gtRosaCreERT2) osteocytes. Secreted factors with high expression levels (>50% of the total probe signals) with fold change values (2expression levels (KO-WT)) of >1.4 (30 gene data sets) or <0.7 (81 gene data sets) were visualized in the heat map. (g) mRNA levels of Cxcl10 were determined from calvarial bone of 3-day-old Men1flox and Men1Dmp1Cre mice by QRT-PCR (n=4 or 5). (h) CXCL10 levels in the supernatant after 5 days of organ culturing of calvaria from neonatal Men1Dmp1Cre mice were determined by ELISA (n=3 or 4). (i) mRNA levels of Cxcl10 were determined by QRT-PCR in 4-OHT-pretreated enriched osteocyte fractions isolated from Men1flox and Men1gtRosaCreERT2 mice (n=3). (j and k) mRNA levels of Men1 (j) and Cxcl10 (k) were determined by QRT-PCR in Men1-overexpressing MLO-Y4 cells after treatment with control media or vitamin D3 (n=3). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean±S.E.M. Scale bar: 25 μm
Mentions: We could not detect differences in Tnfsf11 (RANKL) and Tnfrsf11b (OPG) expression in the bone of Men1Dmp1Cre mice (Supplementary Figures S6a and b). We therefore tested whether other soluble factors derived from Men1-deficient osteocytes affected osteoclastogenesis. Transfer of conditioned medium from cultured Men1-deficient osteocyte-enriched fractions to wild-type osteocyte-enriched fractions/osteoclast co-cultures revealed a higher osteoclast formation than the transfer of medium from wild-type osteocyte-enriched fractions (Figures 4a–c). Furthermore, the conditioned medium from osteocyte-enriched fractions was added to wild-type osteoclast cultures. Again, the conditioned medium from Men1-deficient osteocyte-enriched fractions triggered higher osteoclast formation than the medium from control osteocyte-enriched fractions (Supplementary Figures S6c–e).

View Article: PubMed Central - PubMed

ABSTRACT

During osteoporosis bone formation by osteoblasts is reduced and/or bone resorption by osteoclasts is enhanced. Currently, only a few factors have been identified in the regulation of bone integrity by osteoblast-derived osteocytes. In this study, we show that specific disruption of menin, encoded by multiple endocrine neoplasia type 1 (Men1), in osteoblasts and osteocytes caused osteoporosis despite the preservation of osteoblast differentiation and the bone formation rate. Instead, an increase in osteoclast numbers and bone resorption was detected that persisted even when the deletion of Men1 was restricted to osteocytes. We demonstrate that isolated Men1-deficient osteocytes expressed numerous soluble mediators, such as C-X-C motif chemokine 10 (CXCL10), and that CXCL10-mediated osteoclastogenesis was reduced by CXCL10-neutralizing antibodies. Collectively, our data reveal a novel role for Men1 in osteocyte&ndash;osteoclast crosstalk by controlling osteoclastogenesis through the action of soluble factors. A role for Men1 in maintaining bone integrity and thereby preventing osteoporosis is proposed.

No MeSH data available.


Related in: MedlinePlus