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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 in vivo. (a) Real-time PCR analysis of Men1 mRNA expression in FACS-sorted GFP-positive cells from femoral bone of Men1Dmp1Cre mice crossed to RosamT/mG mice in animals heterozygous and homozygous for Men1flox. (b–f) Micro CT reconstruction (c) of femurs from 12-week-old female Men1flox and Men1Dmp1Cre mice. Cancellous parameters such as BV/TV (b), Tb.Th. (d), Tb.N. (e), and Tb.Sp. (f) were measured from distal femurs of 12-week-old female Men1flox and Men1Dmp1Cre mice by micro CT (n=4 or 5). (g–i) Osteoblast number per bone perimeter (N.Ob/B.Pm) (g), osteoblast surface per bone surface (Ob.S/BS) (h), and osteocyte number per bone area (N.Ot/B.Ar) (i) in trabecular bone of distal femoral sections from 12-week-old female Men1flox and Men1Dmp1Cre mice were measured by histomorphometry (n=4 or 5). (j and k) Fluorescent micrographs of dual calcein labeling (j) and its quantitative analysis of BFR/BS (k) in femoral sections of 12-week-old female Men1flox and Men1Dmp1Cre mice (n=4 or 5). (l) Determination of the serum PINP level in 12-week-old female Men1flox and Men1Dmp1Cre mice (n=5). (m and n) N.Oc/B.Pm (m) and Oc.S/BS (n) in trabecular bone of distal femoral sections from 12-week-old female Men1flox and Men1Dmp1Cre mice were measured by histomorphometry (n=4 or 5). (o) Determination of resorption from the serum CTX level in 12-week-old female Men1flox and Men1Dmp1Cre mice (n=5). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean±S.E.M.
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fig3: Men1 deficiency in osteocytes leads to an enhanced osteoclastogenesis in vivo. (a) Real-time PCR analysis of Men1 mRNA expression in FACS-sorted GFP-positive cells from femoral bone of Men1Dmp1Cre mice crossed to RosamT/mG mice in animals heterozygous and homozygous for Men1flox. (b–f) Micro CT reconstruction (c) of femurs from 12-week-old female Men1flox and Men1Dmp1Cre mice. Cancellous parameters such as BV/TV (b), Tb.Th. (d), Tb.N. (e), and Tb.Sp. (f) were measured from distal femurs of 12-week-old female Men1flox and Men1Dmp1Cre mice by micro CT (n=4 or 5). (g–i) Osteoblast number per bone perimeter (N.Ob/B.Pm) (g), osteoblast surface per bone surface (Ob.S/BS) (h), and osteocyte number per bone area (N.Ot/B.Ar) (i) in trabecular bone of distal femoral sections from 12-week-old female Men1flox and Men1Dmp1Cre mice were measured by histomorphometry (n=4 or 5). (j and k) Fluorescent micrographs of dual calcein labeling (j) and its quantitative analysis of BFR/BS (k) in femoral sections of 12-week-old female Men1flox and Men1Dmp1Cre mice (n=4 or 5). (l) Determination of the serum PINP level in 12-week-old female Men1flox and Men1Dmp1Cre mice (n=5). (m and n) N.Oc/B.Pm (m) and Oc.S/BS (n) in trabecular bone of distal femoral sections from 12-week-old female Men1flox and Men1Dmp1Cre mice were measured by histomorphometry (n=4 or 5). (o) Determination of resorption from the serum CTX level in 12-week-old female Men1flox and Men1Dmp1Cre mice (n=5). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean±S.E.M.

Mentions: The in vitro co-culture experiments suggested that the increased osteoclast number in the mice lacking Men1 in the osteoblastic lineage was conferred by Men1-deficient osteocytes rather than osteoblasts. To confirm this hypothesis in vivo, we generated mice lacking Men1 in osteocytes, but not in early differentiated osteoblasts, by crossing Men1flox mice to Tg(Dmp1-cre)1Jqfe (hereafter designated Dmp1Cre) mice18 to obtain Men1Dmp1Cre mice. Loss of menin in osteocytes in Men1Dmp1Cre mice was confirmed by immunohistochemistry (Supplementary Figure S5d) and by crossing Men1Dmp1Cre mice to RosamT/mG reporter mice that displayed a strongly diminished Men1 expression in GFP-positive cells (Figure 3a and Supplementary Figure S5e). As Dmp1 is also expressed in mature osteoblasts, we stained osteocalcin as a mature osteoblast marker in Men1Dmp1Cre; RosamT/mG reporter mice (Supplementary Figure S5f) to determine the degree of recombination in these cells. We found that 29±9% of osteocalcin-stained mature osteoblasts were EGFP positive, indicating that a minor fraction of the late-stage differentiated osteoblasts is also mutant in addition to osteocytes in Men1Dmp1Cre; RosamT/mG mice. Micro CT analysis revealed a severe osteoporosis in the distal femur of adult Men1Dmp1Cre mice (Figures 3b–f). The decrease of trabecular bone mass in Men1Dmp1Cre mice was because of a low number of trabeculae and an increased trabecular separation. Trabecular thickness was again unchanged. Osteoblast number, osteoblast surface, and osteocyte number were not altered in Men1Dmp1Cre mice (Figures 3g–i). Accordingly, the bone formation rate and serum PINP level were not different between Men1flox mice and Men1Dmp1Cre mice (Figures 3j–l). Osteoclast number, osteoclast surface, and CTX level were significantly elevated in Men1Dmp1Cre mice (Figures 3m–o), whereas RANKL and OPG levels were not changed (Supplementary Figures S5g and h). Thus, a similar bone phenotype was observed in Men1Dmp1Cre mice as shown in Men1Runx2Cre and Men1OsxCre mice. Together with the observed increase of osteoclastogenesis by osteocytes, these observations strongly suggest that osteoporosis by loss of Men1 is executed by osteocytes in these three different conditional knockout mouse strains.


Loss of menin in osteoblast lineage affects osteocyte – osteoclast crosstalk causing osteoporosis
Men1 deficiency in osteocytes leads to an enhanced osteoclastogenesis in vivo. (a) Real-time PCR analysis of Men1 mRNA expression in FACS-sorted GFP-positive cells from femoral bone of Men1Dmp1Cre mice crossed to RosamT/mG mice in animals heterozygous and homozygous for Men1flox. (b–f) Micro CT reconstruction (c) of femurs from 12-week-old female Men1flox and Men1Dmp1Cre mice. Cancellous parameters such as BV/TV (b), Tb.Th. (d), Tb.N. (e), and Tb.Sp. (f) were measured from distal femurs of 12-week-old female Men1flox and Men1Dmp1Cre mice by micro CT (n=4 or 5). (g–i) Osteoblast number per bone perimeter (N.Ob/B.Pm) (g), osteoblast surface per bone surface (Ob.S/BS) (h), and osteocyte number per bone area (N.Ot/B.Ar) (i) in trabecular bone of distal femoral sections from 12-week-old female Men1flox and Men1Dmp1Cre mice were measured by histomorphometry (n=4 or 5). (j and k) Fluorescent micrographs of dual calcein labeling (j) and its quantitative analysis of BFR/BS (k) in femoral sections of 12-week-old female Men1flox and Men1Dmp1Cre mice (n=4 or 5). (l) Determination of the serum PINP level in 12-week-old female Men1flox and Men1Dmp1Cre mice (n=5). (m and n) N.Oc/B.Pm (m) and Oc.S/BS (n) in trabecular bone of distal femoral sections from 12-week-old female Men1flox and Men1Dmp1Cre mice were measured by histomorphometry (n=4 or 5). (o) Determination of resorption from the serum CTX level in 12-week-old female Men1flox and Men1Dmp1Cre mice (n=5). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean±S.E.M.
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fig3: Men1 deficiency in osteocytes leads to an enhanced osteoclastogenesis in vivo. (a) Real-time PCR analysis of Men1 mRNA expression in FACS-sorted GFP-positive cells from femoral bone of Men1Dmp1Cre mice crossed to RosamT/mG mice in animals heterozygous and homozygous for Men1flox. (b–f) Micro CT reconstruction (c) of femurs from 12-week-old female Men1flox and Men1Dmp1Cre mice. Cancellous parameters such as BV/TV (b), Tb.Th. (d), Tb.N. (e), and Tb.Sp. (f) were measured from distal femurs of 12-week-old female Men1flox and Men1Dmp1Cre mice by micro CT (n=4 or 5). (g–i) Osteoblast number per bone perimeter (N.Ob/B.Pm) (g), osteoblast surface per bone surface (Ob.S/BS) (h), and osteocyte number per bone area (N.Ot/B.Ar) (i) in trabecular bone of distal femoral sections from 12-week-old female Men1flox and Men1Dmp1Cre mice were measured by histomorphometry (n=4 or 5). (j and k) Fluorescent micrographs of dual calcein labeling (j) and its quantitative analysis of BFR/BS (k) in femoral sections of 12-week-old female Men1flox and Men1Dmp1Cre mice (n=4 or 5). (l) Determination of the serum PINP level in 12-week-old female Men1flox and Men1Dmp1Cre mice (n=5). (m and n) N.Oc/B.Pm (m) and Oc.S/BS (n) in trabecular bone of distal femoral sections from 12-week-old female Men1flox and Men1Dmp1Cre mice were measured by histomorphometry (n=4 or 5). (o) Determination of resorption from the serum CTX level in 12-week-old female Men1flox and Men1Dmp1Cre mice (n=5). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean±S.E.M.
Mentions: The in vitro co-culture experiments suggested that the increased osteoclast number in the mice lacking Men1 in the osteoblastic lineage was conferred by Men1-deficient osteocytes rather than osteoblasts. To confirm this hypothesis in vivo, we generated mice lacking Men1 in osteocytes, but not in early differentiated osteoblasts, by crossing Men1flox mice to Tg(Dmp1-cre)1Jqfe (hereafter designated Dmp1Cre) mice18 to obtain Men1Dmp1Cre mice. Loss of menin in osteocytes in Men1Dmp1Cre mice was confirmed by immunohistochemistry (Supplementary Figure S5d) and by crossing Men1Dmp1Cre mice to RosamT/mG reporter mice that displayed a strongly diminished Men1 expression in GFP-positive cells (Figure 3a and Supplementary Figure S5e). As Dmp1 is also expressed in mature osteoblasts, we stained osteocalcin as a mature osteoblast marker in Men1Dmp1Cre; RosamT/mG reporter mice (Supplementary Figure S5f) to determine the degree of recombination in these cells. We found that 29±9% of osteocalcin-stained mature osteoblasts were EGFP positive, indicating that a minor fraction of the late-stage differentiated osteoblasts is also mutant in addition to osteocytes in Men1Dmp1Cre; RosamT/mG mice. Micro CT analysis revealed a severe osteoporosis in the distal femur of adult Men1Dmp1Cre mice (Figures 3b–f). The decrease of trabecular bone mass in Men1Dmp1Cre mice was because of a low number of trabeculae and an increased trabecular separation. Trabecular thickness was again unchanged. Osteoblast number, osteoblast surface, and osteocyte number were not altered in Men1Dmp1Cre mice (Figures 3g–i). Accordingly, the bone formation rate and serum PINP level were not different between Men1flox mice and Men1Dmp1Cre mice (Figures 3j–l). Osteoclast number, osteoclast surface, and CTX level were significantly elevated in Men1Dmp1Cre mice (Figures 3m–o), whereas RANKL and OPG levels were not changed (Supplementary Figures S5g and h). Thus, a similar bone phenotype was observed in Men1Dmp1Cre mice as shown in Men1Runx2Cre and Men1OsxCre mice. Together with the observed increase of osteoclastogenesis by osteocytes, these observations strongly suggest that osteoporosis by loss of Men1 is executed by osteocytes in these three different conditional knockout mouse strains.

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