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. 2016 Aug 3;6:30977.
doi: 10.1038/srep30977.

STAT5 Is a Key Transcription Factor for IL-3-mediated Inhibition of RANKL-induced Osteoclastogenesis

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Free PMC article

STAT5 Is a Key Transcription Factor for IL-3-mediated Inhibition of RANKL-induced Osteoclastogenesis

Jongwon Lee et al. Sci Rep. .
Free PMC article

Abstract

Among the diverse cytokines involved in osteoclast differentiation, interleukin (IL)-3 inhibits RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. Here we demonstrate that the activation of signal transducers and activators of transcription 5 (STAT5) by IL-3 inhibits RANKL-induced osteoclastogenesis through the induction of the expression of Id genes. We found that STAT5 overexpression inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate the expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting a key role of STAT5 in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated the expression of Id1 and Id2, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Importantly, microcomputed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in the STAT5 conditional knockout mice than in the wild-type mice after RANKL injection. Taken together, our findings indicate that STAT5 contributes to the remarkable IL-3-mediated inhibition of RANKL-induced osteoclastogenesis by activating Id genes and their associated pathways.

Figures

Figure 1
Figure 1. STAT5A activation reduces RANKL-mediated osteoclastogenesis.
(A) BMMs were cultured with M-CSF and RANKL for the indicated times. Total RNA was collected at each time point. RT-PCR was performed to detect expression of the indicated genes. All gels run under the same experimental conditions and the representative images were cropped and shown. (B,C) BMMs were transduced with pMX-IRES-EGFP (control) or constitutively active STAT5A (STAT5A1*6) retrovirus and cultured with M-CSF in the presence or absence of RANKL for three days. (B) Cultured cells were stained for TRAP. (C) The number of TRAP-positive MNCs per well was counted. Data are represented as the mean ± SD. ***P < 0.001 vs. control; n = 4. (D–F) BMMs were transduced with pMX-IRES-EGFP (control) or STAT5A1*6 retrovirus and cultured with M-CSF and RANKL for the indicated times. (D) mRNA levels of c-fos, Nfatc1, Acp5 and Oscar were assessed by quantitative real-time PCR. Data represent mean ± SD of triplicate samples. **P < 0.01; ***P < 0.001 vs. control. (E,F) Whole cell lysates were harvested from cultured cells and were immunoblotted with the indicated antibodies. All gels run under the same experimental conditions and the representative images were cropped and shown. Bar: 100 μm. All results are representative of at least three independent experiments. Statistical analyses were implemented in TTEST.
Figure 2
Figure 2. STAT5 deficiency does not regulate osteoclastogenesis.
(AE) Bone marrow cells were harvested from long bones of STAT5 conditional knockout (Stat5 cKO) mice or Stat5fl/fl littermates. (A) Total RNA was isolated from BMMs and RT-PCR was performed to detect expression of the indicated genes (upper panel). Whole cell lysates were harvested from BMMs and were immunoblotted with antibodies against STAT5A or actin (lower panel). All gels run under the same experimental conditions and the representative images were cropped and shown. (B,C) BMMs were cultured with M-CSF in the presence or absence of RANKL for three days. (B) Cultured cells were stained for TRAP. (C) The number of TRAP-positive MNCs per well was counted. Data are represented as the mean ± SD. n.s., not significant; n = 4. (D,E) BMMs were cultured with M-CSF and RANKL for the indicated times. (D) mRNA levels of c-fos, Nfatc1, Acp5 and Oscar were assessed by quantitative real-time PCR. Data represent mean ± SD of triplicate samples. n.s., not significant. (F) BMMs were stimulated with RANKL for the indicated times. (G) BMMs were cultured with M-CSF and RANKL for the indicated times. (EG) Whole cell lysates were harvested from cultured cells and were immunoblotted with the indicated antibodies. All gels run under the same experimental conditions and the representative images were cropped and shown. Bar: 100 μm. All results are representative of at least three independent experiments. Statistical analyses were implemented in TTEST.
Figure 3
Figure 3. IL-3 inhibits RANKL-mediated osteoclastogenesis in a STAT5-dependent manner.
Bone marrow cells were harvested from long bones of Stat5 cKO mice or Stat5fl/fl littermates. (A,B) BMMs were cultured with M-CSF and RANKL for three days in various concentrations of IL-3. (A) Cultured cells were stained for TRAP. (B) The number of TRAP-positive MNCs per well was counted. Data are represented as the mean ± SD. ***P < 0.001 vs. Stat5fl/fl control; #P < 0.05 vs. Stat5 cKO control; n.s., not significant; n = 4. (C) BMMs were treated with IL-3 (1 ng/mL). Cells were further cultured in the presence (pOC) or absence (BMM) of RANKL for two days, and subjected to semi-quantitative real-time PCR for the indicated genes. Data represent mean ± SD of triplicate samples. ***P < 0.001; n.s., not significant. Bar: 100 μm. All results are representative of at least three independent experiments. All data were analyzed using ANOVA.
Figure 4
Figure 4. Id1 and Id2 are responsible for STAT5-mediated inhibition f osteoclastogenesis.
(A) BMMs were transduced with pMX-IRES-EGFP (control) or STAT5A1*6 retrovirus and cultured with M-CSF and RANKL for the indicated times. Levels of Id1 and Id2 mRNA were assessed by quantitative real-time PCR. Data represent mean ± SD of triplicate samples. **P < 0.01; ***P < 0.001 vs. control. (BD) Bone marrow cells were harvested from long bones of Stat5 cKO mice or Stat5fl/fl littermates. (B) BMMs were treated with IL-3 (1 ng/mL) and cells were further cultured in the presence (pOC) or absence (BMM) of RANKL for two days. Levels of Id1 and Id2 mRNA were assessed by quantitative real-time PCR. Data represent mean ± SD of triplicate samples. **P < 0.01; ***P < 0.001 vs. control; n.s., not significant. (C,D) Bone marrow cells were harvested from long bones of Stat5 cKO mice or Stat5fl/fl littermates. BMMs were transduced with pMX-IRES-EGFP (control), Id1 or Id2 retrovirus and cultured with M-CSF and RANKL for three days in the presence or absence of IL-3, as indicated. (C) Cultured cells were stained for TRAP. (D) The number of TRAP-positive MNCs per well was counted. Data are represented as the mean ± SD. ***P < 0.001 vs. control; n = 4. Bar: 100 μm. All results are representative of at least three independent experiments. Data were analyzed using TTEST (A) or ANOVA (B,C).
Figure 5
Figure 5. Bone phenotype of STAT5 conditional knockout mice.
(A,B) Long bones obtained from 8-week-old and 16-week-old male Stat5 cKO mice or Stat5fl/fl littermates were subjected to μCT analysis. (A) Representative three-dimensional images of femurs in Stat5 cKO mice or Stat5fl/fl littermates. (B) Bone volume per tissue volume, trabecular bone thickness, trabecular separation and trabecular number were assessed from the μCT measurements. Data are represented as the mean ± SD. *P < 0.05; ***P < 0.001 vs. control; n.s., not significant; 8-week-old Stat5fl/fl, n = 5; 8-week-old Stat5 cKO, n = 5; 16-week-old Stat5fl/fl, n = 9; 16-week-old Stat5 cKO, n = 7. (C,D) Long bones obtained from 16-week-old male Stat5 cKO mice or Stat5fl/fl littermates were subjected to histomorphometric analyses. (C) Hematoxylin and eosin (H&E) and TRAP staining of histological sections of proximal tibiae. (D) Osteoclast surface per bone surface, osteoclast number per bone surface, osteoblast surface per bone surface, and osteoblast number per bone surface were assessed. Data are represented as the mean ± SD. *P < 0.05 vs. control; n.s., not significant; Stat5fl/fl, n = 7; Stat5 cKO, n = 5. Bars: (A) 500 μm; (C) 100 μm. Statistical analyses were implemented in TTEST.
Figure 6
Figure 6. Administration of IL-3 reduces bone loss through STAT5 activation.
(AG) 8-week-old Stat5 cKO mice or Stat5fl/fl littermates were intraperitoneally administrated PBS, RANKL, with or without IL-3. Long bones obtained were subjected to μCT and histomorphometric analysis (A) Illustration of IL-3 administration strategy. (B) Representative three-dimensional images of femurs in Stat5 cKO mice or Stat5fl/fl littermates. (C) Bone volume per tissue volume was assessed from the μCT measurements. Data are represented as the mean ± SD. **P < 0.01; ***P < 0.001 vs. control; n.s., not significant; (PBS Stat5fl/fl n = 8, Stat5 cKO n = 8, RANKL Stat5fl/fl n = 10, Stat5 cKO n = 10, RANKL and IL-3 Stat5fl/fl n = 14, Stat5 cKO n = 11). (D) TRAP staining of histological section of proximal tibiae. (E) Osteoclast number per bone surface was assessed. Data are represented as the mean ± SD. ***P < 0.001 vs. control; **P < 0.01 vs. control; *P < 0.05 vs. control. n.s. not significant; n = 8. (F) Hematoxylin and eosin (H&E) stain of proximal tibiae. (G) Osteoblast number per bone surface was assessed. Data are represented as the mean ± SD. n.s., not significant; n = 8. Bars: (B) 500 μm; (C,D) 100 μm. All data were analyzed using ANOVA.

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