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. 2016 Dec 5:6:38526.
doi: 10.1038/srep38526.

RCANs regulate the convergent roles of NFATc1 in bone homeostasis

Jung Ha Kim  1   2 , Kabsun Kim  1 , Inyoung Kim  1 , Semun Seong  1   2 , Byung-Chul Jeong  1   2 , Kwang-Il Nam  3 , Kyung Keun Kim  1 , Jeffery D Molkentin  4 , Nacksung Kim  1   2
Affiliations

RCANs regulate the convergent roles of NFATc1 in bone homeostasis

Jung Ha Kim et al. Sci Rep. .

Abstract

Activation of calcineurin-dependent nuclear factor of activated T cells c1 (NFATc1) is convergent for normal bone homeostasis. NFATc1 regulates both osteoclastogenesis and osteoblastogenesis. Here we investigated the roles of regulator of calcineurin (RCAN) genes in bone homeostasis. RCANs function as potent physiological inhibitors of calcineurin. Overexpression of RCANs in osteoclast precursor cells attenuated osteoclast differentiation, while their overexpression in osteoblasts enhanced osteoblast differentiation and function. Intriguingly, opposing effects of RCANs in both cell types were shown by blocking activation of the calcineurin-NFATc1 pathway. Moreover, the disruption of RCAN1 or RCAN2 in mice resulted in reduced bone mass, which is associated with strongly increased osteoclast function and mildly reduced osteoblast function. Taken together, RCANs play critical roles in bone homeostasis by regulating both osteoclastogenesis and osteoblastogenesis, and they serve as inhibitors for calcineurin-NFATc1 signaling both in vivo and in vitro.

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Figures

Figure 1
Figure 1. All RCAN genes are expressed during osteoclastogenesis.
(a) BMMs were cultured in the presence of M-CSF and RANKL for the indicated times. (b) BMMs were transduced with pMX-IRES-EGFP (Control) or constitutively active NFATc1 (Ca-NFATc1) retrovirus and cultured in the presence of M-CSF for 4 days. (c) BMMs were cultured in the presence of M-CSF and RANKL with or without cyclosporine A (CsA, 5 μg/ml) for the indicated times. (a–c) Total RNA was harvested from cultured cells, and real-time PCR was performed to analyze expression of TRAP, RCAN1, RCAN2, and RCAN3. Data represent the mean ± SD of triplicate samples. #p < 0.05, *p < 0.01, **p < 0.001 vs. control, n = 3.
Figure 2
Figure 2. RCAN2 inhibits RANKL-induced osteoclast differentiation.
(a–c) BMMs were transduced with pMX-IRES-EGFP (Control) or RCAN2 retrovirus and cultured in the presence of M-CSF and RANKL for 3 days. (a) Cultured cells were fixed and stained for TRAP (left panel). Numbers of TRAP(+) MNCs were counted (right panel). **p < 0.001 vs. control. Bar: 200 μm, n = 3. (b) Total RNA was collected at the indicated time points. Real-time PCR was performed to analyze expression of NFATc1, TRAP, and OSCAR. Data represent the mean ± SD of triplicate samples. #p < 0.05, **p < 0.001 vs. control, n = 3. (c) Cells were harvested at the indicated time points. Cell lysates were analyzed by Western blot analysis using antibodies specific for NFATc1, OSCAR, Flag, and Actin. All gels were run under the same experimental conditions and the representative images were cropped and displayed. Full-length blots are presented in Supplementary Figure 8.
Figure 3
Figure 3. RCAN2 inhibits NFATc1 activation in osteoclasts.
(a) BMMs were transduced with pMX-IRES-EGFP (Control) or RCAN2 retrovirus and cultured in the presence of M-CSF and RANKL for the indicated times. Western blot analysis was performed to analyze NFATc1 expression in nuclear, cytoplasmic, and whole cell fractions. All gels were run under the same experimental conditions and the representative images were cropped and displayed. Full-length blots are presented in Supplementary Figure 9. (b) An OSCAR luciferase reporter was co-transfected with NFATc1 and increasing amounts of RCAN2 into 293 T cells. Luciferase activity was measured a dual-luciferase reporter assay system. Data represent the mean ± SD of triplicate samples. #p < 0.05, *p < 0.01 vs. the control, n = 3.
Figure 4
Figure 4. All RCAN genes expressed during osteoblastogenesis.
Primary osteoblast precursor cells were cultured in osteogenic medium (OGM) containing BMP2, ascorbic acid, and β-glycerophosphate for the indicated times. Total RNA was collected at each time point, and real-time PCR was performed to assess the expression of Runx2, ALP, NFATc1, RCAN1, RCAN2, and RCAN3. Data represent the mean ± SD of triplicate samples, n = 3.
Figure 5
Figure 5. RCAN2 enhances osteoblast differentiation.
(a–c) Osteoblasts were transduced with pMX-IRES-EGFP (control) or RCAN2 retrovirus and cultured in osteogenic medium (OGM). (a) Cells cultured for 3 days were subjected to the alkaline phosphatase activity (ALP) assay. *p < 0.01 vs. the control, n = 3. (b) Cells cultured for 9 days were fixed and stained for Alizarin red (left panel). Alizarin red staining activities was quantified by densitometry at 562 nm (right panel). **p < 0.001 vs. control, n = 3. (c) Cells were cultured for the indicated times, and real-time PCR was performed to analyze expression of Runx2, ALP, and BSP. Data represent the mean ± SD of triplicate samples. #p < 0.05, *p < 0.01, **p < 0.001 vs. the control, n = 3.
Figure 6
Figure 6. RCAN2 inhibits NFATc1 activation in osteoblasts.
(a) Osteoblasts were transduced with pMX-IRES-EGFP (control) or RCAN2 retrovirus and cultured in osteogenic medium (OGM) for 4 days. Western blot analysis was performed to analyze NFATc1 expression in nuclear, cytoplasmic, and whole cell fractions. All gels were run under same experimental conditions and the representative images were cropped and displayed. Full-length blots are presented in Supplementary Figure 10. (b) An ALP luciferase reporter was co-transfected with the indicated plasmids expressing Runx2, NFATc1 and increasing amounts of RCAN2 into 293 T cells. Luciferase activity was measured using a dual-luciferase reporter assay system. Data represent the mean ± SD of triplicate samples. #p < 0.05, *p < 0.01 vs. control, n = 3.
Figure 7
Figure 7. RCAN2 knockout mice exhibit reduced bone mass.
(a) Representative three-dimensional images of femurs in control or RCAN2 knockout mice (left panel). Bone volume per tissue volume, trabecular bone thickness, trabecular separation, and trabecular number were assessed from the μCT measurements (right panel). #p < 0.05, *p < 0.01 vs. control, n = 3 (Male), n = 6 (Female). (b) Hematoxylin/eosin (H&E) and TRAP staining of histological sections of proximal tibiae (upper panel). Osteoclast numbers per bone surface and osteoblast numbers per bone surface were assessed (lower panel). #p < 0.05 vs. control, n = 3.
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