Full Length ArticleInhibition of osteoclasts differentiation by CDC2-induced NFATc1 phosphorylation
Introduction
Bone homeostasis is an important aspect of bone health and regulated by a balance between osteoblast-derived bone formation and osteoclast-derived bone resorption; an imbalance of bone regulation may lead to metabolic bone diseases such as osteoporosis, osteopetrosis, arthritis, and bone fracture [1,2]. In osteoclasts, two haematopoietic factors [i.e., macrophage colony-stimulation factor (M-CSF) and receptor activator of nuclear factor-κB (RANKL)] are necessary and sufficient for osteoclast differentiation and required for proliferation and differentiation of the monocyte/macrophage lineage cells. RANKL-induced osteoclast differentiation is activated via calcium signaling, a process that leads to the activation of calcineurin, a well-known phosphatase. Calcineurin dephosphorylates the transcription factor nuclear activated T cells cytosolic isoform 1 (NFATc1), a key regulator of RANKL-induced osteoclast differentiation belonging to the NFAT family of proteins. Therefore, NFATc1 is translocated to the nucleus and activates osteoclast target genes (e.g., NFATc1, TARP, Cathepsin K, and OSCAR) [3,4].
NFAT proteins were initially identified as inducible nuclear factor in activated T cells; five members have been identified to data [i.e., NFAT1 (NFATc2), NFAT2 (NFATc1), NFAT3 (NFATc4), NFAT4 (NFATc3) and NFAT5 (TonEBP)] [5,6]. These proteins evolved alongside the evolution of vertebrates and are involved in immune responses and other biological systems (e.g., cardiac, muscle, pancreas, skin, bone) [[7], [8], [9], [10]]. NFAT proteins contain three conserved domains: i) a regulatory domain, ii) a DNA binding domain, and iii) a c-terminal domain. The regulatory domain contains many serine-rich motifs that are phosphorylated by various kinases such as casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK), and IκB kinase (IKK)-related kinase (IKKε) [[11], [12], [13], [14]]. NFATc1 (NFAT2) is a key transcription factor regulating osteoclast differentiation and phosphorylated in the cytoplasm under resting conditions. NFATc1 is activated by calcium signaling; calcineurin phosphatase directly dephosphorylates NFATc1, leading to its translocation to the nucleus where it acts as a transcription factor.
Cell differentiation correlates with a lengthening of G1 phase, a process controlled by a family of cyclins and cyclin-dependent kinases (CDKs). During osteoclast differentiation, RANKL-induced cell cycle was arrested in G0-G1 phases, a process associated with the up-regulation of the cyclin-dependent kinase inhibitors p27kip1 and p21cip1 [15]. Additionally, CDK6 is known to be a critical regulator of RANKL-induced osteoclast differentiation [16]. However, the precise mechanism of cell cycle progression to differentiation remains unclear in osteoclast.
Cdc2 is a member of the Ser/Thr protein kinase family it regulated cell cycle progression [17]. Cdc2 is a major enzyme to promote G2/M phase transition at cell cycle [18]. Activation of a Cdc2 requires association with specific cyclin subunit, such as Cdc2-cyclin B [19]. Thereby activated, Cdc2 facilitates mitotic entry through phosphorylation of its substrates. In cancer cells, cdc2 activity related with cell survival so which has been studied as a cancer therapeutic target and thus some inhibitors have been applied to the clinic to verify its effectiveness [20,21].
In this study, we observed that the expression level and the kinase activity of cdc2 are dynamically regulated during RANKL-induced osteoclast differentiation. Of particular, cyclin-dependent kinase 1 (CDK1) or cell division cycle protein 2 (Cdc2) hyper-phosphorylates NFATc1 and regulates its subcellular localization. Our results suggest that regulation of Cdc2 mediated NFATc1 phosphorylation might be a potential target for the treatment of osteoporosis.
Section snippets
Reagents and antibodies
α-MEM, DMEM, fetal bovine serum, and penicillin were purchased from Invitrogen (Carlsbad, CA), and roscovitine and FK506 from Selleck Chemical (Houston, TX) (Darmstadt, GERMANY). TRAP staining solution, A23187 and nocodazole were obtained from Sigma Aldrich (St. Louis, MO). Recombinant human soluble M-CSF and mouse RANKL were purchased from PeproTech EC (London, United Kingdom). Specific antibodies against GST, cyclin B1, cyclin A2, HA, Histone H3, α-tubulin, NFATc1 and GAPDH were obtained from
Cdc2 activity is inversely correlated with NFATc1 activation during osteoclast differentiation
To examine the effect of cell cycle dependent kinases on osteoclast differentiation, we observed the expression of several cyclin dependent kinases (CDKs). Bone marrow macrophage (BMM) cells isolated from mice were stimulated with M-CSF and RANKL for 3 days, and the expression of several cyclin dependent kinases was assessed each time. During osteoclast differentiation, NFATc1 levels started to increase after 36 h of RANKL stimulation. Interestingly, the expression of cell cycle division cycle
Discussion
Cdc2 is the most prominent mitotic kinase regulated by association with both cyclin and multiple phosphorylations, which are essential for G1/S and G2/M phase transitions [34,35]. Although the regulation of cdc2-cyclin complexes in cell mitosis is relatively well understood, little is known about the functional regulation of cellular differentiation. The results presented here surprisingly revealed that the expression levels of cdc2, cyclin B1 and cyclin A2 as well as phosphorylation of cdc2
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by a National Research Council of Science & Technology (NST) grant (CAP-16-03-KRIBB to B.Y.K), the Bio and Medical Technology Development Program (NRF-2016R1A2B3011389 to Y.T.K.), and the KRIBB Research Initiative Program. This work was also supported by the NRF of Korea and the Center for Women In Science, Engineering and Technology (WISET) Grant funded by the Ministry of Science, ICT and Future Planning under the Program for Returners into R&D.
References (51)
- et al.
Cellular and molecular mechanisms of bone remodeling
J. Biol. Chem.
(2010) - et al.
Calcineurin signaling and NFAT activation in cardiovascular and skeletal muscle development
Dev. Biol.
(2004) - et al.
NFATc1 balances quiescence and proliferation of skin stem cells
Cell
(2008) - et al.
Plk1-dependent and -independent roles of an ODF2 splice variant, hCenexin1, at the centrosome of somatic cells
Dev. Cell
(2009) - et al.
CDK inhibitors: cell cycle regulators and beyond
Dev. Cell
(2008) - et al.
Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis
Dev. Cell
(2010) - et al.
IkappaB kinase epsilon is an NFATc1 kinase that inhibits T cell immune response
Cell Rep.
(2016) - et al.
Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos
J. Biol. Chem.
(2004) - et al.
The inhibitors of cyclin-dependent kinases and GSK-3beta enhance osteoclastogenesis
Biochemistry and biophysics reports
(2016) - et al.
Nuclear factor of activated T cells in cancer development and treatment
Cancer Lett.
(2015)
NFAT signaling: choreographing the social lives of cells
Cell
Bone homeostasis
Proc. Natl. Acad. Sci. U. S. A.
Regulation of NFATc1 in osteoclast differentiation
Journal of Bone Metabolism
The role of NFAT in osteoclast formation
Ann. N. Y. Acad. Sci.
NFAT proteins: key regulators of T-cell development and function
Nat. Rev. Immunol.
Transcriptional regulation by calcium, calcineurin, and NFAT
Genes Dev.
Calcineurin/NFAT signalling regulates pancreatic beta-cell growth and function
Nature
NFAT and Osterix cooperatively regulate bone formation
Nat. Med.
NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21
Nature
Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3
Science
A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT
Nature
A conserved docking motif for CK1 binding controls the nuclear localization of NFAT1
Mol. Cell. Biol.
RANKL coordinates cell cycle withdrawal and differentiation in osteoclasts through the cyclin-dependent kinase inhibitors p27KIP1 and p21CIP1
Journal of Bone and Mineral Research : The Official Journal of the American Society for Bone and Mineral Research
Osteoclast differentiation by RANKL requires NF-kappaB-mediated downregulation of cyclin-dependent kinase 6 (Cdk6)
Journal of Bone and Mineral Research : The Official Journal of the American Society for Bone and Mineral Research
Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2
Nature
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The first three authors contributed equally.