Abstract
Sympathetic nervous system stimulation-induced β-adrenergic signal transduction is known to induce bone loss and increase of osteoclast activity. Although isoproterenol, a nonspecific β-adrenergic receptor agonist, has been shown to increase receptor activator of NF-κB ligand (RANKL), the details of the regulatory mechanisms remain unclear. In the present study, we investigated the role of the nuclear factor of activated T-cells (NFAT) in isoproterenol-induced RANKL expression in C2C12 and in primary cultured mouse calvarial cells. Isoproterenol increased nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and RANKL expressions at both mRNA and protein levels and increased NFAT reporter activity. NFATc1 knockdown blocked isoproterenol-mediated RANKL expression. Isoproterenol also promoted cAMP response element-binding protein 1 (CREB1) and activating transcription factor 4 (ATF4) phosphorylation. Isoproterenol-mediated transcriptional activation of NFAT was blocked by protein kinase A (PKA) inhibitor H89. Isoproterenol-induced CREB1, ATF4, NFATc1, and RANKL expressions were suppressed by H89. Mutations in cAMP response element-like or NFAT-binding element suppressed isoproterenol-induced RANKL promoter activity. Chromatin immunoprecipitation analysis demonstrated that isoproterenol increased NFAT-binding and ATF4-binding activities on the mouse RANKL promoter, but did not increase CREB1-binding activity. Association of NFATc1 and ATF4 was not observed in a co-immunoprecipitation study. ATF4 knockdown suppressed isoproterenol-induced NFAT binding to the RANKL promoter, whereas NFATc1 knockdown did not suppress isoproterenol-induced ATF4 binding to the RANKL promoter. ATF4 knockdown suppressed isoproterenol-induced expressions of NFATc1 and RANKL. These results suggest that isoproterenol increases RANKL expression in an ATF4/NFATc1-dependent manner.
Highlights
A line of research has emerged detailing the role that the sympathetic nervous system (SNS) plays in bone-mass regulation
Isoproterenol Induces Expression Levels and Transcriptional Activity of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), Which Is Necessary for Isoproterenol-Induced RANKL Expression
We examined whether isoproterenol induces RANKL expressions and NFATc1 activation in C2C12 cells
Summary
A line of research has emerged detailing the role that the sympathetic nervous system (SNS) plays in bone-mass regulation. The β-adrenergic receptor antagonist mitigated SNS activation-induced metaphyseal bone loss by attenuating declines in osteoblastic cell activity and by abolishing increases in osteoclast surfaces [5,6,7]. It has been demonstrated that downstream of cAMP/protein kinase A (PKA) activation, the activated CREB1 mediates PTH1R-induced RANKL expression by binding to multiple distal enhancers of the RANKL gene [11,12]. ATF4 is a critical transcription factor for osteoblast function and bone formation; a regulatory role in osteoclast differentiation or RANKL expression has not been fully studied. To experimentally address that SNS activation stimulates bone resorption by increasing RANKL expression, a pharmacological model has been developed using β-adrenergic receptor agonists. In the context of SNS activation-induced RANKL expression, the role of the PKA/ATF4 signaling pathway has been demonstrated [3]. We propose another transcriptional regulatory mechanism of β-adrenergic signaling-mediated RANKL expression, in which β-adrenergic signaling-induced NFATc1 upregulates RANKL transcription by directly binding to the mouse RANKL promoter
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