microRNA-433 Inhibits Glucocorticoid and TGF-β Signaling: Impacts on Osteoblast Circadian Rhythm, Commitment, and Differentiation

Lineage commitment and differentiation of skeletal cells requires coordinated regulation of multiple signaling systems by microRNAs (miRNAs). Transforming growth factor β (TGFβ) is important for osteoblastogenesis, chondrogenesis and adipogenesis. Here, we show that miR-433 limits TGFβ signaling and is a negative regulator of osteoblastogenesis and chondrogenesis. miR-433 has also been found to target the glucocorticoid receptor, and rhythmic secretion of glucocorticoids is critical for synchronizing circadian clocks. We hypothesized that miR-433 regulates the circadian clocks by regulating glucocorticoid signaling. In vivo, miR-433 displays robust rhythmicity in mouse calvaria. Its expression pattern was anti-phasic in relation to Bmal1, peaking after light removal. To determine if miR-433 regulates circadian rhythm in vitro, its activity was inhibited using a miR-433 competitive inhibitor (miR-433 decoy) in stably transduced C3H/10T1/2 cells. miR-433 inhibition modestly affected Bmal1 rhythm and it dramatically altered the phase of Per2. Inhibiting miR-433 activity amplified the glucocorticoid responsive genes Dusp1 and Per2, and induced nuclear localization of the glucocorticoid receptor. In vivo inhibition of miR-433 activity using a Col1a1 driven miR-433 decoy transgenic model altered the phase and amplitude of the circadian clocks. Overall, we found that miR-433 displays a circadian rhythm in calvaria, alters the phase of circadian clocks, and regulates sensitivity to glucocorticoids. BMSCs cultured in osteogenic medium caused a progressive decline in miR-433. In contrast, miR-433 was dramatically increased in BMSCs and iPSCs cultured in micromass to induce chondrogenesis. miR-433 levels remained constant during adipogenesis. miR-433 decoy cells were differentiated with BMP2 and miR-433 inhibition induced alkaline phosphatase, Runx2, and osteocalcin mRNAs. In micromass cultures treated with BMP2 and TGFβ, miR-433 inhibition promoted expression of chondrogenic mRNAs, Sox9 and Col2a1. In cells treated with an adipogenic cocktail, miR-433 inhibition failed to alter adipogenic gene markers or Oil-red O staining. Bioinformatic analyses suggested that miR-433 might target critical components of the TGFβ pathway. miR-433 inhibition amplified TGFβ signaling, evidenced by increased activity of a TGFβ-responsive SBE4 luciferase reporter and enhanced TGFβ-induced pSMAD2. To determine underlying mechanisms, we used Luciferase-3’UTR reporter assays, and experimentally validated SMAD2 and TGFBR1 as novel miR-433 targets. Overall, miR-433 attenuates TGF-ß signaling, and restrains osteoblastic and chondrogenic differentiation.