Date of Completion

9-12-2016

Embargo Period

3-2-2017

Keywords

microRNAs, Osteoblasts, Circadian Rhythm, TGF-β, Glucocorticoid, Chondrocytes, Adipocytes, Post-transcriptional

Major Advisor

Dr. Anne Delany

Associate Advisor

Dr. Barbara Kream

Associate Advisor

Dr. Peter Maye

Associate Advisor

Dr. Rosa Guzzo

Associate Advisor

Dr. Mina Mina

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

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.

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