miR-433 targets BMP and Indian hedgehog signaling to coordinate murine postnatal growth plate dynamics

The postnatal growth plate undergoes dynamic morphogenetic changes essential for endochondral bone formation. While morphogen signaling in this context is well studied, microRNA-mediated post-transcriptional control is poorly understood. Here, we identify microRNA-433-3p (miR-433-3p; miR-433) as a key regulator of chondrocyte proliferation and hypertrophy, acting in part through direct targeting of vital chondrocyte genes. miR-433 is evolutionarily conserved and prominently expressed in precursor chondrocytes embryonically and in the proliferating zone of the growth plate postnatally. To interrogate miR-433 function in vivo, we generated a conditional miR-433 tough decoy (TuD) (competitive inhibitor) mouse model to decrease endogenous miR-433 activity in a lineage-restricted manner. Male and female mice expressing miR-433 TuD in Prrx1-expressing skeletal progenitors and their progeny exhibited shortened and narrower femurs, while a significantly decreased trabecular bone volume was only apparent in males. Male miR-433 decoy mice had disorganized growth plates with fewer resting zone cells, abnormal hypertrophic-like cells in the proliferative zone, and delayed secondary ossification center development. These defects were accompanied by elevated expression of Sox9, Ihh, PTHrP, Bmpr1a, as well as increased expression of validated miR-433 targets Runx2, Hdac6, and Hif1a. Tempering miR-433 activity increased proliferation in the resting zone at 1 and 3 weeks of age, and intensified SOX9 immunofluorescence throughout the growth plate, including the hypertrophic zone. The miR-433 target RUNX2 was ectopically expressed within the proliferating zone and showed increased expression in the hypertrophic zone, consistent with premature hypertrophic transition. Luciferase assays confirmed direct targeting of Bmpr1a and Ihh by miR-433. Given that BMP signaling induces Sox9 and Indian hedgehog promotes Runx2 expression, miR-433 may act as a molecular brake on both the BMP and hedgehog signaling axes, contributing to the spatial restriction of transcriptional programs driving chondrocyte maturation, thereby safeguarding orderly chondrocyte differentiation and bone elongation.