The expression and function of microRNAs in chondrogenesis and osteoarthritis

Abstract

Objective To use an in vitro model of chondrogenesis to identify microRNAs (miRNAs) with a functional role in cartilage homeostasis. Methods The expression of miRNAs was measured in the ATDC5 cell model of chondrogenesis using microarray and was verified using quantitative reverse transcription–polymerase chain reaction. MicroRNA expression was localized by in situ hybridization. Predicted miRNA target genes were validated using 3′-untranslated region-Luc reporter plasmids containing either wild-type sequences or mutants of the miRNA target sequence. Signaling through the Smad pathway was measured using a (CAGA)₁₂-Luc reporter. Results The expression of several miRNAs was regulated during chondrogenesis. These included 39 miRNAs that are coexpressed with miRNA-140 (miR-140), which is known to be involved in cartilage homeostasis and osteoarthritis (OA). Of these miRNAs, miR-455 resides within an intron of COL27A1 that encodes a cartilage collagen. When human OA cartilage was compared with cartilage obtained from patients with femoral neck fractures, the expression of both miR-140-5p and miR-455-3p was increased in OA cartilage. In situ hybridization showed miR-455-3p expression in the developing limbs of chicks and mice and in human OA cartilage. The expression of miR-455-3p was regulated by transforming growth factor β (TGFβ) ligands, and miRNA regulated TGFβ signaling. ACVR2B, SMAD2, and CHRDL1 were direct targets of miR-455-3p and may mediate its functional impact on TGFβ signaling. Conclusion MicroRNA-455 is expressed during chondrogenesis and in adult articular cartilage, where it can regulate TGFβ signaling, suppressing the Smad2/3 pathway. Diminished signaling through this pathway during the aging process and in OA chondrocytes is known to contribute to cartilage destruction. We propose that the increased expression of miR-455 in OA exacerbates this process and contributes to disease pathology.