miR-322 stabilizes MEK1 expression to inhibit RAF/MEK/ERK pathway activation in cartilage

Abstract

Cartilage originates from mesenchymal cell condensations that differentiate into chondrocytes of transient growth plate cartilage or permanent cartilage of the articular joint surface and trachea. MicroRNAs fine-tune the activation of entire signaling networks and thereby modulate complex cellular responses, but so far only limited data are available on miRNAs that regulate cartilage development. Here, we characterize a miRNA that promotes the biosynthesis of a key component in the RAF/MEK/ERK pathway in cartilage. Specifically, by transcriptome profiling we identified miR-322 to be upregulated during chondrocyte differentiation. Among the various miR-322 target genes in the RAF/MEK/ERK pathway, only was identified as a regulated target in chondrocytes. Surprisingly, an increased concentration of miR-322 stabilizes mRNA to raise protein levels and dampen ERK1/2 phosphorylation, while cartilage-specific inactivation of miR322 in mice linked the loss of miR-322 to decreased MEK1 levels and to increased RAF/MEK/ERK pathway activation. Such mice died perinatally due to tracheal growth restriction and respiratory failure. Hence, a single miRNA can stimulate the production of an inhibitory component of a central signaling pathway to impair cartilage development.

microRNA-seq of cartilage reveals an over-abundance of miR-140-3p which contains functional isomiRs

“…Even where repression was determined, the effects of miR-140-3p.1 and miR-140-3p.2 were relatively small (up to ~25% repression) when compared to the effects of miR-140-5p on a previously published miR-140-5p direct target, FZD6 (~60% repression) (9). Furthermore, contra to the expected miR-140-3p.1 repression of targets, miR-140-3p.1 increased luciferase for the MARCKSL1, KIAA1199 and MAPILC3A 3'UTR constructs, possibly suggesting stabilisation of transcript, as previously described for miR-322 and MEK1 (52).…”

microRNA-seq of cartilage reveals an overabundance of miR-140-3p which contains functional isomiRs

“…Furthermore, contrary to the expected miR-140-3p.1 repression of targets, miR-140-3p.1 increased luciferase for the MARCKSL1, KIAA1199, and MAPILC3A 3 ′ UTR constructs, possibly suggesting stabilization of transcript, as previously described for miR-322 and MEK1 (Bluhm et al 2017).…”

Respiratory chain inactivation links cartilage-mediated growth retardation to mitochondrial diseases

“…We next generated transgenic mice expressing a patient-derived dominant-negative mutant of the Twinkle helicase in chondrocytes to inhibit the RC and thus determine its function in growth plate–mediated skeletal growth. We crossed R26-K320E-Twinkle loxP/+ mice (Weiland et al, 2018) with mice expressing Cre recombinase driven by the Col2a1 promotor (Cre; Ovchinnikov et al, 2000; Bluhm et al, 2017) and induced the expression of the Twinkle mutant protein in cartilage during early embryonal development. In these mice (CreTW), a marked reduction in the multicopy mtDNA molecules was found and consequently a decrease in index subunits of those RC complexes, which contain essential mtDNA-encoded subunits (complexes I, III, and IV), was observed in cartilage from 1-mo-old mice (Fig.…”