Conditional Disruption of miR17∼92 in Osteoclasts Led to Activation of Osteoclasts and Loss of Trabecular Bone in Part Through Suppression of the miR17‐Mediated Downregulation of Protein‐Tyrosine Phosphatase‐oc in Mice

Abstract

This study sought to understand the regulation of an osteoclastic protein-tyrosine phosphatase (PTP-oc), a positive regulator of osteoclast activaty. Our past studies suggested that PTP-oc is regulated post-transcriptionally. The 3’-UTR of PTP-oc mRNA contains a target site for miR17. During osteoclastic differentiation, there was an inverse relationship between the cellular levels of miR17 (expressed as one of the six cluster genes of miR17~92) and PTP-oc mRNA. Overexpression of pre-miR17~92 in mouse osteoclast precursors reduced PTP-oc mRNA level and the size of the derived osteoclasts; whereas deletion of miR17~92 or inhibition of miR17 resulted in the formation of larger osteoclasts containing more nuclei that expressed higher PTP-oc mRNA levels and created larger resorption pits. Thus, PTP-oc-mediated osteoclast activation is modulated in part by miR17~92, particularly miR17. The miR17~92 osteoclast conditional knockout (cKO) mutants, generated by breeding miR17~92loxp/loxp mice with Ctsk-Cre mice, had lower Tb.BV/TV, Tb.BMD, Tb.Conn-Dens, Tb.N, and Tb.Th, but larger Tb.Sp, and greater bone resorption without a change in bone formation compared to littermate controls. The cKO marrow-derived osteoclasts were twice as large, contained twice as many nuclei, and produced twice as large resorption pits as osteoclasts of littermate controls. The expression of genes associated with osteoclast activation was increased in cKO osteoclasts, suggesting that deletion of miR17~92 in osteoclasts promotes osteoclast activation. The cKO osteoblasts did not show differences in cellular miR17 level, alkaline phosphatase activity, and bone nodule formation ability. In conclusion, miR17-92 negatively regulates the osteoclast activity, in part via the miR17-mediated suppression of PTP-oc in osteoclasts.

A novel miR17 /protein tyrosine phosphatase-oc/EphA4 regulatory axis of osteoclast activity

“…The potential role of miR17 in the regulation of PTP-oc in osteoclastic cells was recently investigated (28), because the expression of a structurally-related PTP (PTPRO) was shown to be negatively regulated by miR17 (100). The miR17 is expressed as one of the six cluster genes of miR17~92 (i.e., miR17 , miR18 , miR19a , miR19b , miR20 , and miR92 ).…”

“…The miR17 is expressed as one of the six cluster genes of miR17~92 (i.e., miR17 , miR18 , miR19a , miR19b , miR20 , and miR92 ). The in vivo evidence that miR17~92 is a negative regulator of osteoclast functions came from the characterization of the miR17~92 osteoclast conditional knockout mice (28), in that the miR17~92 conditional knockout mutants had highly active osteoclasts and lost substantial trabecular bone associated with marked increase in bone resorption, but without an effect on bone formation parameters.…”

“…There are several strong pieces of in vitro evidence supporting the premise that the PTP-oc expression in osteoclasts is regulated in part by miR17 (28): First, an online search with the Targetscan database for potential miRNA target sequences has identified a highly conserved target site for miR17 on the 3′UTR of the PTP-oc mRNA, and the presence of the miR17 target site was confirmed by a miRNA target reporter assay (28). Second, the RANKL-induced osteoclast differentiation upregulated PTP-oc expression with a corresponding time-dependent downregulation of miR17 , and there was an inverse correlation between the levels of miR17 and the levels of PTP-oc mRNA in osteoclasts (28).…”

“…Second, the RANKL-induced osteoclast differentiation upregulated PTP-oc expression with a corresponding time-dependent downregulation of miR17 , and there was an inverse correlation between the levels of miR17 and the levels of PTP-oc mRNA in osteoclasts (28). Third, the suppression of miR17 activity with an anti- miR17-5p locked nucleic acid (LNA) oligonucleotide, but not the suppression of miR19a activity with an anti- miR19a-5p LNA oligonucleotide, increased the cell size and the number of nuclei of osteoclasts, which are indices of activated osteoclasts (28). Fourth, the same resorption activators [i.e., IL-1 and 1,25(OH) 2 D 3 but not IL-6 and TNFα] that increased cellular PTP-oc mRNA and protein levels (21,22) also reduced the cellular miR17 level in osteoclasts (28).…”

“…Intriguingly, PTP-oc also acts directly on EphA4 to suppress its forward signaling (26), which is a negative regulator of the osteoclast activity through inhibition of the integrin β 3 signaling (27), resulting in further enhancement in the osteoclast activity. The PTP-oc expression in osteoclasts is determined by post-transcriptional regulation in part through miR17 (28). Consequently, it appears that the miR17 , the PTP-oc, and the EphA4 represent three novel upstream regulators of the Src and integrin β 3 signaling in osteoclasts.…”