PTH-dependent stabilization of RANKL mRNA is associated with increased phosphorylation of the KH-Type Splicing Regulatory Protein

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Parathyroid hormone (PTH) receptor agonists promote bone formation but also increase osteoclastogenesis, in part by increasing expression of the receptor activator of nuclear factor kappa-Β ligand (RANKL). In addition to activation of transcription, regulation of mRNA stability is another important molecular mechanism controlling mRNA abundance. PTH treatment for 6 hrs. resulted in a 7.4-fold elevation in RANKL mRNA expression in UAMS-32P cells, despite prior inhibition of cellular transcription by thiophosphoryl (TPL). RANKL mRNA, like other TNF family members, contains AU-Rich Elements (AREs) in the 3’ UTR. AU-Rich Element Binding Proteins (ABPs including KSRP, TTP, AUF1 and HuR) bind to AREs and regulate mRNA stability. There was significantly more KSRP bound to RANKL mRNA than any of the other ABPs. PTH did not increase the amount of ABPs bound to the RANKL transcript. However, the level of cellular phosphorylated KSRP was significantly increased in UAMS-32P cells pre-treated with TPL followed by PTH exposure, compared to cells treated with vehicle following TPL. The extent of phosphorylation of cellular AUF1, HuR, and TTP did not increase with PTH treatment. There were no significant changes in the cellular content of total Pin1 and phospho-Pin1 protein with PTH treatment. We conclude that increases in cellular phospho-KSRP following PTH treatment, together with fact that the total amount of the KSRP bound to the RANKL mRNA did not change with PTH-treatment, may indicate that phospho-KSRP plays some role in stabilizing the RANKL transcript.