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Osteoclasts are large multinucleated cells responsible for bone resorption. Excessive inflammatory activation of osteoclasts leads to bony erosions, which are the hallmark of several diseases such as rheumatoid arthritis (RA). Salt-inducible kinases (SIK) constitute a subfamily of kinases comprising three members (SIK1, -2, and -3). Inhibition of SIK kinase activity induces an anti-inflammatory phenotype in macrophages. Since osteoclasts originate from precursors of macrophage origin, we hypothesized a role of SIK in osteoclastogenesis. We analyzed SIK1, -2 and -3 expression and function in osteoclast differentiation using the mouse macrophage cell line RAW264.7 and bone marrow-derived macrophages (BMM). We show that all three SIK are expressed in fully differentiated osteoclasts and that in BMM-derived osteoclasts there is an increased expression of SIK1 and SIK3 proteins. Interestingly, the pan-SIK inhibitor HG-9-91-01 significantly inhibited osteoclastogenesis by dose dependently reducing osteoclast differentiation markers (i.e. CathepsinK, MMP-9 and TRAP) and bone resorbing activity. Analysis of the signaling pathways activated by RANKL in RAW cells showed that SIK inhibitors did not affect RANKL-induced ERK1/2, JNK, p38 or NF-κB activation, but induced a significant downregulation in c-Fos and NFATc1 protein levels, the two main transcription factors involved in the regulation of osteoclast-specific genes. Moreover, SIK inhibition partially increased the proteasome-mediated degradation of c-Fos. SIK2 and SIK3 knockout RAW cells were generated by the CRISPR/Cas9 approach. SIK2 KO and, to a lesser extent, SIK3 KO recapitulated the effect of SIK small molecule inhibitor, thus confirming the specificity of the effect of SIK inhibition on the reduction of osteoclastogenesis. Overall, our results support the notion that the SIK signaling pathway plays a significant role among the check-points controlling osteoclastogenesis. SIK kinase inhibitors could thus represent a potential novel therapy to prevent bone erosions. [ABSTRACT FROM AUTHOR]

The expression of the IL-2R α-chain (IL-2Rα) is regulated at the transcriptional level via TCR- and IL-2R-signaling. The question is how to precede in time the activation signals to induce the IL-2Rα expression in native primary T cells. By comparing the effects of selective drugs on the dynamics of CD25 expression during the mitogen stimulation of human peripheral blood lymphocytes, we identified distinct Src- and JAK-dependent stages of IL-2Rα upregulation. PP2, a selective inhibitor of TCR-associated Src kinase, prevents CD25 expression at initial stages of T cell activation, prior to the cell growth. This early IL-2Rα upregulation underlies the T cell competence and the IL-2 responsiveness. We found that the activated with “weak” mitogen, the population of blood lymphocytes has some pool of competent CD25+ cells bearing a high affinity IL-2R. A distinct pattern of IL-2R signaling in resting and competent T lymphocytes has been shown. Based on the inhibitory effect of WHI-P131, a selective drug of JAK3 kinase activity, we concluded that in quiescent primary T lymphocytes, the constitutive STAT3 and the IL-2-induced prolonged STAT5 activity (assayed by tyrosine phosphorylation) is mostly JAK3-independent. In competent T cells, in the presence of IL-2 JAK3/STAT5 pathway is switched to maintain the higher and sustained IL-2Rα expression as well as cell growth and proliferation. We believe that understanding the temporal coordination of antigen- and cytokine-evoked signals in primary T cells may be useful for improving immunotherapeutic strategies. [ABSTRACT FROM AUTHOR]

Similar to its mammalian counterparts, teleost Toll-like receptor 9 (TLR9) recognizes unmethylated CpG DNA presented in the genome of bacteria or DNA viruses and initiates signaling pathway(s) for immune responses. We have previously shown that the TLR9 pathway in grouper, an economically important teleost, can be debilitated by an inhibitory gTLR9B isoform, whose production is mediated by RNA alternative splicing. However, how does grouper TLR9 (gTLR9) signaling impinge on the RNA splicing machinery to produce gTlr9B is unknown. Here we show that the gTlr9 alternative splicing is regulated through ligand-induced phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). We first observed that ligand-activated NF- κB pathway biased the production of the gTlr9B isoform. Because NF- κB is known to recruit p-TEFb kinase, which phosphorylates the Pol II CTD at Ser2 residues, we examined p-TEFb’s role in alternative splicing. We found that promoting p-TEFb kinase activity significantly favored the production of the gTlr9B isoform, whereas inhibiting p-TEFb yielded an opposite result. We further showed that p-TEFb-mediated production of the gTlr9B isoform down-regulates its own immune responses, suggesting a self-limiting mechanism. Taken together, our data indicate a feedback mechanism of the gTLR9 signaling pathway to regulate the alternative splicing machinery, which in turn produces an inhibitor to the pathway. [ABSTRACT FROM AUTHOR]