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Salt Inducible Kinases (SIKs), of which there are 3 isoforms, are established to play roles in innate immunity, metabolic control and neuronal function, but their role in adaptive immunity is unknown. To address this gap, we used a combination of SIK knockout and kinase-inactive knock-in mice. The combined loss of SIK1 and SIK2 activity did not block T cell development. Conditional knockout of SIK3 in haemopoietic cells, driven by a Vav-iCre transgene, resulted in a moderate reduction in the numbers of peripheral T cells, but normal B cell numbers. Constitutive knockout of SIK2 combined with conditional knockout of SIK3 in the haemopoietic cells resulted in a severe reduction in peripheral T cells without reducing B cell number. A similar effect was seen when SIK3 deletion was driven via CD4-Cre transgene to delete at the DP stage of T cell development. Analysis of the SIK2/3 Vav-iCre mice showed that thymocyte number was greatly reduced, but development was not blocked completely as indicated by the presence of low numbers CD4 and CD8 single positive cells. SIK2 and SIK3 were not required for rearrangement of the TCRβ locus, or for low level cell surface expression of the TCR complex on the surface of CD4/CD8 double positive thymocytes. In the absence of both SIK2 and SIK3, progression to mature single positive cells was greatly reduced, suggesting a defect in negative and/or positive selection in the thymus. In agreement with an effect on negative selection, increased apoptosis was seen in thymic TCRbeta high/CD5 positive cells from SIK2/3 knockout mice. Together, these results show an important role for SIK2 and SIK3 in thymic T cell development.

The salt-inducible kinases, SIK1, SIK2 and SIK3, most closely resemble the AMP-activated protein kinase (AMPK) and other AMPK-related kinases, and like these family members they require phosphorylation by LKB1 to be catalytically active. However, unlike other AMPK-related kinases they are phosphorylated by cyclic AMP-dependent protein kinase (PKA), which promotes their binding to 14-3-3 proteins and inactivation. The most well-established substrates of the SIKs are the CREB-regulated transcriptional co-activators (CRTCs), and the Class 2a histone deacetylases (HDAC4/5/7/9). Phosphorylation by SIKs promotes the translocation of CRTCs and Class 2a HDACs to the cytoplasm and their binding to 14-3-3s, preventing them from regulating their nuclear binding partners, the transcription factors CREB and MEF2. This process is reversed by PKA-dependent inactivation of the SIKs leading to dephosphorylation of CRTCs and Class 2a HDACs and their re-entry into the nucleus. Through the reversible regulation of these substrates and others that have not yet been identified, the SIKs regulate many physiological processes ranging from innate immunity, circadian rhythms and bone formation, to skin pigmentation and metabolism. This review summarises current knowledge of the SIKs and the evidence underpinning these findings, and discusses the therapeutic potential of SIK inhibitors for the treatment of disease.

Idiopathic pulmonary fibrosis is a progressive and normally fatal disease with limited treatment options. The tyrosine kinase inhibitor nintedanib has recently been approved for the treatment of idiopathic pulmonary fibrosis, and its effectiveness has been linked to its ability to inhibit a number of receptor tyrosine kinases including the platelet-derived growth factor, vascular endothelial growth factor, and fibroblast growth factor receptors. We show here that nintedanib also inhibits salt-inducible kinase 2 (SIK2), with a similar IC

Cytokines and chemokines are important regulators of airway hyper-responsiveness, immune cell infiltration, and inflammation and are produced when mast cells are stimulated with interleukin-33 (IL-33). Here, we establish that the salt-inducible kinases (SIKs) are required for the IL-33-stimulated transcription of il13, gm-csf and tnf and hence the production of these cytokines. The IL-33-stimulated secretion of IL-13, granulocyte-macrophage colony stimulating factor, and tumor necrosis factor was strongly reduced in fetal liver-derived mast cells from mice expressing a kinase-inactive mutant of SIK3 and abolished in cells expressing kinase-inactive mutants of SIK2 and SIK3. The IL-33-dependent secretion of these cytokines and several chemokines was also abolished in SIK2/3 double knock-out bone marrow-derived mast cells (BMMC), reduced in SIK3 KO cells but little affected in BMMC expressing kinase-inactive mutants of SIK1 and SIK2 or lacking SIK2 expression. In SIK2 knock-out BMMC, the expression of SIK3 was greatly increased. Our studies identify essential roles for SIK2 and SIK3 in producing inflammatory mediators that trigger airway inflammation. The effects of SIKs were independent of IκB kinase β, IκB kinase β-mediated NF-κB-dependent gene transcription, and activation of the mitogen-activated protein kinase family members p38α and c-jun N-terminal kinases. Our results suggest that dual inhibitors of SIK2 and SIK3 may have therapeutic potential for the treatment of mast cell-driven diseases.

The signalling pathways initiated by members of the transforming growth factor-β (TGFβ) family of cytokines control many metazoan cellular processes, including proliferation and differentiation, epithelial-mesenchymal transition (EMT), and apoptosis. TGFβ signalling is therefore strictly regulated to ensure appropriate context-dependent physiological responses. In an attempt to identify novel regulatory components of the TGFβ signalling pathway, we performed a pharmacological screen using a cell line engineered to report the endogenous transcription of the TGFβ-responsive target genePAI-1. The screen revealed that small-molecular inhibitors of salt-inducible kinases (SIKs) attenuate TGFβ-mediated transcription ofPAI-1without affecting receptor-mediated SMAD phosphorylation, SMAD complex formation or nuclear translocation. We provide evidence that genetic inactivation of SIK isoforms also attenuates TGFβ-dependent transcriptional responses. Pharmacological inhibition of SIKs using multiple small-molecule inhibitors potentiated apoptotic cell death induced by TGFβ stimulation. Our data therefore provides evidence for a novel function of SIKs in modulating TGFβ-mediated transcriptional and cellular responses.

The salt-inducible kinases (SIKs) control a novel molecular switch regulating macrophage polarization. Pharmacological inhibition of the SIKs induces a macrophage phenotype characterized by the secretion of high levels of anti-inflammatory cytokines, including interleukin (IL)-10, and the secretion of very low levels of pro-inflammatory cytokines, such as tumour necrosis factor α. The SIKs, therefore, represent attractive new drug targets for the treatment of macrophage-driven diseases, but which of the three isoforms, SIK1, SIK2 or SIK3, would be appropriate to target remains unknown. To address this question, we developed knock-in (KI) mice for SIK1, SIK2 and SIK3, in which we introduced a mutation that renders the enzymes catalytically inactive. Characterization of primary macrophages from the single and double KI mice established that all three SIK isoforms, and in particular SIK2 and SIK3, contribute to macrophage polarization. Moreover, we discovered that inhibition of SIK2 and SIK3 during macrophage differentiation greatly enhanced the production of IL-10 compared with their inhibition in mature macrophages. Interestingly, macrophages differentiated in the presence of SIK inhibitors, MRT199665 and HG-9-91-01, still produced very large amounts of IL-10, but very low levels of pro-inflammatory cytokines, even after the SIKs had been reactivated by removal of the drugs. Our data highlight an integral role for SIK2 and SIK3 in innate immunity by preventing the differentiation of macrophages into a potent and stable anti-inflammatory phenotype.