Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages

Abstract

Macrophages acquire strikingly different properties that enable them to play key roles during the initiation, propagation, and resolution of inflammation. Classically activated (M1) macrophages produce proinflammatory mediators to combat invading pathogens and respond to tissue damage in the host, whereas regulatory macrophages (M2b) produce high levels of anti-inflammatory molecules, such as IL-10, and low levels of proinflammatory cytokines, like IL-12, and are important for the resolution of inflammatory responses. A central problem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Here, we demonstrate that the salt-inducible kinases (SIKs) restrict the formation of regulatory macrophages and that their inhibition induces striking increases in many of the characteristic markers of regulatory macrophages, greatly stimulating the production of IL-10 and other anti-inflammatory molecules. We show that SIK inhibitors elevate IL-10 production by inducing the dephosphorylation of cAMP response element-binding protein (CREB)-regulated transcriptional coactivator (CRTC) 3, its dissociation from 14-3-3 proteins and its translocation to the nucleus where it enhances a gene transcription program controlled by CREB. Importantly, the effects of SIK inhibitors on IL-10 production are lost in macrophages that express a drug-resistant mutant of SIK2. These findings identify SIKs as a key molecular switch whose inhibition reprograms macrophages to an anti-inflammatory phenotype. The remarkable effects of SIK inhibitors on macrophage function suggest that drugs that target these protein kinases may have therapeutic potential for the treatment of inflammatory and autoimmune diseases.Toll-like receptor | AMPK-related kinases | MRT67307 | MRT199665 |

Small-molecule screening identifies inhibition of salt-inducible kinases as a therapeutic strategy to enhance immunoregulatory functions of dendritic cells

“…1C), which is consistent with the IL-10-potentiating effects of SIK inhibition in activated MΦs (20,21). In agreement with these reports, pretreating BMDCs with HG-9-91-01, a recently described inhibitor of SIK1-3, along with several other kinases (21), results in concentration-dependent potentiation of zymosaninduced IL-10 production with an EC 50 ∼ 200 nM and a maximum effect similar to that observed with PGE 2 (Fig.…”

“…S5B). However, consistent with data indicating that reduction of Sik2 expression by RNAi is well tolerated in macrophages (21), the toxicity of HG-9-91-01 analogs did not correlate with the IC 50 s for SIK1-3 inhibition. The close correlation between the potencies of SIK2 inhibition and IL-10 up-regulation suggests that inhibition of this SIK isoform is primarily responsible for the IL-10-potentiating activity of HG-9-91-01 and structurally related compounds in activated dendritic cells.…”

“…Evaluation of a series of structural analogs of the selective SIK targeting inhibitor HG-9-91-01 demonstrates a strong correlation between the potency of SIK2 inhibition and enhanced IL-10 production. These results, together with data indicating that expression of an HG-9-91-01-resistant SIK2 allele suppresses the IL-10-potentiating effects of this compound in RAW264.7 murine macrophages (21), suggest that SIK2 plays a primary role in restraining IL-10 production in activated myeloid cells. Along with enhanced IL-10 secretion, SIK inhibition converted activated BMDCs to an anti-inflammatory phenotype marked by reduced secretion of the inflammatory cytokines IL-1β, IL-6, IL-12, and TNF-α, and these coordinated effects of SIK inhibition were conserved in human DCs-MΦs.…”

“…In quiescent myeloid cells, SIKs phosphorylate CREBregulated transcriptional coactivator-3 (CRTC3), which results in its sequestration by cytoplasmic 14-3-3 proteins. Following EP2-EP4 stimulation, inhibitory phosphorylation of SIKs by protein kinase A (PKA) leads to decreased CRTC3 phosphorylation, allowing it to enter the nucleus and enhance transcription of CREB target genes including IL-10, and this effect can be recapitulated with small-molecule SIK inhibitors (21). The central role of PKC, GSK-3β, and SIKs in IL-10 production suggests that additional kinase inhibitors are likely to modulate pathways that regulate IL-10 production by DCs-MΦs.…”

“…Conversely, CREB activation by EP2/EP4 prostanoid receptor agonists like prostaglandin E 2 (PGE 2 ) promotes IL-10 production by myeloid cells (19). Recently, salt-inducible kinases (SIKs), which are members of the AMP-activated protein kinase family, have been identified as a key component of the CREB activation cascade (20,21). In quiescent myeloid cells, SIKs phosphorylate CREBregulated transcriptional coactivator-3 (CRTC3), which results in its sequestration by cytoplasmic 14-3-3 proteins.…”