Anti-SIK1 monoclonal antibody, clone 3D23

Macrophages play important roles in the innate immune system during infection and systemic inflammation. When bacterial lipopolysaccharide (LPS) binds to Toll-like receptor 4 on macrophages, several signalling cascades co-operatively up-regulate gene expression of inflammatory molecules. The present study aimed to examine whether salt-inducible kinase [SIK, a member of the AMP-activated protein kinase (AMPK) family] could contribute to the regulation of immune signal not only in cultured macrophages, but also in vivo. LPS up-regulated SIK3 expression in murine RAW264.7 macrophages and exogenously over-expressed SIK3 negatively regulated the expression of inflammatory molecules [interleukin-6 (IL-6), nitric oxide (NO) and IL-12p40] in RAW264.7 macrophages. Conversely, these inflammatory molecule levels were up-regulated in SIK3-deficient thioglycollate-elicited peritoneal macrophages (TEPM), despite no impairment of the classical signalling cascades. Forced expression of SIK3 in SIK3-deficient TEPM suppressed the levels of the above-mentioned inflammatory molecules. LPS injection (10mg/kg) led to the death of all SIK3-knockout (KO) mice within 48hr after treatment, whereas only one mouse died in the SIK1-KO (n=8), SIK2-KO (n=9) and wild-type (n=8 or 9) groups. In addition, SIK3-KO bone marrow transplantation increased LPS sensitivity of the recipient wild-type mice, which was accompanied by an increased level of circulating IL-6. These results suggest that SIK3 is a unique negative regulator that suppresses inflammatory molecule gene expression in LPS-stimulated macrophages.

Objectives Because a newly described salt-inducible kinase 1 (SIK1) network is responsible for increases in active cell sodium transport in response to elevated intracellular sodium, we hypothesized that this network could mediate the effects of the mutant (hypertensive) form of alpha-adducin on Na+,K+-ATPase activity. Methods Studies were performed in normotensive and hypertensive Milan rats and in a cell line of proximal tubule origin expressing transiently variants of alpha-adducin (human G460W/S586C;rat F316Y) that are associated with elevated blood pressure and result in increased Na+,K+-ATPase activity. Na+,K+-ATPase activity was determined as ouabain-sensitive rubidium transport. Results SIK1 activity (T182 phosphorylation) was significantly elevated in renal proximal tubule cells from Milan hypertensive rats (carrying a alpha-adducin mutation) when compared with normotensive controls. Similarly, SIK1 activity (T182 phosphorylation) was elevated in a normal renal proximal tubule cell line when transfected with the alpha-adducin variant carrying the human hypertensive mutation. Blocking the SIK1 network using negative mutants as well as different stages of its activation pathway prevented the effects induced by the hypertensive alpha-adducin. Conclusion The SIK1 network may constitute an alternative target by which agents can modulate active sodium transport in renal epithelia and avoid the increases in systemic blood pressure that are associated with genetic mutations in the human alpha-adducin molecule. J Hypertens 27:2452-2457 (c) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.