[Show abstract][Hide abstract] ABSTRACT: Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3(-/-) mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3(-/-) mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3(-/-) mice. Lipid metabolism disorders in Sik3(-/-) mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice.
PLoS ONE 05/2012; 7(5):e37803. DOI:10.1371/journal.pone.0037803 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chondrocyte hypertrophy is crucial for endochondral ossification, but the mechanism underlying this process is not fully understood. We report that salt-inducible kinase 3 (SIK3) deficiency causes severe inhibition of chondrocyte hypertrophy in mice. SIK3-deficient mice showed dwarfism as they aged, whereas body size was unaffected during embryogenesis. Anatomical and histological analyses revealed marked expansion of the growth plate and articular cartilage regions in the limbs, accumulation of chondrocytes in the sternum, ribs and spine, and impaired skull bone formation in SIK3-deficient mice. The primary phenotype in the skeletal tissue of SIK3-deficient mice was in the humerus at E14.5, where chondrocyte hypertrophy was markedly delayed. Chondrocyte hypertrophy was severely blocked until E18.5, and the proliferative chondrocytes occupied the inside of the humerus. Consistent with impaired chondrocyte hypertrophy in SIK3-deficient mice, native SIK3 expression was detected in the cytoplasm of prehypertrophic and hypertrophic chondrocytes in developing bones in embryos and in the growth plates in postnatal mice. HDAC4, a crucial repressor of chondrocyte hypertrophy, remained in the nuclei in SIK3-deficient chondrocytes, but was localized in the cytoplasm in wild-type hypertrophic chondrocytes. Molecular and cellular analyses demonstrated that SIK3 was required for anchoring HDAC4 in the cytoplasm, thereby releasing MEF2C, a crucial facilitator of chondrocyte hypertrophy, from suppression by HDAC4 in nuclei. Chondrocyte-specific overexpression of SIK3 induced closure of growth plates in adulthood, and the SIK3-deficient cartilage phenotype was rescued by transgenic SIK3 expression in the humerus. These results demonstrate an essential role for SIK3 in facilitating chondrocyte hypertrophy during skeletogenesis and growth plate maintenance.
Development 03/2012; 139(6):1153-63. DOI:10.1242/dev.072652 · 6.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flavonoids, which are plant polyphenols, are now widely used in supplements and cosmetics. Here, we report that 4'-methylflavonoids are potent inducers of melanogenesis in B16F10 melanoma cells and in mice. We recently identified salt inducible kinase 2 (SIK2) as an inhibitor of melanogenesis via the suppression of the cAMP-response element binding protein (CREB)-specific coactivator 1 (TORC1). Using an in vitro kinase assay targeting SIK2, we identified fisetin as a candidate inhibitor, possibly being capable of promoting melanogenesis. However, fisetin neither inhibited the CREB-inhibitory activity of SIK2 nor promoted melanogenesis in B16F10 melanoma cells. Conversely, mono-methyl-flavonoids, such as diosmetin (4'-O-metlylluteolin), efficiently inhibited SIK2 and promoted melanogenesis in this cell line. The cAMP-CREB system is impaired in A(y)/a mice and these mice have yellow hair as a result of pheomelanogenesis, while Sik2(+/-); A(y)/a mice also have yellow hair, but activate eumelanogenesis when they are exposed to CREB stimulators. Feeding Sik2(+/-); A(y)/a mice with diets supplemented with fisetin resulted in their hair color changing to brown, and metabolite analysis suggested the presence of mono-methylfisetin in their feces. Thus, we decided to synthesize 4'-O-methylfisetin (4'MF) and found that 4'MF strongly induced melanogenesis in B16F10 melanoma cells, which was accompanied by the nuclear translocation of TORC1, and the 4'-O-methylfisetin-induced melanogenic programs were inhibited by the overexpression of dominant negative TORC1. In conclusion, compounds that modulate SIK2 cascades are helpful to regulate melanogenesis via TORC1 without affecting cAMP levels, and the combined analysis of Sik2(+/-) mice and metabolites from these mice is an effective strategy to identify beneficial compounds to regulate CREB activity in vivo.
PLoS ONE 10/2011; 6(10):e26148. DOI:10.1371/journal.pone.0026148 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cAMP responsive element-binding protein (CREB) functions in a broad array of biological and pathophysiological processes. We found that salt-inducible kinase 2 (SIK2) was abundantly expressed in neurons and suppressed CREB-mediated gene expression after oxygen-glucose deprivation (OGD). OGD induced the degradation of SIK2 protein concomitantly with the dephosphorylation of the CREB-specific coactivator transducer of regulated CREB activity 1 (TORC1), resulting in the activation of CREB and its downstream gene targets. Ca(2+)/calmodulin-dependent protein kinase I/IV are capable of phosphorylating SIK2 at Thr484, resulting in SIK2 degradation in cortical neurons. Neuronal survival after OGD was significantly increased in neurons isolated from sik2(-/-) mice, and ischemic neuronal injury was significantly reduced in the brains of sik2(-)(/-) mice subjected to transient focal ischemia. These findings suggest that SIK2 plays critical roles in neuronal survival, is modulated by CaMK I/IV, and regulates CREB via TORC1.
[Show abstract][Hide abstract] ABSTRACT: The CREB-specific coactivator TORC2 (also known as CRTC2) upregulates gluconeogenic gene expression in the liver. Salt-inducible kinase (SIK) family enzymes inactivate TORC2 through phosphorylation and localize it in the cytoplasm. Ser(171) and Ser(275) were found to be phosphorylated in pancreatic beta-cells. Calcineurin (Cn) is proposed as the Ser(275) phosphatase, because its inhibitor cyclosporin A (CsA) stabilizes phospho-Ser(275) and retains TORC2 in the cytoplasm. Because the regulation of dephosphorylation at Ser(171) has not been fully clarified, we performed experiments with a range of doses of okadaic acid (OA), an inhibitor of PP2A/PP1, and with overexpression of various phosphatases and found that PP1 functions as an activator for TORC2, whereas PP2A acts as an inhibitor. In further studies using TORC2 mutants, we detected a disassociation between the intracellular distribution and the transcription activity of TORC2. Additional mutant analyses suggested the presence of a third phosphorylation site, Ser(307). The Ser(307)-disrupted TORC2 was constitutively localized in the nucleus, but its coactivator activity was normally suppressed by SIK1 in COS-7 cells. CsA, but not OA, stabilized the phosphogroup at Ser(307), suggesting that differential dephosphorylation at Ser(171) and Ser(307) cooperatively regulate TORC2 activity and that the nuclear localization of TORC2 is insufficient to function as a coactivator. Because the COS-7 cell line may not possess signaling cascades for gluconeogenic programs, we next examined the importance of Ser(307) and Ser(171) for TORC2's function in mouse liver. Levels of phosphorylation at Ser(171) and Ser(307) changed in response to fasting or fed conditions and insulin resistance of the mouse liver, which were modified by treatment with CsA/OA and by overexpression of PP1/PP2A/Cn. These results suggest that multiple phosphorylation sites and their phosphatases may play important roles in regulating TORC2/CREB-mediated gluconeogenic programs in the liver.
[Show abstract][Hide abstract] ABSTRACT: Salt-inducible kinase 2 (SIK2) is expressed abundantly in adipose tissues and represses cAMP-response element-binding protein (CREB)-mediated gene expression by phosphorylating the coactivator transducer of regulated CREB activity (TORC2). Phosphorylation at Ser(587) of SIK2 diminishes its TORC2 phosphorylation activity. In 3T3-L1 white adipocytes, SIK2 downregulates lipogenic gene in response to nutritional stresses. To investigate the impact of SIK2 on the function of brown adipose tissue (BAT), we used T37i brown adipocytes, mice with diet-induced obesity, and SIK2 mutant (S587A) transgenic mice. When T37i adipocytes were treated with insulin, the levels of peroxisome proliferator-activated receptor-coactivator-1alpha (PGC-1alpha) and uncoupling protein-1 (UCP-1) mRNA were increased, and the induction was inhibited by overexpression of SIK2 (S587A) mutant or dominant-negative CREB. Insulin enhanced SIK2 phosphorylation at Ser(587), which was accompanied by decrease in phospho-TORC2. Similarly, the decrease in the level of SIK2 phosphorylation at Ser(587) was observed in the BAT of mice with diet-induced obesity, which was negatively correlated with TORC2 phosphorylation. To confirm the negative correlation between SIK2 phosphorylation at Ser(587) and TORC2 phosphorylation in BAT, SIK2 mutant (S587A) was overexpressed in adipose tissues by using the adipocyte fatty acid-binding protein 2 promoter. The expression of recombinant SIK2 (S587A) was restricted to BAT, and the levels of phospho-TORC2 were elevated in BAT of transgenic mice. Male transgenic mice developed high-fat diet-induced obesity, and their BAT expressed low levels of PGC-1alpha and UCP-1 mRNA, suggesting that SIK2-TORC2 cascade may be important for the regulation of PGC-1alpha and UCP-1 gene expression in insulin signaling in BAT.