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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.

Activation of CRH transcription requires phosphorylation of cAMP response element-binding protein (CREB) and translocation of the CREB coactivator, transducer of regulated CREB activity (TORC) from cytoplasm to nucleus. In basal conditions, transcription is low because TORC remains in the cytoplasm, inactivated by phosphorylation through Ser/Thr protein kinases of the AMP-dependent protein kinases (AMPK) family, including salt-inducible kinase (SIK). To determine which kinase is responsible for TORC phosphorylation in CRH neurons, we measured SIK1 and SIK2 mRNA in the hypothalamic paraventricular nucleus of rats by in situ hybridization. In basal conditions, low mRNA levels of the two kinases were found in the dorsomedial paraventricular nucleus, consistent with location in CRH neurons. One hour of restraint stress increased SIK1 mRNA levels, whereas SIK2 mRNA showed only minor increases. In 4B hypothalamic neurons, or primary cultures, SIK1 mRNA (but not SIK2 mRNA) was inducible by the cAMP stimulator, forskolin. Overexpression of either SIK1 or SIK2 in 4B cells reduced nuclear TORC2 levels (Western blot) and inhibited forskolin-stimulated CRH transcription (luciferase assay). Conversely, the nonselective SIK inhibitor, staurosporine, increased nuclear TORC2 content and stimulated CRH transcription in 4Bcells and primary neuronal cultures (heteronuclear RNA). Unexpectedly, in 4B cells specific short hairpin RNA knockdown of endogenous SIK2 but not SIK1 induced nuclear translocation of TORC2 and CRH transcription, suggesting that SIK2 mediates TORC inactivation in basal conditions, whereas induction of SIK1 limits transcriptional activation. The study provides evidence that SIK represses CRH transcription by inactivating TORC, providing a potential mechanism for rapid on/off control of CRH transcription.

Distinct forms of MEF2 transcription factor act as positive or negative regulators of dendritic spine formation, with MEF2C playing a key regulator role in synapse plasticity. We report here that acute cocaine treatment of rats induced the expression of MEF2C in the striatum through a recently discovered transduction pathway. Repeated injections were found to induce MEF2C to a lesser extent. The mechanism by which MEF2C was induced involves the subsequent activation of the salt-inducible kinase SIK1 and the phosphorylation of HDAC5, a member of the class IIa of HDACs. Cocaine activated SIK1 by phosphorylation on Thr-182 residue, which was accompanied by the nuclear import of the kinase. In the nuclear compartment, SIK1 then phosphorylated HDAC5 causing the shuttling of its phospho-form from the nucleus to the cytoplasm of striatal cells. Activation of SIK1 by cocaine was further validated by the phosphorylation of TORC1/3, which was followed by the shuttling of TORC proteins from the nucleus to the cytoplasm. Activation of MEF2C was assessed by measuring the expression of the MEF2C gene itself, since the gene is known to be under the control of its own product. Since MEF2C plays a key role in memory/learning processes, activation of this pathway by cocaine is probably involved in plasticity mechanisms whereby the drug establishes its long-term effects such as drug dependence., (Copyright © 2011 Wiley Periodicals, Inc.)

Hepatic peroxisomes are essential for lipid conversions that include the formation of mature conjugated bile acids, the degradation of branched chain fatty acids, and the synthesis of docosahexaenoic acid. Through unresolved mechanisms, deletion of functional peroxisomes from mouse hepatocytes (L-Pex5(-/-) mice) causes severe structural and functional abnormalities at the inner mitochondrial membrane. We now demonstrate that the peroxisomal and mitochondrial anomalies trigger energy deficits, as shown by increased AMP/ATP and decreased NAD(+)/NADH ratios. This causes suppression of gluconeogenesis and glycogen synthesis and up-regulation of glycolysis. As a consequence, L-Pex5(-/-) mice combust more carbohydrates resulting in lower body weights despite increased food intake. The perturbation of carbohydrate metabolism does not require a long term adaptation to the absence of functional peroxisomes as similar metabolic changes were also rapidly induced by acute elimination of Pex5 via adenoviral administration of Cre. Despite its marked activation, peroxisome proliferator-activated receptor α (PPARα) was not causally involved in these metabolic perturbations, because all abnormalities still manifested when peroxisomes were eliminated in a peroxisome proliferator-activated receptor α null background. Instead, AMP-activated kinase activation was responsible for the down-regulation of glycogen synthesis and induction of glycolysis. Remarkably, PGC-1α was suppressed despite AMP-activated kinase activation, a paradigm not previously reported, and they jointly contributed to impaired gluconeogenesis. In conclusion, lack of functional peroxisomes from hepatocytes results in marked disturbances of carbohydrate homeostasis, which are consistent with adaptations to an energy deficit. Because this is primarily due to impaired mitochondrial ATP production, these L-Pex5-deficient livers can also be considered as a model for secondary mitochondrial hepatopathies.

Salt-inducible kinase 1 (SIK1) in epithelial cells mediates the increases in active sodium transport (Na(+), K(+)-ATPase-mediated) in response to elevations in the intracellular concentration of sodium. In lung alveolar epithelial cells increases in active sodium transport in response to β-adrenergic stimulation increases pulmonary edema clearance. Therefore, we sought to determine whether SIK1 is present in lung epithelial cells and to examine whether isoproterenol-dependent stimulation of Na(+), K(+)-ATPase is mediated via SIK1 activity. All three SIK isoforms were present in airway epithelial cells, and in alveolar epithelial cells type 1 and type 2 from rat and mouse lungs, as well as from human and mouse cell lines representative of lung alveolar epithelium. In mouse lung epithelial cells, SIK1 associated with the Na(+), K(+)-ATPase α-subunit, and isoproterenol increased SIK1 activity. Isoproterenol increased Na(+), K(+)-ATPase activity and the incorporation of Na(+), K(+)-ATPase molecules at the plasma membrane. Furthermore, those effects were abolished in cells depleted of SIK1 using shRNA, or in cells overexpressing a SIK1 kinase-deficient mutant. These results provide evidence that SIK1 is present in lung epithelial cells and that its function is relevant for the action of isoproterenol during regulation of active sodium transport. As such, SIK1 may constitute an important target for drug discovery aimed at improving the clearance of pulmonary edema., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The steroidogenic acute regulatory (StAR) protein is generated in rodents from 1.6 kb and 3.5 kb mRNA formed by alternative polyadenylation. The zinc finger protein, TIS11B (also Znf36L1), is elevated by cAMP in adrenal cells in parallel with StAR mRNA. TIS11b selectively destabilizes the 3.5 kb mRNA through AU-rich sequences at the end of the 3'UTR. siRNA suppression shows that TIS11b surprisingly increases StAR protein and cholesterol metabolism. StAR transcription is directly activated by PKA phosphorylation. cAMP responsive element binding (CREB) protein 1 phosphorylation is a key step leading to recruitment of the co-activator, CREB binding protein (CBP). A second protein, CREB regulated transcription coactivator (TORC/CRTC), enhances this recruitment, but is inhibited by salt inducible kinase (SIK). Basal StAR transcription is constrained through this phosphorylation of TORC. PKA provides an alternative stimulation by phosphorylating SIK, which prevents TORC inactivation. PKA stimulation of StAR nuclear transcripts substantially precedes TORC recruitment to the StAR promoter, which may, therefore, mediate a later step in mRNA production. Inhibition of SIK by staurosporine elevates StAR transcription and TORC recruitment to maximum levels, but without CREB phosphorylation. TORC suppression by SIK evidently limits basal StAR transcription. Staurosporine and cAMP stimulate synergistically. SIK targets the phosphatase, PP2a (activation), and Type 2 histone de-acetylases (inhibition), which may each contribute to suppression. Staurosporine stimulation through SIK inhibition is repeated in cAMP stimulation of many steroidogenic genes regulated by steroidogenic factor 1 (SF-1) and CREB. TIS11b and SIK may combine to attenuate StAR expression when hormonal stimuli decline., (Copyright © 2011. Published by Elsevier Ireland Ltd.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

cAMP response element-binding protein (CREB) promotes melanogenesis by inducing microphthalmia-associated transcription factor (Mitf ) gene expression. We report here that the CREB-specific coactivator TORC and its repressor, salt-inducible kinase 2 (SIK2), are fundamental determinants of the melanogenic program in mice. Exposure of B16 melanoma cells to ultraviolet (UV) light results in the immediate nuclear translocation of TORC1, which is inhibited by SIK2. Overexpression of dominant-negative TORC1 also inhibits UV-induced Mitf gene expression and melanogenesis. α-MSH signaling regulates hair pigmentation, and the decrease in α-MSH activity in hair follicle melanocytes switches the melanin synthesis from eumelanin (black) to pheomelanin (yellow). Mice with the lethal yellow allele of agouti (A(y)) have yellow hair because of impaired activation of the α-MSH receptor. To examine the involvement of SIK2 in the regulation of the melanogenesis switch in vivo, we prepared SIK2-knockout mice, and the Sik2(-/-) genotype was introduced into A(y)/a mice. The resultant Sik2(-/-); A(y)/a mice had brown hair, indicating that SIK2 represses eumelanogenesis in mice.

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.

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.

Salt inducible kinase (SIK) 1, a member of the AMP-activated kinase (AMPK) family, is activated by the AMPK-activator LKB1 which phosphorylates SIK1 at Thr182. The activated SIK1 then auto-phosphorylates its Ser186 located at the +4 position of Thr182. The phospho-Ser186 is essential for sustained activity of SIK1, which is maintained by sequential phosphorylation at Ser186-Thr182 by glycogen synthase kinase (GSK)-3beta. Meanwhile, SIK1 represses the transcription factor cAMP-response element binding protein (CREB) by phosphorylating its co-activator transducer of regulated CREB activity (TORC). Recently, histone deacetylase (HDAC) 5 was identified as a new substrate of SIK1. Inhibition of SIK1 or AMPK results in the stimulation of glyconeogensis in the liver by enhancing dephosphorylation of TORC2 followed by up-regulation of peroxisome proliferator-activated receptor coactivator (PGC)-1alpha gene expression. However, expression of the PGC-1alpha gene has been found to be repressed in LKB1-defective muscle cells. Our findings show that the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR)-dependent expression of PGC-1alpha is diminished by inhibitors of GSK-3beta or SIKs in C2C12 myoblasts. Treatment with AICAR or the overexpression of SIK1 induces nuclear export of HDAC5 followed by the activation of myogenic transcription factor (MEF)-2C. The levels of phosphorylation at Thr182 and Ser186 of SIK1 in AICAR-treated C2C12 cells are elevated, and GSK-3beta enzyme purified from AICAR-treated cells shows enhanced phosphorylation activity of SIK1 in vitro. These observations suggest that GSK-3 beta and SIK1 may play important roles in the regulation of PGC-1alpha gene expression by inactivating HDAC5 followed by activation of MEF2C.

An analysis of secreted proteins by the signal sequence trap method using a cDNA library of the rat pituitary anlage at embryonic days (E) 13.5 revealed the abundant expression of delta-like protein 1 (Dlk1) in the pituitary gland. Dlk1, an epidermal growth factor-like repeat protein in preadipocytes, functions in maintaining the preadipose state. Expression of Dlk1 mRNA in the pituitary at E13.5 and in the adult pituitary was confirmed by in situ hybridization. The expression pattern of Dlk1 during pituitary development was also studied by immunohistochemistry. Dlk1 protein first appeared in Rathke's pouch and the infundibulum at E11.5; as development proceeded, expression became restricted to the pars distalis and pars tuberalis (PT). Dlk1 was expressed in most ACTH cells during the embryonic stages, but its expression was limited to only a few ACTH cells in the adult pituitary. It was also expressed in a small population of TSH, GTH, and PRL cells throughout development, whereas it was present in the cytoplasm of most GH cells at all developmental stages. Similarly, Dlk1 was localized in the cytoplasm of PT cells during development. These findings provide new insights into the mechanism of Dlk1 regarding its regulation of pituitary hormone-secreting cells during development.

Autophosphorylation is an important mechanism by which protein kinases regulate their own biological activities. Salt inducible kinase 1 (SIK1) is a regulator in the feedback cascades of cAMP-mediated gene expression, while its kinase domain also features autophosphorylation activity. We provide evidence that Ser186 in the activation loop is the site of autophosphorylation and essential for the kinase activity. Ser186 is located at the +4 position of the critical Thr residue Thr182, which is phosphorylated by upstream kinases such as LKB1. The relationship between phosphorylation at Ser186 and at Thr182 in COS-7 cells indicates that the former is a prerequisite for the latter. Glycogen synthase kinase-3beta (GSK-3beta) phosphorylates Ser/Thr residues located at the fourth position ahead of the pre-phosphorylated Ser/Thr residues, and inhibitors of GSK-3beta reduce the phosphorylation at Thr182. The results of an in vitro reconstitution assay also indicate that GSK-3beta could be the SIK1 kinase. However, overexpression and knockdown of GSK-3beta in LKB1-defective HeLa cells suggests that GSK-3beta alone may not be able to phosphorylate or activate SIK1, indicating that LKB1 may play a crucial role by phosphorylating SIK1 at Thr182, possibly as an initiator of the autophosphorylation cascade, and GSK-3beta may phosphorylate SIK1 at Thr182 by recognizing the priming-autophosphorylation at Ser186 in cultured cells. This may also be the case for the other isoform SIK2, but not for SIK3.

Salt inducible kinase (SIK) was identified as a molecule induced in the adrenal glands of rats fed with a high-salt diet. A major downstream of SIK is regulation of camp-responsive element (CRE)-dependent gene expression. SIK represses the activity of CRE-binding protein (CREB) by phosphorylating a CREB-specific co-activator transducer of regulated CREB activity (TORC). When TORC is dephosphorylated it activates CREB in a CREB-phosphorylation independent manner. The importance of the dephosphorylation of TORC has been suggested by the fact that a kinase inhibitor staurosporine induces dephosphorylation of TORC and upregulates the gene expression of CYP11A, CYP11B1, CYP11B2 and StAR in adrenocortical cells. The identification of SIK caused a stir in the field of CREB studies and led to disclosure of cascades hidden behind the classical mechanism for CREB activity.

In mammalian cells, active sodium transport and its derived functions (e.g., plasma membrane potential) are dictated by the activity of the Na(+),K(+)-ATPase (NK), whose regulation is essential for maintaining cell volume and composition, as well as other vital cell functions. Here we report the existence of a salt-inducible kinase-1 (SIK1) that associates constitutively with the NK regulatory complex and is responsible for increases in its catalytic activity following small elevations in intracellular sodium concentrations. Increases in intracellular sodium are paralleled by elevations in intracellular calcium through the reversible Na(+)/Ca(2+) exchanger, leading to the activation of SIK1 (Thr-322 phosphorylation) by a calcium calmodulin-dependent kinase. Activation of SIK1 results in the dephosphorylation of the NK alpha-subunit and an increase in its catalytic activity. A protein phosphatase 2A/phosphatase methylesterase-1 (PME-1) complex, which constitutively associates with the NK alpha-subunit, is activated by SIK1 through phosphorylation of PME-1 and its dissociation from the complex. These observations illustrate the existence of a distinct intracellular signaling network, with SIK1 at its core, which is triggered by a monovalent cation (Na(+)) and links sodium permeability to its active transport.

An 81-year-old man was referred to our hospital with exertional dyspnea following cold-like symptoms. Electrocardiography revealed ST elevation and positive T wave in leads I, II, aVL, aVF, and V2-V6. The diagnosis was acute myocarditis complicating heart failure. He was conservatively managed. On hospital day 8, brain infarction developed and echocardiography disclosed massive mural thrombus in the left ventricle. Left ventriculotomy was performed on hospital day 21 and histological examination showed inflammatory cell infiltration mainly composed of eosinophils and monocytes, degeneration of myocytes with replacement fibrosis, and fresh fibrin thrombus overlaying the endocardium. These findings were compatible with a diagnosis of acute necrotizing eosinophilic myocarditis(ANEM). He recovered uneventfully without specific therapy. This case suggests that a subtype of ANEM might be self-limiting.

The transcription factor cAMP response element-binding protein (CREB) plays important roles in gene expression induced by cAMP signaling and is believed to be activated when its Ser133 is phosphorylated. However, the discovery of Ser133-independent activation by the activation of transducer of regulated CREB activity coactivators (TORC) and repression by salt inducible kinase cascades suggests that Ser133-independent regulation of CREB is also important. The activation and repression are mediated by the basic leucine zipper domain of CREB. In this review, we focus on the basic leucine zipper domain in the regulation of transcriptional activity of CREB and describe the functions of TORC and salt inducible kinase.

During physical exercise, increases in motor neuron activity stimulate the expression of muscle-specific genes through the myocyte enhancer factor 2 (MEF2) family of transcription factors. Elevations in intracellular calcium increase MEF2 activity via the phosphorylation-dependent inactivation of class II histone deacetylases (HDACs). In studies to determine the role of the cAMP responsive element binding protein (CREB) in skeletal muscle, we found that mice expressing a dominant-negative CREB transgene (M-ACREB mice) exhibited a dystrophic phenotype along with reduced MEF2 activity. Class II HDAC phosphorylation was decreased in M-ACREB myofibers due to a reduction in amounts of Snf1lk (encoding salt inducible kinase, SIK1), a CREB target gene that functions as a class II HDAC kinase. Inhibiting class II HDAC activity either by viral expression of Snf1lk or by the administration of a small molecule antagonist improved the dystrophic phenotype in M-ACREB mice, pointing to an important role for the SIK1-HDAC pathway in regulating muscle function.

Cyclic AMP responsive element (CRE) binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. However, transducer of regulated CREB activity (TORC), a CREB specific co-activator, upregulates CREB activity in a phospho-Ser133-independent manner. Interestingly, TORC is also regulated by phosphorylation; the phospho-form is inactive, and the dephospho-form active. When PKA phosphorylates CREB, it inhibits TORC kinases simultaneously and accelerates dephosphorylation of TORC. We show in this report that staurosporine, a kinase inhibitor, induces the expression of the StAR gene in Y1 adrenocortical cells, possibly a result of an increase in the population of dephospho-TORC. The expression of the StAR gene is known to be regulated by SF-1 and CREB, and the co-activators CBP/p300 may mediate the actions of both factors. Our experiments using KG501, a disruptor of the interaction between phospho-CREB and CBP/p300, also support the importance of TORC in the regulation of StAR gene expression.

Late-phase long-term potentiation (L-LTP) and long-term memory depend on the transcription of mRNA of CRE-driven genes and synthesis of proteins. However, how synaptic signals propagate to the nucleus is unclear. Here we report that the CREB coactivator TORC1 (transducer of regulated CREB activity 1) undergoes neuronal activity-induced translocation from the cytoplasm to the nucleus, a process required for CRE-dependent gene expression and L-LTP. Overexpressing a dominant-negative form of TORC1 or down-regulating TORC1 expression prevented activity-dependent transcription of CREB target genes in cultured hippocampal neurons, while overexpressing a wild-type form of TORC1 facilitated basal and activity-induced transcription of CREB target genes. Furthermore, overexpressing the dominant-negative form of TORC1 suppressed the maintenance of L-LTP without affecting early-phase LTP, while overexpressing the wild-type form of TORC1 facilitated the induction of L-LTP in hippocampal slices. Our results indicate that TORC1 is essential for CRE-driven gene expression and maintenance of long-term synaptic potentiation.

Cyclic AMP responsive element (CRE)-binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. Two independent investigations have suggested the presence of Ser133-independent activation. One study identified a kinase, salt-inducible kinase (SIK), which repressed CREB; the other isolated a novel CREB-specific coactivator, transducer of regulated CREB activity (TORC), which upregulated CREB activity. These two opposing signals are connected by the fact that SIK phosphorylates TORC and induces its nuclear export. Because LKB1 has been reported to be an upstream kinase of SIK, we used LKB1-defective HeLa cells to further elucidate TORC-dependent CREB activation. In the absence of LKB1, SIK was unable to phosphorylate TORC, which led to constitutive activation of CRE activity. Overexpression of LKB1 in HeLa cells improved the CRE-dependent transcription in a regulated manner. The inactivation of kinase cascades by 10 nm staurosporine in LKB1-positive HEK293 cells also induced unregulated, constitutively activated, CRE activity. Treatment with staurosporine completely inhibited SIK kinase activity without any significant effect on the phosphorylation level at the LKB1-phosphorylatable site in SIK or the activity of AMPK, another target of LKB1. Constitutive activation of CREB in LKB1-defective cells or in staurosporine-treated cells was not accompanied by CREB phosphorylation at Ser133. The results suggest that LKB1 and its downstream SIK play an important role in silencing CREB activity via the phosphorylation of TORC, and such silencing may be indispensable for the regulated activation of CREB.

11Beta-hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11beta-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolated from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.

The adrenal inner zone antigen (IZA), which reacts specifically with a monoclonal antibody raised against the fasciculata and reticularis zones of the rat adrenal, was previously found to be identical with a protein variously named 25-Dx and membrane-associated progesterone receptor. IZA was purified as a glutathione S-transferase-fused or His(6)-fused protein, and its molecular properties were studied. The UV-visible absorption and EPR spectra of the purified protein showed that IZA bound a heme chromophore in high-spin type. Analysis of the heme indicated that it is of the b type. Site-directed mutagenesis studies were performed to identify the amino-acid residues that bind the heme to the protein. The results suggest that two Tyr residues, Tyr107 and Tyr113, and a peptide stretch, D99-K102, were important for anchoring the heme into a hydrophobic pocket. The effect of IZA on the steroid 21-hydroxylation reaction was investigated in COS-7 cell expression systems. The results suggest that the coexistence of IZA with CYP21 enhances 21-hydroxylase activity.

Glucose homeostasis is regulated systemically by hormones such as insulin and glucagon, and at the cellular level by energy status. Glucagon enhances glucose output from the liver during fasting by stimulating the transcription of gluconeogenic genes via the cyclic AMP-inducible factor CREB (CRE binding protein). When cellular ATP levels are low, however, the energy-sensing kinase AMPK inhibits hepatic gluconeogenesis through an unknown mechanism. Here we show that hormonal and energy-sensing pathways converge on the coactivator TORC2 (transducer of regulated CREB activity 2) to modulate glucose output. Sequestered in the cytoplasm under feeding conditions, TORC2 is dephosphorylated and transported to the nucleus where it enhances CREB-dependent transcription in response to fasting stimuli. Conversely, signals that activate AMPK attenuate the gluconeogenic programme by promoting TORC2 phosphorylation and blocking its nuclear accumulation. Individuals with type 2 diabetes often exhibit fasting hyperglycaemia due to elevated gluconeogenesis; compounds that enhance TORC2 phosphorylation may offer therapeutic benefits in this setting.

The present investigation demonstrates establishment of United Nations Environment Programme (UNEP) priority Persistent Organic Pollutants (POPs) using high-resolution gas chromatography-high-resolution mass spectrometry. Particularly, POPs analytical methods were established using native and (13)C-labeled internal standards of HCHs, HCB, cyclodienes, chlordanes, DDTs, mirex, dioxin-like PCBs, PCDDs, and PCDFs by isotope dilution technique. The relative response factor for 6-point calibration curve native standards (18 replicate analysis) were in the ranges of 0.93-1.43 with relative standard deviation ranges from 1.68 to 4.96%. Instrument detection limit and instrument quantification limit was established for various POPs at femtograms. Concentrations of UNEP-POPs were measured in liver (n = 10) and egg (n = 10) of great cormorants and their major diet, gizzard shad (n = 2), collected in and around Tokyo, Japan. DDTs (ranges in liver and egg, respectively) were predominant accumulants (9800-310,000 and 9600-73,000) followed by dioxin-like PCBs (4500-69,000 and 7900-150,000), chlordanes (2600-16,000 and 700-4,800), cyclodienes (650-4600 and <1-1000), HCB (680-2800 and 180-590), HCHs (230-1800 and 120-490), PCDD/DFs (3.2-27 and 1.7-5.7) on nanogram per gram lipid basis. Concentrations (ranges) of POPs in gizzard shad were in the following order: DDTs (3900-16,000), chlordanes (3400-14,000), cyclodienes (340-1300), HCB (110-480), and HCHs (140-360) on nanogram per gram lipid basis.

Impact of FEBRA (fermented brown rice with Aspergillus oryzae, a rich dietary fiber) intake on the concentrations of polychlorinated-biphenyls (PCBs), -dioxin-like polychlorinated biphenyls (DLPCBs), -dibenzo-p-dioxins (PCDDs), and -dibenzofurans (PCDFs) in blood of nine married Japanese were studied for 2 years. Concentrations of total PCBs (average+/-standard deviation) were greater 326,000+/-263,000 and 207,000+/-89,000 in FEBRA-intake and non-FEBRA-intake groups, respectively, on pg/g lipid (from now onwards data expressed on pg/g lipid). DLPCBs were second major contaminants 94,000+/-77,000 and 60,000+/-28,000, orderly in FEBRA-intake and non-FEBRA-intake groups. The concentrations of PCDD/DFs were several orders of magnitude lower than those of PCBs, whereas PCDDs were greater (433+/-233 and 512+/-511, in FEBRA-intake and non-FEBRA-intake groups, respectively) than PCDFs (56+/-50 and 43+/-35). Calculated toxic equivalency (TEQ) concentrations were 34+/-24 and 23+/-10 in FEBRA-intake and non-FEBRA-intake groups, respectively. In order to corroborate the impact of FEBRA, the changes of PCDD/DFs and PCB concentrations for 0.5-year, 1-year, 1.5-year, and 2-year samples were estimated from average concentration of 1-day before study commences and 1-week study samples. The results explicit FEBRA-intake group eliminated 7%, 33%, 45%, 36%, and 26% and non-FEBRA-intake group eliminated 8%, 28%, 36%, 31%, and 20% of DLPCBs, PCDDs, PCDFs, PCDD/DF TEQ and total TEQ, respectively. This study is the first of its kind showing the possible elimination of PCDDs, PCDFs and their TEQ in humans from Japan after the consumption of FEBRA.

The isotope dilution technique was applied for the analysis of new polybrominated diphenylethers (PBDEs) calibration standard (both labeled and non-labeled) using high-resolution gas chromatography-high-resolution mass spectrometry (HRGC-HRMS). The relative response factor (RRF) and relative standard deviation (RSD) for new calibration standard in Finnigan Thermo Electron (MAT-95XL) and Micromass (Autospec Ultima) were more or less identical with mean RRF (0.9882), RSD (0.0865) and CV% of (8.75). The results also revealed for DeBDE-209 quantification; labeled DeBDE-209 is essential. Furthermore, we recommend on column injection technique with a thin film instead of splitless injection in order to reduce the thermal degradation of DeBDE-209 and formation of octabromodibenzofurans (OBDF). Besides, analysis of human blood (n = 156) of FEBRA-intake and non-FEBRA-intake individuals elucidated frequent detection of eighteen PBDE congeners. The average PBDE concentrations in non-FEBRA intake and FEBRA-intake humans were 6000-11,000 (mean: 8400) and 5400-15,000 (mean: 9900) respectively on pg/g fat basis. Although FEBRA-intake individuals showed slightly greater PBDEs, computer-normalized concentrations of TeBDE-47 corroborate FEBRA-intake individual from four family showed reduced concentrations. The contamination profiles of PBDEs varied in between family, gender as well as geography. International comparison with predominant PBDE congener (TeBDE-47) prevailed lower levels in Japan when compared to Korea, Germany and USA nevertheless, congener specific profiles were different which is in accordance with different technical PBDE usage in between countries.

Salt-inducible kinase-1 (SIK1) is phosphorylated at Ser577 by protein kinase A in adrenocorticotropic hormone-stimulated Y1 cells, and the phospho-SIK1 translocates from the nucleus to the cytoplasm. The phospho-SIK1 is dephosphorylated in the cytoplasm and re-enters the nucleus several hours later. By using green-fluorescent protein-tagged SIK1 fragments, we found that a peptide region (586-612) was responsible for the nuclear localization of SIK1. The region was named the 'RK-rich region' because of its Arg- and Lys-rich nature. SIK1s mutated in the RK-rich region were localized mainly in the cytoplasm. Because SIK1 represses cAMP-response element (CRE)-mediated transcription of steroidogenic genes, the mutants were examined for their effect on transcription. To our surprise, the cytoplasmic mutants strongly repressed the CRE-binding protein (CREB) activity, the extent of repression being similar to that of SIK1(S577A), a mutant localized exclusively in the nucleus. Several chimeras were constructed from SIK1 and from its isoform SIK2, which was localized mainly in the cytoplasm, and they were examined for intracellular localization as well as CREB-repression activity. A SIK1-derived chimera, where the RK-rich region had been replaced with the corresponding region of SIK2, was found in the cytoplasm, its CREB-modulating activity being similar to that of wild-type SIK1. On the other hand, a SIK2-derived chimera with the RK-rich region of SIK1 was localized in both the nucleus and the cytoplasm, and had a CREB-repressing activity similar to that of the wild-type SIK2. Green fluorescent protein-fused transducer of regulated CREB activity 2 (TORC2), a CREB-specific co-activator, was localized in the cytoplasm and nucleus of Y1 cells, and, after treatment with adrenocorticotropic hormone, cytoplasmic TORC2 entered the nucleus, activating CREB. The SIK1 mutants, having a strong CRE-repressing activity, completely inhibited the adrenocorticotropic hormone-induced nuclear entry of green fluorescent protein-fused TORC2. This suggests that SIK1 may regulate the intracellular movement of TORC2, and as a result modulates the CREB-dependent transcription activity. Together, these results indicate that the RK-rich region of SIK1 is important for determining the nuclear localization and attenuating CREB-repressing activity, but the degree of the nuclear localization of SIK1 itself does not necessarily reflect the degree of SIK1-mediated CREB repression.

Elevations in circulating glucose and gut hormones during feeding promote pancreatic islet cell viability in part via the calcium- and cAMP-dependent activation of the transcription factor CREB. Here, we describe a signaling module that mediates the synergistic effects of these pathways on cellular gene expression by stimulating the dephosphorylation and nuclear entry of TORC2, a CREB coactivator. This module consists of the calcium-regulated phosphatase calcineurin and the Ser/Thr kinase SIK2, both of which associate with TORC2. Under resting conditions, TORC2 is sequestered in the cytoplasm via a phosphorylation-dependent interaction with 14-3-3 proteins. Triggering of the calcium and cAMP second messenger pathways by glucose and gut hormones disrupts TORC2:14-3-3 complexes via complementary effects on TORC2 dephosphorylation; calcium influx increases calcineurin activity, whereas cAMP inhibits SIK2 kinase activity. Our results illustrate how a phosphatase/kinase module connects two signaling pathways in response to nutrient and hormonal cues.

The cloning of salt-inducible kinase-1 (SIK1) that was specifically expressed in the adrenal glands of high-salt diet-fed rats led to subsequent cloning of adipose-specific SIK2 and rather ubiquitous SIK3. The three enzymes constitute a novel serine/threonine kinase subfamily, a member of AMP-activated protein kinase (PKA) family. Physiological roles of SIK1 and SIK2 have been investigated. The SIK1 transcript was expressed very early in the ACTH-stimulated Y1 cells, even before the expression of transcripts for CYP11A and StAR protein. Forced expression of SIK1 inhibited the ACTH-dependent expression of CYP11A- and StAR protein-genes. Cotransfection assays employing CRE-reporter gene showed that SIK1 could repress the PKA-dependent activation of CRE by acting on the bZIP domain of the CRE-binding protein (CREB), though the target site of SIK1-mediated phosphorylation has yet to be determined. ACTH/PKA-dependent nucleocytoplasmic shuttling of SIK1 took place in Y1 cells, implying that the intracellular movement of SIK1 might be a physiologically important determining factor for regulation of steroidogenic gene expression in the early phase of ACTH-stimulation. The SIK2 gene was expressed in 3T3-L1 cells at a very early stage of adipogenesis. SIK2 could phosphorylate Ser-794 of human insulin-receptor-substrate-1 (IRS-1) in vitro as well as in vivo. In addition, the SIK2 activity in db/db mice adipose tissues was significantly higher than that in wild-type adipose. These results strongly suggest that SIK2 may play important role(s) in modulating the insulin-signaling cascade of adipocytes, and thus, may be involved in the development of insulin resistance. Taken together, these results suggest that the SIK isoforms regulate hormonal signal transduction in both adrenal and adipose tissues.

Inner zone antigen (IZA) is a protein specifically expressed in the zona fasciculata and reticularis of the adrenal cortex. The cDNA encoding IZA was found to be identical to that encoding the previously reported putative membrane-associated progesterone receptor (MPR) and the TCDD-induced 25kDa protein (25-Dx). From its structure, MPR was classed as a member of a protein family containing a haem-binding domain, and progesterone was proposed to be a ligand of this domain. Indeed, when GST-tagged IZA was expressed in Escherichia coli and purified, the purified GST-IZA had a brown colour with maximum absorbance at 400 nm. The addition of dithionate shifted the absorbance peak to 420 nm, suggesting a haem-binding function. The possible role of IZA in steroidogenesis has been addressed, and the inhibition of adrenal steroidogenesis by the addition of an anti-IZA monoclonal antibody has been reported. When COS-7 cells were transformed with plasmids for appropriate steroidogenic enzymes in the presence or absence of an IZA expression plasmid and tested for their steroidogenic activities, 21-hydroxylation of progesterone was found to be specifically activated by IZA overexpression, suggesting the involvement of IZA in progesterone metabolism. Taken together, the available evidence suggests that IZA may have an important role in the functions of the adrenal zona fasciculata and reticularis.

The salt-inducible kinases (SIKs) are a family of related serine-threonine kinases. In cultured adrenocortical cells, SIK1 is rapidly but transiently induced by adrenocorticotropin (ACTH) treatment, suggesting that it contributes to ACTH-mediated induction of steroidogenic enzymes. However, ACTH treatment of Y1 mouse adrenocortical cells stimulates a rapid translocation of SIK1 from the nucleus to the cytoplasm, and SIK1 represses the transcription of a steroidogenic enzyme by inhibiting the action of cAMP-responsive elements in the promoter. These studies suggest that SIK1 has a role in the fine tuning of steroidogenic enzyme production during the initial phase of steroidogenesis. SIK2 is found in adipocytes and phosphorylates a specific serine residue in insulin receptor substrate-1. This finding, along with the fact that its expression is raised in the white adipose tissue of mice with type 2 diabetes mellitus, suggests that SIK2 might be involved in metabolic regulation in adipose tissue. Thus, members of the SIK family are emerging as important modulators of key processes such as steroid hormone biosynthesis by the adrenal cortex and insulin signaling in adipocytes.

A 66 year-old male patient with rheumatoid arthritis, was admitted due to bronchial pneumonia. Anti-nuclear antibody and anti-DNA antibody were both detected in the patient's serum and many LE-like cells were found in the pleural effusion. In addition, M-proteins(IgG-kappa type, IgM-kappa type) were detected in both serum and pleural effusion by immunofixation electrophoresis. Purified IgG fraction by gel filtration(Superdex 200HR) showed high titer of anti-nuclear antibody(x160) and anti-DNA(39.2 AU/ml) antibody. Furthermore, anti-DNA antibody was detected in the fraction III(fraction of M-protein) by ion-exchange chromatography. In conclusion, these results indicate that M-protein(IgG3-kappa type) contains anti-DNA antibody and it is suggests that M-protein induces the production of LE like cell in this case.

Salt-inducible kinase (SIK), expressed in Y1 mouse adrenocortical tumor cells at an early stage of adrenocorticotropic hormone (ACTH)-stimulation, represses the cAMP-responsive element (CRE)-dependent gene expression of CYP11A and StAR by acting on bZIP domain of CRE-binding protein. ACTH induced the SIK's nuclear to cytosolic translocation in a PKA-dependent manner. A mutant SIK in which the PKA-dependently phosphorylatable Ser577 had been replaced with Ala could not move out of the nucleus. The degree of CRE-reporter repression by SIK was strong as long as SIK was present in the nucleus. These indicated that intracellular translocation of SIK might be an important factor to determine the time-dependent change in the level of steroidogenic gene expression in ACTH-stimulated cells. Promoter analyses suggested that SIK repressed gene expressions not only of CYP11A and StAR but also of CYP11B1, CYP11B2 and SIK itself. We propose here that SIK is one of important molecule regulating expression of steroidogenic genes in the early phase of ACTH treatment.

Salt-inducible kinase (SIK), first cloned from the adrenal glands of rats fed a high salt diet, is a serine/threonine protein kinase belonging to an AMP-activated protein kinase family. Induced in Y1 cells at an early stage of ACTH stimulation, it regulated the initial steps of steroidogenesis. Here we report the identification of its isoform SIK2. When a green fluorescent protein-fused SIK2 was expressed in 3T3-L1 preadipocytes, it was mostly present in the cytoplasm. When coexpressed in cAMP-responsive element-reporter assay systems, SIK2 could repress the cAMP-responsive element-dependent transcription, although the degree of repression seemed weaker than that by SIK1. SIK2 was specifically expressed in adipose tissues. When 3T3-L1 cells were treated with the adipose differentiation mixture, SIK2 mRNA was induced within 1 h, the time of induction almost coinciding with that of c/EBPbeta mRNA. Coexpressed with human insulin receptor substrate-1 (IRS-1) in COS cells, SIK2 could phosphorylate Ser(794) of human IRS-1. Adenovirus-mediated overexpression of SIK2 in adipocytes elevated the level of phosphorylation at Ser(789), the mouse equivalent of human Ser(794). Moreover, the activity and content of SIK2 were elevated in white adipose tissues of db/db diabetic mice. These results suggest that highly expressed SIK2 in insulin-stimulated adipocytes phosphorylates Ser(794) of IRS-1 and, as a result, might modulate the efficiency of insulin signal transduction, eventually causing the insulin resistance in diabetic animals.

Salt-inducible kinase (SIK), a 776 amino acids-protein, contains a kinase domain in the NH2-terminal 278 amino acid residues, and the biological functions of its COOH-terminal half have yet to be clarified. Here we describe the roles played by several domains in the SIK molecule. K56, an amino acid residue found in a region similar to the ATP-binding loop of other protein kinases, was essential for carrying out the SIKs phosphorylation reaction. An SNF-1 homology domain (SNH), identified at a peptide stretch from the 317th to the 346th residues, and conserved among all the sucrose-nonfermenting-1 protein kinase (SNF-1) family protein kinases, was important to maintain the SIKs protein conformation in the cells. S577, an amino acid residue found in one of three consensus PKA-dependent phosphorylation motifs, was indeed phosphorylated by PKA. The phosphorylated SIK was found to move out of the nucleus to the cytoplasm.

Salt-inducible kinase (SIK), a novel serine/threonine protein kinase from adrenal glands of rats fed with a high-salt diet, is induced by ACTH in Y1 mouse adrenocortical tumor cells. Overexpression of SIK repressed ACTH-mediated expression of CYP11A- and Steroidogenic acute regulatory (StAR)-genes by inhibiting CREB bound to their promoters. Immunocytochemical and GFP-fluorocytochemical analyses indicated that SIK was present both in the nucleus and cytosol of resting cells. Responding to ACTH, the nuclear SIK moved to the cytosol. The level of phosphorylation at Ser577, a canonical PKA-phosphorylation site, was elevated by ACTH treatment. The disruption of the serine residue inhibited the nuclear export and enhanced the transcription repression activity of SIK. Various deletion mutants suggested a functional nuclear localization signal was present near Ser577. We conclude that the nucleocytoplasmic shuttling of SIK may play an important role in the transcriptional regulation of the cAMP-responsive element (CRE)-dependent gene expression.

Salt-inducible kinase (SIK), a serine/threonine protein kinase expressed at an early stage of adrenocorticotropic hormone (ACTH) stimulation in Y1 mouse adrenocortical tumor cells, repressed the cAMP-responsive element (CRE)-dependent gene transcription by acting on the basic leucine zipper domain of the CRE-binding protein (Doi, J., Takemori, H., Lin, X.-z., Horike, N., Katoh, Y., and Okamoto, M. (2002) J. Biol. Chem. 277, 15629-15637). The mechanism of SIK-mediated gene regulation has been further explored. Here we show that SIK changes its subcellular location after the addition of ACTH. The immunocytochemical and fluorocytochemical analyses showed that SIK was present both in the nuclear and cytoplasmic compartments of resting cells; when the cells were stimulated with ACTH the nuclear SIK moved into the cytoplasm within 15 min; the level of SIK in the nuclear compartment gradually returned to the initial level after 12 h. SIK translocation was blocked by pretreatment with leptomycin B. A mutant SIK whose Ser-577, the cAMP-dependent protein kinase (PKA)-dependent phosphorylation site, was replaced with Ala could not move out of the nucleus under stimulation by ACTH. As expected, the degree of repression exerted by SIK on CRE reporter activity was weak as long as SIK was present in the cytoplasmic compartment. The same was true for the SIK-mediated repression of a steroidogenic acute regulatory (StAR) protein-gene promoter, which contained a CRE-like sequence at -95 to -85 bp. These results suggest that in the ACTH-stimulated Y1 cells the nuclear SIK was PKA-dependently phosphorylated, and the phosphorylated SIK was then translocated out of the nuclei. This intracellular translocation of SIK, a CRE-repressor, may account for the time-dependent change in the level of ACTH-activated expression of the StAR protein gene.

Salt-inducible kinase (SIK), one of the serine/threonine protein kinases, was transiently expressed in Y1 cells during the early phase of the ACTH/cAMP-dependent protein kinase (PKA)-mediated signal transduction. The overexpression of SIK(N), the SIK's N-terminal kinase domain, repressed the expression of the side chain cleavage cytochrome P450 (CYP11A) gene. To elucidate the mechanism of the repression by SIK, several CYP11A promoter constructs were tested for the promoter activities in the presence of PKA and/or SIK(N). A cAMP-response element (CRE)-like sequence present in the promoter was shown to be responsible not only for the PKA-mediated promoter activation but also for the SIK(N)-mediated repression. When the Gal4 DNA binding domain-linked full-length CRE-binding protein (CREB) construct was cotransfected with Gal4 reporter gene, SIK(N) repressed the PKA-induced reporter gene expression. However, SIK(N) could not repress the PKA-induced reporter activity conferred by Gal4 DNA binding domain-linked basic leucine zipper (bZIP)-less CREB or bZIP-disrupted CREB. On the other hand, SIK(N) could repress the kinase-inducible domain-disrupted CREB-dependent reporter gene expression in the presence of PKA. The in vitro kinase reaction studies showed that SIK(N) could not phosphorylate CREB, and PKA failed to phosphorylate SIK(N). Taken together, these results suggest that SIK(N), cooperating with PKA, may act on the CREB's bZIP domain and repress the CREB-mediated transcriptional activation of the CYP11A gene.

The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase's inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.

Intestinal brush border membrane-associated phospholipase B/lipase (PLB/LIP) consists of four tandem homologous domains (repeats 1 through 4) and a COOH-terminal membrane binding domain, and repeat 2 is the catalytic domain that catalyzes phospholipase A2, lysophospholipase, and lipase activities. We examined the structural basis of the catalysis of PLB/LIP with this unique substrate specificity by site-directed mutagenesis of recombinant repeat 2 enzyme. Ser414 and Ser459 within the active serine-containing consensus sequence G-X-S-X-G in the best-established lipase family were dispensable for activity. In contrast, substitution of Ala for Ser404 almost completely inactivated the three lipolytic activities of PLB/LIP, even though the gross conformation was not altered as determined by CD spectroscopy. Notably, this Ser is located within the conserved G-D-S-L sequence on the NH2-terminal side in lipolytic enzymes of another group proposed recently. Furthermore, mutagenesis and CD spectroscopic analyses suggested that Asp518 and His659, lying within conserved short stretches in the latter group of lipolytic enzymes, were essential for activity. These three essential residues are conserved in the known PLB/LIP enzymes, suggesting that they form the catalytic triad in the active site. These results indicate that PLB/LIP represents a distinct class of the lipase family. PLB/LIP is the first mammalian member of that family. Repeat 2 is equipped with the triad, but not the other repeats, accounting for why only repeat 2 is the catalytic domain. Replacing Thr406 with Gly, matching the enzyme's sequence to the lipase consensus sequence exactly, led to a great decrease in secretion and accumulation of inactive enzyme in the cells, suggesting a role of Thr406 in the structural stability.

We previously reported that cells other than mast cells or neurons could synthesize histamine in response to lipopolysaccharide (LPS) or interleukin 1beta in the rat brain. To identify the responsible cells, we examined histidine decarboxylase (HDC) activity and the expression of HDC mRNA in GMI 6-3 mouse microglial cells. Both the activity and mRNA for HDC in GMI 6-3 cells were induced by LPS treatment, and the induction was sensitive to calmodulin-dependent kinase II inhibitor, KN62. These findings indicate that microglia is a third cell type producing histamine in the brain.

Using immunological methods, a protein specific to the inner zones of the rat adrenal cortex, and called inner zone antigen (IZAg), was previously shown to have two interrelated forms of 26 kDa (IZAg1) and 55-60 kDa (IZAg2), and to have an action on steroid hydroxylation. After two-dimensional gel electrophoresis, and immunoaffinity column purification, N-terminal amino-acid analysis showed that the first 12 amino acids were identical to those of a recently described putative membrane located progesterone receptor (PPMR). RT-PCR was then used to generate the cDNA of this protein, using RNA extracted from rat adrenals. A glutathione S-transferase (GST)-fusion construct was expressed in Escherichia coli, and shown to generate an immunoreactive product of molecular mass consistent with its identification as IZAg1. More detailed examination of the distribution of this protein, not only in the zona fasciculata/reticularis of the adrenal cortex, but also in the Leydig cell, kidney and liver, suggest it may have a role in steroid hormone synthesis and/or metabolism.

Preadipocyte factor-1 (Pref-1) was shown to negatively regulate adipocyte differentiation. We recently reported that ZOG, a rat homolog of Pref-1, was specifically expressed in the adrenal zona glomerulosa. Results of the investigation of Pref-1 expression in preadipocyte and in undifferentiated adrenal cortex suggested that down-regulation of Pref-1 gene was closely correlated with the differentiation process. In this study we demonstrate that an upstream region (from -76 to -47) of the rat Pref-1 gene was essential for its expression in adrenocortical carcinoma-derived H295R cells. A nucleotide sequence found in this region, GCGTGGGCGTGGGCGGGGG (Egr/GC-box), seemed to contain three elements, two early growth response (Egr) elements and one GC-box, overlapping each other. Mutations of four or five nucleotides in a 7-nucleotides-stretch in the midst of the Egr/GC-box eliminated the binding of Sp1/3, abolished the activation by Egr-factor(s) and diminished the Pref-1 promoter activity. When mutations were introduced into the outside of the middle portion, the binding of Sp1/3 to the Egr/GC-box was abolished similarly. However, the decrease in the promoter activity was less than that found with the construct mutated at the middle. These results indicated that an element present at the 7-nucleotides-stretch in the midst of the Egr/GC-box might be important for the Pref-1 promoter activity, and this proximal element was possibly activated by a still-unidentified nuclear factor(s). This element would function as the promoter of the Pref-1 gene in H295R cells, but not in HeLa cells.

Using RT-PCR, a cDNA fragment of NADPH-cytochrome P450 oxidoreductase from silkworm, Bombyx mori, was cloned from three-day-old nondiapause eggs. RACE was used to isolate the ends of the DNA. The full-length cDNA obtained was composed of 3471 bp with an open reading frame encoding a protein of 687 amino-acid residues with a relative molecular mass of 77 700. The protein, fused with glutathione S-transferase, was expressed in Escherichia coli and purified to homogeneity. The fused protein not only had NADPH-dependent cytochrome c-reducing activity, but also acted as an electron carrier from NADPH to bovine adrenal 21-hydroxylase P450 in the steroid hydroxylation reaction, confirming that the protein is the silkworm NADPH-cytochrome P450 oxidoreductase. Ecdysone 20-hydroxylase activity in the nondiapause egg microsomes increased until the fourth day after oviposition, and then decreased, little being detected on the ninth day. An antibody raised against the P450 reductase inhibited the ecdysone hydroxylation. Immunoblot analyses of the microsomes indicated that the P450 reductase protein appeared distinctly in the three-day-old nondiapause eggs and, in contrast to the developmental pattern of ecdysone hydroxylase activity, continued to increase as the embryos developed. These results suggest that ecdysone hydroxylation in the early stage of embryogenesis is dependent on the presence of both P450 reductase and ecdysone 20-hydroxylase P450, but its gradual reduction in the later stage may be due to the decrease in the level of ecdysone 20-hydroxylase P450.

Possible involvement of salt-inducible kinase (SIK), a serine/threonine protein kinase first cloned from high K+-diet treated rat adrenal glands, in the regulation of steroidogenesis was investigated. Y-1 cells, when treated with ACTH, underwent a rapid change in SIK's mRNA content. It reached the maximum within a few hours and returned to the base after 8 h. In contrast, the levels of mRNAs for CYP11A and StAR protein reached the maxima after 8 h. The SIK's mRNA induction failed to occur in ACTH-, forskolin- or 8-Br-cAMP-treated Kin-7 cells, a mutant cell line of Y-1 with defective cAMP-dependent protein kinase A (PKA). Y-1 cells that overexpress SIK, when treated with ACTH, had significantly repressed levels of mRNAs for CYP11A and StAR protein. Therefore, SIK might have a negative effect on the CYP11A- and StAR protein-gene expression in the early phase of ACTH-mediated steroidogenesis. To further explore the mechanisms underlying this phenomenon, we examined intracellular distribution of the green fluorescence protein (GFP)-tagged SIK. When GFP-SIK was introduced into HeLa cells, the fluorescent signals were detected in the nucleus. In Y-1 cells GFP-SIK was detected both in the nucleus and cytosol, and the signal in the former moved to the latter after ACTH-treatment. The nuclear/cytosol re-distribution of GFP-SIK was also observed in forskolin- or 8-Br-cAMP-treated Y-1 cells, but not in Kin-7 cells. These results suggest that the intracellular re-distribution of SIK in Y-1 cells may depend on the cAMP/PKA signaling pathway and has an important regulatory role in the ACTH-mediated steroidogenic gene expression.