Decreased hepatic enzymes reflect the decreased vitamin B6 levels in Parkinson's disease patients.

The study aims to investigate the vitamin B6 levels in Parkinson's disease (PD) patients and their association with liver enzymes and evaluate how much dysregulation is associated with levodopa dose. Furthermore, to evaluate the effect of Opicapone, a catechol-o-methyl-transferase inhibitor, on vitamin B6 levels by monitoring the AST and ALT levels in patients treated with Levodopa-Carbidopa Intestinal Gel Infusion (LCIG). For these aims, serum vitamin B6 levels were measured (PD, n = 72 and controls, n = 31). The vitamin B6 level was compared with the total levodopa dose, clinical parameters, and blood homocysteine, albumin, and hemoglobin levels in PD patients. Correlations between vitamin B6 levels and AST and ALT levels, as well as the ratio ALT/AST, were analyzed. Changes in the AST and ALT levels and ALT/AST were analyzed in the patients treated with LCIG before and after the therapy (n = 24) and in the patients treated with LCIG + Opicapone before and after Opicapone treatment (n = 12). We found vitamin B6 levels were significantly lower in PD patients. Total levodopa dose and albumin levels were independently associated with vitamin B6 levels. Decreased vitamin B6 levels appeared as lower AST and ALT levels and ALT/AS. Treatment with LCIG decreased the AST and ALT levels and ALT/AST. Adjunctive therapy with Opicapone to LCIG ameliorated the decreased ALT and ALT/AST. We conclude that the ALT and ALT/AST can be useful parameters for monitoring vitamin B6 levels and Opicapone can ameliorate the dysregulated vitamin B6 in PD patients.

Lysophagy protects against propagation of α-synuclein aggregation through ruptured lysosomal vesicles.

The neuron-to-neuron propagation of misfolded α-synuclein (αSyn) aggregates is thought to be key to the pathogenesis of synucleinopathies. Recent studies have shown that extracellular αSyn aggregates taken up by the endosomal-lysosomal system can rupture the lysosomal vesicular membrane; however, it remains unclear whether lysosomal rupture leads to the transmission of αSyn aggregation. Here, we applied cell-based αSyn propagation models to show that ruptured lysosomes are the pathway through which exogenous αSyn aggregates transmit aggregation, and furthermore, this process was prevented by lysophagy, i.e., selective autophagy of damaged lysosomes. αSyn aggregates accumulated predominantly in lysosomes, causing their rupture, and seeded the aggregation of endogenous αSyn, initially around damaged lysosomes. Exogenous αSyn aggregates induced the accumulation of LC3 on lysosomes. This LC3 accumulation was not observed in cells in which a key regulator of autophagy, RB1CC1/FIP200, was knocked out and was confirmed as lysophagy by transmission electron microscopy. Importantly, RB1CC1/FIP200-deficient cells treated with αSyn aggregates had increased numbers of ruptured lysosomes and enhanced propagation of αSyn aggregation. Furthermore, various types of lysosomal damage induced using lysosomotropic reagents, depletion of lysosomal enzymes, or more toxic species of αSyn fibrils also exacerbated the propagation of αSyn aggregation, and impaired lysophagy and lysosomal membrane damage synergistically enhanced propagation. These results indicate that lysophagy prevents exogenous αSyn aggregates from escaping the endosomal-lysosomal system and transmitting aggregation to endogenous cytosolic αSyn via ruptured lysosomal vesicles. Our findings suggest that the progression and severity of synucleinopathies are associated with damage to lysosomal membranes and impaired lysophagy.

Phosphatidylinositol-3,4,5-trisphosphate interacts with alpha-synuclein and initiates its aggregation and formation of Parkinson's disease-related fibril polymorphism.

Lipid interaction with α-synuclein (αSyn) has been long implicated in the pathogenesis of Parkinson's disease (PD). However, it has not been fully determined which lipids are involved in the initiation of αSyn aggregation in PD. Here exploiting genetic understanding associating the loss-of-function mutation in Synaptojanin 1 (SYNJ1), a phosphoinositide phosphatase, with familial PD and analysis of postmortem PD brains, we identified a novel lipid molecule involved in the toxic conversion of αSyn and its relation to PD. We first established a SYNJ1 knockout cell model and found SYNJ1 depletion increases the accumulation of pathological αSyn. Lipidomic analysis revealed SYNJ1 depletion elevates the level of its substrate phosphatidylinositol-3,4,5-trisphosphate (PIP3). We then employed Caenorhabditis elegans model to examine the effect of SYNJ1 defect on the neurotoxicity of αSyn. Mutations in SYNJ1 accelerated the accumulation of αSyn aggregation and induced locomotory defects in the nematodes. These results indicate that functional loss of SYNJ1 promotes the pathological aggregation of αSyn via the dysregulation of its substrate PIP3, leading to the aggravation of αSyn-mediated neurodegeneration. Treatment of cultured cell line and primary neurons with PIP3 itself or with PIP3 phosphatase inhibitor resulted in intracellular formation of αSyn inclusions. Indeed, in vitro protein-lipid overlay assay validated that phosphoinositides, especially PIP3, strongly interact with αSyn. Furthermore, the aggregation assay revealed that PIP3 not only accelerates the fibrillation of αSyn, but also induces the formation of fibrils sharing conformational and biochemical characteristics similar to the fibrils amplified from the brains of PD patients. Notably, the immunohistochemical and lipidomic analyses on postmortem brain of patients with sporadic PD showed increased PIP3 level and its colocalization with αSyn. Taken together, PIP3 dysregulation promotes the pathological aggregation of αSyn and increases the risk of developing PD, and PIP3 represents a potent target for intervention in PD.

Two-step screening method to identify α-synuclein aggregation inhibitors for Parkinson's disease.

Parkinson's disease is a neurodegenerative disease characterized by the formation of neuronal inclusions of α-synuclein in patient brains. As the disease progresses, toxic α-synuclein aggregates transmit throughout the nervous system. No effective disease-modifying therapy has been established, and preventing α-synuclein aggregation is thought to be one of the most promising approaches to ameliorate the disease. In this study, we performed a two-step screening using the thioflavin T assay and a cell-based assay to identify α-synuclein aggregation inhibitors. The first screening, thioflavin T assay, allowed the identification of 30 molecules, among a total of 1262 FDA-approved small compounds, which showed inhibitory effects on α-synuclein fibrilization. In the second screening, a cell-based aggregation assay, seven out of these 30 candidates were found to prevent α-synuclein aggregation without causing substantial toxicity. Of the seven final candidates, tannic acid was the most promising compound. The robustness of our screening method was validated by a primary neuronal cell model and a Caenorhabditis elegans model, which demonstrated the effect of tannic acid against α-synuclein aggregation. In conclusion, our two-step screening system is a powerful method for the identification of α-synuclein aggregation inhibitors, and tannic acid is a promising candidate as a disease-modifying drug for Parkinson's disease.

Alternative mitochondrial quality control mediated by extracellular release.

Mitochondrial quality control, which is crucial for maintaining cellular homeostasis, has been considered to be achieved exclusively through mitophagy. Here we report an alternative mitochondrial quality control pathway mediated by extracellular mitochondria release. By performing time-lapse confocal imaging on a stable cell line with fluorescent-labeled mitochondria, we observed release of mitochondria from cells into the extracellular space. Correlative light-electron microscopy revealed that majority of the extracellular mitochondria are in free form and, on rare occasions, some are enclosed in membrane-surrounded vesicles. Rotenone- and carbonyl cyanide m-chlorophenylhydrazone-induced mitochondrial quality impairment promotes the extracellular release of depolarized mitochondria. Overexpression of PRKN (parkin RBR E3 ubiquitin protein ligase), which has a pivotal role in mitophagy regulation, suppresses the extracellular mitochondria release under basal and stress condition, whereas its knockdown exacerbates it. Correspondingly, overexpression of PRKN-independent mitophagy regulators, BNIP3 (BCL2 interacting protein 3) and BNIP3L/NIX (BCL2 interacting protein 3 like), suppress extracellular mitochondria release. Autophagy-deficient cell lines show elevated extracellular mitochondria release. These results imply that perturbation of mitophagy pathway prompts mitochondria expulsion. Presence of mitochondrial protein can also be detected in mouse sera. Sera of PRKN-deficient mice contain higher level of mitochondrial protein compared to that of wild-type mice. More importantly, fibroblasts and cerebrospinal fluid samples from Parkinson disease patients carrying loss-of-function PRKN mutations show increased extracellular mitochondria compared to control subjects, providing evidence in a clinical context. Taken together, our findings suggest that extracellular mitochondria release is a comparable yet distinct quality control pathway from conventional mitophagy.Abbreviations: ACTB: actin beta; ANXA5: annexin A5; ATP5F1A/ATP5A: ATP synthase F1 subunit alpha; ATG: autophagy related; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CM: conditioned media; CSF: cerebrospinal fluid; DMSO: dimethyl sulfoxide; EM: electron microscopy; HSPD1/Hsp60: heat shock protein family D (Hsp60) member 1; KD: knockdown; KO: knockout; MAP1LC3A/LC3: microtubule associated protein 1 light chain 3 alpha; MT-CO1: mitochondrially encoded cytochrome c oxidase I; NDUFB8: NADH:ubiquinone oxidoreductase subunit B8; OE: overexpression; OPA1: OPA1 mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PBS: phosphate-buffered saline; PB: phosphate buffer; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SDHB: succinate dehydrogenase complex iron sulfur subunit B; TOMM20: translocase of outer mitochondrial membrane 20; TOMM40: translocase of outer mitochondrial membrane 40; UQCRC2: ubiquinol-cytochrome c reductase core protein 2; WT: wild-type.

Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes.

Salt-inducible kinases (SIKs), members of the 5'-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes.

Screening of novel malaria DNA vaccine candidates using full-length cDNA library.

No licensed malaria vaccine exists, in spite of intensive development efforts. We have been investigating development of a DNA vaccine to prevent malaria infection. To date, we have established a full-length cDNA expression library from the erythrocytic-stage murine malaria parasite, Plasmodium berghei. We found that immunization of mice with combined 2000 clones significantly prolonged survival after challenge infection and that splenocytes from the immunized mice showed parasite-specific cytokine production. We determined the 5'-end one-pass sequence of these clones and mapped a draft genomic sequence for P. berghei for use in screening vaccine candidates for efficacy. In this study, we annotated these cDNA clones by comparing them with the genomic sequence of Plasmodium falciparum. We then divided them into several subsets based on their characteristics and examined their protective effects against malaria infection. Consequently, we selected 104 clones that strongly induced specific IgG production and decreased the mortality rate in the early phase. Most of these 104 clones coded for unknown proteins. The results suggest that these clones represent potential novel malaria vaccine candidates.

Molecular survey of Tritrichomonas suis (=T. foetus) 'cat' and 'cattle' genotypes in pigs in Japan.

Tritrichomonas suis (=T. foetus) is a protozoan parasite of pigs, cattle and cats. Based on host range and genetic differences, T. suis has been divided into a 'cat genotype' and a 'cattle genotype', with the latter genotype capable of infecting both cattle and pigs. Since no information is currently available on the genetic characteristics of T. suis from pigs in Japan, we conducted a molecular survey of T. suis using fecal DNA from pigs in Japan. Of the 64 pigs examined, nested PCR revealed that 36 (56.3%) were positive for T. suis. Sequence analysis of 8 positive samples showed that 7 of the pig isolates belonged to the 'cattle genotype' and the remaining isolate belonged to the 'cat genotype'. The findings revealed that T. suis infection is common in pigs in Japan and that pigs can be infected by both genotypes.

Involvement of SIK3 in glucose and lipid homeostasis in mice.

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.

Intestinal Tritrichomonas suis (=T. foetus) infection in Japanese cats.

Tritrichomonas suis (=T. foetus) has recently been reported to be a causative agent of chronic large-bowel diarrhea in cats. While the disease was previously attributed to Pentatrichomonas hominis, the etiologic agent for feline trichomonal diarrhea was identified as T. suis. Although feline trichomonosis due to T. suis has been reported at prevalences ranging from 14 to 31% in Europe and the U.S., no reports of the pathogen have been published to date in Japan. In 2008, however, we encountered a case of feline trichomonosis at the Veterinary Teaching Hospital of Hokkaido University. The parasite was identified as T. suis by nested PCR amplification of partial internal transcribed spacer region 1 and 5.8S ribosomal RNA gene sequences with T. suis-specific primers and DNA sequencing of the amplified products. We then conducted surveys for feline trichomonosis in three different animal hospitals using either cultivation and/or PCR-based assays. The results revealed that 13 of 147 samples (8.8%) were positive for T. suis, and that 5 of the 13 infected cats, which ranged between 1 month and 7.5 years-old, showed chronic diarrhea. Seven of the infected cats were purebred and 6 were mixed breed. These findings suggested that feline trichomonosis is prevalent in Japan, and that T. suis may play a role as a causative agent of feline chronic diarrhea.

N-acetylglucosaminyltransferase V-deficiency increases susceptibility to murine malaria.

It is considered that several glycoproteins on erythrocytes in mammalian species are involved in malaria parasite infection. To elucidate the role of N-glycans on malaria parasite infection, we induced experimental murine malaria infection (using Plasmodium berghei ANKA) in mice deficient in N-acetylglucosaminyltransferase V (Mgat5), which is one of the enzymes involved in ß1,6-GlcNAc N-glycan biosynthesis. After infection, Mgat5(-/-) mice showed severe body weight loss and parasitemia compared with wild-type mice. The Mgat5(-/-) mice, but not wild-type mice, also showed severe pathology accompanied by marked infiltration of plasma cells into the lungs and liver. These results suggest that ß1,6-GlcNAc N-glycans on/in host erythrocytes may interfere with invasion of the parasites and progression to severe malaria.

Involvement of SIK2/TORC2 signaling cascade in the regulation of insulin-induced PGC-1alpha and UCP-1 gene expression in brown adipocytes.

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.

Dephosphorylation of TORC initiates expression of the StAR gene.

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.

Silencing the constitutive active transcription factor CREB by the LKB1-SIK signaling cascade.

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.

Molecular identity and gene expression of aldosterone synthase cytochrome P450.

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.

Salt-inducible kinase-1 represses cAMP response element-binding protein activity both in the nucleus and in the cytoplasm.

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.

Salt-inducible kinase (SIK) isoforms: their involvement in steroidogenesis and adipogenesis.

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.

Characterization of the adrenal-specific antigen IZA (inner zone antigen) and its role in the steroidogenesis.

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.

Triiodothyronine but not thyroxine accelerates myofibrillar proteolysis via ATP production in cultured muscle cells.

These experiments were done to clarify that the differential effects of thyroxine (T(4)) and triiodothyronine (T(3)) on skeletal muscle protein turnover are caused by their roles on ATP production. Primary cultured chick muscle cells were treated with a physiological level of T(4) (60 ng/ml), T(3) (12 ng/ml), or ATP (0.5 mM) for 6 days and the protein content, ATP production, proteasome activity, and myofibrillar protein breakdown were measured. The protein content measured as an index of cell growth was not affected by T(4), T(3), or ATP. The cellular ATP level was increased by T(3) and ATP, but not by T(4). Proteasome activity and N(tau)-methylhistidine (MeHis) release measured as an index of myofiblillar protein breakdown was also increased by T(3) and ATP, but not by T(4). These results indicate that T(3) but not T(4) increases ATP production followed by an increase in proteasome activity, and thus stimulates myofibrillar proteolysis.

Salt-inducible kinase-mediated regulation of steroidogenesis at the early stage of ACTH-stimulation.

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.