[Show abstract][Hide abstract] ABSTRACT: Rationale:
In human genetic studies a single nucleotide polymorphism within the salt-inducible kinase 1 (SIK1) gene was associated with hypertension. Lower SIK1 activity in vascular smooth muscle cells (VSMCs) leads to decreased sodium-potassium ATPase activity, which associates with increased vascular tone. Also, SIK1 participates in a negative feedback mechanism on the transforming growth factor-β1 signaling and downregulation of SIK1 induces the expression of extracellular matrix remodeling genes.
To evaluate whether reduced expression/activity of SIK1 alone or in combination with elevated salt intake could modify the structure and function of the vasculature, leading to higher blood pressure.
Methods and results:
SIK1 knockout (sik1(-/-)) and wild-type (sik1(+/+)) mice were challenged to a normal- or chronic high-salt intake (1% NaCl). Under normal-salt conditions, the sik1(-/-) mice showed increased collagen deposition in the aorta but similar blood pressure compared with the sik1(+/+) mice. During high-salt intake, the sik1(+/+) mice exhibited an increase in SIK1 expression in the VSMCs layer of the aorta, whereas the sik1(-/-) mice exhibited upregulated transforming growth factor-β1 signaling and increased expression of endothelin-1 and genes involved in VSMC contraction, higher systolic blood pressure, and signs of cardiac hypertrophy. In vitro knockdown of SIK1 induced upregulation of collagen in aortic adventitial fibroblasts and enhanced the expression of contractile markers and of endothelin-1 in VSMCs.
Vascular SIK1 activation might represent a novel mechanism involved in the prevention of high blood pressure development triggered by high-salt intake through the modulation of the contractile phenotype of VSMCs via transforming growth factor-β1-signaling inhibition.
Full-text · Article · Jan 2015 · Circulation Research
[Show abstract][Hide abstract] ABSTRACT: Cardiac left ventricle hypertrophy (LVH) constitutes a major risk factor for heart failure. Although LVH is most commonly caused by chronic elevation in arterial blood pressure, reduction of blood pressure to normal levels does not always result in regression of LVH, suggesting that additional factors contribute to the development of this pathology. We tested whether genetic preconditions associated with the imbalance in sodium homeostasis could trigger the development of LVH without concomitant increases in blood pressure. The results showed that the presence of a hypertensive variant of α-adducin gene in Milan rats (before they become hypertensive) resulted in elevated expression of genes associated with LVH, and of salt-inducible kinase 2 (SIK2) in the left ventricle (LV). Moreover, the mRNA expression levels of SIK2, α-adducin, and several markers of cardiac hypertrophy were positively correlated in tissue biopsies obtained from human hearts. In addition, we found in cardiac myocytes that α-adducin regulates the expression of SIK2, which in turn mediates the effects of adducin on hypertrophy markers gene activation. Furthermore, evidence that SIK2 is critical for the development of LVH in response to chronic high salt diet (HS) was obtained in mice with ablation of the sik2 gene. Increases in the expression of genes associated with LVH, as well as increases in LV wall thickness upon HS, occurred only in sik2+/+ but not in sik2-/- mice. Thus LVH triggered by HS or the presence of a genetic variant of α-adducin requires SIK2 and is independent of elevated blood pressure. Inhibitors of SIK2 may constitute part of a novel therapeutic regimen aimed at prevention/regression of LVH.
[Show abstract][Hide abstract] ABSTRACT: To uncover the potential cardiovascular effects of human polymorphisms influencing transforming growth factor β1 (TGFβ1) expression, we generated mice with Tgfb1 mRNA expression graded in five steps from 10% to 300% normal. Adrenal expression of the genes for mineralocorticoid-producing enzymes ranged from 50% normal in the hypermorphs at age 12 wk to 400% normal in the hypomorphs accompanied with proportionate changes in plasma aldosterone levels, whereas plasma volumes ranged from 50% to 150% normal accompanied by marked compensatory changes in plasma angiotensin II and renin levels. The aldosterone/renin ratio ranged from 0.3 times normal in the 300% hypermorphs to six times in the 10% hypomorphs, which have elevated blood pressure. Urinary output of water and electrolytes are markedly decreased in the 10% hypomorphs without significant change in the glomerular filtration rate. Renal activities for the Na(+), K(+)-ATPase, and epithelial sodium channel are markedly increased in the 10% hypomorphs. The hypertension in the 10% hypomorphs is corrected by spironolactone or amiloride at doses that do not change blood pressure in wild-type mice. Thus, changes in Tgfb1 expression cause marked progressive changes in multiple systems that regulate blood pressure and fluid homeostasis, with the major effects being mediated by changes in adrenocortical function.
Full-text · Article · Mar 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The salt-inducible kinase 1 (SIK1) network constitutes an alternative target by which agents can modulate active sodium transport in renal epithelia and avoid the increases in systemic blood pressure. SIK1 associates with the plasma membrane Na+,K+-ATPase regulatory network and controls its catalytic activity upon increases in intracellular sodium. The aim of this study was to evaluate the role of SIK1 upon Na+ transporters and transepithelial electrical resistance using an epithelial cell line derived from mouse lung, MLE-12, stably transfected with SIK1-shRNA or with a scrambled shRNA. Normal MLE-12 cells formed tight monolayers; however, MLE-12 cells lacking SIK1 have a significantly lower transepithelial resistant than cells expressing normal levels of SIK1 protein. MLE-12 expressing SIK-shRNA had a significant reduction in Na+,K+-ATPase catalytic activity and it was paralleled by a reduction in the Na+/H+-exchanger activity. Cl-/HCO3- exchanger activity remained unchanged. Similarly, the plasma membrane potential was lower in cells lacking SIK1, and these cells did not exhibit depolarization in response to ouabain, and a change of less magnitude in response to KCL. The levels of intracellular ATP were higher in MLE-12 cells lacking SIK1, most likely due to a reduced consumption (lower Na+,K+-ATPase activity). These results highlight the potential role of SIK1 in controlling the integrity and function of polarized epithelia. Moreover, further insight into the regulation of SIK1 may enable the development of novel drugs to prevent or treat cardiovascular disorders and edema formation states.
No preview · Article · Sep 2012 · Journal of Hypertension
[Show abstract][Hide abstract] ABSTRACT: Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3(-/-) mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3(-/-) mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3(-/-) mice. Lipid metabolism disorders in Sik3(-/-) mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice.
[Show abstract][Hide abstract] ABSTRACT: (A) Effect of 9-cis-RA treatment (0–16 mg kg−1·d−1) on the weight gain of wild-type mice (n = 6). (B) Blood glucose levels before and after treatment are indicated by labels as B and A, respectively. (C) The levels of serum ALP and bile acids were measured before (labeled as B) and after (labeled as A) 9-cis-RA treatment (for 9 days: after the analysis shown in Figure 8E). Ethanol (EtOH, 1%) was used as a solvent. Significant differences before and after treatment in the same group (n = 4) are indicated. Although there were no significant fluctuations in the levels of bile acids, their levels decreased in all Sik3−/− mice after treatment. (D) Effect of 9-cis-RA on gene expression in Sik3−/− mice. At day 7, Sik3−/− mice treated with 9-cis-RA were grouped into sets of 3 (n = 4) and fed a chow, high-Chol, or high-CA diet for an additional 2 days under continuous RA treatment; mRNA levels in the liver were then examined. Significant differences between the chow and special diet groups are indicated.
[Show abstract][Hide abstract] ABSTRACT: (A) Male mice (12 weeks of age, n = 3) were fed diets supplemented with Chol (2%) and cholic acid (0.25%) for 2 days or with fat (60% of calories) for 2 weeks and then sacrificed. The expression of genes for Chol and BA metabolism in the liver was examined using quantitative polymerase chain reaction (normalized by glyceraldehyde 3-phosphate dehydrogenase [GAPDH] levels). Significant differences between wild-type and Sik3−/− mice are shown by *, **, and *** for p<0.05, <0.01, and <0.001, respectively. # indicates a significant difference between the chow and special diet groups. Means and SEM are shown. (B) Expression levels of nuclear receptors. (C) The expression of genes involved in vitamin A metabolism was examined using the liver cDNA in Figure 3A (1-year-old mice, n = 5).
[Show abstract][Hide abstract] ABSTRACT: (A) Body weight curves of wild-type and Sik3 heterozygous mice are also shown (n = 12). (B) Levels of Sik3 mRNA in the livers, brown adipose tissues (BAT), and muscles of wild-type, heterozygous, and Sik3−/− mice (n = 3). The error bars indicate SEM. Levels of SIK3 protein in the livers of wild-type, heterozygous, and Sik3−/− mice. (C) Hepatic parenchymal and non-parenchymal cells were separated by centrifugation, and Sik3 mRNA levels were examined by quantitative PCR. Cyp7α, F4/80, and Desmin were used as markers for parenchymal cells, Kupffer’s cells (non-parenchymal), and hepatic stellate cells (non-parenchymal), respectively. (n = 3: means and SEM are shown). (D) In vitro adipocyte differentiation assay. Preadipocytes were prepared from gonadal fat pads using collagenase and then plated. When the cells reached confluence, the culture medium was changed to Dulbecco’s Modified Eagle’s Medium (high glucose) supplemented with rosiglitazone (Rosi: indicated concentration), and insulin (1 µg/mL). After 8 days (with changes of medium every 2 days), the cells were fixed with 4% paraformaldehyde and stained with Oil Red O. The high magnification images show cells that were differentiated using 3 µM rosiglitazone. (F) Serum adiponectin levels of the mice examined in Figure 3E. Means and SEM are shown. ### indicates p<0.001.
[Show abstract][Hide abstract] ABSTRACT: (A) HE staining of embryo livers. The sets in the left and right panels are the same magnification. The lower panels are a higher magnification of the upper panels. (B) Bile acid was extracted with 95% ethanol/0.5% NH3-water. The numbers of mice (wild-type and Sik3−/−) used for the assay were: E16.5, 11 and 6; E18.5, 16 and 3; P0, 9 and 5; and 12 weeks, 8 and 5, respectively. Means and SEM are shown. Significant differences between wild-type and Sik3−/− mice are shown by * for p<0.05. ## indicates significant differences between P0 and E18.5 or 12 weeks in wild-type mice (p<0.01).
[Show abstract][Hide abstract] ABSTRACT: (A) Most Sik3−/− mice died on the first day after birth. The mating system and time of genotyping are indicated. The percentage and number of mice in the first column indicate the sum of neonates at day 1 and embryos at E17.5–E18.5. Neonates prepared by in vitro fertilization were delivered by cesarean section and living mice were counted without genotyping. However, ∼50% of the mice disappeared by the second day, probably because they were eaten by the foster mice. (B) The difference in the body size of Sik3−/− mice became obvious after 2 weeks. (C) HE staining of gonadal fat of 1-year-old mice. (D) Cholesterol (Chol), triglyceride (TG), and carbohydrate (Carbo) content in feces (from 3 cages). Cholesterol and triglycerides were extracted with methanol/chloroform as described in the Materials and Methods. To extract undigested carbohydrates, the feces were re-digested with amylase at 37°C for 12 h, and the debris was removed by centrifugation. Carbohydrates were stained with a solution of 1 volume of 5% phenol and 5 volumes of sulfuric acid and then detected at 490 nm. (E) After fasting for 4-h fasting, the serum levels of free thyroid hormones (FT3 and FT4) were measured with an automated system for clinical assays. Serum thyroid stimulating hormone (TSH) levels were measured with an ELISA kit from Shibayagi Co., Ltd. (F) Insulin tolerance test (ITT). Mice (male n = 5) were fasted for 2 h and then treated intraperitoneally with 36 µg/kg insulin. All data points are p<0.001.
[Show abstract][Hide abstract] ABSTRACT: The protein kinase liver kinase B1 (LKB1) regulates cell polarity and intercellular junction stability. Also, LKB1 controls the activity of salt-inducible kinase 1 (SIK1). The role and relevance of SIK1 and its downstream effectors in linking the LKB1 signals within these processes are partially understood. We hypothesize that SIK1 may link LKB1 signals to the maintenance of epithelial junction stability by regulating E-cadherin expression. Results from our studies using a mouse lung alveolar epithelial (MLE-12) cell line or human renal proximal tubule (HK2) cell line transiently or stably lacking the expression of SIK1 (using SIK1 siRNAs or shRNAs), or with its expression abrogated (sik1(+/+) vs. sik1(-/-) mice), indicate that suppression of SIK1 (∼40%) increases the expression of the transcriptional repressors Snail2 (∼12-fold), Zeb1 (∼100%), Zeb2 (∼50%), and TWIST (∼20-fold) by activating cAMP-response element binding protein. The lack of SIK1 and activation of transcriptional repressors decreases the availability of E-cadherin (mRNA and protein expression by ∼100 and 80%, respectively) and the stability of intercellular junctions in epithelia (decreases in transepithelial resistance). Furthermore, LKB1-mediated increases in E-cadherin expression are impaired in cells where SIK1 has been disabled. We conclude that SIK1 is a key regulator of E-cadherin expression, and thereby contributes to the stability of intercellular junctions.
Full-text · Article · Apr 2012 · The FASEB Journal
[Show abstract][Hide abstract] ABSTRACT: Cardiac hypertrophy (CH) generally occurs as the result of the sustained mechanical stress caused by elevated systemic arterial blood pressure (BP). However, in animal models, elevated salt intake is associated with CH even in the absence of significant increases in BP. We hypothesize that CH is not exclusively the consequence of mechanical stress but also of other factors associated with elevated BP such as abnormal cell sodium homeostasis. We examined the effect of small increases in intracellular sodium concentration ([Na(+)](i)) on transcription factors and genes associated with CH in a cardiac cell line. Increases in [Na(+)](i) led to a time-dependent increase in the expression levels of mRNA for natriuretic peptide and myosin heavy chain genes and also increased myocyte enhancer factor (MEF)2/nuclear factor of activated T cell (NFAT) transcriptional activity. Increases in [Na(+)](i) are associated with activation of salt-inducible kinase 1 (snflk-1, SIK1), a kinase known to be critical for cardiac development. Moreover, increases in [Na(+)](i) resulted in increased SIK1 expression. Sodium did not increase MEF2/NFAT activity or gene expression in cells expressing a SIK1 that lacked kinase activity. The mechanism by which SIK1 activated MEF2 involved phosphorylation of HDAC5. Increases in [Na(+)](i) activate SIK1 and MEF2 via a parallel increase in intracellular calcium through the reverse mode of Na(+)/Ca(2+)-exchanger and activation of CaMK1. These data obtained in a cardiac cell line suggest that increases in intracellular sodium could influence myocardial growth by controlling transcriptional activation and gene expression throughout the activation of the SIK1 network.
Full-text · Article · Mar 2012 · AJP Heart and Circulatory Physiology
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Characterization of NPSR1 predicted promoter. Left: ARTS-predicted NPSR1 promoter sequence, spanning nucleotides -595 to +1 from the translational start site (ATG). Transcription factors (TFs) predicted to bind to the promoter from MatInspector analysis are also reported, with their recognitions sites underlined. Among these, TFs involved in the modulation of inflammatory responses are shown in blue, while TFs known to regulate neurological functions are shown in green. Four common (MAF>0.02) SNPs mapping within the predicted promoter region are reported in red, together with the corresponding alleles at each site. Right: assessment of the functional activity of NPSR1 predicted promoter in luciferase reporter assays. Colo205 cells were transiently transfected with either a promoterless luciferase vector (pGL3-basic) or the same vector carrying NPSR1 promoter driving luciferase transcription (pNPSR1P). Results, which are representative of 3 independent experiments performed in duplicate, are expressed as fold induction, relative to the luciferase activity obtained for the control transfection (empty vector).
[Show abstract][Hide abstract] ABSTRACT: neuropeptide S (NPS) and its receptor NPSR1 act along the hypothalamic-pituitary-adrenal axis to modulate anxiety, fear responses, nociception and inflammation. The importance of the NPS-NPSR1 signaling pathway is highlighted by the observation that, in humans, NPSR1 polymorphism associates with asthma, inflammatory bowel disease, rheumatoid arthritis, panic disorders, and intermediate phenotypes of functional gastrointestinal disorders. Because of the genetic complexity at the NPSR1 locus, however, true causative variations remain to be identified, together with their specific effects on receptor expression or function. To gain insight into the mechanisms leading to NPSR1 disease-predisposing effects, we performed a thorough functional characterization of all NPSR1 promoter and coding SNPs commonly occurring in Caucasians (minor allele frequency >0.02).
we identified one promoter SNP (rs2530547 [-103]) that significantly affects luciferase expression in gene reporter assays and NPSR1 mRNA levels in human leukocytes. We also detected quantitative differences in NPS-induced genome-wide transcriptional profiles and CRE-dependent luciferase activities associated with three NPSR1 non-synonymous SNPs (rs324981 [Ile107Asn], rs34705969 [Cys197Phe], rs727162 [Arg241Ser]), with a coding variant exhibiting a loss-of-function phenotype (197Phe). Potential mechanistic explanations were sought with molecular modelling and bioinformatics, and a pilot study of 2230 IBD cases and controls provided initial support to the hypothesis that different cis-combinations of these functional SNPs variably affect disease risk.
these findings represent a first step to decipher NPSR1 locus complexity and its impact on several human conditions NPS antagonists have been recently described, and our results are of potential pharmacogenetic relevance.