Salt-inducible Kinase 1 Expression Research Articles

Circ_0078607 inhibits the progression of ovarian cancer via regulating the miR-32-5p/SIK1 network

BackgroundCircular RNA (circRNA) has been shown to be involved in the regulation of human disease progression, including ovarian cancer (OC). Circ_0078607 was found to participate in OC progression. But its function and mechanism in OC deserve further exploration.MethodsThe expression levels of circ_0078607, salt-inducible kinase 1 (SIK1) and microRNA (miR)-32-5p were examined by qRT-PCR. And the protein expression levels of SIK1, metastasis marker and apoptosis marker were determined using western blot analysis. EDU staining, colony formation assay, transwell assay and flow cytometry were used to detect the proliferation, migration, invasion and apoptosis of cells. Moreover, dual-luciferase reporter assay was employed to verify the interaction between miR-32-5p and circ_0078607 or SIK1. Xenograft models were constructed to perform in vivo experiments.ResultsCirc_0078607 and SIK1 were downregulated in OC tissues and cells. Overexpressed circ_0078607 and SIK1 could inhibit OC cell proliferation, migration, invasion, and promote apoptosis. MiR-32-5p could be sponged by circ_0078607, and its overexpression could reverse the suppressive effect of circ_0078607 on OC progression. Furthermore, SIK1 was a target of miR-32-5p, and circ_0078607 could regulate SIK1 by sponging miR-32-5p. The inhibitory effect of circ_0078607 on OC progression also could be reversed by SIK1 silencing. In vivo experiments showed that circ_0078607 reduced OC tumorigenesis by regulating the miR-32-5p/SIK1 axis.ConclusionCirc_0078607 could serve as a sponge of miR-32-5p to regulate SIK1 expression, thereby inhibiting OC progression.

QiShenYiQi ameliorates salt-induced hypertensive nephropathy by balancing ADRA1D and SIK1 expression in Dahl salt-sensitive rats

BackgroundHypertension is a leading risk factor for developing kidney disease. Current single-target antihypertensive drugs are not effective for hypertensive nephropathy, in part due to its less understood mechanism of pathogenesis. We recently showed that QiShenYiQi (QSYQ), a component-based cardiovascular Chinese medicine, is also effective for ischemic stroke. Given the important role of the brain-heart-kidney axis in blood pressure control, we hypothesized that QSYQ may contribute to blood pressure regulation and kidney protection in Dahl salt-sensitive hypertensive rats. MethodsThe therapeutic effects of QSYQ on blood pressure and kidney injury in Dahl salt-sensitive rats fed with high salt for 9 weeks were evaluated by tail-cuff blood pressure monitoring, renal histopathological examination and biochemical indicators in urine and serum. RNA-seq was conducted to identify QSYQ regulated genes in hypertensive kidney, and RT-qPCR, immunohistochemistry, and Western blotting analysis were performed to verify the transcriptomics results and validate the purposed mechanisms. ResultsQSYQ treatment significantly decreased blood pressure in Dahl salt-sensitive hypertensive rats, alleviated renal tissue damage, reduced renal interstitial fibrosis and collagen deposition, and improved renal physiological function. RNA-seq and subsequent bioinformatic analysis showed that the expression of ADRA1D and SIK1 genes were among the most prominently altered by QSYQ in salt-sensitive hypertensive rat kidney. RT-qPCR, immunohistochemistry and Western blotting results confirmed that the mRNA and protein expression levels of alpha-1D adrenergic receptor (ADRA1D) in the kidney tissue of the QSYQ-treated rats were markedly down-regulated, while the mRNA and protein levels of salt inducible kinase 1 (SIK1) were significantly increased. ConclusionQSYQ not only lowered blood pressure, but also alleviated renal damage via reducing the expression of ADRA1D and increasing the expression of SIK1 in the kidney of Dahl salt-sensitive hypertensive rats.

Role of SIK1 in the transition of acute kidney injury into chronic kidney disease

BackgroundAcute kidney injury (AKI), with a high morbidity and mortality, is recognized as a risk factor for chronic kidney disease (CKD). AKI-CKD transition has been regarded as one of the most pressing unmet needs in renal diseases. Recently, studies have showed that salt inducible kinase 1 (SIK1) plays a role in epithelial-mesenchymal transition (EMT) and inflammation, which are the hallmarks of AKI-CKD transition. However, whether SIK1 is involved in AKI-CKD transition and by what mechanism it regulates AKI-CKD transition remains unknown.MethodsWe firstly detected the expression of SIK1 in kidney tissues of AKI patients and AKI mice by immunohistochemistry staining, and then we established Aristolochic acid (AA)-induced AKI-CKD transition model in C57BL/6 mice and HK2 cells. Subsequently, we performed immunohistochemistry staining, ELISA, real-time PCR, Western blot, immunofluorescence staining and Transwell assay to explore the role and underlying mechanism of SIK1 on AKI-CKD transition.ResultsThe expression of SIK1 was down-regulated in AKI patients, AKI mice, AA-induced AKI-CKD transition mice, and HK2 cells. Functional analysis revealed that overexpression of SIK1 alleviated AA-induced AKI-CKD transition and HK2 cells injury in vivo and in vitro. Mechanistically, we demonstrated that SIK1 mediated AA-induced AKI-CKD transition by regulating WNT/β-catenin signaling, the canonical pathway involved in EMT, inflammation and renal fibrosis. In addition, we discovered that inhibition of WNT/β-catenin pathway and its downstream transcription factor Twist1 ameliorated HK2 cells injury, delaying the progression of AKI-CKD transition.ConclusionsOur study demonstrated, for the first time, a protective role of SIK1 in AKI-CKD transition by regulating WNT/β-catenin signaling pathway and its downstream transcription factor Twist1, which will provide novel insights into the prevention and treatment AKI-CKD transition in the future.

SIK1 Regulates CRTC2-Mediated Gluconeogenesis Signaling Pathway in Human and Mouse Liver Cells.

The regulation of hepatic gluconeogenesis is of great significance to improve insulin resistance and benefit diabetes therapy. cAMP-Regulated Transcriptional Co-activator 2 (CRTC2) plays a key role in regulating hepatic gluconeogenesis through controlling the expression of gluconeogenic rate-limiting enzymes such as glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Recently, salt-inducible kinase 1 (SIK1) has been identified to play an important role in glucose metabolism disorders, but whether and how SIK1 regulates the CTRC2 signaling in liver cells under high glucose conditions has rarely been intensively elucidated. Here, we show that high glucose stimulation resulted in time-dependent down-regulated expression of SIK1, phosphorylated SIK1 at T182 site, and phosphorylated CRTC2 at S171 site, as well as upregulated expression of total CRTC2 and its downstream targets G6Pase and PEPCK in the human liver cell line HepG2. The nuclear expression levels of SIK1 and CRTC2 were time-dependently upregulated upon high glucose challenge, which was accompanied by enhanced cytoplasm-to-nucleus translocation of SIK1. Manipulation of SIK1 activity using plasmid-mediated SIK1 over-expression and the use of the SIKs inhibitor HG-9-91-01 confirmed that SIK1 regulated the CRTC2 signaling pathway in HepG2 cells. Furthermore, in mouse primary hepatocytes, high glucose exposure down-regulated SIK1 expression, and promoted SIK1 nuclear accumulation. While HG-9-91-01 treatment suppressed SIK1 expression and released the inhibitory effects of SIK1 on the expressions of key molecules involved in the CRTC2 signaling pathway, additional ectopic expression of SIK1 using adenovirus infection reversed the impacts of HG-9-91-01 on the expressions of these molecules in mouse hepatocytes. Therefore, SIK1 regulates CRTC2-mediated gluconeogenesis signaling pathway under both physiological and high glucose-induced pathological conditions. The modulation of the SIK1-CRTC2 signaling axis could provide an attractive means for treating diabetes.

Liver kinase B1 correlates with prognosis and epithelial-mesenchymal transition of resectable early stage non-small cell lung cancer.

BackgroundRecent evidences support that low expression of liver kinase B1 (LKB1) triggers epithelial-mesenchymal transition (EMT) through induction of Zinc finger E-box binding homeobox 1 (ZEB1) expression, which downregulates E-cadherin in human lung cancer cell lines. However, the clinicopathological significance of LKB1, EMT, salt-inducible kinase 1 (SIK1), ZEB1 and their relationship in early stage non-small cell lung cancer (ES-NSCLC) patients remain to be determined. In this study, the correlation among expression of LKB1, risk of distant metastasis, prognostic significance, and EMT in ES-NSCLC after surgery was investigated by immunohistochemistry.MethodsCase notes and pathology records of 103 patients with ES-NSCLC were retrospectively analyzed. Immunohistochemical staining was employed to detect LKB1, EMT biomarkers (E-cadherin and vimentin), SIK1 and ZEB1 expression in ES-NSCLC tissues.ResultsLKB1 expression is associated with distant metastasis after treatment (P=0.017). LKB1-high expression group has better overall survival (OS) (P=0.000) and disease-free survival (DFS) (P=0.000). LKB1 expression is correlated with E-cadherin (r=0.231, P=0.019), vimentin (r=−0.225, P=0.022), SIK1 (r=0.218, P=0.027) and ZEB1 (r=−0.242, P=0.014).ConclusionsOur findings suggest that LKB1-high expression is possibly associated with favourable prognosis and LKB1 expression is correlated with EMT and expression of SIK1 and ZEB1.

Salt-inducible kinase 1 regulates bone anabolism via the CRTC1\u2013CREB\u2013Id1 axis

New bone anabolic agents for the effective treatment of bone metabolic diseases like osteoporosis are of high clinical demand. In the present study, we reveal the function of salt-inducible kinase 1 (SIK1) in regulating osteoblast differentiation. Gene knockdown of SIK1 but not of SIK2 or SIK3 expression in primary preosteoblasts increased osteoblast differentiation and bone matrix mineralization. SIK1 also regulated the proliferation of osteoblastic precursor cells in osteogenesis. This negative control of osteoblasts required the catalytic activity of SIK1. SIK1 phosphorylated CREB regulated transcription coactivator 1 (CRTC1), preventing CRTC1 from enhancing CREB transcriptional activity for the expression of osteogenic genes like Id1. Furthermore, SIK1 knockout (KO) mice had higher bone mass, osteoblast number, and bone formation rate versus littermate wild-type (WT) mice. Preosteoblasts from SIK1 KO mice showed more osteoblastogenic potential than did WT cells, whereas osteoclast generation among KO and WT precursors was indifferent. In addition, bone morphogenic protein 2 (BMP2) suppressed both SIK1 expression as well as SIK1 activity by protein kinase A (PKA)–dependent mechanisms to stimulate osteogenesis. Taken together, our results indicate that SIK1 is a key negative regulator of preosteoblast proliferation and osteoblast differentiation and that the repression of SIK1 is crucial for BMP2 signaling for osteogenesis. Therefore, we propose SIK1 to be a useful therapeutic target for the development of bone anabolic strategies.

MicroRNA-373 promotes cell migration via targeting salt-inducible kinase 1 expression in melanoma.

It is well established that altered expression of microRNAs (miRs) is critical in numerous human cancer types. Nevertheless, the molecular mechanisms of many miRs are yet to be elucidated. In the present study, reverse transcription-quantitative polymerase chain reaction and western blot analyses, and cell migration assays were performed to verify dysregulation of miR-373 in melanoma and its biological function. The transcriptional level of miR-373 was identified to be upregulated in melanoma tissues and cell lines compared with nevus and normal melanocytes. miR-373 was identified to function as an oncomiR, promoting melanoma cell migration. Notably, miR-373 was observed to suppress its downstream gene salt-inducible kinase 1 (SIK1) through directly binding the 3'-untranslated region of SIK1 expression. Furthermore, reduced SIK1 expression was identified to be responsible for the oncogenic effect of miR-373. In conclusion, the present study indicates that miR-373 functions as an oncomiR to promote melanoma progression through targeting SIK1 expression. This may provide a new therapeutic approach for melanoma.

Abstract 2386: Identification of SIK1 as a potential therapeutic target for desmoplastic small round cell tumor

Abstract Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive malignant cancer caused by the chromosomal translocation of t(11;22)(p13;q12) that produces a novel, aberrant transcription factor, EWS-WT1. EWS-WT1 is essential in the formation of DSRCT as well as for maintaining DSRCT cell growth. However, current research has not elucidated how EWS-WT1 leads to the oncogenesis of DSRCT. Through our integrative gene expression data, we identified Salt Inducible Kinase 1 (SIK1) as one of direct target genes of EWS-WT1 in JN-DSRCT-1 cell line. SIK1 is a member of the AMPK related kinases and is involved in gluconeogenesis and lipogenesis regulation, skeletal myocytes development, p53 dependent anoikis, and cell cycle regulation. Through our ChIP analysis, we showed that EWS-WT1 directly binds to a 2kb proximal promoter region of SIK1. Following SIK1 depletion, JN-DSRCT-1 cell proliferation sharply decreased, similar to the growth inhibition observed when EWS-WT1 is depleted. We further showed that cells do not transit to S phase when SIK1 is depleted, suggesting a critical role of SIK1 in cell cycle regulation in DSRCT. Taken together, we have established that EWS-WT1 directly activates SIK1 expression and promotes cell proliferation through SIK1. Therefore, our work identified SIK1 as a new potential therapeutic target in DSRCT. Citation Format: Alifiani B. Hartono, Sean B. Lee. Identification of SIK1 as a potential therapeutic target for desmoplastic small round cell tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2386.

Salt-Inducible Kinase 1 (SIK1) is Induced by Alcohol and Suppresses Microglia Inflammation via NF-κB Signaling

Background/Aims: Alcohol consumption has been shown to cause neuroinflammation and increase a variety of immune-related signaling processes. Microglia are a crucial part of alcohol-induced neuroinflammation and undergo apoptosis. Even though the importance of these inflammatory processes in the effects of alcohol-related neurodegeneration have been established, the mechanism of alcohol-induced microglia apoptosis is unknown. In prior research, we discovered that alcohol increases expression of salt-inducible kinase 1 (SIK1) in rodent brain tissue. In this study, we sought to determine what role SIK1 expression plays in alcohol-induced neuroinflammation as well as whether and by what mechanism it regulates microglia apoptosis. Methods: Adult C57BL/6 mice were divided into four groups and for 3 weeks treated with either 0%, 5%, 10%, or 15% alcohol during 3 hour periods. The mice were sacrificed and their brains excised for analysis. Additionally, primary microglia were isolated from neonatal mice. SIK1 expression in alcohol-treated brain tissue and microglia was analyzed via RT-PCR and western blotting. TUNEL staining, caspase-3, and caspase-9 activity assays were performed to evaluate microglial apoptosis. Cell fluorescence staining and NF-κB luciferase activity assays were used to evaluate the effects of SIK1 expression on the NF-κB signaling pathway. Results: SIK1 expression was increased in the brains of mice that consumed alcohol, and this effect was seen in mouse primary microglia. SIK1 knockdown in microglia increased alcohol-induced apoptosis in these cells. Furthermore, SIK1 reduced NF-κB signaling pathway factors, and SIK1 knockdown in microglia promoted alcohol-induced NF-κB activity. TUNEL staining, caspase-3, and caspase-9 activity assays consistently revealed that alcohol-induced microglial apoptosis was inhibited by depletion of p65. Finally, we determined that NF-κB signaling is required for alcohol-induced, SIK1-mediated apoptosis in microglia. Conclusion: This study establishes for the first time not only that SIK1 is crucial to regulating alcohol-induced microglial apoptosis, but also that the NF-κB signaling pathway is required for its activity. Overall, our results help elucidate mechanisms of alcohol-induced neuroinflammation.

Activation of the LKB1‑SIK1 signaling pathway inhibits the TGF‑β‑mediated epithelial‑mesenchymal transition and apoptosis resistance of ovarian carcinoma cells.

Ovarian cancer is the most common and lethal type of gynecological malignancy, due to its invasiveness. The present study aimed to analyze the molecular mechanism underlying chemoresistance in ovarian carcinoma cells, which may lead to local migration toward adjacent tissues and long-distance metastasis to other organs. A total of 12 patients with ovarian fibroma were used to evaluate chemoresistance and chemosensitivity. The sensitivity and resistance of ovarian carcinoma cells was measured using apoptosis analysis, morphological observation, survival rate analysis, immunohistochemistry and immunostaining. The mechanism underlying the interaction between the epithelial-mesenchymal transition (EMT) and liver kinase B1 (LKB1)-salt-inducible kinase 1 (SIK1) signaling pathways was additionally investigated in ovarian carcinoma. The results of the present study demonstrated that ovarian carcinoma cells isolated from patients exhibited apoptosis resistance. Inhibition of TGF-β expression led to an inhibition of growth, migration and invasion, in addition to a promotion of apoptosis, in ovarian carcinoma cells treated with paclitaxel. Studies have indicated that the LKB1-SIK1 signaling pathway may be suppressed in ovarian carcinoma cells compared with normal ovarian cells, leading to activation of the EMT signaling pathway. The results of the present study demonstrated that upregulation of LKB1 promoted SIK1 expression and markedly suppressed the growth and aggressiveness of ovarian cancer cells. Upregulation of LKB1 additionally promoted apoptosis in ovarian carcinoma cells. In addition, the results of the present study demonstrated that the knockdown of LKB1 further promoted the expression of transforming growth factor-β and EMT, which downregulated the chemosensitivity of ovarian carcinoma cells. Additionally, overexpression of LKB1 in ovarian carcinoma cells increased chemosensitivity, resulting in a significant inhibition of migration and invasion. The present findings indicated that the enhancement of LKB1-SIK1 suppressed the growth and aggressiveness of ovarian carcinoma cells isolated from clinical patients, which subsequently contributed to an inhibition of metastatic potential. In conclusion, targeting the LKB1-SIK1 signaling pathway to inhibit EMT may provide potential therapeutic benefits in ovarian carcinoma.

Down-regulation of salt-inducible kinase 1 (SIK1) is mediated by RNF2 in hepatocarcinogenesis.

Our previous study reported that down-regulation of SIK1 accelerates the growth and invasion of hepatocellular carcinoma (HCC). However, the underlying mechanism leading to SIK1 down-regulation in HCC largely remains to be determined. Herein, we demonstrated that RNF2 expression is negatively correlated with SIK1 levels in HCC tissues. Kaplan-Meier analysis of tumor samples revealed that high RNF2 expression with concurrent low SIK1 expression is associated with poor overall survival. The down-regulation of RNF2 expression in HCC cells significantly reduces tumor cell growth and metastasis, while the simultaneous down-regulation of both RNF2 and SIK1 restores tumor cell growth in vitro and in tumor xenograft models. Mechanistically, we identified RNF2 as an E3 ligase that targets SIK1 for degradation. We further demonstrated that direct physical interaction between RNF2 and SIK1 triggers SIK1 down-regulation in HCC cells. These data suggest that RNF2 is an important upstream negative regulator of SIK1 and that restoration of SIK1 levels induced by loss of RNF2 inhibited HCC cell growth and promoted apoptosis, which may represent a promising therapeutic strategy for HCC treatment.

A Novel Role of Salt-Inducible Kinase 1 (SIK1) in the Post-Translational Regulation of Scavenger Receptor Class B Type 1 Activity.

Salt-inducible kinase 1 (SIK1) is a serine/threonine kinase that belongs to the stress- and energy-sensing AMPK family of kinases. SIK1 expression is rapidly induced in Y1 adrenal cells in response to ACTH via the cAMP-PKA signaling cascade, and it has been suggested that an increased level of SIK1 expression inhibits adrenal steroidogenesis by repressing the cAMP-dependent transcription of steroidogenic proteins, CYP11A1 and StAR, by attenuating CREB transcriptional activity. Here we show that SIK1 stimulates adrenal steroidogenesis by modulating the selective HDL-CE transport activity of SR-B1. Overexpression of SIK1 increases cAMP-stimulated and SR-B1-mediated selective HDL-BODIPY-CE uptake in cell lines without impacting SR-B1 protein levels, whereas knockdown of SIK1 attenuated cAMP-stimulated selective HDL-BODIPY-CE uptake. SIK1 forms a complex with SR-B1 by interacting with its cytoplasmic C-terminal domain, and in vitro kinase activity measurements indicate that SIK1 can phosphorylate the C-terminal domain of SR-B1. Among potential phosphorylation sites, SIK1-catalyzed phosphorylation of Ser496 is critical for SIK1 stimulation of the selective CE transport activity of SR-B1. Mutational studies further demonstrated that both the intact catalytic activity of SIK1 and its PKA-catalyzed phosphorylation are essential for SIK1 stimulation of SR-B1 activity. Finally, overexpression of SIK1 caused time-dependent increases in SR-B1-mediated and HDL-supported steroid production in Y1 cells; however, these effects were lost with knockdown of SR-B1. Taken together, these studies establish a role for SIK1 in the positive regulation of selective HDL-CE transport function of SR-B1 and steroidogenesis and suggest a potential mechanism for SIK1 signaling in modulating SR-B1-mediated selective CE uptake and associated steroidogenesis.

Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1).

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. 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.

Salt-inducible kinase 1 (SIK1) is induced by gastrin and inhibits migration of gastric adenocarcinoma cells.

Salt-inducible kinase 1 (SIK1/Snf1lk) belongs to the AMP-activated protein kinase (AMPK) family of kinases, all of which play major roles in regulating metabolism and cell growth. Recent studies have shown that reduced levels of SIK1 are associated with poor outcome in cancers, and that this involves an invasive cellular phenotype with increased metastatic potential. However, the molecular mechanism(s) regulated by SIK1 in cancer cells is not well explored. The peptide hormone gastrin regulates cellular processes involved in oncogenesis, including proliferation, apoptosis, migration and invasion. The aim of this study was to examine the role of SIK1 in gastrin responsive adenocarcinoma cell lines AR42J, AGS-GR and MKN45. We show that gastrin, known to signal through the Gq/G11-coupled CCK2 receptor, induces SIK1 expression in adenocarcinoma cells, and that transcriptional activation of SIK1 is negatively regulated by the Inducible cAMP early repressor (ICER). We demonstrate that gastrin-mediated signalling induces phosphorylation of Liver Kinase 1B (LKB1) Ser-428 and SIK1 Thr-182. Ectopic expression of SIK1 increases gastrin-induced phosphorylation of histone deacetylase 4 (HDAC4) and enhances gastrin-induced transcription of c-fos and CRE-, SRE-, AP1- and NF-κB-driven luciferase reporter plasmids. We also show that gastrin induces phosphorylation and nuclear export of HDACs. Next we find that siRNA mediated knockdown of SIK1 increases migration of the gastric adenocarcinoma cell line AGS-GR. Evidence provided here demonstrates that SIK1 is regulated by gastrin and influences gastrin elicited signalling in gastric adenocarcinoma cells. The results from the present study are relevant for the understanding of molecular mechanisms involved in gastric adenocarcinomas.

RETRACTED: SIK1 localizes with nephrin in glomerular podocytes and its polymorphism predicts kidney injury

Mutant α-adducin and endogenous ouabain levels exert a causal role in hypertension by affecting renal Na-K ATPase. In addition, mutant β-adducin is involved in glomerular damage through nephrin down-regulation. Recently, the salt-inducible kinase 1 (SIK1) has been shown to exert a permissive role on mutant α-adducin effects on renal Na-K ATPase activity involved in blood pressure (BP) regulation and a SIK1 rs3746951 polymorphism has been associated with changes in vascular Na-K ATPase activity and BP. Here, we addressed the role of SIK1 on nephrin and glomerular functional modifications induced by mutant β-adducin and ouabain, by using congenic substrains of the Milan rats expressing either mutant α- or β-adducin, alone or in combination, ouabain hypertensive rats (OHR) and hypertensive patients. SIK1 co-localized and co-immunoprecipitated with nephrin from glomerular podocytes and associated with caveolar nephrin signaling. In cultured podocytes, nephrin-gene silencing decreased SIK1 expression. In mutant β-adducin congenic rats and in OHR, the podocyte damage was associated with decreased nephrin and SIK1 expression. Conversely, when the effects of β-adducin on podocytes were blocked by the presence of mutant α-adducin, nephrin and SIK1 expressions were restored. Ouabain effects were also reproduced in cultured podocytes. In hypertensive patients, nephrinuria, but not albuminuria, was higher in carriers of mutant SIK1 rs3746951 than in wild-type, implying a more direct effect of SIK1 on glomerular damage. These results demonstrate that, through nephrin, SIK1 is involved in the glomerular effects of mutant adducin and ouabain and a direct effect of SIK1 is also likely to occur in humans.

Abstract 18975: Increased Arterial Blood Pressure and Vascular Fibrosis/remodeling in Mice Lacking Salt-inducible Kinase 1 (sik1)

Arterial fibrosis is one of the main underlying causes of vascular stiffness and is increasingly recognized as an important prognostic factor in the development of high blood pressure (BP), yet its physiophatology is not fully known. Therefore, elucidating the precise mechanism leading to arterial fibrosis will help improving current anti-hypertensive therapies. We previously described in human genetic studies a single nucleotide polymorphism within the salt-inducible kinase 1 (SIK1) gene, affecting the activity of the protein, which is associated with hypertension (lower SIK1 activity is associated with higher BP). Also, lower SIK1 expression induces the expression of transcription factors commonly involved in fibrosis. Therefore, we hypothesized that SIK1 can regulate BP by reducing vascular fibrosis and hence vascular stiffness. To test this, BP and aortic wall structure/composition was evaluated in sik1 -/- and sik1 +/+ mice challenged with a chronic high salt (HS) or normal salt diet (NS). The results showed that the s ik1 -/- mice challenged with HS developed high BP and presented signs of cardiac hypertrophy (increased left ventricle wall thickness and enhanced expression of hypertrophic genes) when compared to their counterparts on NS. On the contrary, BP and cardiac parameters remained unaffected in the sik1 +/+ mice under HS when compared to NS fed mice. Immunohistochemistry analysis revealed increased intima/media thickness and collagen deposition in the aorta of the sik1 -/- vs. sik1 +/+ mice under NS and before becoming hypertensive. Also, qPCR analysis of the aorta of the sik1 -/- mice showed increased expression of other extracellular matrix (ECM) components such as fibronectin and laminin-β1. In addition, the transient knock-down of the SIK1 gene in vascular smooth muscle cell (VSMC) cultures using siRNAs showed a similar increase in the expression of collagen as well as other ECM genes. The present study shows that SIK1 activity is necessary to prevent the development of hypertension, possibly through an anti-fibrotic mechanism regulating vascular wall structure and function. Therefore, SIK1 could become a novel therapeutic target in the treatment of hypertension and other vascular fibrosis-associated diseases.

Abstract 539: MicroRNA-27b Regulates Salt-Inducible Kinase 1 (SIK1) in Vascular Fibrosis

Arterial fibrosis is one of the main underlying causes of vascular stiffness and various associated diseases, such as hypertension, congestive heart failure, aortic aneurysm development and renal failure. Consequently there is a growing interest in (patho)-mechanisms governing the underlying process of arterial fibrosis and successive vascular stiffness. Recently, a novel class of small noncoding RNAs, termed microRNAs (miRs), have emerged as powerful cellular regulators in disease processes. Furthermore, they have become an intriguing target for therapeutic intervention. Aim of the present study was to explore whether miRs are regulating the expression of salt-inducible kinase 1 (SIK1), which we previously identified as a crucial regulator of fibrosis in heart-/kidney- failure and arterial hypertension. Using different bioinformatic target prediction algorithms, we identified several miRs with predicted conserved sites on the Sik1 gene, including miRs-27a/b, -130a, -148a/b and -152. By analyzing aorta, heart and kidney specimen from Sik-/- and Sik+/+ mice, we were able to detect that miR-27b was the only miR being significantly altered in all three tissues. We proceeded by treating human aortic smooth muscle cells as well as adventitial fibroblasts with TGF-β, to induce a pro-fibrotic response. Interestingly, treatment with TGF-β substantially increased miR-27b expression as well as other components of the extracellular matrix, such as collagen (Col1a1) and fibronectin (Fn1) through repression of SIK1. Overexpression of miR-27b, utilizing a pre-miR precursor, substantially decreased SIK-1 expression and further augmented the pro-fibrotic response, as indicated by raising collagen and Fn1 expressions. The present study explores the regulatory role of miR-27b in different vascular fibrosis associated pathologies. We were able to show that miR-27b, a novel downstream-target of TGF-β signalling pathways, suppresses the expression of SIK1, which itself has been identified as a key regulator of arterial hypertension and consequential heart and kidney failure. On-going studies, utilizing inhibition of miR-27b can become an interesting novel therapeutic strategy to regulate SIK1 levels in vascular fibrosis and arterial stiffness.

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.

Abstract 325: Cardiac Hypertrophy in Spontaneous Hypertensive Rats (SHR) is Associated With Decreased Cardiac Expression of SIK1 and SIK3 Isoforms

Numerous reports have highlighted the importance of protein kinases and transcription factors in cardiogenesis and cardiac hypertrophy. The salt-inducible kinase 1 (SIK1), a protein kinase encoded by the snf1lk gene, is involved in early cardiogenesis in mice. Genotype-phenotype association studies in three Swedish and one Japanese population-based cohorts suggested that 15Ser-SIK1 variant (15Gly-Ser change, T allele), bestow augmented SIK1 activity, was associated with lower blood pressure and decreases in left ventricular (LV) mass. The present study aims to determine the potential role of SIK1 as a mediator of cardiac hypertrophic signaling pathway and to evaluate age-related changes on the cardiac expression of several elements of the SIK network associated with hypertension, in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). For this purpose, mRNA expression levels of skeletal actin (SkA), β-myosin heavy chain (β-MHC) and matrix metalloproteinase-9 (MMP-9) as markers for cardiac hypertrophy, SIK isoforms (SIK1, SIK2 and SIK3), and transcription factors (MEF2C, NFAT5c, KFL4, Snail1 and Snail2) were determined, using Taqman, in 13- , 52- and 91-week old SHR and their normotensive counterparts WKY. Systolic and diastolic blood pressures, determined by the tail-cuff method, were significantly higher in 3- , in 3- , 13- and 21-month old SHR than in age-matched WKY. In the SHR, increases in heart/tibia length ratio were associated to the overexpression of cardiac hypertrophic genes (SkA and β-MHC) and the underexpression of cardiac SIK3, at the age of 3- , 13- and 21-months. Ageing accompanied by a diminished mRNA expression of SIK1 in SHR, relative to WKY (both at 13- and 21-months of age). Whereas, Snail2, MFAT5c and KLF4 mRNA levels decreased with aged SHR, comparing to age-match WKY (both at 13- and 21-months of age). These results indicate that SIK1 and SIK3 isoforms might be upstream mediators of transcriptions factors implicated in the regulation of cardiac hypertrophic growth response. In conclusion, ageing in SHR is accompanied by cardiac alterations that include hypertrophy and dysfunction of the SIK signaling network. Supported by grant PIC/IC/83204/2007.

Salt‐inducible kinase 1 regulates E‐cadherin expression and intercellular junction stability

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.