Ste20-like Kinase Research Articles

MST3 Involvement in Na+ and K+ Homeostasis with Increasing Dietary Potassium Intake.

K+ loading inhibits NKCC2 (Na-K-Cl cotransporter) and NCC (Na-Cl cotransporter) in the early distal tubules, resulting in Na+ delivery to the late distal convoluted tubules (DCTs). In the DCTs, Na+ entry through ENaC (epithelial Na channel) drives K+ secretion through ROMK (renal outer medullary potassium channel). WNK4 (with-no-lysine 4) regulates the NCC/NKCC2 through SAPK (Ste20-related proline-alanine-rich kinase)/OSR1 (oxidative stress responsive). K+ loading increases intracellular Cl−, which binds to the WNK4, thereby inhibiting autophosphorylation and downstream signals. Acute K+ loading-deactivated NCC was not observed in Cl−-insensitive WNK4 mice, indicating that WNK4 was involved in K+ loading-inhibited NCC activity. However, chronic K+ loading deactivated NCC in Cl−-insensitive WNK4 mice, indicating that other mechanisms may be involved. We previously reported that mammalian Ste20-like protein kinase 3 (MST3/STK24) was expressed mainly in the medullary TAL (thick ascending tubule) and at lower levels in the DCTs. MST3−/− mice exhibited higher ENaC activity, causing hypernatremia and hypertension. To investigate MST3 function in maintaining Na+/K+ homeostasis in kidneys, mice were fed diets containing various concentrations of Na+ and K+. The 2% KCl diets induced less MST3 expression in MST3−/− mice than that in wild-type (WT) mice. The MST3−/− mice had higher WNK4, NKCC2-S130 phosphorylation, and ENaC expression, resulting in lower urinary Na+ and K+ excretion than those of WT mice. Lower urinary Na+ excretion was associated with elevated plasma [Na+] and hypertension. These results suggest that MST3 maintains Na+/K+ homeostasis in response to K+ loading by regulation of WNK4 expression and NKCC2 and ENaC activity.

Hippo Signaling Pathway in Gliomas.

The Hippo signaling pathway is a highly conserved pathway involved in tissue development and regeneration that controls organ size through the regulation of cell proliferation and apoptosis. The core Hippo pathway is composed of a block of kinases, MST1/2 (Mammalian STE20-like protein kinase 1/2) and LATS1/2 (Large tumor suppressor 1/2), which inhibits nuclear translocation of YAP/TAZ (Yes-Associated Protein 1/Transcriptional co-activator with PDZ-binding motif) and its downstream association with the TEAD (TEA domain) family of transcription factors. This pathway was recently shown to be involved in tumorigenesis and metastasis in several cancers such as lung, breast, or colorectal cancers but is still poorly investigated in brain tumors. Gliomas are the most common and the most lethal primary brain tumors representing about 80% of malignant central nervous system neoplasms. Despite intensive clinical protocol, the prognosis for patients remains very poor due to systematic relapse and treatment failure. Growing evidence demonstrating the role of Hippo signaling in cancer biology and the lack of efficient treatments for malignant gliomas support the idea that this pathway could represent a potential target paving the way for alternative therapeutics. Based on recent advances in the Hippo pathway deciphering, the main goal of this review is to highlight the role of this pathway in gliomas by a state-of-the-art synthesis.

Serine-Threonine Kinase TAO3-Mediated Trafficking of Endosomes Containing the Invadopodia Scaffold TKS5α Promotes Cancer Invasion and Tumor Growth.

Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. In this study, we used our high-content screening assay to identify kinases whose activity affects invadopodia formation. Among the top hits selected for further analysis was TAO3, an STE20-like kinase of the GCK subfamily. TAO3 was overexpressed in many human cancers and regulated invadopodia formation in melanoma, breast, and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in three-dimensional matrices and in vivo. A novel, potent catalytic inhibitor of TAO3 was developed that inhibited invadopodia formation and function as well as tumor cell extravasation and growth. Treatment with this inhibitor demonstrated that TAO3 activity is required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action. SIGNIFICANCE: An unbiased screening approach identifies TAO3 as a regulator of invadopodia formation and function, supporting clinical development of this class of target.

Long non-coding RNA LOC107985656 represses the proliferation of hepatocellular carcinoma cells through activation of the tumor-suppressive Hippo pathway

ABSTRACT Long non-coding RNAs (lncRNAs) play important regulatory roles in hepatocellular carcinoma (HCC). However, the function of LOC107985656 in HCC progression remains unclear. The lncRNA, mRNA and miRNA levels in HCC tissues or cells were measured using real-time quantitative polymerase chain reaction (RT-qPCR). The proliferation of cancer cells was evaluated using 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) viability and colony formation assays. Bioinformatics prediction, dual luciferase assay and RNA pull-down assay were performed to analyze the relationships between LOC107985656 and miR-106b-5p, or miR-106b-5p and large tumor suppressor 1 (LATS1). The protein expression levels were detected using Western blot. Results showed that LncRNA LOC107985656 was downregulated in HCC tissues and cells. Upregulation of LOC107985656 inhibited the proliferation of HCC cells, whereas its knockdown promoted this phenomenon. LOC107985656 could activate the tumor-suppressive Hippo pathway by repressing yes association protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ) (two homologs of Yki) protein expression in HCC. Further investigation suggested that LOC107985656 regulated the expression of LATS1 by acting as a sponge for absorbing miR-106b-5p in HCC cells. In conclusion, this study unraveled the role of LOC107985656 following a ceRNA (competing endogenous RNAs) mechanism for the miR-106b-5p/LATS1 axis in HCC. The results indicate potential diagnostic and therapeutic applications of LOC107985656 in HCC. Abbreviations: HCC: hepatocellular carcinoma; LncRNA: long non-coding RNA; LATS1: large tumor suppressor 1; MTT: 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; YAP: yes association protein; WWTR1: WW domain-containing transcription regulator protein 1; cDNA: single‐stranded complementary DNA; RT-qPCR: real-time quantitative polymerase chain reaction; Radio-Immunoprecipitation Assay (RIPA); BCA: bicinchoninic acid; ASO: antisense oligonucleotide; MST1/2: Ste20-like kinases 1/2; TEAD: TEA domain transcription factor; ceRNA: competing endogenous RNAs.

Dissecting molecular mechanisms underlying H2O2-induced apoptosis of mouse bone marrow mesenchymal stem cell: role of Mst1 inhibition

BackgroundBone marrow mesenchymal stem cell (BM-MSC) has been shown to treat pulmonary arterial hypertension (PAH). However, excessive reactive oxygen species (ROS) increases the apoptosis of BM-MSCs, leading to poor survival and engraft efficiency. Thus, improving the ability of BM-MSCs to scavenge ROS may considerably enhance the effectiveness of transplantation therapy. Mammalian Ste20-like kinase 1 (Mst1) is a pro-apoptotic molecule which increases ROS production. The aim of this study is to uncover the underlying mechanisms the effect of Mst1 inhibition on the tolerance of BM-MSCs under H2O2 condition.MethodsMst1 expression in BM-MSCs was inhibited via transfection with adenoviruses expressing a short hairpin (sh) RNA directed against Mst1 (Ad-sh-Mst1) and exposure to H2O2. Cell viability was detected by Cell Counting Kit 8 (CCK-8) assay, and cell apoptosis was analyzed by Annexin V-FITC/PI, Caspase 3 Activity Assay kits, and pro caspase 3 expression. ROS level was evaluated by the ROS probe DCFH-DA, mitochondrial membrane potential (ΔΨm) assay, SOD1/2, CAT, and GPx expression. Autophagy was assessed using transmission electron microscopy, stubRFP-sensGFP-LC3 lentivirus, and autophagy-related protein expression. The autophagy/Keap1/Nrf2 signal in H2O2-treated BM-MSC/sh-Mst1 was also measured.ResultsMst1 inhibition reduced ROS production; increased antioxidant enzyme SOD1/2, CAT, and GPx expression; maintained ΔΨm; and alleviated cell apoptosis in H2O2-treated BM-MSCs. In addition, this phenomenon was closely correlated with the autophagy/Keap1/Nrf2 signal pathway. Moreover, the antioxidant pathway Keap1/Nrf2 was also blocked when autophagy was inhibited by the autophagy inhibitor 3-MA. However, Keap1 or Nrf2 knockout via siRNA had no effect on autophagy activation or suppression.ConclusionMst1 inhibition mediated the cytoprotective action of mBM-MSCs against H2O2-induced oxidative stress injury. The underlying mechanisms involve autophagy activation and the Keap1/Nrf2 signal pathway.Graphical abstract

Muscle-specific deletion of SLK/Stk2 enhances p38 activity and myogenesis in mdx mice

Duchenne's muscular dystrophy (DMD) is a severe muscle wasting disorder characterized by the loss of dystrophin expression, muscle necrosis, inflammation and fibrosis. Ongoing muscle regeneration is impaired by persistent cytokine stress, further decreasing muscle function. Patients with DMD rarely survive beyond their early 20s, with cardiac and respiratory dysfunction being the primary cause of death. Despite an increase in our understanding of disease progression as well as promising preclinical animal models for therapeutic intervention, treatment options for muscular dystrophy remain limited and novel therapeutic targets are required. Many reports suggest that the TGFβ signalling pathway is activated in dystrophic muscle and contributes to the pathology of DMD in part by impairing the differentiation of myoblasts into mature myofibers. Here, we show that in vitro knockdown of the Ste20-like kinase, SLK, can partially restore myoblast differentiation downstream of TGFβ in a Smad2/3 independent manner. In an mdx model, we demonstrate that SLK is expressed at high levels in regenerating myofibers. Muscle-specific deletion of SLK reduced leukocyte infiltration, increased myogenin and utrophin expression and enhanced differentiation. This was accompanied by resistance to eccentric contraction-induced injury in slow fiber type-enriched soleus muscles. Finally, we found that these effects were partially dependent on the upregulation of p38 signalling. Collectively, these results demonstrate that SLK downregulation can restore some aspects of disease progression in DMD.

FGFR4 promotes nuclear localization of GABP to inhibit cell apoptosis in uterine leiomyosarcoma.

Fibroblast growth factor receptor 4 (FGFR4) has been indicated as a potential "oncogene" in various types of cancer. However, the effects and underlying mechanisms of FGFR4 on uterine leiomyosarcoma (ULMS) progression remain unclear. In this study, we firstly discovered that FGFR4 was upregulated in ULMS specimens and cell lines and closely associated with poor prognosis of ULMS patients. Cell viability and apoptosis assays showed that FGFR4 deletion inhibited cell proliferation and promoted cell apoptosis. Moreover, FGFR4 silence increased cytoplasmic GABP (GA binding protein) expression, while it decreased the nuclear GABP level to inhibit nuclear localization of GABP. Mechanistically, the inhibition ability of FGFR4 silence on nuclear localization of GABP was mediated via mammalian Ste20-like kinases 1 (MST1) activation, which could promote phosphorylation of large tumor suppressor 1 (LATS1) to reduce nuclear localization of GABP. Gain- and loss-of-functional assays indicated that FGFR4 promoted nuclear localization of GABP to inhibit cell apoptosis in ULMS. In conclusion, our findings indicated that FGFR4 inhibited cell apoptosis in ULMS via the promotion of MST1/LATS1-mediated GABP nuclear localization, shedding light on the underlying mechanism of FGFR4-induced ULMS progression.

MST4 Regulates Epithelial-Mesenchymal Transition of Choriocarcinoma by Mediating TGF-β1 Expression.

BackgroundMammalian Ste20-like kinase 4 (MST4), also known as serine/threonine kinase 26 (STK26), promotes development of several cancers and is found to be highly expressed in the placenta. However, in choriocarcinoma that originated from the placenta, the expression of MST4 was undetermined and its mechanism was unknown. In this study, the expression of MST4 in choriocarcinoma as well as the underlying mechanism was explored.PurposeTo detect the expression of MST4 in patient samples and mechanism of mediating EMT by MST4 in choriocarcinoma.Patients and MethodsThe metastatic lesions of choriocarcinoma (n=17) and volunteer villus (n=17) were collected to determine MST4 expression using immunohistochemistry and H&E staining. We use siRNA and lentiviral vector to knockdown MST4 and use plasmid to overexpress MST4 in choriocarcinoma. Then, we apply real-time polymerase chain reaction (RT-PCR), Western blot assay and immunofluorescence assay to detect target protein expressions. Cell invasion and migration and cell proliferation were detected by transwell assay and wound healing assay and CCK-8 and cell colony formation.ResultsMST4 is lowly expressed in the metastatic lesions of choriocarcinoma patients when compared with normal villus. Knockdown of MST4 activated epithelial–mesenchymal transition (EMT) process, significantly increasing the ability of invasion and migration in choriocarcinoma cell lines (JAR and JEG-3). In contrast, the EMT process was restrained in choriocarcinoma cell lines with overexpressed MST4. Meanwhile, genome-wide gene expression array, Western blot and ELISA revealed that tumor growth factor-beta 1 (TGF-β1) has significantly increased. The EMT process and metastatic prompting biofunction were reversed after using TGF-β1 inhibitor (LY364947) in the choriocarcinoma cell lines with MST4 knockdown.ConclusionOur studies demonstrated that MST4 was lowly expressed in patient samples. Additionally, JAR and JEG-3 increase cell invasion and migration ability while there is no influence on cell proliferation with MST4 knockdown. Conversely, the metastatic ability of JAR and JEG-3 was decreased with overexpressed MST4. Moreover, TGF-β1 was a key factor after MST4 knockdown. In conclusion, MST4 affects choriocarcinoma EMT by mediating TGF-β1 expression.

TRAF6-mediated ubiquitination of MST1/STK4 attenuates the TLR4-NF-κB signaling pathway in macrophages.

Pattern-recognition receptors including Toll-like receptors (TLRs) recognize invading pathogens and trigger an immune response in mammals. Here we show that mammalian ste20-like kinase 1/serine/threonine kinase 4 (MST1/STK4) functions as a negative regulator of lipopolysaccharide (LPS)-induced activation of the TLR4-NF-κB signaling pathway associated with inflammation. Myeloid-specific genetic ablation of MST1/STK4 increased the susceptibility of mice to LPS-induced septic shock. Ablation of MST1/STK4 also enhanced NF-κB activation triggered by LPS in bone marrow-derived macrophages (BMDMs), leading to increased production of proinflammatory cytokines by these cells. Furthermore, MST1/STK4 inhibited TRAF6 autoubiquitination as well as TRAF6-mediated downstream signaling induced by LPS. In addition, we found that TRAF6 mediates the LPS-induced activation of MST1/STK4 by catalyzing its ubiquitination, resulting in negative feedback regulation by MST1/STK4 of the LPS-induced pathway leading to cytokine production in macrophages. Together, our findings suggest that MST1/STK4 functions as a negative modulator of the LPS-induced NF-κB signaling pathway during macrophage activation.

Mammalian Ste20-like kinase 4 inhibits the inflammatory response in Aspergillus fumigatus keratitis

Mammalian Ste20-like kinase 4 (MST4), a new member of the germinal-center kinase STE20 family, was recently demonstrated to be a negative regulator of inflammation. However, whether MST4 participates in the inflammatory response to fungal infection remains unknown. Our study investigated the role and molecular mechanisms of MST4 in mice cornea and corneal epithelial cells exposed to Aspergillus fumigatus (A. fumigatus). Protein level of MST4 was detected in mice corneas and human corneal epithelial cells (HCECs) by Western blot analysis. The MST4 protein level was significantly elevated in mice corneas infected with A. fumigatus and HCECs exposed to A. fumigatus. MST4 expression was also detected in mice corneas by immunofluorescence staining. Furthermore, we found recombinant MST4 inhibited proinflammatory cytokines expressions induced by A. fumigatus at both the mRNA and protein levels in mice corneas and HCECs. To further investigate the mechanism of MST4′s anti-inflammatory effect in A. fumigatus keratitis, we verified recombinant MST4 can inhibit curdlan-mediated proinflammatory cytokines production in HCECs. Surprisingly, recombinant MST4 protein downregulated A. fumigatus-induced Dectin-1 expression in both mRNA and protein levels in mice corneas. Recombinant MST4 can inhibit the mRNA expression level of Dectin-1 which was induced by curdlan in HCECs. MST4 can also inhibit the expression of Dectin-1 in mRNA levels increased by Dectin-1 overexpression plasmid in HCECs. Moreover, A. fumigatus or curdlan significantly induced the phosphorylation of Syk, which was consequently suppressed by recombinant MST4. Finally, recombinant MST4 promotes HCECs proliferation, which contribute to cornea wound healing. Taken together, our results provide evidences that MST4 inhibits inflammatory signaling response in A. fumigatus keratitis by downregulating Dectin-1/p-Syk pathway and simultaneously promotes HCECs proliferation.

STRIPAK regulates Slik localization to control mitotic morphogenesis and epithelial integrity.

Proteins of the ezrin, radixin, and moesin (ERM) family control cell and tissue morphogenesis. We previously reported that moesin, the only ERM in Drosophila, controls mitotic morphogenesis and epithelial integrity. We also found that the Pp1-87B phosphatase dephosphorylates moesin, counteracting its activation by the Ste20-like kinase Slik. To understand how this signaling pathway is itself regulated, we conducted a genome-wide RNAi screen, looking for new regulators of moesin activity. We identified that Slik is a new member of the striatin-interacting phosphatase and kinase complex (STRIPAK). We discovered that the phosphatase activity of STRIPAK reduces Slik phosphorylation to promote its cortical association and proper activation of moesin. Consistent with this finding, inhibition of STRIPAK phosphatase activity causes cell morphology defects in mitosis and impairs epithelial tissue integrity. Our results implicate the Slik-STRIPAK complex in the control of multiple morphogenetic processes.

Downregulation of STK4 promotes colon cancer invasion/migration through blocking β-catenin degradation.

Mammalian STE20-like kinase 1 (MST1/STK4/KRS2) encodes a serine/threonine kinase that is the mammalian homolog of Drosophila Hippo. STK4 plays an important role in controlling cell growth, apoptosis, and organ size. STK4 has been studied in many cancers with previous studies indicating an involvement in colon cancer lymph node metastasis and highlighting its potential as a diagnostic marker for colon cancer. However, the role of STK4 defect in promoting colon cancer progression is still understudied. Here, we found that STK4 was significantly downregulated in colon cancer and was associated with distal metastasis and poor survival. Furthermore, STK4 knockdown enhanced sphere formation and metastasis invitro and promoted tumor development invivo. We found that STK4 colocalized with β-catenin and directly phosphorylated β-catenin resulting in its degradation via the ubiquitin-mediated pathway. This may suggest that STK4 knockdown causes β-catenin phosphorylation failure and subsequently β-catenin accumulation, consequently leading to anchorage-independent growth and metastasis in colon cancer. Our results support that STK4 may act as a potential candidate for the assessment of β-catenin-mediated colon cancer prognosis.

LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.

Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.

Mammalian STE20-Like Kinase 2 Promotes Lipopolysaccharides-Mediated Cardiomyocyte Inflammation and Apoptosis by Enhancing Mitochondrial Fission.

In this study, we analyzed the role of mammalian STE20-like protein kinase 2 (Mst2), a serine-threonine protein kinase, in Lipopolysaccharides (LPS)-mediated inflammation and apoptosis in the H9C2 cardiomyocytes. Mst2 mRNA and protein levels were significantly upregulated in the LPS-treated H9C2 cardiomyocytes. LPS treatment induced expression of IL-2, IL-8, and MMP9 mRNA and proteins in the H9C2 cardiomyocytes, and this was accompanied by increased caspase-3/9 mediating H9C2 cardiomyocyte apoptosis. LPS treatment also increased mitochondrial reactive oxygen species (ROS) and the levels of antioxidant enzymes, such as GSH, SOD, and GPX, in the H9C2 cardiomyocytes. The LPS-treated H9C2 cardiomyocytes showed lower cellular ATP levels and mitochondrial state-3/4 respiration but increased mitochondrial fragmentation, including upregulation of the mitochondrial fission genes Drp1, Mff, and Fis1. LPS-induced inflammation, mitochondrial ROS, mitochondrial fission, and apoptosis were all significantly suppressed by pre-treating the H9C2 cardiomyocytes with the Mst2 inhibitor, XMU-MP1. However, the beneficial effects of Mst2 inhibition by XMU-MP1 were abolished by carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), a potent activator of mitochondrial fission. These findings demonstrate that Mst2 mediates LPS-induced cardiomyocyte inflammation and apoptosis by increasing mitochondrial fission.

Loss of the Ste20-like kinase induces a basal/stem-like phenotype in HER2-positive breast cancers.

HER2 is overexpressed in 20-30% of all breast cancers and is associated with an invasive disease and poor clinical outcome. The Ste20-like kinase (SLK) is activated downstream of HER2/Neu and is required for efficient epithelial-to-mesenchymal transition, cell cycle progression, and migration in the mammary epithelium. Here we show that loss of SLK in a murine model of HER2/Neu-positive breast cancers significantly accelerates tumor onset and decreases overall survival. Transcriptional profiling of SLK knockout HER2/Neu-derived tumor cells revealed a strong induction in the triple-negative breast cancer marker, Sox10, accompanied by an increase in mammary stem/progenitor activity. Similarly, we demonstrate that SLK and Sox10 expression are inversely correlated in patient samples, with the loss of SLK and acquisition of Sox10 marking the triple-negative subtype. Furthermore, pharmacological inhibition of AKT reduces SLK-null tumor growth in vivo and is rescued by ectopic Sox10 expression, suggesting that Sox10 is a critical regulator of tumor growth downstream of SLK/AKT. These findings highlight a role for SLK in negatively regulating HER2-induced mammary tumorigenesis and provide mechanistic insight into the regulation of Sox10 expression in breast cancer.

Ste20-like Kinase-mediated Control of Actin Polymerization Is a New Mechanism for Thin Filament-associated Regulation of Airway Smooth Muscle Contraction.

It has been reported that actin polymerization is regulated by protein tyrosine phosphorylation in smooth muscle on contractile stimulation. The role of protein serine/threonine phosphorylation in modulating actin dynamics is underinvestigated. SLK (Ste20-like kinase) is a serine/threonine protein kinase that plays a role in apoptosis, cell cycle, proliferation, and migration. The function of SLK in smooth muscle is mostly unknown. Here, SLK knockdown (KD) inhibited acetylcholine (ACh)-induced actin polymerization and contraction without affecting myosin light chain phosphorylation at Ser-19 in human airway smooth muscle. Stimulation with ACh induced paxillin phosphorylation at Ser-272, which was reduced in SLK KD cells. However, SLK did not catalyze paxillin Ser-272 phosphorylation in vitro. But, SLK KD attenuated Plk1 (polo-like kinase 1) phosphorylation at Thr-210. Plk1 mediated paxillin phosphorylation at Ser-272 in vitro. Expression of the nonphosphorylatable paxillin mutant S272A (substitution of alanine at Ser-272) attenuated the agonist-enhanced F-actin/G-actin ratios without affecting myosin light chain phosphorylation. Because N-WASP (neuronal Wiskott-Aldrich Syndrome Protein) phosphorylation at Tyr-256 (an indication of its activation) promotes actin polymerization, we also assessed the role of paxillin phosphorylation in N-WASP activation. S272A paxillin inhibited the ACh-enhanced N-WASP phosphorylation at Tyr-256. Together, these results suggest that SLK regulates paxillin phosphorylation at Ser-272 via Plk1, which modulates N-WASP activation and actin polymerization in smooth muscle. SLK-mediated actin cytoskeletal reorganization may facilitate force transmission between the contractile units and the extracellular matrix.

Mammalian STE20‑like kinase1 regulates pancreatic cancer cell survival and migration through Mfn2‑mediated mitophagy.

Mammalian STE20-like kinase 1 (MST1) plays an important role in pancreatic cancer progression, but its downstream targets are still unknown. In the present study, our results indicated that MST1 expression was significantly downregulated in pancreatic cancer cell lines (PANC-1, BxPC-3 and HPAC) compared with that in the normal ductal epithelial cell line (hTERT-HPNE). Moreover, MST1 overexpression in PANC-1 cells led to increased apoptosis as determined by MTT and TUNEL assays and inhibited cellular migration. Mechanistically, upregulation of MST1 expression caused mitochondrial dysfunction, decreased ATP production, and activation of the mitochondrial-dependent apoptotic pathway via inhibition of mitofusin 2 (Mfn2)-mediated mitophagy, which ultimately resulted in increased cellular apoptosis and decreased cellular migration. Collectively, the present study demonstrated that MST1 may regulate pancreatic cancer PANC-1 cell survival, invasion and migration through Mfn2-mediated mitophagy, laying the foundation for the exploration of novel therapeutic targets for pancreatic cancer.

TAOK1 positively regulates TLR4-induced inflammatory responses by promoting ERK1/2 activation in macrophages

Thousand and one amino acid kinase 1 (TAOK1) is a member of Ste20-like kinases, but its function in regulating inflammatory responses remains largely unknown. In this study, we identify TAOK1 as a positive regulator of TLR4-triggered inflammatory responses in macrophages. TAOK1 increases LPS-induced production of pro-inflammatory cytokine such as IL-6, TNF-α and IL12p40 in macrophages. TAOK1 deficient mice showed decreased susceptibility to endotoxin shock, with less pro-inflammatory cytokine production than control mice. TAOK1 promotes LPS-induced activation of ERK1/2 by constitutively interacting with TRAF6 and TPL2. These finding unravel the important role of TAOK1 as a positive regulator of TLR4-induced inflammatory responses.

Genetic ablation of SLK exacerbates glomerular injury in adriamycin nephrosis in mice

The Ste20-like kinase SLK is critical for embryonic development, and may play an important role in wound healing, muscle homeostasis, cell migration and tumor growth. Mice with podocyte-specific deletion of SLK show albuminuria, and damage to podocytes, as they age. The present study addresses the role of SLK in glomerular injury. We induced adriamycin nephrosis in 3-4 month control and podocyte SLK knockout (KO) mice. Compared with control, SLK deletion exacerbated albuminuria and loss of podocytes, synaptopodin and podocalyxin. Glomeruli of adriamycin-treated SLK KO mice showed diffuse increases in matrix and sclerosis, as well as collapse of the actin cytoskeleton. SLK can phosphorylate ezrin. The complex of phospho-ezrin, NHERF2 and podocalyxin in the apical domain of the podocyte is a key determinant of normal podocyte architecture. Deletion of SLK reduced glomerular ezrin and ezrin phosphorylation in adriamycin nephrosis. Also, deletion of SLK reduced the colocalization of ezrin and podocalyxin in the glomerulus. Cultured glomerular epithelial cells with KO of SLK showed reduced ezrin phosphorylation and podocalyxin expression, as well as reduced F-actin. Thus, SLK deletion leads to podocyte injury as mice age, and exacerbates injury in adriamycin nephrosis. The mechanism may at least in part involve ezrin phosphorylation, as well as disruption of the cytoskeleton and podocyte apical membrane structure.

Mst1 knockdown alleviates cardiac lipotoxicity and inhibits the development of diabetic cardiomyopathy in db/db mice

Diabetic cardiomyopathy (DCM) accounts for increasing deaths of diabetic patients, and effective therapeutic targets are urgently needed. Myocardial lipotoxicity, which is caused by cardiac non-oxidative metabolic fatty acids and cardiotoxic fatty acid metabolites accumulation, has gained more attention to explain the increasing prevalence of DCM. However, whether mammalian Ste20-like kinase 1 (Mst1) plays a role in lipotoxicity in type 2 diabetes-induced cardiomyopathy has not yet been illustrated. Here, we found that Mst1 expression was elevated transcriptionally in the hearts of type 2 diabetes mellitus mice and palmitic acid-treated neonatal rat ventricular myocytes. Adeno-associated virus 9 (AAV9)-mediated Mst1 silencing in db/db mouse hearts significantly alleviated cardiac dysfunction and fibrosis. Notably, Mst1 knockdown in db/db mouse hearts decreased lipotoxic apoptosis and inflammatory response. Mst1 knockdown exerted protective effects through inactivation of MAPK/ERK kinase kinase 1 (MEKK1)/c-Jun N-terminal kinase (JNK) signaling pathway. Moreover, lipotoxicity induced Mst1 expression through promoting the binding of forkhead box O3 (FoxO3) and Mst1 promoter. Conclusively, we elucidated for the first time that Mst1 expression is regulated by FOXO3 under lipotoxicity stimulation and downregulation of Mst1 protects db/db mice from lipotoxic cardiac injury through MEKK1/JNK signaling inhibition, indicating that Mst1 abrogation may be a potential treatment strategy for DCM in type 2 diabetic patients.