Mammalian Sterile 20-like Kinase Research Articles

Manipulating the Hippo-Yap signal cascade in stem cells for heart regeneration.

The Hippo-Yap pathway was originally recognized as a crucial signal cascade controlling organ size, and more recently identified as an important component involved in the regulation of cardiomyocyte survival, proliferation, and regeneration. Negative stress responses can activate mammalian sterile 20-like kinase 1 (Mst1) to suppress protective autophagy and promote cardiomyocyte apoptosis via phosphorylation and inhibition of Bcl-xL. Moreover, decreased Yap activity and nuclear entry will decrease upon Mst1 activation, ultimately suppressing cardiomyocytes proliferation and regeneration. Based on these observations, there are potential therapeutic opportunities in cardiac structural and functional regeneration post myocardium infarction to be gained by manipulation of the Hippo-Yap signal cascade. This review will summarize the main components of the Hippo-Yap pathway and their molecular biological functions. It will then highlight the role of these signal modules in the acquisition of stem cell pluripotency, cardiogenic differentiation, cardiomyocyte proliferation and maturation, and mitochondrial biogenesis in cardiac stem cells. Finally, it will discuss the potential for future studies of Hippo-Yap pathway using induced pluripotent stem cell (iPSC) technology.

Differential localization of A-Raf regulates MST2-mediated apoptosis during epithelial differentiation.

A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the MEK-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. We recently identified A-Raf as a potent inhibitor of the proapoptotic mammalian sterile 20-like kinase (MST2) tumor suppressor pathway in several cancer entities including head and neck, colon, and breast. Independent of kinase activity, A-Raf binds to MST2 thereby efficiently inhibiting apoptosis. Here, we show that the interaction of A-Raf with the MST2 pathway is regulated by subcellular compartmentalization. Although in proliferating normal cells and tumor cells A-Raf localizes to the mitochondria, differentiated non-carcinogenic cells of head and neck epithelia, which express A-Raf at the plasma membrane. The constitutive or induced re-localization of A-Raf to the plasma membrane compromises its ability to efficiently sequester and inactivate MST2, thus rendering cells susceptible to apoptosis. Physiologically, A-Raf re-localizes to the plasma membrane upon epithelial differentiation in vivo. This re-distribution is regulated by the scaffold protein kinase suppressor of Ras 2 (KSR2). Downregulation of KSR2 during mammary epithelial cell differentiation or siRNA-mediated knockdown re-localizes A-Raf to the plasma membrane causing the release of MST2. By using the MCF7 cell differentiation system, we could demonstrate that overexpression of A-Raf in MCF7 cells, which induces differentiation. Our findings offer a new paradigm to understand how differential localization of Raf complexes affects diverse signaling functions in normal cells and carcinomas.

The tumor suppressive role of RASSF1A in osteosarcoma through the Wnt signaling pathway.

Ras-association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene and its expression is lost in numerous types of cancer cells, including primary osteosarcoma cells. However, its functional significance in osteosarcoma has not been well defined. The messenger RNA (mRNA) expression of RASSF1A in osteosarcoma tissues and corresponding non-tumoral tissues was measured by real-time PCR. Overexpression of RASSF1A was established by an adenoviral vector expressing RASSF1A. Cell migration and invasion were analyzed in transwells. Apoptosis and cell cycle were analyzed using flow cytometry. Wnt/β-catenin activity was measured by TCF reporter dual-luciferase assay. Cell viability was measured by MTT assay. Protein expression was detected by Western blot. RASSF1A mRNA expression was significantly lower in osteosarcoma tissues than that in the corresponding non-tumoral tissues. The lowered RASSF1A expression correlated with the clinical severity of osteosarcoma. rAd-RASSF1A injection significantly inhibited the growth of xenograft MNNG/HOS tumors in mice. Overexpression of RASSF1A resulted in significant inhibition of the proliferation, migration, and invasion; induced apoptosis; and arrested cell cycle at G0/G1 phase in both the MNNG/HOS and SaOS2 cells. Overexpression of RASSF1A inhibited the Wnt/β-catenin activity, decreased phosphorylation of Akt/glycogen synthase kinase-3-β (GSK3-β), and increased phosphorylation of mammalian sterile 20-like kinase 1 (MST1). Overexpression of RASSF1A downregulated the cyclin D1, c-Myc, and matrix metalloproteinase-7 (MMP-7) protein levels. RASSF1A functions as a tumor suppressor in osteosarcoma and exerts anti-cancer roles through regulating Akt/GSK-3-Wnt/β-catenin signaling.

Mammalian Sterile 20-like Kinase 1 (Mst1) Enhances the Stability of Forkhead Box P3 (Foxp3) and the Function of Regulatory T Cells by Modulating Foxp3 Acetylation

Regulatory T cells (Tregs) play crucial roles in maintaining immune tolerance. The transcription factor Foxp3 is a critical regulator of Treg development and function, and its expression is regulated at both transcriptional and post-translational levels. Acetylation by lysine acetyl transferases/lysine deacetylases is one of the main post-translational modifications of Foxp3, which regulate Foxp3's stability and transcriptional activity. However, the mechanism(s) by which the activities of these lysine acetyl transferases/lysine deacetylases are regulated to preserve proper Foxp3 acetylation during Treg development and maintenance of Treg function remains to be determined. Here we report that Mst1 can enhance Foxp3 stability, its transcriptional activity, and Treg function by modulating the Foxp3 protein at the post-translational level. We discovered that Mst1 could increase the acetylation of Foxp3 by inhibiting Sirt1 activity, which requires the Mst1 kinase activity. We also found that Mst1 could attenuate Sirt1-mediated deacetylation of Foxp3 through directly interacting with Foxp3 to prevent or interfere the interaction between Sirt1 and Foxp3. Therefore, Mst1 can regulate Foxp3 stability in kinase-dependent and kinase-independent manners. Finally, we showed that treatment of Mst1(-/-) Tregs with Ex-527, a Sirt1-specific inhibitor, partially restored the suppressive function of Mst1(-/-) Tregs. Our studies reveal a novel mechanism by which Mst1 enhances Foxp3 expression and Treg function at the post-translational level.

Luteolin alleviates post-infarction cardiac dysfunction by up-regulating autophagy through Mst1 inhibition.

Myocardial infarction (MI), which is characterized by chamber dilation and LV dysfunction, is associated with substantially higher mortality. We investigated the effects and underlying mechanisms of Luteolin on post-infarction cardiac dysfunction. Myocardial infarction was constructed by left anterior descending coronary artery ligation. In vitro, cultured neonatal cardiomyocytes subjected to simulated MI were used to probe mechanism. Luteolin significantly improved cardiac function, decreased cardiac enzyme and inflammatory cytokines release after MI. Enhanced autophagic flux as indicated by more autophagosomes puncta, less accumulation of aggresomes and P62 in the neonatal cardiomyocytes after hypoxia was observed in the Luteolin pre-treatment group. Western blot analysis also demonstrated that Luteolin up-regulated autophagy in the cardiomyocytes subjected to simulated MI injury. Furthermore, Luteolin increased mitochondrial membrane potential, adenosine triphosphate content, citrate synthase activity and complexes I/II/III/IV/V activities in the cardiomyocytes subjected to simulated MI injury. Interestingly, mammalian sterile 20-like kinase 1 (Mst1) knockout abolished the protective effects of Luteolin administration. Luteolin enhances cardiac function, reduces cardiac enzyme and inflammatory markers release after MI. The protective effects of Luteolin are associated with up-regulation of autophagy and improvement of mitochondrial biogenesis through Mst1 inhibition.

Emerging role of Hpo signaling and YAP in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of cancer-related mortality worldwide. Due to the poor prognosis and limited therapeutic options, there is great interest in further understanding better the molecular underpinnings and potential molecular targets associated with HCC. The Hippo (Hpo) signaling pathway and YAP, its principal downstream effector, represent an innovative area of research in HCC. Pioneered in Drosophila melanogaster, the Hpo cascade controls tissue homeostasis including organ size, cell proliferation, apoptosis, as well as cell-cycle regulation and differentiation. This conserved kinase cascade in mammals depends on central control by the tumor suppressor mammalian sterile 20-like kinase 1/2 (Mst1/2). The Mst1/2 commences the downstream kinase cascade, ultimately activating the oncoprotein YAP and allowing its physical association with downstream targets to enhance the gene expression signatures that are involved in proliferation and survival. Alterations in YAP expression and defective regulation of other key Hpo pathway members, such as Mst1/2, Salvador, neurofibromatosis and Mer (Nf2/mer), large tumor suppressor homolog 1/2 (Lats1/2), and Mps one binder kinase activator-like 1A and 1B (Mob1) drive carcinogenesis in animal models. The dysregulation of the Hpo pathway – resulting in an unchecked activation of YAP – culminates in the development of a broad range of human tumor types, including HCC. The abrogation of Mst1/2-mediated YAP phosphorylation permits YAP entry into the nucleus in murine models and functions similarly in human HCCs. Chemoresistance mechanisms displayed by HCC tumors occur in a YAP-dependent manner. The HCC specimens exhibit YAP overexpression, and YAP serves as an independent prognostic marker for disease-free survival and overall survival in patients with HCC. Recently, the small molecule inhibitor, verteporfin has been shown to attenuate YAP activity in murine models, perhaps offering a novel therapeutic approach for patients with advanced HCC.

MiR-155-dependent regulation of mammalian sterile 20-like kinase 2 (MST2) coordinates inflammation, oxidative stress and proliferation in vascular smooth muscle cells

In response to vascular injury, inflammation, oxidative stress, and cell proliferation often occur simultaneously in vascular tissues. We previously observed that microRNA-155 (miR-155), which is implicated in proliferation and inflammation is involved in neointimal hyperplasia; however, the molecular mechanisms by which it regulates these processes remain largely unknown. In this study, we observed that vascular smooth muscle cell (VSMC) proliferation and neointimal formation in wire-injured femoral arteries were reduced by the loss of miR-155 and increased by the gain of miR-155. The proliferative effect of miR-155 was also observed in cultured VSMCs. Notably, expression of the miR-155-target protein mammalian sterile 20-like kinase 2 (MST2) was increased in the injured arteries of miR-155−/− mice. miR-155 directly repressed MST2 and thus activated the extracellular signal-regulated kinase (ERK) pathway by promoting an interaction between RAF proto-oncogene serine/threonine-protein kinase (Raf-1) and mitogen-activated protein kinase kinase (MEK) and stimulating inflammatory and oxidative stress responses; together, these effects lead to VSMC proliferation and vascular remodeling. Our data reveal that MST2 mediates miR-155-promoted inflammatory and oxidative stress responses by altering the interaction of MEK with Raf-1 and MST2 in response to vascular injury. Therefore, suppression of endogenous miR-155 might be a novel therapeutic strategy for vascular injury and remodeling.

Mst2 Controls Bone Homeostasis by Regulating Osteoclast and Osteoblast Differentiation.

Mammalian sterile 20-like kinase 2 (Mst2) plays a central role in the Hippo pathway, controlling cell proliferation, differentiation, and apoptosis during development. However, the roles of Mst2 in osteoclast and osteoblast development are largely unknown. Here, we demonstrate that mice deficient in Mst2 exhibit osteoporotic phenotypes with increased numbers of osteoclasts and decreased numbers of osteoblasts as shown by micro-computed tomography (µCT) and histomorphometric analyses. Osteoclast precursors lacking Mst2 exhibit increased osteoclastogenesis and Nfatc1, Acp5, and Oscar expression in response to receptor activator of NF-κB ligand (RANKL) exposure. Conversely, Mst2 overexpression in osteoclast precursors leads to the inhibition of RANKL-induced osteoclast differentiation. Osteoblast precursors deficient in Mst2 exhibit attenuated osteoblast differentiation and function by downregulating the expression of Runx2, Alpl, Ibsp, and Bglap. Conversely, ectopic expression of Mst2 in osteoblast precursors increases osteoblastogenesis. Finally, we demonstrate that the NF-κB pathway is activated by Mst2 deficiency during osteoclast and osteoblast development. Our findings suggest that Mst2 is involved in bone homeostasis, functioning as a reciprocal regulator of osteoclast and osteoblast differentiation through the NF-κB pathway.

Mst1 inhibition rescues β1-adrenergic cardiomyopathy by reducing myocyte necrosis and non-myocyte apoptosis rather than myocyte apoptosis

It is generally held that inhibition of mammalian sterile 20-like kinase 1 (Mst1) protects the heart through reducing myocyte apoptosis. We determined whether inhibition with a dominant-negative Mst1 (DN-Mst1) would protect against the cardiomyopathy induced by chronic β1-adrenergic receptor (β1-AR) stimulation by preventing myocyte apoptosis. DN-Mst1 mice were mated with β1-AR transgenic (Tg) mice and followed for 20 months. β1-AR Tg mice developed cardiomyopathy as they aged, as reflected by premature mortality and depressed cardiac function, which were rescued in β1-AR × DN-Mst1 bigenic mice. Surprisingly, myocyte apoptosis did not significantly decrease with Mst1 inhibition. Instead, Mst1 inhibition predominantly reduced non-myocyte apoptosis, e.g., fibroblasts, macrophages, neutrophils and endothelial cells. Fibrosis in the hearts with cardiomyopathy increased fivefold and this increase was nearly abolished in the bigenic mice with Mst1 inhibition. Regression analysis showed no correlation between myocyte apoptosis and cardiac function or myocyte number, whereas the latter two correlated significantly, p < 0.05, with fibrosis, which generally results from necrosis. To examine the role of myocyte necrosis, chronic β-AR stimulation with isoproterenol was induced for 24 h and myocyte necrosis was assessed by 1% Evans blue dye. Compared to WT, DN-Mst1 mice showed significant inhibition, p < 0.05, of myocyte necrosis. We confirmed this result in Mst1-knockout mice, which also showed significant protection, p < 0.05, against myocyte necrosis compared to WT. These data indicate that Mst1 inhibition rescued cardiac fibrosis and myocardial dysfunction in β1-AR cardiomyopathy. However, this did not occur through Mst1 inhibition of myocyte apoptosis but rather by inhibition of cardiomyocyte necrosis and non-myocyte apoptosis, features of Mst1 not considered previously.

Role of Mammalian Sterile20-like Kinase 1 and 2 in Oxidative Stress

Mammalian sterile 20 like kinase (Mst) 1 and Mst2 are stress-activated kinases, activated by various stress stimuli including oxidative stress. Mst1/2 act as tumor suppressor proteins and their deregulation is associated with the development of cancers, such as liver cancer and prostate cancer. Cellular oxidative stress is known to upregulate Mst1/2 activity; activation of Mst1/2 induces apoptosis in oxidative stress conditions. Until recently, the exact mechanism/s of Mst1/2 activation and Mst1/2induced apoptosis under oxidative stress conditions was not clear. But some recent reports have begun to elucidate the mechanisms of Mst1/2 activation by oxidative stress. Not only Mst1/2 activity is regulated by oxidative stress, but Mst1/2 are also involved in the regulation of redox levels in cells. In this short report we will discuss various mechanisms of Mst1/2 activation and Mst1/2-dependent apoptosis in response to oxidative stress.

RASSF5 inhibits growth and invasion and induces apoptosis in osteosarcoma cells through activation of MST1/LATS1 signaling.

Ras association (RalGDS/AF-6) domain family member RASSF5 has been implicated in a variety of key biological processes, including cell proliferation, cell cycle regulation and apoptosis. It is believed to play an important role in tumorigenesis as a tumor suppressor in a number of malignancies. Yet, little is known concerning the function and underlying mechanisms of RASSF5 in human osteosarcoma (OS). The expression of RASSF5 was examined by immunohistochemical assay using a tissue microarray in 45cases of OS tissues. A gain-of-function approach was used to observe the effects of lentiviral vector-mediated overexpression of RASSF5 (Lv-RASSF5) on cell growth, invasion and apoptosis, respectively, as indicated by MTT, Transwell and flow cytometry assays, and the expression levels of mammalian sterile 20-like (MST1) kinase, large tumor suppressor1 (LATS1), proliferating cell nuclear antigen (PCNA), matrix metallopeptidase-9 (MMP-9) and p53 were detected by real-time PCR and western blot assays in OS cells (MG-63 and U-2 OS). The results indicated that the expression of RASSF5 protein was significantly downregulated in OS tissues compared to that in adjacent non-cancerous tissues (ANCT) (40.0 vs. 73.3%, P=0.002), and had a negative correlation with distant metastasis of the tumor (P=0.01). Overexpression of RASSF5 markedly suppressed cell proliferation and invasion, and induced cell apoptosis in the OS cell lines with increased expression of MST1, LATS1 and p53 and decreased expression of PCNA and MMP-9. Taken together, our findings demonstrate that RASSF5 expression is negatively correlated with distant metastasis of OS, and RASSF5 may function as a tumor suppressor in OS cells through activation of the MST1/LATS1 pathway.

One Hippo and many masters: differential regulation of the Hippo pathway in cancer.

The Hippo/MST2 (mammalian sterile 20-like kinase 2) pathway is a signalling cascade evolutionarily conserved in its structure. Originally described in Drosophila melanogaster as a regulator of organ size, this pathway has greater functions in mammals. Disturbance of mammalian MST2 pathway is associated with tumorigenesis by affecting apoptosis, cell cycle and polarity. In addition, this pathway has been shown to cross-talk with mitogenic pathways at multiple levels. In the present mini-review, we discuss our contribution highlighting the regulation of MST2 signalling by frequently observed oncogenic perturbations affecting mitogenic pathways. In particular, we review the role of RAS isoforms and PI3K (phosphoinositide 3-kinase)/Akt in the regulation of MST2 activity by phosphorylation. We also put the emphasis on RAF-induced control of MST2 signalling by protein-protein interactions. Finally, we recapitulate some of the direct mechanisms, such as ubiquitin-dependent degradation or gene silencing by promoter hypermethylation, involved in MST2 pathway component down-regulation in cancers.

Genetic deletion of Mst1 alters T cell function and protects against autoimmunity.

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1−/− CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

Gemcitabine-induced pancreatic cancer cell death is associated with MST1/cyclophilin D mitochondrial complexation.

The pancreatic adenocarcinoma remains the most aggressive human malignancy with an extremely low 5-year overall survival. Postoperative gemcitabine could significantly delay recurrence after complete resection of pancreatic cancer. However, the underlying mechanisms are not fully understood. The chemo-resistance factors against gemcitabine still need further characterizations. Here we studied the mechanism of gemcitabine-induced pancreatic cancer cell death by focusing on mammalian sterile 20-like kinase 1 (MST1) and cyclophilin D (Cyp-D). We found that MST1 and Cyp-D expressions were significantly lower in gemcitabine-resistant pancreatic cancer tissues and cell lines. In vitro, gemcitabine activated MST1 through reactive oxygen species (ROS) production, which was prevented by antioxidant n-acetyl-cysteine (NAC). We found that gemcitabine-activated MST1 translocated to mitochondria and formed a complex with the local protein Cyp-D. Gemcitabine-induced cell death was alleviated by MST1 or Cyp-D shRNA silencing, but was aggravated by MST1 or Cyp-D over-expression. Further, cyclosporin A (CsA), the Cyp-D inhibitor, prevented gemcitabine-induced MST1/Cyp-D mitochondrial complexation and cancer cell death. We suggest that gemcitabine-induced death of pancreatic cancer cells requires MST1/Cyp-D mitochondrial complexation.

Inhibition of Mammalian Sterile 20‐like Kinase 1 Rescues Cardiomyopathy through Protection from Myocyte Necrosis as well as Apoptosis (LB700)

Inhibition of Mammalian Sterile 20‐like Kinase 1 (MST1) is known to have cardioprotective effects by preventing myocyte apoptosis. We examined the effects of MST1 inhibition in β1‐adrenergic receptor (β1‐AR) cardiomyopathy by mating dominant negative (DN)‐MST1 mice with β1‐AR mice. As the mice aged, the bigenic mice had higher left ventricular ejection fraction (67+12% vs 43+5%) and decreased cardiac fibrosis (8.8+1.5% vs 19.5+2.7%). It is generally believed that fibrosis in cardiomyopathy is secondary to necrosis, more than apoptosis. Accordingly, we hypothesized that MST1 may also mediate necrosis. We examined necrosis through in‐vivo labeling by Evans Blue Dye in DN‐MST1 mice after continuous isoproterenol (ISO) challenge. At 1 day after ISO the wild type hearts showed massive myocyte necrotic death (0.90+0.37%), which was significantly protected in DN‐MST1 (0.32+0.14% p&lt;0.05), while having no discernible differences in myocyte apoptosis (0.10+0.03% vs 0.08+0.01%). These results suggest that MST1 may also have an important role in myocyte necrosis as well as apoptosis.

MST1 is a key regulator of beta cell apoptosis and dysfunction in diabetes

Apoptotic cell death is a hallmark of the loss of insulin producing beta-cells in all forms of diabetes mellitus. Current treatment fails to halt the decline in functional beta-cell mass. Strategies to prevent beta-cell apoptosis and dysfunction are urgently needed. Here, we identified Mammalian Sterile 20-like kinase 1 (MST1) as a critical regulator of apoptotic beta-cell death and function. MST1 was strongly activated in beta-cells under diabetogenic conditions and correlated with beta-cell apoptosis. MST1 specifically induced the mitochondrial-dependent pathway of apoptosis in beta-cells through up-regulation of the BH3-only protein Bim. MST1 directly phosphorylated PDX1 at Thr11, resulting in its ubiquitination, degradation and impaired insulin secretion. Mst1 deficiency completely restored normoglycemia, beta-cell function and survival in vitro and in vivo. We show MST1 as novel pro-apoptotic kinase and key mediator of apoptotic signaling and beta-cell dysfunction, which may serve as target for the development of novel therapies for diabetes.

The role of reciprocal activation of cAbl and Mst1 in the Oxidative death of cultured astrocytes

The protein kinase Mst1 (mammalian Sterile 20-like kinase 1) likely plays a role in oxidative neuronal cell death as a target of its activator, cAbl. We previously found that H2O2-induced death of astrocytes is mediated by cAbl in a metallothionein-3 (Mt3)-dependent manner. In the present study, we examined a possible role for Mst1 in the oxidative death of astrocytes. Treatment of cortical astrocytes with 170 µM H2O2 activated Mst1. Knockdown of Mst1 reduced H2O2-induced cell death, indicating that Mst1 activation contributes to astrocytic cell death. STI571, an inhibitor of cAbl, blocked induction/activation of Mst1 and H2O2-induced cell death. However, Mst1 silencing also inhibited induction/activation of cAbl, suggesting that the two kinases are regulated by a reciprocal activating mechanism. The zinc chelator TPEN blocked induction/activation of cAbl and Mst1, indicating that these phenomena are dependent on the rise of intracellular zinc. Moreover, H2O2 exposure did not increase free zinc levels in Mt3-null astrocytes, suggesting that the increased levels of free zinc were largely from Mt3. Consistent with the involvement of FoxO1/3, which may play a role in the Mst1-cell death cascade, we found an increase in the level of phosphorylated FoxO1/3 in H2O2-treated astrocytes. Moreover, inhibition of cAbl or Mst1 reversed this effect. The present results suggest the interesting possibility that cAbl and Mst1 are reciprocally activated under oxidative stress conditions in astrocytes. Both kinases appear to be regulated by changes in the levels of free zinc originating from Mt3 and contribute to oxidative cell death through a FoxO-dependent mechanism.

Mammalian sterile 20-like kinase 1/2 inhibits the Wnt/β-catenin signalling pathway by directly binding casein kinase 1ε

The Hippo signalling pathway can suppress the Wnt/β-catenin signalling pathway through the last downstream effectors YAP (Yes-associated protein)/TAZ (tafazzin). MST (mammalian sterile 20-like kinase) 1 functions as the upstream kinase of the Hippo pathway, and CK1ε (casein kinase 1ε) plays roles in the up-stream signal transduction of the Wnt/β-catenin pathway. In the present study, using tandem affinity purification and MS analysis, CK1ε was identified as a novel partner of MST1. Further analysis showed that the interaction between MST1 and CK1ε was mediated by their kinase domains and enhanced by the activation of MST1. To exclude the interference of the phosphorylated YAP/TAZ, the transduction from MST1 to YAP/TAZ was blocked using anti-WW45 shRNA. In the sh-WW45 cells, MST1 still inhibited the Wnt3A-induced phosphorylation of DVL2 (dishevelled 2) and Wnt/β-catenin signalling by disturbing the interaction of DVL2 and CK1ε. The growth-suppressive effect of MST1 in the presence of Wnt3A was effectively relieved by the downstream activation of the Wnt/β-catenin pathway. Moreover, MST2, the close homologue of MST1, also displayed the similar function in suppressing the Wnt/β-catenin pathway. Therefore the results of the present study revealed that, in addition to the phosphorylated YAP/TAZ, the Hippo pathway can suppress the Wnt/β-catenin pathway directly through MST1/2.

MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen species (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.

A DAF‐16/FoxO3a‐dependent longevity signal is initiated by antioxidants

The precise mechanisms of antioxidant-mediated longevity are poorly understood. We show that an antioxidant treatment can extend the lifespan of Caenorhabditis elegans (C. elegans) through the nuclear translocation of the forkhead box O transcription factor (FoxO) homolog DAF-16. This pathway was found to involve 3-phosphoinositide-dependent kinase-1 (PDK-1) and serum- and glucocorticoid-regulated kinase-1 (SGK-1), distinct from the known oxidative stress-mediated mechanism in which FoxO3a translocation is regulated by c-Jun N-terminal kinase (JNK) and mammalian sterile 20-like kinase-1 (MST-1). The differences in the mechanisms of FoxO activation by antioxidants and oxidants result in differences in FoxO phosphorylation and target gene expression. Based on these results, we found that a combination of early antioxidant treatment and late oxidant treatment is most effective for lifespan extension in C. elegans.