Mst1 Deletion Research Articles

Inhibiting endothelial cell Mst1 attenuates acute lung injury in mice.

Lung endothelium plays a pivotal role in the orchestration of inflammatory responses to acute pulmonary insults. Mammalian sterile 20-like kinase 1 (Mst1) is a serine/threonine kinase that has been shown to play an important role in the regulation of apoptosis, stress responses, and organ growth. This study investigated the role of Mst1 in lung endothelial activation and acute lung injury (ALI). We found that Mst1 was significantly activated in inflamed lung endothelial cells (ECs) and mouse lung tissues. Overexpression of Mst1 promoted nuclear factor κ-B (NF-κB) activation through promoting JNK and p38 activation in lung ECs. Inhibition of Mst1 by either its dominant negative form (DN-Mst1) or its pharmacological inhibitor markedly attenuated cytokine-induced expression of cytokines, chemokines, and adhesion molecules in lung ECs. Importantly, in a mouse model of lipopolysaccharide-induced (LPS-induced) ALI, both deletion of Mst1 in lung endothelium and treatment of WT mice with a pharmacological Mst1 inhibitor significantly protected mice from LPS-induced ALI. Together, our findings identified Mst1 kinase as a key regulator in controlling lung EC activation and suggest that therapeutic strategies aimed at inhibiting Mst1 activation might be effective in the prevention and treatment of inflammatory lung diseases.

Abstract 15266: Inhibiting Endothelial Mammalian Sterile 20-Like Kinase 1 Attenuates Acute Lung Injury in Mice

Introduction: Lung endothelium plays a pivotal role in the orchestration of inflammatory and injury responses to acute pulmonary insults. Mammalian sterile 20-like kinase 1 (Mst1), a mammalian homolog of Hippo, is a serine/threonine kinase that is ubiquitously expressed in many tissues and has been shown to play an important role in the regulation of complex biological processes, such as apoptosis, oncogenesis and organ growth. While Mst1 is highly expressed in the lung, its role in the endothelial response to pulmonary insults is largely unknown. Hypothesis: Mst1 regulates inflammatory responses in acute lung injury. Methods: Mst1 activity was assessed in lung endothelium by western blot and immunofluorescent staining. EC activation was measured by nuclear factor kappa B (NF-κB) dependent luciferase assay and the electrophoresis mobility shift assay. Mst1 knockout mice and pharmacological inhibitor were employed to assess the effects of Mst1 on homeostatic and lipopolysaccharide (LPS)-induced endothelial inflammation. Results: In this study, we found that Mst1 activation, as determined by Mst1 phosphorylation and nuclear translocation, was significantly increased in lung ECs after exposure to tumor necrosis factor-alpha (TNF-α) and in lungs of LPS-instilled mice as compared to the vehicle control. Overexpression of either full length Mst1 or its kinase domain promoted NF-κB activation by approximately 6.5-fold and 8-fold, respectively, whereas overexpression of the dominant negative forms of Mst1 attenuated endothelial inflammatory responses to both TNF-α and interleukin-1β stimulation. Consistent with these observations, endothelial specific deletion of Mst1 markedly attenuated LPS-induced acute lung injury in mice. Furthermore, we found that treatment of wild-type mice with Mst1 specific pharmacological inhibitor significantly prevented LPS-induced lung injury. Conclusion: Our findings identified Mst1 kinase as a key regulator in promoting lung EC activation and vascular inflammation, and suggest that therapeutic strategies aimed at inhibiting Mst1 activation might be effective for preventing inflammatory vascular diseases of the lung.

Cerebral ischemia-reperfusion is modulated by macrophage-stimulating 1 through the MAPK-ERK signaling pathway.

Cerebral ischemia-reperfusion (IR) injury is associated with mitochondrial damage. Macrophage-stimulating 1 (MST1) reportedly stimulates mitochondrial apoptosis by suppressing BCL-2. We investigated whether MST1 promotes the progression of cerebral IR injury by inducing mitochondrial dysfunction in vivo and in vitro. Western blot analysis, quantitative polymerase chain reaction, immunofluorescence, and mitochondrial function assays were conducted in cells from wild-type and Mst1-knockout mice subjected to cerebral IR injury. MST1 expression in wild-type glial cells increased following cerebral IR injury. Cerebral IR injury reduced the mitochondrial membrane potential and mitochondrial metabolism in glial cells, while it enhanced mitochondrial reactive oxygen species generation and mitochondrial calcium levels in these cells. The deletion of Mst1 attenuated cerebral IR injury by improving mitochondrial function and reducing mitochondrial damage. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway was suppressed in wild-type glial cell upon cerebral IR injury but was reactivated in Mst1-knockout glial cell. Accordingly, blocking the MAPK/ERK pathway abolished the beneficial effects of Mst1 deletion during cerebral IR injury by inducing mitochondrial damage in glial cells. Our results suggest that cerebral IR injury is associated with MST1 upregulation in the brain, while the genetic ablation of Mst1 can attenuate mitochondrial damage and sustain brain function following cerebral IR injury.

Proinflammation effect of Mst1 promotes BV‐2 cell death via augmenting Drp1‐mediated mitochondrial fragmentation and activating the JNK pathway

Inflammation has been increasingly studied as part of the pathophysiology of neurodegenerative diseases. Mammalian Ste20-like kinase 1 (Mst1), a key factor of the Hippo pathway, is connected to cell death. Unfortunately, little study has been performed to detect the impact of Mst1 in neuroninflammation. The results indicated that Mst1 expression was upregulated because of LPS treatment. However, the loss of Mst1 sustained BV-2 cell viability and promoted cell survival in the presence of LPS treatment. Molecular investigation assay demonstrated that Mst1 deletion was followed by a drop in the levels of mitochondrial fission via repressing Drp1 expression. However, Drp1 adenovirus transfection reduced the protective impacts of Mst1 knockdown on mitochondrial stress and neuronal dysfunction. Finally, our results illuminated that Mst1 affected Drp1 content and mitochondrial fission in a JNK-dependent mechanism. Reactivation of the JNK axis inhibited Mst1 knockdown-mediated neuronal protection and mitochondrial homeostasis. Altogether, our results indicated that Mst1 upregulation and the activation of JNK-Drp1-mitochondrial fission pathway could be considered as the novel mechanism regulating the progression of neuroninflammation. This finding would pave a new road for the treatment of neurodegenerative diseases via modulating the Mst1-JNK-Drp1-mitochondrial fission axis.

Mst1 deletion reduces septic cardiomyopathy via activating Parkin‐related mitophagy

Cardiomyocyte function and viability are highly modulated by mammalian Ste20-like kinase 1 (Mst1)-Hippo pathway and mitochondria. Mitophagy, a kind of mitochondrial autophagy, is a protective program to attenuate mitochondrial damage. However, the relationship between Mst1 and mitophagy in septic cardiomyopathy has not been explored. In the present study, Mst1 knockout mice were used in alipopolysaccharide (LPS)-induced septic cardiomyopathy model. Mitophagy activity was measured via immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay. Pathway blocker and small interfering RNA were used to perform the loss-of-function assay. The results demonstrated that Mst1 was rapidly increased in response to LPS stress. Knockout of Mst1 attenuated LPS-mediated inflammation damage, reduced cardiomyocyte death, and improved cardiac function. At the molecular levels, LPS treatment activated mitochondrial damage, such as mitochondrial respiratory dysfunction, mitochondrial potential reduction, mitochondrial ATP depletion, and caspase family activation. Interestingly, in response to mitochondrial damage, Mst1 deletion activated mitophagy which attenuated LPS-mediated mitochondrial damage. However, inhibition of mitophagy via inhibiting parkin mitophagy abolished the protective influences of Mst1 deletion on mitochondrial homeostasis and cardiomyocyte viability. Overall, our results demonstrated that septic cardiomyopathy is linked to Mst1 upregulation which is followed by a drop in the protective mitophagy.

Mst1 deletion reduces hyperglycemia-mediated vascular dysfunction via attenuating mitochondrial fission and modulating the JNK signaling pathway.

Diabetes is a leading cause of microvascular complications, such as nephropathy and retinopathy. Recent studies have proposed that hyperglycemia-induced endothelial cell dysfunction is modulated by mitochondrial stress. Therefore, our experiment was to detect the upstream mediator of mitochondrial stress in hyperglycemia-treated endothelial cells with a focus on macrophage-stimulating 1 (Mst1) and mitochondrial fission. Our data illuminated that hyperglycemia incubation reduced cell viability, as well as increased apoptosis ratio in endothelial cell, and this alteration seemed to be associated with Mst1 upregulation. Inhibition of Mst1 via transfection of Mst1 siRNA into an endothelial cell could sustain cell viability and maintain mitochondrial function. At the molecular levels, endothelial cell death was accompanied with theactivation of mitochondrial oxidative stress, mitochondrial apoptosis, and mitochondrial fission. Genetic ablation of Mst1 could reduce mitochondrial oxidative injury, block mitochondrial apoptosis, and repress mitochondrial fission. Besides, we also found Mst1 triggered mitochondrial dysfunction as well as endothelial cell damage through augmenting JNK pathway. Suppression of JNK largely ameliorated the protective actions of Mst1 silencing on hyperglycemia-treated endothelial cells and sustain mitochondrial function. The present study identifies Mst1 as a primary key mediator for hyperglycemia-induced mitochondrial damage and endothelial cell dysfunction. Increased Mst1 impairs mitochondrial function and activates endothelial cell death via opening mitochondrial death pathway through JNK.

Mst1 contributes to nasal epithelium inflammation via augmenting oxidative stress and mitochondrial dysfunction in a manner dependent on Nrf2 inhibition.

Nasal epithelium inflammation plays an important role in transmitting and amplifying damage signals for the lower airway. However, the molecular basis of nasal epithelium inflammation damage has not been fully addressed. Mst1 is reported to modulate inflammation via multiple effects. Thus, the aim of our study is to understand the pathological mechanism underlying Mst1-related nasal epithelium inflammation in vitro. Our result indicated that Mst1 expression was rapidly increased in response to tumor necrosis factor-α (TNF-α) treatment in vitro and this effect was a dose-dependent manner. Interestingly, knockdown of Mst1 via transfecting small interfering RNA markedly reversed cell viability in the presence of TNF-α. Further, we found that Mst1 deficiency reduced cellular oxidative stress and attenuated mitochondrial dysfunction, as evidenced by reversed mitochondrial complex-I activity, decreased mitochondrial permeability transition pore opening rate, and stabilized mitochondrial membrane potential. Besides, we found that Nrf2 expression was increased after deletion of Mst1 whereas silencing of Nrf2 abolished the protective effects of Mst1 deletion on nasal epithelium survival and mitochondrial homeostasis. Moreover, Nrf2 overexpression also protected nasal epithelium against TNF-α-induced inflammation damage. Altogether, our data confirm that the Mst1 activation and Nrf2 downregulation seem to be the potential mechanisms responsible for the inflammation-mediated injury in nasal epithelium via mediating mitochondrial damage and cell oxidative stress.

Mst1 facilitates hyperglycemia-induced retinal pigmented epithelial cell apoptosis by evoking mitochondrial stress and activating the Smad2 signaling pathway.

Hyperglycemia induces retinal pigmented epithelial cell apoptosis and mitochondrial stress via poorly understood mechanisms. The goal of our current study is to explore whether mammalian sterile 20-like kinase 1 (Mst1) is involved in the pathogenesis of hyperglycemia-mediated retinal pigmented epithelial cell apoptosis by triggering mitochondrial abnormalities and activating the Smad2 signaling pathway. Retinal pigmented epithelial ARPE-19 cells were presented with a high-glucose challenge in vitro. Cell viability and apoptosis were measured via western blotting, ELISAs, and immunofluorescence assays. Mitochondrial function was detected via JC-1 staining, mitochondrial ROS flow cytometry, western blotting, and ELISAs. Loss- and gain-of-function assays were performed via cell transfection and transduction with Mst1 siRNA and Smad2 adenovirus, respectively. The results indicated that hyperglycemia treatment upregulated the levels of Mst1, an effect that was accompanied by an increase in ARPE-19 cell apoptosis. Loss of Mst1 attenuated hyperglycemia-induced cell apoptosis, and this effect seemed to be associated with mitochondrial protection. In response to hyperglycemia stimulus, mitochondrial stress was noted in ARPE-19 cells, including mitochondrial ROS overproduction, mitochondrial respiratory metabolism dysfunction, mitochondrial fission/fusion imbalance, and mitochondrial apoptosis activation. Further, we provided evidence to support the crucial role played by Smad2 in promoting Mst1-mediated cell apoptosis and mitochondrial stress. Overexpression of Smad2 abrogated the beneficial effects of Mst1 deletion on ARPE-19 cell viability and mitochondrial protection. Altogether, our results identified Mst1 as a novel mediator controlling the fate of retinal pigmented epithelial cells and mitochondrial homeostasis via the Smad2 signaling pathway. Based on this finding, strategies to repress Mst1 upregulation and block Smad2 activation are vital to alleviate hyperglycemia-mediated retinal pigmented epithelial cell damage.

Mst1 promotes cardiac ischemia–reperfusion injury by inhibiting the ERK-CREB pathway and repressing FUNDC1-mediated mitophagy

Cardiac ischemia-reperfusion (I/R) injury results mainly from mitochondrial dysfunction and cardiomyocyte death. Mitophagy sustains mitochondrial function and exerts a pro-survival effect on the reperfused heart tissue. Mammalian STE20-like kinase 1 (Mst1) regulates chronic cardiac metabolic damage and autophagic activity, but its role in acute cardiac I/R injury, especially its effect on mitophagy, is unknown. The aim of this study is to explore whether Mst1 is involved in reperfusion-mediated cardiomyocyte death via modulation of FUN14 domain containing 1 (FUNDC1)-related mitophagy. Our data indicated that Mst1 was markedly increased in reperfused hearts. However, genetic ablation of Mst1 in Mst1-knockout (Mst1-KO) mice significantly reduced the expansion of the cardiac infarction area, maintained myocardial function and abolished I/R-mediated cardiomyocyte death. At the molecular level, upregulation of Mst1 promoted ROS production, reduced mitochondrial membrane potential, facilitated the leakage of mitochondrial pro-apoptotic factors into the nucleus, and activated the caspase-9-related apoptotic pathway in reperfused cardiomyocytes. Mechanistically, Mst1 activation repressed FUNDC1 expression and consequently inhibited mitophagy. However, deletion of Mst1 was able to reverse FUNDC1 expression and thus re-activate protective mitophagy, effectively sustaining mitochondrial homeostasis and blocking mitochondrial apoptosis in reperfused cardiomyocytes. Finally, we demonstrated that Mst1 regulated FUNDC1 expression via the MAPK/ERK-CREB pathway. Inhibition of the MAPK/ERK-CREB pathway prevented FUNDC1 activation caused by Mst1 deletion. Altogether, our data confirm that Mst1 deficiency sends a pro-survival signal for the reperfused heart by reversing FUNDC1-related mitophagy and thus reducing cardiomyocyte mitochondrial apoptosis, which identifies Mst1 as a novel regulator for cardiac reperfusion injury via modulation of mitochondrial homeostasis.

Mst1 regulates post-infarction cardiac injury through the JNK-Drp1-mitochondrial fission pathway

BackgroundPost-infarction cardiac injury is closely associated with cardiac remodeling and heart dysfunction. Mammalian STE20-like kinase 1 (Mst1), a regulator of cellular apoptosis, is involved in cardiac remodeling in post-infarction heart, but the mechanisms remain poorly defined. We aimed to explore the role of Mst1 in regulating chronic post-infarction cardiac injury, with a focus on mitochondrial homoeostasis.MethodsWild-type (WT) and Mst1-knockout mice were as the cardiac myocardial infarction model. Cardiac fibrosis, myocardial inflammation response, heart dysfunction and cardiomyocyte death were measured in vivo using immunohistochemistry, immunofluorescence, western blot, qPCR and TUNEL assays. Cardiomyocytes were isolated from WT and Mst1-knockout mice, and a chronic hypoxia model was used to induce damage. Mitochondrial function was determined via JC1 staining, ROS measurement, cyt-c leakage detection and mitochondrial apoptotic pathways analysis. Mitochondrial fission was observed using immunofluorescence. A pathway activator and inhibitor were applied to establish the signaling pathways involved in regulating mitochondrial homeostasis.ResultsOur study demonstrated that Mst1 expression was significantly upregulated in the heart post-infarction. Activated Mst1 induced cardiac fibrosis, an excessive inflammatory response, and cardiomyocyte death, whereas the genetic ablation of Mst1 protected the myocardium against chronic post-infarction injury. Function assays showed that upregulation of Mst1 activity contributed to JNK pathway activation, which led to Drp1 migration from the cytoplasm onto the surface of the mitochondria, indicative of mitochondrial fission activation. Excessive mitochondrial fission caused mitochondrial fragmentation, resulting in mitochondrial potential collapse, ROS overproduction, mitochondrial pro-apoptotic leakage into the cytoplasm, and the initiation of caspase-9-mediated mitochondrial apoptosis. By contrast, Mst1 deletion helped to maintain mitochondrial structure and function, sending pro-survival signals to the cardiomyocytes.ConclusionsOur results identify Mst1 as a malefactor in the development of post-infarction cardiac injury and that it acts through the JNK-Drp1-mitochondrial fission pathway.

Deletion of Mst1 attenuates neuronal loss and improves neurological impairment in a rat model of traumatic brain injury

Neuronal cell death following traumatic brain injury (TBI) is a considerable contributor to neurological deficits. In our work, we explored the functions of Mammalian STE20-like kinase-1 (Mst1), a apoptosis-promoting kinase and also a pivotal bridgebuilder of apoptotic signaling, in the etiopathogenesis of an experimental rat model of TBI. We found that the phosphorylation level of Mst1 in injured area was significantly increased after TBI. Furthermore, we discovered that inhibition of Mst1 phosphorylation can effectively reduce neuronal cell death by inhibiting the activation of caspase 3 and suppressing the damage of DNA during TBI. In addition, the decreased of Mst1 phosphorylation level, not only reduced brain edema and blood-brain barrier (BBB) damage in injured region but also weakened the impairment of neurologic behavior during TBI. In conclusion, our work demonstrates that Mst1 plays an important role in TBI-induced neuronal cell death, suggesting that Mst1 is expected to be a potential therapeutic target for TBI.

Hepatic Hippo signaling inhibits protumoural microenvironment to suppress hepatocellular carcinoma

ObjectiveHippo signalling is a recently identified major oncosuppressive pathway that plays critical roles in inhibiting hepatocyte proliferation, survival and hepatocellular carcinoma (HCC) formation. Hippo kinase (Mst1 and Mst2) inhibits HCC...

Mammalian sterile 20-like kinase 1 expression and its prognostic significance in patients with breast cancer.

Mammalian sterile 20-like kinase 1 (Mst1) is a major inhibitor of cell proliferation, and is involved in apoptosis, oncogenesis and organ growth via its ubiquitously encoded serine threonine kinase. Previous studies have demonstrated that Mst1 has a tumor suppressor function in human breast cancer. Mst1 deletion or mutation is associated with tumorigenesis, whereas Mst1 overexpression leads to tumor cell apoptosis and decreases proliferation of tumor cells. Our previous study reported the tumor suppressive function of Mst1, and debated Mst1 as a prognostic factor in human breast cancer. In the present study, Mst1 levels were measured in the plasma of patients in order to elucidate their association with overall and disease-free survival. The results of the present study indicated that Mst1 is a strong prognostic and predictive factor in human breast cancer and a promising anticancer target.

Deletion of mammalian sterile 20-like kinase 1 attenuates neuronal loss and improves locomotor function in a mouse model of spinal cord trauma.

Neuronal cell death following spinal cord injury (SCI) is an important contributor to neurological deficits. The purpose of our work was to delineate the function of mammalian sterile 20-like kinase 1 (Mst1), a pro-apoptotic kinase and key mediator of apoptotic signaling, in the pathogenesis of an experimental mouse model of SCI. Male mice received a mid-thoracic spinal contusion injury, and it was found that phosphorylation of Mst1 at the injured site was enhanced significantly following SCI. Furthermore, when compared to the wild-type controls, Mst1-deficient mice displayed improved locomotor function by increased Basso mouse scale score. Deletion of Mst1 in mice attenuated loss of motor neurons and suppressed microglial and glial activation following SCI. Deletion of Mst1 in mice reduced apoptosis via suppressing cytochrome c release and caspase-3 activation following SCI. Deletion of Mst1 attenuated mitochondrial dysfunction and increased ATP formation following SCI. Deletion of Mst1 in mice inhibited local inflammation following SCI, evidenced by reduced activities of myeloperoxidase and protein levels of TNF-α, IL-1β, and IL-6. In conclusion, the present study demonstrated that deletion of Mst1 attenuated neuronal loss and improved locomotor function in a mouse model of SCI, via preserving mitochondrial function, attenuating mitochondria-mediated apoptotic pathway, and suppressing inflammation, at least in part.

Hippo/MST1 signaling mediates microglial activation following acute cerebral ischemia–reperfusion injury

Cerebral ischemia–reperfusion injury is a major public health concern that causes high rates of disability and mortality in adults. Microglial activation plays a crucial role in ischemic stroke-induced alteration of the immune microenvironment. However, the mechanism underlying the triggering of microglial activation by ischemic stroke remains to be elucidated. Previously, we demonstrated that the protein kinase Hippo/MST1 plays an important role in oxidative stress-induced cell death in mammalian primary neurons and that the protein kinase c-Abl phosphorylates MST1 at Y433, which increases MST1 kinase activity. Microglial activation has been implicated as a secondary detrimental cellular response that contributes to neuronal cell death in ischemic stroke. Here, we are the first, to our knowledge, to demonstrate that MST1 mediates stroke-induced microglial activation by directly phosphorylating IκBα at residues S32 and S36. We further demonstrate that Src kinase functions upstream of MST1–IκB signaling during microglial activation. Specific deletion of MST1 in microglia mitigates stroke-induced brain injury. Therefore, we propose that Src–MST1–IκB signaling plays a critical role in stroke-induced microglial activation. Together with our previous work demonstrating that MST1 is important for oxidative stress-induced neuronal cell death, our results indicate that MST1 could represent a potent therapeutic target for ischemic stroke.

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.