Yunfei Yin,Mingyue Tan,Lianhua Han,Lei Zhang,Yue Zhang,Jun Zhang,Wanqian Pan,Jiaxiang Bai,Tingbo Jiang,Hongxia Li
The hippo kinases MST1/2 in cardiovascular and metabolic diseases: A promising therapeutic target option for pharmacotherapy
MST1/2 kinases,Regulation,Apoptosis,Autophagy,Immune,Cardiovascular diseases,Metabolic diseases,Therapy
Cardiovascular diseases (CVDs) and metabolic disorders are major components of noncommunicable diseases, causing an enormous health and economic burden worldwide. There are common risk factors and developmental mechanisms among them, indicating the far-reaching significance in exploring the corresponding therapeutic targets. MST1/2 kinases are well-established proapoptotic effectors that also bidirectionally regulate autophagic activity. Recent studies have demonstrated that MST1/2 influence the outcome of cardiovascular and metabolic diseases by regulating immune inflammation. In addition, drug development against them is in full swing. In this review, we mainly describe the roles and mechanisms of MST1/2 in apoptosis and autophagy in cardiovascular and metabolic events as well as emphasis on the existing evidence for their involvement in immune inflammation. Moreover, we summarize the latest progress of pharmacotherapy targeting MST1/2 and propose a new mode of drug combination therapy, which may be beneficial to seek more effective strategies to prevent and treat CVDs and metabolic disorders.
The hippo kinases MST1/2 in cardiovascular and metabolic diseases: A promising therapeutic target option for pharmacotherapy

Cardiovascular diseases (CVDs) and metabolic disorders are major components of noncommunicable diseases, causing an enormous health and economic burden worldwide. There are common risk factors and developmental mechanisms among them, indicating the far-reaching significance in exploring the corresponding therapeutic targets. MST1/2 kinases are well-established proapoptotic effectors that also bidirectionally regulate autophagic activity. Recent studies have demonstrated that MST1/2 influence the outcome of cardiovascular and metabolic diseases by regulating immune inflammation. In addition, drug development against them is in full swing. In this review, we mainly describe the roles and mechanisms of MST1/2 in apoptosis and autophagy in cardiovascular and metabolic events as well as emphasis on the existing evidence for their involvement in immune inflammation. Moreover, we summarize the latest progress of pharmacotherapy targeting MST1/2 and propose a new mode of drug combination therapy, which may be beneficial to seek more effective strategies to prevent and treat CVDs and metabolic disorders.

Cardiovascular diseases (CVDs) and metabolic disorders, such as diabetes, obesity and fatty liver diseases, are the major component of noncommunicable diseases (NCDs), and they trigger an enormous health and economic burden worldwide. CVDs remain the leading cause of premature death globally, accounting for one-third of all deaths. Obesity and diabetes are well-known risk factors for CVDs,, and greatly increase the risk of acute cardiovascular events. Therefore, CVDs are closely connected to metabolic disorders, indicating the significance of seeking their possible common mechanisms of occurrence and progression. Apoptosis and autophagy have been reported to play integral roles in cardiovascular and metabolic disorders. Apoptosis is a coordinated and orderly biological process involving complex signaling pathways that occurs under physiological and pathological conditions, both deficiency and excess of which can result in diseases5, 6, 7. Autophagy is a core molecular pathway in maintaining cellular and organic homeostasis, and impairing autophagy through pharmacological and genetic interventions is able to promote or aggravate diseases in multiple experimental models8, 9, 10, 11. Meanwhile, overactivation of autophagy in some cases is inclined to accelerate cell death and amplify damage. In addition, immune inflammation is a newly discovered perspective on the pathogenesis of cardiovascular and metabolic diseases. Derailment and overactivation of the immune system induce chronic inflammation and exacerbate the severity of disorders. Studies have shown that a variety of immune cells, such as macrophages, leukocytes, and lymphocytes, can serve as therapeutic targets for improving CVDs and metabolic disorders14, 15, 16, 17, 18. In conclusion, interfering with the processes of apoptosis, autophagy and immune inflammation in cardiovascular and metabolic diseases may be a feasible therapeutic strategy. Mammalian sterile 20-like kinase 1/2 (MST1/2), as serine–threonine kinases, have attracted extensive attention as core components of the Hippo signaling pathway in mammals, which regulates apoptosis and proliferation to maintain organ size and tissue homeostasis,. In the canonical Hippo pathway, MST1/2 activates MOB1A/B and LATS1/2, and then LATS1/2 phosphorylates and inhibits the transcriptional activity of YAP/TAZ, thus restraining the transcription of genes concerned with survival and proliferation. Moreover, it has been illustrated that MST1/2 can phosphorylate and enable the NDR1/2-regulated noncanonical Hippo pathway, as well as other protein-related signaling transduction pathways. Compelling evidence has suggested that MST1/2 are proapoptotic effectors and regulate autophagy in response to stress, which has been extensively explored in various models of cardiovascular and metabolic diseases23, 24, 25, 26, 27. Additionally, the innate and adaptive immune regulation of MST1/2 has been a hotspot of current research. Loss-of-function mutation in the gene encoding MST1 is associated with immunodeficiency and generates recurrent bacterial and viral infections, along with lymphopenia in humans,. Lymphocytes lacking MST1/2 are defective in migration and adhesion, yielding an incremental rate of apoptosis. Besides, MST1 ablation is apt to increase proinflammatory cytokines in macrophages. Consequently, herein, we review the structure of MST1/2 kinases and their regulatory mechanisms while systematically describing their functions in apoptosis and autophagy during cardiovascular and metabolic events. Furthermore, we highlight the existing evidence of immune inflammation where MST1/2 are involved in cardiovascular and metabolic diseases and summarize the latest advances in pharmacotherapy targeting MST1/2 (Fig. 1). Understanding the roles and mechanisms of MST1/2 kinases in these diseases may benefit the development of more effective therapies to prevent CVDs and metabolic disorders.

Human MST1 and MST2 consist of 487 and 491 amino acids, respectively. They are highly homologous, sharing >95% identical catalytic domains and being approximately 75% the same overall. As MST1 and MST2 share common and conserved core components of the Hippo signaling pathway, they function similarly, including antitumor and proapoptotic properties. Of course, in some ways, they differ from each other. It has been reported that MST1 induces a mitochondria-dependent pathway of apoptosis and promotes apoptosis through both caspase-dependent and -independent approaches,, while MST2 upregulates the modulator of apoptosis (PUMA) through the transduction of p53 to trigger apoptosis,. MST1/2 kinases are widely present in various organs of mammals, especially represented by the liver and stomach, required for embryonic development, organ size control and tumor suppression. Among them, in the process of early embryonic growth, one copy of MST1 or MST2 is sufficient to support the normal development of embryo, and their control of organ size is tissue-specific. Hereinafter, we will describe in detail the structure of MST1/2 kinases and their regulation, as well as the summary of signaling network in which MST1/2 are involved.

MST1/2 have a uniform organizational structure, namely, an N-terminal kinase domain and a C-terminal regulatory region, and the latter is further divided into an autoinhibitory domain and a coiled-coil dimerization domain known as the SARAH domain. Between the kinase domain and the autoinhibitory domain of MST1 locate caspase cleavage sites (Fig. 2). Normally, full-length MST1/2 are located in the cytoplasm,. In response to stress and apoptosis stimulation, the autoinhibitory domain and SARAH domain are removed via cleavage of MST1/2 mediated by caspase, which increases the activity by approximately ninefold compared with wild-type MST1/2. At the same time, it raises the affinity for histone 2 B (H2B), the nucleosome component protein, and then the caspase-cleaved version translocates to the nucleus, giving rise to nuclear fragmentation and apoptosis,. Under this condition, MST1/2 themselves can also induce apoptosis, thus constructing a positive feedback loop that further cleaves and actives MST via caspase. Moreover, the activity of MST1/2 is also affected by phosphorylation, dimerization and protein–protein interactions. Faced with stress, multiple-site autophosphorylation of MST1/2 in the activation loop contributes to self-activation, where T183 and T180 sites severally assist their activities45, 46, 47. Meanwhile, MST1/2 are adjusted by the transphosphorylation of the Ste 20-family kinase TAO kinase-3 and other protein kinases, such as protein kinase B (Akt), c-Jun N-terminal kinase (JNK) and cellular Abelson gene product (c-Abl)48, 49, 50. The SARAH domain is vital for dimerization, which mediates the formation of MST1–MST1 and MST2–MST2 homodimers,. MST1/2 kinases can also make heterodimers with other proteins containing the SARAH domain like the Ras-association domain family (RASSF) proteins and salvador homolog 1 (SAV1) to control the status of MST1/2. Finally, some proteins are able to interact with MST1/2, functioning by influencing the levels of homodimerization and autophosphorylation of MST1/2, which is called protein–protein interaction. For example, Raf-1 has been observed to inhibit the activation of MST2 by blocking its homodimerization and recruiting phosphatases to eliminate its activated phosphorylation,.

The signaling pathways that MST1/2 are involved in can be divided into canonical and non-canonical pathways. The canonical pathway is the classical Hippo signaling pathway. In mammals, the core members of Hippo signaling pathway include MST1/2, SAV1, LATS1/2, MOB1 and two downstream transcriptional coactivators YAP and TAZ. Upstream stimulation further phosphorylates and activates the LATS1/2–MOB1A/B complex by exciting the MST1/2–SAV1 complex, finally phosphorylating YAP/TAZ, which are degraded or trapped in the cytoplasm and unable to enter the nucleus to bind to the transcription factor TEAD, thus inhibiting gene transcription that promotes cell survival and proliferation. In addition, MST1/2 participate in signaling regulation through non-canonical pathways as well. The current research is mainly reflected in the promotion of apoptosis and immune regulation. MST1/2 can induce apoptosis in many different ways, such as phosphorylation of Foxo 1/3 to enhance nuclear entry, phosphorylation of histone H2B, promotion of Runx 3 formation and inhibition of Akt activity to antagonize cell survival signals. Moreover, in the immune system, MST1/2 mainly modulate the immune response by interacting with other signaling pathways that regulate immune function, including integrin signal, TCR/BCR signal, cytokine receptor signal, TLR signal, MAPK signal and antiviral signal. In the following paragraphs, we will elaborate the effects of the above regulatory mechanisms on MST1/2 in cardiovascular and metabolic events, along with signaling pathways through which MST1/2 participate in these physiological and pathological processes.

Apoptosis engages in the development, homeostasis and pathological process of the cardiovascular system. Numerous studies have shown that apoptosis of cardiomyocytes plays a role in cardiomyopathy, heart failure and myocardial ischemia/reperfusion injury,. The progression of atherosclerosis comprises apoptosis of endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and macrophages59, 60, 61, 62. Besides, apoptosis of VSMCs and interstitial cells is reported to lead to aortic vascular diseases,. To date, nearly all cardiovascular studies on apoptosis have focused on MST1. At baseline, MST1 is unable to induce apoptosis,, while under stress or in CVDs, MST1 activation plays a crucial role in apoptosis. Some proteins have been identified to regulate the phosphorylation levels of MST1/2 in cardiomyocytes, thus affecting apoptosis. In oxidative stress and arrhythmogenic cardiomyopathy (AC) models, neurofibromin 2 (NF2) has been clarified to phosphorylate and activate MST1 at the T183 site in cardiomyocytes, inhibiting YAP and fostering apoptosis through the Hippo signaling pathway,. Previous studies have shown that the protein kinase Akt is apt to phosphorylate MST1 at the T387 site, preventing MST1-mediated apoptosis, by means of which Lin28a increases the expression of Akt and reduces cardiomyocyte apoptosis in a type one diabetic cardiomyopathy model. Additionally, mechanistic target of rapamycin complex 2 (mTORC2) has been observed to phosphorylate MST1 at the S438 site in the SARAH domain and reduce the activity of MST1. Cardiac-specific destruction of mTORC2 boosts cardiomyocyte apoptosis by enabling MST1. However, the mechanism of apoptosis mediated by mTORC2 is complicated, as in mTOR knockout mice, apoptosis of cardiomyocytes occurs in the early postnatal period without activated MST1. Dimerization in the SARAH domain affects the state of MST1 as well, adjusting apoptosis of cardiomyocytes. It has been reported that RASSF1A can form a heterodimer with MST1 to activate it in cardiomyocytes, promoting apoptosis under pressure overload. Subsequent studies have clarified the concrete mechanism of RASSF1A involved in the control of MST1-mediated apoptosis. Under oxidative stress and other pathological conditions, MST1 is transferred to the mitochondria of cardiomyocytes, combining with RASSF1A and activated K-Ras to form a complex, which enables MST1 to phosphorylate B-cell lymphoma extra large (Bcl-xL) at the S14 site. Considering that Bcl-xL is able to integrate with B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) to curb the Bax-related mitochondrial apoptosis pathway, phosphorylated Bcl-xL dissociates Bax, resulting in the loss of mitochondrial membrane integrity, caspase activation and apoptosis,. Furthermore, it is worth noting that antioxidant enzymes may be critical proteins in the exploration of the interaction with MST1. Peroxiredoxin 1 (Prx 1), combined with MST1, has been proven to be necessary to bring about MST1-mediated apoptosis. Experiments in cardiomyocytes have revealed that overexpression of heat-shock protein B1 (HSPB1) can compete with MST1, binding to oxidized Prx1, which triggers dephosphorylation of MST1 and facilitates the survival of H9C2 cells in response to oxidative stress. Chae et al. have covered another antioxidant enzyme known as thioredoxin (Trx), which inactivates MST1 by arresting its homodimerization and autophosphorylation. A study has reported that MST1 knockout accelerates Trx binding to apoptosis signal-regulating kinase 1 (ASK1) to suppress the activation of ASK1, which reverses angiotensin II (Ang II)-induced cardiomyocyte apoptosis. In the future, they plan to investigate potential sites where MST1 may bind to ASK1/Trx. These results suggest that antioxidant enzymes have immense prospects in groping mechanisms of MST1-related apoptosis despite a lack of direct evidence. Certainly, except for cardiomyocytes, MST1 acts in other cell types, albeit the unclear mechanism for its adjustment and activation. An experiment has made clear that high glucose stimulates and phosphorylates MST1 in cardiac microvascular endothelial cells (CMECs), causing CMEC apoptosis to aggravate microvascular dysfunction. In macrophages, the phosphorylation level of MST1 increases distinctly under high lipid stimulation, and its knockdown/overexpression studies confirm the role of MST1 in provoking macrophage apoptosis in an atherosclerotic model. Moreover, knocking down MST1 in a model of aortic dissection (AD) can decrease apoptosis of VSMCs via the Hippo signaling pathway. In recent years, the mechanism of apoptosis relevant to β-adrenoceptor (β-AR) has been widely studied, and its effect on the Hippo signaling pathway has drawn our attention. Pharmacological or genetic activation of β-AR in the heart induces incremental expression of MST1 and phosphorylation of YAP, which facilitates the upregulation of Bcl-2 interacting mediator of cell death (BIM). Interference of MST1 dampens the growth of BIM expression caused by isoproterenol (ISO). Meanwhile, previous research has revealed that ISO-induced apoptosis is eliminated in the hearts of BIM knockout mice and cultured cardiomyocytes. As is described above, β-AR influences BIM-related cardiomyocyte apoptosis by regulating the expression of MST1 and the phosphorylation of YAP. Moreover, another study has covered that β-AR is able to modulate the phosphorylation and ubiquitin-like modification of NF2 through the cAMP/PKA pathway, thus eliciting the proapoptotic impact of MST1. Nevertheless, Lee et al. has elucidated contradictory consequences where MST1 knockout is unable to suppress cardiomyocyte apoptosis but could attenuate apoptosis of other cell types, such as CMECs and macrophages, due to overexpression of β-AR. As a result, efforts need to be taken to explore and verify the apoptosis-promoting function of β-AR/MST1 in the cardiac microenvironment.

Pancreatic β-cell death is the underlying driver of type one diabetes (T1D), which is caused by activated immune cells and is a major contributor to type two diabetes (T2D)87, 88, 89, 90, where the loss of β cells is significant even in patients with impaired glucose tolerance, which is related to elevated fasting blood glucose levels. There are no current therapeutic strategies to conserve endogenous β cells, while inhibiting proapoptotic or enhancing antiapoptotic signals may be a potential approach to ensure the survival of β cells in diabetes. Recent studies have shown that MST1 is able to play a broad role in the β-cell apoptotic signaling network,. MST1 impairs β-cell function and exerts apoptosis-promoting effects mainly through two mechanisms. One is to activate the integration of the BIM-mediated mitochondrial proapoptotic pathway and caspase machinery, and the other is to directly phosphorylate the key β-cell transcription factor pancreatic and duodenal homeobox 1 (PDX1), leading to its ubiquitin degradation (Fig. 3). It has been reported that pro-diabetic stimuli such as pro-inflammatory cytokines, glycolipotoxicity and oxidative stress can bring about autophosphorylation of MST1 and excitation of caspase in pancreatic β cells. In addition, caspase cleaves MST1 to induce apoptosis. Autophosphorylation MST1 activates a variety of substrates, targeting BIM to raise mitochondrial-dependent apoptosis in β cells, which results in changes of Bax/Bcl-2, release of cytochrome c and subsequent caspase cleavage,,,, amplifying apoptotic signals through the aforementioned positive feedback mechanism. In this process, there is crosstalk between MST1 and other signals, such as JNK and Akt. MST1 has been observed to activate JNK by dint of the MEKK1–MKK4/7–JNK1/2 pathway, supporting it as a mediator of MST1-regulated apoptosis,. JNK can also phosphorylate MST1 at the S82 site as an upstream activator. Persistent JNK-activated and JNK-dependent apoptosis is present in human islets or INS-1E cells that overexpress MST1, and inhibition of JNK blocks MST1 cleavage and β-cell death. Phosphoinositol-3-kinase (PI3K)/Akt is a vital signaling pathway that maintains β-cell survival and insulin gene expression and secretion97, 98, 99. As outlined above, Akt phosphorylates and inactivates MST1 to prevent apoptosis. Conversely, MST1 and its cleaved version acts as inhibitors of Akt. MST1 activation and β-cell apoptosis due to high glucose stimulation are eliminated by unduly activated Akt, while suppression of PI3K–Akt signaling transduction enhances MST1-mediated apoptosis of β-cells. Later, pleckstrin homology domain leucine-rich repeat protein phosphatases one and 2 (PHLPP1/2) have been identified as phosphatases whose expression are upregulated by continuous overactivation of mTORC1 in diabetic models, and they can inactivate Akt and activate MST1, forming a regulatory triangle loop to modulate β-cell apoptosis,. Moreover, activation of MST1 can directly phosphorylate PDX1 and influence β-cell function. PDX1 is a pivotal factor in pancreatic development, and its homozygous mutation gives rise to neonatal diabetes-related pancreatic hypoplasia,. Individuals with PDX1 gene mutations are more likely to suffer from maturity-onset diabetes of the young (MODY4). PDX1 indispensably engages in the control of β-cell survival, possibly affecting its susceptibility to apoptosis105, 106, 107. Its subcellular localization and activity are impacted by posttranslational modifications such as phosphorylation, although the specific role of regulation is not clear108, 109, 110. In normal β cells, the transcription factor PDX1 is located in the nucleus, directly binding to the promoter and inducing the expression of genes encoding glucokinase, glucose transporter 2 (GLUT2) and insulin to maintain the biological functions of cells. In response to pressure, the cleaved MST1 fragment translocates to the nucleus and triggers phosphorylation of PDX1 at the T11 residue, affecting its roles and causing subsequent ubiquitin degradation. In summary, MST1 serves as an important proapoptotic molecule that regulates numerous signaling pathways to trigger apoptosis and dysfunction of β cells. Although the regulation of apoptosis in CVDs and metabolic disorders is complex, these viewpoints provide many ideas to pave the way for detailed MST1/2-based apoptotic means of interference and treatment (Table 1).

Autophagy, a vital regulator for maintaining the homeostasis and function of organs, is a process of lysosome-dependent degradation of cytoplasmic proteins and organelles that is highly conserved in evolution and widely exists in different kinds of cells. Autophagy has emerged as an essential regulator for maintaining cardiac homeostasis and function. It preserves cardiac structure and function under baseline conditions and is activated during stress, mostly limiting damage. Autophagy not only sustains cardiomyocyte survival and protects cardiac function during ischemia but also lessens cardiac remodeling on account of pressure overload by limiting misfolded protein accumulation, mitochondrial dysfunction and oxidative stress,. Nevertheless, it can be excited heavily and lead to cell death under reperfusion injury. Moreover, autophagy engages in the modulation of metabolic activities as well. Diseases characterized by metabolic disorders and intracellular stress often involve the accumulation of damaged cell components, which interferes with the process of autophagy, and impaired autophagy can in turn aggravate the metabolic disorder of target tissues or cells, accelerating the progression of metabolic diseases. Therefore, how to regulate the activity of autophagy is a crucial issue to be considered in cardiovascular and metabolic events. In recent years, MST1/2 signaling has received increasing attention in modulating autophagy. MST1 knockout enhances cardiomyocyte autophagic flux, alleviating post-infarction cardiac remodeling and dysfunction as well as myocardial injury induced by Ang II114, 115, 116, 117, 118. MST1 overexpression suppresses autophagy levels in experimental diabetic myocardium, impairing cardiac function,. Besides, MST1 restrains autophagy in CMECs and macrophages too, which is involved in the progression of vascular lesions,. A growing number of studies have attempted to explore the mechanisms by which MST1 regulates autophagy, although they have not yet been elucidated clearly. In this part, we mainly probe the existing evidence of autophagic mechanisms mediated by MST1/2 and put forward our prospects for future orientation.

In 2013, an article reported that the activation of MST1 in cardiomyocytes boosted the accumulation of p63 and the formation of aggregates with the disappearance of autophagosomes, putting forward that MST1 facilitates the interaction between Beclin 1 and Bcl-2 to restrain autophagy for the first time. Since then, several studies have been verified and expanded in diverse cells and models on this basis,,. Beclin 1 is a Bcl-2-homology (BH)-3 domain-only protein and can form two distinct complexes with the class III-type phosphoinositide 3-kinase (Class III PI3K/Vps34), of which complex I mediates the formation of autophagosomes. In cardiomyocytes, MST1 phosphorylates the T108 residue in the BH3 domain of Beclin1, enhancing the binding force of Beclin1 to Bcl-2 and/or Bcl-xL (based on coimmunoprecipitation data), which stabilizes Beclin1 homodimers and curtails the phosphoinositide3-kinase activity of the Atg 14 L-Beclin1–Vps34 complex to prevent autophagy. Subsequent research have successively provided biophysical and structural evidence that the effect of MST1 attenuating autophagy is mainly due to the enhanced association between Beclin1 and proapoptotic Bcl-2 family proteins,. It should be noted that this binding effect occurs on biofilms, such as the endoplasmic reticulum or mitochondrial membrane,, but it is only observed in the endoplasmic reticulum that MST1 controls autophagy through the combination of Beclin1 to Bcl-2. Thus, we identified a mechanism of crosstalk between apoptosis and autophagy in which MST1 participates. Faced with stress, MST1 phosphorylates Beclin 1, provoking the interaction between Beclin1 and Bcl-2/Bcl-xL to inhibit autophagy,. In the meantime, the binding of Bcl-2 family proteins to Bax is weakened, which induces the Bax-related apoptotic pathway, (Fig. 4). Furthermore, Akt can phosphorylate Beclin1 at the S234 and S295 sites, boosting the formation of the Beclin1/14-3-3/vimentin intermediate filament complex and thus suppressing autophagy. JNK1 is apt to phosphorylate Bcl-2 at multiple sites in nonstructural loops, fostering the dissociation of Beclin1 from Bcl-2, which gives rise to autophagy,. We in front have concluded that JNK and MST1 positively regulate each other, while Akt and MST1 negatively adjust each other, which is contrary to the conclusion of their impacts on autophagy, despite disparate models of diseases. As a result, whether these regulatory effects exist in the autophagic mechanism related to Beclin1 is an issue that deserves further discussion in the future.

Interestingly, a study elucidated that RASSF1A maintains the stability and activity of MST1 by directly combining with it to prevent MST1 from being dephosphorylated. The deletion of RASSF1A in hepatic tissue diminishes the level of MST1 to suppress autophagy, which is opposite to the result that MST1 restrains autophagy through the interaction of Beclin 1 and Bcl-2,. Owing to the negative regulation between MST1 and Akt, along with the adequacy and significance of the PI3K–Akt–mTOR pathway in autophagy131, 132, 133, the experimental group attempted to verify this hypothesis. Consequences have shown that RASSF1A, MST1 and Akt coprecipitate, and inhibition of RASSF1A gives rise to the dephosphorylation of MST1, which reduces the coaction between MST1 and Akt, thus exciting the PI3K–Akt–mTOR pathway to halt autophagy. We can conclude that Akt tends to be an autophagy inhibitor in the overwhelming majority of cases, suppressing autophagy through mTOR or other approaches, such as the autophagy-associated protein Beclin1. The interaction between MST1 and Akt is able to interfere with autophagy induced by Akt/mTOR, but there is a lack of knowledge of the Beclin1 pathway. The contents of these two parts reflect the momentous position of the MST1 regulatory mechanism in exploring its relevant autophagic process.

In addition, light chain 3 (LC3) has been observed to be a new substrate of MST1/2, which plays a pivotal role in many steps of autophagy. It has been reported that MST1/2 can phosphorylate the T50 residue of LC3 to accelerate the fusion of autophagosomes and lysosomes. The latest research complements this mechanism. In the context of lacking LC3B phosphorylation, autophagosomes suffer abnormal transport, including increased transport to the periphery of cells, resulting in a decrease in the colocalization of autophagosomes and lysosomes. MST1/2 phosphorylate LC3B-T50 and lessen LC3B's binding to transport-related protein FYVE and coiled-coil domain-containing protein 1 (FYCO1), promoting directed transport of microtubule-based autophagosomes to meet lysosomes in the perinuclear region, which is a key event to ensure autophagy degradation of cargo, (Fig. 5). This result is fresh evidence that MST1/2 facilitate autophagy, reflecting the complexity and great research value of MST1/2 signaling in autophagic regulation. Therefore, elaborating the different effects of MST1/2 on autophagy in diverse environments is a grand direction for our future experiments.

Based on separate subcellular localization, how MST1 influences mitophagy is also a major focus of research. Mitophagy is a type of selective autophagy that clears aging, damaged, and dysfunctional mitochondria through autophagic lysosomes. Current studies have identified MST1 as an upstream mediator of mitophagy. There are three canonical pathways of mitophagy in mammals, including the PTEN induced putative kinase 1 (PINK1)/Parkin-dependent pathway, FUN14 domain containing protein 1 (FUNDC1)-mediated and Bnip3/Nix-mediated mitophagy pathways. Loss of MST1 has been shown to alleviate nonalcoholic fatty liver disease, septic cardiomyopathy and diabetic cardiomyopathy by activating Parkin-associated mitophagy,138, 139, 140. MST1 can induce myocardial I/R injury by downregulating the extracellular signal-regulated kinase (ERK)-cyclic adenosine monophosphate response element binding protein (CREB) pathway to inactivate FUNDC1-related mitophagy. Moreover, other crucial mediators of mitophagy that MST1/2 affect have been validated in other cellular and pathological conditions, which are worthy of further exploration and supplementation in cardiovascular and metabolic diseases. In response to the stress of colorectal cancer, MST1 activates the JNK/p53 pathway to prevent Bnip3-mediated mitophagy and generates apoptosis. Mitochondrial dynamics-related proteins such as mitofusin-2 (Mfn2) also have an impact on the regulation of autophagy, which embodies the vital concept of mitochondrial quality control. Overexpression of MST1 enhances the antitumor effect of IL-24 by restraining ERK–Mfn2 signaling-dependent mitophagy; MST1 can modulate the survival and migration of pancreatic cancer cells through Mfn2-mediated mitophagy. Generally speaking, autophagy plays a dual role in cardiovascular and metabolic diseases, and how to adjust the level of autophagy is the crux. Current evidence suggests that MST1/2 have a bidirectional regulation of autophagy, which may be concerned with the specific environment of cells. Moreover, the mechanisms that regulate MST1/2 may prominently participate in it, especially those proteins involved in autophagy, which need to be considered and addressed. Ultimately, the crosstalk between apoptosis and autophagy may be the characteristic of MST1/2 signaling, which is reflected to some extent in the existing conclusions of Akt, mTOR, Beclin 1 and RASSF, epitomizing the function of MST1/2 in apoptosis and autophagy as a considerable bridge and mechanism.

The immune system plays a pivotal role in health and diseases, defending the body from internal and external threats through multiple cell types and a variety of secreted factors. According to the present view, dysregulation of the immune system often closely interrelates with the occurrence and progression of cardiovascular and metabolic diseases. The activation of congenital and adaptive immune cells and their infiltration into affected tissues exist in atherosclerotic plaques and ischemic and nonischemic myocardium, which have recently become essential factors in a variety of CVDs, including atherosclerosis, hypertension and heart failure146, 147, 148, 149, 150. The terminology ‘immunometabolism’ is frequently used to describe the relationship between inflammation and metabolic dysfunction. The character, quantity and activity of immune cells in the surroundings of tissues can determine their effects on metabolism, taking the milestone experiments of insulin resistance in adipose tissues induced by macrophages as an example152, 153, 154. Infiltrative immune cells of tumors tend to play a crucial part in resolving the fate of lesions and exert selective pressure on them. In contrast, cancer cells manipulate immune cells to promote the development of conditions, escaping antagonistic activities. Consequently, it is of great concern to hunt for immune regulatory mechanisms and targets of intervention that impact morbid progression. Some compelling evidence for the influence of MST1 signaling in the immune system is derived from clinical case reports in patients with disorders of MST1 expression or function,,156, 157, 158. MST1/2 have been reported to engage in the development, function and homing of lymphocytes, and are compactly bound up with innate and adaptive immune responses. In this section, we focus on the mechanisms of MST1/2 involved in innate and adaptive immune responses in tissues or organs, which are closely linked to inflammation, and we analyze the existing evidence for their role in CVDs and metabolic disorders.

Innate immunity enables the body to quickly recognize and defend against invading pathogens, helping maintain homeostasis, and is the basis for the development of adaptive immunity. Pathogen recognition by the innate immune system relies on pattern recognition receptors (PRRs), which then initiate a series of signaling cascades and exciting transcription factors that induce the synthesis and release of multiple interferons (IFNs), inflammatory cytokines and chemokines. Studies have shown that MST1/2 are related to some specific functions of innate immunity,, which are divided into three categories.

IFN-I, including IFN-α and IFN-β, is a representative marker through which the body defends against viral infection, driving the production of a variety of interferon-stimulated genes (ISGs), which enhance antigen presentation, immune cell function and the generation of cytokines and chemokines to prevent host cells from damage. It has been reported that MST1 augments the secretion of IFN-β in macrophages to prevent hepatocellular carcinoma. MST1 directly combines with IL-1 receptor-associated kinase 1 (IRAK1) and degrades it by phosphorylation. Considering that IRAK1 mediates the formation of INF-β via negatively regulating myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β (TRIF) and retinoic-acid-inducible gene I (RIG-I), which are adaptors to PRRs,, the degradation of IRAK1 mediated by MST1 strengthens the activation of IFN regulatory factor 3 (IRF3) and the generation of IFN-β. Dramatically, innate antiviral immunity itself is also able to influence the status of MST1. Cyclic GMP–AMP synthase (cGAS)-stimulator of interferon gene (STING), acknowledged as a signal transduction pathway that plays a regulatory role in microbial and tumor immunity, is capable of activating TANK-binding kinase 1 (TBK1) and facilitating the phosphorylation and nuclear translocation of IRF3, inducing the expression of effector genes such as IFN-1. Palmitic acid (PA) treatment triggers the release of mitochondrial DNA, which binds to cGAS in the cytoplasm and initiates the cGAS–STING–TBK1–IRF3 cascade to promote MST1 transcription. In addition, another study has manifested that MST1 can directly phosphorylate IRF3 at the T75 and T253 sites to destruct its dimerization and is able to inhibit the activation of TBK1 to further weaken the activity of IRF3, thus suppressing the innate antiviral response. This may be a negative feedback mechanism by which the body avoids excessive activation of innate antiviral immunity. Consequently, there exists a complicated balanced network in host antiviral defense meditated by MST1, which may be related to the immune regulation state of the body.

The antibacterial activity that MST1/2 participates in involves the production of chemokines and ROS. Boro et al. reported that toll-like receptor 2 (TLR2) senses antigens and activates MST1/2 via the TLR2–MyD88–IRAK1/4 axis in Mycobacterium tuberculosis infection, which increases the generation of the inflammatory chemokines CXCL1 and CXCL2 in an IRF3-dependent pathway that is independent of LASTS1/2, further producing antimicrobial and inflammatory factors. ROS can be used to eliminate invading pathogens of innate immunity. Mitochondria is required to coordinate with phagosomes to produce ROS in macrophages to kill pathogens,. Studies have clarified that as TLR is stimulated by bacterial infection, MST1/2 activate and phosphorylate protein kinase C alpha (PKCα), then boosting the PKCα–LyGDI (Rho-specific guanine nucleotide dissociation inhibitor)–Rac cascade to induce the assembly of the TNF receptor associated factor 6 (TRAF6)-evolutionarily conserved signaling intermediate in the Toll pathways (ECSIT) complex, which is necessary to recruit mitochondria to phagosomes and generate ROS,. Increased susceptibility to bacterial infection is observed in MST1-and MST2-deficient mice, as well as a reduction in mitochondrial-phagosome juxtaposition and ROS. Interestingly, MST1/2 can also be excited by ROS. MST1/2 have been shown to regulate the stability of nuclear factor erythroid 2-related factor 2 (Nrf2), an antioxidant transcription factor that protects macrophages from oxidative stress. The release of ROS activates MST1/2, phosphorylating Kelch-like ECH-associated protein 1 (Keap 1) and restraining Nrf2 from degradation to enhance the expression of antioxidant genes and decrease the aging and death of macrophages. As a result, there is mutual activation between ROS and MST1/2 signaling.

Owing to the critical role of the NF-κB signaling pathway in inflammatory and immune responses, strict regulation must be performed to restrict overactivation and subsequent uncontrolled inflammation and immunopathology,. MST1/2 signaling is a promising negative regulatory mechanism of the NF-κB pathway. MST1-deficient mice have shown excessive secretion of proinflammatory cytokines after lipopolysaccharide (LPS) challenge. MST1 can phosphorylate IRAK1 and provoke its degradation, inhibiting TLR4/9–NF-κB signal transduction and the inflammatory response in macrophages. Meanwhile, a research has revealed that TRAF6 mediates the ubiquitination and activation of MST1 at the L221 site under LPS stimulation, and MST1 ablation raises the activation of NF-κB and the susceptibility to septic shock, supporting that MST1 is the negative feedback mechanism of the TLR4–NF-κB pathway associated with macrophage inflammation. Moreover, MST1 is able to attenuate the expression of NF-κB-dependent inflammatory genes induced by TNF-α. Faced with TNF-α stimulation, MST1 is activated and recruited to the TNF-α receptor one signaling complex (TNF–RSC), which binds to and phosphorylates HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC), restraining its activity and related linear ubiquitin, and finally suppressing the activation of NF-κB and the expression of its target gene.

Adaptive immunity refers to an antigen-specific immune response and is divided into cellular immunity and humoral immunity, mediated by T and B cells, respectively. In addition, other cells, such as macrophages and dendritic cells, are required to participate in specific immunity. In the case of antigen stimulation, specific immunity can be classified into the following sections: the stage of antigen processing, presentation and recognition; the stage of proliferation and differentiation of T and B cells and the formation of memory cells; and the stage of effector T cells, antibodies and lymphokines exerting immune effects. Accordingly, we will expound on the role of MST1/2 in adaptive immunity from these perspectives.

Naive T cells can be activated to proliferate and differentiate into memory or effector cells after recognizing the antigens presented by antigen presenting cells (APCs) in the major histocompatibility complex (MHC). Intensive naive T-cell proliferation has been observed in the process of direct stimulation to T-cell receptors in MST1-deficient animal models177, 178, 179. Recently, a study has demonstrated the role of MST1/2 in antigen-specific proliferation, where MST1/2-deficient T cells cannot establish stable contact with dendritic cells (DCs), giving rise to proliferation flaws under antigen-specific stimulation. The concrete mechanism may result from adhesion defects mediated by lymphocyte function-associated antigen-1 (LFA-1) and intercellular cell adhesion molecule-1 (ICAM-1). Here, we need to propose the concept of an “immune synapse (IS)”: lymphocytes can recombine organelles at the antigen recognition site of APC and transfer antigen receptors and integrins to the contact site to form IS, which is a crucial platform for lymphocyte activation and polarizing release of cytotoxic particles181, 182, 183. IS mainly consists of four layers of supramolecular activated clusters (SMAC), among which TCR gathers in the central region of SMAC (cSMAC) and LFA-1/ICAM-1 clusters encircling cSMAC form a peripheral SMAC (pSMAC) where MST1 is located, with a ring structure. MST1/2 deficiency leads to the loss of high affinity binding between LFA-1 and ICAM-1 and eliminates the formation of cSMAC, thereby impairing the interaction between APC and T cells. Therefore, MST1/2 exert vital effects on the adhesion structure necessary for the recognition of T cells. Furthermore, it has been found that regulatory T-cell (Treg)-related contact inhibition plays a significant role in MST1-involved antigen recognition. We failed to observe the formation of mobile immunological synapses on supported planar membranes in MST1-deficient T cells and their interaction with DC damage, resulting in the downregulation of costimulatory molecules and impairment of Treg contact inhibition,. Eventually, it seems that MST1 is of significance in immune synapses referring to B cells, which is capable of positively regulating the costimulatory molecule CD19 of the B-cell receptor through the Hippo signaling pathway and TEA domain family member 2 (TEAD2), and the deletion of MST1 may elicit the interruption of B-cell receptor aggregation and downstream signal transduction, markedly weakening the humoral response.

Under certain environmental stimuli, naive CD4+ T cells are activated and differentiate into specific subtypes, including T helper cells, such as Th1, Th2, Th17, Treg and T follicular helper (Tfh) cells, while naive CD8+ T cells differentiate into cytotoxic T lymphocytes (CTLs). MST1/2 have been clarified to modulate the differentiation and function of effector T cells through transcription factors. MST1 participates in regulating the expression of FOXP3 in Tregs so as to maintain the production and function of Tregs. As is known that transcription factor FOXO combines with the Foxp3 promoter and facilitates its transcription to control the differentiation of Tregs, and MST1 is able to activate FOXO1/3, triggering intensive transcription of Foxop3 in Tregs. Meanwhile, the histone deacetylase Sirtuin 1 (SIRT1) tends to promote the proteasome degradation of FOXP3, and MST1 suppresses the degradation of FOXP3 in Tregs through phosphorylating SIRT1188, 189, 190. Besides, MST1/2 are also associated with IL-2R signaling in Tregs. MST1/2 are stimulated by IL-2, positively regulating the phosphorylation of signal transducer and activator of transcription 5 (STAT5) and maintaining the homeostasis and survival of Tregs, their deficiency bringing about restricted Treg migration and subsequent impairment of IL-2 signaling,. Additionally, the differentiation of CTLs is impacted by MST1, without which the level of FOXO in CD8+ T cells decreases, augmenting cytotoxicity in vitro, along with increasing the expression of T-bet transcription, which is related to the levels of IFN-γ and granzyme B34. In addition, MST1 negatively regulates the amplification of Tfh cells, which get command of survival of B cells and antibody formation. It has been found that more Tfh cells in an MST1-deficient model express higher levels of IL-21, IL-4, CD40L and antibodies. Ultimately, MST1 signal transduction from DCs reveals negative regulation of Th17 differentiation. MST1-deficient DCs boost the activation of p38 MAPK signaling during the process of guiding Th17 cells to differentiate and then mediate the secretion of IL-6, which modulates the activation of IL-6 receptor and STAT3 in CD4+ T cells. To sum up, MST1/2 not only exerts a positive regulatory effect on the differentiation and function of Tregs but also negatively regulates CTLs, Th17 cells and Tfh cells, which can be used to balance the immune activation and tolerance of lymphocytes to avoid an excessive immune response.

In addition to the relevant literature on the above content, there exists evidence that MST1/2 is important in other cells or functions concerning adaptive immunity. The absence of MST1 in B lymphocytes leads to developmental defects of B cells in the marginal region. MST1 is able to positively modulate the signal transduction of B-cell receptors via adjusting the transcription level of CD19, mediating B-cell differentiation in the marginal region and germinal center. Moreover, MST1 is a key regulator in the process of neutrophil extravasation under inflammation. Its deficiency can cause dysfunction of VLA-3, VLA-6 and neutrophil elastase translocation from intracellular vesicles to the neutrophil surface, thereby inhibiting their migration to inflammatory sites. Finally, studies have discovered that MST1 is apt to curb C–C motif receptor 7 (CCR7)-dependent endocytosis, migration and cellular structure in mature DCs, taking part in the initiation of the immune response, and it negatively adjusts DC overactivation by downregulating the response of the Akt/c-myc axis to granulocyte-macrophage colony stimulating factor (GM-CSF).

In view of the role of MST1/2 in innate immunity and adaptive immunity summarized above, we mainly analyze the existing but not abundant evidence of immune inflammation in cardiovascular and metabolic diseases, which has incredible research prospects (Fig. 6).

Ischemic heart disease (IHD) is one of the most common causes of myocardial injury. Regardless of the fact that early revascularization and drug therapy can immensely enhance the individual survival rate, these measures fail to repair necrotic myocardium, inevitably leading to heart failure in many patients in the long-term progression. Consequently, great attention has been given paid to the mechanism of damaged tissue repair, among which the timely control of inflammation is crucial. Acute cardiac inflammation is characterized by the continuous release of inflammatory mediators, which results in the aggregation of polymorphonuclear leukocytes and subsequent phagocytosis of monocytes and macrophages, accompanied by proteolysis, angiogenesis and collagen deposition,. The duration of the inflammatory reaction is closely related to tissue injury, which is regulated by the balance of proinflammatory mediators derived from fatty acids and anti-inflammatory mediators,. Ischemia can stimulate the expression and activity of lipid oxidase that catalyzes arachidonic acid, the precursor of fatty acids, and macrophages are a significant source of these lipid mediators and undergo metabolic reprogramming to the 5-lipoxygenase (5-LOX) pathway in the process of atherosclerosis. Research has shown that pharmacological and genetic inhibition of MST1/2 is able to trigger this outcome, aggravating cardiac insufficiency after myocardial infarction (MI). The specific mechanism is that MST1 in macrophages prevents the interaction between 5-LOX and 5-LOX activating protein (FLAP) in the nuclear membrane through phosphorylating 5-LOX at the T218 residue, thus reducing the production of leukotriene B4 (LTB4), which binds to LBT4 receptor 1 (BLT1) and elicits NF-κB signal transduction. Therefore, the suppression of MST1/2 contributes to the proinflammatory effect induced by LTB4, damaging cardiac repair after MI. Besides, Ilatovskaya et al. summarized the function of the adaptive immune system in the development and prevention of cardiac tissue injury and other CVDs. Considering the essential role of MST1/2 in adaptive immunity, we are apt to further explore their extensive application in CVDs despite no relevant reports.

The evidence is equally strong that the increase of free fatty acids is a common risk factor for obesity, type two diabetes and other metabolic disorders. In patients with diabetes and obesity, the continuous augmentation of free fatty acids and blood glucose can impair vascular mitochondrial function, resulting in endothelial dysfunction and following vascular complications. Studies have reported that free fatty acids are able to destroy mitochondria to mediate the release of mtDNA, which participates in the cGAS–STING–IRF3 pathway to phosphorylate IRF3, thereby combining with the promoter region of MST1 to induce MST1 expression and subsequently inhibiting YAP to suppress angiogenesis. Similarly, the activation of the lipoxygenase approach is also a potentially important source of metabolic disorder inflammation. It has been identified that 12-lipoxygenase (12-LOX) is the main lipoxygenase isomer in human islets, which facilitates the generation of 12(S)-hydroxy eicosatetraenoic acid (12-S-HETE) from arachidonic acid, thus exerting a pathogenic inflammatory effect through downstream inflammatory factors and protein kinases to arouse islet dysfunction and β-cell death. As a result, it is of great significance to probe whether MST1/2 affects 12-LOX, which may be a promising direction of research.

Based on the existing research evidence, MST1/2 kinases are potential intervention targets for the treatment of cardiovascular and metabolic diseases. Pharmacological development for them is in full swing, although there is no drug that has been officially approved to perform functions so far. Studies have shown that pharmacological effects targeting MST1/2 can be divided into two aspects: one is selective small molecular inhibitors of MST1/2, and the other is drugs or even noncoding RNAs that influence MST1/2 kinases. We will emphasize their progress up to date and propose a prospective combination therapy.

A report in 2016 revealed a reversible and selective MST1/2 inhibitor, 4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino)benzenesulfonamide (XMU-MP-1), according to a high-throughput biochemical assay based on an enzyme-linked immunosorbent assay. The eutectic structure and structure–activity relationship confirm that XMU-MP-1 targets MST1/2. XMU-MP-1 is able to block the activity of MST1/2 kinases, which activates downstream YAP and promotes cell growth, showing excellent pharmacokinetics in vivo and enhancing intestinal recovery and liver regeneration in acute and chronic liver injury. Furthermore, subsequent studies have enriched the therapeutic role of XMU-MP-1 in cardiovascular and metabolic diseases. In the case of pressure overload, decreased cardiomyocyte hypertrophy and an increased survival rate by 25%–50% have been observed in the XMU-MP-1 treatment group, accompanied by a significant decrease in the hypertrophic marker BNP, revealing intensive cardiac contractility compared with the control group. Besides, XMU-MP-1 in reperfusion therapy lessens the infarct size, apoptosis and necrosis by regulating the MST1/AMPK signaling pathway and preserves the cardiac function of mice. Additionally, XMU-MP-1 treatment can also ameliorate the survival and function of islet β cells. It has been found that the inhibition of MST1/2 in INS-1 cells by XMU-MP-1 amplifies the number of islets β cells and the area of Langerhans islets in severely diabetic mice, thus improving their glucose tolerance. Dramatically, a study has elucidated that MST1 deficiency and glycemic control synergistically improve myocardial function and reduce cardiomyocyte apoptosis in diabetic mice, both of which are indispensable. The use of XMU-MP-1 after insulin maintenance therapy can alleviate mitochondrial dysfunction to ameliorate cardiac function through activating the AMPK pathway, which indicates that XMU-MP-1 tends to be used as a supplement to insulin in the treatment of diabetic cardiomyopathy. It is worth noting that XMU-MP-1 can also remarkably inhibit 21 other kinases, excluding MST1/2, to cause off-target effects. Researches have demonstrated that XMU-MP-1 has a high affinity for Aurora A/B kinase controlling mitosis and phosphorylation of histone H3, which may be the reason why XMU-MP-1 interferes with the cell cycle process in human hair follicles. Therefore, it is vital to seek MST1/2 inhibitors with high selectivity for kinases. The latest experiments have identified and verified a group of selective MST1/2 inhibitors, such as pyrrolopyridazines and pyrrolopyrimidines, which are derived from Janus kinase (JAK) inhibitors and leucine-rich repeat kinase 2 (LRRK2) inhibitors, respectively. Some of them have higher kinase selectivity than XMU-MP-1, along with lower half maximal inhibitory concentration and MST1/2 activity remaining, which have been characterized and described in prostate cancer (PC) and acute myeloid leukemia (AML) researches,. Pharmacological studies of the above compounds have shown that inhibition of MST1 retards PC cell proliferation and Matrigel invasion, accompanied by cell cycle arrest. Furthermore, they have targeted activity in AML cells, and their single agents restrains cell proliferation in all six AML cell lines in a concentration-dependent manner. The content of this part is of great significance, where understanding and elucidating the schemes to raise kinase selectivity is conducive to developing specific drugs targeting MST1/2 in the future (Fig. 7).

In addition to selective small molecular inhibitors of MST1/2, there are many effective compounds and drugs that affect MST1/2 kinases to play a role in CVDs and metabolic disorders. Moreover, the noncoding RNA that influences MST1/2 signaling has received much widespread attention. Noncoding RNAs account for more than 80% of the genome, including microRNAs (miRNAs), long-chain noncoding RNAs (lncRNAs), small interfering RNAs (siRNAs) and circular RNAs (circRNAs), which are involved in the regulation of multiple cellular activities and diseases. In this section, we summarize the available evidence of these drugs and noncoding RNAs (Table 2213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230).

Numerous advanced studies have concentrated on neratinib, a tyrosine kinase inhibitor primarily used in anticancer therapy, whose function is based on ERBB2/EGFR suppression,. However, clinical observations have shown that it can also alleviate diabetes symptoms, and in-depth validation reveals that neratinib has a strong inhibitory effect on MST1 and improves the survival rate of β cells in vitro under high glucose and high fat conditions,,. Another compound that restrains MST1 is asialo-erythropoietin, a sialic acid residue-free erythropoietin (EPO) derivative that does not exhibit hematopoietic properties and has an immensely short circulating half-life, so it is theoretically safe for application,. Asialo-erythropoietin has been found to possess broad neuroprotective effects. Recent studies have ulteriorly illustrated that it can also protect the islet β cell line RIN-m5F against staurosporine-induced cell death and prevent HL-1 cardiomyocytes from injury by inhibiting MST1,. In addition, melatonin is a widely discussed drug with abundant actions that can participate in multiple signaling pathways to enhance autophagy and reduce apoptosis and mitochondrial dysfunction. Melatonin has been reported to curb MST1 and foster the expression of sirtuin proteins, improving diabetic cardiomyopathy and postinfarction cardiac dysfunction,. Melatonin also rescues damage in CVDs and metabolic disorders by affecting MST1 to regulate the JNK and Parkin pathways,. Moreover, studies have clarified that melatonin effectively inhibits the MST1-mitochondrial elongation factor 1 (MIEF1) axis, thereby enhancing cell survival under TNF α-mediated inflammatory injury to exert endothelial protection. Considering the benefits of melatonin in cardiovascular and metabolic diseases that have been reported, MST1/2 signaling is only one of them, and there may be cross-interactions with other pathways that deserve to be further explored. Certainly, some other drugs, such as irisin, quercetin and luteolin, have been covered to ameliorate CVDs and metabolic disorders through MST1/2 as well,,, and we will not go into detail here. It is remarkable that the combined use of the abovementioned drugs is a topic for future research, where there may be synergistic effects to further strengthen the efficacy of the drugs.

Noncoding RNA mainly regulates the activity of proteins through interactions with proteins to engage in the pathogenesis of various diseases, and research in the field of medicine oriented by it is a hot spot today. It is acknowledged that miRNAs reveal dual action in cancers where it can act as oncogenes or tumor suppressor genes. Studies have pointed out that miR-3910 promotes the growth and migration of liver cancer cells by targeting MST1, and miR-130 b is able to directly target MST1 and SAV1 to boost the self-renewal of glioblastoma,. Growing evidence suggests that lncRNAs can also influence MST1/2 to regulate various cellular functions. Overexpression of LINC01314 in hepatoblastoma enhances the expression of MST, thereby activating the Hippo signaling pathway and inhibiting tumor cell proliferation; TNRC6C-AS1 recruits methyltransferase to methylate the MST1 promoter, thus making MST1 silent, which accelerates the proliferation of thyroid cancer cells and suppresses autophagy,. Furthermore, in hypoxia/reoxygenation-mediated H9C2 cell injury, lncRNA ROR is remarkably upregulated to downregulate miR-138, of which MST1 is revealed to be a negative regulatory target. Finally, incremental expression of circPPP1R12 A can be observed distinctly in colon cancer cells, and circPPP1R12 A-73aa, a protein encoded by circPPP1R12 A, promotes the activation of YAP1 through the inhibition of MST1/2, influencing the proliferation, migration and invasion of colon cancer cells.

It is clear from what we have stated that MST1/2 is a complex paradox. MST1/2 are well-established proapoptotic effectors and are associated with tissue regeneration, so pharmacological inhibition of MST1/2 with XMU-MP-1 reduces apoptosis and enhances tissue regeneration, alleviating oxidative stress and mitochondrial dysfunction under intense pressure. However, since MST1/2 mostly exert anti-inflammatory activity in immune cells, XMU-MP-1 therapy alone may lead to treatment failure due to local persistent tissue inflammation. As a result, a cardiovascular study has proposed a new treatment concept that combines MST1/2 inhibitors with antagonists of downstream immune inflammation. We believe that this model can be extended to cardiovascular and metabolic diseases. Owing to the bidirectional roles of MST1/2 in proapoptotic, autophagy regulation and immunomodulatory functions in disease outcomes, clarifying the mechanisms of MST1/2 in different cells at different stages will help flexibly select MST1/2 activating/inhibiting drugs and immunomodulators. In conclusion, this new mode of drug combination is an inevitable way to improve the efficacy in CVDs and cardiovascular disorders in the future based on extensive and detailed pharmacological and pathogenic studies.

CVDs and metabolic disorders are major components of noncommunicable diseases and cause an enormous health and economic burden worldwide. There are common risk factors and developmental mechanisms among them, and exploring the corresponding intervention targets has urgent and far-reaching significance for treatment and prognosis. MST1/2 kinases have received extensive attention as not only well-established proapoptotic effectors but also bidirectional regulators of autophagic activity. Further studies are needed to elucidate the specific mechanisms by which MST1/2 adjust autophagy during cardiovascular and metabolic events based on disparate circumstances and conditions. In addition, compelling evidence has demonstrated a role of MST1/2 in immunity, which influences immune cell activity and regulates the level and duration of inflammation in tissues and organs to determine the outcome of cardiovascular and metabolic diseases. In recent years, immune inflammation is a research hotspot in the field of CVDs and metabolic disorders. Although there are few related studies focusing on MST1/2, based on the richness of basic research on its immune function, we believe that MST1/2 is the key worthy of further exploration. To sum up, MST1/2 are promising intervention targets for the treatment of CVDs and metabolic disorders. Certainly, drug development for MST1/2 is in full swing. XMU-MP-1 is a selective inhibitor of MST1/2 and has been demonstrated to be useful in multiple models. Nevertheless, XMU-MP-1 can also activate other kinases to produce “off-target effects”, so researchers have subsequently explored and identified new MST1/2 kinase highly selective inhibitors, such as pyrrolopyridazines and pyrrolopyrimidines, and more investments are required to seek and find more suitable small molecule inhibitors of MST1/2 in the future. Unfortunately, the above studies are based on the exploration of in vitro or animal in vivo models, along with no relevant human data, and these studies to explore highly selective inhibitors of MST1/2 kinases have no evidence in the cardiovascular and metabolic fields. In addition, to date no MST1/2 inhibitors have been officially approved to work. What's more, CVDs and metabolic disorders are chronic diseases, which require long-term use of MST1/2 inhibitors in practical applications. It represents a significantly higher drug development and toxicology challenge than administration of these agents for short-term acute diseases. As a result, there is a long way to go for drug research targeting MST1/2. Furthermore, some drugs or compounds with diverse functions and noncoding RNAs also modulate MST1/2 kinase activity and affect cardiovascular and metabolic diseases, which can serve as alternative treatment options. Notably, considering the complex biological roles of MST1/2, a new mode of MST1/2-targeted drugs combined with immunomodulators is an inevitable way to apply in cardiovascular and metabolic diseases in the future on the basis of extensive and detailed pharmacological and pathogenesis research.

This work is supported by grants from the 10.13039/501100001809National Natural Science Foundation of China (Nos. 81770327 and 81100173) and Jiangsu Province Health Care Development Special Fund (M2022038, China).

All authors have read and approved the final review manuscript. Hongxia Li contributed to the conceptualization and preparation of the manuscript; Yunfei Yin, Mingyue Tan and Lianhua Han conceived and wrote this review as well as production of figures and tables; Jun Zhang and Wanqian Pan searched and analyzed the literature; Lei Zhang and Yue Zhang reviewed and edited this review; Hongxia Li, Tingbo Jiang and Jiaxiang Bai supervised this review.

The authors have declared that no competing interest exists.