Mixed lineage kinase domain-like protein in liver diseases: Cell-type-specific functions and dual roles

Abstract

In this letter, we comment on the article by Xuan Yuan et al, published in the recent issue of the World Journal of Gastroenterology. Mixed lineage kinase domain-like protein (MLKL) exhibits cell-type-specific functions in liver parenchymal and non-parenchymal cells, playing dual roles in the pathogenesis of liver diseases. In hepatocytes, MLKL primarily mediates necroptosis and inhibits autophagy, thereby exacerbating liver injury. Conversely, in non-parenchymal liver cells, MLKL modulates inflammatory responses and promotes fibrotic processes, thereby driving disease progression. Notably, MLKL also demonstrates protective functions under specific conditions. For instance, MLKL can inhibit intracellular bacterial replication, promote endosomal trafficking, and facilitate the generation and release of extracellular vesicles, potentially exerting hepatoprotective effects. Understanding these cell-type-specific mechanisms of MLKL action, including its dual roles in promoting injury and providing protection, is crucial for elucidating the complex pathogenesis of liver diseases and developing targeted therapeutic strategies.

Key Words: Cell-type-specific functions; Dual roles; Liver diseases; Mixed lineage kinase domain-like protein; Necroptosis

Core Tip: Mixed lineage kinase domain-like protein (MLKL) functions to mediate necroptosis, inhibit autophagy, and regulate inflammatory responses. The MLKL exhibits different functions in various types of liver cells, leading to diverse manifestations of different liver diseases. This complexity not only brings challenges in understanding the roles of MLKL in liver diseases, but also presents an obstacle in developing therapeutic strategies for targeting MLKL. Here, we briefly review the functions of MLKL and analyze its specific roles in different types of liver cells, aiming to provide clear strategies for precisely targeting MLKL in the treatment of liver diseases.

TO THE EDITOR

Heterogeneity of liver cells and multi-faceted roles of mixed lineage kinase domain-like protein (MLKL) in different liver diseases. The liver is a vital organ composed of a diverse array of specialized parenchymal and non-parenchymal cells, including hepatocytes, Kupffer cells, hepatic stellate cells (HSCs), endothelial cells (ECs), cholangiocytes, natural killer (NK) cells, and T lymphocytes[1,2]. In the physiological state, hepatocytes account for about 80% of the total number of cells in the liver, while Kupffer cells constitute approximately 15% and make up 80%-90% of the body’s mononuclear phagocytes. HSCs and ECs, including the specialized type known as liver sinusoidal ECs (LSECs), account for roughly 5% and 10% of the total hepatic cells, respectively. Cholangiocytes represent about 4% of the total hepatic cells. Additionally, there is a small population of NK cells and T lymphocytes. The composition and proportion of these cells change at different stages of various liver diseases, highlighting the heterogeneity of liver cells and their distinct roles in liver function and pathology.

Among the various cellular components of the liver, MLKL plays a crucial role in liver cell function and is involved in various cellular processes that are vital for maintaining liver health. MLKL primarily functions to mediate necroptosis[3,4]. Additionally, MLKL can inhibit autophagy[5,6] and enhance inflammatory responses by activating the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome[7,8] and fibrosis[9]. Furthermore, MLKL can also induce macrophage polarization[10], promote macrophage extracellular trap release[11], and control the ubiquitination and proteasomal degradation of connexin 43[12]. Activation of MLKL promotes the degradation of myelin[13], inhibition of testosterone production in male mice[14], regulation of endosomal trafficking and extracellular vesicle production[15], and support of hematopoietic and phagocytic functions of splenic ECs[16]. Additionally, MLKL upregulates the expression of adhesion molecules on ECs[17,18], and induces phosphatidylserine externalization[19]. MLKL also promotes the clearance of intracellular bacteria through ubiquitination modification[20,21]. Moreover, MLKL regulates the differentiation, malignant behaviors and therapeutic responses of malignantly transformed parenchymal cells, such as hepatocellular carcinoma (HCC) cells.

ROLES OF MLKL INPARENCHYMAL CELLS

MLKL mediates necroptosis and inhibits autophagy in hepatocytes

Necroptosis is a major type of programmed hepatocyte death that contributes to the pathogenesis of various liver diseases[22], such as acute liver injury, alcoholic liver disease, and non-alcoholic fatty liver disease (NAFLD). This mode of cell death is characterized by increased plasma membrane permeability, the release of cellular components, and cellular lysis, with morphological changes similar to those seen in necrosis[23]. MLKL acts as the executor of necroptosis, and during the executive process of necroptosis, it is phosphorylated by activated receptor-interacting serine/threonine-protein kinases (RIPK) RIPK1 and RIPK3 in the necrosome. Subsequently, the activated MLKL oligomerizes and translocates to the plasma membrane, disrupting it and releasing cytoplasmic contents, thereby exacerbating the inflammatory process[24]. Consequently, necroptosis can lead to chronic inflammation in the liver, which may contribute to chronic liver injury.

Autophagy, a self-cleaning process of cells, maintains cellular health by clearing damaged organelles and proteins to reduce inflammation, and mitigate the risk of fibrosis. Dysfunctional autophagy can lead to cell damage and death. A study has found that MLKL deteriorates insulin resistance and glucose intolerance in obese mouse models[25]. In addition, MLKL also aggravates diet-induced liver injury and inflammatory responses. This occurs because MLKL inhibits autophagy without depending on RIPK3 pathway activation. The mechanism may involve activated MLKL inhibiting autophagic flux through the mammalian target of rapamycin signaling pathway or by directly interfering with the formation of autophagosomes[5,26]. In contrast, in mouse models of alcohol-induced liver injury, inhibiting MLKL can reduce the nuclear translocation of p65, a key transcriptional regulator of NLRP3. This process limits the transcription of NLRP3 message RNA and subsequent NLRP3 expression, thereby suppressing the activation of inflammasomes and related inflammatory responses, ultimately contributing to the reduction of liver damage[8]. Similarly, treatment with MLKL adenosine triphosphate (ATP)-binding pocket inhibitors attenuated inflammation in alcoholic liver disease models by down-regulating the expression of chemokine and adhesion molecules[27].

Dual function of MLKL in HCC

In HCC cells, MLKL-mediated necroptosis serves as an alternative cell death mechanism that can inhibit tumor progression[28] and exert anti-tumor effects by modulating the tumor microenvironment. For instance, necroptosis can promote the polarization of macrophages and the infiltration of cluster of differentiation 8 + T cells within tumor[29,30], thereby enhancing anti-tumor immune responses and suppressing HCC progression. However, the role of MLKL in HCC is complex and dualistic. A recent report suggests that MLKL promotes HCC development by inhibiting the adenosine 5’-monophosphate-activated protein kinaseá1-mediated autophagy[6]. Additionally, MLKL, independent of RIPK3, facilitates immune escape in HCC by regulating parthanatos, a programmed cell death pathway distinct from necroptosis[31].

In addition to this role, MLKL may contribute to maintaining cellular homeostasis by regulating endosomal trafficking and extracellular vesicle generation. This protective function, which limits necroptotic activity[32], can indirectly supports tumor progression by enhancing intercellular communication among liver cell types. Specifically, MLKL’s regulation of extracellular vesicle generation influences the inflammatory microenvironment and the fibrotic response, linking the actions of hepatocytes, macrophages, and HSCs in the pathogenesis of liver disease.

Furthermore, in the context of cancer, MLKL-driven necroptosis also promotes tumor metastasis by inducing the formation of macrophage extracellular traps, thereby evading immune surveillance[11]. Therefore, treatment strategies for HCC need to consider these factors comprehensively. By delving deeper into the interactions and regulatory networks among these mechanisms, more effective and personalized treatment plans should be developed, ultimately enhancing the therapeutic outcomes and survival rates for HCC patients.

ROLES OF MLKL IN NON-PARENCHYMAL CELLS—REGULATING INFLAMMATION AND FIBROSIS

HSCs

HSCs are a unique type of non-parenchymal cells in the liver and play a crucial role in the fibrosis process triggered by liver damage. Following liver injury, quiescent HSCs become activated, proliferate, and transform into myofibroblast-like cells, which produce an excessive amount of extracellular matrix, leading to liver fibrosis[33]. Research has shown that MLKL activation can lead to the formation of ion channels, a function that is positively correlated with its ability to alter membrane potential and induce cell death[34]. Additionally, specific ion channels, such as transient receptor potential melastatin 7, are involved in HSC activation[35]. Moreover, MLKL may mediate the activation of HSCs by modulating the transforming growth factor-â (TGF-â)/SMAD pathway, which is crucial for the transition of quiescent HSCs to myofibroblast-like cells[9]. Liver-specific MLKL knockout can significantly alleviate hepatocyte damage and necroptosis, markedly inhibit HSC activation, and substantially reduce liver injury and fibrosis.

Macrophages

Hepatic macrophages primarily consist of liver-resident Kupffer cells and monocyte-derived macrophages from peripheral blood. MLKL mediates macrophage necroptosis. A previous study reported that mononuclear macrophages and Kupffer cells mitigate acetaminophen-induced liver inflammation and injury[36]. Z-DNA-binding protein 1/p-MLKL-mediated macrophage necroptosis also plays a critical role in the pathogenesis of biliary atresia-associated liver fibrosis. Silencing MLKL specifically in macrophages has been shown to mitigate macrophage necroptosis and bile duct ligation-induced cholestatic liver injury in mice[37].

In addition to necroptosis, MLKL has other functions in macrophages. MLKL deficiency reduces oxidative DNA damage in hepatocytes by promoting the PINK1-mediated mitophagy, thereby inhibiting cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes activation in macrophages and alleviating inflammatory liver ischemia-reperfusion injury[38]. Myeloid MLKL can limit ethanol-induced liver inflammation and injury by regulating the homeostasis of liver immune cells and macrophage phagocytosis[39].

However, as highlighted in the editorial article, Xuan Yuan et al[27] found that RAW 264.7 cells (mouse monocyte-macrophages) did not undergo necroptosis when stimulated with tumor necrosis factor alpha (TNF-á), a known inducer of necroptosis. This may be related to DNA methylation reducing the expression of RIPK3. Additionally, the study revealed that MLKL increased the expression of chemokines, and intercellular adhesion molecules through activating the nuclear factor kappa-B pathway, exacerbating liver inflammation. Notably, this activation process was closely associated with the MLKL ATP pocket, and treatment with an MLKL ATP pocket inhibitor effectively blocked this process, providing new directions for the treatment of liver diseases.

Although current findings indicated that MLKL ATP pocket inhibitors were ineffective in NAFLD models, they exhibited protective effects in alcoholic liver disease models. Thus, while MLKL is known to be crucial for the function of macrophages, further investigations are needed to explore its regulatory role in macrophages during liver disease processes to help translate these findings into clinical treatments.

ECs

ECs, especially LSECs, are crucial for maintaining liver homeostasis and influencing the progression of liver disease. Additionally, they ensure that HSCs and Kupffer cells remain quiescent. With the activation of MLKL under sustained liver injury, LSECs gradually lose their protective phenotype and begin to overexpress adhesion proteins, which promotes HSC activation and leads to liver fibrosis[40,41]. Additionally, LSECs have been shown to produce a series of pro-inflammatory cytokines, such as TNF-á, interleukin (IL)-6, and IL-1, under damaged conditions. These cytokines lead to poor adaptation of the vascular microenvironment in non-alcoholic steatohepatitis (NASH), further exacerbating disease progression[42].

Moreover, the EC-specific deficiency of MLKL effectively alleviates liver fibrosis in NASH by inhibiting the activation of the TGF-â/SMAD 2/3 pathway and disrupting the pro-fibrotic crosstalk between ECs and HSCs[43]. Another study found that MLKL deletion in vascular ECs protects endothelial integrity by blocking necroptosis in a mouse model of systemic inflammatory response syndrome[44]. Therefore, targeting the MLKL-mediated necroptosis pathway may represent a promising strategy for preventing the progression of liver injury and its complications.

Cholangiocytes

Cholangiocytes play a crucial role in the development of cholangiopathy[45], particularly in the context of high necroptosis activity during cholestasis[46]. Prolonged RIPK1-MLKL-mediated necroptosis leads to the destruction of cholangiocytes in patients with primary biliary cholangitis (PBC). The inhibitor of heat shock protein 90 can decrease the half-life of RIPK1 and accelerate its degradation, potentially mitigating PBC by inhibiting the RIPK1-MLKL-mediated necroptosis of cholangiocytes[47].

POTENTIAL PROTECTIVE ROLES OF MLKL IN LIVER DISEASES

Although extensive research has revealed multiple pathogenic mechanisms of MLKL in liver diseases, recent studies suggest that MLKL also plays a multi-dimensional protective role in immune responses, depending on specific signals and modification states within the microenvironment. Specifically, MLKL participates in endosomal trafficking through site-specific ubiquitination, promoting bacterial and lysosomal degradation, thereby maintaining intracellular immune homeostasis and playing a crucial role in combating bacterial infections[20,21]. These findings suggest that MLKL may enhance the ability of Kupffer cells to clear bacteria or gut-derived toxins entering the liver, thereby maintaining hepatic immune homeostasis.

In alcohol-associated liver disease, macrophage-derived MLKL regulates phagocytic function, thereby suppressing alcohol-induced hepatic inflammation and injury, highlighting its protective role in liver immune function[39]. Moreover, studies have demonstrated that MLKL is involved in endosomal trafficking, as well as the generation and release of extracellular vesicles[15,16,32,48]. These vesicles are capable of carrying lipids, proteins, and other signaling molecules, thereby facilitating intercellular communication and modulating the functions of distant cells. By participating in endosome trafficking and the generation and release of extracellular vesicles, MLKL not only contributes to maintaining lipid metabolism balance but also regulates intercellular communication, which is crucial for sustaining cellular homeostasis and function. Notably, the release of extracellular vesicles may act as a self-protective mechanism, helping to prevent the excessive activation of MLKL and thus reduce the occurrence of necroptosis.

Therefore, the precise regulation of MLKL activity should be based on the specific characteristics of the disease stage and microenvironment, rather than simply inhibiting or activating its function.

CONCLUSION

MLKL exhibits significant cell-type-specific functions, and its diverse roles across different cell types highlight its dual role in driving disease progression and exerting protective effects. To effectively develop MLKL-targeted therapeutic strategies, it is essential to gain a deeper understanding of the specific regulatory mechanisms of MLKL in various liver cell types. Additionally, robust clinical data are required to evaluate the efficacy, safety, and potential side effects of MLKL-targeted therapies, alongside challenges such as patient stratification based on disease stage and pathological characteristics. However, MLKL’s mechanisms of action are highly complex, influenced by variations in cell types, disease stages, and experimental models, while its crosstalk with other cell death pathways remains poorly understood. Future research should focus on the following directions: (1) Constructing precise models to systematically explore the dynamic roles of MLKL in liver diseases; (2) Deciphering the MLKL signaling network and its downstream mechanisms to identify potential synergistic targets; and (3) Evaluating the safety and specificity of targeted therapies to facilitate the translation of research findings into clinical applications. Research on MLKL not only deepens the scientific understanding of liver disease mechanisms but also provides new insights and potential therapeutic targets for liver disease treatment.