Dynamic palmitoylation of STX11 controls injury-induced fatty acid uptake to promote muscle regeneration.

Different types of cells uptake fatty acids in response to different stimuli or physiological conditions; however, little is known about context-specific regulation of fatty acid uptake. Here, we show that muscle injury induces fatty acid uptake in muscle stem cells (MuSCs) to promote their proliferation and muscle regeneration. In humans and mice, fatty acids are mobilized after muscle injury. Through CD36, fatty acids function as both fuels and growth signals to promote MuSC proliferation. Mechanistically, injury triggers the translocation of CD36 in MuSCs, which relies on dynamic palmitoylation of STX11. Palmitoylation facilitates the formation of STX11/SNAP23/VAMP4 SANRE complex, which stimulates the fusion of CD36- and STX11-containing vesicles. Restricting fatty acid supply, blocking fatty acid uptake, or inhibiting STX11 palmitoylation attenuates muscle regeneration in mice. Our studies have identified a critical role of fatty acids in muscle regeneration and shed light on context-specific regulation of fatty acid sensing and uptake.

Surplus fatty acid synthesis increases oxidative stress in adipocytes and lnduces lipodystrophy.

Adipocytes are the primary sites for fatty acid storage, but the synthesis rate of fatty acids is very low. The physiological significance of this phenomenon remains unclear. Here, we show that surplus fatty acid synthesis in adipocytes induces necroptosis and lipodystrophy. Transcriptional activation of FASN elevates fatty acid synthesis, but decreases NADPH level and increases ROS production, which ultimately leads to adipocyte necroptosis. We identify MED20, a subunit of the Mediator complex, as a negative regulator of FASN transcription. Adipocyte-specific male Med20 knockout mice progressively develop lipodystrophy, which is reversed by scavenging ROS. Further, in a murine model of HIV-associated lipodystrophy and a human patient with acquired lipodystrophy, ROS neutralization significantly improves metabolic disorders, indicating a causal role of ROS in disease onset. Our study well explains the low fatty acid synthesis rate in adipocytes, and sheds light on the management of acquired lipodystrophy.

Exploring the underlying mechanisms of fisetin in the treatment of hepatic insulin resistance via network pharmacology and in vitro validation.

OBJECTIVE: To characterize potential mechanisms of fisetin on hepatic insulin resistance (IR) using network pharmacology and in vitro validation. METHODS: Putative targets of fisetin were retrieved from the Traditional Chinese Medicine Systems Pharmacology database, whereas the potential genes of hepatic IR were obtained from GeneCards database. A protein-protein interaction (PPI) network was constructed according to the intersection targets of fisetin and hepatic IR using the Venn diagram. The biological functions and potential pathways related to genes were determined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Cell experiments were also conducted to further verify the mechanism of fisetin on hepatic IR. RESULTS: A total of 118 potential targets from fisetin were associated with hepatic IR. The areas of nodes and corresponding degree values of TP53, AKT1, TNF, IL6, CASP3, CTNNB1, JUN, SRC, epidermal growth factor receptor (EGFR), and HSP90AA1 were larger and could be easily found in the PPI network. Furthermore, GO analysis revealed that these key targets were significantly involved in multiple biological processes that participated in oxidative stress and serine/threonine kinase activity. KEGG enrichment analysis showed that the PI3K/AKT signaling pathway was a significant pathway involved in hepatic IR. Our in vitro results demonstrated that fisetin treatment increased the expressions of EGFR and IRS in HepG2 and L02 cells under normal or IR conditions. Western blot results revealed that p-AKT/AKT levels were significantly up-regulated, suggesting that fisetin was involved in the PI3K/AKT signaling pathway to regulate insulin signaling. CONCLUSION: We explored the pharmacological actions and the potential molecular mechanism of fisetin in treating hepatic IR from a holistic perspective. Our study lays a theoretical foundation for the development of fisetin for type 2 diabetes.

Ultra-high static magnetic fields cause immunosuppression through disrupting B-cell peripheral differentiation and negatively regulating BCR signaling.

To increase the imaging resolution and detection capability, the field strength of static magnetic fields (SMFs) in magnetic resonance imaging (MRI) has significantly increased in the past few decades. However, research on the side effects of high magnetic field is still very inadequate and the effects of SMF above 1 T (Tesla) on B cells have never been reported. Here, we show that 33.0 T ultra-high SMF exposure causes immunosuppression and disrupts B cell differentiation and signaling. 33.0 T SMF treatment resulted in disturbance of B cell peripheral differentiation and antibody secretion and reduced the expression of IgM on B cell membrane, and these might be intensity dependent. In addition, mice exposed to 33.0 T SMF showed inhibition on early activation of B cells, including B cell spreading, B cell receptor clustering and signalosome recruitment, and depression of both positive and negative molecules in the proximal BCR signaling, as well as impaired actin reorganization. Sequencing and gene enrichment analysis showed that SMF stimulation also affects splenic B cells' transcriptome and metabolic pathways. Therefore, in the clinical application of MRI, we should consider the influence of SMF on the immune system and choose the optimal intensity for treatment.

SLC38A2 and glutamine signalling in cDC1s dictate anti-tumour immunity.

Cancer cells evade T cell-mediated killing through tumour-immune interactions whose mechanisms are not well understood1,2. Dendritic cells (DCs), especially type-1 conventional DCs (cDC1s), mediate T cell priming and therapeutic efficacy against tumours3. DC functions are orchestrated by pattern recognition receptors3-5, although other signals involved remain incompletely defined. Nutrients are emerging mediators of adaptive immunity6-8, but whether nutrients affect DC function or communication between innate and adaptive immune cells is largely unresolved. Here we establish glutamine as an intercellular metabolic checkpoint that dictates tumour-cDC1 crosstalk and licenses cDC1 function in activating cytotoxic T cells. Intratumoral glutamine supplementation inhibits tumour growth by augmenting cDC1-mediated CD8+ T cell immunity, and overcomes therapeutic resistance to checkpoint blockade and T cell-mediated immunotherapies. Mechanistically, tumour cells and cDC1s compete for glutamine uptake via the transporter SLC38A2 to tune anti-tumour immunity. Nutrient screening and integrative analyses show that glutamine is the dominant amino acid in promoting cDC1 function. Further, glutamine signalling via FLCN impinges on TFEB function. Loss of FLCN in DCs selectively impairs cDC1 function in vivo in a TFEB-dependent manner and phenocopies SLC38A2 deficiency by eliminating the anti-tumour therapeutic effect of glutamine supplementation. Our findings establish glutamine-mediated intercellular metabolic crosstalk between tumour cells and cDC1s that underpins tumour immune evasion, and reveal glutamine acquisition and signalling in cDC1s as limiting events for DC activation and putative targets for cancer treatment.

Liraglutide Accelerates Ischemia-Induced Angiogenesis in a Murine Diabetic Model.

Background Severe hindlimb ischemia is a chronic disease with poor prognosis that can lead to amputation or even death. This study aimed to assess the therapeutic effect of liraglutide on hind-limb ischemia in type 2 diabetic mice and to elucidate the underlying mechanism. Methods and Results Blood flow reperfusion and capillary densities after treatment with liraglutide or vehicle were evaluated in a mouse model of lower-limb ischemia in a normal background or a background of streptozotocin-induced diabetes. The proliferation, migration, and tube formation of human umbilical vein endothelial cells were analyzed in vitro upon treatment with liraglutide under normal-glucose and high-glucose conditions. Levels of phospho-Akt, phospho-endothelial nitric oxide synthase, and phospho-extracellular signal-related kinases 1 and 2 under different conditions in human umbilical vein endothelial cells and in ischemic muscle were determined by western blotting. Liraglutide significantly improved perfusion recovery and capillary density in both nondiabetic and diabetic mice. Liraglutide also promoted, in a concentration-dependent manner, the proliferation, migration, and tube formation of normal glucose- and high glucose-treated human umbilical vein endothelial cells, as well as the phosphorylation of Akt, endothelial nitric oxide synthase, and extracellular signal-related kinases 1 and 2 both in vitro and in vivo. The liraglutide antagonist exendin (9-39) reversed the promoting effects of liraglutide on human umbilical vein endothelial cell functions. Furthermore, exendin (9-39), LY294002, and PD98059 blocked the liraglutide-induced activation of Akt/endothelial nitric oxide synthase and extracellular signal-related kinases 1 and 2 signaling pathways. Conclusions These studies identified a novel role of liraglutide in modulating ischemia-induced angiogenesis, possibly through effects on endothelial cell function and activation of Akt/endothelial nitric oxide synthase and extracellular signal-related kinases 1 and 2 signaling, and suggested the glucagon-like peptide-1 receptor may be an important therapeutic target in diabetic hind-limb ischemia.

Mevalonate metabolism-dependent protein geranylgeranylation regulates thymocyte egress.

Thymocyte egress is a critical determinant of T cell homeostasis and adaptive immunity. Despite the roles of G protein-coupled receptors in thymocyte emigration, the downstream signaling mechanism remains poorly defined. Here, we report the discrete roles for the two branches of mevalonate metabolism-fueled protein prenylation pathway in thymocyte egress and immune homeostasis. The protein geranylgeranyltransferase Pggt1b is up-regulated in single-positive thymocytes, and loss of Pggt1b leads to marked defects in thymocyte egress and T cell lymphopenia in peripheral lymphoid organs in vivo. Mechanistically, Pggt1b bridges sphingosine-1-phosphate and chemokine-induced migratory signals with the activation of Cdc42 and Pak signaling and mevalonate-dependent thymocyte trafficking. In contrast, the farnesyltransferase Fntb, which mediates a biochemically similar process of protein farnesylation, is dispensable for thymocyte egress but contributes to peripheral T cell homeostasis. Collectively, our studies establish context-dependent effects of protein prenylation and unique roles of geranylgeranylation in thymic egress and highlight that the interplay between cellular metabolism and posttranslational modification underlies immune homeostasis.

LKB1 orchestrates dendritic cell metabolic quiescence and anti-tumor immunity.

Dendritic cells (DCs) play a pivotal role in priming adaptive immunity. However, the involvement of DCs in controlling excessive and deleterious T cell responses remains poorly defined. Moreover, the metabolic dependence and regulation of DC function are unclear. Here we show that LKB1 signaling in DCs functions as a brake to restrain excessive tumor-promoting regulatory T cell (Treg) and Th17 cell responses, thereby promoting protective anti-tumor immunity and maintaining proper immune homeostasis. LKB1 deficiency results in dysregulated metabolism and mTOR activation of DCs. Loss of LKB1 also leads to aberrant DC maturation and production of cytokines and immunoregulatory molecules. Blocking mTOR signaling in LKB1-deficient DCs partially rectifies the abnormal phenotypes of DC activation and Treg expansion, whereas uncontrolled Th17 responses depend upon IL-6-STAT3 signaling. By coordinating metabolic and immune quiescence of DCs, LKB1 acts as a crucial signaling hub in DCs to enforce protective anti-tumor immunity and normal immune homeostasis.

Emerging Roles of Cellular Metabolism in Regulating Dendritic Cell Subsets and Function.

Dendritic cells (DCs) are the bridge between innate and T cell-dependent adaptive immunity and are promising therapeutic targets for cancer and immune-mediated disorders. Upon stimulation by pathogen or danger-sensing receptors, DCs become activated and poised to induce T cell priming. Recent studies have identified critical roles of metabolic pathways, including glycolysis, oxidative phosphorylation, and fatty acid metabolism, in orchestrating DC function. In this review, we discuss the shared and distinct metabolic programs shaping the functional specification of different DC subsets, including conventional DCs, bone marrow-derived DCs, and plasmacytoid DCs. We also briefly discuss the signaling networks that tune metabolic programs in DC subsets.

Hippo/Mst signalling couples metabolic state and immune function of CD8α+ dendritic cells.

Dendritic cells orchestrate the crosstalk between innate and adaptive immunity. CD8α+ dendritic cells present antigens to CD8+ T cells and elicit cytotoxic T cell responses to viruses, bacteria and tumours 1 . Although lineage-specific transcriptional regulators of CD8α+ dendritic cell development have been identified 2 , the molecular pathways that selectively orchestrate CD8α+ dendritic cell function remain elusive. Moreover, metabolic reprogramming is important for dendritic cell development and activation3,4, but metabolic dependence and regulation of dendritic cell subsets are largely uncharacterized. Here we use a data-driven systems biology algorithm (NetBID) to identify a role of the Hippo pathway kinases Mst1 and Mst2 (Mst1/2) in selectively programming CD8α+ dendritic cell function and metabolism. Our NetBID analysis reveals a marked enrichment of the activities of Hippo pathway kinases in CD8α+ dendritic cells relative to CD8α- dendritic cells. Dendritic cell-specific deletion of Mst1/2-but not Lats1 and Lats2 (Lats1/2) or Yap and Taz (Yap/Taz), which mediate canonical Hippo signalling-disrupts homeostasis and function of CD8+ T cells and anti-tumour immunity. Mst1/2-deficient CD8α+ dendritic cells are impaired in presentation of extracellular proteins and cognate peptides to prime CD8+ T cells, while CD8α- dendritic cells that lack Mst1/2 have largely normal function. Mechanistically, compared to CD8α- dendritic cells, CD8α+ dendritic cells exhibit much stronger oxidative metabolism and critically depend on Mst1/2 signalling to maintain bioenergetic activities and mitochondrial dynamics for their functional capacities. Further, selective expression of IL-12 by CD8α+ dendritic cells depends on Mst1/2 and the crosstalk with non-canonical NF-κB signalling. Our findings identify Mst1/2 as selective drivers of CD8α+ dendritic cell function by integrating metabolic activity and cytokine signalling, and highlight that the interplay between immune signalling and metabolic reprogramming underlies the unique functions of dendritic cell subsets.

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

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

The non-canonical Hippo/Mst pathway in lymphocyte development and functions.

The canonical Hippo/Mst pathway, originally discovered in Drosophila, is famous for its function in promoting apoptosis, inhibiting cell proliferation and tumorigenesis, and regulating tissue regeneration. However, emerging evidence shows that multiple non-canonical Hippo signaling pathways are also implicated in the regulation of various other biological processes. Recent studies have revealed that Mst1/2, the core kinases of Hippo/Mst pathway are required for T cell development, function, survival, trafficking, and homing, and also involved in regulation of autoimmunity. In this review, we discuss the roles of non-canonical Hippo/Mst signaling pathways in lymphocyte development and functions.

Mst1 and mst2 are essential regulators of trophoblast differentiation and placenta morphogenesis.

The placenta is essential for survival and growth of the fetus because it promotes the delivery of nutrients and oxygen from the maternal circulation as well as fetal waste disposal. Mst1 and Mst2 (Mst1/2), key components of the mammalian hpo/Mst signaling pathway, encode two highly conserved Ser/Thr kinases and play important roles in the prevention of tumorigenesis and autoimmunity, control of T cell development and trafficking, and embryonic development. However, their functions in placental development are not fully understood, and the underlying cellular and molecular mechanisms remain elusive. Here, we investigated the functions of Mst1/2 in mouse placental development using both conventional and conditional (endothelial) Mst1/2 double knockout mice. We found that the number of trophoblast giant cells dramatically increased while spongiotrophoblast cells almost completely disappeared in Mst1/2 deficient placentas. We showed that Mst1/2 deficiency down regulated the expression of Mash2, which is required for suppressing the differentiation of trophoblast giant cells. Furthermore, we demonstrated that endothelial-specific deletion of Mst1/2 led to impaired placental labyrinthine vasculature and embryonic lethality at E11.5, but neither affected vasculature in yolk sac and embryo proper nor endocardium development. Collectively, our findings suggest that Mst1/2 regulate placental development by control of trophoblast cell differentiation and labyrinthine vasculature at midgestation and Mst1/2 control labyrinth morphogenesis in trophoblast- and fetal endothelial-dependent manners. Thus, our studies have defined novel roles of Mst1/2 in mouse placental development.

Mst1/Mst2 regulate development and function of regulatory T cells through modulation of Foxo1/Foxo3 stability in autoimmune disease.

Foxp3 expression and regulatory T cell (Treg) development are critical for maintaining dominant tolerance and preventing autoimmune diseases. Human MST1 deficiency causes a novel primary immunodeficiency syndrome accompanied by autoimmune manifestations. However, the mechanism by which Mst1 controls immune regulation is unknown. In this article, we report that Mst1 regulates Foxp3 expression and Treg development/function and inhibits autoimmunity through modulating Foxo1 and Foxo3 (Foxo1/3) stability. We have found that Mst1 deficiency impairs Foxp3 expression and Treg development and function in mice. Mechanistic studies reveal that Mst1 enhances Foxo1/3 stability directly by phosphorylating Foxo1/3 and indirectly by attenuating TCR-induced Akt activation in peripheral T cells. Our studies have also shown that Mst1 deficiency does not affect Foxo1/3 cellular localization in CD4 T cells. In addition, we show that Mst1(-/-) mice are prone to autoimmune disease, and mutant phenotypes, such as overactivation of naive T cells, splenomegaly, and autoimmune pathological changes, are suppressed in Mst1(-/-) bone marrow chimera by cotransplanted wt Tregs. Finally, we demonstrate that Mst1 and Mst2 play a partially redundant role in Treg development and autoimmunity. Our findings not only identify Mst kinases as the long-searched-for factors that simultaneously activate Foxo1/3 and inhibit TCR-stimulated Akt downstream of TCR signaling to promote Foxp3 expression and Treg development, but also shed new light on understanding and designing better therapeutic strategies for MST1 deficiency-mediated human immunodeficiency syndrome.

Dlic1 deficiency impairs ciliogenesis of photoreceptors by destabilizing dynein.

Cytoplasmic dynein 1 is fundamentally important for transporting a variety of essential cargoes along microtubules within eukaryotic cells. However, in mammals, few mutants are available for studying the effects of defects in dynein-controlled processes in the context of the whole organism. Here, we deleted mouse Dlic1 gene encoding DLIC1, a subunit of the dynein complex. Dlic1(-/-) mice are viable, but display severe photoreceptor degeneration. Ablation of Dlic1 results in ectopic accumulation of outer segment (OS) proteins, and impairs OS growth and ciliogenesis of photoreceptors by interfering with Rab11-vesicle trafficking and blocking efficient OS protein transport from Golgi to the basal body. Our studies show that Dlic1 deficiency partially blocks vesicle export from endoplasmic reticulum (ER), but seems not to affect vesicle transport from the ER to Golgi. Further mechanistic study reveals that lack of Dlic1 destabilizes dynein subunits and alters the normal subcellular distribution of dynein in photoreceptors, probably due to the impaired transport function of dynein. Our results demonstrate that Dlic1 plays important roles in ciliogenesis and protein transport to the OS, and is required for photoreceptor development and survival. The Dlic1(-/-) mice also provide a new mouse model to study human retinal degeneration.

A cell-intrinsic role for Mst1 in regulating thymocyte egress.

The MST1 kinase was recently identified as playing an essential role in the promotion of lymphocyte polarization and adhesion stimulated by chemokines and TCR signaling. However, the physiological relevance of the Mst1 pathway in thymocyte development is not completely understood. In this study, we analyzed the effect of Mst1 disruption on thymocyte development and migration. Mst1-deficient (Mst1(-/-)) mice displayed an accumulation of mature thymocytes in the thymus, a dramatic reduction of lymphocytes in blood and peripheral lymphoid tissues, and a decrease of homing ability to peripheral lymph nodes. Mst1(-/-) thymocytes were impaired in chemotactic response to chemokines, such as CCL19, but not to sphingosine-1-phosphate. Further analyses of Mst1(-/-) mice revealed a severe impairment in the egress of mature T cells from the thymus. T lineage-specific knockout of the Mst1 gene demonstrates a cell-intrinsic role for Mst1 in regulating T cell development. Our study indicates that Mst1 is crucial in controlling lymphocyte chemotaxis and thymocyte emigration.