SLAC2B-dependent microtubule acetylation regulates extracellular matrix-mediated intracellular TM4SF5 traffic to the plasma membranes.

Transmembrane 4 L six family member 5 (TM4SF5) translocates intracellularly and promotes cell migration, but how subcellular TM4SF5 traffic is regulated to guide cellular migration is unknown. We investigated the influences of the extracellular environment and intracellular signaling on the TM4SF5 traffic with regard to migration directionality. Cell adhesion to fibronectin (FN) but not poly-l-lysine enhanced the traffic velocity and straightness of the TM4SF5WT (but not palmitoylation-deficient mutant TM4SF5Pal- ) toward the leading edges, depending on tubulin acetylation. Acetylated-microtubules in SLAC2B-positive cells reached mostly the juxtanuclear regions, but reached-out toward the leading edges upon SLAC2B suppression. TM4SF5 expression caused SLAC2B not to be localized at the leading edges. TM4SF5 colocalization with HDAC6 depended on paxillin expression. The trimeric complex consisting of TM4SF5, HDAC6, and SLAC2B might, thus, be enriched at the perinuclear cytosols toward the leading edges. More TM4SF5WT translocation to the leading edges was possible when acetylated-microtubules reached the frontal edges following HDAC6 inhibition by paxillin presumably at new cell-FN adhesions, leading to persistent cell migration. Collectively, this study revealed that cell-FN adhesion and microtubule acetylation could control intracellular traffic of TM4SF5 vesicles to the leading edges via coordinated actions of paxillin, SLAC2B, and HDAC6, leading to TM4SF5-dependent cell migration.

Differential TM4SF5-mediated SIRT1 modulation and metabolic signaling in nonalcoholic steatohepatitis progression.

Nonalcoholic fatty liver disease is a chronic condition involving steatosis, steatohepatitis and fibrosis, and its progression remains unclear. Although the tetraspanin transmembrane 4 L six family member 5 (TM4SF5) is involved in hepatic fibrosis and cancer, its role in nonalcoholic steatohepatitis (NASH) progression is unknown. We investigated the contribution of TM4SF5 to liver pathology using transgenic and KO mice, diet- or drug-treated mice, in vitro primary cells, and in human tissue. TM4SF5-overexpressing mice exhibited nonalcoholic steatosis and NASH in an age-dependent manner. Initially, TM4SF5-positive hepatocytes and liver tissue exhibited lipid accumulation, decreased Sirtuin 1 (SIRT1), increased sterol regulatory-element binding proteins (SREBPs) and inactive STAT3 via suppressor of cytokine signaling (SOCS)1/3 upregulation. In older mice, TM4SF5 promoted inflammatory factor induction, SIRT1 expression and STAT3 activity, but did not change SOCS or SREBP levels, leading to active STAT3-mediated ECM production for NASH progression. A TM4SF5-associated increase in chemokines promoted SIRT1 expression and progression to NASH with fibrosis. Suppression of the chemokine CCL20 reduced immune cell infiltration and ECM production. Liver tissue from high-fat diet- or CCl4 -treated mice and human patients exhibited TM4SF5-dependent steatotic or steatohepatitic livers with links between TM4SF5-mediated SIRT1 modulation and SREBP or SOCS/STAT3 signaling axes. TM4SF5-mediated STAT3 activation in fibrotic NASH livers increased collagen I and laminin γ2. Both collagen I α1 and laminin γ2 suppression resulted in reduced SIRT1 and active STAT3, but no change in SREBP1 or SOCS, and abolished CCl4 -mediated mouse liver damage. TM4SF5-mediated signaling pathways that involve SIRT1, SREBPs and SOCS/STAT3 promoted progression to NASH. Therefore, TM4SF5 and its downstream effectors may be promising therapeutic targets to treat nonalcoholic fatty liver disease. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

TM4SF5-mediated liver malignancy involves NK cell exhaustion-like phenotypes.

Aberrant extracellular matrix and immune cell alterations within the tumor microenvironment promote the pathological progression of liver carcinogenesis. Although transmembrane 4 L six family member 5 (TM4SF5) is involved in liver fibrosis and cancer, its mechanism avoiding immune surveillance during carcinogenesis remains unknown. We investigated how TM4SF5-mediated signaling caused immune evasion using in vitro primary cells and in vivo liver tissues from genetic or chemically induced mouse models. TM4SF5-transgenic and diethylnitrosamine (DEN)-induced liver cancer mouse models exhibited fibrotic and cancerous livers, respectively, with enhanced TM4SF5, pY705STAT3, collagen I, and laminin γ2 levels. These TM4SF5-mediated effects were abolished by TM4SF5 inhibitor, 4'-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC). TM4SF5-dependent tumorigenesis involved natural killer (NK) cell exhaustion-like phenotypes including the reduction of NK cell number or function, which were blocked with TSAHC treatment. TM4SF5 expression in cancer cells downregulated stimulatory ligands and receptors for NK cell cytotoxicity, including SLAMF6, SLAMF7, MICA/B, and others. TM4SF5 suppression or inhibition reduced STAT3 signaling activity and recovered the receptor levels and NK cell surveillance, leading to reduced fibrotic and cancerous phenotypes, and longer survival. Altogether, these findings suggest that TM4SF5-mediated STAT3 activity for extracellular matrix modulation is involved in the progression of liver disease to HCC and that TM4SF5 appears to suppress NK cells during liver carcinogenesis.

Tetraspanin TM4SF5 in hepatocytes negatively modulates SLC27A transporters during acute fatty acid supply.

Transmembrane 4 L six family member 5 (TM4SF5) is involved in nonalcoholic steatosis and further aggravation of liver disease. However, its mechanism for regulating FA accumulation is unknown. We investigated how TM4SF5 in hepatocytes affected FA accumulation during acute FA supply. TM4SF5-expressing hepatocytes and mouse livers accumulated less FAs, compared with those of TM4SF5 deficiency or inactivation. Binding of TM4SF5 to SLC27A2 increased gradually upon acute FA treatment, whereas TM4SF5 constitutively bound SLC27A5. Suppression of either SLC27A2 or SLC27A5 in hepatocytes expressing TM4SF5 differentially modulated initial and maximal FA uptake levels for a fast turnover of fatty acid. Altogether, TM4SF5 negatively modulates FA accumulation into hepatocytes via association with the transporters for an energy homeostasis, when FA are supplied acutely.

Association between hepatocyte TM4SF5 expression and gut microbiome dysbiosis during non-alcoholic fatty liver disease development.

Gut microbiome dysbiosis is involved in non-alcoholic fatty liver disease (NAFLD) development. Hepatic transmembrane 4 L six family member 5 (TM4SF5) overexpression promotes NAFLD. However, how gut microbiota are associated with TM4SF5-mediated NAFLD remains unexplored. We analyzed the gut microbiome using feces from hepatocyte-specific TM4SF5-overexpressing transgenic (Alb-TGTm4sf5-Flag, TG) or Tm4sf5-/- knock-out (KO) mice fed a normal chow diet (NCD), high-fat diet (HFD) for 2 weeks (HFD2W), or methionine-choline-deficient diet (MCD) for 4 weeks to investigate associations among Tm4sf5 expression, diet, and the gut microbiome. TG-NCD mice showed a higher Firmicutes-to-Bacteroidetes (F/B) ratio, with less enrichment of Akkermansia muciniphila and Lactobacillus reuteri. NASH-related microbiomes in feces were more abundant in TG-HFD2w mice than in KO-HFD2w mice. Further, TG-MCD showed a higher F/B ratio than TG-NCD or KO mice, with decreases or increases in microbiomes beneficial or detrimental to the liver, respectively. Such effects in TG-MCD animals were correlated with functional pathways producing short-chain fatty acids (SCFAs). Furthermore, potential functional pathways of the gut microbiome were metabolically parallel to NAFLD features in TG-MCD mice. These results suggest that hepatocyte Tm4sf5 supports gut microbiome dysbiosis and metabolic activity, leading to SCFA production and hepatic inflammation during NAFLD development.

Glucose-mediated mitochondrial reprogramming by cholesterol export at TM4SF5-enriched mitochondria-lysosome contact sites.

BACKGROUND: Transmembrane 4 L six family member 5 (TM4SF5) translocates subcellularly and functions metabolically, although it is unclear how intracellular TM4SF5 translocation is linked to metabolic contexts. It is thus of interests to understand how the traffic dynamics of TM4SF5 to subcellular endosomal membranes are correlated to regulatory roles of metabolisms. METHODS: Here, we explored the metabolic significance of TM4SF5 localization at mitochondria-lysosome contact sites (MLCSs), using in vitro cells and in vivo animal systems, via approaches by immunofluorescence, proximity labelling based proteomics analysis, organelle reconstitution etc. RESULTS: Upon extracellular glucose repletion following depletion, TM4SF5 became enriched at MLCSs via an interaction between mitochondrial FK506-binding protein 8 (FKBP8) and lysosomal TM4SF5. Proximity labeling showed molecular clustering of phospho-dynamic-related protein I (DRP1) and certain mitophagy receptors at TM4SF5-enriched MLCSs, leading to mitochondrial fission and autophagy. TM4SF5 bound NPC intracellular cholesterol transporter 1 (NPC1) and free cholesterol, and mediated export of lysosomal cholesterol to mitochondria, leading to impaired oxidative phosphorylation but intact tricarboxylic acid (TCA) cycle and ß-oxidation. In mouse models, hepatocyte Tm4sf5 promoted mitophagy and cholesterol transport to mitochondria, both with positive relations to liver malignancy. CONCLUSIONS: Our findings suggested that TM4SF5-enriched MLCSs regulate glucose catabolism by facilitating cholesterol export for mitochondrial reprogramming, presumably while hepatocellular carcinogenesis, recapitulating aspects for hepatocellular carcinoma metabolism with mitochondrial reprogramming to support biomolecule synthesis in addition to glycolytic energetics.

TM4SF5-mediated abnormal food-intake behavior and apelin expression facilitate non-alcoholic fatty liver disease features.

Transmembrane 4 L six family member 5 (TM4SF5) engages in non-alcoholic steatohepatitis (NASH), although its mechanistic roles are unclear. Genetically engineered Tm4sf5 mice fed ad libitum normal chow or high-fat diet for either an entire day or a daytime-feeding (DF) pattern were analyzed for metabolic parameters. Compared to wild-type and Tm4sf5-/- knockout mice, hepatocyte-specific TM4SF5-overexpressing Alb-TGTm4sf5-Flag (TG) mice showed abnormal food-intake behavior during the mouse-inactive daytime, increased apelin expression, increased food intake, and higher levels of NASH features. DF or exogenous apelin injection of TG mice caused severe hepatic pathology. TM4SF5-mediated abnormal food intake was correlated with peroxisomal ß-oxidation, mTOR activation, and autophagy inhibition, with triggering NASH phenotypes. Non-alcoholic fatty liver disease (NAFLD) patients' samples revealed a correlation between serum apelin and NAFLD activity score. Altogether, these observations suggest that hepatic TM4SF5 may cause abnormal food-intake behaviors to trigger steatohepatitic features via the regulation of peroxisomal ß-oxidation, mTOR, and autophagy.

Liver-originated small extracellular vesicles with TM4SF5 target brown adipose tissue for homeostatic glucose clearance.

Transmembrane 4 L six family member 5 (TM4SF5) is involved in chronic liver disease, although its role in glucose homeostasis remains unknown. TM4SF5 deficiency caused age-dependent glucose (in)tolerance with no link to insulin sensitivity. Further, hepatic TM4SF5 binding to GLUT1 promoted glucose uptake and glycolysis. Excessive glucose repletion caused hepatocytes to secrete small extracellular vesicles (sEVs) loaded with TM4SF5 (hep-sEVTm4sf5 ), suggesting a role for sEVTm4sf5 in glucose metabolism and homeostasis. Hep-sEVTm4sf5 were smaller than sEVControl and recruit proteins for efficient organ tropism. Liver-derived sEVs, via a liver-closed vein circuit (LCVC) using hepatic TM4SF5-overexpressing (Alb-Tm4sf5 TG) mice (liv-sEVTm4sf5 ), improved glucose tolerance in Tm4sf5-/- KO mice and targeted brown adipose tissues (BATs), possibly allowing the clearance of blood glucose as heat independent of UCP1. Taken together, hep-sEVTm4sf5 might clear high extracellular glucose levels more efficiently by targeting BAT compared with hep-sEVControl , suggesting an insulin-like role for sEV™4SF5 in affecting age-related metabolic status and thus body weight (BW).

Crosstalk between TM4SF5 and GLUT8 regulates fructose metabolism in hepatic steatosis.

OBJECTIVE: Transmembrane 4 L six family member 5 (TM4SF5) is likely involved in non-alcoholic steatohepatitis, although its roles and cross-talks with glucose/fructose transporters in phenotypes derived from high-carbohydrate diets remain unexplored. Here, we investigated the modulation of hepatic fructose metabolism by TM4SF5. METHODS: Wild-type or Tm4sf5-/- knockout mice were evaluated via different diets, including normal chow, high-sucrose diet, or high-fat diet without or with fructose in drinking water (30% w/v). Using liver tissues and blood samples from the mice or hepatocytes, the roles of TM4SF5 in fructose-mediated de novo lipogenesis (DNL) and steatosis via a crosstalk with glucose transporter 8 (GLUT8) were assessed. RESULTS: Tm4sf5 suppression or knockout in both in vitro and in vivo models reduced fructose uptake, DNL, and steatosis. Extracellular fructose treatment of hepatocytes resulted in an inverse relationship between fructose-uptake activity and TM4SF5-mediated translocalization of GLUT8 through dynamic binding at the cell surface. Following fructose treatment, TM4SF5 binding to GLUT8 transiently decreased with translocation to the plasma membrane (PM), where GLUT8 separated and became active for fructose uptake and DNL. CONCLUSIONS: Overall, hepatic TM4SF5 modulated GLUT8 localization and activity through transient binding, leading to steatosis-related fructose uptake and lipogenesis. Thus, TM4SF5 and/or GLUT8 may be promising treatment targets against liver steatosis resulting from excessive fructose consumption.

Systemic TM4SF5 overexpression in ApcMin/+ mice promotes hepatic portal hypertension associated with fibrosis.

Mutation of the gene for adenomatous polyposis coli (APC), as seen in ApcMin/+ mice, leads to intestinal adenomas and carcinomas via stabilization of ß-catenin. Transmembrane 4 L six family member 5 (TM4SF5) is involved in the development of non-alcoholic fatty liver disease, fibrosis, and cancer. However, the functional linkage between TM4SF5 and APC or ß-catenin has not been investigated for pathological outcomes. After interbreeding ApcMin/+ with TM4SF5-overexpressing transgenic (TgTM4SF5) mice, we explored pathological outcomes in the intestines and livers of the offspring. The intestines of 26-week-old dual-transgenic mice (ApcMin/+:TgTM4SF5) had intramucosal adenocarcinomas beyond the single-crypt adenomas in ApcMin/+ mice. Additional TM4SF5 overexpression increased the stabilization of ß-catenin via reduced glycogen synthase kinase 3ß (GSK3ß) phosphorylation on Ser9. Additionally, the livers of the dualtransgenic mice showed distinct sinusoidal dilatation and features of hepatic portal hypertension associated with fibrosis, more than did the relatively normal livers in ApcMin/+ mice. Interestingly, TM4SF5 overexpression in the liver was positively linked to increased GSK3ß phosphorylation (opposite to that seen in the colon), ß-catenin level, and extracellular matrix (ECM) protein expression, indicating fibrotic phenotypes. Consistent with these results, 78-week-old TgTM4SF5 mice similarly had sinusoidal dilatation, immune cell infiltration, and fibrosis. Altogether, systemic overexpression of TM4SF5 aggravates pathological abnormalities in both the colon and the liver. [BMB Reports 2022; 55(12): 609-614].

TM4SF5-dependent crosstalk between hepatocytes and macrophages to reprogram the inflammatory environment.

Chronic injury to hepatocytes results in inflammation, steatohepatitis, fibrosis, and nonalcoholic fatty liver disease (NAFLD). The tetraspanin TM4SF5 is implicated in fibrosis and cancer. We investigate the role of TM4SF5 in communication between hepatocytes and macrophages (MΦs) and its possible influence on the inflammatory microenvironment that may lead to NAFLD. TM4SF5 induction in differentiated MΦs promotes glucose uptake, glycolysis, and glucose sensitivity, leading to M1-type MΦ activation. Activated M1-type MΦs secrete pro-inflammatory interleukin-6 (IL-6), which induces the secretion of CCL20 and CXCL10 from TM4SF5-positive hepatocytes. Although TM4SF5-dependent secretion of these chemokines enhances glycolysis in M0 MΦs, further chronic exposure reprograms MΦs for an increase in the proportion of M2-type MΦs in the population, which may support diet- and chemical-induced NAFLD progression. We suggest that TM4SF5 expression in MΦs and hepatocytes is critically involved in modulating the inflammatory environment during NAFLD progression.

TM4SF5-mediated CD44v8-10 splicing variant promotes survival of type II alveolar epithelial cells during idiopathic pulmonary fibrosis.

Reactive oxygen species (ROS) regulate cell fate, although signaling molecules that regulate ROS hormesis remain unclear. Here we show that transmembrane 4 L six family member 5 (TM4SF5) in lung epithelial cells induced the alternatively spliced CD44v8-10 variant via an inverse ZEB2/epithelial splicing regulatory proteins (ESRPs) linkage. TM4SF5 formed complexes with the cystine/glutamate antiporter system via TM4SF5- and CD44v8-10-dependent CD98hc plasma-membrane enrichment. Dynamic TM4SF5 binding to CD98hc required CD44v8-10 under ROS-generating inflammatory conditions. TM4SF5 and CD44v8-10 upregulated cystine/glutamate antiporter activity and intracellular glutathione levels, leading to ROS modulation for cell survival. Tm4sf5-null mice exhibited attenuated bleomycin-induced pulmonary fibrosis with lower CD44v8-10 and ESRPs levels than wild-type mice. Primary mouse alveolar epithelial cells (AECs) revealed type II AECs (AECII), but not type I, to adapt the TM4SF5-mediated characteristics, suggesting TM4SF5-mediated AECII survival following AECI injury during idiopathic pulmonary fibrosis (IPF). Thus, the TM4SF5-mediated CD44v8-10 splice variant could be targeted against IPF.

CD133-induced TM4SF5 expression promotes sphere growth via recruitment and blocking of protein tyrosine phosphatase receptor type F (PTPRF).

CD133 is a surface marker of liver cancer stem cells. Transmembrane 4 L six family member 5 (TM4SF5) promotes sphere growth and circulation. However, it is unknown how CD133 and TM4SF5 cross-talk with each other for cancer stem cell properties. Here, we investigated the significance of inter-relationships between CD133, TM4SF5, CD44, and protein tyrosine phosphatase receptor type F (PTPRF) in a three-dimensional (3D) sphere growth system. We found that CD133 upregulated TM4SF5 and CD44, whereas TM4SF5 and CD44 did not affect CD133 expression. Signaling activity following CD133 phosphorylation caused TM4SF5 expression and sphere growth. TM4SF5 bound to CD133 and promoted c-Src activity for CD133 phosphorylation as a positive feedback loop, leading to CD133-mediated sphere growth that was inhibited by TM4SF5 inhibition or suppression. TM4SF5 also bound PTPRF and promoted paxillin phosphorylation. Decreased sphere growth upon CD133 suppression was recovered by TM4SF5 expression and partially by PTPRF suppression. TM4SF5 inhibition enhanced PTPRF levels and abolished PTPRF suppression-mediated sphere growth. Altogether, CD133-induced TM4SF5 expression and function were important for liver cancer sphere growth and may be a promising target to block metastasis.