Systemic knockout of Tmem175 results in aberrant differentiation but no effect on hematopoietic reconstitution.

Lysosomes play crucial roles in regulating cell metabolism, and K+ channels are critical for controlling various aspects of lysosomal function. Additionally, lysosomal activity is essential for maintaining the quiescence of hematopoietic stem cells (HSCs) under both steady-state and stress conditions. Tmem175 is a lysosomal potassium channel protein. To further investigate the role of K+ channels in HSCs, our study employed knockout mice to examine the function of Tmem175. Our research findings demonstrate that the deletion of Tmem175 does not disrupt the functionality of HSCs in both stable and stressed conditions, including irradiation and intraperitoneal 5-FU injections. However, we did observe that the absence of Tmem175 impairs the long-term differentiation capacity of HSCs into myeloid differentiated subpopulation cells（In this paper, it is referred to simply as M cells）in HSC transplantation test, while promoting their differentiation into T cells. This suggests that Tmem175 plays a role in the lineage differentiation of HSCs without being essential for their self-renewal or long-term regenerative capabilities.

mTORC2 Facilitates Liver Regeneration Through Sphingolipid-Induced PPAR-α-Fatty Acid Oxidation.

BACKGROUND & AIMS: During liver regeneration after partial hepatectomy, the function and metabolic pathways governing transient lipid droplet accumulation in hepatocytes remain obscure. Mammalian target of rapamycin 2 (mTORC2) facilitates de novo synthesis of hepatic lipids. Under normal conditions and in tumorigenesis, decreased levels of triglyceride (TG) and fatty acids (FAs) are observed in the mTORC2-deficient liver. However, during liver regeneration, their levels increase in the absence of mTORC2. METHODS: Rictor liver-specific knockout and control mice underwent partial hepatectomy, followed by measurement of TG and FA contents during liver regeneration. FA metabolism was evaluated by analyzing the expression of FA metabolism-related genes and proteins. Intraperitoneal injection of the peroxisome proliferator-activated receptor α (PPAR-α) agonist, p53 inhibitor, and protein kinase B (AKT) activator was performed to verify the regulatory pathways involved. Lipid mass spectrometry was performed to identify the potential PPAR-α activators. RESULTS: The expression of FA metabolism-related genes and proteins suggested that FAs are mainly transported into hepatocytes during liver regeneration. The PPAR-α pathway is down-regulated significantly in the mTORC2-deficient liver, resulting in the accumulation of TGs. The PPAR-α agonist WY-14643 rescued deficient liver regeneration and survival in mTORC2-deficient mice. Furthermore, lipidomic analysis suggested that mTORC2 deficiency substantially reduced glucosylceramide (GluCer) content. GluCer activated PPAR-α. GluCer treatment in vivo restored the regenerative ability and survival rates in the mTORC2-deficient group. CONCLUSIONS: Our data suggest that FAs are mainly transported into hepatocytes during liver regeneration, and their metabolism is facilitated by mTORC2 through the GluCer-PPAR-α pathway, thereby establishing a novel role for mTORC2 in lipid metabolism.

Co-targeting WIP1 and PARP induces synthetic lethality in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most fatal cancers. Due to limited strategies for effective treatments, patients with advanced HCC have a very poor prognosis. This study aims to identify new insights in HCC to develop novel strategies for HCC management. METHODS: The role of WIP1 (wild type p53 induced protein phosphatase1) in HCC was analyzed in HCC cells, xenograft model, DEN (Diethylnitrosamine) induced mice liver cancer model with WIP1 knockout mice, and TCGA database. DNA damage was evaluated by Gene Set Enrichment Analysis, western blotting, comet assay, and Immunofluorescence. RESULTS: High expression of WIP1 is associated with the poor prognosis of patients with HCC. Genetically and chemically suppression of WIP1 drastically reduced HCC cell proliferation. Besides, WIP1 knockout retarded DEN induced mice hepato-carcinogenesis. Mechanically, WIP1 inhibition induced DNA damage by increasing H2AX phosphorylation (γH2AX). Therefore, suppression of WIP1 and PARP induced synthetic lethality in HCC in vitro and in vivo by augmenting DNA damage. CONCLUSION: WIP1 plays an oncogenic effect in HCC development, and targeting WIP1-dependent DNA damage repair alone or in combination with PARP inhibition might be a reasonable strategy for HCC management. Video abstract.

Neuronal Nitric Oxide Synthase Knockdown Within Basolateral Amygdala Induces Autistic-Related Phenotypes and Decreases Excitatory Synaptic Transmission in Mice.

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders characterized by deficits in communication, impaired social interaction, and repetitive or restricted interests and behaviors. We have recently shown that neuronal nitric oxide synthase (nNOS) expression was reduced in the basolateral amygdala of mice after postnatal valproic acid exposure. However, the specific role of nNOS downregulation in mice remains to be elucidated. Herein, we investigated the behavioral alternations of naive mice with a recombinant adeno-associated virus (rAAV)-mediated knockdown of nNOS in a comprehensive test battery, including the social interaction, marble burying, self-grooming, and open field tests. Further, the electrophysiological and surface expression changes induced by nNOS deficiency of the basolateral amygdala in these animals were examined. Our results show that nNOS knockdown displayed typical symptoms of ASD-like behaviors, such as reduced social interaction and communication, elevated stereotypes, and anxiety in mice. Surprisingly, we found that nNOS knockdown exhibited greatly reduced excitatory synaptic transmission concomitant with the lower surface expression of GluN2B-containing N-methyl-D-aspartate receptors and postsynaptic density protein 95 in mice. These findings support a notion that dysregulation of nNOS might contribute to ASD-associated phenotypes, with disease pathogenesis most likely resulting from deficits in excitatory synaptic transmission.

PGC1α protects against hepatic steatosis and insulin resistance via enhancing IL10-mediated anti-inflammatory response.

Inflammatory responses are pivotal incidences in hepatic metabolic derangements. However, the underlying mechanism remains elusive. The present study aimed to evaluate the role of peroxisome proliferator-activated receptor-gamma, coactivator 1 alpha (PGC1α) in IL10-mediated anti-inflammatory response, and its role in hepatic steatosis and insulin resistance. Hepatocyte-specific PGC1α knock-in (LivPGC1α) mice and the control mice were fed high-fat diet (HFD) for 8 weeks. IL-10 neutralizing antibody was injected into the liver of PGC1α mice. A variety of biological and histological approaches were applied to assess hepatic function. We demonstrated that hepatic PGC1α expression was significantly reduced in mice fed HFD. LivPGC1α livers exhibited enhanced gene expressions involving mitochondrial function, and favored an accelerated lipid metabolism upon HFD. Meanwhile, LivPGC1α mice revealed improved hepatic steatosis and insulin resistance. Mechanistically, PGC1α bound and activated the promotor region of IL-10, thereby attenuating inflammatory response in the liver. Administration of IL10 neutralizing antibody to LivPGC1α mice abolished PGC1α-mediated anti-inflammatory effects in mice. Further, IL-10 neutralizing antibody intervention aggravated hepatic steatosis and insulin resistance in LivPGC1α mice. Taken together, our data indicated that hepatic-specific overexpression of PGC1α exerts a beneficial role in the regulation of hepatic steatosis and insulin resistance via enhancing IL10-mediated anti-inflammatory response. Pharmacological activation of PGC1α-IL10 axis may be promising for the treatment of fatty liver diseases.

Hepatic Tmem30a Deficiency Causes Intrahepatic Cholestasis by Impairing Expression and Localization of Bile Salt Transporters.

Mutations in ATP8B1 or ATP11C (members of P4-type ATPases) cause progressive familial intrahepatic cholestasis type 1 in human or intrahepatic cholestasis in mice. Transmembrane protein 30A (TMEM30A), a ß-subunit, is essential for the function of ATP8B1 and ATP11C. However, its role in the etiology of cholestasis remains poorly understood. To investigate the function of TMEM30A in bile salt (BS) homeostasis, we developed Tmem30a liver-specific knockout (LKO) mice. Tmem30a LKO mice experienced hyperbilirubinemia, hypercholanemia, inflammatory infiltration, ductular proliferation, and liver fibrosis. The expression and membrane localization of ATP8B1 and ATP11C were significantly reduced in Tmem30a LKO mice, which correlated with the impaired expression and localization of BS transporters, such as OATP1A4, OATP1B2, NTCP, BSEP, and MRP2. The proteasome inhibitor bortezomib partially restored total protein levels of BS transporters but not the localization of BS transporters in the membrane. Furthermore, the expression of nuclear receptors, including FXRα, RXRα, HNF4α, LRH-1, and SHP, was also down-regulated. A cholic acid-supplemented diet exacerbated the liver damage in Tmem30a LKO mice. TMEM30A deficiency led to intrahepatic cholestasis in mice by impairing the expression and localization of BS transporters and the expression of related nuclear receptors. Therefore, TMEM30A may be a novel genetic determinant of intrahepatic cholestasis.

Phosphatase Wip1 controls antigen-independent B-cell development in a p53-dependent manner.

Wild-type p53-induced phosphatase 1 (Wip1), a phosphatase previously considered as an oncogene, has been implicated in the regulation of thymus homeostasis and neutrophil maturation. However, the role of Wip1 in B-cell development is unknown. We show that Wip1-deficient mice exhibit a significant reduction of B-cell numbers in the bone marrow, peripheral blood, and spleen. A reciprocal transplantation approach revealed a cell-intrinsic defect in early B-cell precursors caused by Wip1 deficiency. Further experiments revealed that Wip1 deficiency led to a sustained activation of p53 in B cells, which led to increased level of apoptosis in the pre-B-cell compartment. Notably, the impairment of B-cell development in Wip1-deficient mice was completely rescued by genetic ablation of p53, but not p21. Therefore, loss of Wip1 phosphatase induces a p53-dependent, but p21-independent, mechanism that impairs B-cell development by enhancing apoptosis in early B-cell precursors. Moreover, Wip1 deficiency exacerbated a decline in B-cell development caused by aging as evidenced in mice with aging and mouse models with serial competitive bone marrow transplantation, respectively. Our present data indicate that Wip1 plays a critical role in maintaining antigen-independent B-cell development in the bone marrow and preventing an aging-related decline in B-cell development.

Inhibition of wild-type p53-induced phosphatase 1 promotes liver regeneration in mice by direct activation of mammalian target of rapamycin.

UNLABELLED: The liver possesses extraordinary regenerative capacity in response to injury. However, liver regeneration (LR) is often impaired in disease conditions. Wild-type p53-induced phosphatase 1 (Wip1) is known as a tumor promoter and enhances cell proliferation, mainly by deactivating antioncogenes. However, in this work, we identified an unexpected role of Wip1 in LR. In contrast to its known role in promoting cell proliferation in extrahepatic tissue, we found that Wip1 suppressed hepatocyte proliferation after partial hepatectomy (PHx). Deletion of Wip1 increased the rate of LR after PHx. Enhanced LR in Wip1-deficient mice was a result of the activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway. Furthermore, we showed that Wip1 physically interacted with and dephosphorylated mTOR. Interestingly, inhibition of Wip1 also activated the p53 pathway during LR. Disruption of the p53 pathway further enhanced LR in Wip1-deficient mice. Therefore, inhibition of Wip1 has a dual role in LR, i.e., promoting hepatocyte proliferation through activation of the mTORC1 pathway, meanwhile suppressing LR through activation of the p53 pathway. However, the proregenerative role of mTORC1 overwhelms the antiproliferative role of p53. Furthermore, CCT007093, a Wip1 inhibitor, enhanced LR and increased the survival rate of mice after major hepatectomy. CONCLUSION: mTOR is a new direct target of Wip1. Wip1 inhibition can activate the mTORC1 pathway and enhance hepatocyte proliferation after hepatectomy. These findings have clinical applications in cases where LR is critical, including acute liver failure, cirrhosis, or small-for-size liver transplantations.

YY1-MIR372-SQSTM1 regulatory axis in autophagy.

Autophagy is a self-proteolytic process that degrades intracellular material to enable cellular survival under unfavorable conditions. However, how autophagy is activated in human carcinogenesis remains largely unknown. Herein we report an epigenetic regulation of autophagy in human cancer cells. YY1 (YY1 transcription factor) is a well-known epigenetic regulator and is upregulated in many cancers. We found that YY1 knockdown inhibited cell viability and autophagy flux through downregulating SQSTM1 (sequestosome 1). YY1 regulated SQSTM1 expression through the epigenetic modulation of the transcription of MIR372 (microRNA 372) which was found to target SQSTM1 directly. During nutrient starvation, YY1 was stimulated to promote SQSTM1 expression and subsequent autophagy activation by suppressing MIR372 expression. Similar to YY1 depletion, MIR372 overexpression blocked autophagy activation and inhibited in vivo tumor growth. SQSTM1 upregulation and competent autophagy flux thus contributed to the oncogenic function of YY1. YY1-promoted SQSTM1 upregulation might be a useful histological marker for cancer detection and a potential target for drug development.

Downregulation of histone deacetylase 1 by microRNA-520h contributes to the chemotherapeutic effect of doxorubicin.

Doxorubicin induces DNA damage to exert its anti-cancer function. Histone deacetylase 1 (HDAC1) can protect the genome from DNA damage. We found that doxorubicin specifically downregulates HDAC1 protein expression and identified HDAC1 as a target of miR-520h, which was upregulated by doxorubicin. Doxorubicin-induced cell death was impaired by exogenous HDAC1 or by miR-520h inhibitor. Moreover, HDAC1 reduced the level of γH2AX by preventing the interaction of doxorubicin with DNA. In summary, doxorubicin downregulates HDAC1 protein expression, by inducing the expression of HDAC1-targeting miR-520h, to exacerbate DNA-doxorubicin interaction. The upregulation of HDAC1 protein may contribute to drug resistance of human cancer cells and targeting HDAC1 is a promising strategy to increase the clinical efficacy of DNA damage-inducing chemotherapeutic drugs.

Regulation and function of mitophagy in development and cancer.

Beyond its role in recycling intracellular components nonselectively to sustain survival in response to metabolic stresses, autophagy can also selectively degrade specific cargoes such as damaged or dysfunctional organelles to maintain cellular homeostasis. Mitochondria, known as the power plant of cells, are the critical and dynamic organelles playing a fundamental role in cellular metabolism. Mitophagy, the selective autophagic elimination of mitochondria, has been identified both in yeast and in mammalian cells. Moreover, defects in mitophagy may contribute to a variety of human disorders such as neurodegeneration and myopathies. However, the role of mitophagy in development and cancer remains largely unclear. In this review, we summarize our current knowledge of the regulation and function of mitophagy in development and cancer.