Transcription Factor EB Research Articles

Beyond a ferroptosis inducer: erastin can suppress nutrient deprivation induced cell death in the intervertebral disc

Background: Erastin has been found to induce ferroptosis; however, whether erastin may have roles other than ferroptosis inducer in cells is unknown. Nutrient deficiency is one of the major causes of many diseases including intervertebral disc (IVD) degeneration. Purpose: The current study investigates the effect of erastin in nucleus pulposus cells under nutrient deprivation condition. Study Design: Experiment in vitro and ex vivo. Methods: The effect of erastin on the cell survival of nucleus pulposus cells was evaluated in fetal bovine serum (FBS) and glucose deprivation condition. RSL3 and ferrostatin-1 were applied to illustrate whether the effect of erastin is ferroptosis dependent. The involvement of solute carrier family 7, membrane 11(SLC7A11), autophagy as well as mechanistic target of rapamycin kinase complex 1(mTORC1) and transcription factor EB (TFEB) were assessed to demonstrate the working mechanism of erastin. Results: Erastin may induce cell death at the concentration of ≥ 5μM; however, it may protect nucleus pulposus cells against nutrient deprivation induced cell death at lower concentration (0.25-1μM) and the effect of erastin is ferroptosis independent. The mechanism study showed that the effect of erastin may relate to its SCL7A11 regulation, as SCL7A11 knock-down may have the similar effect as erastin. Furthermore, it was also demonstrated that mTORC1-TFEB mediated autophagy was involved in protective effect of erastin. Conclusions: Low dose erastin may promote cell survival under nutrient deprivation condition, and its effect is ferroptosis independent; erastin may exert its protective effect through mTORC1-TFEB mediated autophagy regulation. Clinical Significance: Nutrient deprivation is a major contributor to intervertebral disc degeneration. Our in vitro and ex vivo study showed that low dose of erastin may suppress nutrient deprivation induced cell death in IVD degeneration. Although it was not validated in vivo model due to lack of in vivo nutrient deprivation induced IVD degeneration model currently, this study may still provide a potential therapeutic option for IVD degeneration, which of cause need further validation.

HSP90 N-terminal inhibition promotes mitochondria-derived vesicles related metastasis by reducing TFEB transcription via decreased HSP90AA1-HCFC1 interaction in liver cancer

ABSTRACT Cancer cells compensate with increasing mitochondria-derived vesicles (MDVs) to maintain mitochondrial homeostasis, when canonical MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta)-mediated mitophagy is lacking. MDVs promote the transport of mitochondrial components into extracellular vesicles (EVs) and induce tumor metastasis. Although HSP90 (heat shock protein 90) chaperones hundreds of client proteins and its inhibitors suppress tumors, HSP90 inhibitors-related chemotherapy is associated with unexpected metastasis. Herein, we find that HSP90 inhibitor causes mitochondrial damage but stimulates the low LC3-induced MDVs and the release of MDVs-derived EVs. However, why LC3 decreases and what is the transcriptional regulatory mechanism of MDVs formation under HSP90 inhibition remain unknown. Because TFEB (transcription factor EB) is the most important mitophagy transcription factor, and the HSP90 client HCFC1 (host cell factor C1) regulates TFEB transcription, there should be a hidden connection between TFEB, HCFC1 and HSP90 in MDVs formation. Our results support the idea that HSP90 N-terminal inhibition reduces TFEB transcription via decreased HSP90AA1-HCFC1 interaction, which prevents HCFC1 from binding to the TFEB proximal promoter region. Decreased TFEB transcription and consequently reduced LC3, ultimately promoted MDVs formation. Blocking MDVs formation with the microtubule inhibitor nocodazole (NOC) activates the HCFC1-TFEB-LC3 axis, weakens HSP90 inhibitors-induced MDVs and the release of MDVs-derived EVs, inhibits the growth of tumor cell spheres and primary liver tumors, and reduces the extravasation of cancer cells to secondary metastatic sites. Taken together, these data suggest that combination therapy should be used to reduce the metastatic risk of low TFEB-triggered-MDVs formation caused by HSP90 inhibitors.

An mTOR-Tfeb-Fabp7a signaling axis can be harnessed to ameliorate bag3 cardiomyopathy in adult zebrafish.

Dysregulated proteostasis in cardiomyocytes is an important pathological event in BAG3 cardiomyopathy, which can be repaired by inhibiting mechanistic target of rapamycin (mTOR) for cardioprotective effects. Here, we aimed to uncover additional pathological events and therapeutic target genes via leveraging zebrafish genetics. We first assessed transcription factor EB ( tfeb ), a candidate gene that encodes a direct downstream phosphorylation target of mTOR signaling. We found that cardiomyocyte-specific transgenic overexpression of tfeb ( Tg[cmlc2:tfeb] ) is sufficient to repair defective proteostasis, attenuate accelerated cardiac senescence, a previously unrecognized phenotype in the bag3 cardiomyopathy model, and rescue cardiac dysfunction. Next, we compared cardiac transcriptomes between the Tg(cmlc2:tfeb) transgenic fish and the mtor xu015/+ mutant, and tested 4 commonly downregulated lipodystrophy genes using an F0-based genetic assay. We found that inhibition of the fatty acid binding protein a ( fabp7a ) gene, but not the other 3 genes, exerts therapeutic effects on bag3 cardiomyopathy. Conversely, fabp7a expression is elevated in bag3 cardiomyopathy model and cardiomyocyte-specific overexpression of fabp7a resulted in dysregulated proteostasis, accelerated cardiac senescence, as well as cardiac dysfunction. Together, these genetic studies in zebrafish uncovered Fabp7a activation and accelerated cardiac senescence as important pathological events in bag3 cardiomyopathy. The mTOR-Tfeb-Fabp7a signaling axis can be harnessed to repair these pathological changes and exert cardioprotective effects.

Ubiquitin regulatory X (UBX) domain-containing protein 6 is essential for autophagy induction and inflammation control in macrophages.

Ubiquitin regulatory X (UBX) domain-containing protein 6 (UBXN6) is an essential cofactor for the activity of the valosin-containing protein p97, an adenosine triphosphatase associated with diverse cellular activities. Nonetheless, its role in cells of the innate immune system remains largely unexplored. In this study, we report that UBXN6 is upregulated in humans with sepsis and may serve as a pivotal regulator of inflammatory responses via the activation of autophagy. Notably, the upregulation of UBXN6 in sepsis patients was negatively correlated with inflammatory gene profiles but positively correlated with the expression of Forkhead box O3, an autophagy-driving transcription factor. Compared with those of control mice, the macrophages of mice subjected to myeloid cell-specific UBXN6 depletion exhibited exacerbated inflammation, increased mitochondrial oxidative stress, and greater impairment of autophagy and endoplasmic reticulum-associated degradation pathways. UBXN6-deficient macrophages also exhibited immunometabolic remodeling, characterized by a shift to aerobic glycolysis and elevated levels of branched-chain amino acids. These metabolic shifts amplify mammalian target of rapamycin pathway signaling, in turn reducing the nuclear translocation of the transcription factor EB and impairing lysosomal biogenesis. Together, these data reveal that UBXN6 serves as an activator of autophagy and regulates inflammation to maintain immune system suppression during human sepsis.

TFEB signaling promotes autophagic degradation of NLRP3 to attenuate neuroinflammation in diabetic encephalopathy.

Diabetic encephalopathy (DE), a neurological complication of diabetes mellitus, has an unclear etiology. Shreds of evidence show that the Nucleotide-binding oligomerization domain-like receptor family protein 3 (NLRP3) inflammasome-induced neuroinflammation and transcription factor EB (TFEB)-mediated autophagy impairment may take part in DE development. The crosstalk between these two pathways and their contribution to DE remains to be explored. A mouse model of type 2 diabetes mellitus (T2DM) exhibiting cognitive dysfunction was created, along with high glucose (HG) cultured BV2 cells. Following, 3-methyladenine (3-MA) and rapamycin were utilized to modulate autophagy. To evaluate the potential therapeutic benefits of TFEB in DE, we overexpressed and knocked down TFEB in both mice and cells. Autophagy impairment and NLRP3 inflammasome activation were noticed in T2DM mice and HG-cultured BV2 cells. The inflammatory response caused by NLRP3 inflammasome activation was decreased by rapamycin-induced autophagy enhancement, while 3-MA treatment further deteriorated it. Nuclear translocation and expression of TFEB were hampered in HG-cultured BV2 cells and T2DM mice. Exogenous TFEB overexpression boosted NLRP3 degradation via autophagy, which in turn alleviated microglial activation as well as ameliorated cognitive deficits and neuronal damage. Additionally, TFEB knockdown exacerbated neuroinflammation by decreasing autophagy-mediated NLRP3 degradation. Our findings have unraveled the pathogenesis of a previously underappreciated disease, implying that the activation of NLRP3 inflammasome and impairment of autophagy in microglia are significant etiological factors in the DE. The TFEB-mediated autophagy pathway can reduce neuroinflammation by enhancing NLRP3 degradation. This could potentially serve as a viable and innovative treatment approach for DE.

Lysosomal Ca2+ release-facilitated TFEB nuclear translocation alleviates ischemic brain injury by attenuating autophagic/lysosomal dysfunction in neurons

Neuronal death was frequently driven by autophagic/lysosomal dysfunction after ischemic stroke, whereas how to restore the impaired autophagic flux remained elusive. Autophagic/lysosomal signaling could be augmented after transcription factor EB (TFEB) nuclear translocation, which was facilitated by its dephosphorylation. A key TFEB dephosphorylase was calcineurin (CaN), whose activity was drastically regulated by cytosolic calcium ion concentration ([Ca2+]) controlled by lysosomal Ca2+ channel-like protein of TRPML1. Our research shows that ML-SA1, an agonist of the TRPML1 channel, significantly enhanced the lysosomal Ca2+ release and the CaN expression in penumbric neurons, subsequently promoted TFEB nuclear translocation, and greatly reversed autophagy/lysosome dysfunction. Moreover, ML-SA1 treatment significantly reduced neuronal loss, infarct size, and neurological deficits. By contrast, ML-SI3, an inhibitor of TRPML1, inhibited the lysosomal Ca2+ release conversely, aggravated the impairment of autophagic flux and consequentially exacerbated brain stroke lesion. These studies suggest that TRPML1 elevation alleviates ischemic brain injury by restoring autophagic/lysosomal dysfunction via Lysosomal Ca2+ release-facilitated TFEB nuclear translocation in neurons.

Reduced expression of SMAD7 and consequent reduction of autophagy promotes endometrial stromal-myofibroblast transition and fibrosis.

Abnormal autophagy and the transforming growth factor-β (TGFβ)-SMAD3/7 signaling pathway play an important role in the development of intrauterine adhesions (IUA); however, the exact underlying mechanisms remain unclear. In this study, we used IUA patient tissue and SMAD7 conditional knockout mice to detect whether SMAD7 effected IUA via regulation of autophagy and the TGFβ-SMAD3 signaling pathway. We applied a combination of techniques for detection of p-SMAD3, SMAD7, autophagy and fibrosis-related proteins, autophagic flux and analysis of the SMAD3 binding site. Endometrial tissue of patients with IUA exhibited lower expression levels of SMAD7. In endometrial stromal cells, silencing of SMAD7 inhibited autophagic flux, whereas overexpressed SMAD7 promoted autophagic flux. This SMAD7-mediated autophagic flux regulates the stromal-myofibroblast transition, and these phenotypes were regulated by the TGFβ-SMAD3 signaling pathway. SMAD3 directly binds to the 3'-untranslated region of transcription factor EB (TFEB) and inhibits its transcription. SMAD7 promoted autophagic flux by inhibiting SMAD3, thereby promoting the expression of TFEB. In SMAD7 conditional knockout mice, the endometria showed a fibrotic phenotype. Simultaneously, autophagic flux was inhibited. On administering the autophagy activator rapamycin, this endometrial fibrosis phenotype was partially reversed. The loss of SMAD7 promotes endometrial fibrosis by inhibiting autophagic flux via the TGFβ-SMAD3 pathway. Therefore, this study reveals a potential therapeutic target for IUA.

SMURF1 mediates damaged lysosomal homeostasis by ubiquitinating PPP3CB to promote the activation of TFEB

ABSTRACT The calcium-activated phosphatase PPP3/calcineurin dephosphorylates TFEB (transcription factor EB) to trigger its nuclear translocation and the activation of macroautophagic/autophagic targets. However, the detailed molecular mechanism regulating TFEB activation remains poorly understood. Here, we highlighted the importance of SMURF1 (SMAD specific E3 ubiquitin protein ligase 1) in the activation of TFEB for lysosomal homeostasis. SMURF1 deficiency prevents the calcium-triggered ubiquitination of the catalytic subunit of PPP3/calcineurin in a manner consistent with defective autophagic degradation of damaged lysosomes. Mechanically, PPP3CB/CNA2 plays a bridging role in the recruitment of SMURF1 by LGALS3 (galectin 3) upon lysosome damage. Importantly, PPP3CB increases the dissociation of the N-terminal tail (NT) and C-terminal carbohydrate-recognition domain (CRD) of LGALS3, which may promote the formation of open conformers in a PPP3CB dephosphorylation activity-dependent manner. In addition, PPP3CB is ubiquitinated at lysine 146 by the recruited SMURF1 in response to intracellular calcium stimulation. The K63-linked ubiquitination of PPP3CB enhances the recruitment of TFEB. Moreover, TFEB directly interacts with both PPP3CB and the regulatory subunit PPP3R1 which facilitate the conformational correction of TFEB for its activation for the transcription of TFEB-targeted genes. Altogether, our results highlighted a critical mechanism for the regulation of PPP3/calcineurin activity via its ubiquitin ligase SMURF1 in response to lysosomal membrane damage, which may account for a potential target for the treatment of stress-related diseases.Abbreviation AID: autoinhibitory domain; ATG: autophagy related; CD: catalytic domain; CRD: carbohydrate-recognition domain; CsA: cyclosporin A; DMSO: dimethyl sulfoxide; ESCRT: endosomal sorting complexes required for transport; GSK3B: glycogen synthase kinase 3 beta; LAMP1: lysosomal associated membrane protein 1; LGALS3: galectin 3; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; NT: N-terminal tail; PPP3CB: protein phosphatase 3 catalytic subunit beta; PPP3R1: protein phosphatase 3 regulatory subunit B, alpha; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; VCP/p97: valosin containing protein; YWHA/14-3-3: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein.

Transcription factor EB (TFEB) promotes autophagy in early brain injury after subarachnoid hemorrhage in rats.

Subarachnoid hemorrhage (SAH) has high mortality. Early brain injury (EBI) is responsible for unfavorable outcomes for patients with SAH. The protective involvement of autophagy in hemorrhagic stroke has been proposed. The transcription factor EB (TFEB) can increase autophagic flux by promoting autophagosome formation and autophagosome-lysosome fusion, and dysregulation of TFEB activity might induce the development of several diseases. However, the biological functions of TFEB in EBI after SAH remain unknown. We established an animal model of SAH by the modified endovascular perforation method. Expression of TFEB and autophagy required genes was measured by western blotting and immunofluorescence staining. SAH grading, brain water content and neurobehavioral functions were evaluated at 24h post-SAH. Neuronal apoptosis in cerebral cortex was assessed by TUNEL staining and Fluoro Jade B staining. TFEB was downregulated in SAH rats, and its overexpression reduced brain edema and ameliorated neurological deficits of SAH rats. Additionally, the neuronal apoptosis induced by SAH was inhibited by TFEB overexpression. Moreover, TFEB overexpression promoted autophagy after SAH. TFEB overexpression promotes autophagy to inhibit neuronal apoptosis, brain edema and neurological deficits post-SAH.

TFEB agonist clomiphene citrate activates the autophagy-lysosomal pathway and ameliorates Alzheimer's disease symptoms in mice

Autophagy is a conserved eukaryotic cellular clearance and recycling process through the lysosome-mediated degradation of damaged organelles and protein aggregates to maintain homeostasis. Impairment of the autophagy-lysosomal pathway is implicated in the pathogenesis of Alzheimer’s disease (AD). Transcription factor EB (TFEB) is a master regulator of autophagy and lysosomal biogenesis. Therefore, activating TFEB and autophagy provides a novel strategy for AD treatment. We previously described that clomiphene citrate (CC) promotes nuclear translocation of TFEB and increases autophagy and lysosomal biogenesis. In this study, 7 and 3-month-old APP/PS1 mice were treated with TFEB agonist CC and assessed. The behavioral tests were performed using Morris water maze and open field test. Additional changes in Aβ pathology, autophagy and inflammatory response were determined. We found that CC activated TFEB and the autophagy-lysosomal pathway in neuronal cells. Moreover, using mouse model of Alzheimer's disease, CC treatment promoted clearance of Aβ plaques and ameliorated cognitive function in both 7 and 3-month-old APP/PS1 mice. The CC-induced activation of TFEB occurs by promoting acetylation of TFEB for nuclear translocation. These findings provide a molecular mechanism for the TFEB-mediated activation of the autophagy-lysosome pathway by CC, which has the potential to be repurposed and applied in the treatment or prevention of AD.

GSK-3β inhibitor amplifies autophagy-lysosomal pathways by regulating TFEB in Parkinson's disease models

Parkinson's disease (PD), a common neurodegenerative disorder characterized by degeneration of the substantia nigra and a marked increase in Lewy bodies in the brain, primarily manifests as motor dysfunction. Glycogen synthase kinase-3 beta (GSK-3β) is known to play a critical role in various pathological processes of neurodegenerative diseases. However, the impact of GSK-3β inhibitors on PD progression and the underlying molecular mechanisms responsible for the effects have not been fully elucidated. Using in vitro and mouse models of 1-methyl-4-phenylpyridine (MPP+)-or methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD, we found that inhibition of GSK-3β activity alleviated mitochondrial damage, cell apoptosis, and neuronal cell loss by promoting the nuclear translocation of transcription factor EB (TFEB), thereby amplifying the autophagy-lysosomal pathway (ALP). Importantly, siRNA silencing of the TFEB gene impaired the GSK-3β inhibitor-mediated activation of the ALP pathway, thus negating the metabolic support required for neuronal functional improvement. Short-term treatment with the GSK-3β inhibitor significantly ameliorated motor dysfunction and improved motor coordination in model mice with MPTP-induced PD. GSK-3β inhibition increased the ALP and TFEB activities in the mice, thereby reducing α-synuclein aggregation and neuronal damage. In conclusion, our study demonstrates that inhibition of GSK-3β activity can delay the pathological processes of PD via promotion of the TFEB–ALP pathway, potentially providing a novel therapeutic target for this neurodegenerative disorder.

Jiedu recipe, a compound Chinese herbal medicine, suppresses hepatocellular carcinoma metastasis by inhibiting the release of tumor-derived exosomes in a hypoxic microenvironment

ObjectiveTumor-derived exosomes (TDEs) play crucial roles in intercellular communication. Hypoxia in the tumor microenvironment enhances secretion of TDEs and accelerates tumor metastasis. Jiedu recipe (JR), a traditional Chinese medicinal formula, has demonstrated efficacy in preventing the metastasis of hepatocellular carcinoma (HCC). However, the underlying mechanism remains largely unknown. MethodsAnimal experiments were performed to investigate the metastasis-preventing effects of JR. Bioinformatics analysis and in vitro assays were conducted to explore the potential targets and active components of JR. TDEs were assessed using nanoparticle tracking analysis (NTA) and Western blotting (WB). Exosomes derived from normoxic or hypoxic HCC cells (H-TDEs) were collected to establish premetastatic mouse models. JR was intragastrically administered to evaluate its metastasis-preventive effects. WB and lysosomal staining were performed to investigate the effects of JR on lysosomal function and autophagy. Bioinformatics analysis, WB, NTA, and immunofluorescence staining were used to identify the active components and potential targets of JR. ResultsJR effectively inhibited subcutaneous-tumor-promoted lung premetastatic niche development and tumor metastasis. It inhibited the release of exosomes from tumor cells under hypoxic conditions. JR treatment promoted both lysosomal acidification and suppressed secretory autophagy, which were dysregulated in hypoxic tumor cells. Quercetin was identified as the active component in JR, and the epidermal growth factor receptor (EGFR) was identified as a potential target. Quercetin inhibited EGFR phosphorylation and promoted the nuclear translocation of transcription factor EB (TFEB). Hypoxia-impaired lysosomal function was restored, and secretory autophagy was alleviated by quercetin treatment. ConclusionJR suppressed HCC metastasis by inhibiting hypoxia-stimulated exosome release, restoring lysosomal function, and suppressing secretory autophagy. Quercetin acted as a key component of JR and regulated TDE release through EGFR-TFEB signaling. Our study provides a potential strategy for retarding tumor metastasis by targeting H-TDE secretion.

An artificial peptide inhibits autophagy through calcineurin-TFEB pathway

We previously reported that a bioactive peptide (pep3) can potently inhibit the enzyme activity of purified calcineurin (CN). In this paper, we further demonstrate that transfected pep3 can strongly inhibit CN enzyme activity in HEK293 cells. Transcription factor EB (TFEB) plays an important role in the autophagy-lysosome pathway (ALP) as one of the substrates of CN, so we study the effect of pep3 on the CN-TFEB-ALP pathway. Pep3 can significantly inhibit the mRNA levels of the TFEB downstream genes and the expression of the autophagy-associated proteins, and autophagy flux in HEK293 cells. We also validated the inhibitory effect of pep3 on autophagy in mice. These findings may provide a new idea for discovering more CN inhibitors and autophagy inhibitory drugs.

USF2 and TFEB compete in regulating lysosomal and autophagy genes

Autophagy, a highly conserved self-digestion process crucial for cellular homeostasis, is triggered by various environmental signals, including nutrient scarcity. The regulation of lysosomal and autophagy-related processes is pivotal to maintaining cellular homeostasis and basal metabolism. The consequences of disrupting or diminishing lysosomal and autophagy systems have been investigated; however, information on the implications of hyperactivating lysosomal and autophagy genes on homeostasis is limited. Here, we present a mechanism of transcriptional repression involving upstream stimulatory factor 2 (USF2), which inhibits lysosomal and autophagy genes under nutrient-rich conditions. We find that USF2, together with HDAC1, binds to the CLEAR motif within lysosomal genes, thereby diminishing histone H3K27 acetylation, restricting chromatin accessibility, and downregulating lysosomal gene expression. Under starvation, USF2 competes with transcription factor EB (TFEB), a master transcriptional activator of lysosomal and autophagy genes, to bind to target gene promoters in a phosphorylation-dependent manner. The GSK3β-mediated phosphorylation of the USF2 S155 site governs USF2 DNA-binding activity, which is involved in lysosomal gene repression. These findings have potential applications in the treatment of protein aggregation-associated diseases, including α1-antitrypsin deficiency. Notably, USF2 repression is a promising therapeutic strategy for lysosomal and autophagy-related diseases.

CRISPR activation of Tfeb , a master regulator of autophagy and lysosomal biogenesis, in osteoblast lineage cells increases bone mass and strength.

Autophagy is a recycling pathway in which damaged or dysfunctional proteins, protein aggregates, and organelles are delivered to lysosomes for degradation. Insufficiency of autophagy is thought to contribute to several age-related diseases including osteoporosis. Consistent with this, elimination of autophagy from the osteoblast lineage reduces bone formation and causes low bone mass. However, whether increasing autophagy would benefit bone health is unknown. Here, we increased expression of the endogenous Transcription Factor EB gene ( Tfeb ) in osteoblast lineage cells in vivo via CRISPR activation. Tfeb overexpression stimulated autophagy and lysosomal biogenesis in osteoblasts. Tfeb overexpressing male mice displayed a robust increase in femoral and vertebral cortical thickness at 4.5 months of age. Histomorphometric analysis revealed that the increase in femoral cortical thickness was due to increased bone formation at the periosteal surface. Tfeb overexpression also increased femoral trabecular bone volume. Consistent with these results, bone strength was increased in Tfeb overexpressing mice. Female Tfeb overexpressing mice also displayed a progressive increase in bone mass over time and at 12 months of age had high cortical thickness and trabecular bone volume. This increase in vertebral trabecular bone volume was due to elevated bone formation. Osteoblastic cultures showed that Tfeb overexpression increased proliferation and osteoblast formation. Overall, these results demonstrate that stimulation of autophagy in osteoblast lineage cells promotes bone formation and strength and may represent an effective approach to combat osteoporosis.

EGCG inhibits migration, invasion and epithelial-mesenchymal transition of renal cell carcinoma by activating TFEB-mediated autophagy

BackgroundThe incidence of renal cell carcinoma (RCC) is already in the top ten of all types of cancers, with more than 4 %. Epigallocatechin gallate (EGCG), a polyphenolic compound extracted from green tea, has been shown to be effective in the treatment of various tumors. However, limited studies have demonstrated the effect of EGCG on RCC and its underlying molecular mechanisms. MethodsAfter exposure to gradient concentration (0,5,10,20,40,60,80,100 μM) of EGCG, the cell viability of RCC cells was determined by MTT assay. The migration and invasion abilities of RCC cells were investigated by wound healing and transwell assays. The expression levels of proteins involved in the epithelial-mesenchymal transition (EMT) and autophagy were explored by Western blotting assays. The formation of autophagosome was detected by electron microscope and LC3 puncta assays. Nude mouse xenograft model was used as the model system in vivo. ResultsIn the present study, EGCG significantly inhibited the migration, invasion and EMT of RCC cells in a concentrated manner. Further exploration of its mechanism indicated that autophagy is involved in EGCG-mediated metastasis inhibition and EMT inhibition of RCC cells. In addition, EGCG could significantly up-regulate the transcription factor EB (TFEB) and promotes its nuclear localization. The incorporation of TFEB into the nucleus enhanced the transcriptional levels of molecules associated with autophagy. TFEB knockdown inhibited EGCG-mediated autophagy activation, metastasis and EMT inhibition in RCC cells. ConclusionsIn conclusion, these findings demonstrate for the first time that EGCG inhibits migration, invasion, and EMT of RCC by activating TFEB-mediated autophagy. Therefore, the combination of EGCG and TFFB activators or EMT inhibitors is expected to be a promising therapeutic strategy for RCC.

A TBK1-independent primordial function of STING in lysosomal biogenesis

Stimulator of interferon genes (STING) is activated in many pathophysiological conditions, leading to TBK1-dependent interferon production in higher organisms. However, primordial functions of STING independent of TBK1 are poorly understood. Here, through proteomics and bioinformatics approaches, we identify lysosomal biogenesis as an unexpected function of STING. Transcription factor EB (TFEB), an evolutionarily conserved regulator of lysosomal biogenesis and host defense, is activated by STING from multiple species, including humans, mice, and frogs. STING-mediated TFEB activation is independent of TBK1, but it requires STING trafficking and its conserved proton channel. GABARAP lipidation, stimulated by the channel of STING, is key for STING-dependent TFEB activation. STING stimulates global upregulation of TFEB-target genes, mediating lysosomal biogenesis and autophagy. TFEB supports cell survival during chronic sterile STING activation, a common condition in aging and age-related diseases. These results reveal a primordial function of STING in the biogenesis of lysosomes, essential organelles in immunity and cellular stress resistance.

The novel TFEB agonist desloratadine ameliorates hepatic steatosis by activating the autophagy-lysosome pathway.

The autophagy-lysosome pathway plays an essential role in promoting lipid catabolism and preventing hepatic steatosis in non-alcoholic fatty liver disease (NAFLD). Transcription factor EB (TFEB) enhances the autophagy-lysosome pathway by regulating the expression of genes related to autophagy and lysosome biogenesis. Therefore, targeting TFEB provides a novel strategy for the treatment of lipid metabolic diseases. In this study, the antiallergic drug desloratadine was screened and identified as a novel TFEB agonist. Desloratadine effectively induced translocation of TFEB to the nucleus and promoted autophagy and lysosome biogenesis. Desloratadine-induced TFEB activation was dependent on AMPK rather than mTORC1. Moreover, desloratadine treatment enhanced clearance of lipid droplets in cells induced by fatty acids oleate and palmitate. Furthermore, high-fat diet (HFD) induced obesity mouse model experiments indicated treatment with desloratadine markedly reduced the body weight of HFD-fed mice, as well as the levels of hepatic triglycerides and total cholesterol, serum glutamic pyruvic transaminase and glutamic-oxaloacetic transaminase. Oil red O staining showed the liver fat was significantly reduced after desloratadine treatment, and H&E staining analysis demonstrated hepatocellular ballooning was improved. In addition, autophagy and lysosomal biogenesis was stimulated in the liver of desloratadine treated mice. Altogether, these findings demonstrate desloratadine ameliorates hepatic steatosis through activating the TFEB-mediated autophagy-lysosome pathway, thus desloratadine has an exciting potential to be used to treat fatty liver disease.

Lysosomal TFEB-TRPML1 Axis in Astrocytes Modulates Depressive-like Behaviors.

Lysosomes are important cellular structures for human health as centers for recycling, signaling, metabolism and stress adaptation. However, the potential role of lysosomes in stress-related emotions has long been overlooked. Here, it is found that lysosomal morphology in astrocytes is altered in the medial prefrontal cortex (mPFC) of susceptible mice after chronic social defeat stress. A screen of lysosome-related genes revealed that the expression of the mucolipin 1 gene (Mcoln1; protein: mucolipin TRP channel 1) is decreased in susceptible mice and depressed patients. Astrocyte-specific knockout of mucolipin TRP channel 1 (TRPML1) induced depressive-like behaviors by inhibiting lysosomal exocytosis-mediated adenosine 5'-triphosphate (ATP) release. Furthermore, this stress response of astrocytic lysosomes is mediated by the transcription factor EB (TFEB), and overexpression of TRPML1 rescued depressive-like behaviors induced by astrocyte-specific knockout of TFEB. Collectively, these findings reveal a lysosomal stress-sensing signaling pathway contributing to the development of depression and identify the lysosome as a potential target organelle for antidepressants.

HIF-2α drives hepatic Kupffer cell death and proinflammatory recruited macrophage activation in nonalcoholic steatohepatitis.

Proinflammatory hepatic macrophage activation plays a key role in the development of nonalcoholic steatohepatitis (NASH). This involves increased embryonic hepatic Kupffer cell (KC) death, facilitating the replacement of KCs with bone marrow-derived recruited hepatic macrophages (RHMs) that highly express proinflammatory genes. Moreover, phago/efferocytic activity of KCs is diminished in NASH, enhancing liver inflammation. However, the molecular mechanisms underlying these changes in KCs are not known. Here, we show that hypoxia-inducible factor 2α (HIF-2α) mediates NASH-associated decreased KC growth and efferocytosis by enhancing lysosomal stress. At the molecular level, HIF-2α stimulated mammalian target of rapamycin (mTOR)- and extracellular signal-regulated kinase-dependent inhibitory transcription factor EB (TFEB) phosphorylation, leading to decreased lysosomal and phagocytic gene expression. With increased metabolic stress and phago/efferocytic burden in NASH, these changes were sufficient to increase lysosomal stress, causing decreased efferocytosis and lysosomal cell death. Of interest, HIF-2α-dependent TFEB regulation only occurred in KCs but not RHMs. Instead, in RHMs, HIF-2α promoted mitochondrial reactive oxygen species production and proinflammatory activation by increasing ANT2 expression and mitochondrial permeability transition. Consequently, myeloid lineage-specific or KC-specific HIF-2α depletion or the inhibition of mTOR-dependent TFEB inhibition using antisense oligonucleotide treatment protected against the development of NASH in mice. Moreover, treatment with an HIF-2α-specific inhibitor reduced inflammatory and fibrogenic gene expression in human liver spheroids cultured under a NASH-like condition. Together, our results suggest that macrophage subtype-specific effects of HIF-2α collectively contribute to the proinflammatory activation of liver macrophages, leading to the development of NASH.