Transcription Factor EB Overexpression Research Articles

Endothelial TFEB (Transcription Factor EB) Positively Regulates Postischemic Angiogenesis.

Postischemic angiogenesis is critical to limit the ischemic tissue damage and improve the blood flow recovery. The regulation and the underlying molecular mechanisms of postischemic angiogenesis are not fully unraveled. TFEB (transcription factor EB) is emerging as a master gene for autophagy and lysosome biogenesis. However, the role of TFEB in vascular disease is less understood. We aimed to determine the role of endothelial TFEB in postischemic angiogenesis and its underlying molecular mechanism. In primary human endothelial cells (ECs), serum starvation induced TFEB nuclear translocation. VEGF (vascular endothelial growth factor) increased TFEB expression level and nuclear translocation. Utilizing genetically engineered EC-specific TFEB transgenic and KO (knockout) mice, we investigated the role of TFEB in postischemic angiogenesis in the mouse hindlimb ischemia model. We observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic mice compared with the wild-type littermates. Furthermore, blood flow recovery was attenuated in EC-TFEB KO mice compared with control mice. In aortic ring cultures, the TFEB transgene significantly increased vessel sprouting, whereas TFEB deficiency impaired the vessel sprouting. In vitro, adenovirus-mediated TFEB overexpression promoted EC tube formation, migration, and survival, whereas siRNA-mediated TFEB knockdown had the opposite effect. Mechanistically, TFEB activated AMPK (AMP-activated protein kinase)-α signaling and upregulated autophagy. Through inactivation of AMPKα or inhibition of autophagy, we demonstrated that the AMPKα and autophagy are necessary for TFEB to regulate angiogenesis in ECs. Finally, the positive effect of TFEB on AMPKα activation and EC tube formation was mediated by TFEB-dependent transcriptional upregulation of MCOLN1 (mucolipin-1). In summary, our data demonstrate that TFEB is a positive regulator of angiogenesis through activation of AMPKα and autophagy, suggesting that TFEB constitutes a novel molecular target for ischemic vascular disease.

6-Hydroxydopamine induces autophagic flux dysfunction by impairing transcription factor EB activation and lysosomal function in dopaminergic neurons and SH-SY5Y cells

Autophagy deregulation has been implicated in Parkinson’s disease (PD), yet the role of autophagy in neuronal survival remains controversial. In this study, we comprehensively investigated the time-course of autophagy-related markers in 6-OHDA-induced Parkinsonian rat models and assessed its effect on the state of autophagic flux both in vivo and in vitro. We observed an early activation of autophagy followed by autophagic flux impairment, which was confirmed with autophagy inhibitor chloroquine in vivo and Ad-GFP-mCherry-LC3-infected SH-SY5Y cells in vitro. In addition, 6-OHDA not only remarkably reduced the expression level of lysosome-associated membrane protein 1 (Lamp1), but also impaired the hydrolase activities of lysosomal proteases. Transcription factor EB (TFEB), a key transcription factor controlling lysosome biogenesis, was also significantly downregulated by 6-OHDA and its nuclear translocation was inhibited as well, which could account for the impaired lysosomal function. Promoting lysosome biogenesis through TFEB overexpression could protect SH-SY5Y cells against 6-OHDA-induced neurotoxicity. The above findings demonstrated that autophagic flux dysfunction was closely associated with 6-OHDA-induced neurotoxicity and highlighted the importance of functional lysosomes and homeostatic autophagic flux in developing therapeutic agents for PD.

AAV-mediated transcription factor EB (TFEB) gene delivery ameliorates muscle pathology and function in the murine model of Pompe Disease

Pompe disease (PD) is a metabolic myopathy due to acid alpha-glucosidase deficiency and characterized by extensive glycogen storage and impaired autophagy. We previously showed that modulation of autophagy and lysosomal exocytosis by overexpression of the transcription factor EB (TFEB) gene was effective in improving muscle pathology in PD mice injected intramuscularly with an AAV-TFEB vector. Here we have evaluated the effects of TFEB systemic delivery on muscle pathology and on functional performance, a primary measure of efficacy in a disorder like PD. We treated 1-month-old PD mice with an AAV2.9-MCK-TFEB vector. An animal cohort was analyzed at 3 months for muscle and heart pathology. A second cohort was followed at different timepoints for functional analysis. In muscles from TFEB-treated mice we observed reduced PAS staining and improved ultrastructure, with reduced number and increased translucency of lysosomes, while total glycogen content remained unchanged. We also observed statistically significant improvements in rotarod performance in treated animals compared to AAV2.9-MCK-eGFP-treated mice at 5 and 8 months. Cardiac echography showed significant reduction in left-ventricular diameters. These results show that TFEB overexpression and modulation of autophagy result in improvements of muscle pathology and of functional performance in the PD murine model, with delayed disease progression.

Exercise induces TFEB expression and activity in skeletal muscle in a PGC-1α-dependent manner.

The mitochondrial network in muscle is controlled by the opposing processes of mitochondrial biogenesis and mitophagy. The coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) regulates biogenesis, while the transcription of mitophagy-related genes is controlled by transcription factor EB (TFEB). PGC-1α activation is induced by exercise; however, the effect of exercise on TFEB is not fully known. We investigated the interplay between PGC-1α and TFEB on mitochondria in response to acute contractile activity in C2C12 myotubes and following exercise in wild-type and PGC-1α knockout mice. TFEB nuclear localization was increased by 1.6-fold following 2 h of acute myotube contractile activity in culture, while TFEB transcription was also simultaneously increased by twofold to threefold. Viral overexpression of TFEB in myotubes increased PGC-1α and cytochrome- c oxidase-IV gene expression. In wild-type mice, TFEB translocation to the nucleus increased 2.4-fold in response to acute exercise, while TFEB transcription, assessed through the electroporation of a TFEB promoter construct, was elevated by fourfold. These exercise effects were dependent on the presence of PGC-1α. Our data indicate that acute exercise provokes TFEB expression and activation in a PGC-1α-dependent manner and suggest that TFEB, along with PGC-1α, is an important regulator of mitochondrial biogenesis in muscle as a result of exercise.

Silver nanoparticles induce lysosomal-autophagic defects and decreased expression of transcription factor EB in A549 human lung adenocarcinoma cells

Although silver nanoparticles (AgNPs) are widely used in consumer and medical products, the mechanism by which AgNPs cause pulmonary damage is unclear. AgNPs are incorporated into cells and processed via the autophagy pathway. We examined the effects of AgNP exposure on autophagic flux and expression of transcription factor EB (TFEB) in A549 lung adenocarcinoma cells. In cells exposed to citrate-coated 60-nm AgNPs, confocal laser microscopic examination showed a decrease in the LysoTracker fluorescence signal and an increase in that of Cyto-ID, indicating lysosomal pH alkalization and autophagosome formation, respectively. The proteins p62 and microtubule-associated protein light chain 3B-II (LC3B-II) are both degraded by autophagy, and their levels increased depending on AgNP dose. Furthermore, AgNP-induced increase in LC3B-II was not enhanced by treatment with the autophagic inhibitor bafilomycin A1. TFEB mRNA levels, and protein levels in cytosolic and nuclear fractions, were suppressed by exposure to AgNPs, suggesting transcriptional inhibition of TFEB expression. Overexpression of TFEB did not suppress AgNP-induced LC3B-II accumulation and cellular damage, indicating that impairment of autophagic flux and cellular damage by AgNPs might not be primarily caused by reduced TFEB expression. The present study suggests that AgNP-induced lysosomal dysfunction plays a principal role in the autophagic flux defect.

Transcription factor EB is involved in autophagy-mediated chemoresistance to doxorubicin in human cancer cells.

Transcription factor EB (TFEB) is a master regulator of autophagy activity and lysosomal biogenesis, but its role in autophagy-mediated cell survival and chemotherapy resistance is not completely understood. In this study, we explored whether TFEB played an important role in autophagy-mediated chemotherapy resistance in human cancer LoVo and HeLa cells in vitro. Treatment of human colon cancer LoVo cells with doxorubicin (0.5 μmol/L) induced autophagy activation and nuclear translocation of TFEB, which resulted from inactivation of the mTOR pathway. In both LoVo and HeLa cells, overexpression of TFEB enhanced doxorubicin-induced autophagy activation and significantly decreased doxorubicin-induced cell death, whereas knockdown of TFEB with small interfering RNA blocked doxorubicin-induced autophagy and significantly enhanced the cytotoxicity of doxorubicin. In LoVo cells, autophagy inhibition by 3-methyladenine (3-MA) or knockdown of autophagy-related gene Atg5 increased cell death in response to doxorubicin, and abolished TFEB overexpression-induced chemotherapy resistance, suggesting that the inhibition of autophagy made cancer cells more sensitive to doxorubicin. The results demonstrate that TFEB-mediated autophagy activation decreases the sensitivity of cancer cells to doxorubicin.

Transcriptional factor EB regulates macrophage polarization in the tumor microenvironment

ABSTRACTTumor microenvironment (TME) contains a variety of infiltrating immune cells. Among them, tumor-associated macrophages (TAMs) and their alternative activation contribute greatly to the progression of tumors. The mechanisms governing macrophage polarization in the TME are unclear. Here, we show that in TAMs or macrophages under tumor-conditioned medium treatment, the expression of transcription factor EB (TFEB) is reduced and more of the TFEB protein is in an inactive cytosolic form. Transforming growth factor (TGF)-β is identified as a main driving force for the reduced TFEB expression and activity in TAMs via activating ERK signaling. TFEB interference in macrophages significantly enhanced their alternative activation, with reduced expression of MHC-II and co-stimulatory molecule CD80, decreased ability to activate T cells, and increased ability to attract tumor cells. When co-inoculated with tumor cells, macrophages with TFEB knockdown significantly enhanced tumor growth with increased infiltration of M2-like macrophages, reduced infiltration of CD8+ T cells, and enhanced angiogenesis in the tumors. Mechanistic studies revealed that TFEB downregulation resulted in macrophage M2 polarization through reducing SOCS3 production and enhancing STAT3 activation. We further demonstrate that the activation of TFEB by hydroxypropyl-β-cyclodextrin in macrophages suppressed their M2 polarization and tumor-promoting capacity, and that macrophage-specific TFEB overexpression inhibited breast tumor growth in mice. Therefore, our data suggest that TFEB plays critical roles in macrophage polarization, and the downregulation of TFEB expression and activation is an integral part of tumor-induced immune editing in the TME. This study provides a rationale for a new cancer treatment strategy by modulating macrophage polarization through activating TFEB.

Abstract 8: Endothelial TFEB Regulates Postischemic Angiogenesis via AMPKalpha Activation

Rationale: Postischemic angiogenesis is critical to limit the ischemic tissue damage and improve the blood flow recovery. The regulation and the underlying molecular mechanisms of angiogenesis are not fully unraveled. Transcription factor-EB (TFEB) is emerging as a master gene for autophagy and lysosome biogenesis. However, the role of TFEB in the vascular disease is less understood. Objective: We aim to determine the role of endothelial TFEB in postischemic angiogenesis and underlying molecular mechanism. Methods and Results: In a murine hindlimb ischemic model, we demonstrated that TFEB was upregulated in the ischemic skeletal muscle tissue. Utilizing genetically-engineered endothelial cell (EC) specific TFEB transgenic mice, we investigated the function of TFEB in postischemic angiogenesis. We observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic mice compared with the wild-type littermates (n = 8-9 for each group, p < 0.01). Furthermore, we found that blood flow recovery was attenuated in EC-selective TFEB deficient mice compared with control mice (n =8-9 for each group, p< 0.01). In aortic ring cultures, we found that TFEB transgene significantly increased the vessel sprouting. Adenovirus-mediated TFEB overexpression promoted EC tube formation whereas small interfering RNA (siRNA)-mediated TFEB knockdown suppressed tube formation in ECs. Mechanistically, TFEB activated calcium/calmodulin-dependent protein kinase kinase-β and AMP-activated protein kinase (AMPK)-α signaling pathway. Through pharmacological inactivation and siRNA-mediated knockdown of AMPKα, we demonstrated that AMPKα is necessary for TFEB to regulate tube formation in ECs. Conclusions: In summary, our data demonstrate that TFEB is a positive regulator of angiogenesis through activation of AMPKα signaling, suggesting that TFEB constitutes a novel molecular target for ischemic vascular disease.

Janus Kinase 2 Regulates Transcription Factor EB Expression and Autophagy Completion in Glomerular Podocytes.

The nonreceptor kinase Janus kinase 2 (JAK2) has garnered attention as a promising therapeutic target for the treatment of CKD. However, being ubiquitously expressed in the adult, JAK2 is also likely to be necessary for normal organ function. Here, we investigated the phenotypic effects of JAK2 deficiency. Mice in which JAK2 had been deleted from podocytes exhibited an elevation in urine albumin excretion that was accompanied by increased podocyte autophagosome fractional volume and p62 aggregation, which are indicative of impaired autophagy completion. In cultured podocytes, knockdown of JAK2 similarly impaired autophagy and led to downregulation in the expression of lysosomal genes and decreased activity of the lysosomal enzyme, cathepsin D. Because transcription factor EB (TFEB) has recently emerged as a master regulator of autophagosome-lysosome function, controlling the expression of several of the genes downregulated by JAK2 knockdown, we questioned whether TFEB is regulated by JAK2. In immortalized mouse podocytes, JAK2 knockdown decreased TFEB promoter activity, expression, and nuclear localization. In silico analysis and chromatin immunoprecipitation assays revealed that the downstream mediator of JAK2 signaling STAT1 binds to the TFEB promoter. Finally, overexpression of TFEB in JAK2-deficient podocytes reversed lysosomal dysfunction and restored albumin permselectivity. Collectively, these observations highlight the homeostatic actions of JAK2 in podocytes and the importance of TFEB to autophagosome-lysosome function in these cells. These results also raise the possibility that therapeutically modulating TFEB activity may improve podocyte health in glomerular disease.

TFEB inhibits endothelial cell inflammation and reduces atherosclerosis.

Transcription factor EB (TFEB) is a master regulator of autophagy and lysosome biogenesis. We investigated the function of TFEB in vascular biology and pathophysiology and demonstrated that TFEB in endothelial cells inhibited inflammation and reduced atherosclerosis development. Laminar shear stress, which protects against atherosclerosis, increased TFEB abundance in cultured primary human endothelial cells. Furthermore, TFEB overexpression in these cells was anti-inflammatory, whereas TFEB knockdown aggravated inflammation. The anti-inflammatory effect of TFEB was, at least, partially due to reduced oxidative stress because TFEB overexpression in endothelial cells decreased the concentrations of reactive oxygen species and increased the expression of the antioxidant genes HO1 (which encodes heme oxygenase 1) and SOD2 (which encodes superoxide dismutase 2). In addition, transgenic mice with endothelial cell-specific expression of TFEB exhibited reduced leukocyte recruitment to endothelial cells and decreased atherosclerosis development. Our study suggests that TFEB is a protective transcription factor against endothelial cell inflammation and a potential target for treating atherosclerosis and associated cardiovascular diseases.

TFEB-mediated increase in peripheral lysosomes regulates store-operated calcium entry

Lysosomes are membrane-bound organelles mainly involved in catabolic processes. In addition, lysosomes can expel their contents outside of the cell via lysosomal exocytosis. Some of the key steps involved in these important cellular processes, such as vesicular fusion and trafficking, require calcium (Ca2+) signaling. Recent data show that lysosomal functions are transcriptionally regulated by transcription factor EB (TFEB) through the induction of genes involved in lysosomal biogenesis and exocytosis. Given these observations, we investigated the roles of TFEB and lysosomes in intracellular Ca2+ homeostasis. We studied the effect of transient modulation of TFEB expression in HeLa cells by measuring the cytosolic Ca2+ response after capacitative Ca2+ entry activation and Ca2+ dynamics in the endoplasmic reticulum (ER) and directly in lysosomes. Our observations show that transient TFEB overexpression significantly reduces cytosolic Ca2+ levels under a capacitative influx model and ER re-uptake of calcium, increasing the lysosomal Ca2+ buffering capacity. Moreover, lysosomal destruction or damage abolishes these TFEB-dependent effects in both the cytosol and ER. These results suggest a possible Ca2+ buffering role for lysosomes and shed new light on lysosomal functions during intracellular Ca2+ homeostasis.

Transcription Factor EB Expression in Early Breast Cancer Relates to Lysosomal/Autophagosomal Markers and Prognosis

BackgroundDisrupting the autophagic balance to trigger autophagic death may open new strategies for cancer therapy. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and may play a role in cancer biology and clinical behavior. MethodsThe expression of TFEB and the lysosomal cancer cell content (expression of lysosomal associated membrane protein 2a [LAMP2a] and cathepsin D) was studied in a series of 100 T1-stage breast carcinomas. Expression patterns were correlated with autophagy/hypoxia-related proteins, angiogenesis, and clinical outcome. The effect of hypoxic/acidic conditions on TFEB kinetics was studied in the MCF-7 cancer cell line. ResultsOverexpression of TFEB in cancer cell cytoplasm and the perinuclear/nuclear area was noted in 23 (23%) of 100 cases. High LAMP2a and cathepsin D expression was noted in 30 (30%) of 100 and 28 (28%) of 100 cases, respectively. TFEB expression was directly linked with LAMP2a (P < .0001, r = 0.53), cathepsin D (P = .0002, r = 0.36), light chain 3A (LC3A) (P = .02, r = 0.22), and hypoxia-inducible factor 2-alpha (HIF-2α) (P = .01, r = 0.25) expression and inversely with progesterone receptor (P = .01, r = 0.22). High vascular density was directly linked with LAMP2a (P = .05, r = 0.18) and cathepsin D (P = .005, r = 0.28). In Kaplan-Meier survival analysis, TFEB and cathepsin D expression were related to an ominous prognosis (P = .001 and P = .03, respectively). In multivariate analysis, TFEB expression sustained its independent prognostic significance (P = .05, hazard ratio 2.1). In in vitro experiments, acidity triggered overexpression of TFEB and nuclear translocation. ConclusionIntense TFEB expression and lysosomal biogenesis, evident in one fourth of early breast carcinomas, define poor prognosis. Tumor acidity is among the microenvironmental conditions that trigger TFEB overactivity. TFEB is a sound target for the development of lysosomal targeting therapies.

187. TFEB-Mediated Clearance of the Lipofuscin Fluorophore A2E

Abnormal accumulation of various by-products of the visual cycle, including the diretinoid-pyridinium-ethanolamine (A2E), in retinal pigment epithelium (RPE) is an hallmark of both Stargardt disease (STGD) and age-related macular degeneration (AMD). This is responsible for RPE and, consequently, photoreceptor (PR) cell death. A2E storage in RPE lysosomes has been shown to reduce both the capacity of RPE to degrade phagocytized PR outer segments and autophagosome biogenesis, trafficking and autophagic flux. The transcription factor EB (TFEB) is a master regulator of cellular clearance. Here we tested if TFEB overexpression induces A2E clearance from the RPE cells. We have generated a plasmid encoding for a double-serine mutant (S142A, S211A) form of TFEB, which is constitutively active. We have transfected this plasmid in the human RPE-derived ARPE19 cells to evaluate TFEB activation of its transcriptional targets and ability to clear A2E after loading. We found that TFEB overexpression in ARPE19 cells results in the induction of TFEB transcriptional targets involved in cellular clearance. Importantly, TFEB overexpression in ARPE19 cells was associated with reduction of intracellular A2E accumulation. Taken together these results suggest that TFEB overexpression is an effective strategy to promote A2E clearance in vitro. Further studies will clarify if this holds true in vivo. These results may have implications for the therapy of both STGD and the more common AMD.

TFEB Overexpression in the P301S Model of Tauopathy Mitigates Increased PHF1 Levels and Lipofuscin Puncta and Rescues Memory Deficits.

Transcription factor EB (TFEB) was recently shown to be a master regulator of autophagy lysosome pathway. Here, we successfully generated and characterized transgenic mice overexpressing flag-TFEB. Enhanced autophagy in the flag-TFEB transgenic mice was confirmed by an increase in the cellular autophagy markers, as determined by both immunoblots and transmission electron microscopy. Surprisingly, in the flag-TFEB mice we observed increased activity of senescence-associated β-galactosidase by ∼66% of neurons in the cortex (p < 0.001) and 73% of neurons in the hippocampus (p < 0.001). More importantly, flag-TFEB expression remarkably reduced the levels of paired-helical filament (PHF)-tau from 372% in the P301S model of tauopathy to 171% (p < 0.001) in the cortex, and from 436% to 212% (p < 0.001) in the hippocampus. Significantly, reduced levels of NeuN in the cortex (38%, p < 0.001) and hippocampus (25%, p < 0.05) of P301S mice were almost completely restored to WT levels in the P301S/flag-TFEB double-transgenic mice. Also, levels of spinophilin in both the cortex (p < 0.001) and hippocampus (p < 0.001) were restored almost to WT levels. Most importantly, the age-associated lipofuscin granules, which are generally presumed to be nondegradable, were reduced by 57% (p < 0.001) in the cortex and by 55% (p < 0.001) in the hippocampus in the double-transgenic mice. Finally, TFEB overexpression in the P301S mice markedly reversed learning deficits in terms of spatial memory (Barnes maze), as well as working and reference memories (T maze). These data suggest that the overexpression of TFEB can reliably enhance autophagy in vivo, reduce levels of PHF-tau, and thereby reverse the deposition of lipofuscin granules and memory deficits.

Lipolysis and lipophagy in lipid storage myopathies

AimsTriglycerides droplets are massively stored in muscle in Lipid Storage Myopathies (LSM). We studied in muscle regulators of lipophagy, the expression of the transcription factor-EB (TFEB) (a master regulator of lysosomal biogenesis), and markers of autophagy which are induced by starvation and exert a transcriptional control on lipid catabolism. MethodsWe investigated the factors that regulate lipophagy in muscle biopsies from 6 patients with different types of LSM: 2 cases of riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (MADD), 1 case of primary carnitine deficiency (CD), 2 cases of neutral lipid storage myopathy (NLSD-M), 1 case of carnitine–palmitoyl-transferase-II (CPT) deficiency. ResultsConventional morphology and electron microscopy documented the lipid accumulation and its dramatic resolution after treatment. Muscle immunofluorescence showed that while in MADD and NLSD-M there was a co-localized expression of TFEB and p62-SQSTM1 (marker of protein aggregates) in some atrophic fibers, in CD and CPT-II deficiency the reaction was almost normal. In regenerating fibers, TFEB localized in the cytoplasm (inactive form), whereas in atrophic fibers it localized in the nuclei (active form). Lipid-accumulated/atrophic fibers did not display p62-positive protein aggregates, indicating, together with the LC3-II (marker of autophagosomes) and p62-SQSTM1 analysis, that the autophagic flux is often preserved and lipophagy occurs. ConclusionIn atrophic and regenerating fibers of patients with NLSD-M we observed TFEB over-expression; in other conditions autophagy markers are increased, suggesting lipophagy active role on human lipid metabolism.

Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells

Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease characterized by accumulation of cystine into lysosomes secondary to mutations in the cystine lysosomal transporter, cystinosin. The defect initially causes proximal tubular dysfunction (Fanconi syndrome) which in time progresses to end-stage renal disease. Cystinotic patients treated with the cystine-depleting agent, cysteamine, have improved life expectancy, delayed progression to chronic renal failure, but persistence of Fanconi syndrome. Here, we have investigated the roleofthe transcription factor EB (TFEB), a master regulator ofthe autophagy-lysosomal pathway, in conditionally immortalized proximal tubular epithelial cells derived from the urine of a healthy volunteer or a cystinotic patient. Lack of cystinosin reduced TFEB expression and induced TFEB nuclear translocation. Stimulation of endogenous TFEB activity by genistein, or overexpression of exogenous TFEBlowered cystine levels within 24 hours in cystinotic cells. Overexpression of TFEB also stimulated delayed endocytic cargo processing within 24 hours. Rescue of otherabnormalities of the lysosomal compartment was observed but required prolonged expression of TFEB. These abnormalities could not be corrected with cysteamine. Thus, these data show that the consequences of cystinosin deficiency are not restricted to cystine accumulation and support the role of TFEB as a therapeutic target for the treatment of lysosomal storage diseases, in particular of cystinosis.

The Transcription Factor EB (TFEB) Regulates Osteoblast Differentiation Through ATF4/CHOP-Dependent Pathway.

Osteoblasts are bone-forming cells that produce large amounts of collagen type I and various bone matrix proteins. Although osteoblast differentiation is highly regulated by various factors, it remains unknown whether lysosomes are directly involved in osteoblast differentiation. Here, we demonstrate the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, modulates osteoblast differentiation. The expression levels of TFEB as well as those of endosomal/lysosomal proteins were up-regulated during osteoblast differentiation using mouse osteoblastic MC3T3-E1 cells. By gene knockdown (KD) experiments with small interfering RNA (siRNA), TFEB depletion caused markedly reduced osteoblast differentiation as compared with the control cells. Conversely, overexpression (OE) of TFEB resulted in strikingly enhanced osteoblastogenesis compared to the control cells. By analysis of down-stream effector molecules, TFEB KD was found to cause marked up-regulation of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP), both of which are essential factors for osteoblastogenesis. In contrast, TFEB OE promoted osteoblast differentiation through reduced expression of ATF4 and CHOP without differentiation agents. Given the importance of ATF4 and CHOP in osteoblastogenesis, it is clear that the TFEB-regulated signaling pathway for osteoblast differentiation is involved in ATF4/CHOP-dependent signaling pathway.

Transition metals activate TFEB in overexpressing cells.

Transition metal toxicity is an important factor in the pathogenesis of numerous human disorders, including neurodegenerative diseases. Lysosomes have emerged as important factors in transition metal toxicity because they handle transition metals via endocytosis, autophagy, absorption from the cytoplasm and exocytosis. Transcription factor EB (TFEB) regulates lysosomal biogenesis and the expression of lysosomal proteins in response to lysosomal and/or metabolic stresses. Since transition metals cause lysosomal dysfunction, we proposed that TFEB may be activated to drive gene expression in response to transition metal exposure and that such activation may influence transition metal toxicity. We found that transition metals copper (Cu) and iron (Fe) activate recombinant TFEB and stimulate the expression of TFEB-dependent genes in TFEB-overexpressing cells. In cells that show robust lysosomal exocytosis, TFEB was cytoprotective at moderate levels of Cu exposure, decreasing oxidative stress as reported by the expression of heme oxygenase-1 (HMOX1) gene. However, at high levels of Cu exposure, particularly in cells with low levels of lysosomal exocytosis, activation of overexpressed TFEB was toxic, increasing oxidative stress and mitochondrial damage. Based on these data, we conclude that TFEB-driven gene network is a component of the cellular response to transition metals. These data suggest limitations and disadvantages of TFEB overexpression as a therapeutic approach.

Altered TFEB-mediated lysosomal biogenesis in Gaucher disease iPSC-derived neuronal cells.

Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase). The severe forms of GD are associated with neurodegeneration with either rapid (Type 2) or slow progression (Type 3). Although the neurodegenerative process in GD has been linked to lysosomal dysfunction, the mechanisms involved are largely unknown. To identify the lysosomal alterations in GD neurons and uncover the mechanisms involved, we used induced pluripotent stem cells (iPSCs) derived from patients with GD. In GD iPSC-derived neuronal cells (iPSC-NCs), GBA1 mutations caused widespread lysosomal depletion, and a block in autophagic flux due to defective lysosomal clearance of autophagosomes. Autophagy induction by rapamycin treatment in GD iPSC-NCs led to cell death. Further analysis showed that in GD iPSC-NCs, expression of the transcription factor EB (TFEB), the master regulator of lysosomal genes, and lysosomal gene expression, were significantly downregulated. There was also reduced stability of the TFEB protein and altered lysosomal protein biosynthesis. Treatment of mutant iPSC-NCs with recombinant GCase (rGCase) reverted the lysosomal depletion and autophagy block. The effect of rGCase on restoring lysosomal numbers in mutant cells was enhanced in the presence of overexpressed TFEB, but TFEB overexpression alone did not reverse the lysosomal depletion phenotype. Our results suggest that GBA1 mutations interfere with TFEB-mediated lysosomal biogenesis, and that the action of GCase in maintaining a functioning pool of lysosomes is exerted in part through TFEB. The lysosomal alterations described here are likely to be a major determinant in GBA1-associated neurodegeneration.

Increased expression of transcription factor EB (TFEB) is associated with autophagy, migratory phenotype and poor prognosis in non-small cell lung cancer.

We investigated the role of lysosomal biogenesis and hydrolase activity in the clinical behavior and postoperative outcome of lung cancer. Using immunohistochemistry we investigated the expression of the transcription factor EB (TFEB) which orchestrates lysosomal biogenesis, the lysosome membrane protein LAMP2a and of the lysosomal hydrolase cathepsin D in a series of 98 non-small cell lung carcinomas (NSCLC) treated with surgery alone. In vitro experiments with the A549 and H1299 lung cancer cell lines were also performed. Overexpression of TFEB, LAMP2a and Cathepsin D was noted in 47/98 (47.9%), 43/98 (43.9%) and 39/98 (39.8%) cases, respectively, and were significantly correlated with each other and with adenocarcinomas. High LAMP2a was related to high histology grade. Linear regression analysis confirmed significant association of TFEB with BNIP3 (p=0.0003, r=0.35) and LC3A with LAMP2a expression (p=0.0002, r=0.37). An inverse association of Cathepsin D expression with stone-like structures (SLS) was recorded (p=0.02, r=0.22). On univariate analysis all three lyososomal variables were associated with poor prognosis (p=0.05, 0.04 and 0.01, for TFEB, Cathepsin D and LAMP2a, respectively). Multivariate analysis showed that the SLS number (p=0.0001, HR5.37), Cathepsin D expression (p=0.01, HR=2.2) and stage (p=0.01, HR=1.5) were independent prognostic variables. Silencing of TFEB with siRNAs in the A549 and H1299 lung cancer cell lines did not affect proliferation but resulted in reduced migration ability. Lysosomal biogenesis is linked to autophagosomal protein expression in NSCLC and characterizes subgroups of high risk patients after complete surgical lung tumor resection.