TMEM55B links autophagy flux, lysosomal repair, and TFE3 activation in response to oxidative stress.

Lysosomes have emerged as critical regulators of cellular homeostasis. Here we show that the lysosomal protein TMEM55B contributes to restore cellular homeostasis in response to oxidative stress by three different mechanisms: (1) TMEM55B mediates NEDD4-dependent PLEKHM1 ubiquitination, causing PLEKHM1 proteasomal degradation and halting autophagosome/lysosome fusion; (2) TMEM55B promotes recruitment of components of the ESCRT machinery to lysosomal membranes to stimulate lysosomal repair; and (3) TMEM55B sequesters the FLCN/FNIP complex to facilitate translocation of the transcription factor TFE3 to the nucleus, allowing expression of transcriptional programs that enable cellular adaptation to stress. Knockout of tmem55 genes in zebrafish embryos increases their susceptibility to oxidative stress, causing early death of tmem55-KO animals in response to arsenite toxicity. Altogether, our work identifies a role for TMEM55B as a molecular sensor that coordinates autophagosome degradation, lysosomal repair, and activation of stress responses.

Beta-coronaviruses exploit cellular stress responses by modulating TFEB and TFE3 activity.

Beta-coronaviruses have emerged as a severe threat to global health. Undercovering the interplay between host and beta-coronaviruses is essential for understanding disease pathogenesis and developing efficient treatments. Here we report that the transcription factors TFEB and TFE3 translocate from the cytosol to the nucleus in response to beta-coronavirus infection by a mechanism that requires activation of calcineurin phosphatase. In the nucleus, TFEB and TFE3 bind to the promoter of multiple lysosomal and immune genes. Accordingly, MHV-induced upregulation of immune regulators is significantly decreased in TFEB/TFE3-depleted cells. Conversely, over-expression of either TFEB or TFE3 is sufficient to increase expression of several cytokines and chemokines. The reduced immune response observed in the absence of TFEB and TFE3 results in increased cellular survival of infected cells but also in reduced lysosomal exocytosis and decreased viral infectivity. These results suggest a central role of TFEB and TFE3 in cellular response to beta-coronavirus infection.

p38 MAPK-dependent phosphorylation of TFEB promotes monocyte-to-macrophage differentiation.

The transcription factor EB (TFEB) regulates energy homeostasis and cellular response to a wide variety of stress conditions, including nutrient deprivation, oxidative stress, organelle damage, and pathogens. Here we identify S401 as a novel phosphorylation site within the TFEB proline-rich domain. Phosphorylation of S401 increases significantly in response to oxidative stress, UVC light, growth factors, and LPS, whereas this increase is prevented by p38 MAPK inhibition or depletion, revealing a new role for p38 MAPK in TFEB regulation. Mutation of S401 in THP1 cells demonstrates that the p38 MAPK/TFEB pathway plays a particularly relevant role during monocyte differentiation into macrophages. TFEB-S401A monocytes fail to upregulate the expression of multiple immune genes in response to PMA-induced differentiation, including critical cytokines, chemokines, and growth factors. Polarization of M0 macrophages into M1 inflammatory macrophages is also aberrant in TFEB-S401A cells. These results indicate that TFEB-S401 phosphorylation links differentiation signals to the transcriptional control of monocyte differentiation.

The FACT complex facilitates expression of lysosomal and antioxidant genes through binding to TFEB and TFE3.

TFEB (transcription factor EB) and TFE3 (transcription factor binding to IGHM enhancer 3) orchestrate the cellular response to a variety of stressors, including nutrient deprivation, oxidative stress and pathogens. Here we describe a novel interaction of TFEB and TFE3 with the FAcilitates Chromatin Transcription (FACT) complex, a heterodimeric histone chaperone consisting of SSRP1 and SUPT16H that mediates nucleosome disassembly and assembly, thus facilitating transcription. Extracellular stimuli, such as nutrient deprivation or oxidative stress, induce nuclear translocation and activation of TFEB and TFE3, which then associate with the FACT complex to regulate stress-induced gene transcription. Depletion of FACT does not affect TFEB activation, stability, or binding to the promoter of target genes. In contrast, reduction of FACT levels by siRNA or treatment with the FACT inhibitor curaxin, severely impairs induction of numerous antioxidant and lysosomal genes, revealing a crucial role of FACT as a regulator of cellular homeostasis. Furthermore, upregulation of antioxidant genes induced by TFEB over-expression is significantly reduced by curaxin, consistent with a role of FACT as a TFEB transcriptional activator. Together, our data show that chromatin remodeling at the promoter of stress-responsive genes by FACT is important for efficient expression of TFEB and TFE3 targets, thus providing a link between environmental changes, chromatin modifications and transcriptional regulation.Abbreviations: ADNP2, ADNP homeobox 2; ATP6V0D1, ATPase H+ transporting V0 subunit d1; ATP6V1A, ATPase H+ transporting V1 subunit A; ATP6V1C1, ATPase H+ transporting V1 subunit C1; CSNK2/CK2, casein kinase 2; CLCN7, chloride voltage-gated channel 7; CTSD, cathepsin D; CTSZ, cathepsin Z; EBSS, earle's balanced salt solution; FACT complex, facilitates chromatin transcription complex; FOXO3, forkhead box O3; HEXA, hexosaminidase subunit alpha; HIF1A, hypoxia inducible factor 1 subunit alpha; HMOX1, heme oxygenase 1; LAMP1, lysosomal associated membrane protein 1; MAFF, MAF bZIP transcription factor F; MAFG, MAF bZIP transcription factor G; MCOLN1, mucolipin TRP cation channel 1; MTORC1, mechanistic target of rapamycin kinase complex 1; NaAsO2, sodium arsenite; POLR2, RNA polymerase II; PPARGC1A, PPARG coactivator 1 alpha; PYROXD1, pyridine nucleotide-disulfide oxidoreductase domain 1; RRAGC, Ras related GTP binding C; SEC13, SEC13 homolog, nuclear pore and COPII coat complex component; SLC38A9, solute carrier family 38 member 9; SSRP1, structure specific recognition protein 1; SUPT16H, SPT16 homolog, facilitates chromatin remodeling subunit; TFEB, transcription factor EB; TFE3, transcription factor binding to IGHM enhancer 3; TXNRD1, thioredoxin reductase 1; UVRAG, UV radiation resistance associated; WDR59, WD repeat domain 59.

Early estrogen-induced gene 1 facilitates osteoclast formation through the inhibition of interferon regulatory factor 8 expression.

Osteoclast-mediated inflammatory bone resorption is a major cause of many inflammatory bone disorders, including rheumatoid arthritis and periodontitis. However, the mechanisms regulating osteoclast differentiation in inflammatory settings are not well understood. We demonstrate here that early estrogen-induced gene 1 (EEIG1)-deficient mice are protected from inflammatory bone loss as determined with the use of models of lipopolysaccharide (LPS)-induced bone destruction. EEIG1-deficient macrophages markedly decreased RANKL- and TNFα-mediated osteoclastogenesis due to the downregulation of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which is an essential transcription factor for osteoclast formation. In contrast, expression of interferon regulatory factor 8 (IRF8), a transcriptional repressor that blocks osteoclast differentiation, is elevated in EEIG1-deficient macrophages relative to wild-type cells. We found that reduced expression of B lymphocyte-induced maturation protein-1 (Blimp1) by siRNA downregulated RANKL-induced EEIG1 levels, whereas overexpression of Blimp1 potentiated EEIG1 levels. Mechanistic studies revealed that EEIG1 forms a complex with Blimp1 to negatively regulate the expression of the anti-osteoclastogenic gene, Irf8. We elucidated a novel mechanism by which EEIG1 restricts IRF8 expression and function, thereby enhancing the osteoclast formation by contributing to Blimp1-mediated IRF8 regulation. Together, these findings identify EEIG1 as a key regulator of osteoclastogenesis and a possible therapeutic target for pathological bone destruction.

MiT/TFE Family of Transcription Factors: An Evolutionary Perspective.

Response and adaptation to stress are critical for the survival of all living organisms. The regulation of the transcriptional machinery is an important aspect of these complex processes. The members of the microphthalmia (MiT/TFE) family of transcription factors, apart from their involvement in melanocyte biology, are emerging as key players in a wide range of cellular functions in response to a plethora of internal and external stresses. The MiT/TFE proteins are structurally related and conserved through evolution. Their tissue expression and activities are highly regulated by alternative splicing, promoter usage, and posttranslational modifications. Here, we summarize the functions of MiT/TFE proteins as master transcriptional regulators across evolution and discuss the contribution of animal models to our understanding of the various roles of these transcription factors. We also highlight the importance of deciphering transcriptional regulatory mechanisms in the quest for potential therapeutic targets for human diseases, such as lysosomal storage disorders, neurodegeneration, and cancer.

RACK1 interaction with c-Src is essential for osteoclast function.

The scaffolding protein receptor for activated C-kinase 1 (RACK1) mediates receptor activator of nuclear factor κΒ ligand (RANKL)-dependent activation of p38 MAPK in osteoclast precursors; however, the role of RACK1 in mature osteoclasts is unclear. The aim of our study was to identify the interaction between RACK1 and c-Src that is critical for osteoclast function. A RACK1 mutant protein (mutations of tyrosine 228 and 246 residues to phenylalanine; RACK1 Y228F/Y246F) did not interact with c-Src. The mutant retained its ability to differentiate into osteoclasts; however, the integrity of the RANKL-mediated cytoskeleton, bone resorption activity, and phosphorylation of c-Src was significantly decreased. Importantly, lysine 152 (K152) within the Src homology 2 (SH2) domain of c-Src is involved in RACK1 binding. The c-Src K152R mutant (mutation of lysine 152 into arginine) impaired the resorption of bone by osteoclasts. These findings not only clarify the role of the RACK1-c-Src axis as a key regulator of osteoclast function but will also help to develop new antiresorption therapies to prevent bone loss-related diseases.

The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage.

The transcription factors TFE3 and TFEB cooperate to regulate autophagy induction and lysosome biogenesis in response to starvation. Here we demonstrate that DNA damage activates TFE3 and TFEB in a p53 and mTORC1 dependent manner. RNA-Seq analysis of TFEB/TFE3 double-knockout cells exposed to etoposide reveals a profound dysregulation of the DNA damage response, including upstream regulators and downstream p53 targets. TFE3 and TFEB contribute to sustain p53-dependent response by stabilizing p53 protein levels. In TFEB/TFE3 DKOs, p53 half-life is significantly decreased due to elevated Mdm2 levels. Transcriptional profiles of genes involved in lysosome membrane permeabilization and cell death pathways are dysregulated in TFEB/TFE3-depleted cells. Consequently, prolonged DNA damage results in impaired LMP and apoptosis induction. Finally, expression of multiple genes implicated in cell cycle control is altered in TFEB/TFE3 DKOs, revealing a previously unrecognized role of TFEB and TFE3 in the regulation of cell cycle checkpoints in response to stress.

STAC2 negatively regulates osteoclast formation by targeting the RANK signaling complex.

The receptor activator of nuclear factor-κB (RANK) protein activates various protein kinase signaling cascades, including those involving NF-κB, mitogen-activated protein kinase (MAPK), and Bruton tyrosine kinase (Btk)/tyrosine-protein kinase Tec. However, the mechanism underlying the negative regulation of RANK by downstream signaling molecules remains unclear. Here, we report that Src homology 3 domain and cysteine-rich domain-containing protein 2 (STAC2) is a novel RANK ligand-inducible protein that negatively regulates RANK-mediated osteoclast formation. STAC2 physically interacts with RANK and inhibits the formation of the RANK signaling complex, which contains Grb-2-associated binder 2 (Gab2) and phospholipase Cγ2 (PLCγ2), thus leading to the suppression of RANK-mediated NF-κB and MAPK activation. Furthermore, STAC2 overexpression limits Btk/Tec-mediated PLCγ2 phosphorylation via the interaction between STAC2 and Btk/Tec. Taken together, our results reveal a novel mechanism whereby RANK signaling is restricted by its physical interaction with STAC2.