TFEB and TFE3: The art of multi-tasking under stress conditions

ABSTRACT

Cellular adaptation response to a myriad of stressors is key for survival. The lysosomal/autophagy pathway is inextricably connected to the stress response regulation. Two transcription factors, TFEB and TFE3, have recently emerged as master regulators of this degradative pathway. Their function modulating different cellular pathways will be discussed.

Introduction

Lysosomes are the main degradative membrane-bound organelles in all cells. Lysosomes contain over 50 enzymes capable of hydrolyzing a variety of substrates in their acidic environment.¹ Lysosomes receive substrates destined for degradation through endocytosis, phagocytosis, and autophagy.² Intralysosomal accumulation of undigested materials due to inability of lysosomes to break down cellular components is the cause of over 50 human diseases collectively known as Lysosomal Storage Disorders (LSDs).

Lysosomes have been traditionally regarded as the end point of the endocytic pathway. However, this paradigm has changed since the discovery of mTORC1, a lysosomal membrane-associated multi-protein complex that couples growth signals, nutrients, and energy to cellular growth and proliferation.^3-5 The current view of lysosomes as signaling hubs whose function is important for the maintenance of cellular homeostasis was also spearheaded by the finding of a transcription-dependent mechanism regulating lysosomal biogenesis and autophagy.^6,7

Autophagy is a critical catabolic process responsible for the lysosome-mediated degradation of misfolded/aggregated proteins, damaged organelles, and the elimination of intracellular pathogens. As an essential mechanism involved in maintaining cell homeostasis, dysregulated autophagy is responsible for a variety of clinical manifestations associated with neurodegenerative and metabolic diseases.⁸

A recently described nutrient-dependent cross-talk between lysosomes, autophagy, and the nucleus is mediated by TFEB^9-11 and TFE3¹², two transcription factors that belong to the MITF/TFE family of basic helix-loop-helix/leucine zipper (bHLH-LZ) transcription factors, composed also of MITF and TFEC.¹³

Several studies have revealed an amino acid-dependent and mTORC1-regulated mechanism of TFEB and TFE3 activation. Here, we discuss this process as well as recent findings describing mTORC1-independent activation of these transcription factors and their broader role in cellular response and adaptation to stress.

Nutrient-dependent mechanism of TFEB activation

The highly conserved serine/threonine kinase mTOR is a key regulator of a signaling pathway that couples nutrient availability, energy, and growth factors to protein synthesis, cellular growth, autophagy, and survival.^14,15 mTOR is present in a multi-protein complex named mTORC1, which is recruited to the cytoplasmic surface of late endosomes/lysosomes in response to amino acid stimulation,^3,16 where it is phosphorylated and activated by the GTPase Rheb.^17,18

The amino acid-dependent recruitment of mTORC1 to the endo/lysosomal membranes is mediated by the active heterodimeric Rag GTPases complex, which is anchored to the membranes by the Ragulator complex.^19,20 Rag GTPases are obligate heterodimers formed by the combinations of either RagA or RagB bound to Rag C or RagD.^21,22 In conditions, where nutrients are plentiful, active Rags (GTP-bound RagA/B and GDP-bound RagC/D) recruit mTORC1 to lysosomes leading to its activation. Active mTORC1 then promotes amino acid synthesis and cellular growth as autophagy is suppressed.^23,24 Conversely, nutrient deprivation results in an inactive conformation of Rags (GDP-bound RagA/B and GTP-bound RagC/D) promoting lysosomal dissociation and inactivation of mTORC1.²⁰ mTORC1 inactivation results in protein synthesis inhibition, induction of autophagy, and increased lysosomal degradation.²⁵

A new mechanism of autophagy regulation and lysosomal biogenesis mediated by the transcriptional control of autophagic and lysosomal genes has been recently uncovered involving TFEB as a key player in this process.⁷ Through binding to one or more ten-base pair motifs named Coordinated Lysosomal Expression and Regulation (CLEAR) elements found in the promoter regions of different lysosomal and autophagic genes, TFEB induces transcriptional activation of these genes, thus resulting in autophagy activation as well as an increase in lysosome number and activity.^6,7

Understanding how activated TFEB controls the lysosomal/autophagy pathway was crucial for understanding cellular adaptation to nutrient levels within the cell. One important observation toward that aim was the finding that the subcellular localization of TFEB is regulated by amino acid availability and mTORC1 activity.^9-11 Under nutrient-rich conditions, mTORC1 is active and directly phosphorylates TFEB on several residues, including serine 211. This phosphorylation promotes binding of TFEB to the chaperone-like cytosolic protein 14–3–3, thus leading to the cytoplasmic retention of TFEB. Conversely, in amino acid deprived conditions, inactive mTORC1 can no longer phosphorylate TFEB, resulting in dissociation of the TFEB/14–3–3 complex and subsequent transport of TFEB to the nucleus.^9,10 In the nucleus, TFEB promotes transcriptional activation of multiple lysosomal, autophagic, and metabolic genes, which helps cells adapt and survive fasting.²⁶

The identification of the molecular machinery involved in TFEB recruitment to lysosomes was critical for understanding the mechanism of TFEB regulation by mTORC1. In full nutrient conditions, TFEB recruitment to the lysosomal surface is mediated by its interaction with active Rag GTPase heterocomplex.²⁷ Recruitment of TFEB to lysosomes facilitates its phosphorylation and subsequent inactivation by mTORC1. In contrast, amino acid deprivation conditions that lead to Rag GTPases inactivation, as well as overexpression of inactive Rags or depletion of RagC, result in TFEB dephosphorylation, TFEB/14–3–3 dissociation, and TFEB nuclear translocation (Fig. 1A).^12,27,10,11

Figure 1.

TFEB and TFE3 are the master regulators of cellular adaptation to stress. (A) Starvation-dependent activation of TFEB promoting lysosomal biogenesis and autophagy. (B) TFEB and TFE3 cooperate in the cellular response to a variety of stressors modulating different stress pathways.

Even though TFEB was initially identified as the only member of the MITF/TFE family with the capability of regulating the expression of numerous lysosomal and autophagic genes, our recent findings revealed that TFE3 is also a master regulator of lysosomal biogenesis and autophagy.¹² The mechanism of TFE3 activation in response to nutrient levels, its binding to CLEAR elements and its ability to induce lysosomal biogenesis and autophagy are characteristics shared with TFEB.¹² In some cell types, TFEB and TFE3 seem partially redundant in terms of their ability to induce lysosomal biogenesis and both must be present for a maximal response.^12,28 However, some TFEB and TFE3 biological functions may be unique, as the expression of these transcription factor is differentially regulated during development and is cell type specific.^29,30

It is important to note that the mechanism of nutrient regulation of TFEB and TFE3 appears to be conserved across different species,^31,32 suggesting the existence of an evolutionarily process dedicated to the metabolic control of the cellular response to stress mediated by the cross-talk between the lysosome and nucleus.^30,11

Role of TFEB and TFE3 in cellular response to other types of stress

In addition to the participation of TFEB and TFE3 in the nutrient-dependent regulation of lysosomal function and autophagy, recent observations have demonstrated a surprisingly broader role of these transcription factors in the response to a variety of insults, including protein misfolding, pathogen infection, and mitochondrial damage (Fig. 1B). Notably, whereas the activation of TFEB and TFE3 by amino acid depletion is mediated by mTORC1, the mechanism of regulation of TFEB and TFE3 activity triggered by other stressors seems to be independent of mTORC1 activity.

When the homeostasis of the ER is perturbed by the aberrant accumulation of misfolded proteins, cells trigger the unfolded protein response (UPR) aimed to cope and restore normal cellular functions.³³ It was recently shown that activation of TFEB and TFE3 is achieved upon induction of ER stress.³⁴ TFE3 nuclear translocation under ER stress is mTORC1 independent but requires PERK, an ER integral membrane protein that senses protein misfolding in the ER lumen and activates UPR. Following activation, TFE3 targets a variety of genes involved not only in lysosomal biogenesis and autophagy but also critical regulators of ER homeostasis and apoptosis.³⁴ Particularly, interesting is the ability of TFE3 to induce transcriptional upregulation of the Activating Transcription Factor 4 (ATF4). ATF4 is an essential transcription factor that integrates the UPR and Integral Stress Responses, aiming to restore ER homeostasis and ultimately support cell survival. However, under severe ER stress conditions, ATF4 can promote expression of genes involved in cell death.³³ It is important to note that under prolong exposure to ER stress inducers, activated TFE3 increases the ATF4-dependent expression of pro-apoptotic genes, ultimately inducing cell death.³⁴ Accordingly, it was reported that the depletion of both TFEB and TFE3 renders cells more resistant to apoptosis under condition of chronic ER stress or prolonged starvation.³⁴ Therefore, these observations suggest a novel dual role of TFEB and TFE3 in cell fate. In early stages of cellular stress, TFEB and TFE3 promote cell survival, however, under chronic stress conditions, their activation may enhance mechanisms leading to cell death. We suggest that the final outcome on cell viability may depend on the cell type as well as the duration and intensity of the insult. Furthermore, under stress conditions, TFEB and TFE3 regulate expression of genes involved in multiple cellular pathways, suggesting that these transcription factors modulate a very complex transcriptional network and should be considered as master regulators of the cellular stress response.³⁴

Recent evidence shows that TFEB and TFE3 may also play an important role in the transcriptional regulation of the innate immune response. In cellular immunity, many different signaling pathways are interconnected to monitor and respond to infectious and pathogenic agents. There is growing body of evidence suggesting that lysosome activity and autophagy operate as a stress integrator mechanism in innate and adaptive immunity. The study of the role of TFEB and TFE3 in these processes was, thereby, key to understand how cells and organisms adapt to immunological challenges. We and other have shown that TFEB and TFE3 participate in the modulation of the innate immune response to pathogen infection. In macrophages, both transcription factors are activated in response to toll-like receptor ligand stimulation or pathogenic bacteria infection through a mechanism which does not require mTORC1 inactivation.²⁸ Autophagy induction and lysosomal biogenesis are significantly impaired both in Raw cells depleted of TFEB and TFE3 and in bone marrow-derived macrophages isolated from TFEB/TFE3 double knockout animals. Depletion of both transcription factors also dramatically affects the typical expansion of the endo-lysosomal compartment observed in wild-type activated macrophages.²⁸ Unexpectedly, transcriptional upregulation and secretion of many inflammatory cytokines and immune regulators are severely compromised following TFEB/TFE3 depletion both in vitro and in vivo, revealing a more direct role of these transcription factors in immune response.²⁸

The mechanism of control of the host immune response is evolutionarily conserved since HLH-30, the C. elegans ortholog of TFEB and TFE3, functions in the regulation of genes involved in the host defense response to bacterial infection.³⁵ A role of TFEB and TFE3 in adaptive immune response has also been suggested by several reports showing TFEB function in antigen presentation³⁶ and HIV infection.³⁷ In addition, defective B cell activation and antibody production has been observed in animals lacking TFE3.³⁸ Overall, these observations revealed a more general function of TFEB and TFE3 in the regulation of adaptation to stress than previously anticipated and suggested that these transcription factors may have critical and specialized roles depending of the cell, tissue, and type of stress.

Finally, TFEB and TFE3 may also regulate mitochondrial homeostasis. Several metabolic and neurodegenerative diseases are thought to be associated with severe mitochondrial dysfunction,³⁹ suggesting that mitochondria play an important role in maintaining cellular homeostasis. Accordingly, many studies have now linked defective lysosomal function and aberrant autophagic flux in many LSDs to phenotypes associated with mitochondrial diseases.⁴⁰ It has been recently described that PINK and Parkin are involved in the mitophagy process responsible for the elimination of malfunctioning mitochondria.⁴¹ The connection between the lysosomal/autophagy pathway and mitochondrial quality control mechanism was strengthened by recent observations showing that TFEB and TFE3 are activated by mitochondrial damage in an mTORC1-independent process that involves PINK and Parkin.⁴² Furthermore, depletion of all members of the MITF/TFE family renders cells unable to eliminate damaged mitochondria due to defective mitophagy.⁴² In addition, TFEB participates in the transcriptional control of a PGC1-α-dependent mechanism dedicated to eliminate dysfunctional mitochondria.⁴³ TFE3 has been also described as potential transcriptional regulator of PGC1-α in muscle.⁴⁴ PGC1-α is a critical regulator of mitochondrial biogenesis that can control mitophagy through the transactivation of TFEB.⁴³ Likewise, it has been suggested that TFEB plays an important role in coordinating mitochondria formation/degradation through PGC1-α transcriptional upregulation followed Pink/Parkin-dependent mitophagy induction.⁴⁵

Therefore, it is becoming evident that many processes, such as protein misfolding/aggregation, pathogen infection, or mitochondrial damage, can induce TFEB and TFE3 activation independently of mTORC1 inactivation. Although the mechanism/s of activation is/are not yet well understood and need to be further elucidated, the participation of several kinases and phosphatases in TFEB/TFE3 regulation has been recently suggested.^46-49

In summary, TFEB was identified as the first member of the MITF/TFE family of transcription factors with the ability to sense intracellular levels of amino acid and transcriptionally upregulate the expression of many genes involved in the regulation of autophagy and lysosomal biogenesis (Fig. 1A). However, recent observations demonstrating that TFE3 is also capable of responding to amino acid deprivation have changed our view of TFEB as the “only” master regulator of the lysosomal/autophagic pathway (Fig. 1B). In addition, work from several groups supports the idea that both transcription factors cooperate in the cellular response to a variety of intracellular and extracellular stressors and can be activated through different mechanisms. Finally, it has become evident that, in addition to lysosomal and autophagy genes, TFEB and TFE3 control the expression of key genes involved in the modulation of the immune response, metabolism, mitochondrial homeostasis, and unfolded protein response, indicating that TFEB and TFE3 function as critical master integrators of different stress pathways (Fig. 1B).

Concluding thoughts

Cells must respond and adapt to adverse conditions to survive. The modulation of the lysosomal/autophagy pathway has been shown to be critical for adaptation to stress. The mechanism of stress response has recently become the focus of much attention owing to the identification of TFEB and TFE3 as master modulators of lysosomal biogenesis and autophagy with capability to upregulate hundreds of genes involved in intracellular clearance, catabolism, metabolic processes, and cellular homeostasis. This interest is further strengthened by the observation that different types of stress, including nutrient limitation, aberrant protein accumulation, mitochondrial damage, and pathogen infection, induce TFEB/TFE3 activation, thus revealing a more complex network of regulation than previously anticipated.

The ability of TFEB and TFE3 to sense cellular stress and mediate homeostasis restoration makes them ideal candidates for palliative strategies intended for human disease. However, the precise molecular mechanism of TFEB/TFE3 activation is not well defined yet; elucidating this process will shed light on our current understanding of the important role played by these transcription factors in the well-orchestrated mechanism adopted by cells to respond and adapt to stress.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed

Funding

This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute of the NIH.