cells

Review
TFEB; Beyond Its Role as an Autophagy and
Lysosomes Regulator
Berenice Franco-Juárez 1 , Cristina Coronel-Cruz 2 , Beatriz Hernández-Ochoa 3 , Saúl Gómez-Manzo 4 ,
Noemi Cárdenas-Rodríguez 5,6 , Roberto Arreguin-Espinosa 7 , Cindy Bandala 6,8 ,
Luis Miguel Canseco-Ávila 9 and Daniel Ortega-Cuellar 10, *

1 Departamento de Neurodesarrollo y Fisiología, División de Neurociencias, Instituto de Fisiología Celular,

UNAM, Mexico City 04510, Mexico
2 Departamento de Biología Celular y Tisular, Facultad de Medicina, UNAM, Mexico City 04510, Mexico
3 Laboratorio de Inmunoquímica, Hospital Infantil de México Federico Gómez, Secretaría de Salud,
Mexico City 06720, Mexico
4 Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, Secretaría de Salud,
Mexico City 04530, Mexico
5 Laboratorio de Neurociencias, Instituto Nacional de Pediatría, Secretaría de Salud, Mexico City 04530, Mexico
6 Escuela Superior de Medicina, Instituto Politécnico Nacional, Mexico City 11340, Mexico
7 Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de
Mexico, Mexico City 04510, Mexico
8 Neurociencias Básicas, Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra, SSa,
Mexico City 14389, Mexico
9 Facultad de Ciencias Químicas, Campus IV, Universidad Autónoma de Chiapas,
Tapachula Chiapas 30700, Mexico
10 Laboratorio de Nutrición Experimental, Instituto Nacional de Pediatría, Secretaría de Salud,
Mexico City 04530, Mexico
* Correspondence: [email protected] or [email protected]; Tel.: +52-55-1084-0900 (ext. 1876)
Citation: Franco-Juárez, B.;
Coronel-Cruz, C.; Hernández-Ochoa,
Abstract: Transcription factor EB (TFEB) is considered the master transcriptional regulator of au-
B.; Gómez-Manzo, S.;
tophagy and lysosomal biogenesis, which regulates target gene expression through binding to CLEAR
Cárdenas-Rodríguez, N.;
Arreguin-Espinosa, R.; Bandala, C.;
motifs. TFEB dysregulation has been linked to the development of numerous pathological condi-
Canseco-Ávila, L.M.; Ortega-Cuellar, tions; however, several other lines of evidence show that TFEB might be a point of convergence
D. TFEB; Beyond Its Role as an of diverse signaling pathways and might therefore modulate other important biological processes
Autophagy and Lysosomes Regulator. such as cellular senescence, DNA repair, ER stress, carbohydrates, and lipid metabolism and WNT
Cells 2022, 11, 3153. https://doi.org/ signaling-related processes. The regulation of TFEB occurs predominantly at the post-translational
10.3390/cells11193153 level, including phosphorylation, acetylation, SUMOylating, PARsylation, and glycosylation. It
Academic Editor: Tuula Kallunki
is noteworthy that TFEB activation is context-dependent; therefore, its regulation is subjected to
coordinated mechanisms that respond not only to nutrient fluctuations but also to stress cell programs
Received: 2 September 2022 to ensure proper cell homeostasis and organismal health. In this review, we provide updated insights
Accepted: 5 October 2022
into novel post-translational modifications that regulate TFEB activity and give an overview of TFEB
Published: 7 October 2022
beyond its widely known role in autophagy and the lysosomal pathway, thus opening the possibility
Publisher’s Note: MDPI stays neutral of considering TFEB as a potential therapeutic target.
with regard to jurisdictional claims in
published maps and institutional affil- Keywords: transcriptional factor EB (TFEB); cellular senescence; DNA damage repair and cell cycle;
iations. WNT signaling; endoplasmic reticulum stress; carbohydrate; lipids; metabolism

Copyright: © 2022 by the authors.

1. Introduction
Licensee MDPI, Basel, Switzerland.
This article is an open access article The transcriptional factor EB (TFEB) belongs to the basic helix–loop–helix (bHLH)
distributed under the terms and leucine zipper transcription factor, a member of the microphthalmia family (MiT/TFE),
conditions of the Creative Commons which includes microphthalmia-associated transcription factor (MITF), transcription factor
Attribution (CC BY) license (https:// E3 (TFE3), and transcription factor EC (TFEC). The members of the MiT/TFE family are
creativecommons.org/licenses/by/ proteins with a basic helix–loop–helix leucine zipper region that allows them to form ho-
4.0/). modimers or heterodimers between members of the same family of MiT/TFE transcription

Cells 2022, 11, 3153. https://doi.org/10.3390/cells11193153 https://www.mdpi.com/journal/cells

Cells 2022, 11, 3153 2 of 20

factors; the DNA regions recognized by MiT/TFE members are the Ephrussi boxes (E-box)
formed by the sequence CANNTG. Specifically, TFEB recognizes E-boxes present on the
promoter of autophagy genes. Moreover, E-box is present in the CLEAR motif (Coordinat-
ed Lysosomal Expression and Regulation), a 10-base-pair motif (GTCACGTGAC) located
in the promoter region of lysosomal genes [1–3]. As a protein that is capable of being
imported or exported to the nucleus, TFEB possesses a nuclear localization signal (NLS)
identified between amino acids 241 and 252, while the nuclear export signal (NES) is located
at 140–150 amino acids of the human TFEB [4,5].
TFEB is considered a master regulator of autophagy and lysosomal function and is
conserved through evolution [6–8]. TFEB also regulates another cellular process, which
we will discuss later. TFEB increases autophagic flux by promoting the biogenesis of
lysosomes, autophagosomes, and fusion with lysosomes, to efficiently degrade complex
molecules [9–12]. TFEB activation depends on cellular nutrients and general cell status.
Thus, under nutrient-rich conditions, TFEB is located in the cytoplasm under basal cellular
conditions and translocates to the nucleus in response to starvation, lysosomal stress,
pathogen infections, ER stress, and exercise to promote organismal homeostasis [9,12,13].
Once activated, TFEB directly binds to the promoter sequences to augment the expression of
autophagy–lysosome-related genes, and its dysregulation of TFEB activity might contribute
to the development of several diseases, including hepatic steatosis, neurodegenerative
diseases, cancer, and inflammatory diseases [14–20]. Therefore, TFEB might serve as a
potential therapeutic target for the treatment of human diseases.

2. Post-Translational Control of TFEB Activity

2.1. Phosphorylation
The main function of TFEB known to date is the transcription of genes involved in
diverse cellular processes. The activity of TFEB is dependent on cellular localization, which
is controlled by translational modifications (phosphorylation, acetylation, etc.); thus, under
cellular stress conditions such as starvation, DNA damage, or oxidative stress, TFEB may
be located in the nucleus to promote its activity. Nevertheless, under basal conditions,
phosphorylated TFEB lies inactive in the cytoplasm, where it is further degraded by the
ubiquitin–proteasome pathway [21]. The phosphorylation of diverse serine residues is
the main post-translational modification (PTM) that maintains TFEB in the cytoplasm; the
main negative regulator of TFEB activity is the mammalian target of Rapamycin (mTorc1),
which phosphorylates TFEB in at least three serines, S122, S142, and S211, although other
kinases have been shown to promote TFEB phosphorylation (Figure 1) [13,22,23]. Glucose
synthase kinase 3 beta (GSK3β) phosphorylates S134 and S138 to promote TFEB lysosomal
surface localization together with small GTPases Rag, while serine-threonine kinase AKT
(also named protein kinase B (PKB) has been shown to drive TFEB phosphorylation at
S467 [24,25]. Although the phosphorylation of TFEB prevents its nuclear import, when the
cellular context requires TFEB nuclear translocation, TFEB is dephosphorylated mainly
by two phosphatases: Calcineurin and Protein Phosphatase 2 (PP2A) (Figure 1). Under
starvation, the calcium stored in lysosomes effluxes, increasing cytosolic calcium concen-
tration, thereby promoting the activation of CaN, a phosphatase activated by Ca2+ , which
removes phosphates from S142 and S211, favoring TFEB nuclear translocation [26]. PP2A
is a phosphatase that is considered the master regulator of the cell cycle, as its dephos-
phorylate proteins are involved throughout cell cycle stages. Nevertheless, it has been
demonstrated that under oxidative stress, PP2A dephosphorylates TFEB at several serine
residues (S109, S114, and S122), including S211, the target serine of mTOR [27,28]. While
starvation induces TFEB activation by inactivating mTOR and activating CaN, it has been
demonstrated that TFEB can be activated independently of mTOR activity; this requires the
removal of inactivating phosphorylated sites by phosphatases such as PP2A, suggesting
that TFEB activity is subjected not only to one exclusive pathway but can be activated by
the cell if a mechanism dependent on TFEB needs to be executed.
 activating CaN, it has been demonstrated that TFEB can be activated independently of mTOR
activity; this requires the removal of inactivating phosphorylated sites by phosphatases such
Cells 2022, 11, 3153 as PP2A, suggesting that TFEB activity is subjected not only to one exclusive pathway but
3 ofcan
20
be activated by the cell if a mechanism dependent on TFEB needs to be executed.

Figure1.1.The
Figure TheTFEB
TFEBactivity
activityisisregulated
regulatedbybypost-translational
post-translationalmodifications.
modifications. TFEB
TFEB isis mainly
mainly phos-
phos-
phorylated
phorylatedtotoavoid
avoidits
itsnuclear
nucleartranslocation;
translocation;however,
however,depending
dependingon
onthe
thecellular
cellularcontext,
context,TFEB
TFEBcan
can
bedephosphorylated,
be dephosphorylated,acetylated,
acetylated,glycosylated,
glycosylated,or
orPARsylated.
PARsylated.

Although
Althoughthe thecytoplasm
cytoplasmretention
retentionof ofTFEB
TFEBisiswell
welldocumented,
documented,and andititoccurs
occursthrough
through
phosphorylation
phosphorylation events,events, once TFEB TFEB hashasbeen
beentranslocated
translocatedtotothe thenucleus
nucleus and
and develops
develops its
its transcriptional activity, it is exported outside by the exportin
transcriptional activity, it is exported outside by the exportin CRM1 (Chromosomal CRM1 (Chromosomal
maintenance
maintenance 1) 1) (Figure
(Figure 1)1) [29].
[29]. The
The first
first serine
serine residue
residue toto be
be identified
identified as as necessary
necessary for for
nuclear export was S142, as it serves as a prime site for subsequent S138
nuclear export was S142, as it serves as a prime site for subsequent S138 phosphorylation phosphorylation by
GSK3β.
by GSK3β.Moreover,
Moreover, further studies
further havehave
studies revealed that residues
revealed S114,S114,
that residues S142, S142,
T331, T331,
and S467
and
are
S467phosphorylated
are phosphorylated by cyclin-dependent
by cyclin-dependent kinase 4/6 (CDK4/CDK6),
kinase 4/6 (CDK4/CDK6), although
although the the
critical
crit-
residue that favors
ical residue nuclear
that favors export
nuclear seems
export to betoS142
seems [30].[30].
be S142 Phosphorylation
Phosphorylation appears to be
appears to
the main PTM that prevents the activation of TFEB; however, Paquette
be the main PTM that prevents the activation of TFEB; however, Paquette et al. showed et al. showed that
once
that TFEB is translocated
once TFEB to nuclei,
is translocated the AMP-activated
to nuclei, proteinprotein
the AMP-activated kinase (AMPK) contributes
kinase (AMPK) con-
to TFEB transcriptional activity as it phosphorylates TFEB at a serine cluster
tributes to TFEB transcriptional activity as it phosphorylates TFEB at a serine cluster (S466, (S466, S467,
and
S467,S469)
and (Figure 1). Although
S469) (Figure 1). Althoughthe AMPKthe AMPKtargettarget
cluster is notisindispensable
cluster not indispensable for nuclear
for nu-
translocation, the phosphorylation
clear translocation, the phosphorylation of S466, of S467,
S466, and
S467,S469
andisS469
necessary to sustain
is necessary TFEB
to sustain
activity, as changes in the serine cluster by alanine prevent the transcription of TFEB
TFEB activity, as changes in the serine cluster by alanine prevent the transcription of TFEB
target genes [31]. While some evidence suggests that the presence of a nuclear AMPK
target genes [31]. While some evidence suggests that the presence of a nuclear AMPK
leads to an increase in nuclear TFEB, further studies need to be undertaken in order to
leads to an increase in nuclear TFEB, further studies need to be undertaken in order to
elucidate whether TFEB is phosphorylated by nuclear or cytoplasmic AMPK [32]. To date,
elucidate whether TFEB is phosphorylated by nuclear or cytoplasmic AMPK [32]. To date,
the phosphorylation status of TFEB seems to be the major mechanism that regulates its
the phosphorylation status of TFEB seems to be the major mechanism that regulates its
transcriptional activity; nevertheless, the activation of TFEB can be orchestrated, according
transcriptional activity; nevertheless, the activation of TFEB can be orchestrated, accord-
to the cellular context, by other PTMs to ensure the proper function of TFEB.
ing to the cellular context, by other PTMs to ensure the proper function of TFEB.
2.2. Acetylation
TFEB acetylation has recently been proposed as a novel PTM that controls TFEB ac-
tivity depending on the acetylated lysines (Figure 1). Acetylation at K116 blocks TFEB
activity, as the substitution of K116Q (acetylation mimic) prevents degradation by the
autophagy–lysosomal pathway of fibrillar amyloid-β (Aβ) by microglia [33]. Although
Cells 2022, 11, 3153 4 of 20

there is no evidence that only the acetylation of K116 influences the subcellular localiza-
tion of TFEB, acetylation at K116, together with K91, K103, and K430, are important for
nuclear translocation and transcriptional activity, as mutations of lysine (K) to arginine (R)
(deacetylated mimic mutation) decrease nuclear abundance, concomitant with autophagy
flux reduction [34]. Other reports suggest that TFEB acetylation by the general control of
amino acid synthesis 5 (CGN5) impairs the dimerization of TFEB and its binding to target
promoter regions when TFEB is acetylated at K116, K274, and K279 [35]. Conversely, it
was observed that the cytosolic deacetylase histone deacetylase 6 (HDAC6) could deacety-
late TFEB and limit its activity by sequestering in the cytosol, suggesting that the TFEB
acetylation pattern affects its transcriptional activation and that there exists a regulatory
relationship between HDAC6 and TFEB [36]. However, whether the acetylation of TFEB
promotes its transcriptional activity or not is still a matter of debate, but it seems to be a
novel mechanism that might influence its activity.

2.3. Other Post-Translational Modifications of TFEB

Beyond phosphorylation and acetylation, TFEB has been shown to be modified by
SUMOylating, PARsylation, glycosylation, and cysteine oxidation. The addition of small
ubiquitin-like modifiers (SUMO) at the lysine present in the consensus sequences ΨKXE
occurs on members of the MiT/TFE family; although TFEB preserves one of the consen-
sus sequences, and SUMOylating arises at K316, we have yet to determine the effect of
SUMOylation on TFEB activity and the cellular context that could mediate the addition of
SUMO on TFEB [37].
Poly-ADP-ribosylation (PARsylation) is a reversible PTM executed by poly (ADP-
ribose) polymerases (PARPs) that mediates the transfer of ADP-ribose from Nicotinamide
adenine dinucleotide (NAD+) to the acceptor proteins [38]. Tankyrase 1 (TNKS1), also
known as (PARP5A), is a PARP that PARsylate TFEB when the WNT/β-catenin signaling
pathway is activated; intriguingly, the PARsylation of TFEB leads to the formation of a
trimeric complex with the coactivator β-catenin and the transcription factors TCF1/LCF1 to
induce the transcription of WNT target genes (Figure 1) [39,40]. To date, TFEB PARsylation
is the only PTM that prevents the canonical transcription of TFEB target genes, suggesting
that TFEB activity is not subjected exclusively to the transcription of autophagy and
lysosomal genes.
TFEB glycosylation has been documented to occur during host infection by
Legionella pneumophila as a mechanism to ensure nutrient availability by activating catabolic
pathways such as autophagy. SetA is a glucosyltransferase from L. pneumophila that directly
glycosylates TFEB at S138 and hampers S138 phosphorylation by GSK3β; therefore, NES
is disrupted, and TFEB is not exported outside the nucleus (Figure 1). Likewise, glycosy-
lation at S195 or S196, T201 or S203, and T208 disrupts the binding of 14-3-3 probably by
masking the phosphorylation site of a target mTOR site, the serine 211 [41]. It is worth
noting that, until now, the glycosylation of TFEB has not been shown to be mediated
by the glucosyl–transferase property of the cell and has been proven to occur only dur-
ing the infection of L. pneumophila; nevertheless, it might be possible that under specific
cell circumstances, TFEB could be a target of glycosylation, which would promote its
nuclear retention.
The oxidation of cysteine (Cys) residues by reactive oxygen species (ROS), specifically
by hydrogen peroxide (H2 O2 ), has been considered a mechanism that controls intracellular
signaling cascades, as Cys oxidation promotes the formation of disulfide bonds with thiol
groups, thereby changing the protein function [42]. Although Cys residues are not very
abundant on proteins, they are present in functional regions. Interestingly, Martina et al. [43]
showed that TFEB contains a Cys residue at position 212 of human TFEB, which is oxidized
and favors the formation of oligomers with other TFEB molecules (Figure 1). The formation
of oligomers prevents the deactivation of TFEB by mTOR, presumably by preventing the
access of the kinase to its target sites [43]. The presence of many post-translational modi-
Cells 2022, 11, 3153 5 of 20

fications over TFEB highlights the importance of the fine regulation of its transcriptional
activity, allowing the cell to execute the mechanisms that define its cellular fate.

3. TFEB on Senescence
Senescence is a form of stable cell cycle arrest that prevents the proliferation of cells
with unresolved or accumulated damage, such as telomere attrition, mitochondrial dysfunc-
tion, DNA damage, perturbed proteostasis, loss of nuclear integrity, oncogene activation,
or autophagy atrophy [44,45]. Although the cell has stopped proliferating, it remains viable
and metabolically active as senescent cells secrete a plethora of cytokines, chemokines,
matrix metalloproteinases (MMPs), and growth factors, collectively known as senescence-
associated secretory phenotype (SASP). It is noteworthy that SASP secretion can induce
senescence in a paracrine fashion in neighboring cells [46,47]. Cellular senescence is distin-
guished by the expression of antiproliferative proteins (p16INK4A and p21CIP1), increased
senescence-associated β-galactosidase activity, the persistent activation of DNA damage
response (expression of histone family member X gamma and γ-H2AX), the stabilization
of lamin A/C, and the decreased expression of lamin B, among other things [46,48,49].
The induction of senescence and the concomitant secretion of SASP can play beneficial
roles when senescence is transient, as it contributes to embryonic development and wound
healing and limits tumor progression; however, the accumulation of senescent cells can
contribute to tissue dysfunction, leading to the development of age-related pathologies
such as arthritis, neurodegeneration, diabetes mellitus, and cancer progression [50–55].
Dysfunctional autophagy has been considered a cellular senescence inducer as dam-
aged proteins or organelles cannot be removed; in line with this, the knockdown of au-
tophagy genes ATG7 and ATG5 in the human primary fibroblast leads to the increased
expression of p16INK4A and p21CIP1 and increases β-galactosidase activity. Interestingly,
the knockdown of ATG genes induces the accumulation of damaged mitochondria and in-
creases ROS production [56]. The induction of senescence after H2 O2 treatment in NIH3T3
cells impairs autophagic flux and decreases lysosomal activity. Nonetheless, the activa-
tion of AMPK, a kinase that activates autophagy, restores autophagy flux and lysosomal
activity and prevents senescence development [57]; however, autophagy activation has
also been shown to promote senescence. As such, the relationship between autophagy
and senescence might need further investigation [58]. TFEB, as the main controller of
autophagy and lysosome biogenesis, has been linked to senescence in both in vivo and
in vitro models of intervertebral disc degeneration, where TFEB decreases its nuclear abun-
dance, leads to lysosomal dysfunction, and decreases autophagy flux. As a consequence,
β-galactosidase activity increases, and the expressions of p16INK4A and markers of SASP,
such as interleukin-6 (IL-6) and matrix metalloproteinase 3 (MMP3), increase as well.
However, when TFEB is overexpressed, senescence is not induced, and autophagy flux is re-
stored (Figure 2) [59]. The accumulation of senescent cells in aged organisms is considered
one of many contributors to aging [60]. In the brains of mice at 18 months, specifically in the
hippocampus and cortex, Wang et al. [61] showed the decreased expression of nuclear TFEB
compared with mice aged 6 months, as well as the increased expression of p16INK4A and
γ-H2AX; however, they also found that the overexpression of TFEB ameliorated senescence
markers and improved learning and memory skills in mice of the same age [61]. Similar
findings were made by Gorostieta et al. [62] in young and old mice hippocampus and
cortex; notably, they found that the decreased nuclear TFEB was due to the increased
expression of CRM1, which exports TFEB from the nucleus, and this was correlated with
the expression of senescence markers in old mice. Interestingly, when they inhibited CRM1
in vitro with leptomycin, nuclear TFEB was augmented, and β-galactosidase activity was
reduced; nevertheless, no changes were observed in lamin A/C and lamin B expression
after TFEB nuclear restoration [62]. In a cellular model of chronic obstructive pulmonary
disease (COPD) emphysema, β-galactosidase activity dropped when cells were treated
with gemfibrozil to stimulate TFEB activity, suggesting that TFEB activation could prevent
senescence induction. Nevertheless, additional senescence markers need to be outlined in
 inhibited CRM1 in vitro with leptomycin, nuclear TFEB was augmented, and β-galacto-
sidase activity was reduced; nevertheless, no changes were observed in lamin A/C and
lamin B expression after TFEB nuclear restoration [62]. In a cellular model of chronic ob-
structive pulmonary disease (COPD) emphysema, β-galactosidase activity dropped when
Cells 2022, 11, 3153 cells were treated with gemfibrozil to stimulate TFEB activity, suggesting that TFEB 6acti- of 20
vation could prevent senescence induction. Nevertheless, additional senescence markers
need to be outlined in order to propose senescence evasion by the activation of TFEB [63].
Although autophagy
order to propose induction
senescence after TFEB
evasion by theoverexpression
activation of TFEBor activation has been
[63]. Although shown
autophagy
as the main mechanism of evading senescence, we cannot discard additional
induction after TFEB overexpression or activation has been shown as the main mechanism operating
mechanisms that TFEB we
of evading senescence, cancannot
orchestrate independently
discard of autophagy
additional operating to delaythat
mechanisms senescence.
TFEB can
As we will discuss
orchestrate in the following
independently section,
of autophagy to TFEB
delay induces
senescence. the transcription of genes
As we will discuss in re-
the
lated to DNA
following repair;
section, as the
TFEB induction
induces the of senescence of
transcription is the
genesresult of unresolved
related DNA dam-
to DNA repair; as the
age, overexpressing
induction TFEBismight
of senescence contribute
the result to a betterDNA
of unresolved cell response
damage,after a cell stress insult
overexpressing TFEB
and therefore contribute to the evasion of senescence.
might contribute to a better cell response after a cell stress insult and therefore contribute
to the evasion of senescence.

Figure 2. The TFEB activation can influence the induction of senescence. Decreased nuclear TFEB
Figure 2. The TFEB activation can influence the induction of senescence. Decreased nuclear TFEB
localization may be one of many causes of senescence; however, restoring TFEB nuclear activity could
localization may be one of many causes of senescence; however, restoring TFEB nuclear activity
contribute
could to avoiding
contribute cellular
to avoiding senescence,
cellular perhaps
senescence, through
perhaps the induction
through of autophagy
the induction or DNA
of autophagy or
repair genes.
DNA repair genes.
4. TFEB on DNA Damage Repair and Cell Cycle
4. TFEB on DNA Damage Repair and Cell Cycle
DNA integrity is essential to maintaining cellular and organismal health; therefore,
DNA integrity is essential to maintaining cellular and organismal health; therefore,
mechanisms that ensure the proper repair and elimination of DNA damage under de-
mechanisms that ensure the
termined circumstances needproper
to berepair and elimination
provided. The DNA of DNA damage
damage response under
(DDR) deter-
is a
mined
well-described pathway that senses and responds to different types of DNA damagewell-
circumstances need to be provided. The DNA damage response (DDR) is a [64].
described pathway that
p53, considered thesenses
“Guardianand responds to different
of the Genome”, acts astypes of DNA damage
an integrator of many [64].
programs
p53, considered the “Guardian of the Genome”, acts as an integrator
that define the fate of the cell according to the nature, intensity, and duration of many pro-
of stress,
grams that define the fate of the cell according to the nature, intensity, and
leading to cell cycle arrest or the activation of cell death programs [65]. For example, when duration of
stress,
DNA leading
damagetooccurs
cell cycle arrest
during theorG1
thephase,
activation of stabilized,
p53 is cell death programs
leading to[65].
the For example,
transcription
when DNA damage occurs during the G1 phase, p53 is stabilized, leading
of P21, an inhibitor of CDK4,6/cyclin-D, CDK2/cyclin-E, and CDK2/cyclin-A activity, to the transcrip-
tion of P21,
thereby an inhibitor
arresting of CDK4,6/cyclin-D,
the cell CDK2/cyclin-E,
cycle [66,67]. Interestingly, TFEBandhas CDK2/cyclin-A activity,
been recently proposed
as a transcriptional amplifier in response to DNA damage through the activation and
stabilization of p53 [68]. Brady et al. [68] showed that the induction of DNA damage
with etoposide increased the nuclear translocation of TFEB, causing mTOR activity to
decrease; interestingly, TFEB activation is p53-dependent, as the p53 knockout of mouse
embryonic fibroblasts (MEFs) failed to activate TFEB. Intriguingly, the knockout of TFEB
downregulates the expression of DDR genes after exposure to etoposides, including the p53
transcript. Moreover, p53 is less stable when TFEB is not expressed, suggesting that p53
Cells 2022, 11, 3153 7 of 20

and TFEB might regulate each other in response to DNA damage. The reduced levels of p53
might be due to the increased levels of MDM2, an E3 ubiquitin ligase that renders p53 for
proteasome degradation; nevertheless, the increased levels of MDM2 seem not to be TFEB-
dependent, and importantly, overexpressing the active TFEB increases the stability of p53 as
its half-life is extended. Brady et al. [68] also proposed TFEB as a mediator of cell fate after
DNA damage, as its expression is necessary for apoptosis cell death. Similar findings were
made by Slade et al. [69] in relation to breast cancer cells, where TFEB is activated and favors
the transcription of DNA repair genes, specifically the homologous recombination genes,
thereby increasing the DNA damage repair capacity of breast cancer cells. Remarkably, the
activity of the phosphatase calcineurin is necessary for TFEB activation, but the induction
of TFEB activity results in the evasion of apoptosis cell death [69].
p21, a target of p53, can be regulated positively by TFEB [70]. Analyses of the promoter
of p21 found one motif region recognized by TFEB. Moreover, the chromatin immunopre-
cipitation of TFEB on HeLa cells stably expressing active TFEB demonstrates the binding of
TFEB to the promoter region of p21. Interestingly, the knockout or knockdown of TFEB
suggests p21CIP1 downregulation, indicating a TFEB requirement; nonetheless, the tran-
scription of p21 after DNA damage is not exclusively due to TFEB, as its expression still
requires p53 [70]. Contrary to treatment with etoposide, where mTOR activity was reduced,
and therefore TFEB was translocated to the nucleus, doxorubicin treatment did not lead to
mTOR inhibition, suggesting that TFEB activation is independent of mTOR [70]. Notably,
the depletion of TFEB and treatment of cells with doxorubicin increase the number of
cells at the G2-phase of the cell cycle. Nonetheless, the overexpression of TFEB and the
concomitant increase in p21 CIP1 lead to an increased population of cells at the G1-phase,
suggesting that, in the absence of TFEB and p21 after DNA damage, cells are arrested at the
G2-phase [70]. The work of Brady and colleagues also shows the relevance of TFEB in the
expression of cell cycle genes, specifically the genes involved in cell cycle checkpoints [68];
interestingly, further reports made by Doronzo et al. [71] show that, in endothelial cells
(ECs), TFEB drives the expression of genes related to cell cycle, cell division, G1/S transi-
tion, and DNA replication, among other genes; notably, when TFEB is silenced, ECs halts
proliferation at the G1-S cycle transition; interestingly, one of the direct targets of TFEB is
the cyclin-dependent kinase 4 (CDK4), important for G1-S transition of the cell cycle [71].
Similar results were further reported in TFEB-depleted hepatoblasts, in which the authors
showed a block of the G1-S cycle transition [72]. Together, the recent evidence shows the
important role of TFEB in cell cycle control and progression.

5. TFEB on WNT Signaling

WNT/β-catenin signaling is an evolutionarily conserved pathway that controls cell
proliferation, differentiation, and cell fate determination during embryogenesis and adult-
hood tissue homeostasis [73]. The control of WNT pathway activity is well documented,
and two defined mechanisms control its activity: the WNT canonical (β-catenin-dependent)
pathway and the noncanonical pathway (β-catenin-independent activity) [74]. The canon-
ical pathway is initiated by the binding of WNT ligands to Frizzled receptors present
on the cell surface. Then, β-catenin disassembles from the destruction complex contain-
ing Axin, adenomatous polyposis coli (APC), and GSK3β and accumulates; afterward,
β-catenin is translocated to the nucleus and, as a transcriptional coactivator, it binds to
and modulates the activity of the T-cell factor/lymphoid enhancer factor (TCF/LEF) tran-
scription factors [73–75]. Interestingly, the activation of the WNT/β-catenin pathway with
Wnt3a-conditioned media induces the nuclear translocation of TFEB independently of
the phosphorylation status of TFEB, and surprisingly, nuclear TFEB after WNT/β-catenin
stimulation does not transcribe lysosomal genes but promotes the transcription of WNT
target genes [40]. Kim et al. [40] found the first evidence of the importance of TFEB PAR-
sylation to the formation of a complex between TFEB and transcription factors of WNT
signaling as a mechanism to promote the expression of WNT genes. That said, previous
reports made by Li et al. [76] had found similar results regarding the relevant role of TFEB
Cells 2022, 11, 3153 8 of 20

in activating TCF/LEF1 on gastric cancer cells to enhance migration and invasiveness,

suggesting a positive loop within the TFEB and WNT/β-catenin signaling pathway, as
WNT inhibitors decrease the protein stability of TFEB [76]. WNT signaling has been found
to be dysregulated in many types of cancer; for example, mutations on regulatory proteins
such as APC prevent the ubiquitination and subsequent degradation of β-catenin, leading
to the sustained transcription of WNT target genes in colorectal cancer cells. Interestingly,
previous reports made by Liang et al. [77] gave evidence on the role of TFEB in cancer pro-
gression, as TFEB promotes cell proliferation and migration; however, autophagy induction
as a consequence of TFEB activation was suggested to be the main mechanism for tumor
growth and metastasis [75,77]. In this context, recent studies indicate the involvement of
TFEB in the control of cell proliferation and cell motility in endometrial, lung, pancreatic,
prostate, and endothelial cancer cells [78–82]. The recent findings open the possibility of
further research into how TFEB can contribute to the activity and regulation of additional
pathways not related to autophagy.

6. Endoplasmic Reticulum Stress and TFEB

The endoplasmic reticulum (ER) is a metabolic organelle responsible for the synthesis
of proteins, lipids, carbohydrates, and calcium storage. The ER is highly sensitive to
cellular changes, and its homeostasis may be disrupted by several insults, including an
imbalance between the rate of protein synthesis and folding capacity, dysregulation in
calcium homeostasis, oxidative stress, and redox imbalance in the ER lumen. Perturbations
in the functions of the ER provoke the accumulation of unfolded proteins and trigger ER
stress, which in several contexts might lead to apoptosis when the response is sustained for
a long time [83,84]. ER stress activates an adaptive signaling pathway called the unfolded
protein response (UPR), which transduces signals related to the protein folding status of the
ER lumen to the nucleus in order to trigger the expression of specific transcription factors
to cope with stress conditions. Thus, UPR plays an important role in the pathogenesis
of various metabolic diseases, such as diabetes, obesity, cancer, infectious diseases, and
inflammatory diseases, among others [85–87]. Therefore, UPR components offer interesting
targets for therapeutic intervention that may reduce stress levels and offer therapeutic
benefits that improve human health [88].
UPR signaling is initiated by the activation of three distinct types of stress sensors
located at the ER membrane, including the protein kinase RNA-like endoplasmic reticulum
kinase (PERK), the inositol-requiring enzyme 1 (IRE1), and the activating transcription
factor 6 (ATF6) [89,90]. Briefly, the three UPR transmembrane sensors are normally bound
to the ER-resident chaperone GRP78/BiP (glucose-regulated protein, 78kDa/Binding im-
munoglobulin Protein) to form a stable complex with the luminal domain, keeping them
in an inactive monomeric state. However, under conditions of ER stress, GRP78/BiP is
released from the UPR sensors and binds to the accumulated unfolded proteins, thus acti-
vating the signaling pathways mediated by PERK, IRE1α, and ATF6 [89]. After GRP78/BiP
is dissociated from PERK, it becomes activated and oligomerized, phosphorylates itself,
and in turn, phosphorylates the α-subunit of eukaryotic initiation factor 2α (eIF2α) at serine
51, and then halts translation by preventing ribosome assembly. This reduces the overload
of proteins entering the ER of a stressed cell, which leads to attenuation in protein synthesis.
In summary, this pathway inhibits mRNA translation, reduces the flux of proteins in ER,
and ameliorates ER stress [91]. On the other hand, the low expression of eIF2α activates
the transcription factor ATF4, which induces cell survival by increasing the expression
of genes related to oxidative stress and amino acid synthesis [92]. However, if ER stress
persists, ATF4 induces the transcription of a pro-apoptotic transcription factor CHOP
(C/EBP homologous protein), which may trigger apoptosis. In addition, CHOP promotes
the expression of GADD34 (growth-arrest and DNA damage-inducible 34), which dephos-
phorylates eIF2α, reinitiating translation and sensitizing cells to apoptotic signals [83,93].
IRE1α is a bi-functional transmembrane kinase/endoribonuclease that, in response to UPR,
is activated, dimerized, trans-autophosphorylated, and undergoes a conformational change
Cells 2022, 11, 3153 9 of 20

that activates its RNase domain. This, in turn, cleaves the intron of mRNA to XBP1 (X-box
binding protein) in sXBP1, an active transcription factor that promotes the expression of
genes associated with the UPR. XBP1s regulate the expression of genes encoding factors
that modulate protein folding, secretion, ER-associated degradation (ERAD), and protein
translocation into the ER [94,95]. ATF6 is also an ER transmembrane protein that contains a
bZIP transcription factor on its cytosolic domain. Upon the excessive loading of misfolded
proteins, ATF6 is sent to the Golgi apparatus for further processing and activation, where
the proteases S1P and S2P release the N-terminal cytosolic portion that is translocated
further to the nucleus, where ATF6 binds to ER stress response elements and stimulates
the transcription of a subset of UPR target genes, such as DNA damage-inducible tran-
script 3 (DDIT3), chaperones BiP, and GRP94 (glucose-regulated protein 94) [93,96]. ATF6
exhibits cross talk with XBP1s by forming heterodimers, which may drive specific gene
expression programs to reach ER proteostasis [97]. As previously mentioned, the cellular
localization of TFEB is dependent on the cellular context; once in the nucleus, TFEB trig-
gers changes in gene expression to cope with cellular stress. Initially, Martina et al. [98]
showed that TFEB participates in UPR signaling since the induction of ER stress with
tunicamycin or brefeldin A promotes the nuclear localization of TFEB in a PERK- and
calcineurin-dependent fashion. Interestingly, the activation of TFEB induces not only the
transcription of lysosomal- and autophagy-related genes but also those related to ER home-
ostasis and apoptosis, such as the transcription factor 4 (ATF4) and its targets genes (CHOP
Cells 2022, 11, 3153 and GADD34), which are key deciding factors that integrate the UPR and integrated10stressof 20
response. Thus, under ER stress conditions, the TFEB-mediated activation of ATF4 seems
to occur via its direct binding to the CLEAR element in the promoter region of ATF4 in
order to restore ER homeostasis and support cell survival (Figure 3) [98].

Figure 3. The TFEB regulates the expression of several regulators of the ER stress response. ER stress,
Figure 3. The TFEB regulates the expression of several regulators of the ER stress response. ER
triggered by several compounds, promotes the nuclear localization of TFEB in a PERK-, ATF6-, IRE1α-,
stress, triggered by several compounds, promotes the nuclear localization of TFEB in a PERK-,
and calcineurin-dependent manner that consequently induces the transcription of UPR-related genes to
ATF6-, IRE1α-,
restore ER and calcineurin-dependent
homeostasis manner
and support cell survival that consequently
or apoptosis, if the stress induces the transcription of
is persistent.
UPR-related genes to restore ER homeostasis and support cell survival or apoptosis, if the stress
is persistent.
It has been shown that carbon monoxide (CO) may protect cells against ER stress via
the activation of the PERK-dependent pathway [99]. The treatment of hepatic or HeLa
cells with CO is able to promote TFEB nuclear translocation. In fact, a mild increase in
mitochondrial ROS levels in response to CO treatment leads to PERK activation, Ca2+ re-
lease, and calcineurin-dependent TFEB nuclear translocation, which, in turn, promotes
mitochondrial homeostasis through mitophagy and mitochondrial biogenesis (Figure 3)
[100]. Similar findings emerged with the PERK activator (SB202190) in neuroblastoma
2+
Cells 2022, 11, 3153 10 of 20

It has been shown that carbon monoxide (CO) may protect cells against ER stress via
the activation of the PERK-dependent pathway [99]. The treatment of hepatic or HeLa cells
with CO is able to promote TFEB nuclear translocation. In fact, a mild increase in mitochon-
drial ROS levels in response to CO treatment leads to PERK activation, Ca2+ release, and
calcineurin-dependent TFEB nuclear translocation, which, in turn, promotes mitochondrial
homeostasis through mitophagy and mitochondrial biogenesis (Figure 3) [100]. Similar
findings emerged with the PERK activator (SB202190) in neuroblastoma cells, where ac-
tivated PERK led to an increase in cytosolic Ca2+ levels that subsequently promoted the
translocation of TEFB into the nucleus via the calcineurin-dependent dephosphorylation of
TFEB. These findings suggest that the activation of PERK–TFEB signaling could decrease
amyloidogenesis in neurodegenerative diseases [101]. It is well documented that UPR
increases ER cross talk with autophagy in each of the three canonical branches of the UPR,
and it activates autophagy, which permits the restoration of ER homeostasis. Recent work
by Zhang et al. [102] showed that the promoter of Tfeb possesses a consensus sequence
recognized by the transcription factor sXBP1. Interestingly, a ChIP assay showed the enrich-
ment of sXBP1 on the promoter of Tfeb in primary hepatocytes treated with thapsigargin
and in the livers of fasted mice. Moreover, the overexpression of sXBP1 increases the
nuclear localization of TFEB and induces autophagy flux; together, the data demonstrated
that the ER and autophagy are functionally coupled at the transcriptional level via the
XBP1-mediated activation of TFEB (Figure 3) [102]. Similar findings were made when
several cell line cultures or rodent models were treated with distinct compounds such as
Kazinol C, Isobacachalcone, DFS, or Guanabenz, respectively; these were able to activate
ER stress-associated UPR components such as PERK, ATF6, IRE1α phosphorylation, BiP,
CHOP, ATG7, GADD34, and sXBP1, as well as the nuclear translocation of TFEB and
autophagy. However, the underlying molecular details remain unknown [103–106].
The transforming growth factor-beta 1 (TGF-β1) significantly promotes protein syn-
thesis, causing an excessive demand for the protein folding capacity of ER, resulting in ER
stress and the concomitant activation of three branches (PERK, IRE1α, and ATF6) of UPR.
Similar to other reports, here, the activation of UPR led to dependent TFEB autophagy in-
duction. However, TFEB activation induces fibroblast differentiation and collagen secretion,
processes that are linked to the formation of hypertrophic skin scars [107]. Similar findings
have been reported in the osteoblast since TFEB may regulate its differentiation through
the ATF4/CHOP signaling pathway in response to ER stress [108]. Together, the reports
suggest that TFEB might ameliorate ER stress but can also influence cell differentiation
together with the UPR signaling pathway.

7. TFEB and Its Influence on the Metabolism

In general, all living things are exposed to constant changes in nutrient supply in
their life activities; therefore, to reach metabolic homeostasis, they must undergo the con-
stant adjustment of internal metabolic activities in response to nutritional states. Energy
metabolism is regulated through several aspects of cellular machinery that catalyze chem-
ical reactions in order to generate energy as ATP that is consumed by anabolic reactions
and supplied to organelles; this involves the conversion of simple chemical compounds
into complex ones that are essential for cell growth. Some components involved in this
regulation include enzymes and transcriptional factors that play an important role in this
process. Relatively recent works suggest that TFEB could influence mammalian metabolism
to maintain energy balance.

7.1. Role in Glucose Metabolism

Glucose homeostasis is critical to human health due to its central importance as a
source of energy. For utilization, glucose is transported by members of the facilitative
glucose transporter (SLC2) and by the sodium–glucose cotransporter (SGLT) family [109].
Glucose is metabolized via glycolysis and provides the energy molecule ATP and several
metabolites for other metabolic pathways.
Cells 2022, 11, 3153 11 of 20

It has been reported that in the skeletal muscles of mice overexpressing TFEB might
control several genes related to glucose metabolism. Specifically, TFEB overexpression is
correlated with an increase in the expression of two genes for glucose transport (GLUT1
and GLUT4), genes for glucose phosphorylation (hexokinase 1 and 2), and an increase in
glucose uptake. Interestingly, these genes contain a CLEAR sequence, suggesting that their
regulation is TFEB-dependent and highlighting the importance of TFEB in the regulation of
glucose homeostasis in skeletal muscle [110]. Consistent with these findings, the treatment
of tumor-associated macrophages (TAMs) with chloroquine, an antimalarial drug, promotes
lysosomal calcium release that contributes to TFEB activation and increases the presence of
genes involved in glycolysis, such as GLUT1 and GLUT4, phosphofructokinase (PFKM),
and pyruvate kinase (PKLR); these effects are TFEB-dependent since the regulation was
blocked after TFEB knockdown. Collectively, these data suggest that TFEB reprograms the
macrophage metabolism, from oxidative phosphorylation to glycolysis [111].
Strikingly, and contrary to the above-described findings, the genetic loss of TFEB
(TFEB−/− ) in murine cardiomyocytes led to an enrichment of genes related to glycolysis
and gluconeogenesis, such as phosphofructokinase (Pfkp), glycerol-3-phosphate dehy-
drogenase (Gpd1), glucose 6-phosphate translocase (slc37a4), hexokinase 1 (hk1), and
phosphoglucomutase (Pgm5), suggesting that glucose oxidation processes are dampened
in TFEB−/− cardiomyocytes [4]. In general, cancer cells require a key regulator to induce
metabolic reprogramming, which sustains their cell growth. An analysis of the role of
TFEB in metabolic processes in lymphoma cells found that its activation significantly re-
duces basal levels of glycolysis, glucose uptake, and some of its encoding genes, such as
lactate dehydrogenase (Ldha) and phosphoglycerate kinase (Pgk), as well as the metabolite
2-phosphoglycerate (Figure 4). Conversely, there was an accumulation of glyceralde-
hyde 3-phosphate and glucose-6-phosphate that was consistent with reduced glycolytic
flux [112]. These findings suggest that the effect of TFEB on the regulation of glucose
metabolism might be tissue-dependent.
Insulin-activated Akt signaling in vascular endothelial cells (ECs) is involved in the
regulation of systemic glucose metabolism and vascular homeostasis [113]. In ECs, insulin
signaling starts with the binding of the hormone to the insulin receptor (IR) and the activa-
tion of insulin receptor substrate (IRS1, 2) by phosphorylation, which follows the activation
of the PI3-kinase/Akt/eNOS pathway, where protein kinase B (AKT) phosphorylates
the endothelial nitric oxide synthase (eNOS) at serine 1177, resulting in increased nitric
oxide production and vasodilation [114]. In recent work, Sun et al. [115] studied the role
of endothelial TFEB in glucose metabolism. The results showed that endothelial TFEB
improves systemic glucose tolerance in mice via the activation of Akt-IRS1, 2. In fact, the
overexpression of endothelial TFEB is related to upregulation at the transcriptional level of
IRS2, which consequently augments the amount of IRS protein via the downregulation of
several microRNAs (mirR-335-3p, miR-495-3p, and miR-548o-3p), leading to the activation
of Akt signaling and glucose uptake through GLUT1 in ECs (Figure 4) [115]. Whether this
mechanism of TFEB occurs in other cell types warrants future investigation. Interestingly,
it has also been shown that the overexpression of TFEB reverses the suppressed glucose
uptake of palmitate-induced insulin resistance in C2C12 myotubes via the restoration
of the insulin-dependent GLUT4 signaling pathway and the activation of AMPK, a key
energy sensor, which regulates glucose metabolism and has been shown to protect against
insulin resistance. Therefore, it is proposed that TFEB acts as a critical regulator of glucose
homeostasis in skeletal muscle cells [116].
Cells 2022, 11, 3153 12 of 20

Cells 2022, 11, 3153 flux [112]. These findings suggest that the effect of TFEB on the regulation of glucose me-
12 of 20
tabolism might be tissue-dependent.

Figure 4.
Figure 4. The
The TFEB
TFEB regulates
regulates genes
genes involved
involved in
in carbohydrate
carbohydrate metabolism,
metabolism, lipid
lipid metabolism,
metabolism, and
and
insulin signaling. Once active, TFEB may promote energy utilization through the activation of
insulin signaling. Once active, TFEB may promote energy utilization through the activation of several sev-
eral related
related genes.
genes. SomeSome
of theofproposed
the proposed metabolic
metabolic processes
processes regulated
regulated by TFEB
by TFEB are shown,
are shown, together
together with
with the relevant substrates.
the relevant substrates.

Insulin-activated
7.2. Role Akt signaling in vascular endothelial cells (ECs) is involved in the reg-
in Lipid Metabolism
ulation of systemic glucose
Fatty acids are molecules metabolism
that canandbe vascular
obtained homeostasis [113]. intake
from dietary In ECs, andinsulin
designal-
novo
ing starts with the binding of the hormone to the insulin receptor
synthesis. Their catabolism generates small molecules that modulate several cell signal- (IR) and the activation of
insulin receptor substrate (IRS1, 2) by phosphorylation, which follows
ing pathways and play a critical role in transcriptional regulation [117]. Initially, Set- the activation of the
PI3-kinase/Akt/eNOS
tembre et al. [118] showedpathway,thatwhere
inducingprotein kinase B or
starvation (AKT) phosphorylates
overexpressing TFEB theinendothelial
the liver
nitric oxide synthase (eNOS) at serine 1177, resulting in increased
upregulates genes that encode for lipid metabolism, such as the transport of fatty acid nitric oxide production and
vasodilation [114]. In recent work, Sun et al. [115] studied the role
chains across the plasma membrane (Cd36 and Fabps) for the oxidation of free fatty acids of endothelial TFEB in glu-
cose metabolism.
(FFA) in mitochondriaThe results
(Cpt1, showed that endothelial
Crat, Acadl, Acads, and TFEB improves
Hdad) and systemic
peroxisomes glucose toler-
(Cyp4a)
ance in mice via the activation of Akt-IRS1, 2. In fact, the overexpression
(Figure 4). Similarly, TFEB overexpression in inguinal white adipose tissue (iWAT) leads to of endothelial TFEB
aisclear
related to upregulation
induction of adipose at tissue
the transcriptional
browning-related level of IRS2,including
genes, which consequently
uncouplingaugments
protein 1
the amount
(Ucp1), which of encodes
IRS protein viathermogenic
a key the downregulation
protein,of andseveral microRNAs
mitochondrial and (mirR-335-3p,
lipid metabolism miR-
495-3p,such
genes, and asmiR-548o-3p), leading DFFA-like
cell death-inducing to the activation
effectorofa (CIDEA),
Akt signaling and glucose
aconitase 2 (ACO2),uptake
car-
through GLUT1 in ECs (Figure 4) [115]. Whether this mechanism
nitine palmitoyltransferase 1B (CPT1B), cytochrome c oxidase subunit 5B (COX5B), and of TFEB occurs in other cell
types warrants future investigation. Interestingly, it has also been shown
citrate synthase (CS). It is noteworthy that most of the effects of TFEB on lipid metabolism that the overexpres-
sion oftoTFEB
seem reverses the
be mediated by suppressed glucose uptake
the direct regulation exertedof palmitate-induced insulin resistance
by TFEB on the regulators of fat
in C2C12 myotubes
metabolism, such asvia thethe restorationproliferator-activated
peroxisome of the insulin-dependent GLUT4 signaling
receptor-gamma pathway
coactivator
(PGC1α) and peroxisome proliferator-activated receptor (PPARα) because, without PGC-α,
the abilities of TFEB overexpression to drive TFEB-induced browning and to elicit beneficial
metabolic effects were blunted [118,119]. Additionally, TFEB overexpression can rescue
Cells 2022, 11, 3153 13 of 20

the obese phenotype and the glucose intolerance in knockout mice of the transcription
factor E3 (TFE3), another member of the MiT family helix–loop–helix leucine zipper, sug-
gesting that these transcription factors work together and cooperatively control the energy
metabolism [120]. These effects appear to be evolutionarily conserved since HLH-30, the
Caenorhabditis elegans homolog to TFEB, has been shown to activate the transcription
of lipid catabolism genes and promote fat utilization upon food withdrawal [121,122].
Conversely, the genetic loss of TFEB−/− in cardiomyocytes has different effects on lipid-
related genes; specifically, the genes required for processing lipid intermediates (cholesterol,
phospholipid, and sphingolipid) such as 2,4-dienoyl-CoA reductase 2 (Decr2), acyl-CoA
synthetase family member 2 (Acsf2), ceramide-1-phosphate transfer protein (Cptp), lipase a
(Lipa), StAR-related lipid transfer domain containing 3 (Stard3), sphingosine-1-phosphate
lyase-1 (Sgpl1), and apolipoprotein E (ApoE) were upregulated, whereas those for lipid
transport and metabolism genes, such as the cluster of differentiation 36 (CD36) and
angiopoietin Like 3 (Angptl3), were downregulated [4].
It has been shown that TFEB can be dysregulated in lipid-related diseases. In nonalco-
holic fatty liver disease (NAFLD) patients, hepatic steatosis alters the subcellular location
of TFEB, being mainly cytosolic; as TFEB promotes genes related to lipid metabolism, TFEB
activation could be an important factor in determining the severity of hepatic steatosis in
NAFLD patients and might conceivably be related to reduced lipophagy activity [123]. Ad-
ditionally, TFEB has been identified as an inductor of hepatic bile acid synthesis; in fact, bile
acid synthesis is a major mechanism for preventing intrahepatic cholesterol accumulation
and hypercholesterolemia. Wang et al. [124] found that excessive intracellular cholesterol
accumulation causes lysosomal stress and subsequent TFEB nuclear translocation. TFEB
activation induces cholesterol 7α-hydroxylase (CYP7A1) to promote bile acid synthesis,
which promotes cholesterol catabolism and elimination (Figure 5). In addition, bile acids
activate FXR to induce intestinal FGF15/19 and, thus, feedback, and they inhibit TFEB by
causing TFEB phosphorylation and cytosolic retention (Figure 5) [124]. Furthermore, recent
work shows that genetic loss of TFEB causes the reduction in the total cellular cholesterol in
ECs, suggesting that TFEB is involved in cholesterol synthesis via the transcriptional mod-
ulation of genes encoding the sterol regulatory element-binding protein (SREBP-2), a key
regulator of cholesterol, the SREBP cleavage-activating protein sterol sensor (SCAP), and
the SREBP-2 target gene HMGCR (β-Hydroxy β-methylglutaryl-CoA reductase). Therefore,
TFEB promotes the transcription of genes that drive the synthesis of cholesterol [82].
Generally, it is accepted that autophagy is activated under nutrient deprivation condi-
tions and repressed in feeding conditions. Intriguingly, recent data show that after feeding,
autophagy/lipophagy is activated in the intestine via the orphan nuclear receptor, the
small heterodimer partner (SHP/NR0B2), the gut hormone, fibroblast growth factor-15/19
(FGF15/19), and the TFEB axis (Figure 5). After feeding, FGF15/19 is activated, and in turn,
the nuclear localization of SHP and TFEB is increased via PKC-mediated phosphorylation,
which consequently activates the transcription of genes (Ulk1 and Atgl) that are essential
for lipophagy. This activation may reduce postprandial lipids via lipophagy and may
provide novel targets for treating obesity-related metabolic disorders [125]. Intriguingly,
TFEB expression was increased in macrophages of white adipose tissue (WAT) from obese
mice and humans, whereas macrophage TFEB overexpression protects against obesity and
insulin resistance. The livers of mice with HFD-induced weight gain and lipid accumula-
tion were markedly reduced, probably because of the increased expressions of the fatty acid
oxidation-related gene and a decrease in lipogenesis-related gene expression. Moreover,
macrophage TFEB protected against HFD-induced obesity by inducing growth differen-
tiation factor 15 (GDF15), suggesting that the activation of the TFEB–GDF15 axis could
play a crucial role in regulating obesity-induced metabolic diseases (Figure 5) [126]. The
estrogen-related receptor α (ERRα) drives lipid metabolism, together with PGC1α/PPAR;
nevertheless, recent evidence shows that ERRα is a direct target of TFEB, and both are
overexpressed in patients with endometrial cancer [41]. Intriguingly, TFEB and ERRα favor
the invasion and metastasis of endometrial cancer cells by modulating lipid metabolism
 against HFD-induced obesity by inducing growth differentiation factor 15 (GDF15), suggest-
ing that the activation of the TFEB–GDF15 axis could play a crucial role in regulating obesity-
induced metabolic diseases (Figure 5) [126]. The estrogen-related receptor α (ERRα) drives
lipid metabolism, together with PGC1α/PPAR; nevertheless, recent evidence shows that
Cells 2022, 11, 3153 ERRα is a direct target of TFEB, and both are overexpressed in patients with endometrial 14
can-
of 20
cer [41]. Intriguingly, TFEB and ERRα favor the invasion and metastasis of endometrial cancer
cells by modulating lipid metabolism and increasing unsaturated fatty acid phosphatidylcho-
line, phosphatidylglycerol, and the ratio phosphatidylcholine/sphingomyelin, which together
and increasing unsaturated fatty acid phosphatidylcholine, phosphatidylglycerol, and the
enhance membrane fluidity via epithelial–mesenchymal transformation signaling and pro-
ratio phosphatidylcholine/sphingomyelin, which together enhance membrane fluidity via
mote EC progression [78].
epithelial–mesenchymal transformation signaling and promote EC progression [78].

Figure
Figure 5. 5.
TheThe TFEB
TFEB is isexpressed
expressedininseveral
severaltissues,
tissues,including
including the
the liver,
liver, intestine,
intestine, and
andwhite
whiteadipose
adiposetis-
sue. In
tissue. In these
thesecell
celltypes,
types,ininresponse
responsetotoseveral
several stimuli, TFEB
stimuli, TFEBis activated
is activated andand
induces the the
induces expression
expres- of
several
sion genes,genes,
of several as indicated in the figure.
as indicated In the liver,
in the figure. In thecholesterol accumulation
liver, cholesterol causes lysosomal
accumulation stress,
causes lyso-
somal
whichstress, which consequently
consequently induces TFEBinduces TFEB
nuclear nuclear translocation,
translocation, activating activating the expression
the expression of
of cholesterol
cholesterol 7α-hydroxylase
7α-hydroxylase (CYP7A1)(CYP7A1)
to producetohepatic
produce bilehepatic bile acid
acid synthesis synthesis
that, that, in cells,
in the intestine the intestine
promotes
the production of the hormone fibroblast growth factor-15/19 (FGF15/19), and this consequently
activates both the activate orphan nuclear receptor, the small heterodimer partner (SHP/NR0B2) and
TFEB, to activates genes (Ulk1 and Atgl) essential for lipophagy. TFEB activation by overexpression or
the occurrence of lysosomal stress in fat-resident macrophages protects against diet-induced weight
gain and adiposity through the induction of growth differentiation factor 15 (GDF15)-enhanced
adipose lipid catabolism, and it reduces inflammation.

8. Conclusions and Future Perspectives

Accumulated evidence has underscored the role of TFEB as a master regulator of
lysosome biogenesis and autophagy. TFEB activation positively regulates the expression of
autophagy and lysosomal biogenesis-related genes that promote the clearance of intracellu-
lar substrates through lysosomal exocytosis. However, increasing evidence suggests the
crucial role of the TFEB protein in the control of several other vital cellular processes, such
as cellular senescence, DNA repair, ER stress, carbohydrates metabolism, lipid metabolism,
and WNT signaling-related processes that position TFEB as a key regulator responding to a
variety of environmental cues. The newly identified noncanonical roles of TFEB have re-
vealed how it is able to integrate multiple upstream stimuli to mediate specific downstream
responses that conceivably could have medical relevance. Therefore, pharmacological
strategies for TFEB activation or mitigation might be a promising approach in specific
disease conditions, as is the case of metabolic or neurodegenerative diseases, and may hold
relevance for a larger number of human diseases.
Cells 2022, 11, 3153 15 of 20

Author Contributions: Conceptualization, B.F.-J. and D.O.-C.; methodology, software, validation,

formal analysis, investigation, writing—original draft preparation, writing—review and editing,
B.F.-J., C.C.-C., B.H.-O., S.G.-M., N.C.-R., R.A.-E., C.B., L.M.C.-Á. and D.O.-C.; supervision, B.F.-J. and
D.O.-C.; project administration, D.O.-C. All authors have read and agreed to the published version of
the manuscript.
Funding: We appreciate the support of the E022 Program, National Institute of Pediatrics, Mexico
City, Mexico (Recursos Fiscales para la Investigación). D.O.-C was supported by 033/2022. S.G.-M
was supported by 034/2022. CONACyT doctoral fellowship was awarded to B.F-J (857097).
Acknowledgments: SNI-CONACYT fellow (Beatriz Hernández-Ochoa, Saúl Gómez-Manzo, Noemi
Cárdenas-Rodríguez, Roberto Arreguin-Espinosa, Cindy Bandala and Daniel Ortega-Cuellar.
Conflicts of Interest: The authors declare no conflict of interest.

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