Mol. Cells 2021; 44(5): 342-355
Published online May 11, 2021
https://doi.org/10.14348/molcells.2021.0067
© The Korean Society for Molecular and Cellular Biology
Correspondence to : youmekim@kaist.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The microphthalmia-associated transcription factor family (MiT family) proteins are evolutionarily conserved transcription factors that perform many essential biological functions. In mammals, the MiT family consists of MITF (microphthalmia-associated transcription factor or melanocyte-inducing transcription factor), TFEB (transcription factor EB), TFE3 (transcription factor E3), and TFEC (transcription factor EC). These transcriptional factors belong to the basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor family and bind the E-box DNA motifs in the promoter regions of target genes to enhance transcription. The best studied functions of MiT proteins include lysosome biogenesis and autophagy induction. In addition, they modulate cellular metabolism, mitochondria dynamics, and various stress responses. The control of nuclear localization via phosphorylation and dephosphorylation serves as the primary regulatory mechanism for MiT family proteins, and several kinases and phosphatases have been identified to directly determine the transcriptional activities of MiT proteins. In different immune cell types, each MiT family member is shown to play distinct or redundant roles and we expect that there is far more to learn about their functions and regulatory mechanisms in host defense and inflammatory responses.
Keywords autophagy, immune cells, lysosome, metabolism, microphthalmia-associated transcription factor (MITF), MiT family transcription factors, mitochondria, stress response, transcription factor E3 (TFE3), transcription factor EB (TFEB), transcription factor EC
The microphthalmia-associated transcription factor family (MiT family) consists of four transcription factors: MITF (microphthalmia-associated transcription factor or melanocyte-inducing transcription factor), TFEB (transcription factor EB), TFE3 (transcription factor E3), and TFEC (transcription factor EC) (Goding and Arnheiter, 2019; Napolitano and Ballabio, 2016; Oppezzo and Rosselli, 2021). The
MiT family members share a common basic helix-loop-helix-leucine zipper (bHLH-LZ) domain, which serves as a DNA binding and dimerization domain, and bind DNA as a homodimer or heterodimer (Goding and Arnheiter, 2019). Unlike other bHLH-LZ family transcription factors, MiT family proteins seem to form heterodimers only with transcription factors within the MiT family and not with other bHLH-LZ family proteins (Hemesath et al., 1994). MiT family proteins bind the palindromic, canonical E-box motif (CACGTG) as well as asymmetric M-box sequence (CATGTG) (Aksan and Goding, 1998). The bHLH-LZ domain is well conserved among all members of the MiT family but regulatory regions outside the bHLH-LZ domain show considerable variabilities (Fig. 1). TFEC is the most divergent member of the family and was originally suggested to inhibit TFE3-dependent transcription activation (Zhao et al., 1993).
TFEB and TFE3 are ubiquitously expressed whereas MITF and TFEC show more tissue-specific expression patterns. Additionally, the
Here, we will first briefly summarize diverse functions of MiT family proteins outside the immune system and their common regulatory mechanisms. Then, we will describe the known roles of individual MiT transcription factors in various immune cell types.
As mentioned before, mutations of
MITF, along with TFEB and TFEC, has also been implicated in lysosome biogenesis and autophagy. When MITF-M was over-expressed in melanoma cell lines, the transcription of numerous lysosomal genes and synthesis of lysosomal proteins were enhanced (Ploper et al., 2015). Moreover, microarray analysis of 51 human melanoma cell lines demonstrated that high MITF expression is highly correlated with upregulation of lysosomal genes. MITF also upregulates the transcription of v-ATPase components, which are essential for the acidification of endolysosomes and activation of lysosomal functions (Zhang et al., 2015). Recently, MITF was shown to play an important role in autophagy induction in melanoma (Moller et al., 2019). Autophagosome formation induced by either starvation or mTORC1 inhibition was significantly perturbed and expression of autophagy-related proteins such as LC3B and SQSTM1 was significantly decreased in MITF-deficient cells.
Notably, genomic amplification of
Systemic ablation of
Soon after the discovery of TFEB as a master regulator of lysosome biogenesis, its role in autophagy was also revealed (Palmieri et al., 2011; Settembre et al., 2011). Many autophagy-associated genes, such as
Aside from an induction by starvation or deposit of undesirable macromolecules and damaged organelles in the cytoplasm, autophagy can also be induced by an accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) (Rashid et al., 2015). In cells where ER stress was triggered by treatment with brefeldin A and tunicamycin, TFEB and TFE3 become activated and directly induce transcription of ATF4, one of the major transcription factors controlling the unfolded protein response (UPR), and other UPR-associated genes (Martina et al., 2016). TFEB and TFE3 are also activated by genotoxic stress and DNA damage. In TFEB/TFE3 double-knockout cells exposed to DNA damaging conditions, the half-life of p53 is significantly shortened and cell cycle checkpoint gene induction and apoptosis responses are dysregulated (Brady et al., 2018). Therefore, TFEB and TFE3 seem to play critical roles in integrated stress responses in general under various cellular stress conditions such as nutrient deprivation, ER stress, and DNA damage.
TFEB also controls cellular metabolism. Overexpression of TFEB in mouse liver results in major expression changes of genes in cellular lipid metabolic processes via direct transcriptional control of
The function of TFEB in cancer has been studied in several cancer types; renal cell carcinoma (RCC) in particular is one of the most studied (Perera et al., 2019; Puertollano et al., 2018). TFEB overexpression, due to chromosomal translocation or genetic amplification, was found in a group of RCC patients, and the TFEB overexpression seems to be associated with poor prognosis (Argani et al., 2016). Moreover, a positive correlation between TFEB and PD-L1 expression has been found in human primary RCC and murine RCC xenograft models and TFEB was suggested to mediate immune evasion and resistance to mTOR inhibition (Zhang et al., 2019). In addition, TFEB has been implicated in specific killing of B cell non-Hodgkin lymphoma (B-NHL) by apilimod, a PIKfyve kinase inhibitor. Apilimod induces B-NHL cell death by destabilization of lysosomes. Interestingly, TFEB is most highly expressed in B-NHL compared to other cancer types and deletion of
The tissue expression pattern and known biological functions of TFE3 highly overlap with those of TFEB, even though
Like TFEB, TFE3 has been implicated in development and progress of various tumors (Perera et al., 2019; Puertollano et al., 2018). Chromosomal translocation and fusion of
TFEC was initially described as lacking the acidic domain (AD) required for transcriptional transactivation and inhibit TFE3-mediated transcriptional activation (Zhao et al., 1993). However, a more recent study found that TFEC functions as a transcriptional activator of the non-muscle myosin II heavy chain A gene in transfected cells (Chung et al., 2001). TFEC expression is highly restricted in macrophages and its physiological roles has been little studied (Rehli et al., 1999).
The control of intracellular localization serves as a major regulatory mechanism for the function of MiT family transcription factors. Transcriptional factors need to be inside the nucleus to be functional and the nuclear localization of MiT proteins is mainly controlled by phosphorylation and dephosphorylation events (Puertollano et al., 2018). Various kinases and phosphatases have been identified to control the nuclear localization of MITF, TFEB, and TFE3 and phosphorylation target sequences and regulatory mechanisms seem to be well conserved among the three proteins (Fig. 2). Therefore, here we will mainly describe the regulatory mechanisms of TFEB as representative examples.
Regulation of TFEB localization by phosphorylation/dephosphorylation was first clearly demonstrated in cells undergoing starvation (Settembre et al., 2011). TFEB is normally phosphorylated at multiple serine residues and stays in the cytoplasm. Upon starvation, it becomes dephosphorylated and translocates into the nucleus to induce autophagy. Among several possible phosphorylation sites, Ser-142 was first identified as a key residue for controlling the nuclear localization. The phospho-mimetic mutation at Ser-142 (S142D) of TFEB inhibits the nuclear translocation and autophagy induction whereas TFEB with the S142A mutation is constitutively localized in the nucleus and induces autophagy even without starvation. Based on a bioinformatics analysis, ERK2 was suggested as the kinase responsible for the phosphorylation of Ser-142 (Settembre et al., 2011). Ser-142 in TFEB corresponds to Ser-73 in MITF-M and the phosphorylation at Ser-73 was also shown to be mediated by ERK2 (Hemesath et al., 1998; Ngeow et al., 2018). Subsequent studies have demonstrated that mTOR kinase couples nutrient conditions to the TFEB subcellular localization (Settembre et al., 2012). In a normal, nutrient-sufficient condition, mTOR is active and phosphorylates TFEB, thereby sequestering it in the cytoplasm. At least three serine residues, Ser-122, 142, and 211, are identified as the phosphorylation target sites of mTOR and inhibition of mTOR induces nuclear translocation of TFEB even in the nutrient-rich conditions (Martina et al., 2012; Roczniak-Ferguson et al., 2012; Vega-Rubin-de-Celis et al., 2017). Moreover, similar to S142A mutation, S211A mutation alone can induce constitutive nuclear localization of TFEB whereas S122A mutation by itself is insufficient in driving nuclear localization of TFEB. Therefore, dephosphorylation at either S142 or S211 seems to be a critical step for nuclear translocation of TFEB. Interestingly, Ser-142 and 211 are conserved in MITF and TFE3, but Ser-122 is only found in TFEB (Fig. 2). Phosphorylation of Ser-211 induces a physical interaction between TFEB and cytosolic 14-3-3 protein, potentially explaining the cytosolic retention of TFEB (Martina et al., 2012; Roczniak-Ferguson et al., 2012). Similarly, phosphorylation of Ser-173 in MITF and Ser-321 in TFE3, each corresponding to Ser-211 in TFEB, facilitates the interaction of MITF and TFE3 with 14-3-3 and retains the proteins in the cytoplasm (Bronisz et al., 2006; Hsu et al., 2018; Martina et al., 2016).
The mTOR-dependent regulation of TFEB has been elucidated more precisely (Raben and Puertollano, 2016). Lysosomal nutrient sensing mechanisms activate Ragulator, a guanine nucleotide exchange factor for Rag GTPases, located on the lysosomal membrane. Rag GTPases are small GTPases normally present as heterodimers of RagA/B - RagC/D. The activated Ragulator promotes the GDP/GTP exchange of RagA/B to form a RagA/BGTP-RagC/DGDP state, which is an active state of the Rag heterodimer. The activated Rag heterodimer recruits mTORC1 and TFEB onto the lysosomal surface, enabling mTORC1-mediated phosphorylation of TFEB. The first 30 amino acids of TFEB are shown to be essential for Rag-TFEB interaction (Martina and Puertollano, 2013). In starvation conditions, Rag GTPases return to an inactive RagA/BGDP-RagC/DGTP form, which cannot recruit mTORC1 for TFEB phosphorylation, and TFEB is subsequently released from cytosolic 14-3-3 protein and translocates to the nucleus for autophagy induction. A recent study further showed that the interaction of TFEB with the Ragulator/Rag GTPase/mTORC1 complex depends on Ser-3 phosphorylation by MAP4K3 and the MAP4K3-mediated Ser-3 phosphorylation precedes the phosphorylation of Ser-211 by mTORC1 (Hsu et al., 2018). Ser-3 is also conserved in TFE3 and MITF-A, suggesting that both proteins may be subject to the same mode of regulation by MAP4K3 (Fig. 2). In energy-deficient conditions, AMPK becomes activated and promotes nuclear translocation of TFEB and TFE3 without affecting the mTORC1 signaling pathway. However, it is unclear if AMPK directly phosphorylates TFEB and TFE3 and how AMPK facilitates their nuclear translocation (El-Houjeiri et al., 2019).
Several other kinases have been identified to phosphorylate TFEB in a mTOR-independent manner. GSK3β phosphorylates Ser-134 and Ser-138 and inhibits nuclear translocation of TFEB (Li et al., 2016). Accordingly, inhibition of GSK3β results in TFEB nuclear localization and autophagy induction (Marchand et al., 2015; Parr et al., 2012). A recent study revealed that phosphorylation of TFEB at Ser-142 primes for GSK3β-mediated phosphorylation at Ser-138. Phosphorylation of both sites, but neither alone, promotes CRM1 (also called as XPO1)-mediated nuclear export of TFEB (Li et al., 2018). Similarly, Ser-69 in MITF-M, a counterpart of Ser-138 in TFEB, is also phosphorylated by GSK3, contributing to the nuclear export of MITF-M (Ngeow et al., 2018). AKT-mediated phosphorylation of TFEB at Ser-467 also causes the cytoplasmic retention of TFEB while AKT inhibition causes nuclear translocation of TFEB even in the presence of constitutively active mTORC1 (Palmieri et al., 2017). Trehalose, a disaccharide having an autophagy-inducing activity, has been shown to activate TFEB via AKT inhibition and pharmacological inhibition of AKT was suggested as a potential therapeutic strategy for neurodegenerative storage diseases (Palmieri et al., 2017; Sarkar et al., 2007). CDK4 and CDK6, which are located in the nucleus, also phosphorylate TFEB and TFE3 at Ser-142 and Ser-246, respectively and promote their nuclear export (Yin et al., 2020). The CDK4/6-mediated phosphorylation of TFEB and TFE3 is suggested to regulate cell cycle-dependent lysosome biogenesis, and a CDK4/6 inhibitor promotes lysosomal activation and cellular clearance of various substrates.
In contrast to phosphorylation by mTOR, GSK3β, AKT, and CDK4/6, which inhibit nuclear translocation and transcription-activating function of TFEB, PKCβ-mediated phosphorylation of multiple serine residues (Ser-462, Ser-463, Ser-467, Ser-469) in the C-terminal tail of TFEB stabilizes the protein and induces its nuclear localization, resulting in lysosomal biogenesis (Ferron et al., 2013). The C-terminal serine residues are conserved in all four MiT family members. However, Ser-405 in MITF-M (corresponding to Ser-463 in TFEB), along with Ser-397 and Ser-401, seems to be phosphorylated by GSK3 and inhibition of GSK3-mediated phosphorylation promotes stabilization and nuclear translocation of MITF-M, which enhances endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells (Ploper et al., 2015).
As discussed above, dephosphorylation of TFEB at a few key serine residues induces translocation of TFEB into the nucleus where TFEB performs its transcription-activating functions. A siRNA-based screen has identified calcineurin as the major phosphatase for TFEB dephosphorylation (Medina et al., 2015). Similarly, calcineurin also plays a critical role in nuclear localization and activation of TFE3 upon ER stress (Martina et al., 2016). Calcineurin is activated by high intracellular calcium concentration and, therefore, calcium ionophores facilitate nuclear localization of TFEB whereas calcium chelators inhibit it (Medina et al., 2015). In cells under nutrient starvation, the lysosomal calcium channel MCOLN1, also called TRPML1, becomes activated and releases calcium into cytosol from lysosomes (Wang et al., 2015). This results in activation of calcineurin and TFEB translocation. Accordingly, TFEB-mediated enhancement of lysosomal functions is inhibited by blocking MCOLN1 channel expression or activity. PP2A can also dephosphorylate TFEB and TFE3 (Martina and Puertollano, 2018). Activation of TFEB and TFE3 upon acute oxidative stress caused by sodium arsenite treatment involves dephosphorylation of Ser-211 of TFEB and Ser-321 of TFE3. PP2A seems to be able to dephosphorylate TFEB at additional residues, including Ser-109, Ser-114, and Ser-122.
In addition to the localization-based regulation by phosphorylation/dephosphorylation, TFEB activity is subjected to various alternative post-translational regulatory mechanisms. An E3 ubiquitin ligase STUB1 targets phosphorylated, inactive TFEB for degradation via the ubiquitin/proteasome pathway (Sha et al., 2017). In STUB1-deficient cells, phosphorylated TFEB accumulates in the cytoplasm, resulting in reduced TFEB activity. Regulation of protein stability by the ubiquitin/proteasome pathway is also demonstrated for MITF-M, and phosphorylation at Ser-73 and Ser-409 seems to be critical for ubiquitination and degradation of MITF-M (Wu et al., 2000; Xu et al., 2000; Zhao et al., 2011). MITF-M is also regulated by another ubiquitination-like modification, sumoylation, and the sumoylation regulates MITF-M’s transcriptional activity on a subset of target genes (Murakami and Arnheiter, 2005). A histone acetyltransferase GCN5 functions as a TFEB-specific acetyltransferase and acetylation of TFEB by GCN5 decreases TFEB transcriptional activity, thereby inhibiting autophagy induction (Wang et al., 2020). Transcriptional activity of TFEB is also controlled by liquid-liquid phase separation, which promotes the formation of transcriptional condensates for efficient gene expression. Inositol polyphosphate multikinase, IPMK was shown to directly interact with TFEB and inhibit liquid-liquid phase separation of TFEB in the nucleus. Consequently, depletion of IPMK leads to increased TFEB activity and promotion of autophagy and lysosomal functions (Chen et al., 2020).
MiT family proteins are expressed in various immune cell types. Analysis of mRNA expression shows that TFEB and TFE3 are expressed in most of immune cell types whereas MITF expression is restricted to myeloid cells with the highest expression in mast cells. TFEC is highly expressed in a subset of macrophages but not in other immune cell types (Fig. 3, Supplementary Table S1). Below we summarize the known function of MiT family proteins in individual immune cell types (Fig. 4).
Phagocytosis is one of the most important functions of macrophages and MiT family transcription factors regulate lysosome biogenesis, phagosome-lysosome fusion and autophagy in macrophages as in other cell types. In addition, they also regulate cell death and expression of cytokines and chemokines in macrophages. One of the earliest studies on TFEB in macrophages shows that cell death responses induced by co-stimulation with lipopolysaccharide (LPS) and palmitate are preceded by a significant depletion of lysosomes, and TFEB overexpression can rescue the lipotoxicity-induced cell death by preventing lysosome depletion (Schilling et al., 2013). The follow-up study by the same group found that inhibition of mTOR induces nuclear localization of TFEB and protects macrophages from lipotoxic cell death. The protective effect of mTOR inhibition was autophagy-independent and was surprisingly not affected by siRNA-mediated TFEB knockdown (He et al., 2016). However, it is not clear whether a residual presence of TFEB due to an incomplete depletion by the siRNA-mediated approach is enough to mediate the beneficial effect of mTOR inhibition. Treatment of palmitate in LPS-stimulated macrophages also results in increased secretion of proinflammatory cytokines. Ezetimibe, a cholesterol transporter blocker and U.S. Food and Drug Administration (FDA)-approved lipid lowering drug, increases nuclear localization of TFEB, induces autophagy, and reduces both inflammatory cytokine production and NLRP3 inflammasome activation in macrophages co-stimulated with LPS and palmitate (Kim et al., 2017a). These findings suggest that TFEB may exert anti-inflammatory functions in macrophages via autophagy induction in a lipotoxic condition.
In contrast, TFEB and TFE3 are required for production of proinflammatory cytokines and chemokines in responses to pathogen stimulation. Infection of macrophages with
HLH-30, a sole MiT protein in
During infection by various intracellular microorganisms, TFEB plays a critical role in antimicrobial responses by enhancing lysosomal functions and autophagy induction. On the other hand, many pathogens themselves seem to either activate or inhibit TFEB for their own benefit. Interferon (IFN)-γ-stimulated macrophages restrict HIV-1 infection by expressing apolipoprotein L1 (APOL1), which promotes nuclear translocation of TFEB and degradation of viral proteins Gag and Vif. Conversely, HIV-1 Nef protein activates mTOR and sequesters TFEB in the cytoplasm to inhibit lysosomal degradation and autophagy for establishment of permissive infection (Campbell et al., 2015; Taylor et al., 2014).
In the case of
The role of TFEB has also been studied in tumor-associated macrophages (TAMs) in mouse breast cancer models. TAMs isolated from orthotopic breast cancer tissues express lower levels of TFEB and peritoneal macrophages co-cultured with tumor cell-conditioned media (TCM) downregulate expression of TFEB, but not TFE3 and MITF (Fang et al., 2017). Among cancer-derived molecules, transforming growth factor β (TGF-β), but not IL-4 and IL-10, was identified to inhibit TFEB expression and its nuclear translocation. Moreover, TFEB depletion via shRNA led to increased expression of Arg1 and YM1, markers of M2-polarized macrophages, in IL-4- or TCM-treated macrophages, indicating that TFEB potentially inhibits M2-like polarization of macrophages. Indeed, TFEB depletion in macrophages decreased their ability to activate T cells and enhanced cancer cell growth. Conversely, macrophage-specific overexpression of TFEB suppressed breast tumor growth in mice (Fang et al., 2017). A follow-up study by the same group has demonstrated that TFEB activation without TFEB overexpression by trehalose treatment also inhibits tumor growth and TFEB expression is a positive prognostic marker for breast cancer in humans (Li et al., 2020).
TFEC is highly and specifically expressed in macrophages and mice genetically lacking TFEC develop normally and show no obvious defects (Rehli et al., 1999; 2005). TFEC expression in macrophages is further upregulated by IL-4 in a STAT6-dependent manner and a small number of IL-4-induced genes, including
Osteoclasts, specialized cells differentiated from monocyte/macrophage precursors in the bone tissues, degrade and reabsorb old bone matrix to maintain bone homeostasis. Osteopetrosis (‘stony bone’ or bone hardening) has long been observed in many but not all of the mice with various
Dendritic cells (DCs) bridge innate and adaptive immune responses. They capture pathogens in the peripheral tissues, migrate into regional draining lymph nodes, and present pathogen-derived peptide antigens loaded on MHC I and MHC II to cognate T cells for initiation of T cell-dependent immune responses. The classical MHC I pathway mediates presentation of intracellular pathogen-derived peptides, that are synthesized inside DCs, to CD8+ T cells. In contrast, the MHC II pathway presents extracellular antigens, which gain entry to the endolysosomal compartments of DCs via endocytosis or phagocytosis, and activates CD4+ T cells. In addition to the classical MHC I and II pathways, a specific subset of DCs—termed cDC1—can also display peptides from extracellular antigens onto MHC I and present them to CD8+ T cells via the cross-presentation pathway. The cross-presentation pathway in cDC1 involves endocytosis/phagocytosis of extracellular antigens and their transport from endolysosomal compartments into the cytoplasm. For efficient cross-presentation, antigenic peptides need to be preserved inside the endolysosomes without too much degradation. Therefore, the lysosomal pH and proteolytic capacity of cDC1 need to be carefully regulated and, in general, a milder pH and a weaker proteolysis in the endolysosomes are preferred for cross-presentation (Joffre et al., 2012). Accordingly, TFEB expression is usually lower in cDC1 compared to other DC subsets. Furthermore, overexpression of TFEB results in decreased lysosomal pH and increased expression of proteases (CatD, CatL, and CatS), leading to an inefficient cross-presentation and a rather enhanced MHC II pathway-mediated presentation of extracellular antigens. Consequently, TFEB overexpression in DCs reduces extracellular antigen-induced CD8+ T cell activation
TFEB also plays a role in DC migration. After pathogen sensing in the periphery, mature DCs migrate towards lymph nodes in a continuous, directional manner, via activation and positioning of actin-based motor protein myosin II at the cell rear. This event is regulated by lysosomal calcium release through MCOLN1 located on the lysosomal membrane. LPS-activated TFEB induces MCOLN1 expression and is required for fast DC migration (Bretou et al., 2017).
In the case of MITF, it was shown to be phosphorylated and to translocate into the nucleus in monocyte-derived DCs after treatment of IL-10 or an AKT inhibitor, resulting in the expression of a coinhibitory molecule GPNMB (Gutknecht et al., 2015). However, the physiological role of MITF in DCs has not been extensively studied.
The original
In case of TFE3, it was initially suggested to play a role in B cell activation because chimeric mice generated by injection of embryonic stem cells having a defective
In T cells, TFE3 is constitutively expressed while TFEB expression is post-transcriptionally induced by T cell receptor activation (Huan et al., 2006). Mice expressing a dominant negative mutant TFE3 protein, containing the HLH-LZ domain without the DNA-binding basic region and the transcriptional activation domain, in T cells show defective germinal center responses and a hyper-IgM syndrome due to an impaired expression of CD40L in CD4+ T cells. Notably,
MITF is most highly expressed in mast cells and various mast cell defects were identified in several MITF mutant mouse strains (Kitamura et al., 2002). MITF deficiency in either mast cells themselves or in the surrounding tissue environment has been shown to impact the mast cell development, indicating both intrinsic and extrinsic roles of MITF in mast cell development (Morii et al., 2004). Specifically, MITF has been shown to enhance expression of key molecules for mast cell development and function, including c-Kit (colony-stimulating factor receptor), tryptases, chymases, and tryptophan hydroxylase (Kitamura et al., 2002). Recently, it has been demonstrated that mast cells differentiate from ‘pre-basophil and mast cell progenitors (pre-BMPs)’ while antagonistic regulation by MITF and C/EBPα specifies the mast cell versus basophil fate. MITF directs the differentiation of pre-BMPs into mast cells and represses basophil development by inhibiting expression of C/EBPα. Conversely, C/EBPα promotes basophil development and prevents mast cell development by inhibiting MITF expression. Importantly, overexpression of MITF alone in committed basophil progenitors is sufficient in redirecting the cellular programming into mast cell development (Qi et al., 2013).
TFE3 has also been implicated in mast cell function. TFE3-deficient mice show normal mast cell numbers in various tissues but expression of FcεRI and c-Kit is significantly lower in TFE3-deficient mast cells. Moreover, TFE3-deficient mast cells display defective functionalities including impaired degranulation and cytokine releases
Since the first discovery of MITF almost 30 years ago, the biological roles of the MiT family transcriptional factors and their regulatory mechanisms have been studied by many cell biologists. Thanks to their successful efforts, we now know that MiT family proteins perform several important functions in a variety of cellular processes such as proliferation, differentiation, lysosome biogenesis, autophagy, metabolism, and stress responses. However, despite the fact that MiT family proteins perform essential and evolutionarily conserved host defense responses in lower organisms, our understanding of the roles of MiT family transcriptional factors in mammalian immune cell functions are still limited. Considering the growing attention on the roles of lysosomal functions and cellular metabolism on immune cell regulation, we anticipate that far more will soon be revealed on how MiT family proteins control the immune system and the homeostasis of organisms at large.
We apologize for not being able to describe and cite many important works related to biology of the MiT family proteins due to space limitation. We thank Hojune Kwak for English editing. This work was supported by grants from the National Research Foundation of Korea (2016M3A9D3918546, 2020R1A2C2011307) and Korea Advanced Institute of Science and Technology (KAIST).
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2021; 44(5): 342-355
Published online May 31, 2021 https://doi.org/10.14348/molcells.2021.0067
Copyright © The Korean Society for Molecular and Cellular Biology.
Seongryong Kim1,3 , Hyun-Sup Song1,3
, Jihyun Yu1
, and You-Me Kim1,2,*
1Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea, 2The Center for Epidemic Preparedness, KAIST, Daejeon 34141, Korea, 3These authors contributed equally to this work.
Correspondence to:youmekim@kaist.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The microphthalmia-associated transcription factor family (MiT family) proteins are evolutionarily conserved transcription factors that perform many essential biological functions. In mammals, the MiT family consists of MITF (microphthalmia-associated transcription factor or melanocyte-inducing transcription factor), TFEB (transcription factor EB), TFE3 (transcription factor E3), and TFEC (transcription factor EC). These transcriptional factors belong to the basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor family and bind the E-box DNA motifs in the promoter regions of target genes to enhance transcription. The best studied functions of MiT proteins include lysosome biogenesis and autophagy induction. In addition, they modulate cellular metabolism, mitochondria dynamics, and various stress responses. The control of nuclear localization via phosphorylation and dephosphorylation serves as the primary regulatory mechanism for MiT family proteins, and several kinases and phosphatases have been identified to directly determine the transcriptional activities of MiT proteins. In different immune cell types, each MiT family member is shown to play distinct or redundant roles and we expect that there is far more to learn about their functions and regulatory mechanisms in host defense and inflammatory responses.
Keywords: autophagy, immune cells, lysosome, metabolism, microphthalmia-associated transcription factor (MITF), MiT family transcription factors, mitochondria, stress response, transcription factor E3 (TFE3), transcription factor EB (TFEB), transcription factor EC
The microphthalmia-associated transcription factor family (MiT family) consists of four transcription factors: MITF (microphthalmia-associated transcription factor or melanocyte-inducing transcription factor), TFEB (transcription factor EB), TFE3 (transcription factor E3), and TFEC (transcription factor EC) (Goding and Arnheiter, 2019; Napolitano and Ballabio, 2016; Oppezzo and Rosselli, 2021). The
MiT family members share a common basic helix-loop-helix-leucine zipper (bHLH-LZ) domain, which serves as a DNA binding and dimerization domain, and bind DNA as a homodimer or heterodimer (Goding and Arnheiter, 2019). Unlike other bHLH-LZ family transcription factors, MiT family proteins seem to form heterodimers only with transcription factors within the MiT family and not with other bHLH-LZ family proteins (Hemesath et al., 1994). MiT family proteins bind the palindromic, canonical E-box motif (CACGTG) as well as asymmetric M-box sequence (CATGTG) (Aksan and Goding, 1998). The bHLH-LZ domain is well conserved among all members of the MiT family but regulatory regions outside the bHLH-LZ domain show considerable variabilities (Fig. 1). TFEC is the most divergent member of the family and was originally suggested to inhibit TFE3-dependent transcription activation (Zhao et al., 1993).
TFEB and TFE3 are ubiquitously expressed whereas MITF and TFEC show more tissue-specific expression patterns. Additionally, the
Here, we will first briefly summarize diverse functions of MiT family proteins outside the immune system and their common regulatory mechanisms. Then, we will describe the known roles of individual MiT transcription factors in various immune cell types.
As mentioned before, mutations of
MITF, along with TFEB and TFEC, has also been implicated in lysosome biogenesis and autophagy. When MITF-M was over-expressed in melanoma cell lines, the transcription of numerous lysosomal genes and synthesis of lysosomal proteins were enhanced (Ploper et al., 2015). Moreover, microarray analysis of 51 human melanoma cell lines demonstrated that high MITF expression is highly correlated with upregulation of lysosomal genes. MITF also upregulates the transcription of v-ATPase components, which are essential for the acidification of endolysosomes and activation of lysosomal functions (Zhang et al., 2015). Recently, MITF was shown to play an important role in autophagy induction in melanoma (Moller et al., 2019). Autophagosome formation induced by either starvation or mTORC1 inhibition was significantly perturbed and expression of autophagy-related proteins such as LC3B and SQSTM1 was significantly decreased in MITF-deficient cells.
Notably, genomic amplification of
Systemic ablation of
Soon after the discovery of TFEB as a master regulator of lysosome biogenesis, its role in autophagy was also revealed (Palmieri et al., 2011; Settembre et al., 2011). Many autophagy-associated genes, such as
Aside from an induction by starvation or deposit of undesirable macromolecules and damaged organelles in the cytoplasm, autophagy can also be induced by an accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) (Rashid et al., 2015). In cells where ER stress was triggered by treatment with brefeldin A and tunicamycin, TFEB and TFE3 become activated and directly induce transcription of ATF4, one of the major transcription factors controlling the unfolded protein response (UPR), and other UPR-associated genes (Martina et al., 2016). TFEB and TFE3 are also activated by genotoxic stress and DNA damage. In TFEB/TFE3 double-knockout cells exposed to DNA damaging conditions, the half-life of p53 is significantly shortened and cell cycle checkpoint gene induction and apoptosis responses are dysregulated (Brady et al., 2018). Therefore, TFEB and TFE3 seem to play critical roles in integrated stress responses in general under various cellular stress conditions such as nutrient deprivation, ER stress, and DNA damage.
TFEB also controls cellular metabolism. Overexpression of TFEB in mouse liver results in major expression changes of genes in cellular lipid metabolic processes via direct transcriptional control of
The function of TFEB in cancer has been studied in several cancer types; renal cell carcinoma (RCC) in particular is one of the most studied (Perera et al., 2019; Puertollano et al., 2018). TFEB overexpression, due to chromosomal translocation or genetic amplification, was found in a group of RCC patients, and the TFEB overexpression seems to be associated with poor prognosis (Argani et al., 2016). Moreover, a positive correlation between TFEB and PD-L1 expression has been found in human primary RCC and murine RCC xenograft models and TFEB was suggested to mediate immune evasion and resistance to mTOR inhibition (Zhang et al., 2019). In addition, TFEB has been implicated in specific killing of B cell non-Hodgkin lymphoma (B-NHL) by apilimod, a PIKfyve kinase inhibitor. Apilimod induces B-NHL cell death by destabilization of lysosomes. Interestingly, TFEB is most highly expressed in B-NHL compared to other cancer types and deletion of
The tissue expression pattern and known biological functions of TFE3 highly overlap with those of TFEB, even though
Like TFEB, TFE3 has been implicated in development and progress of various tumors (Perera et al., 2019; Puertollano et al., 2018). Chromosomal translocation and fusion of
TFEC was initially described as lacking the acidic domain (AD) required for transcriptional transactivation and inhibit TFE3-mediated transcriptional activation (Zhao et al., 1993). However, a more recent study found that TFEC functions as a transcriptional activator of the non-muscle myosin II heavy chain A gene in transfected cells (Chung et al., 2001). TFEC expression is highly restricted in macrophages and its physiological roles has been little studied (Rehli et al., 1999).
The control of intracellular localization serves as a major regulatory mechanism for the function of MiT family transcription factors. Transcriptional factors need to be inside the nucleus to be functional and the nuclear localization of MiT proteins is mainly controlled by phosphorylation and dephosphorylation events (Puertollano et al., 2018). Various kinases and phosphatases have been identified to control the nuclear localization of MITF, TFEB, and TFE3 and phosphorylation target sequences and regulatory mechanisms seem to be well conserved among the three proteins (Fig. 2). Therefore, here we will mainly describe the regulatory mechanisms of TFEB as representative examples.
Regulation of TFEB localization by phosphorylation/dephosphorylation was first clearly demonstrated in cells undergoing starvation (Settembre et al., 2011). TFEB is normally phosphorylated at multiple serine residues and stays in the cytoplasm. Upon starvation, it becomes dephosphorylated and translocates into the nucleus to induce autophagy. Among several possible phosphorylation sites, Ser-142 was first identified as a key residue for controlling the nuclear localization. The phospho-mimetic mutation at Ser-142 (S142D) of TFEB inhibits the nuclear translocation and autophagy induction whereas TFEB with the S142A mutation is constitutively localized in the nucleus and induces autophagy even without starvation. Based on a bioinformatics analysis, ERK2 was suggested as the kinase responsible for the phosphorylation of Ser-142 (Settembre et al., 2011). Ser-142 in TFEB corresponds to Ser-73 in MITF-M and the phosphorylation at Ser-73 was also shown to be mediated by ERK2 (Hemesath et al., 1998; Ngeow et al., 2018). Subsequent studies have demonstrated that mTOR kinase couples nutrient conditions to the TFEB subcellular localization (Settembre et al., 2012). In a normal, nutrient-sufficient condition, mTOR is active and phosphorylates TFEB, thereby sequestering it in the cytoplasm. At least three serine residues, Ser-122, 142, and 211, are identified as the phosphorylation target sites of mTOR and inhibition of mTOR induces nuclear translocation of TFEB even in the nutrient-rich conditions (Martina et al., 2012; Roczniak-Ferguson et al., 2012; Vega-Rubin-de-Celis et al., 2017). Moreover, similar to S142A mutation, S211A mutation alone can induce constitutive nuclear localization of TFEB whereas S122A mutation by itself is insufficient in driving nuclear localization of TFEB. Therefore, dephosphorylation at either S142 or S211 seems to be a critical step for nuclear translocation of TFEB. Interestingly, Ser-142 and 211 are conserved in MITF and TFE3, but Ser-122 is only found in TFEB (Fig. 2). Phosphorylation of Ser-211 induces a physical interaction between TFEB and cytosolic 14-3-3 protein, potentially explaining the cytosolic retention of TFEB (Martina et al., 2012; Roczniak-Ferguson et al., 2012). Similarly, phosphorylation of Ser-173 in MITF and Ser-321 in TFE3, each corresponding to Ser-211 in TFEB, facilitates the interaction of MITF and TFE3 with 14-3-3 and retains the proteins in the cytoplasm (Bronisz et al., 2006; Hsu et al., 2018; Martina et al., 2016).
The mTOR-dependent regulation of TFEB has been elucidated more precisely (Raben and Puertollano, 2016). Lysosomal nutrient sensing mechanisms activate Ragulator, a guanine nucleotide exchange factor for Rag GTPases, located on the lysosomal membrane. Rag GTPases are small GTPases normally present as heterodimers of RagA/B - RagC/D. The activated Ragulator promotes the GDP/GTP exchange of RagA/B to form a RagA/BGTP-RagC/DGDP state, which is an active state of the Rag heterodimer. The activated Rag heterodimer recruits mTORC1 and TFEB onto the lysosomal surface, enabling mTORC1-mediated phosphorylation of TFEB. The first 30 amino acids of TFEB are shown to be essential for Rag-TFEB interaction (Martina and Puertollano, 2013). In starvation conditions, Rag GTPases return to an inactive RagA/BGDP-RagC/DGTP form, which cannot recruit mTORC1 for TFEB phosphorylation, and TFEB is subsequently released from cytosolic 14-3-3 protein and translocates to the nucleus for autophagy induction. A recent study further showed that the interaction of TFEB with the Ragulator/Rag GTPase/mTORC1 complex depends on Ser-3 phosphorylation by MAP4K3 and the MAP4K3-mediated Ser-3 phosphorylation precedes the phosphorylation of Ser-211 by mTORC1 (Hsu et al., 2018). Ser-3 is also conserved in TFE3 and MITF-A, suggesting that both proteins may be subject to the same mode of regulation by MAP4K3 (Fig. 2). In energy-deficient conditions, AMPK becomes activated and promotes nuclear translocation of TFEB and TFE3 without affecting the mTORC1 signaling pathway. However, it is unclear if AMPK directly phosphorylates TFEB and TFE3 and how AMPK facilitates their nuclear translocation (El-Houjeiri et al., 2019).
Several other kinases have been identified to phosphorylate TFEB in a mTOR-independent manner. GSK3β phosphorylates Ser-134 and Ser-138 and inhibits nuclear translocation of TFEB (Li et al., 2016). Accordingly, inhibition of GSK3β results in TFEB nuclear localization and autophagy induction (Marchand et al., 2015; Parr et al., 2012). A recent study revealed that phosphorylation of TFEB at Ser-142 primes for GSK3β-mediated phosphorylation at Ser-138. Phosphorylation of both sites, but neither alone, promotes CRM1 (also called as XPO1)-mediated nuclear export of TFEB (Li et al., 2018). Similarly, Ser-69 in MITF-M, a counterpart of Ser-138 in TFEB, is also phosphorylated by GSK3, contributing to the nuclear export of MITF-M (Ngeow et al., 2018). AKT-mediated phosphorylation of TFEB at Ser-467 also causes the cytoplasmic retention of TFEB while AKT inhibition causes nuclear translocation of TFEB even in the presence of constitutively active mTORC1 (Palmieri et al., 2017). Trehalose, a disaccharide having an autophagy-inducing activity, has been shown to activate TFEB via AKT inhibition and pharmacological inhibition of AKT was suggested as a potential therapeutic strategy for neurodegenerative storage diseases (Palmieri et al., 2017; Sarkar et al., 2007). CDK4 and CDK6, which are located in the nucleus, also phosphorylate TFEB and TFE3 at Ser-142 and Ser-246, respectively and promote their nuclear export (Yin et al., 2020). The CDK4/6-mediated phosphorylation of TFEB and TFE3 is suggested to regulate cell cycle-dependent lysosome biogenesis, and a CDK4/6 inhibitor promotes lysosomal activation and cellular clearance of various substrates.
In contrast to phosphorylation by mTOR, GSK3β, AKT, and CDK4/6, which inhibit nuclear translocation and transcription-activating function of TFEB, PKCβ-mediated phosphorylation of multiple serine residues (Ser-462, Ser-463, Ser-467, Ser-469) in the C-terminal tail of TFEB stabilizes the protein and induces its nuclear localization, resulting in lysosomal biogenesis (Ferron et al., 2013). The C-terminal serine residues are conserved in all four MiT family members. However, Ser-405 in MITF-M (corresponding to Ser-463 in TFEB), along with Ser-397 and Ser-401, seems to be phosphorylated by GSK3 and inhibition of GSK3-mediated phosphorylation promotes stabilization and nuclear translocation of MITF-M, which enhances endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells (Ploper et al., 2015).
As discussed above, dephosphorylation of TFEB at a few key serine residues induces translocation of TFEB into the nucleus where TFEB performs its transcription-activating functions. A siRNA-based screen has identified calcineurin as the major phosphatase for TFEB dephosphorylation (Medina et al., 2015). Similarly, calcineurin also plays a critical role in nuclear localization and activation of TFE3 upon ER stress (Martina et al., 2016). Calcineurin is activated by high intracellular calcium concentration and, therefore, calcium ionophores facilitate nuclear localization of TFEB whereas calcium chelators inhibit it (Medina et al., 2015). In cells under nutrient starvation, the lysosomal calcium channel MCOLN1, also called TRPML1, becomes activated and releases calcium into cytosol from lysosomes (Wang et al., 2015). This results in activation of calcineurin and TFEB translocation. Accordingly, TFEB-mediated enhancement of lysosomal functions is inhibited by blocking MCOLN1 channel expression or activity. PP2A can also dephosphorylate TFEB and TFE3 (Martina and Puertollano, 2018). Activation of TFEB and TFE3 upon acute oxidative stress caused by sodium arsenite treatment involves dephosphorylation of Ser-211 of TFEB and Ser-321 of TFE3. PP2A seems to be able to dephosphorylate TFEB at additional residues, including Ser-109, Ser-114, and Ser-122.
In addition to the localization-based regulation by phosphorylation/dephosphorylation, TFEB activity is subjected to various alternative post-translational regulatory mechanisms. An E3 ubiquitin ligase STUB1 targets phosphorylated, inactive TFEB for degradation via the ubiquitin/proteasome pathway (Sha et al., 2017). In STUB1-deficient cells, phosphorylated TFEB accumulates in the cytoplasm, resulting in reduced TFEB activity. Regulation of protein stability by the ubiquitin/proteasome pathway is also demonstrated for MITF-M, and phosphorylation at Ser-73 and Ser-409 seems to be critical for ubiquitination and degradation of MITF-M (Wu et al., 2000; Xu et al., 2000; Zhao et al., 2011). MITF-M is also regulated by another ubiquitination-like modification, sumoylation, and the sumoylation regulates MITF-M’s transcriptional activity on a subset of target genes (Murakami and Arnheiter, 2005). A histone acetyltransferase GCN5 functions as a TFEB-specific acetyltransferase and acetylation of TFEB by GCN5 decreases TFEB transcriptional activity, thereby inhibiting autophagy induction (Wang et al., 2020). Transcriptional activity of TFEB is also controlled by liquid-liquid phase separation, which promotes the formation of transcriptional condensates for efficient gene expression. Inositol polyphosphate multikinase, IPMK was shown to directly interact with TFEB and inhibit liquid-liquid phase separation of TFEB in the nucleus. Consequently, depletion of IPMK leads to increased TFEB activity and promotion of autophagy and lysosomal functions (Chen et al., 2020).
MiT family proteins are expressed in various immune cell types. Analysis of mRNA expression shows that TFEB and TFE3 are expressed in most of immune cell types whereas MITF expression is restricted to myeloid cells with the highest expression in mast cells. TFEC is highly expressed in a subset of macrophages but not in other immune cell types (Fig. 3, Supplementary Table S1). Below we summarize the known function of MiT family proteins in individual immune cell types (Fig. 4).
Phagocytosis is one of the most important functions of macrophages and MiT family transcription factors regulate lysosome biogenesis, phagosome-lysosome fusion and autophagy in macrophages as in other cell types. In addition, they also regulate cell death and expression of cytokines and chemokines in macrophages. One of the earliest studies on TFEB in macrophages shows that cell death responses induced by co-stimulation with lipopolysaccharide (LPS) and palmitate are preceded by a significant depletion of lysosomes, and TFEB overexpression can rescue the lipotoxicity-induced cell death by preventing lysosome depletion (Schilling et al., 2013). The follow-up study by the same group found that inhibition of mTOR induces nuclear localization of TFEB and protects macrophages from lipotoxic cell death. The protective effect of mTOR inhibition was autophagy-independent and was surprisingly not affected by siRNA-mediated TFEB knockdown (He et al., 2016). However, it is not clear whether a residual presence of TFEB due to an incomplete depletion by the siRNA-mediated approach is enough to mediate the beneficial effect of mTOR inhibition. Treatment of palmitate in LPS-stimulated macrophages also results in increased secretion of proinflammatory cytokines. Ezetimibe, a cholesterol transporter blocker and U.S. Food and Drug Administration (FDA)-approved lipid lowering drug, increases nuclear localization of TFEB, induces autophagy, and reduces both inflammatory cytokine production and NLRP3 inflammasome activation in macrophages co-stimulated with LPS and palmitate (Kim et al., 2017a). These findings suggest that TFEB may exert anti-inflammatory functions in macrophages via autophagy induction in a lipotoxic condition.
In contrast, TFEB and TFE3 are required for production of proinflammatory cytokines and chemokines in responses to pathogen stimulation. Infection of macrophages with
HLH-30, a sole MiT protein in
During infection by various intracellular microorganisms, TFEB plays a critical role in antimicrobial responses by enhancing lysosomal functions and autophagy induction. On the other hand, many pathogens themselves seem to either activate or inhibit TFEB for their own benefit. Interferon (IFN)-γ-stimulated macrophages restrict HIV-1 infection by expressing apolipoprotein L1 (APOL1), which promotes nuclear translocation of TFEB and degradation of viral proteins Gag and Vif. Conversely, HIV-1 Nef protein activates mTOR and sequesters TFEB in the cytoplasm to inhibit lysosomal degradation and autophagy for establishment of permissive infection (Campbell et al., 2015; Taylor et al., 2014).
In the case of
The role of TFEB has also been studied in tumor-associated macrophages (TAMs) in mouse breast cancer models. TAMs isolated from orthotopic breast cancer tissues express lower levels of TFEB and peritoneal macrophages co-cultured with tumor cell-conditioned media (TCM) downregulate expression of TFEB, but not TFE3 and MITF (Fang et al., 2017). Among cancer-derived molecules, transforming growth factor β (TGF-β), but not IL-4 and IL-10, was identified to inhibit TFEB expression and its nuclear translocation. Moreover, TFEB depletion via shRNA led to increased expression of Arg1 and YM1, markers of M2-polarized macrophages, in IL-4- or TCM-treated macrophages, indicating that TFEB potentially inhibits M2-like polarization of macrophages. Indeed, TFEB depletion in macrophages decreased their ability to activate T cells and enhanced cancer cell growth. Conversely, macrophage-specific overexpression of TFEB suppressed breast tumor growth in mice (Fang et al., 2017). A follow-up study by the same group has demonstrated that TFEB activation without TFEB overexpression by trehalose treatment also inhibits tumor growth and TFEB expression is a positive prognostic marker for breast cancer in humans (Li et al., 2020).
TFEC is highly and specifically expressed in macrophages and mice genetically lacking TFEC develop normally and show no obvious defects (Rehli et al., 1999; 2005). TFEC expression in macrophages is further upregulated by IL-4 in a STAT6-dependent manner and a small number of IL-4-induced genes, including
Osteoclasts, specialized cells differentiated from monocyte/macrophage precursors in the bone tissues, degrade and reabsorb old bone matrix to maintain bone homeostasis. Osteopetrosis (‘stony bone’ or bone hardening) has long been observed in many but not all of the mice with various
Dendritic cells (DCs) bridge innate and adaptive immune responses. They capture pathogens in the peripheral tissues, migrate into regional draining lymph nodes, and present pathogen-derived peptide antigens loaded on MHC I and MHC II to cognate T cells for initiation of T cell-dependent immune responses. The classical MHC I pathway mediates presentation of intracellular pathogen-derived peptides, that are synthesized inside DCs, to CD8+ T cells. In contrast, the MHC II pathway presents extracellular antigens, which gain entry to the endolysosomal compartments of DCs via endocytosis or phagocytosis, and activates CD4+ T cells. In addition to the classical MHC I and II pathways, a specific subset of DCs—termed cDC1—can also display peptides from extracellular antigens onto MHC I and present them to CD8+ T cells via the cross-presentation pathway. The cross-presentation pathway in cDC1 involves endocytosis/phagocytosis of extracellular antigens and their transport from endolysosomal compartments into the cytoplasm. For efficient cross-presentation, antigenic peptides need to be preserved inside the endolysosomes without too much degradation. Therefore, the lysosomal pH and proteolytic capacity of cDC1 need to be carefully regulated and, in general, a milder pH and a weaker proteolysis in the endolysosomes are preferred for cross-presentation (Joffre et al., 2012). Accordingly, TFEB expression is usually lower in cDC1 compared to other DC subsets. Furthermore, overexpression of TFEB results in decreased lysosomal pH and increased expression of proteases (CatD, CatL, and CatS), leading to an inefficient cross-presentation and a rather enhanced MHC II pathway-mediated presentation of extracellular antigens. Consequently, TFEB overexpression in DCs reduces extracellular antigen-induced CD8+ T cell activation
TFEB also plays a role in DC migration. After pathogen sensing in the periphery, mature DCs migrate towards lymph nodes in a continuous, directional manner, via activation and positioning of actin-based motor protein myosin II at the cell rear. This event is regulated by lysosomal calcium release through MCOLN1 located on the lysosomal membrane. LPS-activated TFEB induces MCOLN1 expression and is required for fast DC migration (Bretou et al., 2017).
In the case of MITF, it was shown to be phosphorylated and to translocate into the nucleus in monocyte-derived DCs after treatment of IL-10 or an AKT inhibitor, resulting in the expression of a coinhibitory molecule GPNMB (Gutknecht et al., 2015). However, the physiological role of MITF in DCs has not been extensively studied.
The original
In case of TFE3, it was initially suggested to play a role in B cell activation because chimeric mice generated by injection of embryonic stem cells having a defective
In T cells, TFE3 is constitutively expressed while TFEB expression is post-transcriptionally induced by T cell receptor activation (Huan et al., 2006). Mice expressing a dominant negative mutant TFE3 protein, containing the HLH-LZ domain without the DNA-binding basic region and the transcriptional activation domain, in T cells show defective germinal center responses and a hyper-IgM syndrome due to an impaired expression of CD40L in CD4+ T cells. Notably,
MITF is most highly expressed in mast cells and various mast cell defects were identified in several MITF mutant mouse strains (Kitamura et al., 2002). MITF deficiency in either mast cells themselves or in the surrounding tissue environment has been shown to impact the mast cell development, indicating both intrinsic and extrinsic roles of MITF in mast cell development (Morii et al., 2004). Specifically, MITF has been shown to enhance expression of key molecules for mast cell development and function, including c-Kit (colony-stimulating factor receptor), tryptases, chymases, and tryptophan hydroxylase (Kitamura et al., 2002). Recently, it has been demonstrated that mast cells differentiate from ‘pre-basophil and mast cell progenitors (pre-BMPs)’ while antagonistic regulation by MITF and C/EBPα specifies the mast cell versus basophil fate. MITF directs the differentiation of pre-BMPs into mast cells and represses basophil development by inhibiting expression of C/EBPα. Conversely, C/EBPα promotes basophil development and prevents mast cell development by inhibiting MITF expression. Importantly, overexpression of MITF alone in committed basophil progenitors is sufficient in redirecting the cellular programming into mast cell development (Qi et al., 2013).
TFE3 has also been implicated in mast cell function. TFE3-deficient mice show normal mast cell numbers in various tissues but expression of FcεRI and c-Kit is significantly lower in TFE3-deficient mast cells. Moreover, TFE3-deficient mast cells display defective functionalities including impaired degranulation and cytokine releases
Since the first discovery of MITF almost 30 years ago, the biological roles of the MiT family transcriptional factors and their regulatory mechanisms have been studied by many cell biologists. Thanks to their successful efforts, we now know that MiT family proteins perform several important functions in a variety of cellular processes such as proliferation, differentiation, lysosome biogenesis, autophagy, metabolism, and stress responses. However, despite the fact that MiT family proteins perform essential and evolutionarily conserved host defense responses in lower organisms, our understanding of the roles of MiT family transcriptional factors in mammalian immune cell functions are still limited. Considering the growing attention on the roles of lysosomal functions and cellular metabolism on immune cell regulation, we anticipate that far more will soon be revealed on how MiT family proteins control the immune system and the homeostasis of organisms at large.
We apologize for not being able to describe and cite many important works related to biology of the MiT family proteins due to space limitation. We thank Hojune Kwak for English editing. This work was supported by grants from the National Research Foundation of Korea (2016M3A9D3918546, 2020R1A2C2011307) and Korea Advanced Institute of Science and Technology (KAIST).
The authors have no potential conflicts of interest to disclose.
Seung-Min Yoo, and Yong-Keun Jung
Mol. Cells 2018; 41(1): 18-26 https://doi.org/10.14348/molcells.2018.2277Jinyoung Kim, Yu-Mi Lim, and Myung-Shik Lee
Mol. Cells 2018; 41(1): 11-17 https://doi.org/10.14348/molcells.2018.2228Tomoyuki Fukuda, and Tomotake Kanki
Mol. Cells 2018; 41(1): 35-44 https://doi.org/10.14348/molcells.2018.2214