The Novel Inducer of Innate Immunity HO53 Stimulates Autophagy in Human Airway Epithelial Cells.

Aroylated phenylenediamines (APDs) are novel modulators of innate immunity with respect to enhancing the expression of antimicrobial peptides and maintaining epithelial barrier integrity. Here, we present a new study on induction of autophagy in human lung epithelial cells by the APD HO53. Interestingly, HO53 affected autophagy in a dose-dependent manner, demonstrated by increased microtubule-associated proteins 1A/1B light-chain 3B (LC3B) processing in mature polarized bronchial epithelial cells. The quantification of LC3B puncta showed increased autophagy flux and formation of autophagosomes visualized by transmission electron microscopy. The phenotypic changes indicated that autophagy induction was associated with activation of 5' adenosine monophosphate-activated protein kinase (AMPK), nuclear translocation of transcription factor EB (TFEB), and changes in expression of autophagy-related genes. The kinetics of the explored signaling pathways indicated on activation of AMPK followed by the nuclear translocation of TFEB. Moreover, our data suggest that HO53 modulates epigenetic changes related to induction of autophagy manifested by transcriptional regulation of histone-modifying enzymes. These changes were reflected by decreased ubiquitination of histone 2B at the lysine 120 residue that is associated with autophagy induction. Taken together, HO53 modulates autophagy, a part of the host defense system, through a complex mechanism involving several pathways and epigenetic events.

MITF reprograms the extracellular matrix and focal adhesion in melanoma.

The microphthalmia-associated transcription factor (MITF) is a critical regulator of melanocyte development and differentiation. It also plays an important role in melanoma where it has been described as a molecular rheostat that, depending on activity levels, allows reversible switching between different cellular states. Here, we show that MITF directly represses the expression of genes associated with the extracellular matrix (ECM) and focal adhesion pathways in human melanoma cells as well as of regulators of epithelial-to-mesenchymal transition (EMT) such as CDH2, thus affecting cell morphology and cell-matrix interactions. Importantly, we show that these effects of MITF are reversible, as expected from the rheostat model. The number of focal adhesion points increased upon MITF knockdown, a feature observed in drug-resistant melanomas. Cells lacking MITF are similar to the cells of minimal residual disease observed in both human and zebrafish melanomas. Our results suggest that MITF plays a critical role as a repressor of gene expression and is actively involved in shaping the microenvironment of melanoma cells in a cell-autonomous manner.

MITF and TFEB cross-regulation in melanoma cells.

The MITF, TFEB, TFE3 and TFEC (MiT-TFE) proteins belong to the basic helix-loop-helix family of leucine zipper transcription factors. MITF is crucial for melanocyte development and differentiation, and has been termed a lineage-specific oncogene in melanoma. The three related proteins MITF, TFEB and TFE3 have been shown to be involved in the biogenesis and function of lysosomes and autophagosomes, regulating cellular clearance pathways. Here we investigated the cross-regulatory relationship of MITF and TFEB in melanoma cells. Like MITF, the TFEB and TFE3 genes are expressed in melanoma cells as well as in melanoma tumors, albeit at lower levels. We show that the MITF and TFEB proteins, but not TFE3, directly affect each other's mRNA and protein expression. In addition, the subcellular localization of MITF and TFEB is subject to regulation by the mTOR signaling pathway, which impacts their cross-regulatory relationship at the transcriptional level. Our work shows that the relationship between MITF and TFEB is multifaceted and that the cross-regulatory interactions of these factors need to be taken into account when considering pathways regulated by these proteins.

NBEAL1 controls SREBP2 processing and cholesterol metabolism and is a susceptibility locus for coronary artery disease.

Dysregulated cholesterol homeostasis promotes the pathology of atherosclerosis, myocardial infarction and strokes. Cellular cholesterol is mainly regulated at the transcriptional level by SREBP2, but also through uptake of extracellular cholesterol from low density lipoproteins (LDL) via expression of LDL receptors (LDLR) at the cell surface. Identification of the mechanisms involved in regulation of these processes are thus key to understand the pathology of coronary artery disease. Here, we identify the large and poorly characterized BEACH domain protein Neurobeachin-like (NBEAL) 1 as a Golgi- associated protein required for regulation of cholesterol metabolism. NBEAL1 is most abundantly expressed in arteries. Genetic variants in NBEAL1 are associated with decreased expression of NBEAL1 in arteries and increased risk of coronary artery disease in humans. We show that NBEAL1 regulates cholesterol metabolism by modulating LDLR expression in a mechanism involving interaction with SCAP and PAQR3 and subsequent SREBP2-processing. Thus, low expression of NBEAL1 may lead to increased risk of coronary artery disease by downregulation of LDLR levels.

Mechanism of conditional partner selectivity in MITF/TFE family transcription factors with a conserved coiled coil stammer motif.

Interrupted dimeric coiled coil segments are found in a broad range of proteins and generally confer selective functional properties such as binding to specific ligands. However, there is only one documented case of a basic-helix-loop-helix leucine zipper transcription factor-microphthalmia-associated transcription factor (MITF)-in which an insertion of a three-residue stammer serves as a determinant of conditional partner selectivity. To unravel the molecular principles of this selectivity, we have analyzed the high-resolution structures of stammer-containing MITF and an engineered stammer-less MITF variant, which comprises an uninterrupted symmetric coiled coil. Despite this fundamental difference, both MITF structures reveal identical flanking in-phase coiled coil arrangements, gained by helical over-winding and local asymmetry in wild-type MITF across the stammer region. These conserved structural properties allow the maintenance of a proper functional readout in terms of nuclear localization and binding to specific DNA-response motifs regardless of the presence of the stammer. By contrast, MITF heterodimer formation with other bHLH-Zip transcription factors is only permissive when both factors contain either the same type of inserted stammer or no insert. Our data illustrate a unique principle of conditional partner selectivity within the wide arsenal of transcription factors with specific partner-dependent functional readouts.

The microphthalmia-associated transcription factor (Mitf) gene and its role in regulating eye function.

Mutations in the microphthalmia-associated transcription factor (Mitf) gene can cause retinal pigment epithelium (RPE) and retinal dysfunction and degeneration. We examined retinal and RPE structure and function in 3 month old mice homo- or heterozygous or compound heterozygous for different Mitf mutations (Mitfmi-vga9/+, Mitfmi-enu22(398)/Mitfmi-enu22(398), MitfMi-Wh/+ and MitfMi-Wh/Mitfmi) which all have normal eye size with apparently normal eye pigmentation. Here we show that their vision and retinal structures are differentially affected. Hypopigmentation was evident in all the mutants while bright-field fundus images showed yellow spots with non-pigmented areas in the Mitfmi-vga9/+ mice. MitfMi-Wh/+ and MitfMi-Wh/Mitfmi mice showed large non-pigmented areas. Fluorescent angiography (FA) of all mutants except Mitfmi-vga9/+ mice showed hyperfluorescent areas, whereas FA from both Mitf-Mi-Wh/+ and MitfMi-Wh/Mitfmi mice showed reduced capillary network as well as hyperfluorescent areas. Electroretinogram (ERG) recordings show that MitfMi-Wh/+ and MitfMi-Wh/Mitfmi mice are severely impaired functionally whereas the scotopic and photopic ERG responses of Mitfmi-vga9/+ and Mitfmi-enu22(398)/Mitfmi-enu22(398) mice were not significantly different from wild type mice. Histological sections demonstrated that the outer retinal layers were absent from the MitfMi-Wh/+ and MitfMi-Wh/Mitfmi blind mutants. Our results show that Mitf mutations affect eye function, even in the heterozygous condition and that the alleles studied can be arranged in an allelic series in this respect.

Subcellular localization and stability of MITF are modulated by the bHLH-Zip domain.

Microphthalmia-associated transcription factor (MITF) is a member of the basic helix-loop-helix leucine zipper (bHLH-Zip) family and functions as the master regulator of the melanocytic lineage. MITF-M is the predominant isoform expressed in melanocytes and melanoma cells, and, unlike other MITF isoforms, it is constitutively nuclear. Mutational analysis revealed three karyophilic signals in the bHLH-Zip domain of MITF-M, spanning residues 197-206, 214-217, and 255-265. Structural characterization of the MITF protein showed that basic residues within these signals are exposed for interactions in the absence of DNA. Moreover, our data indicate that neither DNA binding nor dimerization of MITF-M are required for its nuclear localization. Finally, dimerization-deficient MITF-M mutants exhibited a significantly reduced stability in melanoma cells when compared to the wild-type protein. Taken together, we have shown that, in addition to its well-established role in DNA binding and dimer formation, the bHLH-Zip domain of MITF modulates the transcription factor's subcellular localization and stability.

Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth.

The mechanistic target of rapamycin complex 1 (mTORC1) is recruited to the lysosome by Rag guanosine triphosphatases (GTPases) and regulates anabolic pathways in response to nutrients. We found that MiT/TFE transcription factors-master regulators of lysosomal and melanosomal biogenesis and autophagy-control mTORC1 lysosomal recruitment and activity by directly regulating the expression of RagD. In mice, this mechanism mediated adaptation to food availability after starvation and physical exercise and played an important role in cancer growth. Up-regulation of MiT/TFE genes in cells and tissues from patients and murine models of renal cell carcinoma, pancreatic ductal adenocarcinoma, and melanoma triggered RagD-mediated mTORC1 induction, resulting in cell hyperproliferation and cancer growth. Thus, this transcriptional regulatory mechanism enables cellular adaptation to nutrient availability and supports the energy-demanding metabolism of cancer cells.

Mitf is a master regulator of the v-ATPase, forming a control module for cellular homeostasis with v-ATPase and TORC1.

The v-ATPase is a fundamental eukaryotic enzyme that is central to cellular homeostasis. Although its impact on key metabolic regulators such as TORC1 is well documented, our knowledge of mechanisms that regulate v-ATPase activity is limited. Here, we report that the Drosophila transcription factor Mitf is a master regulator of this holoenzyme. Mitf directly controls transcription of all 15 v-ATPase components through M-box cis-sites and this coordinated regulation affects holoenzyme activity in vivo. In addition, through the v-ATPase, Mitf promotes the activity of TORC1, which in turn negatively regulates Mitf. We provide evidence that Mitf, v-ATPase and TORC1 form a negative regulatory loop that maintains each of these important metabolic regulators in relative balance. Interestingly, direct regulation of v-ATPase genes by human MITF also occurs in cells of the melanocytic lineage, showing mechanistic conservation in the regulation of the v-ATPase by MITF family proteins in fly and mammals. Collectively, this evidence points to an ancient module comprising Mitf, v-ATPase and TORC1 that serves as a dynamic modulator of metabolism for cellular homeostasis.