HECT E3 Ubiquitin Ligase Itch Functions as a Novel Nega

The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases

Induction of the Cytochrome P450 Gene CYP26 during Mucous Cell Differentiation of Normal Human Tracheobronchial Epithelial Cells

ABSTRACT In this study, the expression of CYP26 is examined in relation to retinoid-induced mucosecretory differentiation in human tracheobronchial epithelial (HTBE) cells and compared with that in human lung carcinoma cell lines. In HTBE cells, retinoic acid (RA) inhibits squamous differentiation and induces mucous cell differentiation as indicated by the suppression of transglutaminase I and increased expression of the mucin gene MUC2. The latter is accompanied by increased expression of CYP26 mRNA. RA is required but not sufficient to induce RAR␤, CYP26, and MUC2 mRNA because induction is only observed in confluent but not in logarithmic cultures, suggesting that additional factors are critical in their regulation. CYP26 mRNA can be induced by the RAR-selective retinoid 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)-benzoic acid (TTAB) but not by the RXR-selective retinoid SR11217 or the antiactivator-protein 1-selective retinoid SR11302. RAR␣-, ␤-, and ␥-selective retinoids are able to induce CYP26; this induction is inhibited by the RAR␣-selective antagonist Ro41-5253. TTAB is able to induce CYP26 mRNA expression in only a few of the lung carcinoma cell lines tested. The lack of CYP26 induction in many carcinoma cell lines may relate to previously reported defects in the retinoid-signaling pathway. The induction of CYP26 correlated with increased metabolism of RA into 18-hydroxy-, 4-oxo-, and 4-hydroxy-RA. The latter metabolite was shown to be able to induce MUC2 and MUC5AC expression in HTBE cells. Our results demonstrate that in normal HTBE cells, CYP26 expression is closely associated with mucous cell differentiation and that many lung carcinoma cells exhibit increased RA metabolism and a defective regulation of CYP26

11β-Hydroxysteroid dehydrogenases control access of 7β,27-dihydroxycholesterol to retinoid-related orphan receptor γ

Oxysterols previously were considered intermediates of bile acid and steroid hormone biosynthetic pathways. However, recent research has emphasized the roles of oxysterols in essential physiologic processes and in various diseases. Despite these discoveries, the metabolic pathways leading to the different oxysterols are still largely unknown and the biosynthetic origin of several oxysterols remains unidentified. Earlier studies demonstrated that the glucocorticoid metabolizing enzymes, 11β-hydroxysteroid dehydrogenase (11β-HSD) types 1 and 2, interconvert 7-ketocholesterol (7kC) and 7β-hydroxycholesterol (7βOHC). We examined the role of 11β-HSDs in the enzymatic control of the intracellular availability of 7β,27-dihydroxycholesterol (7β27OHC), a retinoid-related orphan receptor γ (RORγ) ligand. We used microsomal preparations of cells expressing recombinant 11β-HSD1 and 11β-HSD2 to assess whether 7β27OHC and 7-keto,27-hydroxycholesterol (7k27OHC) are substrates of these enzymes. Binding of 7β27OHC and 7k27OHC to 11β-HSDs was studied by molecular modeling. To our knowledge, the stereospecific oxoreduction of 7k27OHC to 7β27OHC by human 11β-HSD1 and the reverse oxidation reaction of 7β27OHC to 7k27OHC by human 11β-HSD2 were demonstrated for the first time. Apparent enzyme affinities of 11β-HSDs for these novel substrates were equal to or higher than those of the glucocorticoids. This is supported by the fact that 7k27OHC and 7β27OHC are potent inhibitors of the 11β-HSD1-dependent oxoreduction of cortisone and the 11β-HSD2-dependent oxidation of cortisol, respectively. Furthermore, molecular docking calculations explained stereospecific enzyme activities. Finally, using an inducible RORγ reporter system, we showed that 11β-HSD1 and 11β-HSD2 controlled RORγ activity. These findings revealed a novel glucocorticoid-independent prereceptor regulation mechanism by 11β-HSDs that warrants further investigation

Generation of T Follicular Helper Cells Is Mediated by Interleukin-21 but Independent of T Helper 1, 2, or 17 Cell Lineages

SummaryAfter activation, CD4+ helper T (Th) cells differentiate into distinct effector subsets. Although chemokine (C-X-C motif) receptor 5-expressing T follicular helper (Tfh) cells are important in humoral immunity, their developmental regulation is unclear. Here we show that Tfh cells had a distinct gene expression profile and developed in vivo independently of the Th1 or Th2 cell lineages. Tfh cell generation was regulated by ICOS ligand (ICOSL) expressed on B cells and was dependent on interleukin-21 (IL-21), IL-6, and signal transducer and activator of transcription 3 (STAT3). However, unlike Th17 cells, differentiation of Tfh cells did not require transforming growth factor β (TGF-β) or Th17-specific orphan nuclear receptors RORα and RORγ in vivo. Finally, naive T cells activated in vitro in the presence of IL-21 but not TGF-β signaling preferentially acquired Tfh gene expression and promoted germinal-center reactions in vivo. This study thus demonstrates that Tfh is a distinct Th cell lineage

Endogenously produced nonclassical vitamin D hydroxy-metabolites act as "biased" agonists on VDR and inverse agonists on RORα and RORγ

The classical pathway of vitamin D activation follows the sequence D3→25(OH)D3→1,25(OH)(2)D3 with the final product acting on the receptor for vitamin D (VDR). An alternative pathway can be started by the action of CYP11A1 on the side chain of D3, primarily producing 20(OH)D3, 22(OH)D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3 and 17,20,23(OH)(3)D3. Some of these metabolites are hydroxylated by CYP27B1 at C1α, by CYP24A1 at C24 and C25, and by CYP27A1 at C25 and C26. The products of these pathways are biologically active. In the epidermis and/or serum or adrenals we detected 20(OH)D3, 22(OH)D3, 20,22(OH)(2)D3, 20,23(OH)(2)D3, 17,20,23(OH)(3)D3, 1,20(OH)(2)D3, 1,20,23(OH)(3)D3, 1,20,22(OH)(3)D3, 20,24(OH)(2)D3, 1,20,24(OH)(3)D3, 20,25(OH)(2)D3, 1,20,25(OH)(3)D3, 20,26(OH)(2)D3 and 1,20,26(OH)(3)D3. 20(OH)D3 and 20,23(OH)(2)D3 are non-calcemic, while the addition of an OH at C1α confers some calcemic activity. Molecular modeling and functional assays show that the major products of the pathway can act as “biased” agonists for the VDR with high docking scores to the ligand binding domain (LBD), but lower than that of 1,25(OH)(2)D3. Importantly, cell based functional receptor studies and molecular modeling have identified the novel secosteroids as inverse agonists of both RORα and RORγ receptors. Specifically, they have high docking scores using crystal structures of RORα and RORγ LBDs. Furthermore, 20(OH)D3 and 20,23(OH)(2)D3 have been tested in cell model that expresses a Tet-on RORα or RORγ vector and a RORE-LUC reporter (ROR-responsive element), and in a mammalian 2-hybrid model that test interactions between an LBD-interacting LXXLL-peptide and the LBD of RORα/γ. These assays demonstrated that the novel secosteroids have ROR-antagonist activities that were further confirmed by the inhibition of IL17 promoter activity in cells overexpressing RORα/γ. In conclusion, endogenously produced novel D3 hydroxy-derivatives can act both as “biased” agonists of the VDR and/or inverse agonists of RORα/γ. We suggest that the identification of large number of endogenously produced alternative hydroxy-metabolites of D3 that are biologically active, and of possible alternative receptors, may offer an explanation for the pleiotropic and diverse activities of vitamin D, previously assigned solely to 1,25(OH)(2)D3 and VDR

Functional analysis of the zinc finger and activation domains of Glis3 and mutant Glis3(NDH1)

The Krüppel-like zinc finger protein Gli-similar 3 (Glis3) plays a critical role in pancreatic development and has been implicated in a syndrome with neonatal diabetes and hypothyroidism (NDH). In this study, we examine three steps critical in the mechanism of the transcriptional regulation by Glis3: its translocation to the nucleus, DNA binding and transcriptional activity. We demonstrate that the putative bipartite nuclear localization signal is not required, but the tetrahedral configuration of the fourth zinc finger is essential for the nuclear localization of Glis3. We identify (G/C)TGGGGGGT(A/C) as the consensus sequence of the optimal, high-affinity Glis3 DNA-binding site (Glis-BS). All five zinc finger motifs are critical for efficient binding of Glis3 to Glis-BS. We show that Glis3 functions as a potent inducer of (Glis-BS)-dependent transcription and contains a transactivation function at its C-terminus. A mutation in Glis3 observed in NDH1 patients results in a frameshift mutation and a C-terminal truncated Glis3. We demonstrate that this truncation does not effect the nuclear localization but results in the loss of Glis3 transactivating activity. The loss in Glis3 transactivating function may be responsible for the abnormalities observed in NDH1

Regulation of the Transglutaminase I gene

The transglutaminase I (TGase I) gene encodes an enzyme that catalyzes the cross-linking of structural proteins involved in the formation of the cornified envelope during squamous cell differentiation. To identify DNA elements important for the transcriptional control of the TGase I gene, we analyzed the ability of a 2.9-kilobase pair (kb) upstream regulatory region to control the expression of a reporter gene in vivo and in vitro. Transgenic mice bearing the pTG(-2.9kb)CAT construct exhibited the same pattern of tissue-specific expression of CAT as reported for TGase I. Deletion analysis in transiently transfected rabbit tracheal epithelial cells indicated that two sequences from bp -490 to -470 and from -54 to -37 are involved in the activation of TGase I transcription. Point mutation analysis and mobility shift assays showed that the sequence located between -54 and -37 is a functional Sp1-like transcription element. Sp1 and Sp3, but not Sp2, are part of nuclear protein complexes from differentiated RbTE cells binding to this site. The element TGATGTCA between bp -490 and -470 is contained in a larger 22-bp palindrome and resembles the consensus cAMP response element-binding protein (CREB)/AP-1 element recognized by dimeric complexes of members of the CREB, ATF, Fos, and Jun families. Mutations in this sequence greatly reduced promoter activity. Supershift analysis identified CREB1, JunB, c-Fos, Fra-1, and c-Jun in protein complexes isolated from differentiated rabbit tracheal epithelial cells binding to this site. Our study shows that the Sp1- and CREB/AP-1-like sites act in concert to stimulate transcription of the TGase I gene. The 2.9-kb promoter region could guide expression of specific genes in the granular layer of the epidermis and could be useful in gene therapy