Transcriptional Repressor Hes1 Research Articles

Sox10 Activity and the Timing of Schwann Cell Differentiation Are Controlled by a Tle4-Dependent Negative Feedback Loop.

The HMG-domain containing transcription factor Sox10 plays a crucial role in regulating Schwann cell survival and differentiation and is expressed throughout the entire Schwann cell lineage. While its importance in peripheral myelination is well established, little is known about its role in the early stages of Schwann cell development. In a search for direct target genes of Sox10 in Schwann cell precursors, the transcriptional co-repressor Tle4 was identified. At least two regions upstream of the Tle4 gene appear involved in mediating the Sox10-dependent activation. Once induced, Tle4 works in tandem with the bHLH transcriptional repressor Hes1 and exerts a dual inhibitory effect on Sox10 by preventing the Sox10 protein from transcriptionally activating maturation genes and by suppressing Sox10 expression through known enhancers of the gene. This mechanism establishes a regulatory barrier that prevents premature activation of factors involved in differentiation and myelin formation by Sox10 in immature Schwann cells. The identification of Tle4 as a critical downstream target of Sox10 sheds light on the gene regulatory network in the early phases of Schwann cell development. It unravels an elaborate regulatory circuitry that fine-tunes the timing and extent of Schwann cell differentiation and myelin gene expression.

Abstract 5831: The TLE1 transcriptional and epigenetic machinery contributes to EGFR targeted therapy resistance in human lung adenocarcinoma

Abstract In human lung adenocarcinoma that carry Epidermal Growth Factor Receptor (EGFR) sensitizing mutations, targeting the EGFR pathway with the use of EGFR tyrosine kinase inhibitors (EGFR-TKIs) has emerged as a promising therapeutic strategy providing initial clinical benefit to many patients. Unfortunately, resistance to EGFR-TKIs eventually develops leading to disease progression. In addition to genetic mutations, transcriptional and epigenetic mediated changes in gene expression remain an important mechanism contributing to EGFR-TKI resistance. Here, we report a novel function of the transcriptional corepressor Transducin-Like Enhancer of Split-1 (TLE1) in mediating EGFR-TKI resistance in EGFR mutant LUAD cells through its survival promoting gene transcriptional program. In EGFR mutant, EGFR TKI sensitive LUAD cells, sole activation of the TLE1 nuclear function attenuates EGFR TKI sensitivity with concomittant reduction of EGFR-TKI-mediated apoptosis. Conversely, downregulation of endogenous TLE1 expression via siRNAs/shRNAs and deletion of TLE1 gene via CRISPR-Cas9 in EGFR-mutant, EGFR-TKI resistant cells partially restore EGFR-TKI sensitivity via induction of EGFR-TKI apoptosis. Mechanistically, the anti-apoptosis promoting gene transcriptional program of TLE1 is dependent on its functional interaction with the transcriptional repressor HES-1, a downstream Notch target which has been associated with relapse in LUAD patients following EGFR-TKI treatment. Collectively, these studies indicate the TLE1 corepressor as a determinant of resistance to EGFR targeted therapy and raise the possibility that inhibiting TLE1 nuclear function may serve as an adjuct therapy to circumvent EGFR-TKI resistance. Citation Format: Hector Ramos Biliran, Xin Yao, Nasir Roberts, Prince Iheukwumere, Renwei Chen, Ma. Carmela Dela Cruz. The TLE1 transcriptional and epigenetic machinery contributes to EGFR targeted therapy resistance in human lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5831.

#3048 INDOXYL SULFATE INHIBITS OSTEOGENESIS OF BONE MARROW DERIVED MESENCHYMAL STEM CELLS THROUGH AHR/HES1/BMP2 PATHWAY

Abstract Background and Aims There is a high global burden of chronic kidney disease (CKD). The increased risk of bone fracture in patients with CKD suggests that CKD-associated factors, such as uremic toxins, might be the cause. Recent studies demonstrated that low turnover osteodystrophy and bone formation defect develop in early CKD, which indicates that uremic toxins, particularly in relatively low concentrations, can potentially be a cause. Thus far, studies about defected osteogenesis adopted high concentrations of uremic toxins (for example, indoxyl sulfate (ISx) >100 μM). Furthermore, the pathology is not yet fully investigated. Uremic toxin induced aryl hydrocarbon receptor (AHR) has been linked to bone formation defect, however, the mechanisms are incompletely understood. AHR inhibit osteogenesis during early processes of differentiation of early cells (stem cells), but whether uremic toxins induced AHR to affect stem cell in osteogenesis lineage has yet to be studied. The present study aims to clarify whether ISx at concentrations approximate to average in CKD patients (20 μM in free form) affect osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs), and further survey the underline signal transduction of AHR-induced defected osteogenesis. Method Mineralization of D1 cells (mouse BMSCs) was evaluated by alizarin red S staining (ARS). The mRNA expressions and protein levels of osteogenic markers including Runx2, BMP2, ALP, and osteocalcin were determined by quantitative real-time polymerase chain reaction (RT-PCR) and western blotting. AHR translocation into the nucleus was evaluated by an immunofluorescence confocal microscope. Reporter gene assays were adopted to study the transcriptional regulation of Runx2 and BMP2. Results ISx at concentrations ≤50 μM significantly downregulated mineralization of BMSCs with the most influence in the early stage of osteogenesis. ISx decreased the mRNA expressions and protein levels of osteogenic markers Runx2, BMP2, ALP, and osteocalcin. ISx of 50 μM induced AHR translocation into the nucleus and upregulated AHR target genes CYP1a1 and CYP1b1. The ISx-induced mineralization defects of BMSCs were counteracted by AHR antagonist CH223191, while downregulated gene expressions of osteogenic markers were recovered by AHR antagonist and knockdown (KD) of AHR. In mechanistic studies, ISx upregulated mRNA levels of hairy and enhancer of split (HES1), which is a primary target of NOTCH signaling. Although there were no significant differences in expressions of Notch1∼4, the upregulated Hes1 was attenuated by the antagonist and KD of AHR, which indicated that Hes1 was directly regulated by AHR. We identified putative binding sites of transcription repressor Hes1 in the promoter area of BMP2 and Runx2. Following experimental treatment of ISx, expression levels of BMP2 and Runx2 monitored by reporter assays were downregulated, which indicated that Hes1 plays as a transcription repressor to downregulated BMP2 and Runx2. Conclusion ISx under clinically relevant concentrations attenuated mineralization of BMSCs, particularly in the early stage of osteogenesis. ISx-induced AHR upregulated Hes1, which inhibited expressions of not only Runx2, but also BMP2, which has not been published before. A study of the underline signal transduction pathways relevant to the AHR genomic control system may provide potential therapy for low bone formation in early CKD.

Transcriptional profiling of β-2M-SPα-6+THY1+ spermatogonial stem cells in human spermatogenesis.

SummaryMale infertility is responsible for approximately half of all cases of reproductive issues. Spermatogenesis originates in a small pool of spermatogonial stem cells (SSCs), which are of interest for therapy of infertility but remain not well defined in humans. Using multiparametric analysis of the side population (SP) phenotype and the α-6 integrin, THY1, and β-2 microglobulin cell markers, we identified a population of human primitive undifferentiated spermatogonia with the phenotype β-2 microglobulin (β-2M)−SPα-6+THY1+, which is highly enriched in stem cells. By analyzing the expression signatures of this SSC-enriched population along with other germinal progenitors, we established an exhaustive transcriptome of human spermatogenesis. Transcriptome profiling of the human β-2M−SPα-6+THY1+ population and comparison with the profile of mouse undifferentiated spermatogonia provide insights into the molecular networks and key transcriptional regulators regulating human SSCs, including the basic-helix-loop-helix (bHLH) transcriptional repressor HES1, which we show to be implicated in maintenance of SSCs in vitro.

Hes1 oscillation frequency correlates with activation of neural stem cells

Quiescent neural stem cells (NSCs) are occasionally activated to undergo proliferation and subsequent neuronal differentiation. It was previously shown that the transcriptional repressor Hes1 is involved in both active and quiescent states of NSCs: when Hes1 expression oscillates, it periodically represses the proneural gene Ascl1, thereby driving Ascl1 oscillations, which regulate the active state, while sustained Hes1 expression continuously suppresses Ascl1, promoting quiescence. However, it remains to be analyzed how the transition from quiescent to active states of NSCs is controlled. Here, we found that overexpression of the active form of Notch1 significantly activates NSCs in both in-vitro and in-vivo conditions and that its levels are proportional to NSC activation. The active form of Notch1 induces a burst of Hes1 oscillations in quiescent NSCs, and the frequency of Hes1 oscillations, rather than the Hes1 peak levels, correlates with the efficiency of NSC activation. These results raised the possibility that bursting Hes1 oscillations could increase the chance of Ascl1 oscillations in quiescent NSCs, suggesting that Notch1-induced Hes1 oscillation is a cue for a transition from quiescent to active states of NSCs.

TNF-α inhibits GDNF levels in Sertoli cells, through a NF-κB-dependent, HES1-dependent mechanism.

Glial cell line-derived neurotrophic factor (GDNF) is a soluble molecule crucial for the regulation of the spermatogonial stem cells (SSC) of the testis. The effects of GDNF on target cells have been extensively described, but mechanisms underlying GDNF regulation are currently under investigation. In the nervous system, GDNF expression is regulated by pro-inflammatory cytokines including lipopolysaccharide (LPS), interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α) but the effect of these cytokines on GDNF expression in the testis is unclear. The aim of the present study was to investigate the impact of TNF-α on GDNF expression levels using primary murine Sertoli cells as experimental model. The expression of TNF-α-regulated genes including Gdnf in different culture conditions was determined by real-time PCR. GDNF protein levels were determined by ELISA. The activation of the NF-κb pathway and HES1 levels were assessed by Western Blot analysis and immunofluorescence. HES1 expression was downregulated by RNAi. In primary Sertoli cells, TNF-α downregulates GDNF levels through a nuclear factor-κB (NF-κB)-dependent mechanism. Mechanistically, TNF-α induces the transcriptional repressor HES1 by a NF-Κb-dependent mechanism, which in turn downregulates GDNF. Under physiological conditions, TNF-α is secreted by germ cells suggesting that this cytokine plays a role in the paracrine control of SSC niche by modulating GDNF levels. HES1, a well-known target of the Notch pathway, is implicated in the regulation of GDNF expression. In Sertoli cells, TNF-α and Notch signaling may converge at molecular level, to regulate the expression of HES1 and HES1- target genes, including GDNF. Because of the importance of GDNF for spermatogonial stem cell self-renewal and proliferation, this data may give important insights on how cytokine signals in the testis modulate the expression of niche-derived factors.

Oscillatory expression of Hes1 regulates cell proliferation and neuronal differentiation in the embryonic brain.

The expression of the transcriptional repressor Hes1 oscillates in many cell types, including neural progenitor cells (NPCs), but the significance of Hes1 oscillations in development is not fully understood. To examine the effect of altered oscillatory dynamics of Hes1, we generated two types of Hes1 knock-in mice, a shortened (type-1) and an elongated (type-2) Hes1 gene, and examined their phenotypes focusing on neural development. Although both mutations affected Hes1 oscillations, the type-1 mutation dampened Hes1 oscillations more severely, resulting in much lower amplitudes. The average levels of Hes1 expression in type-1 mutant NPCs were also lower than in wild-type NPCs but similar to or slightly higher than those in Hes1 heterozygous mutant mice, which exhibit no apparent defects. Whereas type-2 mutant mice were apparently normal, type-1 mutant mice displayed smaller brains than wild-type mice and upregulated proneural gene expression. Furthermore, proliferation of NPCs decreased and cell death increased in type-1 mutant embryos. When Hes3 and Hes5 were additionally deleted, neuronal differentiation was also accelerated, leading to microcephaly. Thus, robust Hes1 oscillations are required for maintenance and proliferation of NPCs and the normal timing of neurogenesis, thereby regulating brain morphogenesis.

Notch inhibition overcomes resistance to tyrosine kinase inhibitors in EGFR-driven lung adenocarcinoma.

EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFRT790M and EGFRC797S. It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (p-STAT3) in EGFRT790M and EGFRC797S tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a p-STAT3-dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor-resistant tumors, with EGFRT790M and EGFRC797S mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib or osimertinib, respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib-treated patients after disease progression.

Transcription Repressor Hes1 Contributes to Neuropathic Pain Development by Modifying CDK9/RNAPII-Dependent Spinal mGluR5 Transcription.

Diverse transcriptional controls in the dorsal horn have been observed in pain hypersensitivity. However, the understanding of the exact causes and mechanisms of neuropathic pain development is still fragmentary. Here, the results demonstrated nerve injury decreased the expression of spinal hairy and enhancer of split 1 (Hes1), a transcriptional repressor, and enhanced metabotropic glutamate receptor subtype 5 (mGluR5) transcription/expression, which was accompanied with behavioral allodynia. Moreover, nerve injury decreased Hes1 levels and reciprocally increased cyclin dependent kinase-9 (CDK9) levels and recruited CDK9 to phosphorylate RNA polymerase II (RNAPII) in the promoter fragments of mGluR5, thereby enhancing mGluR5 transcription/expression in the dorsal horn. These effects were also induced by intrathecally administering naïve rats with Hes1 small interfering RNA (siRNA). Conversely, Hes1 overexpression using intrathecal lentiviral vectors in nerve injury rats produced reversal of pain behavior and reversed protein expressions, phosphorylation, and coupling to the promoter segments in the dorsal horn. Collectively, the results in this study indicated nerve injury diminishes spinal Hes1-dependent suppression of CDK9-dependent RNAPII phosphorylation on the mGluR5 promoter that possibly enhances mGluR5 transcription/expression for neuropathic pain development.

Abstract 1164: Expression of Notch and Nodal embryonic components in BRAF- and RAS-like thyroid cancer cell lines

Abstract TCGA genomic study in papillary thyroid carcinoma (PTC) samples highlights two groups of tumors: BRAF-like and RAS-like, whose genotype might influence its biological behavior. The mechanisms behind progressive loss of thyroid differentiation, observed mainly in the most aggressive histotype anaplastic thyroid carcinoma (ATC), could be associated with the reactivation of embryonic signaling and acquisition of stem cell-like phenotype. In this study we focused on Notch and Nodal embryonic signaling pathways, which are crucial for stemness maintenance in embryonic cells and has also been linked to cancer cell stemness. By assessing the expression of Notch and Nodal pathway components in BRAF- and RAS-like cell lines, we sought to investigate if the reactivation of these pathways is involved in dedifferentiation mechanisms observed in ATC. For that purpose, RNA and protein were extracted from three PTC (TPC-1, BCPAP and K1), six ATC (KTC2, SW1736, C643, Hth7, Hth74 and Hth83) and non-tumoral (Nthy-ori 3-1) cell lines. For gene expression analysis, cDNA generate from mRNA was amplified by qPCR using specific primers for NOTCH1, NOTCH4, HES1, NUMB, CER1 and LEFTY1/2. For protein expression analysis, SDS-PAGE-fractionated proteins were transferred onto nitrocellulose membrane, incubated with anti-Nodal antibody and visualized by chemoluminescence. NOTCH1 was downregulated in BRAF-like PTC cell lines, while slightly overexpressed in RET/PTC-containing TPC-1. On the other hand, NOTCH1 was overexpressed in Hth83 and C643 ATC lines, both HRAS-mutated, and also in BRAF-mutated SW1736. NOTCH4 was overexpressed in PI3K-mutated K1 and all ATC cell lines. Transcription repressor HES1, which prevents cell cycle arrest by inhibiting p27, was highly expressed in all cell lines compared to Nthy. Levels of NUMB expression were elevated in all ATC cell lines with no difference between BRAF- and RAS-mutated, but were inversely related to those of HES1, as expected for a Notch pathway inhibitor. CER1 and LEFTY1/2, which encode Nodal inhibitors, were highly expressed in ATC cell lines (4/6 and 5/6, respectively) but with no apparent distinction between BRAF- and RAS-mutated cells. At the same time, Nodal protein levels are only upregulated in TP53-mutated BCPAP and C643, suggesting that alterations in MAPK components are not sufficient to promote Nodal overexpression. Our results show re-expression of Notch embryonic signaling components in both PTC and ATC cell lines with either BRAF or RAS oncogenes. Moreover, additional genetic alterations such as in PI3K and TP53 might also interfere with the expression of both Notch and Nodal components. We therefore highlight the importance of tumor genotype, which might determine the modulation of embryonic-related genes and therefore contribute to the undifferentiated phenotype in thyroid cancer. Financial support: FAPESP and CNPq. Citation Format: Paola M. Dantonio, Cesar Seigi Fuziwara, Kelly C. Saito, Edna T. Kimura. Expression of Notch and Nodal embryonic components in BRAF- and RAS-like thyroid cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1164.

Nutrient Sensing by the Intestinal Epithelium Orchestrates Mucosal Antimicrobial Defense via Translational Control of Hes1

Metabolic programs and host defense are highly integrated to ensure proper immune responses during stress. Central to these responses, mTOR regulates immune functions by sensing and integrating environmental cues, yet how these systems are coordinated at the intestinal surface remains undefined. We show that the antimicrobial peptide α-defensin is functionally sustained during nutrient deprivation because of regulation of the defensin-processing enzyme MMP7 by microbiota- and host-derived factors. Unlike other antimicrobial peptides, the MMP7-α-defensin axis remains active during nutrient fluctuations, providing essential protection against enteric pathogens. Sustained Mmp7 expression requires themicrobiota and is mediated by de-repression of the transcription activator Atoh1 upon attenuation of the transcriptional repressor Hes1 in intestinal epithelial cells. Hes1 levels are regulated via mTORand controlled translationally, constituting a metabolism-translation-transcription loop. Disrupting this loop by supplying nutrients paradoxically compromises antibacterial defense. Together, these results uncover a regulatory circuit that couples host nutrient status to epithelial antimicrobial immunity.

Myeloid Notch1 deficiency activates the RhoA/ROCK pathway and aggravates hepatocellular damage in mouse ischemic livers.

Myeloid Notch1 deficiency activates the ras homolog gene family member A/ROCK pathway and exacerbates hepatocellular injury by inhibiting transcriptional repressor Hes1 and inducing scaffold protein JSAP1 in IR-triggered liver inflammation; our findings underscore the crucial role of the Notch-Hes1 axis as a novel regulator of innate immunity-mediated inflammation and imply the therapeutic potential for the management of organ IR injury in transplant recipients. (Hepatology 2018;67:1041-1055).

Alteration in MicroRNA Expression Governs the Nature and Timing of Cellular Fate Commitment.

In the central nervous system, the expression level of transcriptional repressor Hes1 (hairy and enhancer of split-1) tightly controls the alternative cell fate commitment during differentiation as well as the time required for such cellular transitions. A microRNA, miR-9, that interacts with Hes1 in a mutually antagonistic manner, influences both the process of lineage specification and timing of differentiation significantly, but the impact of the miR-9 in guiding these events still remains poorly understood. Here, we proposed a stochastic mathematical model of the miR-9/Hes1 double-negative feedback interaction network that at the outset shows how alternative cell fate such as quiescence, progenitor, and neuronal states can be accomplished through fine-tuning the Hes1 dynamics by altering the expression level of miR-9. The model simulations further foretell a correlated variation of the period of oscillation of Hes1, and the time delay observed between Hes1 mRNA and protein as the transcription rate of miR-9 increases during the neural progenitor state attainment. Importantly, the model simulations aided by the systematic sensitivity analysis predict that the timing of differentiation to the neuronal state crucially depends on the negative regulators (miR-9 and Hes6) of the Hes1. Our results indicate that miR-9/Hes1 interaction network can be effectively exploited for an efficient and well-timed neuronal transformation.

Pro-invasive properties of Snail1 are regulated by sumoylation in response to TGFβ stimulation in cancer.

Transforming growth factor β (TGFβ) is a key regulator of epithelial-to-mesenchymal transition (EMT) during embryogenesis and in tumors. The effect of TGFβ, on ΕΜΤ, is conveyed by induction of the pro-invasive transcription factor Snail1. In this study, we report that TGFβ stimulates Snail1 sumoylation in aggressive prostate, breast and lung cancer cells. Sumoylation of Snail1 lysine residue 234 confers its transcriptional activity, inducing the expression of classical EMT genes, as well as TGFβ receptor I (TβRI) and the transcriptional repressor Hes1. Mutation of Snail1 lysine residue 234 to arginine (K234R) abolished sumoylation of Snail1, as well as its migratory and invasive properties in human prostate cancer cells. An increased immunohistochemical expression of Snail1, Sumo1, TβRI, Hes1, and c-Jun was observed in aggressive prostate cancer tissues, consistent with their functional roles in tumorigenesis.

Chronic stress and intestinal barrier dysfunction: Glucocorticoid receptor and transcription repressor HES1 regulate tight junction protein Claudin-1 promoter

Chronic stress and elevated glucocorticoid hormone are associated with decreases in the intestinal epithelial tight junction protein claudin-1 (CLDN1). Human/rat CLDN1 promoters contain glucocorticoid response elements (GREs) and adjacent transcription repressor HES1 binding N-boxes. Notch signaling target HES1 expression was high and glucocorticoid receptor (NR3C1) low at the crypt base and the pattern reversed at the crypt apex. Chronic stress reduced overall rat colon HES1 and NR3C1 that was associated with CLDN1 downregulation. Chromatin-immunoprecipitation experiments showed that HES1 and NR3C1 bind to the CLDN1 promoter in rat colon crypts. The binding of NR3C1 but not HES1 to CLDN1 promoter significantly decreased in chronically stressed animals, which was prevented by the NR3C1 antagonist RU486. We employed the 21-day Caco-2/BBe cell model to replicate cell differentiation along the crypt axis. HES1 siRNA treatment early in differentiation increased CLDN1. In contrast, stress levels of cortisol decreased CLDN1 in late differentiation stage but not in the early stage. HES1 was high, whereas NR3C1 and CLDN1 were low in the early stage which reversed in the late stage, e.g. HES1/NR3C1 binding to CLDN1 promoter demonstrates a dynamic and reciprocal pattern. These results suggest that chronic stress impairs colon epithelium homeostasis and barrier function via different mechanisms along the crypt axis.

The transcriptional repressor Hes1 attenuates inflammation by regulating transcription elongation.

Most of the known regulatory mechanisms that curb inflammatory gene expression target pre-transcription initiation steps and evidence for regulation of inflammatory gene expression post initiation remains scarce. Here we show that transcription repressor hairy and enhancer of split 1 (Hes1) suppresses production of CXCL1, a chemokine crucial for recruiting neutrophils. Hes1 negatively regulates neutrophil recruitment in vivo in a manner that is dependent on macrophage-produced CXCL1 and attenuates severity of inflammatory arthritis. Mechanistically, inhibition of Cxcl1 expression by Hes1 does not involve modification of transcription initiation. Instead, Hes1 inhibits signal-induced recruitment of positive transcription elongation complex P-TEFb, thereby preventing phosphorylation of RNA polymerase II on serine-2 and productive elongation. Thus, our results identify Hes1 as a homeostatic suppressor of inflammatory responses which exerts its suppressive function by regulating transcription elongation.

Sumoylation of Hes6 Regulates Protein Degradation and Hes1-Mediated Transcription.

BackgroundHes6 is a transcriptional regulator that induces transcriptional activation by binding to transcription repressor Hes1 and suppressing its activity. Hes6 is controlled by the ubiquitin-proteosome-mediated degradation system. Here we investigated the sumoylation of Hes6 and its functional role in its rhythmic expression.MethodsHes6, SUMO, and ubiquitin were transfected into HeLa cells and the expression pattern was observed by Western blot and immunoprecipitation. To confirm the effect of sumoylation on the rhythmic expression of Hes6, we generated mouse Hes6 promoter-driven GFP-Hes6 fusion constructs and expressed these constructs in NIH 3T3 cells.ResultsOverexpression of SUMO led to sumoylation of Hes6 at both lysine 27 and 30. Protein stability of Hes6 was decreased by sumoylation. Moreover, expression of a Hes6 sumoylation-defective mutant, the 2KR (K27/30R) mutant, or co-expression of SUMO protease SUSP1 with native Hes6, strongly reduced ubiquitination. In addition, sumoylation was associated with both the rhythmic expression and transcriptional regulation of Hes6. Wild type Hes6 showed oscillatory expression with about 2-hour periodicity, whereas the 2KR mutant displayed a longer period. Furthermore, sumoylation of Hes6 derepressed Hes1-induced transcriptional repression.ConclusionHes6 sumoylation plays an important role in the regulation of its stability and Hes1-mediated transcription. These results suggest that sumoylation may be crucial for rhythmic expression of Hes6 and downstream target genes.

Abstract 1248: The Gemini vitamin D analogue BXL0124 inhibits Notch signaling via HES1, resulting in the reduction of CD44+/CD24-/low subpopulation and proliferation of MCF10DCIS cells

Abstract Activation of Notch signaling is correlated with poor prognosis and decreased survival of breast cancer patients. Recent studies reported that Notch signaling plays an important role for the maintenance of tumor-initiating cells in breast cancer. Using MCF10DCIS.com human breast cancer cells (MCF10DCIS), which contain tumor-initiating subpopulation (CD44+/CD24-/low), we demonstrated that the Gemini vitamin D analog, BXL0124, reduced the tumor-initiating subpopulation in vitro and repressed the growth of MCF10DCIS xenograft tumors in vivo. In the present study, we investigated Notch signaling in CD44+/CD24-/low and CD44+/CD24+ subpopulations of MCF10DCIS cells and determined whether BXL0124 represses tumor-initiating subpopulation of breast cancer by targeting Notch signaling. CD44+/CD24-/low subpopulation showed higher Notch1 activation and cell proliferation than CD44+/CD24+ subpopulation in MCF10DCIS cells. To investigate the effects of BXL0124 on Notch signaling, the protein levels of Notch receptors and their ligands were determined. BXL0124 decreased activated Notch1 (c-Notch1) as well as its ligands, Jagged1, Jagged2 and DLL1. However, total Notch1 was not affected by BXL0124, suggesting that BXL0124 inhibits Notch signaling by decreasing Notch ligands. A downstream target of Notch signaling, cMyc, and cell proliferation were reduced by BXL0124. The inhibition of Notch1 activation by BXL0124 was blocked with knock-down of the vitamin D receptor (VDR), indicating that it is a VDR-dependent event. In a time course study, the effects of BXL0124 on the mRNA expression of Notch1, Notch ligands and downstream targets were investigated. BXL0124 decreased the mRNA levels of Jagged1, Jagged2 and DLL1 starting at 8 h which followed by the repression of cMyc mRNA at 16 h. The mRNA level of a transcriptional repressor HES1, which is a key downstream target of Notch signaling, was rapidly induced by BXL0124 as early as 30 min. To determine the involvement of HES1 in Notch signaling, both knock-down and overexpression of HES1 were performed by using HES1 siRNA and HES1-expression vector, respectively. The inhibition of Notch signaling by BXL0124 was reversed by the knock-down of HES1, while the overexpression of HES1 inhibited Notch activation, reduced CD44+/CD24-/low population and decreased cell proliferation of MCF10DCIS cells. These results suggest that HES1 functions as a repressor of Notch signaling, and BXL0124 inhibits Notch signaling in a HES1-dependent manner. The present study demonstrates that BXL0124 inhibits Notch signaling by rapid induction of HES1 which results in the reduction of CD44+/CD24-/low subpopulation and proliferation of MCF10DCIS cells. Our study suggests the Gemini vitamin D analog, BXL0124, as a potential agent to repress the tumor-initiating subpopulation of breast cancer by targeting Notch signaling. Citation Format: Jae Young So, David M. Salerno, Hubert Maehr, Milan Uskokovic, Nanjoo Suh. The Gemini vitamin D analogue BXL0124 inhibits Notch signaling via HES1, resulting in the reduction of CD44+/CD24-/low subpopulation and proliferation of MCF10DCIS cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1248. doi:10.1158/1538-7445.AM2014-1248

Non-neural tyrosine hydroxylase, via modulation of endocrine pancreatic precursors, is required for normal development of beta cells in the mouse pancreas

Aims/hypothesisApart from transcription factors, little is known about the molecules that modulate the proliferation and differentiation of pancreatic endocrine cells. The early expression of tyrosine hydroxylase (TH) in a subset of glucagon+ cells led us to investigate whether catecholamines have a role in beta cell development.MethodsWe studied the immunohistochemical characteristics of TH-expressing cells in wild-type (Th +/+) mice during early pancreas development, and analysed the endocrine pancreas phenotype of TH-deficient (Th −/−) mice. We also studied the effect of dopamine addition and TH-inhibition on insulin-producing cells in explant cultures.ResultsIn the mouse pancreas at embryonic day (E)12.5–E13.5, the ∼10% of early glucagon+ cells that co-expressed TH rarely proliferated and did not express the precursor marker neurogenin 3 at E13.5. The number of insulin+ cells in the Th −/− embryonic pancreas was decreased as compared with wild-type embryos at E13.5. While no changes in pancreatic and duodenal homeobox 1 (PDX1)+-progenitor cell number were observed between groups at E12.5, the number of neurogenin 3 and NK2 homeobox 2 (NKX2.2)-expressing cells was reduced in Th −/− embryonic pancreas, an effect that occurred in parallel with increased expression of the transcriptional repressor Hes1. The potential role of dopamine as a pro-beta cell stimulus was tested by treating pancreas explants with this catecholamine, which resulted in an increase in total insulin content and insulin+ cells relative to control explants.Conclusions/interpretationA non-neural catecholaminergic pathway appears to modulate the pancreatic endocrine precursor and insulin producing cell neogenesis. This finding may have important implications for approaches seeking to promote the generation of beta cells to treat diabetes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-014-3341-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Transcription repressor Hes1 is a selective regulator of TLR-induced CXCL1 expression and neutrophil responses (IRM5P.708)

Abstract Chemokine-mediated neutrophil recruitment to sites of inflammation is a critical initial step in host defense and inflammatory responses. However, neutrophil recruitment must be tightly regulated to avoid inappropriate immune activation which may lead to autoimmune and inflammatory diseases. Here we show that transcription repressor hairy and enhancer of split 1 (Hes1) suppresses TLR-induced expression of CXCL1, a chemokine crucial for recruiting neutrophils. Interestingly, Hes1 deficiency does not affect expression of other pro-inflammatory cytokines such as Tnf and Il1b. Hes1 negatively regulates Cxcl1 expression and neutrophil recruitment in vivo in LPS-induced peritonitis. In addition, deficiency of Hes1 exacerbated severity of K/BxN serum-induced arthritis in which neutrophils play a key pathogenic role. Mechanistically, regulation of Cxcl1 expression by Hes1 is at the transcriptional level but not via modification of TLR-induced canonical signaling events. Instead, Hes1 represses Cxcl1 productive transcription by attenuating occupancy of a positive-transcription elongation complex P-TEFb at the Cxcl1 gene locus and thus preventing subsequent recruitment of serine 2-phosphorylated RNA polymerase II. In summary, our results identify Hes1 as a novel homeostatic regulator of CXCL1 expression and neutrophil responses and suggest that targeting Hes1-mediated repressive pathway may represent a new therapeutic approach to selectively curbing neutrophil-mediated inflammation.