Transcription Factor GATA Binding Protein Research Articles

#489 UCP1 regulates cardiolipin metabolism and mediates mitochondrial homeostasis maintenance of ANXA1 in diabetic nephropathy

Abstract Background and Aims Diabetic nephropathy (DN) is accounted for a large proportion of chronic kidney disease (CKD) and is the leading cause of end-stage renal disease (ESRD) nowadays. The complex pathogenesis of DN has hindered the development of a single effective medicine, so it is of great significance to seek new therapeutic targets. Uncoupling of mitochondrial respiration by chemical uncouplers has proven effective in ameliorating obesity, insulin resistance, and hyperglycemia, which were risk factors for DN. Recently, we found that ANXA1 could improve mitochondrial function to mitigate DN progression. However, the underlying mechanism is not fully clear yet. Method We used kidney samples from patients in our hospital. ANXA1-KO mice with local renal overexpression of UCP1 were constructed, UCP1-KO mice were constructed for STZ/HFD modeling, and db/db mice were treated with CL316243. UCP1-KO mice with renal overexpression of CRLS1 were constructed. Mice with PTECs-specific deletion of ANXA1 were constructed. Cell lines with UCP1/ANXA1/GATA3 knockdown and UCP1/ANXA1/GATA3 overexpression were constructed, and cell reversal tests were performed using Ac2-26, CL316243 and siRNA targeting the above genes. Results Here, we identified uncoupling protein 1 (UCP1), an inner membrane protein of mitochondria, as a key to mitochondrial homeostasis improved by ANXA1. Specifically, ANXA1 attenuated mitochondrial dysfunction via appropriately upregulating UCP1 by stabilizing its transcription factor GATA binding protein 3 (GATA3) through combining with thioredoxin. Moreover, specific overexpression of UCP1 in renal cortex rescued renal injuries in diabetic Anxa1-KO mice. UCP1 deletion aggravated renal injuries in HFD/STZ-induced diabetic mice. Mechanistically, UCP1 reduced mitochondrial fission through the aristaless-related homeobox (ARX)/cardiolipin synthase 1 (CRLS1) pathway. Therapeutically, CL316243, a UCP1 agonist, could attenuate established DN in db/db mice. Conclusion Thus, our data unraveled specific mechanism by which UCP1 mediated mitochondrial homeostasis maintenance via the ANXA1-GATA3-UCP1-ARX-CRLS1 signaling pathway. This work established a novel principle to harness the power of uncouplers for the treatment of DN.

Abstract PO2-06-08: Suppression of GATA2/3-FOXA1-HER3 Axis by Histone Deacetylase (HDAC) Inhibitors shows Antitumor Activity in Basal-like Breast Cancer

Abstract Introduction: Basal-like breast cancer (BLBC) represents an aggressive subtype of triple-negative breast cancer (TNBC) that exhibits a high risk of recurrence and resistance to treatment. Histone deacetylase (HDAC) inhibitors (HDACis), including panobinostat and romidepsin, have obtained approval for the treatment of hematopoietic malignancies and demonstrated effectiveness in TNBC cells. We recently found that panobinostat and romidepsin potently induced TNBC cell growth inhibition and apoptosis via downregulation of HER3 through suppression of forkhead box protein A1 (FOXA1), a pioneering transcription factor. Nonetheless, the underlying mechanism through which HDACis suppress FOXA1, thereby inhibiting HER3 expression and its downstream signaling in FOXA1/HER3 co-expressing TNBC remains elusive. Methods: Colony formation, MTS, and LIVE/DEAD cell staining assays were used to detect cell viability. Apoptosis was detected by flow cytometry assays. QRT-PCR, western blots, and immunohistochemistry were performed to determine the expression and activation of genes and/or proteins. Co-immunoprecipitation was performed to assess the interaction between proteins. Lentivirus vectors containing cDNA or shRNAs were used to overexpress or knockdown gene expression. Chromatin immunoprecipitation-quantitative PCR and dual luciferase reporter assays were performed to elucidate the regulatory role of gene transcription. Results: Elevated expression of FOXA1 was observed in BLBC specimens and cell lines tested. FOXA1 was co-expressed with HER3 and transcriptionally activated the HER3 expression in BLBC cells. HER3 formed heterodimers with epidermal growth factor receptor (EGFR), activating downstream signaling pathways in BLBC cells. Treatment with panobinostat and romidepsin resulted in growth inhibition and apoptosis in BLBC cells by downregulating FOXA1 expression and inhibiting HER3 signaling. Notably, the combination of HDACis with an EGFR inhibitor (gefitinib) or an anti-HER3 antibody synergistically enhanced the anti-survival effects on BLBC cells. Additionally, gene expression profiling datasets revealed a significant positive correlation between FOXA1 expression and the transcription factors GATA-Binding Protein 2/3 (GATA2/3). Patients with high GATA2/3-FOXA1 expression exhibited worse Relapse-Free Survival (RFS) compared to those with low expressions in BLBC via Kaplan-Meier analysis. Further investigations showed that GATA2/3 directly activated FOXA1 transcription by binding to its promoter. Moreover, ectopic expression of GATA2/3 not only restored FOXA1 expression downregulated by HDACis but also attenuated the anti-survival effects of HDACis on BLBC cells. Conclusion: HDACis exhibit potent inhibitory effects on BLBC cells via downregulation of GATA2/3-mediated repression of FOXA1 gene transcription, which in turn suppresses HER3 expression and signaling. Our findings indicate that epigenetic targeting of the GATA2/3-FOXA1-HER3 axis may be an effective therapeutic strategy for the eradication of BLBC tumors. Keywords: FOXA1, GATA2/3, HER3, HDAC inhibitors, Basal-like breast cancer Citation Format: Bolin Liu, Congcong Tan, Hui Lyu. Suppression of GATA2/3-FOXA1-HER3 Axis by Histone Deacetylase (HDAC) Inhibitors shows Antitumor Activity in Basal-like Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-06-08.

Modulating Th1/Th2 drift in asthma-related immune inflammation by enhancing bone mesenchymal stem cell homing through targeted inhibition of the Notch1/Jagged1 signaling pathway

Asthma, a disease intricately linked to immune inflammation, is significantly influenced by the immune regulatory effect of bone mesenchymal stem cells (BMSCs). This study aims to investigate changes in the homing of BMSCs in bronchial asthma, focusing on the Notch homolog (Notch)1/Jagged1 signaling pathway’s role in regulating T helper 1(Th1)/T helper 2(Th2) drift. Additionally, we further explore the effects and mechanisms of homologous BMSCs implantation in asthma-related immune inflammation. Following intervention with BMSCs, a significant improvement in the pathology of rats with asthma was observed. Simultaneously, a reduction in the expression of inflammatory cells and inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin(IL)-4, and IL-13 was observed in bronchoalveolar lavage fluid (BALF). Furthermore, there was an increase in the expression of Th1 cytokine Interferon-γ（IFN-γ）and the transcription factor T-box expressed in T cell (T-bet), while the expression of Th2 cytokine IL-13 and transcription factor GATA binding protein (GATA)-3 decreased in lung tissue. This indicates that the Th1/Th2 drift leans towards Th1, which a crucial in ameliorating asthma inflammation. Importantly, inhibition of the Notch1 signaling pathway led to an increased expression of the Stromal cell-derived factor-1(SDF-1)/C-X-C motif chemokine receptor (CXCR)4 chemokine axis. Consequently, the homing ability of bone marrow mesenchymal stem cells to asthma-affected lung tissue was significantly enhanced. BMSCs demonstrated heightened efficacy in regulating the cytokine/chemokine network and Th1/Th2 balance, thereby restoring a stable state during the immune response process in asthma. In conclusion, inhibiting the Notch signaling pathway enhances the expression of the SDF-1 and CXCR4 chemokine axis, facilitating the migration of allogeneic BMSCs to injured lung tissues. This, in turn, promotes immune regulation and improves the Th1/Th2 imbalance, thereby enhancing the therapeutic effect on asthmatic airway inflammation.

Therapeutic effect of notoginseng saponins before and after fermentation on blood deficiency rats.

Notoginseng saponins (NS) are the active ingredients in Panax notoginseng (Burk.) F.H. Chen (PN). NS can be transformed depending on how the extract is processed. Fermentation has been shown to produce secondary ginsenosides with increased bioavailability. However, the therapeutic effect of fermented NS (FNS) requires further study. The present study compared the compositions and activities of FNS and NS in blood deficiency rats, which resembles the symptoms of anemia in modern medicine, induced by acetylphenylhydrazine and cyclophosphamide. A total of 32 rats were randomly divided into control, model, FNS and NS groups. A blood deficiency model was established and then treatment was orally administered for 21 days. The results of component analysis indicated that some saponins transformed during the fermentation process resulting in a decrease of notoginsenoside R1, and ginsenosides Rg1, Rb1 and Re, and an increase in ginsenosides Rd, Rh2, compound K, protopanaxadiol and protopanaxatriol. The animal results showed that both FNS and NS increased the number of white blood cells (WBCs), red blood cells, hemoglobin, platelets and reticulocytes, and the levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO) and thrombopoietin (TPO), decreased the G0/G1 phase and increased G2/M phase, and decreased the apoptosis rate of bone marrow (BM) cells, which suggested a contribution to the recovery of hematopoietic function of the BM cells. FNS and NS increased the protein expression levels of the cytokines IL-4, IL-10, IL-12, IL-13, TGF-β, IL-6, IFN-γ and TNF-α, and the mRNA expression levels of transcription factors GATA binding protein 3 and T-box expressed in T cell (T-bet). FNS and NS treatment also increased the number of CD4+ T cells, and decreased the enlargement of the rat spleen and thymus atrophy, which indicated a protective effect on the organs of the immune system. The results of the present study demonstrated that compared with NS, FNS showed an improved ability to increase the levels of WBCs, lymphocytes, GM-CSF, EPO, TPO, aspartate aminotransferase, IL-10, IL-12, IL-13 and TNF-α, and the mRNA expression levels of T-bet, and decrease alanine aminotransferase levels. The differences seen for FNS treatment could arise from their improved bioavailability compared with NS, due to the larger proportion of hydrophobic ginsenosides produced during fermentation.

Two regulatory T cell populations in the visceral adipose tissue shape systemic metabolism.

Visceral adipose tissue (VAT) is an energy store and endocrine organ critical for metabolic homeostasis. Regulatory T (Treg) cells restrain inflammation to preserve VAT homeostasis and glucose tolerance. Here, we show that the VAT harbors two distinct Treg cell populations: prototypical serum stimulation 2-positive (ST2+) Treg cells that are enriched in males and a previously uncharacterized population of C-X-C motif chemokine receptor 3-positive (CXCR3+) Treg cells that are enriched in females. We show that the transcription factors GATA-binding protein 3 and peroxisome proliferator-activated receptor-γ, together with the cytokine interleukin-33, promote the differentiation of ST2+ VAT Treg cells but repress CXCR3+ Treg cells. Conversely, the differentiation of CXCR3+ Treg cells is mediated by the cytokine interferon-γ and the transcription factor T-bet, which also antagonize ST2+ Treg cells. Finally, we demonstrate that ST2+ Treg cells preserve glucose homeostasis, whereas CXCR3+ Treg cells restrain inflammation in lean VAT and prevent glucose intolerance under high-fat diet conditions. Overall, this study defines two molecularly and developmentally distinct VAT Treg cell types with unique context- and sex-specific functions.

GATA4 inhibits odontoblastic differentiation of dental pulp stem cells through targeting IGFBP3

ObjectiveThe odontogenic differentiation of human dental pulp stem cells (HDPSCs) is associated with reparative dentinogenesis. Transcription factor GATA binding protein 4 (GATA4) is proved to be essential for osteoblast differentiation and bone remodeling. This study clarified the function of GATA4 in HDPSCs odontoblast differentiation. MethodsThe change in GATA4 expression during reparative dentin formation was detected by immunohistochemistry staining. The expression of GATA4 during HDPSCs odontoblastic differentiation was detected by western blot and quantitative polymerase chain reaction. The effect of GATA4 on odontoblast differentiation was investigated following overexpression lentivirus transfection. RNA sequencing, dual luciferase assay and chromatin immunoprecipitation (CHIP) were conducted to verify downstream targets of GATA4. GATA4 overexpression lentivirus and small interference RNA targeting IGFBP3 were co-transfected to investigate the regulatory mechanism of GATA4. ResultsUpregulated GATA4 was observed during reparative dentin formation in vivo and the odontoblastic differentiation of HDPSCs in vitro. GATA4 overexpression suppressed the odontoblastic potential of HDPSCs, demonstrated by decreased alkaline phosphatase activity (p < 0.0001), mineralized nodules formation (p < 0.01), and odonto/osteogenic differentiation markers levels (p < 0.05). RNA sequencing revealed IGFBP3 was a potential target of GATA4. CHIP and dual luciferase assays identified GATA4 could activate IGFBP3 transcription. Additionally, IGFBP3 knockdown recovered the odontoblastic differentiation defect caused by GATA4 overexpression (p < 0.05). ConclusionsGATA4 inhibited odontoblastic differentiation of HDPSCs via activating the transcriptional activity of IGFBP3, identifying its promising role in regulating HDPSCs odontoblast differentiation and reparative dentinogenesis.

ARHGAP18 is Upregulated by Transcription Factor GATA1 Promotes the Proliferation and Invasion in Hepatocellular Carcinoma.

Rho GTPase activating protein 18 (ARHGAP18), a member of the RhoGAP gene family that increases GTP hydrolysis and inhibits RhoGTPase, was recently discovered to play a role in the development of breast cancer. However, its exact biological role in hepatocellular carcinoma (HCC) remains unclear. In our present study, we comprehensively assessed ARHGAP18 expression and its correlation with the prognostic value of cancer patients in databases. Cell proliferation and colony formation assays were employed to monitor cell growth. Luciferase reporter assay, Chromatin immunoprecipitation qPCR (ChIP-qPCR), immunofluorescence were performed for mechanism research. The expression of genes and proteins was detected by real-time PCR and western blotting. According to the findings of this research, ARHGAP18 protein levels are increased in HCC tissues compared to adjacent nontumor tissues, and ARHGAP18 overexpression is associated with poor survival. The results of a gain- and loss-of-function experiment with HCC cells in vitro demonstrated that ARHGAP18 stimulated cell proliferation, migration, and invasion. Mechanistically, we found that the transcription factor GATA binding protein 1 (GATA1) could bind to the ARHGAP18 promoter and facilitate ARHGAP18 expression. Further studies revealed that the effects of ARHGAP18 silencing on HCCLM3 and Bel-7402 cells were blocked by GATA1 overexpression. In conclusion, GATA1-mediated ARHGAP18 up-regulation plays an important role in HCC tumorigenesis and might be a potential therapeutic target for HCC.

Erianin alleviated liver steatosis by enhancing Nrf2-mediated VE-cadherin expression in vascular endothelium

Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common chronic liver disease and is closely associated with metabolic syndrome. Endothelial dysfunction was involved in many metabolic diseases, but the concrete participation of hepatic vascular endothelial dysfunction in liver steatosis that is an early stage of NAFLD is still unclear. In this study, the formation of liver steatosis and the elevation of serum insulin content were observed accompanying with the decreased vascular endothelial cadherin (VE-cadherin) expression in hepatic vessels from db/db mice, Goto-Kakizaki (GK) and high-fat diet (HFD)-fed rats. Liver steatosis was obviously enhanced in mice after the application of VE-cadherin neutralizing antibody. In vitro results showed that insulin decreased VE-cadherin expression and caused endothelial barrier breakdown. Furthermore, the alteration of VE-cadherin expression was found to be positively related with the transcriptional activation of nuclear erythroid 2-related factor 2 (Nrf2), and chromatin immunoprecipitation (ChIP) assay displayed that Nrf2 could directly regulate VE-cadherin expression. Insulin reduced Nrf2 activation by decreasing sequestosome-1 (p62/SQSTM1) expression downstream of insulin receptor. Moreover, the p300-mediated Nrf2 acetylation was weakened by enhancing the competitive binding of transcription factor GATA-binding protein 4 (GATA4) to p300. Finally, we found that erianin, a natural compound, could promote VE-cadherin expression by inducing Nrf2 activation, thereby alleviating liver steatosis in GK rats. Our results suggest that hepatic vascular endothelial dysfunction owing to the VE-cadherin deficiency dependent on the reduced Nrf2 activation promoted liver steatosis, and erianin alleviated liver steatosis through enhancing Nrf2-mediated VE-cadherin expression.

Overexpression of GATA binding protein 4 and myocyte enhancer factor 2C induces differentiation of mesenchymal stem cells into cardiac-like cells.

BACKGROUNDHeart diseases are the primary cause of death all over the world. Following myocardial infarction, billions of cells die, resulting in a huge loss of cardiac function. Stem cell-based therapies have appeared as a new area to support heart regeneration. The transcription factors GATA binding protein 4 (GATA-4) and myocyte enhancer factor 2C (MEF2C) are considered prominent factors in the development of the cardiovascular system.AIMTo explore the potential of GATA-4 and MEF2C for the cardiac differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs).METHODShUC-MSCs were characterized morphologically and immunologically by the presence of specific markers of MSCs via immunocytochemistry and flow cytometry, and by their potential to differentiate into osteocytes and adipocytes. hUC-MSCs were transfected with GATA-4, MEF2C, and their combination to direct the differentiation. Cardiac differentiation was confirmed by semiquantitative real-time polymerase chain reaction and immunocytochemistry.RESULTShUC-MSCs expressed specific cell surface markers CD105, CD90, CD44, and vimentin but lack the expression of CD45. The transcription factors GATA-4 and MEF2C, and their combination induced differentiation in hUC-MSCs with significant expression of cardiac genes i.e., GATA-4, MEF2C, NK2 homeobox 5 (NKX2.5), MHC, and connexin-43, and cardiac proteins GATA-4, NKX2.5, cardiac troponin T, and connexin-43.CONCLUSIONTransfection with GATA-4, MEF2C, and their combination effectively induces cardiac differentiation in hUC-MSCs. These genetically modified MSCs could be a promising treatment option for heart diseases in the future.

GATA3 expression loss is linked to stage progression but is unrelated to prognosis in muscle-invasive urothelial carcinoma of the bladder

The transcription factor GATA binding protein 3 (GATA3) is commonly used in surgical pathology as a diagnostic marker to distinguish urothelial carcinomas from other cancer entities. However, the clinical relevance of GATA3 expression in urothelial bladder cancer is not completely clarified. In this study, we investigated GATA3 immunostaining on 2710 urothelial bladder carcinomas on a tissue microarray platform by using two different antibodies to better understand its impact in relation to pathological parameters of disease progression and patient outcome. Nuclear GATA3 immunostaining was regularly seen in normal urothelium and found in 74%/82% of interpretable urothelial neoplasms depending on the antibody used. Within pTa tumors, the rate of GATA3 positive tumors decreased with advancing grade. GATA3 positivity was seen in 98.6%/99.8% of pTaG2 low-grade, 98.6%/100% of pTaG2 high-grade, and 94.9%/99.2% of pTaG3 high-grade tumors (P=.0002). As compared to pTa tumors, GATA3 positivity was markedly less common in muscle-invasive urothelial carcinoma (59.9%/71.6%; P<.0001). Within pT2-4 cancers, high-level GATA3 immunostaining was associated with the presence of lymph node metastasis (P=.0034), and blood vessel (P=.0290) or lymphatic invasion (P=.0005) but unrelated to pT stage. GATA3 immunostaining results for both antibodies were not associated with overall survival in 586 patients treated by cystectomy for pT2-4 urothelial carcinoma. The results of our study identify GATA3 expression as a frequent event in noninvasive urothelial carcinomas with favorable tumor features. Loss of GATA3 immunostaining is linked with muscle-invasive disease but is largely unrelated to pathological parameters and patient prognosis.

GATA1 regulates the microRNA‑328‑3p/PIM1axis via circular RNA ITGB1 to promote renal ischemia/reperfusion injury in HK‑2 cells.

Acute kidney injury (AKI) is caused by renal ischemia/reperfusion injury (IRI) during kidney transplantation. The levels of both circular RNAs (circRNAs) and microRNAs (miRNAs/miR) appear to be critical for AKI detection. While several RNA interactions in AKI have been found, the regulatory mechanisms between the molecules remain to be fully elucidated. In the present study, miRNA expression profiling analysis was conducted using an online dataset to identify the differentially expressed miRNAs in rats with IRI. miR-328-3p was also found to be downregulated in human kidney-2 (HK-2) cells subjected to hypoxia/reperfusion (H/R), and its overexpression targeting pim-1 proto-oncogene (PIM1) resulted in an increased viability and a reduced apoptosis, as well as in the decreased expression of inflammatory factors upon H/R exposure. Putative targets and circRNAs of miR-328-3p were identified using publically available databases. The inhibition of circRNA integrin beta 1 (ITGB1; circITGB1) suppressed the inflammatory response induced by H/R by sponging miR-328-3p in HK-2 cells. Furthermore, a sequence of the functional ITGB1 promoter was studied for transcription factor GATA binding protein 1 (GATA1) binding sites. GATA1 binds to the ITGB1 promoter, leading to the expression of circITGB1. On the whole, the findings of the present study revealed a regulatory pathway modulating miR-328-3p in IRI, demonstrating that the GATA1-mediated regulation of circITGB1 enhanced the H/R-induced inflammatory response via the miR-328-3p/PIM1 axis.

Transcription factor GATA4 drives RNA polymerase III-directed transcription and transformed cell proliferation through a filamin A/GATA4/SP1 pathway

RNA polymerase III (pol III) products play fundamental roles in a variety of cellular processes, including protein synthesis and cancer cell proliferation. In addition, dysregulation of pol III-directed transcription closely correlates with tumorigenesis. It is therefore of interest to identify novel pathways or factors governing pol III-directed transcription. Here, we show that transcription factor (TF) GATA binding protein 4 (GATA4) expression in SaOS2 cells was stimulated by the silencing of filamin A (FLNA), a repressor of pol III-directed transcription, suggesting that GATA4 is potentially associated with the regulation of pol III-directed transcription. Indeed, we show that GATA4 expression positively correlates with pol III-mediated transcription and tumor cell proliferation. Mechanistically, we found that GATA4 depletion inhibits the occupancies of the pol III transcription machinery factors at the loci of pol III target genes by reducing expression of both TFIIIB subunit TFIIB-related factor 1 and TFIIIC subunit general transcription factor 3C subunit 2 (GTF3C2). GATA4 has been shown to activate specificity factor 1 (Sp1) gene transcription by binding to the Sp1 gene promoter, and Sp1 has been confirmed to activate pol III gene transcription by directly binding to both Brf1 and Gtf3c2 gene promoters. Thus, the findings from this study suggest that GATA4 links FLNA and Sp1 signaling to form an FLNA/GATA4/Sp1 axis to modulate pol III-directed transcription and transformed cell proliferation. Taken together, these results provide novel insights into the regulatory mechanism of pol III-directed transcription.

Targeting GATA1 and p2x7r Locus Binding in Spinal Astrocytes Suppresses Chronic Visceral Pain by Promoting DNA Demethylation

Irritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread, chronic abdominal pain associated with altered bowel movements. Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases. In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1 (GATA1) in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation (NCI). The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay, chromatin immunoprecipitation, patch clamp, and interference in vitro and in vivo. In addition, a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island. We showed that NCI caused the induction of GATA1, Ten-eleven translocation 3 (TET3), and purinergic receptors (P2X7Rs) in astrocytes of the spinal dorsal horn, and demonstrated that inhibiting these molecules markedly increased the pain threshold, inhibited the activation of astrocytes, and decreased the spinal sEPSC frequency. NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1–TET3 physical interaction and GATA1 binding at the p2x7r promoter. Importantly, we showed that demethylation of the p2x7r locus (and the attendant increase in P2X7R expression) was reversed upon knockdown of GATA1 or TET3 expression, and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter. These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes, and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.

COMMD10 is critical for Kupffer cell survival and controls Ly6Chi monocyte differentiation and inflammation in the injured liver.

Liver-resident macrophages Kupffer cells (KCs) and infiltrating Ly6Chi monocytes both contribute to liver tissue regeneration in various pathologies but also to disease progression upon disruption of orderly consecutive regeneration cascades. Little is known about molecular pathways that regulate their differentiation, maintenance, or inflammatory behavior during injury. Here, we show that copper metabolism MURR1 domain (COMMD)10-deficient KCs adopt liver-specific identity. Strikingly, COMMD10 deficiency in KCs and in other tissue-resident macrophages impedes their homeostatic survival, leading to their continuous replacement by Ly6Chi monocytes. While COMMD10 deficiency in KCs mildly worsens acetaminophen-induced liver injury (AILI), its deficiency in Ly6Chi monocytes results in exacerbated and sustained hepatic damage. Monocytes display unleashed inflammasome activation and a reduced type I interferon response and acquire "neutrophil-like" and lipid-associated macrophage differentiation fates. Collectively, COMMD10 appears indispensable for KC and other tissue-resident macrophage survival and is an important regulator of Ly6Chi monocyte fate decisions and reparative behavior in the diseased liver.

Baoyuan decoction (BYD) attenuates cardiac hypertrophy through ANKRD1-ERK/GATA4 pathway in heart failure after acute myocardial infarction

BackgroundThe pathological cardiac functions of ankyrin repeat domain 1 (ANKRD1) in left ventricle can directly aggravate cardiac hypertrophy (CH) and fibrosis through the activation of extracellular signal-regulated kinase (ERK)/ transcription factor GATA binding protein 4 (GATA4) pathway, and subsequently contribute to heart failure (HF). Baoyuan Decoction (BYD), which is a famous classic Chinese medicinal formulation, has shown impressive cardioprotective effects clinically and experimentally. However, the knowledge is still limited in its underlying mechanisms against HF. PurposeTo explore whether BYD plays a protective role against HF by attenuating CH via the ANKRD1-ERK/GATA4 pathway. MethodsIn vivo, HF rat models were prepared using left anterior descending coronary artery (LADCA) ligation. Rats in the BYD group were administered a dosage of 2.57 g/kg of BYD for 28 days, while in the positive control group rats were given 4.67 mg/kg of Fosinopril. In vitro, a hypertrophic model was constructed by stimulating H9C2 cells with 1 uM Ang II. An ANKRD1-overexpressing cell model was established through transient transfection of ANKRD1 plasmid into H9C2 cells. Subsequently, BYD intervention was performed on the cell models to further elucidate its effects and underlying mechanism. ResultsIn vivo results showed that BYD significantly improved cardiac function and inhibited pathological hypertrophy and fibrosis in a rat model of HF post-acute myocardial infarction (AMI). Noticeably, label-free proteomic analysis demonstrated that BYD could reverse the CH-related biological turbulences, mainly through ANKRD1-ERK/GATA4 pathway. Further in vitro results validated that the hypertrophy was attenuated by BYD through suppression of AT1R, ANKRD1, Calpain1, p-ERK1/2 and p-GATA4. The results of in vitro model indicated that BYD could reverse the outcome of transfected over-expression of ANKRD1, including down-regulated expressions of ANKRD1, p-ERK1/2 and p-GATA4. ConclusionBYD ameliorates CH and improves HF through the ANKRD1-ERK/GATA4 pathway, providing a novel therapeutic option for the treatment of HF.

Triptolide impairs glycolysis by suppressing GATA4/Sp1/PFKP signaling axis in mouse Sertoli cells

Triptolide (TP), a primary bioactive ingredient isolated from the traditional Chinese herbal medicine Tripterygium wilfordii Hook. F. (TWHF), has attracted great interest for its therapeutic biological activities in inflammation and autoimmune disease. However, its clinical use is limited by severe testicular toxicity, and the underlying mechanism has not been elucidated. Our preliminary evidence demonstrated that TP disrupted glucose metabolism and caused testicular toxicity. During spermatogenesis, Sertoli cells (SCs) provide lactate as an energy source to germ cells by glycolysis. The transcription factors GATA-binding protein 4 (GATA4) and specificity protein 1 (Sp1) can regulate glycolysis. Based on this evidence, we speculate that TP causes abnormal glycolysis in SCs by influencing the expression of the transcription factors GATA4 and Sp1. The mechanism of TP-induced testicular toxicity was investigated in vitro and in vivo. The data indicated that TP decreased glucose consumption, lactate production, and the mRNA levels of glycolysis-related transporters and enzymes. TP also downregulated the protein expression of the transcription factors GATA4 and Sp1, as well as the glycolytic enzyme phosphofructokinase platelet (PFKP). Phosphorylated GATA4 and nuclear GATA4 protein levels were reduced in a dose- and time-dependent manner after TP incubation. Similar effects were observed in shGata4-treated TM4 cells and BALB/c mice administered 0.4 mg/kg TP for 28 days, and glycolysis was also inhibited. Gata4 knockdown downregulated Sp1 and PFKP expression. Furthermore, the Sp1 inhibitor plicamycin inhibited PFKP protein levels in TM4 cells. In conclusion, TP inhibited GATA4-mediated glycolysis by suppressing Sp1-dependent PFKP expression in SCs and caused testicular toxicity.

FOXP3 and GATA3 Polymorphisms, Vitamin D3 and Multiple Sclerosis.

Background: Regulatory T cells (Tregs) alterations have been implicated in the pathogenesis of Multiple Sclerosis (MS). Recently, a crucial role of the X-Linked Forkhead Box P3 (FoxP3) for the development and the stability of Tregs has emerged, and FOXP3 gene polymorphisms have been associated with the susceptibility to autoimmune diseases. The expression of Foxp3 in Tregs is regulated by the transcription factor GATA binding-protein 3 (GATA3) and vitamin D3. The aim of this retrospective case-control study was to investigate the potential association between FOXP3 and GATA3 genetic variants, Vitamin D3, and MS risk. Methods: We analyzed two polymorphisms in the FOXP3 gene (rs3761547 and rs3761548) and a polymorphism in the GATA3 gene (rs3824662) in 106 MS patients and 113 healthy controls. Serum 25(OH)D3 was also measured in all participants. Results: No statistically significant genotypic and allelic differences were found in the distribution of FOXP3 rs3761547 and rs3761548, or GATA3 rs3824662 in the MS patients, compared with controls. Patients that were homozygous for rs3761547 had lower 25(OH)D3 levels. Conclusions: Our findings did not show any association among FOXP3 and GATA3 SNPs, vitamin D3, and MS susceptibility.

The M310T mutation in the GATA4 gene is a novel pathogenic target of the familial atrial septal defect

BackgroundAlthough most cases of atrial septal defect (ASD) are sporadic, familial cases have been reported, which may be caused by mutation of transcription factor GATA binding protein 4 (GATA4). Herein we combined whole-exome sequencing and bioinformatics strategies to identify a novel mutation in GATA4 accounting for the etiology in a Chinese family with ASD.MethodsWe identified kindred spanning 3 generations in which 3 of 12 (25.0%) individuals had ASD. Punctilious records for the subjects included complete physical examination, transthoracic echocardiography, electrocardiograph and surgical confirming. Whole-exome capture and high-throughput sequencing were performed on the proband III.1. Sanger sequencing was used to validate the candidate variants, and segregation analyses were performed in the family members.ResultsDirect sequencing of GATA4 from the genomic DNA of family members identified a T-to-C transition at nucleotide 929 in exon 5 that predicted a methionine to threonine substitution at codon 310 (M310T) in the nuclear localization signal (NLS) region. Two affected members (II.2 and III.3) and the proband (III.1) who was recognized as a carrier exhibited this mutation, whereas the other unaffected family members or control individuals did not. More importantly, the mutation GATA4 (c.T929C: p.M310T) has not been reported previously in either familial or sporadic cases of congenital heart defects (CHD).ConclusionsWe identified for the first time a novel M310T mutation in the GATA4 gene that is located in the NLS region and leads to family ASD with arrhythmias. However, the mechanism by which this pathogenic mutation contributes to the development of heart defect and tachyarrhythmias remains to be ascertained.

Spinal transcription factor GATA4 mediated adenosine A1 receptor activation contributes to electroacupuncture analgesia in neuropathic pain rats

To investigate the effect of electroacupuncture (EA) on pain behaviors and expression of spinal transcription factor GATA-binding Protein 4 (GATA4) and adenosine A1 receptor in neuropathic pain rats, so as to explore its mechanism underlying pain relief. The present study includes 2 parts. In the first part, 18 SD rats were randomly divided into control, adenovirus short-hairpin interference RNA for GATA4 (AV-shGATA4 RNA) and adenovirus empty vector (AV-control short-hairpin RNA, AV-shCTRL) groups, with 6 rats in each group. The expression of GATA4 protein in the lumbar spinal cord (L4-L6) was detected to evaluate the transfection efficiency of AV-shGATA4 RNA (silencing GATA4 expression). In the second part, thirty SD rats were randomly divided into 5 groups, namely sham operation, CCI model, EA, EA+AV-shGATA4 RNA, and EA+AV-shCTRL groups, with 6 rats in each group. The neuropathic pain model was established by chronic constriction injury (CCI) of the right sciatic nerve. On the 7th day following modeling, EA was applied to the right "Zusanli"(ST36) and "Taichong"(LR3) (1 mA，2 Hz /100 Hz) for 30 min. Rats of the EA+AV-shGATA4 RNA and EA+AV-shCTRL groups received intrathecal injection of AV-shGATA4 RNA and AV-shCTRL(1×1011 PFU/mL，10 μL)at the spinal L4-L6 segments, separately, 48 h before EA intervention. The mechanical pain threshold and thermal pain threshold of the affected limb were detected before molding, 7 days following molding and 60 min after EA. The expressions of adenosine A1 receptor and GATA4 protein in the spinal cord (L4-L6) were detected by Western blot. Outcomes of the first part showed that compared with the control group, no significant changes were found in the mechanical and thermal pain thresholds in both AV-shCTRL and AV-shGATA4 RNA groups and in the expression of spinal GATA4 protein of the AV-shCTRL group (P＞0.05). The expression of spinal GATA4 protein of the AV-shGATA4 RNA group was significantly lower than that of the AV-shCTRL group (P<0.05). In the second part of the study, before CCI modeling, there were no significant differences among the five groups in the mechanical and thermal pain thre-sholds (P> 0.05). On the 7th day following modeling, the mechanical and thermal pain thresholds were significantly lowered in compa-rison with their own pre-modeling of each group and with the sham operation group (P<0.05). At 60 min after EA and compared with the model group, the mechanical and thermal pain thresholds were significantly increased in both the EA and EA+AV-shCTRL groups (P<0.05) but not in the EA+AV-shGATA4 RNA group (P>0.05), suggesting a critical involvement of GATA4 in EA analgesia. The expression levels of adenosine A1 receptor and GATA4 protein were significantly increased in the model group than in the sham operation group (P<0.05), and considerably further up-regulated in both EA and EA+AV-shCTRL groups (P< 0.05), rather than in the EA+AV-shGATA4 RNA group (P>0.05), suggesting that the effects of EA in up-regulating the expression of A1 receptor and GATA4 were eliminated after silencing GATA4 protein. EA of ST36 and LR3 can relieve pain by increasing the expression of adenosine A1 receptor of the lumbar spinal cord in neuropathic pain rats, which is probably mediated by GATA4 protein.

Bone Morphogenetic Protein 9 Is a Paracrine Factor Controlling Liver Sinusoidal Endothelial Cell Fenestration and Protecting Against Hepatic Fibrosis.

Bone morphogenetic protein 9 (BMP9) is a circulating factor produced by hepatic stellate cells that plays a critical role in vascular quiescence through its endothelial receptor activin receptor-like kinase 1 (ALK1). Mutations in the gene encoding ALK1 cause hereditary hemorrhagic telangiectasia type 2, a rare genetic disease presenting hepatic vessel malformations. Variations of both the circulating levels and the hepatic mRNA levels of BMP9 have been recently associated with various forms of hepatic fibrosis. However, the molecular mechanism that links BMP9 with liver diseases is still unknown. Here, we report that Bmp9 gene deletion in 129/Ola mice triggers hepatic perisinusoidal fibrosis that was detectable from 15weeks of age. An inflammatory response appeared within the same time frame as fibrosis, whereas sinusoidal vessel dilation developed later on. Proteomic and mRNA analyses of primary liver sinusoidal endothelial cells (LSECs) both revealed that the expression of the LSEC-specifying transcription factor GATA-binding protein 4 was strongly reduced in Bmp9 gene knockout (Bmp9-KO) mice as compared with wild-type mice. LSECs from Bmp9-KO mice also lost the expression of several terminal differentiation markers (Lyve1, Stab1, Stab2, Ehd3, Cd209b, eNos, Maf, Plvap). They gained CD34 expression and deposited a basal lamina, indicating that they were capillarized. Another main characteristic of differentiated LSECs is the presence of permeable fenestrae. LSECs from Bmp9-KO mice had a significantly reduced number of fenestrae. This was already observable in 2-week-old pups. Moreover, we could show that addition of BMP9 to primary cultures of LSECs prevented the loss of their fenestrae and maintained the expression levels of Gata4 and Plvap. Conclusion: Taken together, our observations show that BMP9 is a key paracrine regulator of liver homeostasis, controlling LSEC fenestration and protecting against perivascular hepatic fibrosis.