CD19-chimeric antigen receptor-invariant natural killer T cells transactivate NK cells and reduce alloreactivity.

Invariant natural killer T (iNKT) cells are a small fraction of T lymphocytes with strong cytotoxic and immunoregulatory properties. We previously showed that human culture-expanded iNKT cells prevent alloreactivity and lyse primary leukemia blasts. Here, iNKT cells have several advantages over T cells based on their immunoregulatory capabilities. Since chimeric antigen receptors (CARs) increase the benefit of immune effector cells, they play a crucial role in improvement of cytotoxic abilities of novel cellular therapeutics such as iNKT cells. In the present study, we investigated transactivation of NK cells and prevention of alloreactivity through iNKT cells transduced with a CD19-directed CAR. iNKT cells were isolated by magnetic cell separation from peripheral blood mononuclear cells and transduced with a CD19-CAR retrovirus. Transduction efficiency, purity and cell subsets were measured by flow cytometry. Transactivation and cytotoxicity assays have been established to investigate the ability of CD19-CAR-iNKT cells to transactivate primary NK cells. A mixed lymphocyte reaction (MLR) was performed to explore the inhibition of alloreactive CD3+ T cells by CD19-CAR-iNKT cells. CD19-CAR-iNKT cells are able to transactivate NK cells independent of cell contact: The expression of activation marker CD69 was significantly increased and also production of the proinflammatory cytokine interferon-gamma was higher in NK cells pretreated with CD19-CAR-iNKT cells. Consequently, the cytotoxic activity of such NK cells was significantly increased being able to lyse leukemia cells more effectively than without prior transactivation. Adding CD19-CAR-iNKT cells to an MLR resulted in a decreased expression of the T cell activation marker CD25 on alloreactive CD3+ T lymphocytes stimulated with HLA mismatched dendritic cells. Also, the proliferation of alloreactive CD3+ T lymphocytes was significantly reduced in this setting. We demonstrate that CD19-CAR-iNKT cells keep their immunoregulatory properties despite transduction with a CAR making them an attractive effector cell population for application after allogeneic hematopoietic cell transplantation. By transactivating NK cells, increasing their cytotoxic activity and suppressing alloreactive T cells, they might further improve outcomes through prevention of both relapse and graft-versus-host disease.

Activation of automethylated PRC2 by dimerization on chromatin.

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator that trimethylates lysine 27 of histone 3 (H3K27me3) and is essential for embryonic development and cellular differentiation. H3K27me3 is associated with transcriptionally repressed chromatin and is established when PRC2 is allosterically activated upon methyl-lysine binding by the regulatory subunit EED. Automethylation of the catalytic subunit enhancer of zeste homolog 2 (EZH2) stimulates its activity by an unknown mechanism. Here, we show that human PRC2 forms a dimer on chromatin in which an inactive, automethylated PRC2 protomer is the allosteric activator of a second PRC2 that is poised to methylate H3 of a substrate nucleosome. Functional assays support our model of allosteric trans-autoactivation via EED, suggesting a previously unknown mechanism mediating context-dependent activation of PRC2. Our work showcases the molecular mechanism of auto-modification-coupled dimerization in the regulation of chromatin-modifying complexes.

Ophiopogonin D' inhibited tumour growth and metastasis of anaplastic thyroid cancer by modulating JUN/RGS4 signalling.

Anaplastic thyroid cancer (ATC), an aggressive malignancy with virtually 100% disease-specific mortality, has long posed a formidable challenge in oncology due to its resistance to conventional treatments and the severe side effects associated with current regimens such as doxorubicin chemotherapy. Consequently, there was urgent need to identify novel candidate compounds that could provide innovative therapeutic strategies for ATC. Ophiopogonin D' (OPD'), a triterpenoid saponin extracted, yet its roles in ATC has not been reported. Our data demonstrated that OPD' potently inhibited proliferation and metastasis of ATC cells, promoting cell cycle arrest and apoptosis. Remarkably, OPD' impeded growth and metastasis of ATC in vitro and in vivo, displaying an encouraging safety profile. Regulator of G-protein signalling 4 (RGS4) expression was significantly up-regulated in ATC compared to normal tissues, and this upregulation was suppressed by OPD' treatment. Mechanistically, we elucidated that the transcription factor JUN bound to the RGS4 promoter, driving its transactivation. However, OPD' interacted with JUN, attenuating its transcriptional activity and thereby disrupting RGS4 overexpression. In summary, our research revealed that OPD' bound with JUN, which in turn resulted in the suppression of transcriptional activation of RGS4, thereby eliciting cell cycle arrest and apoptosis in ATC cells. These findings could offer promise in the development of high-quality candidate compounds for treatment in ATC.

Valsartan as a prophylactic treatment against breast cancer development and niche activation: What molecular sequels follow chronic AT-1R blockade?

AIMS: Transactivation of insulin-growth-factor-receptor (IGF-1R) by angiotensin-II-type-1-receptor (AT-1R) was only demonstrated in vascular-smooth-muscle cells and has never been tested in breast-cancer (BC). This investigation addressed the impact of chronic AT-1R blockade by valsartan (Val) on possible concurrent AT-1R/IGF-1R signaling inhibition, regressing BC-tumor-microenvironment (TME) cellular components activation, and hindering BC development. MAIN METHODS: The effect of different Val doses (10, 20, 40 & 80 mg/kg/day for 490 days) was tested on dimethylbenz(a)anthracene (DMBA)-induced progesterone-promoted-BC in rats. The influence on intratumoral/circulating angiotensin-II (ANG-II) levels and AT-1R/Mas-R immunofluorescent-expression were assessed. The potential AT-1R/IGF-1R crosstalk within TME-BC-stem-cells (BCSCs) and cancer-associated-fibroblasts (CAFs) was evaluated by fluorescently marking these cells and locating the immunofluorescently-stained AT-1R/IGF-1R in them using confocal-laser-microscopy and further quantified by flow cytometry. In addition, the molecular alterations following blocking AT-1R were inspected including determining Src; crucial for IGF-1R transactivation by AT-1R, Notch-1; IGF-IR transcriptional-regulator, and PI3K/Akt &IL-6/STAT expression. Further, the suppression of CSCs' capabilities to maintain pluripotency, stemness features, epithelial-to-mesenchymal-transition (EMT), and angiogenesis was evaluated by assessing NANOG gene, aldehyde-dehydrogenase (ALDH), N-cadherin and vascular-endothelial-growth-factor (VEGF), respectively. Furthermore, the proliferative marker; Ki-67, was detected by immunostaining, and tumors were histologically graded using Elston-Ellis-modified-Scarff-Bloom-Richardson method. KEY FINDINGS: Prophylactic Val significantly reduced tumor size, prolonged latency, reduced tumor histopathologic grade, decreased circulating/intratumoral-ANG-II levels, increased Mas-R, and decreased AT1R expression. AT-1R/IGF-1R were co-expressed with a high correlation coefficient on CAFs/BCSCs. Moreover, Val significantly attenuated IGF-1R transactivation and transcriptional regulation via Src and Notch-1 genes' downregulation and reduced Src/IGF-IR-associated PI3K/Akt and IL-6/STAT3 signaling. Further, Val significantly decreased intratumoral NANOG, ALDH, N-cadherin, VEGF, and Ki-67 levels. SIGNIFICANCE: Chronic Val administration carries a potential for repurposing as adjuvant or conjunct therapy for patients at high risk for BC.

Synthesis and Anti-Cancer Activity of the Novel Selective Glucocorticoid Receptor Agonists of the Phenylethanolamine Series.

Glucocorticoids (GCs) are widely used for treating hematological malignancies despite their multiple adverse effects. The biological response to GCs relies on glucocorticoid receptor (GR) transrepression (TR) that mediates the anticancer effects and transactivation (TA) associated with the side effects. Selective GR agonists (SEGRAs) preferentially activating GR TR could offer greater benefits in cancer treatment. One of the well-characterized SEGRAs, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride (CpdA), exhibited anticancer activity; however, its translational potential is limited due to chemical instability. To overcome this limitation, we obtained CpdA derivatives, CpdA-01-CpdA-08, employing two synthetic strategies and studied their anti-tumor activity: 4-(1-hydroxy-2-(piperidin-1-yl)ethyl)phenol or CpdA-03 demonstrated superior GR affinity and stability compared to CpdA. In lymphoma Granta and leukemia CEM cell lines, CpdA-03 ligand exhibited typical SEGRA properties, inducing GR TR without triggering GR TA. CpdA-03 effects on cell viability, growth, and apoptosis were similar to the reference GR ligand, dexamethasone (Dex), and the source compound CpdA. In vivo testing of CpdA-03 activity against lymphoma on the transplantable P388 murine lymphoma model showed that CpdA-03 reduced tumor volume threefold, outperforming Dex and CpdA. In conclusion, in this work, we introduce a novel SEGRA CpdA-03 as a promising agent for lymphoma treatment with fewer side effects.

Protopanaxadiol derivative: A plant origin of novel selective glucocorticoid receptor modulator with anti-inflammatory effect.

Constant efforts have been made to move towards maintaining the positive anti-inflammatory functions of glucocorticoids (GCs) while minimizing side effects. The anti-inflammatory effect of GCs is mainly attributed to the inhibition of major inflammatory pathways such as NF-κB through GR transrepression, while its side effects are mainly mediated by transactivation. Here, we investigated the selective glucocorticoid receptor modulator (SGRM)-like properties of a plant-derived compound. In this study, glucocorticoid receptor (GR)-mediated alleviation of inflammation by SP-8 was investigated by a combination of in vitro, in silico, and in vivo approaches. Molecular docking and cellular thermal shift assay suggested that SP-8 bound stably to the active site of GR via hydrogen bonding and hydrophobic interactions. SP-8 activated GR, induced GR nuclear translocation, and inhibited NF-κB pathway activation. Furthermore, SP-8 did not up-regulate the gene and protein expression of PEPCK and TAT in HepG2 cells, and it did not induce fat deposition like GC and has little effect on bone metabolism. Interestingly, SP-8 upregulated GR protein expression and did not cause GR phosphorylation at Ser211 in RAW264.7 cells. This work proved that SP-8 dissociated characteristics of transrepression and transactivation can be separated. In addition, the in vitro and in vivo anti-inflammatory effects of SP-8 were confirmed in LPS-induced RAW 264.7 cells and in a mouse model of DSS-induced ulcerative colitis, respectively. In conclusion, SP-8 might serve as a potential SGRM and might hold great potential for therapeutic use in inflammatory diseases.

Activation of the CDK7 Gene, Coding for the Catalytic Subunit of the Cyclin-Dependent Kinase (CDK)-Activating Kinase (CAK) and General Transcription Factor II H, by the Trans-Activator Protein Tax of Human T-Cell Leukemia Virus Type-1.

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The trans-activator protein Tax of HTLV-1 plays crucial roles in leukemogenesis by promoting proliferation of virus-infected cells through activation of growth-promoting genes. However, critical target genes are yet to be elucidated. We show here that Tax activates the gene coding for cyclin-dependent kinase 7 (CDK7), the essential component of both CDK-activating kinase (CAK) and general transcription factor TFIIH. CAK and TFIIH play essential roles in cell cycle progression and transcription by activating CDKs and facilitating transcriptional initiation, respectively. Tax induced CDK7 gene expression not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs) along with increased protein expression. Tax stimulated phosphorylation of CDK2 and RNA polymerase II at sites reported to be mediated by CDK7. Tax activated the CDK7 promoter through the NF-κB pathway, which mainly mediates cell growth promotion by Tax. Knockdown of CDK7 expression reduced Tax-mediated induction of target gene expression and cell cycle progression. These results suggest that the CDK7 gene is a crucial target of Tax-mediated trans-activation to promote cell proliferation by activating CDKs and transcription.

PAR2 activation on human tubular epithelial cells engages converging signaling pathways to induce an inflammatory and fibrotic milieu.

Key features of chronic kidney disease (CKD) include tubulointerstitial inflammation and fibrosis. Protease activated receptor-2 (PAR2), a G-protein coupled receptor (GPCR) expressed by the kidney proximal tubular cells, induces potent proinflammatory responses in these cells. The hypothesis tested here was that PAR2 signalling can contribute to both inflammation and fibrosis in the kidney by transactivating known disease associated pathways. Using a primary cell culture model of human kidney tubular epithelial cells (HTEC), PAR2 activation induced a concentration dependent, PAR2 antagonist sensitive, secretion of TNF, CSF2, MMP-9, PAI-1 and CTGF. Transcription factors activated by the PAR2 agonist 2F, including NFκB, AP1 and Smad2, were critical for production of these cytokines. A TGF-ß receptor-1 (TGF-ßRI) kinase inhibitor, SB431542, and an EGFR kinase inhibitor, AG1478, ameliorated 2F induced secretion of TNF, CSF2, MMP-9, and PAI-1. Whilst an EGFR blocking antibody, cetuximab, blocked PAR2 induced EGFR and ERK phosphorylation, a TGF-ßRII blocking antibody failed to influence PAR2 induced secretion of PAI-1. Notably simultaneous activation of TGF-ßRII (TGF-ß1) and PAR2 (2F) synergistically enhanced secretion of TNF (2.2-fold), CSF2 (4.4-fold), MMP-9 (15-fold), and PAI-1 (2.5-fold). In summary PAR2 activates critical inflammatory and fibrotic signalling pathways in human kidney tubular epithelial cells. Biased antagonists of PAR2 should be explored as a potential therapy for CKD.

Post-translational modifications of p65: state of the art.

P65, a protein subunit of NF-κB, is a widely distributed transcription factor in eukaryotic cells and exerts diverse regulatory functions. Post-translational modifications such as phosphorylation, acetylation, methylation and ubiquitination modulate p65 transcriptional activity and function, impacting various physiological and pathological processes including inflammation, immune response, cell death, proliferation, differentiation and tumorigenesis. The intricate interplay between these modifications can be antagonistic or synergistic. Understanding p65 post-translational modifications not only elucidates NF-κB pathway regulation but also facilitates the identification of therapeutic targets and diagnostic markers for associated clinical conditions.

Signaling by Neutrophil G Protein-Coupled Receptors that Regulate the Release of Superoxide Anions.

In human peripheral blood, the neutrophil granulocytes (neutrophils) are the most-abundant white blood cells. These professional phagocytes are rapidly recruited from the bloodstream to inflamed tissues by chemotactic factors that signal danger. Neutrophils, which express many receptors that are members of the large family of G protein-coupled receptors (GPCRs), are critical for the elimination of pathogens and inflammatory insults, as well as for the resolution of inflammation leading to tissue repair. Danger-signaling molecular patterns such as the N-formylated peptides that are formed during bacterial and mitochondrial protein synthesis and recognized by formyl peptide receptors (FPRs) and free fatty acids recognized by free fatty acid receptors (FFARs) regulate neutrophil functions. Short peptides and short chain fatty acids activate FPR1 and FFA2R, respectively, while longer peptides and fatty acids activate FPR2 and GPR84, respectively. The activation profiles of these receptors include the release of reactive oxygen species (ROS) generated by the NADPH oxidase. Activation of the oxidase and the production of ROS are processes that are regulated by proinflammatory mediators, including TNFα and GM-CSF. The receptors have signaling and functional similarities, although there are also important differences, not only between the two closely related neutrophil FPRs, but also between the FPRs and the FFARs. In neutrophils, these receptors never walk alone, and additional mechanistic insights into the regulation of the GPCRs and the novel regulatory mechanisms underlying the activation of NADPH oxidase advance our understanding of the role of receptor transactivation in the regulation of inflammatory reactions.

Fusion of a rice endogenous N-methylpurine DNA glycosylase to a plant adenine base transition editor ABE8e enables A-to-K base editing in rice plants.

Engineering of a new type of plant base editor for simultaneous adenine transition and transversion within the editing window will greatly expand the scope and potential of base editing in directed evolution and crop improvement. Here, we isolated a rice endogenous hypoxanthine excision protein, N-methylpurine DNA glycosylase (OsMPG), and engineered two plant A-to-K (K = G or T) base editors, rAKBE01 and rAKBE02, for simultaneous adenine transition and transversion base editing in rice by fusing OsMPG or its mutant mOsMPG to a plant adenine transition base editor, ABE8e. We further coupled either OsMPG or mOsMPG with a transactivation factor VP64 to generate rAKBE03 and rAKBE04, respectively. Testing these four rAKBEs, at five endogenous loci in rice protoplasts, indicated that rAKBE03 and rAKBE04 enabled higher levels of A-to-G base transitions when compared to ABE8e and ABE8e-VP64. Furthermore, whereas rAKBE01 only enabled A-to-C/T editing at one endogenous locus, in comparison with rAKBE02 and rAKBE03, rAKBE04 could significantly improve the A-to-C/T base transversion efficiencies by up to 6.57- and 1.75-fold in the rice protoplasts, respectively. Moreover, although no stable lines with A-to-C transversion were induced by rAKBE01 and rAKBE04, rAKBE04 could enable simultaneous A-to-G and A-to-T transition and transversion base editing, at all the five target loci, with the efficiencies of A-to-G transition and A-to-T transversion editing ranging from 70.97 to 92.31% and 1.67 to 4.84% in rice stable lines, respectively. Together, these rAKBEs enable different portfolios of editing products and, thus, now expands the potential of base editing in diverse application scenario for crop improvement. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00138-8.

Receptor mediated biological activities of phytoestrogens.

Phytoestrogens are plant-derived compounds that have chemical structures and functions similar to estrogen. Phytoestrogens act as ligand-inducible transcription factors involved in cellular growth by binding to estrogen receptors (ERs), specifically ER alpha (ERα) and beta (ERß). Through this mechanism, phytoestrogens have a physiological function similar to that of the female hormone 17ß-estradiol (E2), which can be useful in treating osteoporosis, cardiovascular disease, and cancer. Furthermore, phytoestrogens have been found to elicit various cellular responses depending on their affinity for ERs; in particular, they show a greater affinity with for ERß. This study aimed to comprehensively analyze the mode of action of eight phytoestrogens, namely kaempferol, coumestrol, glycitein, apigenin, daidzein, genistein, equol, and resveratrol, by evaluating their estrogenic activity as ER ligands. Based on the bioluminescence resonance energy transfer (BRET)-based ER dimerization and transactivation assay results, all the phytoestrogens tested were identified as estrogen agonists by mediating ERα and ERß dimerization. The specific binding and functions of ERα and ERß were distinguished by differentiating between their dimerization activity. In addition, this study contributes to advancing our understanding of the overall mechanism of action involving both ERs.

Inhibitors of the transactivation domain of androgen receptor as a therapy for prostate cancer.

The androgen receptor (AR) is a modular transcription factor which functions as a master regulator of gene expression. AR protein is composed of three functional domains; the ligand-binding domain (LBD); DNA-binding domain (DBD); and the intrinsically disordered N-terminal transactivation domain (TAD). AR is transactivated upon binding to the male sex hormone testosterone and other androgens. While the AR may tolerate loss of its LBD, the TAD contains activation function-1 (AF-1) that is essential for all AR transcriptional activity. AR is frequently over-expressed in most prostate cancer. Currently, androgen deprivation therapy (ADT) in the form of surgical or chemical castration remains the standard of care for patients with high risk localized disease, advanced and metastatic disease, and those patients that experience biochemical relapse following definitive primary treatment. Patients with recurrent disease that receive ADT will ultimately progress to lethal metastatic castration-resistant prostate cancer. In addition to ADT not providing a cure, it is associated with numerous adverse effects including cardiovascular disease, osteoporosis and sexual dysfunction. Recently there has been a renewed interest in investigating the possibility of using antiandrogens which competitively bind the AR-LBD without ADT for patients with hormone sensitive, non-metastatic prostate cancer. Here we describe a class of compounds termed AR transactivation domain inhibitors (ARTADI) and their mechanism of action. These compounds bind to the AR-TAD to inhibit AR transcriptional activity in the absence and presence of androgens. Thus these inhibitors may have utility in preventing prostate cancer growth in the non-castrate setting.

The cellular Notch1 protein promotes KSHV reactivation in an Rta-dependent manner.

The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi's sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi's sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma-secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. Reduction of KSHV production is associated with gene-specific reduction of viral transcription in both Vero and PEL cells. Specific inhibition of Notch1 by siRNA partially reduces the production of infectious KSHV, and NICD1 forms promoter-specific complexes with viral DNA during reactivation. We conclude that constitutive Notch activity is required for the robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk/DNA complex during viral reactivation. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA-binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces the expression of specific viral genes yet does not affect the growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta's ability to redistribute RBP-Jk DNA binding to the virus during reactivation.

Trans-Activation of the Coactivator-Associated Arginine Methyltransferase 1 (Carm1) Gene by the Oncogene Product Tax of Human T-Cell Leukemia Virus Type 1.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the Carm1 gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene IL-2Rα, concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of IL-2Rα and Cyclin D2 gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the Carm1 gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the Carm1 gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.

Caveolin-1 differentially regulates the transforming growth factor-ß and epidermal growth factor signaling pathways in MDCK cells.

Caveolin-1 is critical for interacting with the TGF-ß receptor (TGFßR) and EGF receptor (EGFR) signaling, often observed in advanced cancers and tissue fibrosis. However, the mechanism underlying caveolin-1-mediated transactivation of TGFßR and EGFR signaling remains unclear. Therefore, we sought to determine whether caveolin-1 is involved in canonical and non-canonical TGFßR and EGFR signaling transactivation in this study. Methyl-ß-cyclodextrin (MßCD) was used to disrupt the cholesterol-containing membranes domains, and the caveolin-1 scaffolding domain (CSD) peptide was used to mimic the CSD of caveolin-1. Additionally, we transfected the Madin-Darby canine kidney cells with wild-type or phosphorylation-defective caveolin-1. We discovered that tyrosine 14 of caveolin-1 was critical for the negative regulation of TGFßR and EGFR canonical signaling. On the contrary, caveolin-1 inhibited TGF-ß1-induced ERK2 activation independent of tyrosine 14 phosphorylation. Although EGF failed to induce Smad3 phosphorylation in caveolin-1 knockdown cells, it activated Smad3 upon MßCD co-treatment, indicating that caveolin-1 indirectly regulated the non-canonical pathway of EGF. In conclusion, caveolin-1 differentially modulates TGFßR and EGFR signaling. Thus, targeting caveolin-1 is a potential strategy for treating diseases involving TGF-ß1 and EGF signaling.

Cannabidiol mitigates radiation-induced intestine ferroptosis via facilitating the heterodimerization of RUNX3 with CBFß thereby promoting transactivation of GPX4.

Radiation enteritis remains a major challenge for radiotherapy against abdominal and pelvic malignancies. Nevertheless, there is no approved effective therapy to alleviate irradiation (IR)-induced gastrointestinal (GI) toxicity. In the current study, Cannabidiol (CBD) was found to mitigate intestinal injury by GPX4-mediated ferroptosis resistance upon IR exposure. RNA-sequencing was employed to investigate the underlying mechanism involved in the radio-protective effect of CBD, wherein runt-related transcription factor 3 (RUNX3) and its target genes were changed significantly. Further experiment showed that the transactivation of GPX4 triggered by the direct binding of RUNX3 to its promoter region, or by stimulating the transcriptional activity of NF-κB via RUNX3-mediated LILRB3 upregulation was critical for the anti-ferroptotic effect of CBD upon IR injury. Specially, CBD was demonstrated to be a molecular glue skeleton facilitating the heterodimerization of RUNX3 with its transcriptional chaperone core-biding factor ß (CBFß) thereby promoting their nuclear localization and the subsequent transactivation of GPX4 and LILRB3. In short, our study provides an alternative strategy to counteract IR-induced enteritis during the radiotherapy on abdominal/pelvic neoplasms.

Transient expression analysis of promoters of okra enation leaf curl virus in Nicotiana benthamiana, cotton and okra plants.

Viral promoters can be used to drive heterologous gene expression in transgenic plants. As part of our quest to look for new promoters, we have explored, for the first time, the promoters of okra enation leaf curl virus (OELCuV), a begomovirus infecting okra (Abelmoschus esculentus). The Rep and CP promoters of OELCuV fused with the gfp reporter gene, were expressed transiently in the natural host okra and the laboratory host cotton and Nicotiana benthamiana. The expression levels of the promoters were quantified through confocal laser scanning microscopy and GFP assay in N. benthamiana and okra. The results indicated that the Rep promoter was more active than the CP promoter, whose activity was similar to that of CaMV 35S promoter. Additionally, the Rep and CP promoters showed increase of expression, probably due to transactivation, when assayed following inoculation of OELCuV and betasatellite DNAs in cotton plants. A moderate increase in promoter activity in N. benthamiana was also seen, when assayed following the inoculation of the heterologous begomovirus Sri Lankan cassava mosaic virus.

DELLA proteins recruit the Mediator complex subunit MED15 to coactivate transcription in land plants.

DELLA proteins are negative regulators of the gibberellin response pathway in angiosperms, acting as central hubs that interact with hundreds of transcription factors (TFs) and regulators to modulate their activities. While the mechanism of TF sequestration by DELLAs to prevent DNA binding to downstream targets has been extensively documented, the mechanism that allows them to act as coactivators remains to be understood. Here, we demonstrate that DELLAs directly recruit the Mediator complex to specific loci in Arabidopsis, facilitating transcription. This recruitment involves DELLA amino-terminal domain and the conserved MED15 KIX domain. Accordingly, partial loss of MED15 function mainly disrupted processes known to rely on DELLA coactivation capacity, including cytokinin-dependent regulation of meristem function and skotomorphogenic response, gibberellin metabolism feedback, and flavonol production. We have also found that the single DELLA protein in the liverwort Marchantia polymorpha is capable of recruiting MpMED15 subunits, contributing to transcriptional coactivation. The conservation of Mediator-dependent transcriptional coactivation by DELLA between Arabidopsis and Marchantia implies that this mechanism is intrinsic to the emergence of DELLA in the last common ancestor of land plants.

Dichotomous transactivation domains contribute to growth inhibitory and promotion functions of TAp73.

The transcription factor p73, a member of the p53 tumor-suppressor family, regulates cell death and also supports tumorigenesis, although the mechanistic basis for the dichotomous functions is poorly understood. We report here the identification of an alternate transactivation domain (TAD) located at the extreme carboxyl (C) terminus of TAp73ß, a commonly expressed p73 isoform. Mutational disruption of this TAD significantly reduced TAp73ß's transactivation activity, to a level observed when the amino (N)-TAD that is similar to p53's TAD, is mutated. Mutation of both TADs almost completely abolished TAp73ß's transactivation activity. Expression profiling highlighted a unique set of targets involved in extracellular matrix-receptor interaction and focal adhesion regulated by the C-TAD, resulting in FAK phosphorylation, distinct from the N-TAD targets that are common to p53 and are involved in growth inhibition. Interestingly, the C-TAD targets are also regulated by the oncogenic, amino-terminal-deficient DNp73ß isoform. Consistently, mutation of C-TAD reduces cellular migration and proliferation. Mechanistically, selective binding of TAp73ß to DNAJA1 is required for the transactivation of C-TAD target genes, and silencing DNAJA1 expression abrogated all C-TAD-mediated effects. Taken together, our results provide a mechanistic basis for the dichotomous functions of TAp73 in the regulation of cellular growth through its distinct TADs.