Pharmacological Inhibition of STAT6 Ameliorates Myeloid Fibroblast Activation and Alternative Macrophage Polarization in Renal Fibrosis.

A hallmark of chronic kidney disease is renal fibrosis, which can result in progressive loss of kidney function. Currently, there is no effective therapy for renal fibrosis. Therefore, there is an urgent need to identify potential drug targets for renal fibrosis. In this study, we examined the effect of a selective STAT6 inhibitor, AS1517499, on myeloid fibroblast activation, macrophage polarization, and development of renal fibrosis in two experimental murine models. To investigate the effect of STAT6 inhibition on myeloid fibroblast activation, macrophage polarization, and kidney fibrosis, wild-type mice were subjected to unilateral ureteral obstruction or folic acid administration and treated with AS1517499. Mice treated with vehicle were used as control. At the end of experiments, kidneys were harvested for analysis of myeloid fibroblast activation, macrophage polarization, and renal fibrosis and function. Unilateral ureteral obstruction or folic acid administration induced STAT6 activation in interstitial cells of the kidney, which was significantly abolished by AS1517499 treatment. Mice treated with AS1517499 accumulated fewer myeloid fibroblasts and myofibroblasts in the kidney with ureteral obstruction or folic acid nephropathy compared with vehicle-treated mice. Moreover, AS1517499 significantly suppressed M2 macrophage polarization in the injured kidney. Furthermore, AS1517499 markedly reduced the expression levels of extracellular matrix proteins, and development of kidney fibrosis and dysfunction. These findings suggest that AS1517499 inhibits STAT6 activation, suppresses myeloid fibroblast activation, reduces M2 macrophage polarization, attenuates extracellular matrix protein production, and preserves kidney function. Therefore, targeting STAT6 with AS1517499 is a novel therapeutic approach for chronic kidney disease.

A non-canonical type 2 immune response coordinates tuberculous granuloma formation and epithelialization.

The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-sequencing analysis of zebrafish granulomas and analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.

Activation of adenosine A3 receptor reduces early brain injury by alleviating neuroinflammation after subarachnoid hemorrhage in elderly rats.

The incidence of subarachnoid hemorrhage (SAH) and hazard ratio of death increase with age. Overactivation of microglia contributes to brain damage. This study aimed to investigate the effects of A3 adenosine receptors (A3R) activation on neurofunction and microglial phenotype polarization in the context of SAH in aged rats. The A3R agonist (CI-IB-MECA) and antagonist (MRS1523) were used in the SAH model. Microglia were cultured to mimic SAH in the presence or absence of CI-IB-MECA and/or siRNA for A3R. The neurofunction and status of the microglial phenotype were evaluated. The P38 inhibitor SB202190 and the STAT6 inhibitor AS1517499 were used to explore the signaling pathway. The results showed that SAH induced microglia to polarize to the M(LPS) phenotype both in vivo and in vitro. CI-IB-MECA distinctly skewed microglia towards the M(IL-4) phenotype and ameliorated neurological dysfunction, along with the downregulation of inflammatory cytokines. Knockdown of A3R or inhibition of P38 and/or STAT6 weakened the effects of CI-IB-MECA on microglial phenotypic shifting. Collectively, our findings suggest that activation of A3R exerted anti-inflammatory and neuroprotective effects by regulating microglial phenotype polarization through P38/STAT6 pathway and indicated that A3R agonists may be a promising therapeutic options for the treatment of brain injury after SAH.

IL-4/IL-13 upregulates Sonic hedgehog expression to induce allergic airway epithelial remodeling.

We have previously demonstrated that upregulation of Sonic hedgehog (SHH) expression in allergic airway epithelia essentially contributes to the goblet cell metaplasia and mucous hypersecretion. However, the mechanism underlying the upregulation of SHH expression remains completely unknown. In cultured human airway epithelial cells, IL-4/IL-13 but not IL-5 robustly induces the mRNA and protein expression of SHH and in turn activates SHH signaling by promoting the JAK/STAT6-controlling transcription of SHH gene. Moreover, intratracheal instillation of IL-4 and/or IL-13 robustly activates STAT6 and concomitantly upregulates SHH expression in mouse airway epithelia, whereas, in Club cell 10-kDa protein (CC10)-positive airway epithelial cells of children with asthma, activated STAT6 closely correlates with the increased expression of SHH and high activity of SHH signaling. Finally, intratracheal inhibition of STAT6 by AS-1517499 significantly diminished the allergen-induced upregulation of SHH expression, goblet cell phenotypes, and airway hyperresponsiveness, in an ovalbumin- or house dust mite-induced mouse model with allergic airway inflammation,. Together, upregulation of SHH expression by IL-4/IL-13-induced JAK/STAT6 signaling contributes to allergic airway epithelial remodeling, and this study thus provides insight into how morphogen signaling is coordinated with Th2 cytokine pathways to regulate tissue remodeling in chronic airway diseases.

Cytochrome P450 1A1 enhances Arginase-1 expression, which reduces LPS-induced mouse peritonitis by targeting JAK1/STAT6.

The polarization of macrophages is critical to inflammation and tissue repair, with unbalanced macrophage polarization associated with critical dysfunctions of the immune system. Cytochrome P450 1A1 (CYP1A1) is a hydroxylase mainly controlled by the inflammation-limiting aryl hydrocarbon receptor (AhR), which plays a critical role in mycoplasma infection, oxidative stress injury, and cancer. Arginase-1 (Arg-1) is a surrogate for polarized alternative macrophages and is important to the production of nitric oxide (NO) by the modulation of arginine. In the present study, we found CYP1A1 to be upregulated in IL-4-stimulated mouse peritoneal macrophages (PMs) and human peripheral blood monocytes. Using CYP1A1-overexpressing RAW264.7 cells (CYP1A1/RAW) we found that CYP1A1 augmented Arg-1 expression by strengthening the activation of the JAK1/STAT6 signaling pathway in macrophages treated with IL-4. 15(S)-HETE, a metabolite of CYP1A1 hydroxylase, was elevated in IL-4-induced CYP1A1/RAW cells. Further, in macrophages, the loss-of-CYP1A1-hydroxylase activity was associated with reduced IL-4-induced Arg-1 expression due to impaired 15(S)-HETE generation. Of importance, CYP1A1 overexpressing macrophages reduced the inflammation associated with LPS-induced peritonitis. Taken together, these findings identified a novel signaling axis, CYP1A1-15(S)-HETE-JAK1-STAT6, that may be a promising target for the proper maintenance of macrophage polarization and may also be a means by which to treat immune-related disease due to macrophage dysfunction.

STAT3/5 Inhibitors Suppress Proliferation in Bladder Cancer and Enhance Oncolytic Adenovirus Therapy.

The JAK-STAT signalling pathway regulates cellular processes like cell division, cell death and immune regulation. Dysregulation has been identified in solid tumours and STAT3 activation is a marker for poor outcome. The aim of this study was to explore potential therapeutic strategies by targeting this pathway in bladder cancer (BC). High STAT3 expression was detected in 51.3% from 149 patient specimens with invasive bladder cancer by immunohistochemistry. Protein expression of JAK, STAT and downstream targets were confirmed in 10 cell lines. Effects of the JAK inhibitors Ruxolitinib and BSK-805, and STAT3/5 inhibitors Stattic, Nifuroxazide and SH-4-54 were analysed by cell viability assays, immunoblotting, apoptosis and cell cycle progression. Treatment with STAT3/5 but not JAK1/2 inhibitors reduced survival, levels of phosphorylated STAT3 and Cyclin-D1 and increased apoptosis. Tumour xenografts, using the chicken chorioallantoic membrane (CAM) model responded to Stattic monotherapy. Combination of Stattic with Cisplatin, Docetaxel, Gemcitabine, Paclitaxel and CDK4/6 inhibitors showed additive effects. The combination of Stattic with the oncolytic adenovirus XVir-N-31 increased viral replication and cell lysis. Our results provide evidence that inhibitors against STAT3/5 are promising as novel mono- and combination therapy in bladder cancer.

Non-lethal proteasome inhibition activates pro-tumorigenic pathways in multiple myeloma cells.

Multiple myeloma (MM) is a haematological malignancy being characterized by clonal plasma cell proliferation in the bone marrow. Targeting the proteasome with specific inhibitors (PIs) has been proven a promising therapeutic strategy and PIs have been approved for the treatment of MM and mantle-cell lymphoma; yet, while outcome has improved, most patients inevitably relapse. As relapse refers to MM cells that survive therapy, we sought to identify the molecular responses induced in MM cells after non-lethal proteasome inhibition. By using bortezomib (BTZ), epoxomicin (EPOX; a carfilzomib-like PI) and three PIs, namely Rub999, PR671A and Rub1024 that target each of the three proteasome peptidases, we found that only BTZ and EPOX are toxic in MM cells at low concentrations. Phosphoproteomic profiling after treatment of MM cells with non-lethal (IC10 ) doses of the PIs revealed inhibitor- and cell type-specific readouts, being marked by the activation of tumorigenic STAT3 and STAT6. Consistently, cytokine/chemokine profiling revealed the increased secretion of immunosuppressive pro-tumorigenic cytokines (IL6 and IL8), along with the inhibition of potent T cell chemoattractant chemokines (CXCL10). These findings indicate that MM cells that survive treatment with therapeutic PIs shape a pro-tumorigenic immunosuppressive cellular and secretory bone marrow microenvironment that enables malignancy to relapse.

Non-hematopoietic STAT6 induces epithelial tight junction dysfunction and promotes intestinal inflammation and tumorigenesis.

Enhanced gut permeability due to dysregulated epithelial tight junction is often associated with inflammatory bowel diseases (IBD), which have a greater risk for developing colorectal cancer. STAT6 activation was detected in inflamed colonic epithelium of active IBD patients, suggesting a role of epithelial STAT6 in colitis development. Here, we demonstrated that non-hematopoietic STAT6, but not hematopoietic STAT6, triggered DSS-induced colitis and subsequent tumorigenesis. This could be due to the enhancing-effect of STAT6 on gut permeability and microbiota translocation via interruption of epithelial tight junction integrity. Mechanistically, long-myosin light-chain kinase (MLCK1) was identified as a target of STAT6, leading to epithelial tight junction dysfunction and microbiota-driven colitis. Furthermore, neutralization of IL-13, which was primarily derived from type 2 innate lymphoid cells (ILC2) in a microbiota-dependent way, inhibited epithelial STAT6 activation and improved gut permeability and DSS-induced colitis. Importantly, pharmacological inhibition of STAT6 reduces murine intestinal tumor formation, and tumoral p-STAT6 levels positively correlated to the clinical stage and poor prognosis of human colorectal cancer. Thus, our study reveals a direct role of STAT6 in the disruption of epithelial tight junction integrity and colitis development, and suggests STAT6 as a potential therapeutic and prophylactic target for IBD and colitis-associated cancer.

STAT6 inhibitory peptide reduces dendritic cell migration to the lymph nodes to control Th2 adaptive immunity in the mouse lung.

Type 2 immunity in the lung is promoted through the release of innate cytokines, including TSLP, from lung structural cells. These cytokines drive Type 2 immunity in part through upregulation of OX40L on dendritic cells (DCs). DCs expressing OX40L are potent inducers of Th2 differentiation. We have shown previously that STAT6 inhibitory peptide (STAT6-IP), a cell penetrating peptide designed to inhibit the STAT6 transcription factor, reduces the induction of Th2 adaptive immunity in murine models of respiratory syncytial virus infection. Here we show that intranasal administration of STAT6-IP at the time of antigen priming with ovalbumin (OVA), in conjunction with the Nod2 agonist, MDP, reduced frequencies of CD11b+ lung DCs expressing OX40L. Consistent with these reductions, fewer activated DCs were localized to the lung draining lymph nodes in STAT6-IP-treated mice. Upon OVA challenge four weeks later, mice treated with STAT6-IP at the time of OVA/MDP priming did not develop airway hyperresponsiveness (AHR) and had reduced influx of eosinophils into the airways, mucus production, and serum OVA-specific IgE levels. Our findings provide evidence that the long-lasting inhibitory effects of STAT6-IP are due in part to inhibition of DC responses that drive maladaptive Th2 adaptive immunity and allergic airways disease.

A hispanolone-derived diterpenoid inhibits M2-Macrophage polarization in vitro via JAK/STAT and attenuates chitin induced inflammation in vivo.

Macrophages are highly plastic cells that adopt different functional phenotypes in response to environmental signals. Classically activated macrophages (M1) exhibit a pro-inflammatory role, mediating host defense against microorganisms or tumor cells; whereas alternatively activated macrophages (M2) perform a range of physiological processes, including inflammation, wound repair and tissue remodeling. Interestingly, M2 macrophages have been involved in pathological settings such as tumor progression, parasitic infection and respiratory disorders. Consequently, the search of new agents able to control macrophage polarization is on the basis of new therapeutic strategies. In the present study, we have evaluated the effect of the hispanolone derivative 8,9-dehydrohispanolone-15,16-lactol (DHHL) on M2 macrophage polarization. Our results reveal that DHHL significantly inhibited IL-4- or IL-13-stimulated M2 macrophage activation, as showed by reduced expression of M2 markers. In addition, DHHL suppressed IL-4-induced STAT-6 and JAK-1 tyrosine phosphorylation, suggesting that this compound inhibited M2 polarization by suppressing the JAK-STAT signaling pathway. Finally, DHHL prevented eosinophil recruitment and the presence of F4/80+-CD206+ M2-like macrophages in an in vivo model of M2 polarization via administration of chitin. Collectively, these results confirm DHHL as a novel regulator of macrophage polarization suitable to design future therapies towards M2-macrophages mediated pathologies.

A STAT6 Inhibitor AS1517499 Reduces Preventive Effects of Apoptotic Cell Instillation on Bleomycin-Induced Lung Fibrosis by Suppressing PPARγ.

BACKGROUND/AIMS: The signal transducer and activator of transcription 6 (STAT6) transcription factor mediates PPARγ-regulated gene expression in macrophages. However, it remains largely unknown how proximal membrane signaling events initiated by apoptotic cell recognition upregulate PPARγ expression and activate the lung homeostatic program. METHODS: The STAT6 inhibitor AS1517499 was used to determine the role of STAT6 in mediating PPARγ activity, anti-inflammatory effects, and anti-fibrotic effects induced by apoptotic cell instillation after bleomycin treatment into C57BL/6 mice. Bronchoalveolar lavage fluid, alveolar macrophages and lungs were harvested at days 2, 7, and 14 and then analyzed by real-time PCR, immunoblotting, ELISA, immunocytochemistry and immunohistochemistry assays. RESULTS: Our data demonstrate that apoptotic cell instillation after bleomycin results in prolonged enhancement of STAT6 phosphorylation in alveolar macrophages and lung. Co-administration of the STAT6 inhibitor, AS1517499, reversed the enhanced PPARγ expression and activity induced by apoptotic cell instillation after bleomycin treatment. By reducing the expression of PPARγ target genes, including CD36, macrophage mannose receptor, and arginase 1, AS1517499 inhibited efferocytosis and restored pro-inflammatory cytokine expression, neutrophil recruitment, protein levels, hydroxyproline content, and expression of fibrosis markers, including type 1 collagen α2, fibronectin, and α-smooth muscle actin. STAT6 inhibition reversed the expression profile of hepatocyte growth factor and interleukin-10. CONCLUSION: These results indicate that prolonged STAT6 activation following one-time apoptotic cell instillation facilitates continuous PPARγ activation, resulting in the resolution of bleomycin-induced lung inflammation and fibrosis.

Blocking Interleukin (IL)4- and IL13-Mediated Phosphorylation of STAT6 (Tyr641) Decreases M2 Polarization of Macrophages and Protects Against Macrophage-Mediated Radioresistance of Inflammatory Breast Cancer.

PURPOSE: To determine the role of macrophage polarization on the response of inflammatory breast cancer (IBC) cells to radiation and whether modulation of macrophage plasticity can alter radiation response. METHODS AND MATERIALS: The human THP-1 monocyte cell line and primary human monocytes isolated from peripheral blood mononuclear cells were differentiated into macrophages and polarized to either an "antitumor" (M1) or a "protumor" (M2) phenotype. These polarized macrophages were co-cultured with IBC cells (SUM149, KPL4, MDA-IBC3, or SUM190) without direct contact for 24 hours, then subjected to irradiation (0, 2, 4, or 6 Gy). Interleukin (IL)4/IL13-induced activation of STAT6 signaling was measured by Western blotting of phospho-STAT6 (Tyr641), and expression of M2 polarization gene markers (CD206, fibronectin, and CCL22) was measured by quantitative polymerase chain reaction. RESULTS: Expression of M2 polarization markers was higher in M2-polarized macrophages after IL4/IL13 treatment than in control (M0) or M1-polarized macrophages. Co-culture of IBC cell lines with M1-polarized THP-1 macrophages mediated radiosensitivity of IBC cells, whereas co-culture with M2-polarized macrophages mediated radioresistance. Phosphopeptide mimetic PM37, targeting the SH2 domain of STAT6, prevented and reversed IL4/IL13-mediated STAT6 phosphorylation (Tyr641) and decreased the expression of M2 polarization markers. Pretreatment of M2-THP1 macrophages with PM37 reduced the radioresistance they induced in IBC cells after co-culture. Targeted proteomics analysis of IBC KPL4 cells using a kinase antibody array revealed induction of protein kinase C zeta (PRKCZ) in these cells only after co-culture with M2-THP1 macrophages, which was prevented by PM37 pretreatment. KPL4 cells with stable short hairpin RNA knockdown of PRKCZ exhibited lower radioresistance after M2-THP1 co-culture. CONCLUSIONS: These data suggest that inhibition of M2 polarization of macrophages by PM37 can prevent radioresistance of IBC by down-regulating PRKCZ.

Inhibition of STAT6/Anoctamin-1 Activation Suppresses Proliferation and Invasion of Gastric Cancer Cells.

BACKGROUND: Gastric carcinoma is the most popular cancer worldwide. Anoctamin-1 is a calcium-activated channel and highly expressed in various tumors. A previous study indicated that suppressed Anoctamin-1 expression decreased cancer cell proliferation or migration. As a signal transduction and transcription activator, STAT6 is a novel agonist for Anoctamin-1 promoter. However, its role in tumor cell proliferation or migration remains unclear. Therefore, this study aimed to suppress STAT6 and Anoctamin-1 protein expression in gastric cancer cells to test the inhibitory effects on gastric cancer cell migration or invasion. MATERIALS AND METHODS: MTT colorimetry was used to test cell proliferation. Western blot was used to measure STAT6 and Anoctamin-1 expression before and after small interfering RNA (siRNA) treatment. A scratch assay was performed to measure cell migration, followed by Transwell chamber assay analysis of cell invasion. RESULTS: After STAT6 siRNA interference, the expression of STAT6 and Anoctamin-1 was significantly decreased in the gastric carcinoma cell line. Anoctamin-1 siRNA interference only decreased its protein expression, but not STAT6 protein expression. Interference of STAT6 or Anoctamin-1 reduced their protein expression and inhibited proliferation, migration, or invasion of gastric cancer cells. CONCLUSIONS: Inhibition of STAT6/Anoctamin-1 activation decreased proliferation, migration, or invasion of gastric cancer cells, suggesting that the STAT6/Anoctamin-1 pathway might be a novel target for treating gastric cancer.

STATs in Lung Development: Distinct Early and Late Expression, Growth Modulation and Signaling Dysregulation in Congenital Diaphragmatic Hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a life-threatening developmental anomaly, intrinsically combining severe pulmonary hypoplasia and hypertension. During development, signal transducers and activators of transcription (STAT) are utilized to elicit cell growth, differentiation, and survival. METHODS: We used the nitrofen-induced CDH rat model. At selected gestational time points, lungs were divided into two experimental groups, i.e., control or CDH. We performed immunohistochemistry and western blotting analysis to investigate the developmental expression profile of the complete family of STATs (STAT1-6), plus specific STATs activation (p-STAT3, p-STAT6) and regulation by SOCS (SOCS3) in normal lungs against those of diseased lungs. The normal fetal lung explants were treated with piceatannol (STAT3 inhibitor) in vitro followed by morphometrical analysis. RESULTS: Molecular profiling of STATs during the lung development revealed distinct early and late expression signatures. Experimental CDH altered the STATs expression, activation, and regulation in the fetal lungs. In particular, STAT3 and STAT6 were persistently over-expressed and early over-activated. Piceatannol treatment dose-dependently stimulated the fetal lung growth. CONCLUSION: These findings suggest that STATs play an important role during normal fetal lung development and CDH pathogenesis. Moreover, functionally targeting STAT signaling modulates fetal lung growth, which highlights that STAT3 and STAT6 signaling might be promising therapeutic targets in reducing or preventing pulmonary hypoplasia in CDH.

Partial enteral nutrition increases intestinal sIgA levels in mice undergoing parenteral nutrition in a dose-dependent manner.

BACKGROUND AND AIMS: Partial enteral nutrition (PEN) protects parenteral nutrition (PN) induced gut mucosal immunity impairment. However, the gastrointestinal function of most patients with PN is too poor to tolerate full dosage of PEN and no guidelines recommend PEN dose. We aimed to identify an optimal PEN dose and to understand the protective mechanism. METHODS: Mice were assigned to groups with total parenteral nutrition (TPN), total enteral nutrition (TEN), or various degrees of PEN with PN. Additionally, AS1517499 was used to inhibit STAT6. Five days after treatment, secretory immunoglobulin A (sIgA) levels of luminal washing fluid and JAK1-STAT6 signalling in ileum tissue of different groups were assessed. RESULTS: We found that TPN lowered luminal sIgA and down-regulated pIgR, phosphorylated JAK1 and STAT6, IL-4 and IL-13 as well relative to TEN. Moreover, 40% EN were lowest dose that reversed these detrimental consequences of PN to an equivalent level as TEN. The rescue of pIgR and luminal sIgA expression was decreased when the JAK1-STAT6 pathway was inhibited. CONCLUSION: We conclude that 40% EN is the optimal PEN dose that reverses PN-induced impairment of gut mucosal immunity. Additionally, we hypothesise that this benefit involves activation of the JAK1-STAT6 pathway.

Renoprotective effect of erythropoietin via modulation of the STAT6/MAPK/NF-κB pathway in ischemia/reperfusion injury after renal transplantation.

Ischemia/reperfusion injury (IRI) commonly occurs in renal transplantation. Erythropoietin (EPO) exerts a protective effect in IRI. To investigate the underlying molecular mechanism, rat models of renal IRI were established and treated with EPO and/or lentivirus­mediated EPO-siRNA, the signal transducer and activator of transcription 6 (STAT6) inhibitor AS1517499, the JNK inhibitor SP600125, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, and the nuclear factor (NF)-κB inhibitor lactacystin. Histological examination revealed that EPO protected the kidney from IRI, through decreasing the extent of tissue congestion and inflammatory cell infiltration; however, EPO siRNA did not exert the same protective effect. In addition, the EPO level was inversely associated with renal IRI. EPO downregulated the expression of interferon-γ, interleukin (IL)-4, creatinine and caspase-3, and upregulated the expression of IL-10, thymic stromal lymphopoietin, STAT6, p-JNK and p-p38, while the opposite effects were observed with the administration of EPO-siRNA and the specific respective inhibitors. Further results revealed that MAPK (p-JNK and p-p38) acted upstream of NF-κB, and that NF-κB signaling regulated the expression of caspase-1 and -3, which may be responsible for the cytotoxicity associated with IRI. Taken together, the results of the present study demonstrated that EPO exerted a protective effect in renal IRI via the STAT6/MAPK/NF-κB pathway. This protective effect of EPO may improve reperfusion tolerance in ischemic kidneys and benefit transplant recipients.

Targeted Inhibition of the NCOA1/STAT6 Protein-Protein Interaction.

The complex formation between transcription factors (TFs) and coactivator proteins is required for transcriptional activity, and thus disruption of aberrantly activated TF/coactivator interactions could be an attractive therapeutic strategy. However, modulation of such protein-protein interactions (PPIs) has proven challenging. Here we report a cell-permeable, proteolytically stable, stapled helical peptide directly targeting nuclear receptor coactivator 1 (NCOA1), a coactivator required for the transcriptional activity of signal transducer and activator of transcription 6 (STAT6). We demonstrate that this stapled peptide disrupts the NCOA1/STAT6 complex, thereby repressing STAT6-mediated transcription. Furthermore, we solved the first crystal structure of a stapled peptide in complex with NCOA1. The stapled peptide therefore represents an invaluable chemical probe for understanding the precise role of the NCOA1/STAT6 interaction and an excellent starting point for the development of a novel class of therapeutic agents.

1,6-O,O-Diacetylbritannilactone Inhibits Eotaxin-1 and ALOX15 Expression Through Inactivation of STAT6 in A549 Cells.

1,6-O,O-Diacetylbritannilactone (OODBL), a plant sesquiterpene lactone, was previously reported to show multiple pharmacological effects such as anti-cancer and anti-inflammatory activities. In this study, we investigated the anti-inflammatory effect of OODBL on interleukin (IL)-4-induced signal transducer and activator of transcription 6 (STAT6) signaling pathway in human lung A549 cells. We found that OODBL dramatically inhibited IL-4-induced messenger RNA (mRNA) expression of eotaxin-1 and arachidonate 15-lipoxygenase-1 (ALOX15) in a dose-dependent manner. To clarify the action mechanism of OODBL, we examined the effect of OODBL on activation of STAT6. OODBL decreased both STAT6 phosphorylation and reporter gene activity. Furthermore, OODBL suppressed phosphorylation of Janus Kinase 3 (JAK3) without affecting JAK1. Taken together, OODBL abolished IL-4-induced eotaxin-1 and ALOX15 mRNA expressions by repressing the activation of STAT6 and JAK3. These results suggest that OODBL has a potential therapeutic efficacy on inflammatory diseases especially allergic airway disease as a lead compound.

STAT6 silencing induces hepatocellular carcinoma-derived cell apoptosis and growth inhibition by decreasing the RANKL expression.

Signal transducer and activator of transcription-6 (STAT6) is highly expressed in various human cancers and considered a regulator of multiple biological processes in cancers, including cell apoptosis. Evidence has indicated that STAT6 predicts a worse prognosis in hepatocellular carcinoma (HCC) patients. The objective of this study was to investigate the effects and mechanism of STAT6 in human HCC cells. We found that STAT6 silencing significantly inhibited HepG2 and Hep3B cell survival and proliferation. We observed that depletion of STAT6 increased HepG2 and Hep3B cell apoptosis by using a histone DNA ELISA detection kit. STAT6 silencing induced expression of apoptosis-associated genes Bax and caspase-3/7 and inhibited anti-apoptosis gene Bcl-2 levels. We also observed that STAT6 silencing downregulated the expression of receptor activator of NF-κB ligand (RANKL). Our results demonstrated that treatment with pcDNA3.1-RANKL abolished STAT6 depletion-induced HepG2 and Hep3B cell apoptosis and growth inhibition. Based on these findings, we believe that RANKL plays a major role in STAT6-induced HCC cell apoptosis.

Aggravated myocardial infarction-induced cardiac remodeling and heart failure in histamine-deficient mice.

Histamine has pleiotropic pathophysiological effects, but its role in myocardial infarction (MI)-induced cardiac remodeling remains unclear. Histidine decarboxylase (HDC) is the main enzyme involved in histamine production. Here, we clarified the roles of HDC-expressing cells and histamine in heart failure post-MI using HDC-EGFP transgenic mice and HDC-knockout (HDC-/-) mice. HDC+CD11b+ myeloid cell numbers markedly increased in the injured hearts, and histamine levels were up-regulated in the circulation post-MI. HDC-/- mice exhibited more adverse cardiac remodeling, poorer left ventricular function and higher mortality by increasing cardiac fibrogenesis post-MI. In vitro assays further confirmed that histamine inhibited heart fibroblast proliferation. Furthermore, histamine enhanced the signal transducer and activator of transcription (STAT)-6 phosphorylation level in murine heart fibroblasts, and the inhibitive effects of histamine on fibroblast proliferation could be blocked by JAK3/STAT6 signaling selective antagonist. STAT6-knockout (STAT6-/-) mice had a phenotype similar to that of HDC-/- mice post-MI; however, in contrast to HDC-/- mice, the beneficial effects of exogenous histamine injections were abrogated in STAT6-/- mice. These data suggest that histamine exerts protective effects by modulating cardiac fibrosis and remodeling post-MI, in part through the STAT6-dependent signaling pathway.