[Tofacitinib inhibits the transformation of lung fibroblasts into myofibroblasts through JAK/STAT3 pathway].

OBJECTIVE: To investigate the effect of tofacitinib, a pan-Janus kinase (JAK) inhibitor, on transforming growth factor-beta 1 (TGF-ß1)-induced fibroblast to myofibroblast transition (FMT) and to explore its mechanism. To provide a theoretical basis for the clinical treatment of connective tissue disease-related interstitial lung disease (CTD-ILD). METHODS: (1) Human fetal lung fibroblast 1 (HFL-1) were cultured in vitro, and 6 groups were established: DMSO blank control group, TGF-ß1 induction group, and TGF-ß1 with different concentrations of tofacitinib (0.5, 1.0, 2.0, 5.0 µmol/L) drug intervention experimental groups. CCK-8 was used to measure the cell viability, and wound-healing assay was performed to measure cell migration ability. After 48 h of combined treatment, quantitative real-time PCR (RT-PCR) and Western blotting were used to detect the gene and protein expression levels of α-smooth muscle actin (α-SMA), fibronectin (FN), and collagen type Ⅰ (COL1). (2) RT-PCR and enzyme-linked immunosorbnent assay (ELISA) were used to detect the interleukin-6 (IL-6) gene and protein expression changes, respectively. (3) DMSO carrier controls, 1.0 µmol/L and 5.0 µmol/L tofacitinib were added to the cell culture media of different groups for pre-incubation for 30 min, and then TGF-ß1 was added to treat for 1 h, 6 h and 24 h. The phosphorylation levels of Smad2/3 and signal transducer and activator of transcription 3 (STAT3) protein were detected by Western blotting. RESULTS: (1) Tofacitinib inhibited the viability and migration ability of HFL-1 cells after TGF-ß1 induction. (2) The expression of α-SMA, COL1A1 and FN1 genes of HFL-1 in the TGF-ß1-induced groups was significantly up-regulated compared with the blank control group (P < 0.05). Compared with the TGF-ß1 induction group, α-SMA expression in the 5.0 µmol/L tofacitinib intervention group was significantly inhi-bited (P < 0.05). Compared with the TGF-ß1-induced group, FN1 gene was significantly inhibited in each intervention group at a concentration of 0.5-5.0 µmol/L (P < 0.05). Compared with the TGF-ß1-induced group, the COL1A1 gene expression in each intervention group did not change significantly. (3) Western blotting results showed that the protein levels of α-SMA and FN1 in the TGF-ß1-induced group were significantly higher than those in the control group (P < 0.05), and there was no significant difference in the expression of COL1A1. Compared with the TGF-ß1-induced group, the α-SMA protein level in the intervention groups with different concentrations decreased. And the differences between the TGF-ß1-induced group and 2.0 µmol/L or 5.0 µmol/L intervention groups were statistically significant (P < 0.05). Compared with the TGF-ß1-induced group, the FN1 protein levels in the intervention groups with different concentrations showed a downward trend, but the difference was not statistically significant. There was no difference in COL1A1 protein expression between the intervention groups compared with the TGF-ß1-induced group. (4) After TGF-ß1 acted on HFL-1 cells for 48 h, the gene expression of the IL-6 was up-regulated and IL-6 in culture supernatant was increased, the intervention with tofacitinib partly inhibited the TGF-ß1-induced IL-6 gene expression and IL-6 in culture supernatant. TGF-ß1 induced the increase of Smad2/3 protein phosphorylation in HFL-1 cells for 1 h and 6 h, STAT3 protein phosphorylation increased at 1 h, 6 h and 24 h, the pre-intervention with tofacitinib inhibited the TGF-ß1-induced Smad2/3 phosphorylation at 6 h and inhibited TGF-ß1-induced STAT3 phosphorylation at 1 h, 6 h and 24 h. CONCLUSION: Tofacitinib can inhibit the transformation of HFL-1 cells into myofibroblasts induced by TGF-ß1, and the mechanism may be through inhibiting the classic Smad2/3 pathway as well as the phosphorylation of STAT3 induced by TGF-ß1, thereby protecting the disease progression of pulmonary fibrosis.

DYRK1B-STAT3 Drives Cardiac Hypertrophy and Heart Failure by Impairing Mitochondrial Bioenergetics.

BACKGROUND: Heart failure is a global public health issue that is associated with increasing morbidity and mortality. Previous studies have suggested that mitochondrial dysfunction plays critical roles in the progression of heart failure; however, the underlying mechanisms remain unclear. Because kinases have been reported to modulate mitochondrial function, we investigated the effects of DYRK1B (dual-specificity tyrosine-regulated kinase 1B) on mitochondrial bioenergetics, cardiac hypertrophy, and heart failure. METHODS: We engineered DYRK1B transgenic and knockout mice and used transverse aortic constriction to produce an in vivo model of cardiac hypertrophy. The effects of DYRK1B and its downstream mediators were subsequently elucidated using RNA-sequencing analysis and mitochondrial functional analysis. RESULTS: We found that DYRK1B expression was clearly upregulated in failing human myocardium and in hypertrophic murine hearts, as well. Cardiac-specific DYRK1B overexpression resulted in cardiac dysfunction accompanied by a decline in the left ventricular ejection fraction, fraction shortening, and increased cardiac fibrosis. In striking contrast to DYRK1B overexpression, the deletion of DYRK1B mitigated transverse aortic constriction-induced cardiac hypertrophy and heart failure. Mechanistically, DYRK1B was positively associated with impaired mitochondrial bioenergetics by directly binding with STAT3 to increase its phosphorylation and nuclear accumulation, ultimately contributing toward the downregulation of PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α). Furthermore, the inhibition of DYRK1B or STAT3 activity using specific inhibitors was able to restore cardiac performance by rejuvenating mitochondrial bioenergetics. CONCLUSIONS: Taken together, the findings of this study provide new insights into the previously unrecognized role of DYRK1B in mitochondrial bioenergetics and the progression of cardiac hypertrophy and heart failure. Consequently, these findings may provide new therapeutic options for patients with heart failure.

RP11-296E3.2 acts as an important molecular chaperone for YBX1 and promotes colorectal cancer proliferation and metastasis by activating STAT3.

BACKGROUND: RP11-296E3.2 is a novel long noncoding RNA (lncRNA) associated with colorectal cancer (CRC) metastasis, that was reported in our previous clinical studies. However, the mechanisms of RP11-296E3.2 in colorectal tumorigenesis remain elusive. METHODS: RNA sequencing (RNA-seq), Fluorescence in situ hybridization (FISH), Transwell assays and others, were performed to evaluate the function of RP11-296E3.2 for proliferation and metastasis in vitro. In situ and metastatic tumor models were performed to evaluate the function of RP11-296E3.2 for proliferation and metastasis in vivo. RNA-pulldown, RNA-interacting protein immunoprecipitation (RIP), tissue microarray (TMA) assay, a luciferase reporter assay, chromatin immunoprecipitation (ChIP) and others were performed to explore the mechanisms by which RP11-296E3.2 regulates CRC tumorigenesis. RESULTS: RP11-296E3.2 was confirmed to be associated with CRC cell proliferation and metastasis in vitro and in vivo. Mechanistically, RP11-296E3.2 directly bound to recombinant Y-Box Binding Protein 1 (YBX1) and enhanced signal transducer and activator of transcription 3 (STAT3) transcription and phosphorylation. YBX1 promoted the CRC cell proliferation and migration, while knockdown of RP11-296E3.2 attenuated the effects of YBX1 on CRC cell proliferation, and metastasis and the expression of several related downstream genes. We are the first to discover and confirm the existence of the YBX1/STAT3 pathway, a pathway dependent on RP11-296E3.2. CONCLUSION: Together, these novel findings show that the RP11-296E3.2/YBX1 pathway promotes colorectal tumorigenesis and progression by activating STAT3 transcription and phosphorylation, and suggest that RP11-296E3.2 is a potential diagnostic biomarker and therapeutic target in CRC.

Canonical and noncanonical regulatory roles for JAK2 in the pathogenesis of rheumatoid arthritis-associated interstitial lung disease and idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) and rheumatoid arthritis-associated interstitial lung disease (RA-ILD) are two fibrotic interstitial lung diseases that share the usual interstitial pneumonia (UIP) injury pattern. Here, we report that RNA sequencing of lung biopsies from patients with RA-ILD and IPF revealed shared and distinct disease-causing pathways. Analysis of transcriptomic data identified a JAK2 related JAK/STAT signaling pathway gene signature that distinguishes RA-UIP from idiopathic UIP. This was further confirmed by immunohistostaining, which identified JAK2 phosphorylation with two distinct forms of activation: a cytoplasmic form of JAK2 activation in most IPF cases (13/20) and a nuclear form of p-JAK2 in RA-UIP (5/5) and a minority of IPF (6/20) cases. Further immunohistostaining identified STAT5A&B as the downstream transcriptional activator for JAK2-mediated canonical signal transduction and phosphorylation of Tyr41 on histone H3 (H3Y41ph) as the downstream epigenetic regulation site for JAK2-mediated noncanonical signal transduction. Gene Set Enrichment Analysis (GSEA) of the RNA-Seq data further supported this shared pathogenic mechanism for the two diseases with the enrichment of STAT5A&B target gene sets as well as the JAK2 regulated H3Y41ph target gene set. This regulatory role of JAK2 in the pathogenesis of pulmonary fibrosis was further demonstrated by the attenuation of bleomycin-induced murine pulmonary fibrosis using a JAK2-selective pharmacological inhibitor CEP33779. In vitro studies with normal and IPF derived lung fibroblasts revealed a central role for JAK2 as an essential intermediary molecule in TGF-ß-mediated myofibroblast trans-differentiation, proliferation, and extracellular matrix protein production. These observations support a crucial role for JAK2 as an intermediary molecule in fibrotic lung disease development.

Nanoparticle Delivery of STAT3 Alleviates Pulmonary Hypertension in a Mouse Model of Alveolar Capillary Dysplasia.

BACKGROUND: Pulmonary hypertension (PH) is a common complication in patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with mutations in the FOXF1 gene. Although the loss of alveolar microvasculature causes PH in patients with ACDMPV, it is unknown whether increasing neonatal lung angiogenesis could prevent PH and right ventricular (RV) hypertrophy. METHODS: We used echocardiography, RV catheterization, immunostaining, and biochemical methods to examine lung and heart remodeling and RV output in Foxf1WT/S52F mice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1WT/S52F mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating Stat3 expression vector was used to improve neonatal pulmonary angiogenesis in Foxf1WT/S52F mice and determine its effects on PH and RV hypertrophy. RESULTS: Foxf1WT/S52F mice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1WT/S52F mice directly correlated with mortality, low body weight, pulmonary artery muscularization, and increased collagen deposition in the lung tissue. Increased fibrotic remodeling was found in human ACDMPV lungs. Mouse embryonic stem cells carrying the S52F Foxf1 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that Foxf1WT/S52F embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1WT/S52F mice from RV hypertrophy and PH, improved survival, and decreased fibrotic lung remodeling. CONCLUSIONS: Nanoparticle therapies increasing neonatal pulmonary angiogenesis may be considered to prevent PH in ACDMPV.

The ALK inhibitors, alectinib and ceritinib, induce ALK-independent and STAT3-dependent glioblastoma cell death.

Glioblastoma (GBM) is the most common, but extremely malignant, brain tumor; thus, the development of novel therapeutic strategies for GBMs is imperative. Many tyrosine kinase inhibitors (TKIs) have been approved for various cancers, yet none has demonstrated clinical benefit against GBM. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is confirmed only during the embryonic development period in humans. In addition, various ALK gene alterations are known to act as powerful oncogenes and therapeutic targets in various tumors. The antitumor activity of various TKIs was tested against three human GBM cell lines (U87MG, LN229, and GSC23), which expressed substantially low ALK levels; second-generation ALK inhibitors, alectinib and ceritinib, effectively induced GBM cell death. In addition, treatment with either alectinib or ceritinib modulated the activation of various molecules downstream of RTK signaling and induced caspase-dependent/-independent cell death mainly by inhibiting signal transducer and activator of transcription 3 activation in human GBM cells. In addition, alectinib and ceritinib also showed antitumor activity against a U87MG cell line with acquired temozolomide resistance. Finally, oral administration of alectinib and ceritinib prolonged the survival of mice harboring intracerebral GBM xenografts compared with controls. These results suggested that treatment with the second-generation ALK inhibitors, alectinib and ceritinib, might serve as a potent therapeutic strategy against GBM.

Efficacy of tocilizumab therapy in a patient with severe pancytopenia associated with a STAT3 gain-of-function mutation.

BACKGROUND: We aimed to report the clinical characteristics, immunological features, and treatment of one patient with a de novo STAT3 gain-of-function mutation identified by next generation sequencing. We investigated the efficacy of tocilizumab therapy in immune dysregulation diseases caused by STAT3 mutation. RESULTS: The patient was a 16-year-old girl. She presented with recurrent respiratory infections and chronic diarrhea after birth. She had life-threatening autoimmune pancytopenia at 14 years old. After receiving glucocorticoid therapy, she developed diabetes. However, her pancytopenia relapsed when the glucocorticoid was tapered. Next-generation sequencing showed a de novo heterozygous mutation in the STAT3 gene, c.1261G > A (p. G421R), which was previously described as a gain-of-function mutation. After tocilizumab therapy, her pancytopenia fully resolved, and insulin and glucocorticoid therapies were gradually discontinued within 12 months. She had lymphopenia and an inverted CD4/CD8 ratio before therapy. Lymphocyte subpopulation analysis indicated an expansion of effector memory CD4+, effector memory CD8+ and central memory CD4+ T cells. The proportions of memory B cells and naive CD4+ T cells were decreased, and the proportion of naïve B cells was increased. None of the abnormal lymphocytic changes improved significantly. STAT3 GOF mutations were identified by next gene sequencing in those with early-onset multi-organ autoimmunity. Including our patient, 13 patients with STAT3 GOF mutations received targeted treatment. Twelve of them were treated with tocilizumab alone or combination tocilizumab with JAK inhibitor, and ten patients improved. CONCLUSIONS: Gene sequencing should be performed for patients with early-onset refractory or multiorgan immune dysregulation diseases. Targeted drugs can effectively improve the clinical problems associated with STAT3 gain-of-function mutations, while nontargeted immunosuppressive therapy is usually insufficient.

Novel Paracrine Functions of Smooth Muscle Cells in Supporting Endothelial Regeneration Following Arterial Injury.

RATIONALE: Regeneration of denuded or injured endothelium is an important component of vascular injury response. Cell-cell communication between endothelial cells and smooth muscle cells (SMCs) plays a critical role not only in vascular homeostasis but also in disease. We have previously demonstrated that PKCδ (protein kinase C-delta) regulates multiple components of vascular injury response including apoptosis of SMCs and production of chemokines, thus is an attractive candidate for a role in SMC-endothelial cells communication. OBJECTIVE: To test whether PKCδ-mediated paracrine functions of SMCs influence reendothelialization in rodent models of arterial injury. METHODS AND RESULTS: Femoral artery wire injury was performed in SMC-conditional Prkcd knockout mice, and carotid angioplasty was conducted in rats receiving transient Prkcd knockdown or overexpression. SMC-specific knockout of Prkcd impaired reendothelialization, reflected by a smaller Evans blue-excluding area in the knockout compared with the wild-type controls. A similar impediment to reendothelialization was observed in rats with SMC-specific knockdown of Prkcd. In contrast, SMC-specific gene transfer of Prkcd accelerated reendothelialization. In vitro, medium conditioned by AdPKCδ-infected SMCs increased endothelial wound closure without affecting their proliferation. A polymerase chain reaction-based array analysis identified Cxcl1 and Cxcl7 among others as PKCδ-mediated chemokines produced by SMCs. Mechanistically, we postulated that PKCδ regulates Cxcl7 expression through STAT3 (signal transducer and activator of transcription 3) as knockdown of STAT3 abolished Cxcl7 expression. The role of CXCL7 in SMC-endothelial cells communication was demonstrated by blocking CXCL7 or its receptor CXCR2, both significantly inhibited endothelial wound closure. Furthermore, insertion of a Cxcl7 cDNA in the lentiviral vector that carries a Prkcd shRNA overcame the adverse effects of Prkcd knockdown on reendothelialization. CONCLUSIONS: SMCs promote reendothelialization in a PKCδ-dependent paracrine mechanism, likely through CXCL7-mediated recruitment of endothelial cells from uninjured endothelium.

Application of decoy oligodeoxynucleotides strategy for inhibition of cell growth and reduction of metastatic properties in nonresistant and erlotinib-resistant SW480 cell line.

Signal transducer and activator of transcription 3 (STAT3) is a critical regulator for angiogenesis, cell cycle progression, apoptosis, and drug resistance. Resistance toward EGF receptor (EGFR) inhibitors is a significant clinical concern for metastatic colon cancer patients. The present study aimed to evaluate the blocking influences of STAT3 decoy oligodeoxynucleotides (ODNs) on the STAT3 survival signaling pathway in nonresistant and erlotinib-resistant SW480 colon cancer cells. First, STAT3 decoy and scramble ODNs were designed according to STAT3 elements in the promoter region of MYCT1 gene and tested for the interaction of STAT3 protein with designed ODNs via in silico molecular docking study. Then, the efficiency of transfection and subcellular localization of ODNs were assessed using flow cytometry and fluorescence microscopy, respectively. Cell viability, cell cycle, and apoptosis tests, scratch and colony formation assays, and real-time PCR were also used to study the cancerous properties of cells. A considerable decrease in proliferation of colon cancer cells was observed with blockade of STAT3 signaling due to cell cycle arrest and induced apoptosis via downregulation of cyclin D1 and Bcl-XL, respectively. Furthermore, upon transfecting STAT3 decoy ODNs, colony formation potential and migration activity in both SW480 colon cancer cell lines were decreased compared to the control groups. From this study, it could be concluded that STAT3 is critical for cell growth inhibition and metastatic properties reduction of resistant SW480 colon cancer cells; therefore, STAT3 decoy ODNs could be considered as potential therapeutics along with current remedies for treating drug-resistant colon cancer.

Macrophages Promote Aortic Valve Cell Calcification and Alter STAT3 Splicing.

OBJECTIVE: Macrophages have been described in calcific aortic valve disease, but it is unclear if they promote or counteract calcification. We aimed to determine how macrophages are involved in calcification using the Notch1+/- model of calcific aortic valve disease. Approach and Results: Macrophages in wild-type and Notch1+/- murine aortic valves were characterized by flow cytometry. Macrophages in Notch1+/- aortic valves had increased expression of MHCII (major histocompatibility complex II). We then used bone marrow transplants to test if differences in Notch1+/- macrophages drive disease. Notch1+/- mice had increased valve thickness, macrophage infiltration, and proinflammatory macrophage maturation regardless of transplanted bone marrow genotype. In vitro approaches confirm that Notch1+/- aortic valve cells promote macrophage invasion as quantified by migration index and proinflammatory phenotypes as quantified by Ly6C and CCR2 positivity independent of macrophage genotype. Finally, we found that macrophage interaction with aortic valve cells promotes osteogenic, but not dystrophic, calcification and decreases abundance of the STAT3ß isoform. CONCLUSIONS: This study reveals that Notch1+/- aortic valve disease involves increased macrophage recruitment and maturation driven by altered aortic valve cell secretion, and that increased macrophage recruitment promotes osteogenic calcification and alters STAT3 splicing. Further investigation of STAT3 and macrophage-driven inflammation as therapeutic targets in calcific aortic valve disease is warranted.

Suppressing the metastatic properties of the breast cancer cells using STAT3 decoy oligodeoxynucleotides: A promising approach for eradication of cancer cells by differentiation therapy.

Due to the presence of cancer stem cells (CSCs), breast cancer often relapsed after conventional therapies. Strategies that induce differentiation of CSCs will be helpful in eradication of tumor cells, so we designed an oligodeoxynucleotide (ODNs) for targeting of signal transducer and activator of transcription 3 (STAT3) transcription factor which is involved in stemness, and constitutively activated in triple-negative breast cancer. Molecular docking and electrophoretic mobility shift assay analysis showed that decoy ODN bound specifically to the DNA binding site of STAT3 protein. The prevalent uptake of Cy3-labeled ODNs is in the cytoplasm and the nucleus of MDA-MB-231 treated cells. STAT3 decoy ODNs treatment showed cell growth inhibition by decreasing cell viability (17%), increasing the percentage of arrested cells in G0/G1 phases (18%), and triggering apoptosis (29%). Migration and invasion potential decreased from 10.77 to 6.76 µm/hr, by wound closure rate, and migrated/invaded percentage by 26.4% and 15.4% in the transwell assays, respectively. CD44 protein expression level on the cell surface also decreased, while CD24 increased. Mammosphere formation efficiency reduced in terms of tumorsphere size by 47%, while the required time increased. Cells morphology was changed, and lipid droplets were accumulated in the cytoplasm compared to the control and scrambled groups, in all assays (repeated triplicate). Furthermore, the gene expression of all downstream targets significantly decreased owing to suppressing the STAT3 transcription factor. Overall, the results confirmed the antitumor effects of STAT3 decoy in MDA-MB-231 cells. Thus, it seems that STAT3 decoy ODNs might be considered as an auxiliary tool for breast cancer eradicating by the differentiation therapy approach.

Yes-Associated Protein Promotes Angiogenesis via Signal Transducer and Activator of Transcription 3 in Endothelial Cells.

RATIONALE: Angiogenesis is a complex process regulating endothelial cell (EC) functions. Emerging lines of evidence support that YAP (Yes-associated protein) plays an important role in regulating the angiogenic activity of ECs. OBJECTIVE: The objective of this study was to specify the effect of EC YAP on angiogenesis and its underlying mechanisms. METHOD AND RESULTS: In ECs, vascular endothelial growth factor reduced YAP phosphorylation time and dose dependently and increased its nuclear accumulation. Using Tie2Cre-mediated YAP transgenic mice, we found that YAP promoted angiogenesis in the postnatal retina and tumor tissues. Mass spectrometry revealed signal transducer and activator of transcription 3 (STAT3) as a potential binding partner of YAP in ECs. Western blot and immunoprecipitation assays indicated that binding with YAP prolonged interleukin 6-induced STAT3 nuclear accumulation by blocking chromosomal maintenance 1-mediated STAT3 nuclear export without affecting its phosphorylation. Moreover, angiopoietin-2 expression induced by STAT3 was enhanced by YAP overexpression in ECs. Finally, a selective STAT3 inhibitor or angiopoietin-2 blockage partly attenuated retinal angiogenesis in Tie2Cre-mediated YAP transgenic mice. CONCLUSIONS: YAP binding sustained STAT3 in the nucleus to enhance the latter's transcriptional activity and promote angiogenesis via regulation of angiopoietin-2.

Evaluation of STAT3 decoy oligodeoxynucleotides' synergistic effects on radiation and/or chemotherapy in metastatic breast cancer cell line.

Resistance to radiotherapy and chemotherapy has been a major problem of conventional cancer therapies, which consequently leads to cancer relapse and cancer-related death. It is known that cancer stem cells (CSCs) play a key role in therapy resistance and CSC-based targeted therapies have been considered as a powerful tool for cancer treatment. In the current study, we investigated the synergistic effects of suppressing signal transducer and activator of transcription (STAT3) function by decoy ODNs on X-irradiation (XI) and methotrexate (MTX) exposure as a combinational therapy in triple-negative breast cancer (TNBC) MDA-MB-231 cells. Lipofectamine 2000® was used as a transfecting agent and the cells treated with Scramble ODNs (SCR) and decoy ODNs were subjected to irradiation with 2 Gy at single/fractionated (XI group) doses, different concentration of MTX group, and X-irradiation-methotrexate (XI/MTX group). Synergistic effects of STAT3 SCR and decoy ODNs on cells were investigated by cell viability (MTT), cell cycle profile, apoptosis rate, migration, and invasion assays. Statistical analysis of obtained data showed that STAT3 decoy ODNs significantly decreased the cell viability, arrested the growth at G0/G1 phase, increased apoptosis rate, and reduced migrated and invaded cells through transwell membrane, in XI, MTX, and XI/MTX exposed groups. Since STAT3 is a master transcription factor in breast cancer cells stemness, aggressiveness, TNBC's heterogeneity, and therapy resistance; therefore, inhibition of this transcription factor by decoy ODNs could increase antitumor efficiencies of XI and MTX exposure strategies. Accordingly, this method could have the potential to increase the efficiency of combination therapies.

Neuroprotective potential of imatinib in global ischemia-reperfusion-induced cerebral injury: possible role of Janus-activated kinase 2/signal transducer and activator of transcription 3 and connexin 43.

The present study was aimed to explore the neuroprotective role of imatinib in global ischemia-reperfusion-induced cerebral injury along with possible mechanisms. Global ischemia was induced in mice by bilateral carotid artery occlusion for 20 min, which was followed by reperfusion for 24 h by restoring the blood flow to the brain. The extent of cerebral injury was assessed after 24 h of global ischemia by measuring the locomotor activity (actophotometer test), motor coordination (inclined beam walking test), neurological severity score, learning and memory (object recognition test) and cerebral infarction (triphenyl tetrazolium chloride stain). Ischemia-reperfusion injury produced significant cerebral infarction, impaired the behavioral parameters and decreased the expression of connexin 43 and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in the brain. A single dose administration of imatinib (20 and 40 mg/kg) attenuated ischemia-reperfusion-induced behavioral deficits and the extent of cerebral infarction along with the restoration of connexin 43 and p-STAT3 levels. However, administration of AG490, a selective Janus-activated kinase 2 (JAK2)/STAT3 inhibitor, abolished the neuroprotective actions of imatinib and decreased the expression of connexin 43 and p-STAT3. It is concluded that imatinib has the potential of attenuating global ischemia-reperfusion-induced cerebral injury, which may be possibly attributed to activation of JAK2/STAT3 signaling pathway along with the increase in the expression of connexin 43.

AP-1 and the injury response of the GFAP gene.

Increased GFAP gene expression is a common feature of CNS injury, resulting in its use as a reporter to investigate mechanisms producing gliosis. AP-1 transcription factors are among those proposed to participate in mediating the reactive response. Prior studies found a consensus AP-1 binding site in the GFAP promoter to be essential for activity of reporter constructs transfected into cultured cells, but to have little to no effect on basal transgene expression in mice. Since cultured astrocytes display some properties of reactive astrocytes, these findings suggested that AP-1 transcription factors are critical for the upregulation of GFAP in injury, but not for its resting level of expression. We have examined this possibility by comparing the injury response in mice of lacZ transgenes driven by human GFAP promoters that contain the wild-type AP-1 binding site to those in which the site is mutated. An intact AP-1 site was found critical for a GFAP promoter response to the three different injury models used: physical trauma produced by cryoinjury, seizures produced by kainic acid, and chronic gliosis produced in an Alexander disease model. An unexpected additional finding was that the responses of the lacZ transgenes driven by the wild-type promoters were substantially less than that of the endogenous mouse GFAP gene. This suggests that the GFAP gene has previously unrecognized injury-responsive elements that reside further upstream of the transcription start site than the 2.2 kb present in the GFAP promoter segments used here.

Defective TLR9-driven STAT3 activation in B cells of patients with CVID.

B cell activation by Toll-like receptor 9 (TLR9) ligands is dependent on STAT3 and is important for optimal antibody responses to microbial antigens. B cells from patients with common variable immune deficiency (CVID) have impaired proliferation and differentiation in response to the TLR9 ligand CpG, despite normal levels of TLR9 expression. We demonstrate that CpG-driven STAT3 phosphorylation, but not activation of NFκB and p38, is selectively impaired in B cells from CVID patients. These results suggest that defective STAT3 activation contributes to the defective TLR9 and antibody response of B cells in CVID.

The effects of signal transducer and activator of transcription three mutations on human platelets.

Involvement of signal transducer and activator of transcription 3 (STAT3) in inflammation is well known. Recently, a role for STAT3 in platelet activation and platelet production has been suggested. Platelets exhibit important immune functions and engagement of STAT3 in platelet physiology may link inflammation and hemostasis. This study investigated the effects of STAT3 loss-of-function mutations and single nucleotide polymorphisms (SNPs) in STAT3 on glycoprotein VI (GPVI)-mediated platelet activation and platelet numbers in humans. Two cohorts were studied. The first cohort concerned patients with STAT3 loss-of-function mutations. Platelet numbers were investigated in eight patients and GPVI-mediated platelet activation was functionally tested in four patients. Additional experiments were performed to investigate underlying mechanisms. The second cohort concerned 334 healthy volunteers and investigated the consequences of SNPs in STAT3 on GPVI-mediated platelet activation and platelet numbers. Platelet activation was lower in STAT3 loss-of-function patients at baseline and after stimulation of the GPVI receptor, reflected by decreased P-selectin expression. This was independent of gene transcription. Blockade of the adenosine di-phosphate (ADP) pathway resulted in a further decrease of P-selectin expression, particularly in STAT3 loss-of-function patients. In contrast, the SNPs in STAT3 did not influence GPVI-mediated platelet activation. Also, platelet numbers were not affected by STAT3 loss-of-function mutations, nor was there an association with the SNPs. In conclusion, STAT3 signaling does not seem to play a major role in thrombopoiesis. We confirm that STAT3 is involved in GPVI-mediated platelet activation in humans, independent of gene transcription. GPVI-mediated platelet activation is highly dependent on secondary ADP release. Our findings suggest that STAT3 modulation may affect inflammation, hemostasis, and their interaction.

Critical Roles of STAT3 in ß-Adrenergic Functions in the Heart.

BACKGROUND: ß-Adrenergic receptors (ßARs) play paradoxical roles in the heart. On one hand, ßARs augment cardiac performance to fulfill the physiological demands, but on the other hand, prolonged activations of ßARs exert deleterious effects that result in heart failure. The signal transducer and activator of transcription 3 (STAT3) plays a dynamic role in integrating multiple cytokine signaling pathways in a number of tissues. Altered activation of STAT3 has been observed in failing hearts in both human patients and animal models. Our objective is to determine the potential regulatory roles of STAT3 in cardiac ßAR-mediated signaling and function. METHODS AND RESULTS: We observed that STAT3 can be directly activated in cardiomyocytes by ß-adrenergic agonists. To follow up this finding, we analyzed ßAR function in cardiomyocyte-restricted STAT3 knockouts and discovered that the conditional loss of STAT3 in cardiomyocytes markedly reduced the cardiac contractile response to acute ßAR stimulation, and caused disengagement of calcium coupling and muscle contraction. Under chronic ß-adrenergic stimulation, Stat3cKO hearts exhibited pronounced cardiomyocyte hypertrophy, cell death, and subsequent cardiac fibrosis. Biochemical and genetic data supported that Gαs and Src kinases are required for ßAR-mediated activation of STAT3. Finally, we demonstrated that STAT3 transcriptionally regulates several key components of ßAR pathway, including ß1AR, protein kinase A, and T-type Ca(2+) channels. CONCLUSIONS: Our data demonstrate for the first time that STAT3 has a fundamental role in ßAR signaling and functions in the heart. STAT3 serves as a critical transcriptional regulator for ßAR-mediated cardiac stress adaption, pathological remodeling, and heart failure.

Quercetin inhibits multiple pathways involved in interleukin 6 secretion from human lung fibroblasts and activity in bronchial epithelial cell transformation induced by benzo[a]pyrene diol epoxide.

The interaction between epithelial and stromal cells through soluble factors such as cytokines plays an important role in carcinogenesis. Breaking this cancer-promoting interaction poses an opportunity for cancer prevention. The tumor-promoting function of interleukin 6 (IL-6) has been documented; however, the underlying mechanisms of this function in lung carcinogenesis are not well elucidated. Here, we show that benzo[a]pyrene diol epoxide (BPDE, the active metabolite of cigarette smoke carcinogen benzo[a]pyrene)-induced human bronchial epithelial cell (HBEC) transformation was enhanced by IL-6 in vitro. The carcinogen/IL-6-transformed cells exhibited higher expression of STAT3 (signal transducer and activator of transcription 3) when compared with cells transformed by BPDE alone. Constitutive STAT3 activation drove cell proliferation and survival through anti-apoptosis gene expression. We further show that quercetin, a dietary compound having preventive properties for lung cancer, decreased BPDE-stimulated IL-6 secretion from human lung fibroblasts through inhibition of the NF-κB and ERK pathways. The inhibition was accomplished at clinically achievable concentrations of the compound. Finally, quercetin blocked IL-6-induced STAT3 activation in HBECs, and IL-6 enhancement of HBEC transformation by BPDE was abolished by quercetin treatment. Altogether, our data reveal novel mechanisms for IL-6 in lung carcinogenesis and for the preventive role of quercetin in the process. © 2015 Wiley Periodicals, Inc.

Enzymes and signal pathways in the pathogenesis of alcoholic cardiomyopathy.

Owing to its acute psychotropic effects, ethanol is the most frequently consumed toxic agent worldwide. However, excessive alcohol intake results in an array of health, social, and economic consequences, which are related to its property as an addictive substance. It has been well established that exposure to high levels of alcohol for a long period leads to the onset and progression of nonischemic cardiomyopathy through direct toxic mechanisms of ethanol and its metabolite, acetaldehyde. Excessive alcohol ingestion causes myocardial damage including disruptions of the myofibrillar architecture and is associated with reduced myocardial contractility and decreased ejection volumes. Key features of alcoholic cardiomyopathy are cardiac hypertrophy and ventricular dilatation, and the disease is manifested mainly as cardiomegaly, congestive heart failure, and even cardiac death. Mechanisms that have been postulated to underlie the pathogenesis of alcoholic cardiomyopathy include apoptosis, mitochondrial alterations, acetaldehyde protein adduct formation, oxidative stress, and imbalances in fatty acid metabolism. In the following, we give a brief overview of the molecular effects of ethanol-metabolizing enzymes and their impact on myocardial signal transduction pathways.