Combined p53- and PTEN-deficiency activates expression of mesenchyme homeobox 1 (MEOX1) required for growth of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive cancer subtype for which effective therapies are unavailable. TNBC has a high frequency of tumor protein p53 (Tp53/p53)- and phosphatase and tensin homolog (PTEN) deficiencies, and combined p53- and PTEN-deficiency is associated with poor prognosis and poor response to anticancer therapies. In this study, we discovered that combined p53- and PTEN-deficiency in TNBC activates expression of the transcription factor mesenchyme homeobox 1 (MEOX1). We found that MEOX1 is expressed only in TNBC cells with frequent deficiencies in p53 and PTEN, and that its expression is undetectable in luminal A, luminal B, and HER2+ subtypes, as well as in normal breast cells with wild-type (WT) p53 and PTEN. Notably, siRNA knockdown of both p53 and PTEN activated MEOX1 expression in breast cancer cells, whereas individual knockdowns of either p53 or PTEN had only minimal effects on MEOX1 expression. MEOX1 knockdown abolished cell proliferation of p53- and PTEN-deficient TNBC in vitro and inhibited tumor growth in vivo, but had no effect on the proliferation of luminal and HER2+ cancer cells and normal breast cells. RNA-Seq and immunoblotting analyses showed that MEOX1 knockdown decreased expression of tyrosine kinase 2 (TYK2), signal transducer and activator of transcription 5B (STAT5B), and STAT6 in p53- and PTEN-deficient TNBC cells. These results reveal the effects of combined p53- and PTEN-deficiency on MEOX1 expression and TNBC cell proliferation, suggesting that MEOX1 may serve as a potential therapeutic target for managing p53- and PTEN-deficient TNBC.

Decreased expression of STAT5A predicts poor prognosis in osteosarcoma.

BACKGROUND: Signal transducer and activator of transcription 5 (STAT5) plays a key role in the malignancy of many tumors and has been identified as a therapeutic target. However, the role of STAT5A in osteosarcoma is still unclear. METHODS: 98 osteosarcoma patients were obtain from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET). The relationship between STAT5A and clinical features was analyzed using the Wilcoxon signed-rank test and logistic regression. Kaplan-Meier method, univariate and multivariate Cox regression analyses were performed to assess the prognostic value in event-free survival (EFS) and overall survival (OS). Gene Set Enrichment Analysis (GSEA) was performed. RESULTS: STAT5A low expression was not linked to age, gender, tumor site, surgical approach, tumor region, histologic response, and metastasis, but was correlated with progression (OR = 5.2, P = 0.012). Kaplan-Meier survival curve showed that patients with STAT5A low expression had worse EFS and OS than those with STAT5A high expression (P < 0.01). Furthermore, the multivariate analysis revealed STAT5A was an independent prognostic factor for poor OS (HR = 3.29, P = 0.0408)) and EFS (HR = 7.29, P = 0.0025). GSEA showed that the complemen, metabolism, apoptosis, interferon-gamma response, inflammatory response, Notch, Kras, reactive oxygen species, VEGF, IL-6/JAK/STAT3, IL-2/Stat5, B-cell receptor, and p53 pathways were significantly associated with the STAT5A gene. CONCLUSIONS: STAT5A may be a novel prognostic factor for osteosarcoma and may act as a molecular target in the treatment of osteosarcoma.

STAT5 expression correlates with recurrence and survival in melanoma patients treated with interferon-α.

Interferons (IFN) have a direct growth-inhibiting effect on tumor cells through Janus kinase-dependent activation of the transcription factor signal transducer and activator of transcription (STAT1). In vitro, signaling through STAT5 has been demonstrated to counteract this effect and lead to IFN resistance of melanoma cell lines. In 32 patients treated with IFN-α in an adjuvant setting, we investigated paraffin-embedded tumor tissue from primary melanomas and melanoma metastases for expression of STAT3 and STAT5, by immunohistochemistry, and for expression of phosphorylated signaling transduction activating transcription factor (pSTAT)3 and pSTAT5, by immunofluorescence. Tumor cell expression levels of these proteins were correlated with patient characteristics and clinical outcomes. The patient cohort consisted of 12 (37.5%) patients at AJCC stage I/II (primary melanoma) and 20 (62.5%) at stage III/IV (metastatic melanoma). Recurrence was observed for 25 (78.1%) either during or after IFN-α therapy. χ Correlation of staining intensities with clinical data revealed association of pSTAT3 and STAT5 expression with sex (P=0.003 and 0.016, respectively) and of STAT3 with tumor stage (P=0.019). Recurrence of melanoma was found to be associated with high STAT5 expression (P=0.017). Multivariable regression analysis revealed STAT5 expression as an independent factor for predicting progression-free survival (P<0.0001) and overall survival (P=0.022). In summary, high expression of STAT5 correlated with melanoma recurrence and survival of patients treated with IFN-α in the adjuvant setting. Recently, it has been suggested that mutations of Janus kinases are involved in resistance to immune checkpoint blocker treatments implying a possible role of STAT5 for immune checkpoint resistance.

Indole-3-carbinol induces apoptosis of chronic myelogenous leukemia cells through suppression of STAT5 and Akt signaling pathways.

Signal transducer and activator of transcription 5 and Akt pathways, implicated in signaling transduction downstream of BCR-ABL, play critical roles in the pathogenesis of chronic myeloid leukemia. Therefore, idenication of novel compounds that modulate the activity of such pathways could be a new approach in the treatment of chronic myeloid leukemia. Previous studies have demonstrated that indole-3-carbinol inhibits the proliferation and induces apoptosis of various tumor cells. However, its anticancer activity against chronic myeloid leukemia cells and the underlying mechanism remain unclear. Our data revealed that indole-3-carbinol promoted mitochondrial apoptosis of chronic myeloid leukemia-derived K562 cells, as evidenced by the activation of caspases and poly (ADP-ribose) polymerase cleavage. Treatment with indole-3-carbinol was found to be associated with a decrease in the cellular levels of phospho-Akt and phospho-signal transducer and activator of transcription 5. In addition, real-time polymerase chain reaction analysis showed that the downregulation of genes is regulated by Akt and signal transducer and activator of transcription 5. We also found that treatment with indole-3-carbinol resulted in the activation of the p38 mitogen-activated protein kinase and reduced expression of human telomerase and c-Myc. Collectively, these results demonstrate that the oncogenic signal transducer and activator of transcription 5/Akt pathway is a cellular target for indole-3-carbinol in chronic myeloid leukemia cells. Thus, this clinically tested natural compound can be a potential candidate in the treatment of chronic myeloid leukemia following confirmation with clinical studies.

Epo reprograms the epigenome of erythroid cells.

The hormone erythropoietin (Epo) is required for erythropoiesis, yet its molecular mechanism of action remains poorly understood, particularly with respect to chromatin dynamics. To investigate how Epo modulates the erythroid epigenome, we performed epigenetic profiling using an ex vivo murine cell system that undergoes synchronous erythroid maturation in response to Epo stimulation. Our findings define the repertoire of Epo-modulated enhancers, illuminating a new facet of Epo signaling. First, a large number of enhancers rapidly responded to Epo stimulation, revealing a cis-regulatory network of Epo-responsive enhancers. In contrast, most of the other identified enhancers remained in an active acetylated state during Epo signaling, suggesting that most erythroid enhancers are established at an earlier precursor stage. Second, we identified several hundred super-enhancers that were linked to key erythroid genes, such as Tal1, Bcl11a, and Mir144/451. Third, experimental and computational validation revealed that many predicted enhancer regions were occupied by TAL1 and enriched with DNA-binding motifs for GATA1, KLF1, TAL1/E-box, and STAT5. Additionally, many of these cis-regulatory regions were conserved evolutionarily and displayed correlated enhancer:promoter acetylation. Together, these findings define a cis-regulatory enhancer network for Epo signaling during erythropoiesis, and provide the framework for future studies involving the interplay of epigenetics and Epo signaling.

Strategies for over-expression and purification of recombinant full length STAT5B in Escherichia coli.

STAT5B, a ubiquitious transcription factor, has been implicated in the onset and progression of several cancers. Since the inhibition of STAT activity holds significant therapeutic potential, there is a need to develop high-throughput biophysical screening platforms to rapidly identify high affinity binders of STATs. Biophysical assays would benefit from the efficient and cost-effective production of high purity, full-length STAT proteins. Herein, we have sampled a large region of protein expression and purification space that has substantially increased recombinant STAT5B protein yields from Escherichia coli. The identity of STAT5B was confirmed by Western blotting analysis, while the results of a fluorescence polarization assay indicated that the purified protein is correctly folded and functional. A thermal shift assay was employed to assess the effect of various osmolytes on the stability of the protein. The protein expression conditions identified in this study allowed for more efficient and higher recovery of soluble STAT5B protein, which will enable a broad range of biophysical studies and facilitate high-throughput STAT5B drug screening.

GRAM domain-containing protein 1A (GRAMD1A) promotes the expansion of hepatocellular carcinoma stem cell and hepatocellular carcinoma growth through STAT5.

Hepatocellular carcinoma (HCC) is the leading cause for cancer death worldwide, new prognostic factors and targets are critical for HCC treatment. Here, we found GRAMD1A was upregulated in HCC tissues, patients with high GRAMD1A levels had poor outcome, statistical analyses found GRAMD1A expression was positively correlated with pathologic differentiation and survival or mortality. It was an unfavorable prognostic factor for HCC patients. Functional analyses revealed GRAMD1A contributed to the self-renewal of HCC stem cells, resistance to chemotherapy and tumor growth of HCC determined by hepatosphere formation assay, side population (SP) analysis, TUNEL assay, soft agar growth ability assay and tumor growth model in vivo. Mechanism analyses found signal transducer and activator of transcription 5 (STAT5) was the target of GRAMD1A, GRAMD1A regulated the target genes of STAT5 and the transcriptional activity of STAT5. Inhibition of STAT5 in indicated HCC cells overexpressing GRAMD1A suppressed the effects of GRAMD1A on the self-renewal of HCC stem cell, resistance to chemotherapy and tumor growth, suggesting GRAMD1A promoted the self-renewal of HCC stem cells and the development of HCC by increasing STAT5 level. GRAMD1A might be a useful biomarker and target for HCC.

Exploring the mechanism of non-small-cell lung cancer cell lines resistant to epidermal growth factor receptor tyrosine kinase inhibitor.

PURPOSE: Here we aimed to explore the possible mechanism and potential regulatory relationships in which the non.small.cell lung cancer. (NSCLC)-resisted epidermal growth factor receptor. (EGFR) tyrosine kinase inhibitor erlotinib. MATERIALS AND METHODS: GSE38310, the gene expression profiles of NSCLC cell lines treated with dimethylsulfoxide or erlotinib, including HCC827, ER3, and T15-2, were downloaded from Gene Expression Omnibus database and preprocessed by normalization. Basing on the regulatory relationships of transcriptional factors obtained from University of California Santa Cruz. (UCSC) database, the differentially expressed genes. (DEGs) were screened using limma package in R with. |logFC| >1 and P < 0.05, and regulatory networks of these DEGs were built with supervised inference of regulatory networks (SIRENE). Subsequently, differentially regulatory networks were compared basing on Limit Fold Change. (LFC) method. RESULTS: Totally 24,380 genes were obtained, 1,531 DEGs were identified in HCC827 cell lines, 37 DEGs in ER3 cell lines, 156 DEGs in T15-2 cell lines. After removing the redundancy genes, 1,575 differentially expressed genes were got at last. Basing on three regulatory networks of HCC827 cell lines, ER3 cell lines and T15-2 cell lies, sex-determining region Y (SRY).related high mobility group-box gene 9. (SOX9) and Suppressor of cytokine signaling 3 (STAT3) were identified by comparing with HCC827 and ER3 networks. And suppressor of cytokine signaling 5 B (STAT5B), early growth response-1 (EGR1) and STAT6 were obtained in comparison of HCC827 and T15-2 networks. CONCLUSIONS: The regulatory edges with remarkable changes between HCC827 and ER3, HCC827 and T15.2 included some transcription factors and genes. (e. g., STAT3 and SOX9). STAT3, SOX9, STAT5B, EGR1, and STAT6 might affect the resistance of NSCLC to erlotinib.

Prolactin inhibits a major tumor-suppressive function of wild type BRCA1.

Even though mutations in the tumor suppressor, BRCA1, markedly increase the risk of breast and ovarian cancer, most breast and ovarian cancers express wild type BRCA1. An important question is therefore how the tumor-suppressive function of normal BRCA1 is overcome during development of most cancers. Because prolactin promotes these and other cancers, we investigated the hypothesis that prolactin interferes with the ability of BRCA1 to inhibit the cell cycle. Examining six different cancer cell lines with wild type BRCA1, and making use of both prolactin and the growth-inhibiting selective prolactin receptor modulator, S179D PRL, we demonstrate that prolactin activation of Stat5 results in the formation of a complex between phospho-Stat5 and BRCA1. Formation of this complex does not interfere with nuclear translocation or binding of BRCA1 to the p21 promoter, but does interfere with the ability of BRCA1 to transactivate the p21 promoter. Overexpression of a dominant-negative Stat5 in prolactin-stimulated cells resulted in increased p21 expression. We conclude that prolactin inhibits a major tumor-suppressive function of BRCA1 by interfering with BRCA1's upregulation of expression of the cell cycle inhibitor, p21.

The BH3-only protein BIM contributes to late-stage involution in the mouse mammary gland.

After cessation of lactation, involution of the mouse mammary gland proceeds in two distinct phases, a reversible and an irreversible one, which leads to the death and removal of alveolar cells. Cell death is preceded by the loss of STAT5 activity, which abrogates cell differentiation and gain of STAT3 activity. Despite early observations implicating BCL2 (B cell lymphoma 2) family proteins in this process, recent evidence suggests that STAT3-controlled cathepsin activity is most critical for cell death at the early stage of involution. Somewhat surprisingly, this cell death associates with but does not depend on the activation of pro-apoptotic effector caspases. However, transgenic overexpression of BCL2, that blocks caspase activation, delays involution while conditional deletion of BclX accelerates this process, suggesting that BCL2 family proteins are needed for the effective execution of involution. Here, we report on the transcriptional induction of multiple pro-apoptotic BCL2 family proteins of the 'BH3-only' subgroup during involution and the rate-limiting role of BIM in this process. Loss of Bim delayed epithelial cell clearance during involution after forced weaning in mice, whereas the absence of related Bmf had minor and loss of Bad or Noxa no impact on this process. Consistent with a contribution of BCL2 family proteins to the second wave of cell death during involution, loss of Bim reduced the number of apoptotic cells in this irreversible phase. Notably, the expression changes observed within the BCL2 family did not depend on STAT3 signalling, in line with its initiating role early in the process, but rather appear to result from relief of repression by STAT5. Our findings support the existence of a signalling circuitry regulating the irreversible phase of involution in mice by engaging BH3-only protein-driven mitochondrial apoptosis.

Rankl Impairs Lactogenic Differentiation Through Inhibition of the Prolactin/Stat5 Pathway at Midgestation.

Prolactin and progesterone both orchestrate the proliferation and differentiation of the mammary gland during gestation. Differentiation of milk secreting alveoli depends on the presence of prolactin receptor, the downstream Jak2-Stat5 pathway and the transcription factor Elf5. A strict regulation of Rank signaling is essential for the differentiation of the mammary gland and in particular for alveolar commitment. Impaired alveologenesis and lactation failure are observed in both, knockout and Rank overexpressing mice; however, the underlying molecular mechanism responsible for these phenotypes remains largely unknown. Using genome-wide expression analyses and functional studies, we show here that Rankl (RL) exposure leads to impaired secretory differentiation of alveolar cells not only in MMTV-RANK but also in wild-type (WT) mammary acini. Conversely, pharmacological blockage of Rank signaling at midgestation in WT mice leads to precocious and exacerbated lactogenesis. Mechanistically, RL negatively regulates Stat5 phosphorylation and Elf5 expression at the onset of lactogenesis. Continuous RL exposure leads to the expansion of basal and bipotent cells in WT and MMTV-RANK acini. Overall, we demonstrate that enhanced Rank signaling impairs secretory differentiation during pregnancy by inhibition of the prolactin/p-Stat5 pathway.

Thalidomide can promote erythropoiesis by induction of STAT5 and repression of external pathway of apoptosis resulting in increased expression of GATA-1 transcription factor.

BACKGROUND: Thalidomide was shown to stimulate erythropoiesis and increase hemoglobin level in multiple myeloma patients, but way of such activity remains unclear. The aim of the study was to investigate the mechanisms of thalidomide stimulating effect on erythroid differentiation. METHODS: Hematopoietic stem cells were isolated from bone marrow aspirates taken from myeloma patients and cultured with or without thalidomide. Then the generation of erythroid cells and the expression of STAT5, GATA-1, GATA-2, selected caspases and Bcl-2 family proteins in erythroid cells were assessed using flow cytometry and real-time PCR. RESULTS: The generation of erythroblasts was higher in thalidomide than in control cultures (63.9% vs. 55.8%, p < 0.001). The expression of caspase 3 (cytometry 947.3 vs. 1021.0, p = 0.025; PCR 12.9 vs. 16.3, p = 0.025) and caspase 8 (cytometry 1050.8 vs. 1168.5, p = 0.033; PCR 16.2 vs. 17.8, p = 0.004) was significantly lower in thalidomide than in control cultures. The expression of STAT5 (cytometry 331.5 vs. 276.1, p = 0.015; PCR 24.3 vs. 21.1, p = 0.003) and GATA-1 (cytometry 259.7 vs. 232.0, p = 0.027; PCR 18.9 vs. 16.5, p = 0.003) was higher in thalidomide than in control cultures. CONCLUSION: Our results suggest that thalidomide enhances expression of STAT5 in response of erythroid cells to erythropoietin and as a result of caspase 3 suppression. Moreover it may exert inhibitory effect on an external pathway of caspases activation with consequent decreased degradation of GATA-1 transcription factor by downstream caspases.

Reassessment of H&E stained clot specimens and immunohistochemistry of phosphorylated Stat5 for histological diagnosis of MDS/MPN.

Few studies have comprehensively analysed histopathological findings of bone marrow clots for diagnosis of haematopoietic cell dysplasia. In particular, a limited number of studies have assessed the use of haematoxylin and eosin (H&E) staining, which is generally considered less informative than May-Giemsa staining. In the current study, the utility of bone marrow clot specimens for diagnosis was examined using H&E staining and immunohistochemistry. Patients with myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasm (MDS/MPN), including chronic myelomonocytic leukaemia (CMML), atypical chronic myeloid leukaemia (aCML) lacking Philadelphia chromosome, and juvenile myelomonocytic leukaemia (JMML), were selected for histological evaluation. H&E stained specimens were advantageous for observation of atypical basophilic staining of the cytoplasm and nucleus related to dysplasia. This finding was significantly supported for both MDS and MDS/MPN (p < 0.05 versus May-Giemsa staining); therefore, we concluded that H&E staining could be used for identification of dysplastic cells. In addition, despite the loss of tissue structure, phosphorylated Stat5 immunostaining was sufficiently useful for the observation of myelodysplastic blasts. Thus, clot specimens are useful for diagnosis of haematopoietic dysplasia by pathologists.

ABL-N may induce apoptosis of human prostate cancer cells through suppression of KLF5, ICAM-1 and Stat5b, and upregulation of Bax/Bcl-2 ratio: An in vitro and in vivo study.

Identification of novel botanicals that can selectively induce apoptosis and arrest growth of cancer cells without producing cytotoxic effects is highly appreciable for cancer therapy. The present study aimed to investigate the possibility of acetylbritannilactone (ABL) derivative 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methylene-2-oxo-2,3,3a,4,7,7a­hexahydroben-zofuran-4-yl2-(6-methoxynaphthalen-2-yl) propanoate (ABL-N) as a therapeutic agent in human prostate cancer and potential mechanisms. Human prostate cancer cells were treated with ABL-N of different concentrations (0, 5, 10, 20, 30 and 40 µmol/l). Cell viability, migration and apoptosis were determined. Activities of caspases were assayed, as well as protein expression of cancer­related proteins KLF5, Stat5b and ICAM-1 in PC3 cells. The therapeutic effect of ABL-N was further evaluated in our tumor xenografts. ABL-N inhibited growth of prostate cancer cells in a dose-dependent manner, without obvious effect on normal human prostate epithelial PrEC cells. ABL-N administration induced apoptosis of PC3 cells in a dose-dependent manner, along with the enhanced activity of caspases and increased Bax/Bcl-2 ratio. Expression of KLF5, Stat5b and ICAM-1 was significantly downregulated in PC3 cells. Our in vivo study further confirmed that ABL-N significantly inhibited the tumor growth of PC3 cells in the xenograft mouse model. ABL-N induces apoptosis of prostate cancer cells through activation of caspases, increasing the ratio of Bax/Bcl-2, as well as suppression of KLF5, Stat5b and ICAM-1 expressions. The present study indicated that ABL-N may be a potential therapeutic drug for human prostate cancer, and our data supported further studies to explore the therapeutic potential of ABL-N in other types of human cancer.

The Transcription Factor Ehf Is Involved in TGF-ß-Induced Suppression of FcεRI and c-Kit Expression and FcεRI-Mediated Activation in Mast Cells.

FcεRI, which is composed of α, ß, and γ subunits, plays an important role in IgE-mediated allergic responses. TGF-ß1 has been reported to suppress FcεRI and stem cell factor receptor c-Kit expression on mast cell surfaces and to suppress mast cell activation induced by cross-linking of FcεRI. However, the molecular mechanism by which these expressions and activation are suppressed by TGF-ß1 remains unclear. In this study, we found that the expression of Ets homologous factor (Ehf), a member of the Ets family transcriptional factors, is upregulated by TGF-ß/Smad signaling in mouse bone marrow-derived mast cells (BMMCs). Forced expression of Ehf in BMMCs repressed the transcription of genes encoding FcεRIα, FcεRIß, and c-Kit, resulting in a reduction in cell surface FcεRI and c-Kit expression. Additionally, forced expression of Ehf suppressed FcεRI-mediated degranulation and cytokine production. Ehf inhibited the promoter activity of genes encoding FcεRIα, FcεRIß, and c-Kit by binding to these gene promoters. Furthermore, the mRNA levels of Gata1, Gata2, and Stat5b were lower in BMMCs stably expressing Ehf compared with control cells. Because GATA-1 and GATA-2 are positive regulators of FcεRI and c-Kit expression, decreased expression of GATAs may be also involved in the reduction of FcεRI and c-Kit expression. Decreased expression of Stat5 may contribute to the suppression of cytokine production by BMMCs. In part, mast cell response to TGF-ß1 was mimicked by forced expression of Ehf, suggesting that TGF-ß1 suppresses FcεRI and c-Kit expression and suppresses FcεRI-mediated activation through upregulation of Ehf.

The Status of STAT3 and STAT5 in Human Breast Atypical Ductal Hyperplasia.

Signal Transducer and Activation of Transcription factors (STAT3 and STAT5) play important roles in breast epithelial cell differentiation, proliferation, and apoptosis. They have been investigated extensively in established breast cancer, but their activation status in precancerous lesions has not been reported. Formalin-fixed, paraffin-embedded archival tissues from 59 cases of atypical ductal hyperplasia (ADH) and 31 cases of normal human breast tissue as well as 21 cases of usual ductal hyperplasias (UDH) were obtained from the First Hospital of Jilin University, China, and stained for pSTAT3 and pSTAT5 by immunohistochemistry. The median percentage of pSTAT5+ cells in ADH was 12%, not significantly deviant from that in normal breast. The median percentage of pSTAT3+ cells in ADH was 30%, significantly higher than that of normal breast. pSTAT3 and pSTAT5 were exclusive of each other--they were detected in different ADHs or in different cells within the same ADHs. In addition, both pSTAT3 and pSTAT5 were produced in similar percentages of cells in ADHs from cancer-free patients vs. ADHs that were adjacent to an invasive cancer. Our finding of a complementary expression pattern of pSTAT3 and pSTAT5 in ADH suggests that these two transcription factors may have feedback inhibitory effects on each other during early stages of breast cancer evolution, and that disruption of this inverse relationship may be important in the progression from early lesions to cancer, which exhibits positive association between pSTAT3 and pSTAT5.

Age-Associated Failure To Adjust Type I IFN Receptor Signaling Thresholds after T Cell Activation.

With increasing age, naive CD4 T cells acquire intrinsic defects that compromise their ability to respond and differentiate. Type I IFNs, pervasive constituents of the environment in which adaptive immune responses occur, are known to regulate T cell differentiation and survival. Activated naive CD4 T cells from older individuals have reduced responses to type I IFN, a defect that develops during activation and that is not observed in quiescent naive CD4 T cells. Naive CD4 T cells from young adults upregulate the expression of STAT1 and STAT5 after activation, lowering their threshold to respond to type I IFN stimulation. The heightened STAT signaling is critical to maintain the expression of CD69 that regulates lymphocyte egress and the ability to produce IL-2 and to survive. Although activation of T cells from older adults also induces transcription of STAT1 and STAT5, failure to exclude SHP-1 from the signaling complex blunts their type I IFN response. In summary, our data show that type I IFN signaling thresholds in naive CD4 T cells after activation are dynamically regulated to respond to environmental cues for clonal expansion and memory cell differentiation. Naive CD4 T cells from older adults have a defect in this threshold calibration. Restoring their ability to respond to type I IFN emerges as a promising target to restore T cell responses and to improve the induction of T cell memory.

Interference of STAT 5b expression enhances the chemo-sensitivity of gastric cancer cells to gefitinib by promoting mitochondrial pathway-mediated cell apoptosis.

Signal transducer and activator of transcription (STAT) 5, including STAT 5a and STAT 5b, was reported to play important roles in the malignant biological behaviors of tumors. However, their roles in gastric cancer, especially for STAT 5b remain unknown. This study aimed to detect the expression of STAT 5b in gastric cancer cells and analyze the role and possible mechanism of STAT 5b in the chemo-sensitivity of gastric cancer cells to gefitinib. A total of 69 patients with gastric carcinomas were analyzed for the expression of STAT 5b in carcinomas and para-carcinomas by immunohistochemistry. Cultured MGC-803 and MKN-45 cells were exposed to gefitinib and/or STAT 5b siRNA. Mitochondrial proteins including Bcl-2, Bax, caspase-3 and caspase-9 were extracted using special kits for detecting mitochondrial pathway-related apoptosis proteins. The results showed that STAT 5b expression was significantly increased in gastric carcinomas compared with para-carcinomas, with a positive rate of 49/69 in carcinomas and 27/69 in para-carcinomas (P=0.001). Gefitinib exposure reduced the relative viabilities of MGC-803 and MKN-45 cells in a concentration- and time-dependent manner, and cell apoptosis increased significantly (P<0.05) with gefitinib treatment (4 mM, 24 h). STAT 5b expression was significantly downregulated by treatment with gefitinib (4 mM, 24 h). Interference of STAT 5b expression by siRNA targeting enhanced the chemo-sensitivity of gastric cancer cells to gefitinib by promoting mitochondrial pathway-mediated apoptosis. Bax, caspase-3 and caspase-9 expression were upregulated, and Bcl-2 expression was downregulated in the combined treatment group (gefitinib+siRNA) compared with the gefitinib (4 mM, 24 h) only group in the MGC-803 and MKN-45 cells (P<0.05). Overall, STAT 5b was upregulated in gastric carcinomas compared with para-carcinomas. Interference of STAT 5b expression by siRNA targeting enhanced the chemo-sensitivity of gastric cancer cells to gefitinib by promoting mitochondrial pathway-mediated cell apoptosis. These findings may be useful for developing new approaches for the treatment of gastric cancer.

Erythropoietin acts as an anti-inflammatory signal on murine mast cells.

Recently it was found that the erythropoietin receptor (EpoR) is expressed on innate immune cells, such as dendritic cells and macrophages. We found that murine bone marrow-derived mast cells express the EpoR and that its expression is increased under hypoxic conditions. Interestingly, Epo stimulation of the cells did not activate signal transducer and activator of transcription molecules, nor did we find differences in the expression of typical STAT-dependent genes, the proliferation rate, and the ability to differentiate or to protect the cells from apoptosis. Instead, we demonstrate that stimulation of mast cells with Epo leads to phosphorylation of the receptor tyrosine kinase c-kit. We hypothesize that this is due to the formation of a receptor complex between the EpoR and c-kit. The common beta chain of the IL-3 receptor family, which was described as part of the tissue protective receptor (TPR) on other non-erythroid cells, however is not activated. To investigate whether the EpoR/c-kit complex has tissue protective properties, cells were treated with the Toll-like receptor ligand LPS. Combined Epo and LPS treatment downregulated the inflammatory response of the cells as detected by a decrease in IL-6 and TNF-α secretion.

Influence of resistance exercise intensity and metabolic stress on anabolic signaling and expression of myogenic genes in skeletal muscle.

INTRODUCTION: We investigated the effect of resistance exercise intensity and exercise-induced metabolic stress on the activation of anabolic signaling and expression of myogenic genes in skeletal muscle. METHODS: Ten strength-trained athletes performed high-intensity [HI, 74% of 1-repetition maximum (RM)], middle-intensity (MI, 54% 1RM), or middle-intensity (54% 1RM) no-relaxation exercise (MIR). Kinase phosphorylation level and myogenic gene expression in muscle samples were evaluated before, 45 min, 5 h, and 20 h after exercise. RESULTS: The lactate concentration in MI was approximately 2-fold lower than in the 2 other sessions, and was highest in MIR. The phosphorylation level of extracellular kinase 1/2Thr202/Tyr204 after exercise was related to metabolic stress. Metabolic stress induced a decrease in myostatin mRNA expression, whereas mechano-growth factor mRNA level depended on exercise intensity. CONCLUSIONS: This study demonstrates that both intensity and exercise-induced metabolic stress can be manipulated to affect muscle anabolic signaling.