Stromal STAT5-Mediated Trophic Activity Regulates Hematopoietic Niche Factors.

Signal transducer and activator of transcription 5 (STAT5a and STAT5b) are intrinsically critical for normal hematopoiesis but are also expressed in stromal cells. Here, STAT5ab knockout (KO) was generated with a variety of bone marrow hematopoietic and stromal Cre transgenic mouse strains. Vav1-Cre/+STAT5abfl/fl, the positive control for loss of multipotent hematopoietic function, surprisingly dysregulated niche factor mRNA expression, and deleted STAT5ab in CD45neg cells. Single-cell transcriptome analysis of bone marrow from Vav1-Cre/+ wild-type or Vav1-Cre/+STAT5abfl/fl mice showed hematopoietic stem cell (HSC) myeloid commitment priming. Nes+ cells were detected in both CD45neg and CD45+ clusters and deletion of STAT5ab with Nes-Cre caused hematopoietic repopulating defects. To follow up on these promiscuous Cre promoter deletions in CD45neg and CD45+ bone marrow cell populations, more stroma-specific Cre strains were generated and demonstrated a reduction in multipotent hematopoietic progenitors. Functional support for niche-supporting activity was assessed using STAT5-deficient mesenchymal stem cells (MSCs). With Lepr-Cre/+STAT5abfl/fl, niche factor mRNAs were downregulated with validation of reduced IGF-1 and CXCL12 proteins. Furthermore, advanced computational analyses revealed a key role for STAT5ab/Cish balance with Cish strongly co-expressed in MSCs and HSCs primed for differentiation. Therefore, STAT5ab-associated gene regulation supports the bone marrow microenvironment.

Effect of PGE2 on TT cells viability and division.

Previous studies have shown prostaglandin E2 (PGE2) produced a marked increase in calcitonin secretion in human C-cells derived from medullary thyroid carcinoma. However, it's unclear whether PGE2 can increase the growth of C cells. In this study, we use TT cells as a C cell model to investigate the effect of PGE2 on the growth of C cells. The results revealed that both PGE2 and arachidonic acid (AA) significantly increased the count of TT cells, whereas indomethacin and Dup697 reduced this count. Notably, an increase in the level of AA was associated with an increase in the number of proliferating TT cells, indicating a dose-response relationship. PGE2 and its receptor agonists (sulprostone and butaprost) enhanced the proliferation of TT cells. By contrast, 17-phenyl-trinor-PGE2 exerted no significant effect on TT cell proliferation, whereas L161982 suppressed it. The positive effect of AA on TT cell proliferation was inhibited by indomethacin, NS398, Dup697 (complete inhibition), and SC560. Both PGE2 and AA increased the level of p-STAT5a. The positive effect of AA on p-STAT5a was completely inhibited by Dup697 but not indomethacin, NS398, or SC560. Treatment with indomethacin or Dup697 alone reduced the level of STAT5a in TT cells. AA increased the level of STAT5a, but this effect was inhibited by indomethacin, NS398, and Dup697. Overall, this study confirms the effect of PGE2 on the proliferation of TT cells. This effect is likely mediated through EP2, EP3, and EP4 receptors and associated with an increase in p-STAT5a level within TT cells.

Nuclear translocation of STAT5 initiates iron overload in huntington's disease by up-regulating IRP1 expression.

Mutant huntingtin (mHtt) proteins interact to form aggregates, disrupting cellular functions including transcriptional dysregulation and iron imbalance in patients with Huntington's disease (HD) and mouse disease models. Previous studies have indicated that mHtt may lead to abnormal iron homeostasis by upregulating the expression of iron response protein 1 (IRP1) in the striatum and cortex of N171-82Q HD transgenic mice, as well as in HEK293 cells expressing the N-terminal fragment of mHtt containing 160 CAG repeats. However, the mechanism underlying the upregulation of IRP1 remains unclear. We investigated the levels and phosphorylation status of signal transducer and activator of transcription 5 (STAT5) in the brains of N171-82Q HD transgenic mice using immunohistochemistry staining. We also assessed the nuclear localization of STAT5 protein through western blot and immunofluorescence, and measured the relative RNA expression levels of STAT5 and IRP1 using RT-PCR in both N171-82Q HD transgenic mice and HEK293 cells expressing the N-terminal fragment of huntingtin. Our findings demonstrate that the transcription factor STAT5 regulates the transcription of the IPR1 gene in HEK293 cells. Notably, both the brains of N171-82Q mice and 160Q HEK293 cells exhibited increased nuclear content of STAT5, despite unchanged total STAT5 expression. These results suggest that mHtt promotes the nuclear translocation of STAT5, leading to enhanced expression of IRP1. The nuclear translocation of STAT5 initiates abnormal iron homeostatic pathways, characterized by elevated IRP1 expression, increased levels of transferrin and transferrin receptor, and iron accumulation in the brains of HD mice. These findings provide valuable insights into potential therapeutic strategies targeting iron homeostasis in HD.

The Mechanism of Leptin Resistance in Obesity and Therapeutic Perspective.

Leptin resistance is induced via leptin signaling blockade by chronic overstimulation of the leptin receptor and intracellular signaling defect or increased hypothalamic inflammation and suppressor of cytokine signaling (SOCS)-3 expression. High-fat diet triggers leptin resistance induced by at least two independent causes: first, the limited ability of peripheral leptin to activate hypothalamic signaling transducers and activators of transcription (STAT) signaling and secondly a signaling defect in leptin-responsive hypothalamic neurons. Central leptin resistance is dependent on decreased leptin transport efficiency across the blood brain barrier (BBB) rather than hypothalamic leptin insensitivity. Since the hypothalamic phosphorylated STAT3 (pSTAT3) represents a sensitive and specific readout of leptin receptor-B signaling, the assessment of pSTAT3 levels is the gold standard. Hypertriglyceridemia is one of important factors to inhibit the transport of leptin across BBB in obesity. Mismatch between high leptin and the amount of leptin receptor expression in obesity triggers brain leptin resistance via increasing hypothalamic inflammation and SOCS-3 expression. Therapeutic strategies that regulate the passage of leptin to the brain include the development of modifications in the structure of leptin analogues as well as the synthesis of new leptin receptor agonists with increased BBB permeability. In the hyperleptinemic state, polyethylene glycol (PEG)-modified leptin is unable to pass through the BBB. Peripheral histone deacetylase (HDAC) 6 inhibitor, tubastatin, and metformin increase central leptin sensitization. While add-on therapy with anagliptin, metformin and miglitol reduce leptin concentrations, the use of long-acting leptin analogs, and exendin-4 lead to the recovery of leptin sensitivity. Contouring surgery with fat removal, and bariatric surgery independently of the type of surgery performed provide significant improvement in leptin concentrations. Although approaches to correcting leptin resistance have shown some success, no clinically effective application has been developed to date. Due to the impairment of central and peripheral leptin signaling, as well as the extensive integration of leptin-sensitive metabolic pathways with other neurons, the effectiveness of methods used to eliminate leptin resistance is extremely limited.

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Signal transducer and activator of transcription 5 (STAT5) can be involved in the processes such as cell proliferation, differentiation, apoptosis, and hematopoiesis, and its dysregulation is closely associated with the development and progression of malignant tumors including leukemia and may affect the treatment outcome and prognosis of pediatric patients. Identification of STAT5 can facilitate targeted therapy to improve the response rate of children with acute lymphoblastic leukemia. This article reviews the impact of STAT5 on the development/progression, targeted therapy strategies and the prognosis of childhood acute lymphoblastic leukemia.

A crucial role of fibroblast growth factor 2 in the differentiation of hair follicle stem cells toward endothelial cells in a STAT5-dependent manner.

Fibroblast growth factor (FGF2) is reported to affect the proliferation, differentiation, and survival abilities of stem cells. In this study, we hypothesize that FGF2 might promote the differentiation of hair follicle stem cell (HFSCs) into endothelial cells (ECs), in a manner dependent on STAT5 activation. We first treated human HFSCs with recombinant human FGF2 to determine the involvement of FGF2 in the differentiation of HFSCs. Then the expression of EC-specific markers including von Willebrand factor (vWF), VE-cadherin, CD31, FLT-1, KDR and Tie2 was evaluated using immunofluorescence and flow cytometry, while the expression of HFSC-specific markers such as K15, K19, Lgr5, Sox9 and Lhx2 was determined by flow cytometry. Next, in vitro tube formation was performed to confirm the function of FGF2, and low-density lipoprotein (LDL) uptake by ECs and HFSCs was studied by Dil-acetylated LDL assay. In addition, we transduced FGF2-treated HFSCs with constitutive-active or dominant-negative STAT5A adenovirus vectors. FGF2 up-regulated the expression of EC-specific markers, and promoted the differentiation of HFSCs into ECs, tube formation and LDL uptake. The phosphorylated STAT5 was translocated into the nucleus of HFSCs after FGF2 treatment, but this translocation was blocked by the dominant-negative STAT5A mutant. FGF2 increased the differentiation potential through the activation of STAT5 in vivo. Taken together, we find that FGF2 promotes the differentiation of HFSCs into ECs via activated STAT5, which gives a new perspective on the role of FGF2 in the development of ischemic vascular disease.

Rationale for a Combination Therapy with the STAT5 Inhibitor AC-4-130 and the MCL1 Inhibitor S63845 in the Treatment of FLT3-Mutated or TET2-Mutated Acute Myeloid Leukemia.

The FMS-like tyrosine kinase 3 (FLT3) gene is mutated in one-third of patients with de novo acute myeloid leukemia (AML). Mutated FLT3 variants are constitutively active kinases signaling via AKT kinase, MAP kinases, and STAT5. FLT3 inhibitors have been approved for the treatment of FLT3-mutated AML. However, treatment response to FLT3 inhibitors may be short-lived, and resistance may emerge. Compounds targeting STAT5 may enhance and prolong effects of FLT3 inhibitors in this subset of patients with FLT3-mutated AML. Here STAT5-inhibitor AC-4-130, FLT3 inhibitor midostaurin (PKC412), BMI-1 inhibitor PTC596, MEK-inhibitor trametinib, MCL1-inhibitor S63845, and BCL-2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Synergistic effects on cell viability were detected in both FLT3-mutated and FLT3-wild-type AML cells treated with AC-4-130 in combination with the MCL1 inhibitor S63845. AML patient samples with a strong response to AC-4-130 and S63845 combination treatment were characterized by mutated FLT3 or mutated TET2 genes. Susceptibility of AML cells to AC-4-130, PTC596, trametinib, PKC412, and venetoclax was altered in the presence of HS-5 stroma. Only the MCL1 inhibitor S63845 induced cell death with equal efficacy in the absence or presence of bone marrow stroma. The combination of the STAT5-inhibitor AC-4-130 and the MCL1 inhibitor S63845 may be an effective treatment targeting FLT3-mutated or TET2-mutated AML.

Usefulness of immunohistochemistry to distinguish between secretory carcinoma and acinic cell carcinoma in the salivary gland.

Secretory carcinoma (SC) of the salivary gland is a relatively newly described disease, separate from acinic cell carcinoma (ACC), which frequently displays ETV6-NTRK3 gene fusion. However, the differences between SC and ACC remain unclear. Here, histological reevaluation of 12 formerly diagnosed ACC cases was performed, which yielded a new diagnosis of SC in four cases due to a lack of obvious acinar-like cells. Immunohistochemically, phosphorylated signal transducer and activator of transcription 5 (p-STAT5) was expressed in SC but not in ACC, whereas discovered on GIST-1 (DOG1) was expressed in ACC but not in SC. Molecular analysis was possible in three SC cases, of which two showed the ETV6-NTRK3 fusion transcript on reverse-transcription polymerase chain reaction, as well as breaks in the ETV6 gene on fluorescence in situ hybridization. However, the remaining SC cases did not show this fusion transcript. Recently, several reports have suggested that SC might not be adequately diagnosed if the focus is placed solely on the ETV6-NTRK3 fusion gene due to genetic diversity. In this regard, immunohistochemistry of p-STAT5 and DOG1 is expected to be a useful alternative diagnostic tool to discriminate SC from ACC.

Emodin Inhibits Resistance to Imatinib by Downregulation of Bcr-Abl and STAT5 and Allosteric Inhibition in Chronic Myeloid Leukemia Cells.

Imatinib-resistance is a significant concern for Bcr-Abl-positive chronic myelogenous leukemia (CML) treatment. Emodin, the predominant compound of traditional medicine rhubarb, was reported to inhibit the multidrug resistance by downregulating P-glycoprotein of K562/ADM cells with overexpression of P-glycoprotein in our previous studies. In the present study, we found that emodin can be a potential inhibitor for the imatinib-resistance in K562/G01 cells which are the imatinib-resistant subcellular line of human chronic myelogenous leukemia cells with overexpression of breakpoint cluster region-abelson (Bcr-Abl) oncoprotein. Emodin greatly enhanced cell sensitivity to imatinib, suppressed resistant cell proliferation and increased potentiated apoptosis induced by imatinib in K562/G01 cells. After treatment of emodin and imatinib together, the levels of p-Bcr-Abl and Bcr-Abl were significantly downregulated. Moreover, Bcr-Abl important downstream target, STAT5 and its phosphorylation were affected. Furthermore, the expression of Bcr-Abl and signal transducers and activators of transcription 5 (STAT5) related molecules, including c-MYC, MCL-1, poly(ADP-ribose)polymerase (PARP), Bcl-2 and caspase-3, were changed. Emodin also decreased Src expression and its phosphorylation. More importantly, emodin simultaneously targeted both the ATP-binding and allosteric sites on Bcr-Abl by molecular docking, with higher affinity with the myristoyl-binding site for enhanced Bcr-Abl kinase inhibition. Overall, these data indicated emodin might be an effective therapeutic agent for inhibiting resistance to imatinib in CML treatment.

Thymic stromal lymphopoietin epigenetically upregulates Fc receptor γ subunit-related receptors on antigen-presenting cells and induces TH2/TH17 polarization through dectin-2.

BACKGROUND: Fc receptor γ subunit (FcRγ)-related receptors expressed on antigen-presenting cells (APCs) enhance allergen sensitization and allergic inflammation. DNA demethylation of the high-affinity IgE receptor γ subunit gene (FCER1G) leads to FcRγ and FcεRI overexpression on monocytes from patients with atopic dermatitis. OBJECTIVE: We investigated epigenetic mechanisms underlying FCER1G demethylation and upregulation of FcRγ-related receptors on APCs and the consequent effect on allergic responses. METHODS: Effects of thymic stromal lymphopoietin (TSLP) on expression of FcRγ and its related receptors and methylation or hydroxymethylation of FCER1G in human monocytes were assessed. Recruitment of ten-eleven translocation protein (TET) 2 to FCER1G by TSLP-activated phosphorylated signal transducer and activator of transcription 5 (pSTAT5) was evaluated. Effects of TSLP on expression of FcRγ-related receptors and costimulatory receptors on monocyte-derived dendritic cells (DCs) and the ability of DCs to take up ovalbumin were analyzed. TSLP-induced TH polarization and related cytokine production were also analyzed. RESULTS: pSTAT5 activation by TSLP resulted in TET2 recruitment to FCER1G, leading to FCER1G demethylation and subsequent upregulation of FcRγ-related receptors on monocytes. TSLP not only stimulated monocyte-derived DC maturation but also maintained their allergen uptake ability, likely through maintenance and upregulation of FcRγ-related receptors. Allergen sensitization and upregulation of TH2/TH17-related cytokines contributed to TSLP-DC-induced TH2/TH17 polarization. The latter was attenuated on neutralization with a dectin-2 antibody. CONCLUSIONS: TSLP mediated upregulation of FcRγ-related receptors on APCs through activation of pSTAT5, which recruited TET2 to induce FCER1G demethylation. TSLP-induced allergic TH2/TH17 polarization likely depends on dectin-2-mediated allergen sensitization and upregulation of TH2/TH17-related cytokines.

Siglec-F is induced by granulocyte-macrophage colony-stimulating factor and enhances interleukin-4-induced expression of arginase-1 in mouse macrophages.

Siglecs are cell surface lectins that recognize sialic acids and are primarily expressed in hematopoietic cells. Previous studies showed that some Siglecs regulate macrophage function. In the present study, we examined the induction and putative roles of mouse Siglec-F in bone-marrow-derived macrophages in mice. A quantitative RT-PCR analysis showed that the basal expression of Siglec-F was weak in bone-marrow-derived macrophages differentiated by macrophage colony-stimulating factor. However, a 24-hr stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced Siglec-F expression. GM-CSF also enhanced Siglec-F expression in thioglycollate-induced peritoneal macrophages. The inhibition of signal transducer and activator of transcription 5 (STAT5), but not that of phosphoinositide 3-kinase or mitogen-activated protein kinase kinase, significantly reduced the induction of Siglec-F. Interleukin-3, which uses a common ß-chain shared with the GM-CSF receptor to stimulate the STAT5 pathway, also enhanced Siglec-F expression. The knockdown of Siglec-F by a specific small interfering RNA enhanced GM-CSF-induced STAT5 phosphorylation, suggesting that Siglec-F down-regulates its own expression upon prolonged GM-CSF stimulation. Furthermore, the knockdown of Siglec-F reduced the STAT6 phosphorylation and expression of arginase-1 in interleukin-4-stimulated macrophages. These results suggest that Siglec-F fine-tunes the immune responses of macrophages.

Ginkgetin, a biflavone from Ginkgo biloba leaves, prevents adipogenesis through STAT5-mediated PPARγ and C/EBPα regulation.

Adipogenesis involved in hypertrophy and hyperplasia of adipocytes is responsible for expanding the mass of adipose tissues in obese individuals. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) are two principal transcription factors induced by delicate signaling pathways, including signal transducer and activator of transcription 5 (STAT5), in adipogenesis. Here, we demonstrated a novel role of ginkgetin, a biflavone from Ginkgo biloba leaves, as a STAT5 inhibitor that blocks the differentiation of preadipocytes into adipocytes. During the differentiation of 3T3-L1 cells, ginkgetin treatment during the first 2 days markedly inhibited the formation of lipid-bearing adipocytes. PPARγ and C/EBPα expression was decreased in 3T3-L1 cells during adipogenesis following ginkgetin treatment, whereas no change was observed in C/EBPß or C/EBPδ expression. Inhibition of PPARγ and C/EBPα expression by ginkgetin occurred through the prevention of STAT5 activation during the initiation phase of adipogenesis. In addition, ginkgetin-mediated the inhibition of adipogenesis was recapitulated in the differentiation of primary preadipocytes. Lastly, we confirmed the inhibitory effects of ginkgetin on the hypertrophy of white adipose tissues from high-fat diet-fed mice. These results indicate that ginkgetin is a potential anti-adipogenesis and anti-obesity drug.

CircScd1 Promotes Fatty Liver Disease via the Janus Kinase 2/Signal Transducer and Activator of Transcription 5 Pathway.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in affluent countries. Recent studies have reported that circular RNAs (circRNAs) are important regulators of hepatic steatosis. However, the role and mechanism of circRNA in NAFLD are poorly understood. AIMS: This study is to reveal the role and mechanism of circRNA in NAFLD. METHODS: Through NAFLD-related circRNA microarrays, we used real-time quantitative reverse transcription-polymerase chain reaction to screen circScd1 levels in control and test groups of mice fed a high-fat diet. RNA interference and over-expression plasmid vectors were used to manipulate the expression of circScd1, and the biological effects were evaluated by oil red staining, triglyceride detection, and western blot analysis. RESULTS: CircScd1 expression was significantly lower in NAFLD tissues than in control tissues. Moreover, over-expression of circScd1 significantly inhibited the formation of lipid droplets. Western blot analyses showed that the protein levels of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) were significantly increased. However, knockdown of circScd1 significantly promoted the degree of hepatocellular lipidosis and reduced the expression levels of JAK2 and STAT5. CONCLUSIONS: Aberrant expression of circScd1 affects the extent of hepatocellular lipidosis in NAFLD and promotes fatty liver disease via the JAK2/STAT5 pathway.

Steroid resistance of airway type 2 innate lymphoid cells from patients with severe asthma: The role of thymic stromal lymphopoietin.

BACKGROUND: Type 2 innate lymphoid cells (ILC2s) represent an important type 2 immune cell. Glucocorticoid regulation of human ILC2s is largely unknown. OBJECTIVE: We sought to assess steroid resistance of human blood and airway ILC2s from asthmatic patients and to examine its mechanism of induction. METHODS: We studied human blood and lung ILC2s from asthmatic patients and control subjects using flow cytometry and ELISA. RESULTS: Dexamethasone inhibited (P = .04) chemoattractant receptor-homologous molecule expressed on TH2 lymphocytes and type 2 cytokine expression by blood ILC2s stimulated with IL-25 and IL-33. However, it did not do so when ILC2s were stimulated with IL-7 and thymic stromal lymphopoietin (TSLP), 2 ligands of IL-7 receptor α. Unlike blood ILC2s, bronchoalveolar lavage (BAL) fluid ILC2s from asthmatic patients were resistant to dexamethasone. BAL fluid from asthmatic patients had increased TSLP but not IL-7 levels. BAL fluid TSLP levels correlated (r = 0.74) with steroid resistance of ILC2s. TSLP was synergistically induced in epithelial cells by IL-13 and human rhinovirus. Mechanistically, dexamethasone upregulated ILC2 expression of IL-7 receptor α, which augmented and sustained signal transducer and activator of transcription (STAT) 5 signaling by TSLP. TSLP induced mitogen-activated protein kinase kinase (MEK), c-Fos, inhibitor of DNA binding 3, phosphorylated signal transducer and activator of transcription (pSTAT) 3, and pSTAT5, molecules linked to steroid resistance. Dexamethasone inhibited c-Fos, inhibitor of DNA binding 3, and pSTAT3 but not pSTAT5 and MEK. The MEK inhibitor trametinib, the Janus kinase-STAT inhibitor tofacitinib, and the STAT5 inhibitor pimozide reversed steroid resistance of BAL ILC2s. CONCLUSIONS: Dexamethasone inhibited type 2 cytokine production by blood ILC2s. IL-7 and TSLP abrogated this inhibition and induced steroid resistance of ILC2s in a MEK- and STAT5-dependent manner. BAL fluid ILC2s from asthmatic patients with increased TSLP levels were steroid resistant, which was reversed by clinically available inhibitors of MEK and STAT5.

Three Tyrosine Residues in the Erythropoietin Receptor Are Essential for Janus Kinase 2 V617F Mutant-induced Tumorigenesis.

The erythropoietin receptor (EpoR) regulates development of blood cells, and its full activation normally requires the cytokine erythropoietin (Epo). In the case of myeloproliferative neoplasms (MPN), Epo-independent signaling through EpoR can be caused by a point mutation, V617F, in the EpoR-interacting tyrosine kinase Janus kinase 2 (JAK2). In cells expressing the JAK2 V617F mutant, eight tyrosine residues in the intracellular domain of EpoR are phosphorylated, but the functional role of these phosphorylations in oncogenic signaling is incompletely understood. Here, to evaluate the functional consequences of the phosphorylation of these tyrosine residues, we constructed an EpoR-8YF mutant in which we substituted all eight tyrosine residues with phenylalanine. Co-expression of EpoR-8YF with the JAK2 V617F mutant failed to induce cytokine-independent cell proliferation and tumorigenesis, indicating that JAK2-mediated EpoR phosphorylation is the reason for JAK2 V617F mutant-induced oncogenic signaling. An exhaustive mutational analysis of the eight EpoR tyrosine residues indicated that three of these residues, Tyr-343, Tyr-460, and Tyr-464, are required for the JAK2 V617F mutant to exhibit its oncogenic activity. We also showed that phosphorylation at these three residues was necessary for full activation of the transcription factor STAT5, which is a critical downstream factor of JAK2 V617F-induced oncogenic signaling. In contrast, Epo stimulation could moderately stimulate the proliferation of cells expressing wild type JAK2 and EpoR-8YF, suggesting that the requirement of the phosphorylation of these three tyrosine residues seems to be specific for the oncogenic proliferation provoked by V617F mutation. Collectively, these results have revealed that phosphorylation of Tyr-343, Tyr-460, and Tyr-464 in EpoR underlies JAK2 V617F mutant-induced tumorigenesis. We propose that the targeted disruption of this pathway has therapeutic utility for managing MPN.

Impaired natural killer cell functions in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations.

BACKGROUND: Gain-of-function (GOF) mutations affecting the coiled-coil domain or the DNA-binding domain of signal transducer and activator of transcription 1 (STAT1) cause chronic mucocutaneous candidiasis disease. This condition is characterized by fungal and bacterial infections caused by impaired generation of TH17 cells; meanwhile, some patients with chronic mucocutaneous candidiasis disease might also have viral or intracellular pathogen infections. OBJECTIVE: We sought to investigate the effect of STAT1 GOF mutations on the functioning of natural killer (NK) cells. METHODS: Because STAT1 is involved in the signaling response to several cytokines, we studied NK cell functional activities and STAT1 signaling in 8 patients with STAT1 GOF mutations. RESULTS: Functional analysis of NK cells shows a significant impairment of cytolytic and degranulation activities in patients with STAT1 GOF mutations. Moreover, NK cells from these patients display lower production of IFN-γ in response to IL-15 and reduced proliferation after stimulation with IL-2 or IL-15, suggesting that STAT5 signaling is affected. In addition, signaling studies demonstrate that the increased phosphorylation of STAT1 in response to IFN-α is associated with detectable activation of STAT1 and increased STAT1 binding to the interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) promoter in response to IL-15, whereas STAT5 phosphorylation and DNA binding to IL-2 receptor α (IL2RA) are reduced or not affected in response to the same cytokine. CONCLUSION: These observations suggest that persistent activation of STAT1 might affect NK cell proliferation and functional activities.

Identification of an Essential Cytoplasmic Region of Interleukin-7 Receptor α Subunit in B-Cell Development.

Interleukin-7 (IL-7) is essential for lymphocyte development. To identify the functional subdomains in the cytoplasmic tail of the IL-7 receptor (IL-7R) α chain, here, we constructed a series of IL-7Rα deletion mutants. We found that IL-7Rα-deficient hematopoietic progenitor cells (HPCs) gave rise to B cells both in vitro and in vivo when a wild-type (WT) IL-7Rα chain was introduced; however, no B cells were observed under the same conditions from IL-7Rα-deficient HPCs with introduction of the exogenous IL-7Rα subunit, which lacked the amino acid region at positions 414⁻441 (d414⁻441 mutant). Signal transducer and activator of transcription 5 (STAT5) was phosphorylated in cells with the d414⁻441 mutant, similar to that in WT cells, in response to IL-7 stimulation. In contrast, more truncated STAT5 (tSTAT5) was generated in cells with the d414⁻441 mutant than in WT cells. Additionally, the introduction of exogenous tSTAT5 blocked B lymphopoiesis but not myeloid cell development from WT HPCs in vivo. These results suggested that amino acids 414⁻441 in the IL-7Rα chain formed a critical subdomain necessary for the supportive roles of IL-7 in B-cell development.

The Role of Signal Transducer and Activator of Transcription 5 and Transforming Growth Factor-ß1 in Hepatic Fibrosis Induced by Chronic Hepatitis C Virus Infection in Egyptian Patients.

OBJECTIVE: To investigate the possible role of signal transducer and activator of transcription 5 (STAT5) in the pathogenesis of liver fibrosis in Egyptian patients with chronic hepatitis C (CHC) virus infection and its relation to hepatic stellate cells (HSC). SUBJECTS AND METHODS: Sixty-five patients (46 males and 19 females) were divided into 4 groups based on the severity of fibrosis as detected by Fibroscan as follows: F1, n = 15; F2, n = 21; F3, n = 13; and F4, n = 16. Twenty age- and gender-matched healthy persons volunteered as controls. The serum levels of STAT5, TGF-ß1, α-smooth muscle actin (α-SMA), fasting blood sugar, and fasting insulin, as well as homeostasis model assessment of insulin resistance (HOMA-IR), were determined and compared for all groups. The usefulness of the studied serum biomarkers for predicting liver fibrosis was evaluated using a receiver operating characteristic curve. RESULTS: Serum levels of STAT5 were significantly lower in patients compared to controls (9.69 ± 5.62 vs. 14.73 ± 6.52, p ≤ 0.001); on the contrary, TGF-ß1, α-SMA, and HOMA-IR were significantly higher in patients compared to controls (mean: 1,796.04 vs. 1,636.94; 14.94 vs. 8.1; and 7.91 vs. 4.18; p ≤ 0.01 and 0.001, respectively). TGF-ß1 and α-SMA showed a progressive increase with advancing severity of hepatic fibrosis (mean TGF-ß1: 2,058.4 in F1-F2 and 1,583.8 in F3-F4, p ≤ 0.04; mean α-SMA: 13.59 in F1-F2 and 16.62 in F3-F4, p ≤ 0.05). STAT5 had a significant negative correlation with TGF-ß1 (p ≤ 0.001), while no correlation was detected with α-SMA (p ≤ 0.8). CONCLUSIONS: STAT5 may play a significant role in hepatic fibrogenesis through the induction of TGF-ß1 but not through the activation of hepatic stellate cells.

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.

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.