Prolonged STAT3 Activation Research Articles

Abstract 13640: Endothelial Dysfunction in Chronic Stress-Related Cardiovascular Disease May Be Driven by Epinephrine Exacerbating TNFα Effects in a JAK/Stat3-Dependent Manner

Background: Chronic psychosocial and environmental stress (PSES) impairs endothelial function and accelerates cardiovascular disease (CVD); however, underlying signaling pathways are less understood. Studies have independently shown that chronic PSES increases catecholamine levels (e.g. epinephrine) and induces a low-grade chronic inflammation by cytokines (e.g. tumor necrosis factor α - TNFα). The potential impact of a combination of increased levels of epinephrine and TNFα on the endothelium has not been fully investigated. We sought to elucidate if epinephrine and TNFα synergistically impair endothelial function and which signaling pathways might underlie this phenomenon. Methods: Human aortic endothelial cells (HAoEC) were treated with 10μM epinephrine (E), 10ng/ml TNFα (T), or their combination (E+T). Endothelial monolayer integrity was measured in a transwell filter setup and visualized by immunofluorescence. Signaling pathway analysis was performed by western blotting and PCR. Various pathway inhibitors were deployed to identify key pathways for the observed effects. Results: The addition of epinephrine exacerbated the observed TNFα-induced loss of the adherens junction protein VE-cadherin accompanied with accelerated intercellular gap formation and loss of endothelial barrier integrity. Signaling pathway analysis revealed that E+T induced delayed and prolonged Stat3 activation lasting up to 24h of treatment, which could not be observed in the individual E or T treatments. Inhibition of the Stat3 pathway using Tofacetinib, an inhibitor of JAK/Stat signaling, prevented the E+T-induced activation of Stat3, loss in VE-cadherin, E+T-induced gap formation and loss in barrier integrity, indicating the crucial importance of JAK/Stat signaling in the E+T-induced effects on the endothelium. Conclusion: These data indicate long-term PSES-induced CVD might in part be mediated by a combined effect of epinephrine and TNFα that can induce endothelial dysfunction dependent on a JAK/Stat3 pathway. Understanding pathways involved in PSES-related CVD is crucial to elucidate the biology of adversity in CVD health disparities, or mechanisms by which populations most impacted by PSES are disproportionately susceptible to CVD.

Oncostatin M Receptor-Targeted Antibodies Suppress STAT3 Signaling and Inhibit Ovarian Cancer Growth.

Although patients with advanced ovarian cancer may respond initially to treatment, disease relapse is common, and nearly 50% of patients do not survive beyond five years, indicating an urgent need for improved therapies. To identify new therapeutic targets, we performed single-cell and nuclear RNA-seq data set analyses on 17 human ovarian cancer specimens, revealing the oncostatin M receptor (OSMR) as highly expressed in ovarian cancer cells. Conversely, oncostatin M (OSM), the ligand of OSMR, was highly expressed by tumor-associated macrophages and promoted proliferation and metastasis in cancer cells. Ovarian cancer cell lines and additional patient samples also exhibited elevated levels of OSMR when compared with other cell types in the tumor microenvironment or to normal ovarian tissue samples. OSMR was found to be important for ovarian cancer cell proliferation and migration. Binding of OSM to OSMR caused OSMR-IL6ST dimerization, which is required to produce oncogenic signaling cues for prolonged STAT3 activation. Human monoclonal antibody clones B14 and B21 directed to the extracellular domain of OSMR abrogated OSM-induced OSMR-IL6ST heterodimerization, promoted the internalization and degradation of OSMR, and effectively blocked OSMR-mediated signaling in vitro. Importantly, these antibody clones inhibited the growth of ovarian cancer cells in vitro and in vivo by suppressing oncogenic signaling through OSMR and STAT3 activation. Collectively, this study provides a proof of principle that anti-OSMR antibody can mediate disruption of OSM-induced OSMR-IL6ST dimerization and oncogenic signaling, thus documenting the preclinical therapeutic efficacy of human OSMR antagonist antibodies for immunotherapy in ovarian cancer. SIGNIFICANCE: This study uncovers a role for OSMR in promoting ovarian cancer cell proliferation and metastasis by activating STAT3 signaling and demonstrates the preclinical efficacy of antibody-based OSMR targeting for ovarian cancer treatment.

Bazedoxifene inhibits sustained STAT3 activation and increases survival in GBM

An important factor correlated with poor survival in glioblastoma (GBM) is the aberrant and persistent activation of STAT3, a critical transcription factor that regulates multiple genes with key roles in cell survival, proliferation, resistance to chemotherapy, and stem cell maintenance. The Interleukin-6 (IL6)-STAT3 signaling axis has been studied extensively in inflammation and cancer. However, it is not completely understood how high levels of activated STAT3 are sustained in tumors. Previously, we identified a novel mechanism of biphasic activation of STAT3 in response to gp130-linked cytokines, including IL6, in which activation of STAT3 is prolonged by circumventing the negative regulatory mechanisms induced by its initial activationTo target prolonged STAT3 activation, we used the small molecule inhibitor bazedoxifene (BZA), which blocks formation of the IL6 receptor-gp130 complex. Glioma stem-like cells (GSCs) are more tumorigenic and more resistant to therapy. STAT3 is a key driver of the expression of stem cell transcription factors, making it a therapeutically important target in GBM. We show that treating GSCs with BZA decreases their self-renewal capacity and the expression of GSC markers in vitro. Additionally, BZA crosses the blood-brain barrier and confers a survival advantage in an orthotopic syngeneic mouse model of GBM. Although IL6-STAT3 signaling is important for GSC survival, a therapeutic agent that inhibits this pathway without toxicity has yet to be identified. Our findings reveal a mechanism of sustained STAT3 signaling in GBM and reveal its role in GSC maintenance, and we identify BZA as a novel candidate for treating GBM.

Inhibition of heat shock protein (HSP) 90 reverses signal transducer and activator of transcription (STAT) 3-mediated muscle wasting in cancer cachexia mice.

Cancer cachexia is a common cause of death among cancer patients with no currently effective treatment available. In animal models, aberrant activation of STAT3 in skeletal muscle contributes to muscle wasting. However, clinically the factors regulating STAT3 activation and the molecular mechanisms involved remain incompletely understood. The expression of HSP90 and the activation of STAT3 were detected in muscle from the patients with cancer cachexia or the tumour-bearing cachectic mice. HSP90 inhibitors, including 17DMAG (alvespimycin) and PU-H71, were administered to cachexic mice and cachexia parameters, weight loss, food intake, survival rate, body composition, serum metabolites, muscle wasting pathology and catabolic activation were analysed. The co-culture of C2C12 myotube cells with C26 conditioned media was performed to investigate the pathological mechanism involved in catabolic muscle wasting. The roles of HSP90, STAT3 and FOXO1 in myotube atrophy were explored via overexpression or knockdown. An enhanced interaction between activated STAT3 and HSP90 in the skeletal muscle of cancer cachexia patients, is a crucial for the development of cachectic muscle wasting. HSP90 inhibitors 17DMAG and PU-H71 alleviated the muscle wasting in C26 and models or the myotube atrophy of C2C12 cells induced by C26 conditional medium. Prolonged STAT3 activation transactivated FOXO1 by binding directly to its promoter and triggered the muscle wasting in a FOXO1-dependent manner in muscle cells. The HSP90/STAT3/FOXO1 axis plays a critical role in cachectic muscle wasting, which might be a potential therapeutic target for the treatment of cancer cachexia.

Endothelial dysfunction may be driven by a synergism of epinephrine and TNFα via prolonged STAT3 activation

Background and Aims: Epidemiologic studies have highlighted the association between chronic psychosocial stressors and adverse cardiovascular outcomes; however, the mechanistic relationship between psychosocial stress and atherosclerosis is not well understood. Recent studies have found that epinephrine and tumor necrosis factor-alpha (TNFα) are upregulated in individuals reporting higher levels of chronic stress. Our aim was to evaluate a potential synergistic effect of epinephrine and TNFα (E/T) on human aortic endothelial cells (HAoEC) and elucidate the molecular signaling events that potentially drive stress-induced endothelial dysfunction.

Prolonged Activation of STAT3 Mediates the IL6‐Induced Loss of Stress Fibers and Increase in Endothelial Permeability

Interleukin 6 (IL6) promotes endothelial barrier breakdown in vitro, as well as, in vivo. However, the signaling mechanisms that mediate this response are not fully understood. IL6 trans‐signaling promotes a prolonged activation of JAK/STAT3 in human umbilical vein endothelial cells (HUVECs) that lasts for at least 24 hours, leading to a sustained increase in endothelial permeability. IL6‐induced STAT3 phosphorylation and loss of barrier function is prevented when cells are pre‐treated with the JAK inhibitor, ruxolitinib, suggesting that JAK activity is required for these responses. Furthermore, siRNA‐mediated STAT3 knockdown attenuates the loss of IL6‐induced loss of barrier function, demonstrating a critical role for STAT3 in this response. Immunofluorescence analysis showed that this change in barrier function correlates with a marked loss of junctional ZO‐1 (but not changes in ZO‐1 protein expression). Phalloidin staining demonstrates that IL‐6 also promotes a JAK, and STAT3, dependent loss of actin stress fibers and a marked reduction of the cortical actin signal. A prolonged activation of STAT3 is required for this response, because treatment of HUVECs with ruxolitinib up to 4 hours after activation of IL6 trans‐signaling, completely reverses the IL6‐induced loss of barrier function. STAT3 activation kinetics are regulated by a negative feedback loop that is mediated by the suppressor of cytokine signaling 3 (SOCS3). Consistently, siRNA‐mediated SOCS3 knockdown increases IL6‐induced STAT3 phosphorylation in HUVECs and exacerbates the IL6‐induced increase in permeability. Collectively, our data shows that prolonged activation of STAT3 is required for the IL6‐induced barrier breakdown, and loss of junctional ZO‐1, cortical actin and actin stress fibers in endothelial cells.Support or Funding InformationThis project was supported by an American Heart Association Science Development Grant 13SDG17100110 and funds from the Department of Molecular and Cellular Physiology, Albany Medical Center.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Myeloid-Derived Suppressor Cells Endow Stem-like Qualities to Breast Cancer Cells through IL6/STAT3 and NO/NOTCH Cross-talk Signaling.

Myeloid-derived suppressor cells (MDSC) contribute to immune suppression in cancer, but the mechanisms through which they drive metastatic progression are not fully understood. In this study, we show how MDSC convey stem-like qualities to breast cancer cells that coordinately help enable immune suppression and escape. We found that MDSC promoted tumor formation by enhancing breast cancer cell stem-like properties as well as by suppressing T-cell activation. Mechanistic investigations indicated that these effects relied upon cross-talk between the STAT3 and NOTCH pathways in cancer cells, with MDSC inducing IL6-dependent phosphorylation of STAT3 and activating NOTCH through nitric oxide leading to prolonged STAT3 activation. In clinical specimens of breast cancer, the presence of MDSC correlated with the presence of cancer stem-like cells (CSC) and independently predicted poor survival outcomes. Collectively, our work revealed an immune-associated mechanism that extrinsically confers cancer cell stemness properties and affects patient outcome. We suggest that targeting STAT3-NOTCH cross-talk between MDSC and CSC could offer a unique locus to improve cancer treatment, by coordinately targeting a coupled mechanism that enables cancer stemness and immune escape. Cancer Res; 76(11); 3156-65. ©2016 AACR.

Abstract 3452: N-glycosylation on lung cancer cell-secreted IL-6 prolongs its activation on JAK/STAT pathway

Abstract Interleukin-6 (IL-6) is overexpressed in various cancer cells, intriguingly contributing to both tumor growth and modulating anti-tumor immunity. Cell-type-specific glycosylations on IL-6 have been deduced in early studies; however, the glycosylation pattern and biological function of cancer cell-secreted IL-6 remain unclear. Here, we describe the glycosylation pattern of lung cancer cell-secreted IL-6 and its impact on the activation of JAK/STAT pathway. IL-6 molecules with different molecular weights were detected in the conditional media of IL-6-overexpressed lung cancer cell lines by immunoblot. Because in NetNGlyc 1.0 Server, one possible N-glycosylation site has been predicted at N73 on IL-6, we used treatment of N-glycosylation-specific inhibitors and site-directed mutagenesis on N73 to demonstrate that lung cancer cell-secreted IL-6 is modified by N-glycosylation. To examine which glycotransferases may participate in the modification, we had screened the expression of glycosyltransferases using qPCR and found that the expression of fucosyltransferase 8 (FUT8), responsible for core fucosylation on N-glycosylated proteins, was higher in lung cancer cells compared to normal bronchial cell. We then reduced core fucosylation on lung cancer cell-secreted IL-6 by silencing FUT8 with shRNA transduction. Subsequently, cells were treated with conditional media containing fully-glycosylated or core fucose-depleted IL-6 to uncover the potential influences on cellular signaling from glycosylation on IL-6. The fully-gycosylated IL-6 induced prolonged STAT3Y705 phosphorylation and distinct gene population compared to core fucose-depleted IL-6. The nuclear retention of STAT3 was concordant with the prolonged STAT3 activation in cells treated with fully-gycosylated IL-6. In paired normal (N) and tumor (T) tissues from lung cancer patients, higher FUT8 mRNA was detected in tumor part than normal part. Besides, we found similar glycosylation pattern in the secreted IL-6 of short-term cultured lung cancer cells derived from malignant pleural effusions. Together, we report the presence of specific IL-6 glycoforms secreted from lung cancer cell lines and lung cancer cells from clinical samples. Moreover, the glycosylation on IL-6 changes its activity on the regulation of JAK/STAT pathway. Citation Format: Chun-Hua Hung, Hsuan-Heng Yeh, Hao-Chen Wang, Chien-Chung Lin, Tsung-Lin Tsai, Wei-Lun Huang, Chuan-Fa Chang, Wu-Chou Su. N-glycosylation on lung cancer cell-secreted IL-6 prolongs its activation on JAK/STAT pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3452. doi:10.1158/1538-7445.AM2014-3452

STAT3 activation in response to IL-6 is prolonged by the binding of IL-6 receptor to EGF receptor

The activation of STAT3 by tyrosine phosphorylation, essential for normal development and for a normal inflammatory response to invading pathogens, is kept in check by negative regulators. Abnormal constitutive activation of STAT3, which contributes to the pathology of cancer and to chronic inflammatory diseases such as rheumatoid arthritis, occurs when negative regulation is not fully effective. SOCS3, the major negative regulator of STAT3, is induced by tyrosine-phosphorylated STAT3 and terminates STAT3 phosphorylation about 2 h after initial exposure of cells to members of the IL-6 family of cytokines by binding cooperatively to the common receptor subunit gp130 and JAKs 1 and 2. We show here that when the epidermal growth factor receptor (EGFR) is present and active, STAT3 is rephosphorylated about 4 h after exposure of cells to IL-6 or oncostatin M and remains active for many hours. Newly synthesized IL-6 drives association of the IL-6 receptor and gp130 with EGFR, leading to EGFR-dependent rephosphorylation of STAT3, which is not inhibited by the continued presence of SOCS3. This second wave of STAT3 activation supports sustained expression of a subset of IL-6-induced proteins, several of which play important roles in inflammation and cancer, in which both IL-6 secretion and EGFR levels are often elevated.

Role of IL-6 in the resolution of pancreatitis in obese mice

Obesity increases severity of acute pancreatitis and risk of pancreatic cancer. Pancreatitis and obesity are associated with elevated IL-6, a cytokine involved in inflammation and tumorigenesis. We studied the role of IL-6 in the response of lean and obese mice to pancreatitis induced by IL-12 + IL-18. Lean and diet-induced obese (DIO) WT and IL-6 KO mice and ob/ob mice pretreated with anti-IL-6 antibodies were evaluated at Days 1, 7, and 15 after induction of pancreatitis. Prolonged elevation of IL-6 in serum and visceral adipose tissue was observed in DIO versus lean WT mice, whereas circulating sIL-6R declined in DIO but not lean mice with pancreatitis. The severe inflammation and lethality of DIO mice were also observed in IL-6 KO mice. However, the delayed resolution of neutrophil infiltration; sustained production of CXCL1, CXCL2, and CCL2; prolonged activation of STAT-3; and induction of MMP-7 in the pancreas, as well as heightened induction of serum amylase A of DIO mice, were blunted significantly in DIO IL-6 KO mice. In DIO mice, production of OPN and TIMP-1 was increased for a prolonged period, and this was mediated by IL-6 in the liver but not the pancreas. Results obtained in IL-6 KO mice were confirmed in ob/ob mice pretreated with anti-IL-6 antibodies. In conclusion, IL-6 does not contribute to the increased severity of pancreatitis of obese mice but participates in delayed recovery from acute inflammation and may favor development of a protumorigenic environment through prolonged activation of STAT-3, induction of MMP-7, and sustained production of chemokines.

STAT transcription in the ischemic heart

All seven STAT proteins are expressed in the heart, and in this review we will focus on their contribution to cardiac physiology and to ischemic heart disease and its consequences. A substantial literature has focused on the roles of STAT1 and STAT3 in ischemic heart disease, where, at least in the acute phase, they appear to have a yin-yang relationship. STAT1 contributes to the loss of irreplaceable cardiac myocytes both by increasing apoptosis and by reducing cardioprotective autophagy. In contrast, STAT3 is cardioprotective, since STAT3-deficient mice have larger infarcts following ischemic injury, and a number of cardioprotective agents have been shown to act, at least partly, through STAT3 activation. STAT3 is also absolutely required for preconditioning—a process where periods of brief ischemia protect against a subsequent or previous prolonged ischemic episode. Prolonged activation of STAT3, however, is strongly implicated in the post-infarction remodeling of the heart which leads to heart failure, where, possibly together with STAT5, it augments activation of the renin-angiotensin system.

Cannabinoid 1 Receptor and Interleukin-6 Receptor Together Induce Integration of Protein Kinase and Transcription Factor Signaling to Trigger Neurite Outgrowth

Activation of the G(o/i)-coupled cannabinoid 1 receptor (CB1R) has been shown to induce neurite outgrowth in Neuro2A cells through activation of Src kinase and STAT3 transcription factor. Signaling by the interleukin 6 receptor (IL-6R) also activates STAT3 through Jak kinase. We studied if signals from the two pathways could be integrated in a synergistic manner to trigger neurite outgrowth in Neuro2A cells. At low concentrations, when agonist at either receptor by itself has no effect, we found that CB1R and IL-6R stimulation together induced synergistic neurite outgrowth. Signal integration requires activation of transcription factors by Src, Jak, and mitogen-activated protein kinases. Mitogen-activated protein kinase can be activated by both receptors and shows enhanced early activation in the presence of both ligands. CREB and STAT3 transcription factors are required for synergy and show enhanced DNA-binding activity when both receptors are activated. STAT3 plays a critical role in integration of the signals downstream of the two receptors. When both pathways are activated, STAT3 phosphorylation is sustained for 6 h. This prolonged activation of STAT3 requires deactivation of SHP2 phosphatase. Reduction of SHP2 levels by RNA interference results in greater synergy in neurite outgrowth. Simultaneous knockdown of both SHP2 and STAT3 blocks the synergistic triggering of neurite outgrowth, indicating that STAT3 is downstream of SHP2. CB1R and IL-6R co-stimulation enhanced the differentiation of rat cortical neuron primary cultures. These results provide a mechanism where multiple protein kinases and transcription factors interact to integrate signals from G protein-coupled and cytokine receptor to evoke neurite outgrowth in Neuro2A cells.

STAT3-Mediated Up-Regulation of BLIMP1 Is Coordinated with BCL6 Down-Regulation to Control Human Plasma Cell Differentiation

STAT family members have been implicated in regulating the balance between B cell lymphoma (BCL)6 and B lymphocyte induced maturation protein (BLIMP)1 to control plasma cell differentiation. We previously showed that STAT5 induces BCL6 to block plasma cell differentiation and extend the life span of human B cells. The heterogeneity in STAT activation by cytokines and their effects on B cell differentiation prompted us to investigate the effect of STAT3 activation in plasma cell differentiation. First stimulation with IL-21, which promotes plasma cell differentiation, induced robust and prolonged STAT3 activation in primary human B cells. We then investigated effects of direct STAT3 activation on regulation of plasma cell genes, cellular phenotype, and Ig production. Activation of a tamoxifen-regulated STAT3-estrogen receptor fusion protein triggered BLIMP1 mRNA and protein up-regulation, plasma cell phenotypic features, and Ig secretion. When STAT3 was activated by IL-21 in B cells ectopically expressing BCL6, BLIMP1 was up-regulated, but only partial plasma cell differentiation was achieved. Lastly, through coexpression of BCL6 and STAT3-ER, we verified that STAT3 activation functionally mimicked IL-21 treatment and that STAT3-mediated BLIMP1 up-regulation occurred despite high BCL6 expression levels indicating that BCL6 is not the dominant repressor of BLIMP1. Thus, up-regulation of BLIMP1 alone is not sufficient for differentiation of primary human B cells into plasma cells; concomitant down-regulation of BCL6 is absolutely required for completion of the plasma cell differentiation program.

Cr(VI)-stimulated STAT3 tyrosine phosphorylation and nuclear translocation in human airway epithelial cells requires Lck

Chronic inhalation of low amounts of Cr(VI) promotes pulmonary diseases and cancers through poorly defined mechanisms. SFKs (Src family kinases) in pulmonary airway cells may mediate Cr(VI) signalling for lung injury, although the downstream effectors of Cr(VI)-stimulated SFKs and how they relate to pathogenic gene induction are unknown. Therefore SFK-dependent activation of transcription factors by non-cytotoxic exposure of human bronchial epithelial cells to Cr(VI) was determined. Protein-DNA binding arrays demonstrated that exposing BEAS 2B cells to 5 microM Cr(VI) for 4 and 24 h resulted in increased protein binding to 25 and 43 cis-elements respectively, while binding to 12 and 16 cis-elements decreased. Of note, Cr(VI) increased protein binding to several STAT (signal transducer and activator of transcription) cis-elements. Cr(VI) stimulated acute tyrosine phosphorylation and nuclear translocation of STAT1 over a 4 h period and a prolonged activation of STAT3 that reached a peak between 48 and 72 h. This prolonged activation was observed for both STAT3alpha and STAT3beta. Immunofluorescent confocal microscopy confirmed that Cr(VI) increased nuclear localization of phosphorylated STAT3 for more than 72 h in both primary and BEAS 2B human airway cells. Cr(VI) induced transactivation of both a STAT3-driven luciferase reporter construct and the endogenous inflammatory gene IL-6 (interleukin-6). Inhibition with siRNA (small interfering RNA) targeting the SFK Lck, but not dominant-negative JAK (Janus kinase), prevented Cr(VI)-stimulated phosphorylation of both STAT3 isoforms and induction of IL-6. The results suggest that Cr(VI) activates epithelial cell Lck to signal for prolonged STAT3 activation and transactivation of IL-6, an important immunomodulator of lung disease progression.

Loss of SOCS3 Gene Expression Converts STAT3 Function from Anti-apoptotic to Pro-apoptotic

The transcription factor STAT3 is activated by interleukin-6-related cytokines and has been implicated as an oncogene; it promotes cell proliferation and is anti-apoptotic. However, in some cases, STAT3 has been shown to be pro-apoptotic, especially in mammary epithelial cells. In this report, we generated SOCS3-deficient murine embryonic fibroblasts (MEFs), in which STAT3 activation is extremely enhanced and prolonged. We found that LIF induces caspase-3 activation and apoptosis of SOCS3(-/-) MEFs. Exogenous expression of the dominant negative form of STAT3 but not STAT1 suppressed LIF-induced apoptosis of SOCS3(-/-) MEFs, indicating that STAT3 plays a critical role in apoptosis induction. As shown in mammary gland epithelial cells, expression of the phosphatidylinositol 3-kinase regulatory subunits p50alpha and p55alpha was induced in response to LIF in SOCS3(-/-) MEFs but not in wild-type MEFs, and Akt/protein kinase B activity was substantially reduced in SOCS3(-/-) MEFs. Furthermore, we found that some of the STAT3 target genes related to apoptosis and proliferation, such as Bcl-2 and cyclin D1, were repressed upon LIF treatment in SOCS3(-/-) cells. Not only the up-regulation of p50alpha and p55alpha but also the repression of cyclin D1 and Bcl-2 in SOCS3(-/-) MEFs was inhibited by dominant negative STAT3. These data suggest that prolonged activation of STAT3 could induce apoptosis/growth arrest rather than anti-apoptosis and proliferation in certain cases, and SOCS3 is a critical regulator of this balance.

SOCS3 Is a Critical Physiological Negative Regulator of G-CSF Signaling and Emergency Granulopoiesis

To determine the importance of suppressor of cytokine signaling-3 (SOCS3) in the regulation of hematopoietic growth factor signaling generally, and of G-CSF-induced cellular responses specifically, we created mice in which the Socs3 gene was deleted in all hematopoietic cells. Although normal until young adulthood, these mice then developed neutrophilia and a spectrum of inflammatory pathologies. When stimulated with G-CSF in vitro, SOCS3-deficient cells of the neutrophilic granulocyte lineage exhibited prolonged STAT3 activation and enhanced cellular responses to G-CSF, including an increase in cloning frequency, survival, and proliferative capacity. Consistent with the in vitro findings, mutant mice injected with G-CSF displayed enhanced neutrophilia, progenitor cell mobilization, and splenomegaly, but unexpectedly also developed inflammatory neutrophil infiltration into multiple tissues and consequent hind-leg paresis. We conclude that SOCS3 is a key negative regulator of G-CSF signaling in myeloid cells and that this is of particular significance during G-CSF-driven emergency granulopoiesis.

SOCS as Immune Modulator

Suppressor of cytokine signaling (SOCS) proteins inhibit signaling through cytokine receptors by inhibiting Janus protein kinases (JAKs) and thus blocking activation of STAT (signal transducers and activators of transcription) transcription factors. Although experiments with overexpression of the SOCS3 molecule have indicated that it might suppress signaling by several cytokines, three new papers on the effects of tissue-specific loss of SOCS3 function in vivo in macrophages show that physiological regulation by SOCS3 is focused on regulation of signaling by interleukin 6 (IL-6) and the gp130 receptor subunit. Furthermore, the results presented show that loss of SOCS3 not only modulates the strength of IL-6 signals (which are normally inflammatory), but also causes qualitative changes in the signal such that the spectrum of gene expression induced by IL-6 is shifted and resembles more closely an immunosuppressive response to interferons. The new results help explain how similar signaling mechanisms can be coupled to distinct, even opposing, functional outputs. IL-6 and IL-10 normally have pro- and anti-inflammatory actions, respectively, even though they both activate STAT3. But in macrophages lacking SOCS3, IL-6 produces a more prolonged activation of STAT3, similar to that produced by IL-10, and also produces an anti-inflammatory biological response. Thus, SOCS3 seems to be necessary to prevent anti-inflammatory signaling by IL-6. Because IL-6 is implicated in diseases associated with excessive inflammation, like arthritis, inhibition of SOCS3 activity in macrophages could be used to therapeutic effect. The three papers are discussed in context by Johnston and O'Shea. H. Yasukawa, M. Ohishi, H. Mori, M. Murakami, T. Chinen, D. Aki, T. Hanada, K. Takeda, S. Akira, M. Hoshijima, T. Hirano, K. R. Chien, A. Yoshimura, IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat. Immunology 4 , 551-556 (2003). [Online Journal] R. Lang, A. -L. Pauleau, E. Parganas, Y. Takahashi, J. Mages, J. N. Ihle, R. Rutschman, P. J. Murray, SOCS3 regulates the plasticity of gp130 signaling. Nat. Immunology 4 , 546-550 (2003). [Online Journal] B. A. Croker, D. L. Krebs, J. -G. Zhang, S. Wormald, T. A. Willson, E. G. Stanley, L. Robb, C. J. Greenhalgh, I. Förster, B. E. Clausen, N. A. Nicola, D. Metcalf, D. J. Hilton, A. W. Roberts, W. S. Alexander, SOCS3 negatively regulates IL-6 signaling in vivo . Nat. Immunology 4 , 540-545 (2003). [Online Journal] J. A. Johnston, J. J. O'Shea, Matching SOCS with function. Nat. Immunology 4 , 507-509 (2003). [Online Journal]

SOCS as Immune Modulator

Suppressor of cytokine signaling (SOCS) proteins inhibit signaling through cytokine receptors by inhibiting Janus protein kinases (JAKs) and thus blocking activation of STAT (signal transducers and activators of transcription) transcription factors. Although experiments with overexpression of the SOCS3 molecule have indicated that it might suppress signaling by several cytokines, three new papers on the effects of tissue-specific loss of SOCS3 function in vivo in macrophages show that physiological regulation by SOCS3 is focused on regulation of signaling by interleukin 6 (IL-6) and the gp130 receptor subunit. Furthermore, the results presented show that loss of SOCS3 not only modulates the strength of IL-6 signals (which are normally inflammatory), but also causes qualitative changes in the signal such that the spectrum of gene expression induced by IL-6 is shifted and resembles more closely an immunosuppressive response to interferons. The new results help explain how similar signaling mechanisms can be coupled to distinct, even opposing, functional outputs. IL-6 and IL-10 normally have pro- and anti-inflammatory actions, respectively, even though they both activate STAT3. But in macrophages lacking SOCS3, IL-6 produces a more prolonged activation of STAT3, similar to that produced by IL-10, and also produces an anti-inflammatory biological response. Thus, SOCS3 seems to be necessary to prevent anti-inflammatory signaling by IL-6. Because IL-6 is implicated in diseases associated with excessive inflammation, like arthritis, inhibition of SOCS3 activity in macrophages could be used to therapeutic effect. The three papers are discussed in context by Johnston and O'Shea.H. Yasukawa, M. Ohishi, H. Mori, M. Murakami, T. Chinen, D. Aki, T. Hanada, K. Takeda, S. Akira, M. Hoshijima, T. Hirano, K. R. Chien, A. Yoshimura, IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat. Immunology 4, 551-556 (2003). [Online Journal]R. Lang, A. -L. Pauleau, E. Parganas, Y. Takahashi, J. Mages, J. N. Ihle, R. Rutschman, P. J. Murray, SOCS3 regulates the plasticity of gp130 signaling. Nat. Immunology 4, 546-550 (2003). [Online Journal]B. A. Croker, D. L. Krebs, J. -G. Zhang, S. Wormald, T. A. Willson, E. G. Stanley, L. Robb, C. J. Greenhalgh, I. Förster, B. E. Clausen, N. A. Nicola, D. Metcalf, D. J. Hilton, A. W. Roberts, W. S. Alexander, SOCS3 negatively regulates IL-6 signaling in vivo. Nat. Immunology 4, 540-545 (2003). [Online Journal]J. A. Johnston, J. J. O'Shea, Matching SOCS with function. Nat. Immunology 4, 507-509 (2003). [Online Journal]

Novel variant isoform of G-CSF receptor involved in induction of proliferation of FDCP-2 cells: relevance to the pathogenesis of myelodysplastic syndrome.

Recent studies have shown that point mutations in granulocyte colony-stimulating factor receptor (G-CSFR) are involved in the pathogenesis of severe congenital neutropenia (SCN) and in the transformation of SCN to acute myelogenous leukemia (AML). It is reasonably speculated that the abnormalities in the signal transduction pathways for G-CSF could be partly responsible for the pathogenesis and the development to AML in patients with myelodysplastic syndromes (MDS). Therefore, we investigated the structural and functional abnormalities of the G-CSFR in 14 patients with MDS and 10 normal subjects. In in vitro colony forming assay, MDS samples showed reduced response to growth factors. However, G-CSF, but not GM-CSF and IL-3, enhanced clonal growth in three cases of high risk patients with MDS (RAEB, RAEB-t, and MDS having progressed to acute myeloid leukemia (AML)) and one low risk patient (RA). Eight out of 14 patients including above 4 patients demonstrated a common deletion of the G-CSFR cDNA; a deletion of three nucleotides (2128-2130) in the juxtamembrane domain of the G-CSFR, which resulted in a conversion of Asn(630)Arg(631) to Lys(630). To assess the functional activities of this deletion in the G-CSFR isoform, a mutant with the same three-nucleotide deletion was constructed by site-directed mutagenesis. FDCP-2 cells expressing the G-CSFR isoform responded to G-CSF, and exhibited proliferative responses than did those cells having wild-type G-CSFR. Moreover, these isoforms showed prolonged activation of STAT3 in response to G-CSF than did the wild-type. These results suggest that the deletion in the juxtamembrane domain of the G-CSFR gives a growth advantage to abnormal MDS clones and may contribute to the pathogenesis of MDS.

Prolonged STAT3 activation inhibits hepatocyte proliferation after hepatectomy

Prolonged STAT3 activation inhibits hepatocyte proliferation after hepatectomy