Suppressor Of Cytokine Signaling 3 Protein Expression Research Articles

SOCS3 Protein Developmentally Regulates the Chemokine Receptor CXCR4-FAK Signaling Pathway during B Lymphopoiesis

The chemokine CXCL12 induces prolonged focal adhesion kinase (FAK) phosphorylation and sustained proadhesive responses in progenitor bone-marrow (BM) B cells, but not in mature peripheral B cells. Here we demonstrate that suppressor of cytokine signaling 3 (SOCS3) regulated CXCL12-induced FAK phosphorylation through the ubiquitin-proteasome pathway. CXCL12 triggered increased FAK ubiquitination in mature B cells, but not in progenitor B cells. Accordingly, SOCS3 expression was low in progenitor B cells, increased in immature B cells, and highest in mature B cells. SOCS3 overexpression in pro-B cells impaired CXCL12-induced FAK phosphorylation and proadhesive responses. Conversely, SOCS3-deficient mature B cells from Cre(MMTV)Socs3(fl/fl) mice exhibited prolonged FAK phosphorylation and adhesion to VCAM-1. In contrast to wild-type mice, Cre(MMTV)Socs3(fl/fl) mice had a 2-fold increase in immature B cells, which were evenly distributed in endosteal and perisinusoidal BM compartments. We propose that the developmental regulation of CXCR4-FAK signaling by SOCS3 is an important mechanism to control the lodgement of B cell precursors in the BM microenvironment.

SOCS3 regulates B lymphopoiesis at the immature B cell stage (86.4)

Abstract CXCL12-mediated FAK phosporylation and sustained pro-adhesive responses occur uniquely in progenitor bone marrow (BM) B cells, but not in mature peripheral B cells. Since CXCR4 receptor expression is similar at all B cell stages, we hypothesized that progenitor and mature B cells possess different mechanisms to control FAK phosphorylation. Here we demonstrate that Suppressor of Cytokine Signaling 3 (SOCS3) regulates CXCL12-induced FAK phosphorylation through the ubiquitin-proteosome pathway. CXCL12 triggers increased FAK ubiquitination in peripheral B cells and spleen B cells, but not in BM B cells. In accordance with this finding, SOCS3 expression is relatively low in progenitor BM B cells and high in peripheral blood and spleen B cells. Over-expression of SOCS3 results in decreased CXCL12-induced FAK phosphorylation and impaired adhesion to VCAM1. Next, we examined B lineage cells from MMTV-Cre SOCS3fl/flmice in which SOCS3 protein expression was reduced greater than 95%. SOCS3 deficient splenic B cells have increased and prolonged FAK phosphorylation and enhanced sustained adhesion to VCAM-1, similar to progenitor B cells. MMTV-Cre SOCS3fl/flmice have a two-fold increase in the absolute number of IgM+,IgD−, immature B cells in BM compared to wild type mice (n=8, p<0.01). IL-7 dependent pre-B cell proliferation was unaffected by SOCS3 deficiency. However, withdrawal of IL-7 induced the generation of immature B cells that survived longer in culture compared with wild type cells. Taken together, our results suggest that SOCS3 may be an important negative regulator of immature B cells by affecting their survival and pro-adhesive responses in the bone marrow microenvironment.

Regulation of Suppressor of Cytokine Signaling 3 (SOCS3) mRNA Stability by TNF-α Involves Activation of the MKK6/p38MAPK/MK2 Cascade

The potential of some proinflammatory mediators to inhibit gp130-dependent STAT3 activation by enhancing suppressor of cytokine signaling (SOCS) 3 expression represents an important molecular mechanism admitting the modulation of the cellular response toward gp130-mediated signals. Thus, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by proinflammatory mediators. In this study, we investigate SOCS3 expression initiated by the proinflammatory cytokine TNF-alpha. In contrast to IL-6, TNF-alpha increases SOCS3 expression by stabilizing SOCS3 mRNA. Activation of the MAPK kinase 6 (MKK6)/p38(MAPK)-cascade is required for TNF-alpha-mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression. In fibroblasts or macrophages deficient for MAPK-activated protein kinase 2 (MK2), a downstream target of the MKK6/p38(MAPK) cascade, basal SOCS3-expression is strongly reduced and TNF-alpha-induced SOCS3-mRNA stabilization is impaired, indicating that MK2 is crucial for the control of SOCS3 expression by p38(MAPK)-dependent signals. As a target for SOCS3 mRNA stability-regulating signals, a region containing three copies of a pentameric AUUUA motif in close proximity to a U-rich region located between positions 2422 and 2541 of the 3' untranslated region of SOCS3 is identified. One factor that could target this region is the zinc finger protein tristetraprolin (TTP), which is shown to be capable of destabilizing SOCS3 mRNA via this region. However, data from TTP-deficient cells suggest that TTP does not play an irreplaceable role in the regulation of SOCS3 mRNA stability by TNF-alpha. In summary, these data indicate that TNF-alpha regulates SOCS3 expression on the level of mRNA stability via activation of the MKK6/p38(MAPK) cascade and that the activation of MK2, a downstream target of p38(MAPK), is important for the regulation of SOCS3 expression.

SOCS3 is required to temporally fine-tune photoreceptor cell differentiation

Suppressor of cytokine signaling 3 (SOCS3) is an intracellular, ligand-induced negative feedback modulator of STAT3 activation that acts during inflammation. Here, we demonstrate that SOCS3 expression is important for normal retinal development in the perinatal period. STAT3 is highly activated in the late-embryonic retina, then downregulated at postnatal day 0 (P0), presumably by the depletion of upstream ligands. We found that SOCS3 was required after P0 to shut down the residual STAT3 activation; this loss of activated STAT3 leads to Rhodopsin expression and rod photoreceptor cell differentiation. SOCS3 deficiency failed to terminate STAT3 activation, thereby delaying expression of Rhodopsin and its upstream transcription factor, crx. Development subsequently continued, but its course was temporally erratic, probably because of faulty compensation. Interestingly, SOCS3 protein expression was first detected postnatally, after STAT3 activation was mostly downregulated. It initially appeared in some of the presumptive photoreceptor cells and gradually spread. SOCS3 mRNA level was constant from the late-embryonic to early-postnatal period. Post-transcriptional inhibition of SOCS3 protein expression maintains a high STAT3 activation during late embryogenesis, and after P0, releasing from the inhibition promptly terminates STAT3 activation. Thus, SOCS3 can act as a temporal fine-tuner of STAT3 activation during photoreceptor cell differentiation.

A novel role for suppressor of cytokine signaling 3 in cartilage destruction via induction of chondrocyte desensitization toward insulin‐like growth factor

An important mechanism contributing to cartilage destruction in arthritis is chondrocyte desensitization toward its main anabolic factor, insulin-like growth factor 1 (IGF-1). In this study, we sought to determine the role of suppressor of cytokine signaling 3 (SOCS-3) in the induction of IGF-1 desensitization of murine chondrocytes. Chondrocyte responsiveness to IGF-1 was assessed by 35S-sulfate incorporation into proteoglycans (PGs), via aggrecan messenger RNA expression, using quantitative real-time polymerase chain reaction or insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation (Western blot analysis). IGF-1 desensitization of patellar chondrocytes was studied in zymosan-induced arthritis. IGF-1 desensitization was induced in patellar cartilage explants or the H4 chondrocyte cell line, exposed to interleukin-1alpha (IL-1alpha). SOCS-3 protein expression was assessed by immunohistochemistry or by Western blot analysis of protein extracts. The role of SOCS-3 in IGF-1 signaling was elucidated by adenoviral overexpression. Exposure of murine articular cartilage to IL-1 caused a significant decrease in IGF-1-induced PG synthesis. This effect also occurred in inducible nitric oxide synthase-knockout mice, revealing the involvement of a secondary IL-1-induced factor other than nitric oxide. We showed that IL-1 significantly up-regulated SOCS-3 transcription and protein synthesis in H4 chondrocytes. In contrast, IL-18 was unable to induce SOCS-3 expression and failed to induce chondrocyte IGF-1 desensitization. Histologic analysis of samples from arthritic knee joints revealed high expression of SOCS-3 in chondrocytes. Through adenoviral overexpression of SOCS-3, we obtained direct evidence that SOCS-3 inhibits IGF-1-mediated cell signaling, since IRS-1 phosphorylation was reduced. This study demonstrates that IL-1-induced SOCS-3 expression is a novel mechanism of IGF-1 desensitization in chondrocytes; in conjunction with nitric oxide it can contribute to cartilage damage during arthritis.

Bcl2 Associated Induction of the Suppressor of Cytokine Signaling-3 (SOCS3) Gene Involves Activation of the p44/42 MAPK Pathway.

SOCS3 has been shown to be an essential regulator of IL-6 signaling in hepatocytes and macrophages, as well as a negative regulator of G-CSF signaling in myeloid cells, and SOCS3 expression appears to be STAT3-dependent. However, the underlying cellular pathways which regulate SOCS3 expression in B cells remain to be clearly defined. We previously showed that polyclonal CD19+ B cells isolated from Bcl2 overexpressing Eμ-Bcl2 transgenic mice overexpress SOCS3 protein and that exogenous expression of Bcl2 in hematopoietic and fibroblast cell lines induces SOCS3 protein expression. We also found that a distinct subset of follicular lymphomas (FL) exhibit combined overexpression of Bcl2 and SOCS3. We hypothesized that Bcl2-mediated induction of SOCS3 in B cells may occur indirectly via deregulation of Bcl2-associated cell signaling pathways. In the current study, IL-3-dependent BaF3 pro-B cells were stably transduced with either a 717bp human Bcl2α cDNA (BaF3/Bcl2) or vector only control (Baf3Δ). Cell lines were initially grown in the presence of IL-3, with IL-3 removed 48 hours prior to protein measurements. Western analysis using Bcl2 antisera revealed high level Bcl2 expression in transduced cells (BaF3/Bcl2) and absence of Bcl2 in BaF3Δ control cells. When probed with SOCS3 antisera, BaF3/Bcl2 cells exhibited marked overexpression of SOCS3 protein whereas BaF3Δ control cells did not express SOCS3. To assess whether Bcl2-associated induction of SOCS3 is STAT3-dependent, we measured phospho-STAT3 levels relative to STAT3 in BaF3/Bcl2 cells. Interestingly, when probed with phospho-STAT3 and STAT3 antisera, BaF3/Bcl2 and BaF3Δ cells did not reveal phospho-STAT3 protein but revealed equivalent STAT3 protein levels. Since we had previously noted overexpression of p44/42 Map kinase (MAPK) in CD19+ B cells from Eμ-Bcl2 transgenic mice relative to littermate controls, we next wanted to determine whether p44/42MAPK signaling played a role in Bcl2-mediated SOCS3 induction. Western analysis using p44/42MAPK antisera revealed overexpression of p44/42MAPK in BaF3/Bcl2 cells relative to BaF3Δ control cells. BaF3/Bcl2 and BaF3Δ cells were then grown in the presence or absence of a specific p44/42MAPK pathway inhibitor (MEK 1/2 inhibitor U0126), and whole cell lysates were analyzed for SOCS3 expression by Western analysis. When probed with SOCS3 antisera, BaF3/Bcl2 cells selectively grown in the presence of the p44/42MAPK inhibitor revealed complete abrogation of SOCS3 protein expression while those grown in the absence of inhibitor continued to harbor SOCS3 protein. As expected, ERK1/2 protein levels were selectively decreased in response to treatment with the p44/42MAPK inhibitor, indicating specific inhibition of the p44/42MAPK pathway. Bcl2 protein levels in BaF3/Bcl2 cells remained unchanged in the presence or absence of the p44/42MAPK inhibitor. As controls, inhibitors of p38MAPK (SB203580), AKT (Triciribine), and PI3K (LY294002) were evaluated and none were found to affect SOCS3 protein levels in BaF3/Bcl2 cells. These data indicate that Bcl2-associated induction of SOCS3 in B cells involves activation of the p44/42MAPK cell signaling pathway and is independent of STAT3 activation. These studies illustrate a novel and previously unappreciated Bcl2-associated signaling pathway involving SOCS3 induction in B cells that may play an important role in B cell biology and in Bcl2-associated hematologic malignancies.

Bcl-2 Overexpression Leads to Increases in Suppressor of Cytokine Signaling-3 Expression in B Cells and De novo Follicular Lymphoma

Abstract The t(14;18)(q32;q21), resulting in deregulated expression of B-cell-leukemia/lymphoma-2 (Bcl-2), represents the genetic hallmark in human follicular lymphomas. Substantial evidence supports the hypothesis that the t(14;18) and Bcl-2 overexpression are necessary but not solely responsible for neoplastic transformation and require cooperating genetic derangements for neoplastic transformation to occur. To investigate genes that cooperate with Bcl-2 to influence cellular signaling pathways important for neoplastic transformation, we used oligonucleotide microarrays to determine differential gene expression patterns in CD19+ B cells isolated from Eμ-Bcl-2 transgenic mice and wild-type littermate control mice. Fifty-seven genes were induced and 94 genes were repressed by ≥2-fold in Eμ-Bcl-2 transgenic mice (P < 0.05). The suppressor of cytokine signaling-3 (SOCS3) gene was found to be overexpressed 5-fold in B cells from Eμ-Bcl-2 transgenic mice. Overexpression of Bcl-2 in both mouse embryo fibroblast-1 and hematopoietic cell lines resulted in induction of SOCS3 protein, suggesting a Bcl-2-associated mechanism underlying SOCS3 induction. Immunohistochemistry with SOCS3 antisera on tissue from a cohort of patients with de novo follicular lymphoma revealed marked overexpression of SOCS3 protein that, within the follicular center cell region, was limited to neoplastic follicular lymphoma cells and colocalized with Bcl-2 expression in 9 of 12 de novo follicular lymphoma cases examined. In contrast, SOCS3 protein expression was not detected in the follicular center cell region of benign hyperplastic tonsil tissue. These data suggest that Bcl-2 overexpression leads to the induction of activated signal transducer and activator of transcription 3 (STAT3) and to the induction of SOCS3, which may contribute to the pathogenesis of follicular lymphoma.

Analysis of SOCS-3 Promoter Responses to Interferon γ

SOCS-3 (suppressor of cytokine signaling 3) is an intracellular protein that is selectively and rapidly induced by appropriate agonists and that modulates responses of immune cells to cytokines by interfering with the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway. On the basis of the observations that interferon gamma (IFNgamma) up-regulates SOCS-3 gene and protein expression in primary mouse macrophages, J774 macrophage cell line and embryonal fibroblasts, we investigated which sequences of the 5' SOCS-3 gene are responsive to IFNgamma. By promoter deletion analysis we identified a functional IFNgamma-responsive element, located at nucleotides -72/-64 upstream from the transcription initiation, whose presence and integrity is necessary to ensure responsiveness to IFNgamma. This element contains a STAT consensus binding sequence (SOCS-3/STAT-binding element (SBE)) whose specific mutation totally abolished the responsiveness to IFNgamma. In contrast, discrete deletion of other 5' regions of the SOCS-3 promoter did not substantially modify the inducibility by IFNgamma. Electromobility shift assay analyses revealed that IFNgamma promotes specific DNA binding activities to an oligonucleotide probe containing the SOCS-3/SBE sequence. Even though IFNgamma triggered tyrosine phosphorylation of both STAT1 and STAT3 in macrophages and J774 cells, only STAT1 was appropriately activated and thus found to specifically bind to the SOCS-3/SBE oligonucleotide probe. Accordingly, IFNgamma-induced SOCS-3 protein expression was not impaired in STAT3-deficient embryonal fibroblasts. Taken together, these results demonstrate that the induction of SOCS-3 by IFNgamma depends upon the presence of a STAT-binding element in the SOCS-3 promoter that is specifically activated by STAT1.

Down-regulation of SHP1 and up-regulation of negative regulators of JAK/STAT signaling in HTLV-1 transformed cell lines and freshly transformed human peripheral blood CD4+ T-Cells

Adult T-cell leukemia (ATL) is an aggressive malignancy that is associated with human T-cell lymphotropic virus I (HTLV-I) infection. HTLV-I transformed T-cell lines and fresh ATL cells are characterized by constitutive activation of the interleukin-2 receptor (IL-2R) signaling pathway however, the mechanism(s) responsible for constitutive IL-2R activation are unknown. To further examine the cause of this signaling pathway deregulation, we measured mRNA and protein expression levels by real-time PCR and Western blots, respectively, of four negative regulators of the IL-2R signaling pathway including src homology 2 (SH2)-containing phosphatase (SHP1), cytokine-inducible (CIS) SH2-containing protein, suppressor of cytokine signaling-1 (SOCS1) and protein inhibitor of activated signal transducer and activator of transcription 3 (STAT3) (PIAS3) in six HTLV-1 negative and seven HTLV-1 positive T-cell leukemia lines. The activation status of the JAK/STAT pathway was also examined. SHP1 mRNA and protein expression levels were selectively down regulated in all HTLV-1-infected transformed cell lines, while CIS, SOCS1, and PIAS3 protein expression were markedly but variably upregulated and the cells showed evidence of constitutive STAT3 activation. In acutely HTLV-1 infected primary CD4+ T-cells there was a gradual loss of SHP1 expression over 10 weeks in culture which correlated with progression from immortalization to transformation and loss of IL-2 dependence for growth. Two transformed cell lines that were established following HTLV-1 infection showed loss of SHP1 expression and overexpression of CIS, SOCS1, PIAS3. However, this overexpression was not adequate to block constitutive activation of the JAK/STAT pathway. Thus, multiple levels of IL-2 receptor signal deregulation are found in HTLV-1 transformed cells, which may be a result of early loss of SHP1 expression.

Suppressor of cytokine signaling-1 regulates acute inflammatory arthritis and T cell activation.

Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of cytokine signaling. To investigate the role of SOCS-1 in regulating inflammatory and immune responses in disease, acute inflammatory arthritis was induced in mice lacking SOCS-1. Expression of SOCS-1 protein was detected within synovial granulomas and pannus tissue of WT mice by day 7 following induction of acute arthritis. The severity of synovial inflammation and joint destruction at the peak of disease was greater in the absence of SOCS-1, although disease resolution occurred normally. There was an increased percentage of myeloid cells infiltrating the synovium in mice lacking SOCS-1, and SOCS-1 promoter activity was present in synovial macrophages, lymphocytes, and fibroblasts, but not granulocytes. The T cell response in draining LNs was also dysregulated, as popliteal LNs from mice lacking SOCS-1 contained approximately fivefold more cells at the peak of acute arthritis. These cells were hyperproliferative on exposure to antigen in vitro, and purified splenic CD4(+) T cells from mice lacking SOCS-1 proliferated more strongly in response to stimulation with anti-CD3. Reporter gene expression was detected in CD4(+) T cells bearing the activation markers CD25, CD44, and CD69. SOCS-1 is therefore expressed in hematopoietic and nonhematopoietic cell types in vivo and is an important regulator of acute inflammatory arthritis and of CD4(+) T cell activation.

Suppressor of cytokine signaling-1 regulates acute inflammatory arthritis and T cell activation

Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of cytokine signaling. To investigate the role of SOCS-1 in regulating inflammatory and immune responses in disease, acute inflammatory arthritis was induced in mice lacking SOCS-1. Expression of SOCS-1 protein was detected within synovial granulomas and pannus tissue of WT mice by day 7 following induction of acute arthritis. The severity of synovial inflammation and joint destruction at the peak of disease was greater in the absence of SOCS-1, although disease resolution occurred normally. There was an increased percentage of myeloid cells infiltrating the synovium in mice lacking SOCS-1, and SOCS-1 promoter activity was present in synovial macrophages, lymphocytes, and fibroblasts, but not granulocytes. The T cell response in draining LNs was also dysregulated, as popliteal LNs from mice lacking SOCS-1 contained approximately fivefold more cells at the peak of acute arthritis. These cells were hyperproliferative on exposure to antigen in vitro, and purified splenic CD4+ T cells from mice lacking SOCS-1 proliferated more strongly in response to stimulation with anti-CD3. Reporter gene expression was detected in CD4+ T cells bearing the activation markers CD25, CD44, and CD69. SOCS-1 is therefore expressed in hematopoietic and nonhematopoietic cell types in vivo and is an important regulator of acute inflammatory arthritis and of CD4+ T cell activation.

Thermal injury-induced increases of hepatocyte SOCS3 lead to decreases in STAT3.

Previous work in this laboratory has shown an increase of both mRNA and protein for suppressor of cytokine signaling 3 (SOCS3) in rat liver after thermal injury. This study identifies which liver cell type (parenchymal or non-parenchymal) is responsible for the postburn increase in SOCS3 and how this increase is connected to the signal transducer and activator of transcription (STAT) pathway. Parenchymal (hepatocytes) and non-parenchymal cells were isolated by Liberase digestion from postburn day 1 (PBD1) rats (including sham controls) and were analyzed for the expression of SOCS3 mRNA and protein and STAT3 and p-STAT3 protein. Reverse transcriptase (RT)-PCR performed on the isolated cells showed a significant increase of SOCS3 in the hepatocytes, but not in the non-parenchymal cells. When isolated hepatocytes from rats and the human hepatocyte cell line, HepG2, were cultured in the presence of IL-6, both showed an increase in SOCS3 mRNA expression. Anti-SOCS3, anti-STAT3, and anti-phosphorylated STAT3 labeling in both postburn rat liver and isolated hepatocyte cells that were cultured in the presence of IL-6 revealed that an increase in SOCS3 protein was accompanied by decrease in STAT3 protein. We propose that thermal injury stimulates non-parenchymal cells to produce cytokines, including IL-6, which in tum stimulate the Jak/STAT pathway in hepatocytes. The signal transduction pathway triggered by non-parenchymal cells causes an increase in SOCS3 production, which in turn induces the reduction of STAT3 protein in the hepatocytes.