Cell proliferation and STAT6 pathways are negatively regulated in T cells by STAT1 and suppressors of cytokine signaling.

Suppressor of cytokine signaling (SOCS) proteins have emerged as important regulators of cytokine signals in lymphocytes. In this study, we have investigated regulation of SOCS expression and their role in Th cell growth and differentiation. We show that SOCS genes are constitutively expressed in naive Th cells, albeit at low levels, and are differentially induced by Ag and Th-polarizing cytokines. Whereas cytokines up-regulate expression of SOCS1, SOCS2, SOCS3, and cytokine-induced Src homology 2 protein, Ags induce down-regulation of SOCS3 within 48 h of Th cell activation and concomitantly up-regulate SOCS1, SOCS2, and cytokine-induced Src homology 2 protein expression. We further show that STAT1 signals play major roles in inducing SOCS expression in Th cells and that induction of SOCS expression by IL-4, IL-12, or IFN-gamma is compromised in STAT1-deficient primary Th cells. Surprisingly, IL-4 is a potent inducer of STAT1 activation in Th2 but not Th1 cells, and SOCS1 or SOCS3 expression is dramatically reduced in STAT1(-/-) Th2 cells. To our knowledge, this is the first report of IL-4-induced STAT1 activation in Th cells, and suggests that its induction of SOCS, may in part, regulate IL-4 functions in Th2 cells. In fact, overexpression of SOCS1 in Th2 cells represses STAT6 activation and profoundly inhibits IL-4-induced proliferation, while depletion of SOCS1 by an anti-sense SOCS1 cDNA construct enhances cell proliferation and induces constitutive activation of STAT6 in Th2 cells. These results are consistent with a model where IL-4 has dual effects on differentiating T cells: it simulates proliferation/differentiation through STAT6 and autoregulates its effects on Th2 growth and effector functions via STAT1-dependent up-regulation of SOCS proteins.

Activation of STAT signaling pathways and induction of suppressors of cytokine signaling (SOCS) proteins in mammalian lens by growth factors.

PURPOSE: This study was conducted to examine whether the effects of growth factors are mediated in the lens by Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways and whether they induce expression of suppressors of cytokine signaling (SOCS), a novel family of feedback regulators of cytokine and growth factor activities. METHODS: STAT activation and SOCS expression were analyzed in transgenic or wild-type mouse lens and lens epithelial cells stimulated with growth factors by immunohistochemistry, RT-PCR, Northern, Western, proliferation, or transient reporter assays. RESULTS: STATs were constitutively expressed at low levels and activated by insulin-like growth factor (IGF)-1, platelet-derived growth factor (PDGF)-aa, and FGF-1 or -2 in the lens. The Intensity of STAT signaling increased at high FGF-2 concentration and FGFs act in synergy with IGF-1 or PDGFaa to enhance STAT signaling and SOCS expression. Growth factor-induced proliferation of lens cells is inhibited by AG-490, a specific inhibitor of JAK2/STAT3. CONCLUSIONS: This is the first report that FGFs activate STAT pathways in the lens and that SOCS proteins are constitutively expressed and upregulated by growth factors in this tissue. Physiological relevance of STAT pathways in the lens is underscored by inhibition of lens cell proliferation by inhibitors of JAK/STAT pathways and by the aberrant proliferation of lens epithelium in the posterior pole of transgenic mice with constitutively activated STAT1 in the lens. Common activation of STAT pathways by FGF-1, FGF-2, IGF-1, or PDGFaa and their synergistic activation of STATs and SOCS in lens cells suggest that activities and crosstalk between these factors are sensitive to the steady state levels of activated STATs in the lens and may be under feedback regulation by SOCS family proteins.

Dendritic cell maturation requires STAT1 and is under feedback regulation by suppressors of cytokine signaling.

In this study we show that activation of STAT pathways is developmentally regulated and plays a role in dendritic cell (DC) differentiation and maturation. The STAT6 signaling pathway is constitutively activated in immature DC (iDC) and declines as iDCs differentiate into mature DCs (mDCs). However, down-regulation of this pathway during DC differentiation is accompanied by dramatic induction of suppressors of cytokine signaling 1 (SOCS1), SOCS2, SOCS3, and cytokine-induced Src homology 2-containing protein expression, suggesting that inhibition of STAT6 signaling may be required for DC maturation. In contrast, STAT1 signaling is most robust in mDCs and is not inhibited by the up-regulated SOCS proteins, indicating that STAT1 and STAT6 pathways are distinctly regulated in maturing DC. Furthermore, optimal activation of STAT1 during DC maturation requires both IL-4 and GM-CSF, suggesting that synergistic effects of both cytokines may in part provide the requisite STAT1 signaling intensity for DC maturation. Analyses of STAT1(-/-) DCs reveal a role for STAT1 in repressing CD86 expression in precursor DCs and up-regulating CD40, CD11c, and SOCS1 expression in mDCs. We further show that SOCS proteins are differentially induced by IL-4 and GM-CSF in DCs. SOCS1 is primarily induced by IL-4 through a STAT1-dependent mechanism, whereas SOCS3 is induced mainly by GM-CSF. Taken together, these results suggest that cytokine-induced maturation of DCs is under feedback regulation by SOCS proteins and that the switch from constitutive activation of the STAT6 pathway in iDCs to predominant use of STAT1 signals in mDC is mediated in part by STAT1-induced SOCS expression.

Characterization of the roles of STAT1 and STAT3 signal transduction pathways in mammalian lens development.

PURPOSE: IGF-1 and PDGF are implicated in regulating lens proliferation and/or providing spatial cues that restrict lens proliferation to germinative and transition zones of the lens. However, very little is known about how IGF-1- or PDGF-induced signals are transduced and coupled to gene transcription in lens cells. Therefore, we examined whether these growth factors mediate their effects in the lens through the evolutionarily conserved JAK/STAT signal transduction pathway and if STAT signaling is essential for mammalian lens development. METHODS: Expression of STAT1 and STAT3 was analyzed in mouse lens and lens epithelial cells by RT-PCR and western blot analysis. Activation of the STAT signaling pathway was examined by a combination of gel-shift, super-shift, and western blotting assays. Regulation of lens proliferation and gene transcription by STAT pathways was assessed by 3H-Thymidine incorporation or RT-PCR assays with lens explants treated or untreated with Genistein or the JAK2 and STAT3 inhibitor, AG-490. Mice with targeted deletion of STAT3 in the lens were generated by Cre/lox recombination and STAT1-/-, STAT3-/- deficient as well as normal lenses were characterized by histology. RESULTS: We show that PDGF and IGF1 induce proliferation in 1AMLE6 lens cells and couple their extracellular signals to gene transcription, in part through activation of STAT3 and to a lesser extent STAT1 signal transduction pathways. We further show that targeted deletion of STAT3 in E10.5 lens does not produce overt developmental lens defects. STAT1 knockout mice also exhibit a normal lens phenotype. CONCLUSIONS: Our results showing that deletion of either STAT1 or STAT3 does not affect the normal development of the lens is surprising in view of the fact that STAT pathways are activated in developing chick or mouse lens and inappropriate activation of STAT1 pathway in the lens by ectopic lens expression of IFN? inhibits lens differentiation and induces cataract in transgenic mice. Our data thus suggest that although STAT-signaling pathways may contribute to activation of gene transcription in the lens, it may not be essential for normal lens development or STAT proteins may be functionally redundant during lens development. However, because several growth factors and cytokines present in the lens activate STATs in mouse lens explants and 1AMLE6 lens epithelial cells, it may well be that this evolutionarily conserved signaling pathway is under stringent control in the mammalian lens. Whereas deficiency in any particular STAT pathway can be compensated for by any of the functionally redundant STAT proteins induced by a wide array of growth factors in the lens, chronic or prolonged activation of a particular STAT protein may perturb homeostatic balance in STAT-dependent growth factor signaling, culminating in pathologic lens changes.

Suppressor of cytokine signaling 3 regulates proliferation and activation of T-helper cells.

Suppressors of cytokine signaling (SOCS) have been implicated in regulation of T-cell activation and cytokine-mediated differentiation of T-helper cells. In this study we have characterized the pattern of SOCS expression in naïve and activated primary T-helper cells, examined whether expression of SOCS genes is regulated by cytokine or T-cell receptor signaling, and analyzed the function of SOCS in differentiated T-cells. We show that SOCS1, SOCS2, SOCS3, CIS (cytokine-induced SH2 protein) genes are constitutively expressed in naïve T-helper cells, with SOCS3 being the most abundant. Antigen stimulation of naïve T-helper cells down-regulates SOCS3 expression and concomitantly up-regulates SOCS1, SOCS2, and CIS gene transcription, suggesting that SOCS genes are regulated differentially by T-cell activation. Down-regulation of SOCS3 expression is subsequently followed by gradual increase in SOCS3 level and corresponding decline in interleukin 2 (IL-2) secretion. In fact, SOCS3 mRNA levels are inversely correlated with the amount of IL-2 secretion and proliferative responses of differentiating T-helper cells, suggesting mutually antagonistic effects of SOCS3 and IL-2 and feedback regulation of T-cell activation by SOCS3. Furthermore, the degree of SOCS3 inhibition is antigen concentration-dependent and is mediated in part by growth factor independence-1, a T-cell transcription factor that regulates S-phase entry in T-cells. Forced overexpression of SOCS3 inhibits proliferation of T-helper cells, whereas depletion of endogenous SOCS3 by antisense SOCS3 cDNA enhances T-cell receptor- and cytokine-induced proliferation. Taken together, these results suggest a role for SOCS3 in maintaining T-helper cells in a quiescent state. Transient inhibition of SOCS3 by antigen stimulation may therefore be essential in allowing activation of resting T-cells.

Keratinocyte growth factor pretreatment is associated with decreased macrophage inflammatory protein-2alpha concentrations and reduced neutrophil recruitment in acid aspiration lung injury.

A two-hit model of acid aspiration was used to examine the effect of keratinocyte growth factor (KGF) on chemokine levels and neutrophil recruitment into the lung. Mice were subjected to cecal ligation and puncture and then either KGF or saline, intratracheally (i.t.). Forty-eight hours later, the mice were given i.t. acid. After 8 h, neutrophil counts in bronchoalveolar lavage (BAL) fluid were significantly decreased in animals pretreated with KGF (23 +/- 4 x 10(3)/mouse) compared with saline (74 +/- 2 x 10(3)/mouse). In addition, the BAL fluid IL-6 levels were decreased in the KGF-treated group (88+/- 44 pg/mL) compared with the saline group (166 +/- 34 pg/mL). To examine the mechanism behind the KGF-induced reduction in neutrophil influx, the murine chemokines KC and macrophage inflammatory protein (MIP)-2alpha were measured. KC levels in plasma and BAL fluid were not significantly different between the treatment groups. Likewise, levels of MIP-2alpha in plasma were not affected by KGF treatment. However, 8 h after acid aspiration, MIP-2alpha concentrations were significantly lower in the KGF-treated group. The ratio of MIP-2alpha in BAL fluid versus plasma was lower in the KGF group (0.72 +/- 0.28) than in the saline group at 3 h (2.23 +/- 0.93) and also significantly lower in the KGF group (3.02 +/- 0.78) compared with the saline group (6.23 +/- 1.19) at 8 h. In this study, KGF pretreatment after acid aspiration was associated with reduced neutrophil recruitment into the lung and a decrease in MIP-2alpha gradients between BAL fluid and plasma.

Calcitonin gene-related peptide partially reverses decreased production of chemokines KC and MIP-2 following murine sepsis.

The secretion of calcitonin gene-related peptide (CGRP) and the chemokines KC and MIP-2 are increased in the animal models of endotoxemic and septic shock. We tested whether CGRP could modulate KC and MIP-2 secretion from different sources of macrophages after murine sepsis induced by cecal ligation and puncture (CLP). Macrophages were obtained from the peritoneal exudate and lung of female BALB/c mice 16 h after CLP and plated in culture with CGRP and/or LPS for 12 h. The results showed that peritoneal macrophage production of the chemokines (KC, MIP-2) and cytokines (TNF-alpha, IL-6) was markedly decreased in CLP mice. Alveolar macrophages did not display decreased cytokine/chemokines production after CLP. CGRP (0.1 nM-10 nM) partially reversed this decreased production of LPS-induced KC and MIP-2 from peritoneal macrophages. These results suggest that CGRP might be intimately involved in recruitment of neutrophils by promoting local production of the chemokines KC and MIP-2 in murine sepsis.