Signaling Invivo Research Articles

SOCS-1 Binding to Tyrosine 441 of IFN-γ Receptor Subunit 1 Contributes to the Attenuation of IFN-γ Signaling In Vivo

Suppressor of cytokine signaling (SOCS)-1 is a critical inhibitor of IFN-gamma signal transduction in vivo, but the precise biochemical mechanism of action of SOCS-1 is unclear. Studies in vitro have shown that SOCS-1 binds to Jaks and inhibits their catalytic activity, but recent studies indicate SOCS-1 may act in a similar manner to SOCS-3 by firstly interacting with cytokine receptors and then inhibiting Jak activity. Here, we have generated mice, termed Ifngr1(441F), in which a putative SOCS-1 binding site, tyrosine 441 (Y441), on the IFN-gamma receptor subunit 1 (IFNGR1) is mutated. We confirm that SOCS-1 binds to IFNGR1 in wild-type but not mutant cells. Mutation of Y441 results in impaired negative regulation of IFN-gamma signaling. IFN-gamma-induced STAT1 activation is prolonged in Ifngr1(441F) cells, but not to the extent seen in cells completely lacking SOCS-1, suggesting that SOCS-1 maintains activity to modulate IFN-gamma signaling via other mechanisms. Despite this, we show that hypersensitivity to IFN-gamma results in enhanced innate tumor protection in Ifngr1(441F) mice in vivo, and unregulated expression of an IFN-gamma-dependent chemokine, monokine-induced by IFN-gamma. Collectively, these data indicate that Y441 contributes to the regulation of signaling through IFNGR1 via the recruitment of SOCS-1 to the receptor.

Growth Hormone Signaling in Vivo in Human Muscle and Adipose Tissue: Impact of Insulin, Substrate Background, and Growth Hormone Receptor Blockade

GH induces insulin resistance in muscle and fat, and in vitro data indicate that this may involve cross-talk between the signaling pathways of the two hormones. Our objective was to investigate GH and insulin signaling in vivo in human muscle and fat tissue in response to GH, GH receptor blockade, and insulin stimulation. We conducted two randomized crossover studies. Sixteen healthy males participated. GH was administered as a bolus (n = 8) and constant infusion (n = 8). The bolus study included three arms: 1) control (saline), 2) GH (0.5 mg iv), and 3) GH blockade (pegvisomant 30 mg sc), each combined with a hyperinsulinemic glucose clamp. The infusion study included two arms: 1) GH infusion (45 ng/.kg.min, 5.5 h) and 2) saline infusion (5.5 h) combined with a hyperinsulinemic glucose clamp during the final 2.5 h. Muscle and fat biopsies were subjected to Western blotting for expression of Stat5/p-Stat5, Akt/p-Akt, and ERK1/2/p-ERK1/2 and to real-time RT-PCR for expression of SOCS1-3 and IGF-I mRNA. GH significantly reduced insulin sensitivity. The GH bolus as well as GH infusion induced phosphorylation of Stat5 in muscle and fat, and SOCS3 and IGF-I mRNA expression increased after GH infusion. Hyperinsulinemia induced Akt phosphorylation in both tissues, irrespective of GH status. In muscle, ERK1/2 phosphorylation was increased by insulin, but insulin per se did not induce phosphorylation of Stat5. GH exposure associated with insulin resistance acutely translates into GH receptor signaling in human muscle and fat without evidence of cross-talk with insulin signaling pathways. The molecular mechanisms subserving GH-induced insulin resistance in humans remain unclarified.

IL-4 Expression and Constitutive Activation of Erk Signaling In Vivo in Follicular Lymphoma.

Follicular lymphoma (FL) is characterized by constitutive expression of the Bcl-2 oncoprotein as a consequence of the t(14;18) BCL2/JH translocation in germinal center B-cells. While Bcl-2 confers survival advantage through anti-apoptosis mechanisms, the t(14;18) alone is not sufficient for lymphomagenesis. Evidence suggests that additional factors in the lymph node microenvironment, related to tumor infiltrating T-cells and macrophages, may cooperate with Bcl-2 in orchestrating the malignant phenotype in vivo. In this study we generated proteomic cytokine profiles of patient lymph node tissue lysates from FL (N=53) grades 1–3, and the non-malignant normal counterpart follicular hyperplasia (FH; N=24). Using an ultrasensitive multiplex elisa system we assayed a panel of 11 cytokines known to be secreted by T-cells and/or macrophages including IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-12p70, TNF-alpha, and INF gamma. With the exception of IL-4, all cytokines measured showed higher concentrations in FH than FL. In particular, IL-1 beta demonstrated levels 20 times higher in FH; IL-8 levels were 30 times higher; and IL-13 levels were 9 times higher in FH than in FL (p values < 0.005). In contrast, IL-4 levels were 5 times higher in FL than in FH (p < 0.0054). Interestingly, IL-4 levels were highest in FL grade 1 (N=20) in comparison to grade 2 (N=18) and grade 3 (N=12). Immunohistochemical stains for CD3 showed no significant difference in the numbers of CD3+ T-cells in FL samples over FH lymph nodes; or in FL grade 1 vs. grade 2 or grade 3. Hence the differences observed are not due to numerical differences in the total number of T-cells present in FL and FH. We evaluated the activation state of the mitogen activated protein (MAP) kinase signaling pathway, which is activated in response to stimulation by most cytokines in our panel via PI3Kinase. Western blot analysis showed that Erk was constitutively phosphorylated in 14 out of 15 (93%) of FL cases in contrast to only 3 out of 9 (33%) of FH cases. These findings demonstrate that tumor growth in FL is characterized overall by low levels of cytokines within the lymph node microenvironment in comparison to growth associated with the benign counterpart FH. The T-cell secreted cytokine, IL-4, which drives B-cell proliferation, was the only cytokine that showed statistically significant higher expression in the lymph node microenvironment of FL in comparison to FH. Moreover, the intracellular MAP kinase signaling pathway was constitutively activated in vivo in patient FL samples as compared to FH, despite higher overall cytokine levels in FH. These findings suggest that the IL-4 cytokine, and intracellular Erk activation in FL cells, may 1) play a role, in cooperation with Bcl-2, in driving the malignant phenotype in FL and 2) may serve as effective molecular targets for future therapies in FL.

Engineering the Melanocortin-4 Receptor to Control Constitutive and Ligand-Mediated Gs Signaling In Vivo

The molecular and functional diversity of G protein–coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein–mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of Gs signaling in vivo. We used naturally occurring human mutations to develop two Gs-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our Gs-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone α-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the Gs pathway in vivo. These RASSLs can be used to activate Gs signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering.

Specific Polyunsaturated Fatty Acids Drive TRPV-Dependent Sensory Signaling In Vivo

A variety of lipid and lipid-derived molecules can modulate TRP cation channel activity, but the identity of the lipids that affect TRP channel function in vivo is unknown. Here, we use genetic and behavioral analysis in the nematode C. elegans to implicate a subset of 20-carbon polyunsaturated fatty acids (PUFAs) in TRPV channel-dependent olfactory and nociceptive behaviors. Olfactory and nociceptive TRPV signaling are sustained by overlapping but nonidentical sets of 20-carbon PUFAs including eicosapentaenoic acid (EPA) and arachidonic acid (AA). PUFAs act upstream of TRPV family channels in sensory transduction. Short-term dietary supplementation with PUFAs can rescue PUFA biosynthetic mutants, and exogenous PUFAs elicit rapid TRPV-dependent calcium transients in sensory neurons, bypassing the normal requirement for PUFA synthesis. These results suggest that a subset of PUFAs with omega-3 and omega-6 acyl groups act as endogenous modulators of TRPV signal transduction.

Pregnancy-specific changes in uterine artery endothelial cell signaling in vivo are both programmed and retained in primary culture.

Ovine uterine artery (UA) endothelial cells (UAEC) maintained in culture to passage 4 retain pregnancy-specific changes in vasodilator production, which in turn is associated with differences in Ca(2+) and ERK 1/2 signaling. The question remains whether this is an accurate portrayal of the situation in vivo, or more simply whether these same signaling responses seen at passage 4 accurately reflect those functioning in the cells in vivo. Small groups of endothelial nitric oxide synthase-positive cells from both pregnant and nonpregnant ewes were freshly isolated and used to image changes in the intracellular free calcium concentration ([Ca(2+)](i)) using fura 2 and to detect ERK 1/2 phosphorylation by immunocytochemistry. Furthermore, detailed comparisons of mRNA species were made between freshly isolated and cultured (passage 4) cells using cDNA microarray analysis and verified, where possible, using PowerBlot analysis. Freshly isolated cells showed no detectable [Ca(2+)](i) elevation in response to angiotensin II, epidermal growth factor, basic fibroblast growth factor, or vascular endothelial growth factor but did respond to ATP in a dose-dependent (1-300 microM) manner. At higher doses of ATP, [Ca(2+)](i) elevation was sustained longer and showed a high incidence of regular oscillations in cells from pregnant compared with nonpregnant ewes. Also, ATP and basic fibroblast growth factor treatment caused activation of ERK 1/2 in significantly greater numbers of freshly isolated cells from pregnant than from nonpregnant ewes. cDNA microarray analysis showed results consistent with endothelium but revealed few differences in mRNA species and levels between freshly isolated and passage 4 cells or between the pregnant and nonpregnant ewes. In conclusion, our data show for the first time that pregnancy-specific changes in Ca(2+) and ERK 1/2 signaling are indeed observed in freshly isolated UA endothelium. This suggests in turn that such pregnancy-specific changes in UA endothelial function in vivo in response to a variety of agonists during pregnancy are both programmed at the level of cell signaling and retained in culture.

SOCS-1/SSI-1-Deficient NKT Cells Participate in Severe Hepatitis through Dysregulated Cross-Talk Inhibition of IFN-γ and IL-4 Signaling In Vivo

Suppressor of cytokine signaling-1 (SOCS-1), also known as STAT-induced STAT inhibitor-1 (SSI-1), is a negative feedback molecule for cytokine signaling, and its in vivo deletion induces fulminant hepatitis. However, elimination of the STAT1 or STAT6 gene or deletion of NKT cells substantially prevented severe hepatitis in SOCS-1-deficient mice, while administration of IFN-γ and IL-4 accelerated its development. SOCS-1 deficiency not only sustained IFN-γ/IL-4 signaling but also eliminated the cross-inhibitory action of IFN-γ on IL-4 signaling. These results suggest that SOCS-1 deficiency-induced persistent activation of STAT1 and STAT6, which would be inhibited by SOCS-1 under normal conditions, may induce abnormal activation of NKT cells, thus leading to lethal pathological changes in SOCS-1-deficient mice.

High specificity of Müllerian-inhibiting substance signaling in vivo.

Female transgenic mice that ectopically express high levels of human Müllerian-inhibiting substance (hMIS) under the control of the mouse metallothionein (MT) promoter lack a uterus, oviducts, and ovaries. The loss of the uterus and oviducts is consistent with the known activities for MIS. However, it is not clear if the loss of the ovaries in these transgenic females is caused by interactions of MIS with its normal receptor signaling pathway or by abnormal interactions with other transforming growth factor-beta (TGF-beta) super family receptor signaling pathways. To address this question, female mice carrying the MT-hMIS transgene that were also homozygous for a targeted deletion of the MIS type II receptor gene were generated. Although these females had high levels of circulating hMIS, they had normal reproductive tracts and ovaries with germ cells. In addition, these females were able to become pregnant and gave birth to pups. These findings demonstrate that all of the abnormalities of the reproductive system that are found in female transgenic mice that ectopically express high levels of hMIS are caused by signaling through the MIS type II receptor. These in vivo data demonstrate a high specificity for MIS and its receptor.