Suppressor of cytokine signaling-3 is a glucagon-inducible inhibitor of PKA activity and gluconeogenic gene expression in hepatocytes

Abstract

SOCS3 is a cytokine-inducible negative regulator of cytokine receptor signaling. Recently, SOCS3 was shown to be induced by a cAMP-dependent pathway involving exchange protein directly activated by cAMP (Epac). We observed in livers of fasted mice that Socs3 mRNA was increased 4-fold compared with refed mice, suggesting a physiologic role for SOCS3 in the fasted state that may involve glucagon and Epac. Treating primary hepatocytes with glucagon resulted in a 4-fold increase in Socs3 mRNA levels. The Epac-selective cAMP analog 8-4-(chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate, acetoxymethyl ester (cpTOME) increased Socs3 expression comparably. In gain-of-function studies, adenoviral expression of SOCS3 in primary hepatocytes caused a 50% decrease in 8-br-cAMP-dependent PKA phosphorylation of the transcription factor CREB. Induction of the gluconeogenic genes Ppargc1a, Pck1, and G6pc by glucagon or 8-br-cAMP was suppressed nearly 50%. In loss-of-function studies, hepatocytes from liver-specific SOCS3 knock-out mice responded to 8-br-cAMP with a 200% greater increase in Ppargc1a and Pck1 expression, and a 30% increase in G6pc expression, relative to wild-type cells. Suppression of SOCS3 by shRNA in hepatocytes resulted in a 60% increase in cAMP-dependent G6pc and Pck1 expression relative to control cells. SOCS3 expression also inhibited cAMP-dependent phosphorylation of the IP3 receptor but did not inhibit nuclear localization of the catalytic subunit of PKA. Using an in vitro kinase assay, cAMP-dependent PKA activity was reduced by 80% in hepatocytes expressing ectopic SOCS3. These data indicate that cAMP activates both the PKA and Epac pathways with induction of SOCS3 by the Epac pathway negatively regulating the PKA pathway.

FIGURE 1.

Glucagon-dependent induction of Socs3 in primary hepatocytes. A, primary hepatocytes were treated with IL-6 (20 ng/ml) or 8-br-cAMP (0.1 mm) for 2 h. Relative Socs3 expression was measured by qRT-PCR. Time-dependent (B) and concentration-dependent (C) expression of Socs3, Pck1, and G6pc in response to glucagon (10 nm) were measured. D, Socs3 and Pck1 mRNA levels were measured in liver from mice fasted 24 h, or refed for 4 h following a 24 h fast. Data are normalized to fed mice. Data represent the mean ± S.D., n = 3–6. **, p < 0.01.

FIGURE 2.

Glucagon-dependent induction of Socs3 is mediated by Epac. Primary hepatocytes were treated with glucagon (10 nm), the Epac-selective analog cpTOME (5 μm), or the PKA inhibitor H-89 (10 and 30 μm) for 2 h. mRNA levels were determined for Socs3 (A) and Pck1 (B). C, Socs3 mRNA was measured after a 2 h glucagon treatment in primary hepatocytes infected with RapGAP1-expressing adenovirus (AV-RapGap) (200 moi) or an adenovirus-GFP (AV-GFP) control. Bar graph in C is normalized to GFP with no treatment. Data represent the mean ± S.D., n = 3–6. **, p < 0.01.

FIGURE 3.

PLCϵ activation is not required for Epac-mediated SOCS3 induction. A, WT and PLCϵ^−/− primary hepatocytes were treated with glucagon (10 nm) or cpTOME (5 μm) for 2 h. SOCS3 mRNA levels were measured by qRT-PCR. B, WT primary hepatocytes were treated with 8-br-cAMP (0.1 mm). Phosphorylation of p44/p42 MAP kinase (ERK) was detected by Western blot analysis with a phosphoERK antibody (Thr-202/Tyr-204). Data are normalized to no treatment controls. Data represent the mean ± S.D., n = 5–8.

FIGURE 4.

Epac activation inhibits PKA-mediated CREB phosphorylation and gluconeogenic gene expression. cpTOME (5 μm) was added to primary hepatocytes for 1 h (A and B, bottom graph) or 2 h (B, top graph) followed by a 30 min (A) or 2 h (B, bottom graph) incubation with sp-CAMP (10 μm). A, CREB phosphorylation was determined by Western blot analysis using a phosphoCREB-specific (Ser-133) antibody. B, Ppargc1a, G6pc, and Pck1 mRNA levels were determined by qRT-PCR. In the bottom graph, fold activation over basal were 16-, 233-, and 347-fold for ppargc1a, g6pc, and pck1, respectively. Data are normalized to sp-cAMP treated controls. Data represent the mean ± S.D., n = 6. *, p < .05; **, p < 0.01.

FIGURE 5.

SOCS3 expression inhibits CREB phosphorylation and gluconeogenic gene expression. Primary hepatocytes were infected with either adeno-SOCS3 or adeno-LacZ (control) (200 moi) for 24 h. A, after a 30 min 8-br-cAMP (0.1 mm) treatment, CREB phosphorylation was measured by Western blot analysis. A representative blot is shown. Bar graph represents quantitation of pSer133-CREB relative to AV-LacZ untreated controls. A time course of CREB phosphorylation in the absence of adenovirus is also shown. B, Pck1, G6pc, and Ppargc1a induction was measured by qRT-PCR after a 2 h 8-br-cAMP (0.1 mm) treatment. Data are normalized to AV-LacZ cells treated with 8-br-cAMP. C, Pck1, G6pc, and Ppargc1a induction was measured by qRT-PCR after a 2-h stimulation with glucagon (10 nm) or glucagon plus H-89 (10 μm). Data are normalized to glucagon treated AV-LacZ controls. Data represent the mean ± S.D., n = 6. *, p < 0.05; **, p < .01.

FIGURE 6.

Loss of Socs3 expression increases gluconeogenic gene expression. Primary hepatocytes were infected with adenovirus (200 moi) expressing either shSOCS3 or shLuciferase (control). Socs3 (A) or Pck1, G6pc, and Ppargc1a (B) were measured by qRT-PCR after a 2-h treatment with 8-br-cAMP (0.1 mm). Data represent the mean ± S.D., n = 6. C, primary hepatocytes from liver-specific SOCS3 knock-out mice or their littermate controls were treated with 8-br-cAMP (0.1 mm) or IL-6 (20 ng/ml) for 6 h. Socs3 was measured by qRT-PCR. D, Pck1, G6pc, and Ppargc1a induction was measured by qRT-PCR after a 6-h 8-br-cAMP treatment. Data are normalized to 8-br-cAMP-treated WT controls. Data represent the mean ± S.D., n = 3. *, p < .05; **, p < .01.

FIGURE 7.

SOCS3 inhibits PKA-mediated Ser-1756 phosphorylation of the IP3R. Primary hepatocytes expressing adeno-SOCS3 or adeno-LacZ (200 moi) were treated with 8-br-cAMP (0.1 mm) for 30 min. Phosphorylation of the IP3 receptor was measured by Western blot analysis using a phosphoIP3R (Ser-1756) antibody. Bar graph represents quantitation of pSer1756-IP3R relative to AV-LacZ-nontreated controls. Data represent the mean ± S.D., n = 3. **, p < 0.01.

FIGURE 8.

SOCS3 inhibits PKA activity. A, primary hepatocytes were treated with 8-br-cAMP (0.1 mm) for 30 min with or without a 1 h cpTOME (5 μm) pretreatment. PKA activity was measured in cell lysates using a fluorescent peptide substrate assay (Promega). B, primary hepatocytes expressing adeno-SOCS3 (200 moi) were treated with 8-br-cAMP (0.1 mm) for 30 min. PKA activity was measured in cell lysates. Values are relative to PKA activity (units/ml) with no treatment. Data represent the mean ± S.D., n = 4–6. *, p < 0.05; **, p < .01.

FIGURE 9.

SOCS3 does not inhibit the translocation of the PKA catalytic subunit to the nucleus. Primary hepatocytes expressing adeno-LacZ or adeno-SOCS3 (200 moi) were treated with 8-br-cAMP (0.1 mm) for 30 min. Cells were subsequently lysed and separated into cytosolic and nuclear fractions. Protein levels were detected by Western blot analysis. β-Actin and histone H1 were used as cytosolic and nuclear markers, respectively. A representative experiment is shown, n = 5.

FIGURE 10.

Crosstalk between the Epac and PKA pathways. Glucagon-dependent activation of adenylate cyclase and generation of cAMP activates both the PKA and Epac pathways. The catalytic subunit of PKA enters the nucleus, and rapidly phosphorylates and activates the transcription factor CREB. The gluconeogenic genes are induced by this pathway. Activation of the Epac pathway leads to induction of SOCS3. SOCS3, acting as a feedback inhibitor, interacts with and inhibits activity of the catalytic subunit of PKA. Thus, the cAMP-Epac-SOCS3 pathway modulates activity of the cAMP-PKA-CREB pathway.