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.
Highlights
From glycogen and increasing de novo glucose production [1, 2]
We demonstrate that glucagon induces SOCS3 in hepatocytes via exchange protein directly activated by cAMP (Epac) activation, and identify a mechanism of inhibitory crosstalk between Epac and protein kinase A (PKA) that is mediated by SOCS3 and may contribute to regulation of gluconeogenic gene expression
To determine if SOCS3 is involved in the Epac-dependent inhibition of cAMP response element-binding protein (CREB) phosphorylation and gluconeogenic gene expression, SOCS3 was expressed in primary hepatocytes using a SOCS3 adenoviral construct
Summary
Materials—Phosphospecific CREB (Ser-133) and CREB antibodies were purchased from Millipore (Billerica, MA). Adenovirus constructs: the mouse SOCS3 transcript was amplified from mouse cDNA by PCR with primers: SOCS3-forward: 5Ј-GGAATTCGCCACCATGGTCACCCACAGCAAG-3Ј and SOCS3-reverse: 5Ј-CCGCTCGAGTTAAAGTGGAGCATCATACTG3Ј. The mouse SOCS3 PCR product (678 bp, GenBankTM accession number NM007707) was digested with EcoR I and XhoI and ligated into the pShuttle-CMV vector (Stratagene, La Jolla, CA 92037). The pShuttle-CMV-mSOCS construct was verified by restriction and sequence analysis (ABI 3700, ACGT Inc., Wheeling, IL). Recombinant adenovirus was generated by homologous recombination of pShuttle-U6-shmSOCS3 or pShuttle-U6shluciferase constructs with pAdEasy in BJ5183 cells following the manufacturer’s protocol (Stratagene). Adenovirus constructs were administered to primary hepatocytes after overnight incubation at 200 viral particles per cell. Cells were exposed to serum-free Williams medium containing 1% BSA 1 h before treatments with 0.1 mM 8-bromo-cAMP, 10 nM glucagon, 20 ng/ml IL-6, 10 M H-89, or 10 M cpTOME. Experimental means were compared using ANOVA where sample means from four groups were compared and Student’s t test for comparing two groups
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