Cyclic AMP (cAMP)-Mediated Stimulation of Adipocyte Differentiation Requires the Synergistic Action of Epac- and cAMP-Dependent Protein Kinase-Dependent Processes

Abstract

Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of cAMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response.Adipocytes are derived from multipotent mesenchymal stem cells in a process involving commitment to the adipocyte lineage followed by terminal differentiation of the committed preadipocytes. The process is regulated via complex interaction of external and internal clues, where cell shape and cytoskeletal tension converging on regulation of Rho and Rhokinase activity have been demonstrated to play pivotal roles (48,63). Whereas our understanding of the early steps of lineage determination still is limited, regulatory cascades controlling terminal adipocyte differentiation have been elucidated in great detail, particularly the sequential action of different transcription factors culminating in the expression of adipocyte-specific genes (25,30,58). Much information on terminal adipocyte differentiation has been obtained using model cell lines such as 3T3-L1 and 3T3-F442A or mouse embryo fibroblasts (MEFs). In both MEFs and 3T3-L1 preadipocytes, terminal differentiation is initiated upon treatment with fetal calf serum, glucocorticoids, and high levels of insulin or physiological concentrations of insulin-like growth factor 1 (IGF-1). Factors that increase cellular cyclic AMP (cAMP), such as isobutylmethylxanthine (IBMX) or forskolin, strongly accelerate the initiation of the differentiation program (for review, see references 25 and 45).Elevation of cellular cAMP concentration has been associated with crucial events in the early program of differentiation, such as suppression of Wnt10b (5) and Sp1 (64) and induction of CCAAT/enhancer-binding protein ␤ (C/EBP␤) (10,29,70). Moreover, the transcriptional activity of peroxisome proliferator-activated receptor ␦ (PPAR␦) is regulated synergistically by ligands and cAMP (32). In addition, cAMP has been implicated in the production of endogenous PPAR␥ ligand(s) occurring during the initial stages of differentiation (46, 67). The cAMP-responsive element-binding protein (CREB) is a central transcriptional activator of the adipocyte differentiation program. Activated CREB induces expres...

Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation

“…We observed the same We have recently shown that the adipogenic effect of elevated cAMP levels relies on the activation of both protein kinase A (PKA) and the exchange protein activated by cAMP (Epac). 28 Using selective activators of both PKA and Epac, we show that only the PKA activator augmented expression of C/EBPd ( Figure 3d). …”

“…In fact, it is the mitogen-activated protein kinase (MAPK) that is responsible for CREB phosphorylation during the initiation of adipocyte differentiation. 28 An important function of PKA during adipose conversion is inhibition of the Rho kinase 28 that in turn can inhibit adipogenesis by blocking the MAPK-mediated phosphorylation of CREB. 31 To ensure that the failure to induce C/EBPd in the absence of Mdm2 was not a result of uncontrolled Rho-kinase activity, we concomitantly treated the p53 À/À ;mdm2 À/À MEFs with IBMX and increasing doses of a Rho-kinase inhibitor.…”

Regulation of MKL1 via actin cytoskeleton dynamics drives adipocyte differentiation

“…Both IBMX and dexamethasone trigger the activation of protein kinase A by increasing the intracellular concentration of cAMP, thereby promoting adipocyte differentiation 36 . The major role of protein kinase A in adipogenesis has been thought to lie in downregulation of RhoA and ROCK activity 37 . In the present study, we found that Rho activity is rapidly reduced after treatment of DFAT cells with the adipogenic cocktail, and the downregulation of RhoA-ROCK signalling then promotes adipocyte differentiation via modulation of remodelling of the actin cytoskeleton.…”

TLR4-Dependent Tumor-Initiating Stem Cell-Like Cells (TICs) in Alcohol-Associated Hepatocellular Carcinogenesis

“…This approach is prompted by the observation that TLR4 expression is induced in the liver by hepatocyte-specifi c NS5A expression [ 28 ]. As endotoxin-TLR4 pathway is established in the pathogenesis of ALD [ 29 , 30 ], a synergism between alcohol-mediated endotoxemia and NS5A-induced TLR4 overexpression was predicted.…”