Hormonal induction of adipogenesis induces Skp2 expression through PI3K and MAPK pathways.
We have previously shown that the F-box protein, S-phase kinase-associated protein (Skp2) plays a mechanistic role in targeting the cell-cycle inhibitor, p27 for degradation by the 26S proteasome during early stages of 3T3-L1 adipocyte differentiation. Here, we demonstrate that protein levels of Skp2 and its accessory protein, Cks1 increased as density-arrested preadipocytes re-entered the cell cycle during clonal expansion, decreased with differentiation-induced growth arrest, and became refractory to hormonal stimulation following the onset of terminal adipocyte differentiation. Component analysis revealed that while maximal Skp2/Cks1 protein accumulation required the complete differentiation cocktail, that insulin was principally involved. Skp2 mRNA accumulation was found to precede the increase in Skp2 protein and succeed the activation of Akt and Erk1/2, mediators of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signal transduction pathways, respectively. Using specific inhibitors, we found that while activation of both pathways was required for maximal expression, PI3K signaling was primarily responsible for the increase in Skp2/Cks1 accumulation. The increase in Skp2 mRNA was notable 4 h following hormonal stimulation, plateaued by 12 h during mid-G1 phase progression, and occurred without change to mRNA stability. We further demonstrate that luciferase activity, originating from a pGL3 vector containing 2.4 kb of the Skp2 promoter, increased 2.5-fold with hormonal stimulation. This increase in promoter activity was markedly suppressed following PI3K and MAPK blockade. Deletion studies indicate that responsive elements were located within the proximal Skp2 promoter. These data demonstrate that Skp2 is transcriptionally regulated by PI3K and MAPK pathways as 3T3-L1 preadipocytes transition from quiescence to proliferation during adipocyte hyperplasia.
Auld
Corinth A
CA
Department of Nutrition, The University of North Carolina at Greensboro, Greensboro, North Carolina 27402, USA.
Caccia
Carla D
CD
Morrison
Ron F
RF
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism / Q000473:pathology*; D050156:Adipogenesis; D000818:Animals; D042846:CDC2-CDC28 Kinases / Q000378:metabolism; D002453:Cell Cycle; D002454:Cell Differentiation; D049109:Cell Proliferation; D003907:Dexamethasone / Q000494:pharmacology; D006728:Hormones / Q000494:pharmacology*; D006965:Hyperplasia; D007328:Insulin / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020871:RNA Stability; D044786:S-Phase Kinase-Associated Proteins / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic
D015056:1-Methyl-3-isobutylxanthine; D042846:CDC2-CDC28 Kinases; D003907:Dexamethasone; D006728:Hormones; D007328:Insulin; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D044786:S-Phase Kinase-Associated Proteins; C474110:Cks1 protein, mouse