Novel effect of helenalin on Akt signaling and Skp2 expression in 3T3-L1 preadipocytes

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Abstract

We have previously shown that the F-box protein, Skp2, is highly regulated during preadipocyte proliferation and plays a mechanistic role in p27 degradation during cell cycle progression. Data presented here demonstrate that the anti-inflammatory, anti-carcinogenic phytochemical, helenalin is a potent inhibitor of periodic Skp2 protein accumulation during early phases of 3T3-L1 adipocyte differentiation. Furthermore, helenalin was shown to completely block p27 degradation, cyclin A accumulation, and G1/S transition resulting in G1 arrest. Helenalin was also shown to block Skp2 mRNA accumulation in a concentration-dependent manner and to completely suppress hormonally induced Skp2 promoter activity suggesting transcriptional mechanisms were involved. Examination of signaling events previously determined to be important for Skp2 upregulation during adipogenesis revealed impaired Akt phosphorylation immediately preceding the inhibitory effect of helenalin on Skp2 mRNA accumulation. These studies demonstrate a novel effect of helenalin on Skp2 regulation and growth factor receptor signaling during early stages of adipocyte differentiation.

Section snippets

Materials and methods

Materials. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Cellgro by Mediatech. Calf bovine serum, fetal bovine serum (FBS), and trypsin-EDTA were from Invitrogen Corporation. Propidium iodide and RNase A were purchased from Sigma. Helenalin was purchased from Biomol. The following antibodies were used for immunoblotting: phospho-Akt (Ser473), total Akt, phospho-Erk1/2 (Thr202/Tyr204), and total Erk1/2 (Cell Signaling); Skp2, Skp1, and cyclin A (Santa Cruz Biotechnology); and p27

Helenalin inhibits hormonally induced Skp2 accumulation, p27 degradation, and G1/S phase transition

We have previously determined that Skp2 protein periodically accumulates during S/G2 phase progression of 3T3-L1 preadipocyte differentiation. To examine the effect of helenalin on Skp2 accumulation, density-arrested 3T3-L1 preadipocytes were stimulated with MDI in the presence or absence of helenalin (3 μM). Whole cell lysates were collected at 0 h (density arrest) and 20 h post-MDI and immunoblotted as indicated in Fig. 1A. We demonstrate that helenalin completely suppressed hormonally induced

Discussion

Positive energy balance can cause preadipocytes to undergo a transition from quiescence to proliferation during the development of hyperplastic obesity [14], [15]. The cyclin-dependent kinase inhibitors (CKIs), p27 and p21, govern this transition as potent inhibitors of the G1/S phase transition. Recent studies underscore the importance of these CKIs in adipose tissue development as mice defective in p27/p21 gene expression were shown to have a disproportionate increase in adipose tissue mass

Acknowledgments

We are grateful to Howard Green (Harvard Medical School) for the murine 3T3-L1 cell line. This work was supported by grants from the American Heart Association (0265418U) and National Institutes of Health (1R21DK072067-01) to R.F.M.

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