Activation of Erk1/Erk2 and transiently increased p53 levels together may account for p21 expression associated with phorbol ester-induced transient growth inhibition in HepG2 cells

doi:10.1016/S0898-6568(01)00229-7

Cellular Signalling

Volume 14, Issue 2, February 2002, Pages 115-121

https://doi.org/10.1016/S0898-6568(01)00229-7 Get rights and content

Abstract

In HepG2 cells grown in the presence of serum, enhanced Raf-activation correlated with transient growth inhibition. The activation of Raf was increased either by the phorbol ester-induced activation of protein kinase C (PKC) or by the addition of the PKC inhibitor bisindolylmaleimide I (BIM). Either of these treatments increased the cellular levels of p21 by an Erk1/Erk2 MAP kinase cascade-dependent way, since this increase was prevented by the MEK-inhibitor PD98059. Nevertheless, the growth inhibition correlated with the transient increase of p53 levels as well. Either the activation of PKC with phorbol ester or the addition of BIM to cells growing in serum induced a rapid but transient increase of p53 levels, which preceded growth inhibition. This increase of p53 levels was probably due to the transient stabilisation of p53 and did not require the activation of Erk1/Erk2.

Introduction

Phorbol ester or hepatocyte growth factor/scatter factor (HGF) induces the scattering of HepG2 human hepatoma cells. Both phorbol ester [1] and HGF have been reported to induce also the transient growth inhibition of these cells. Increased p21/Cip1/WAF1 expression has been suggested to be responsible for the growth inhibition caused by HGF [2]. High cellular levels of the cyclin-dependent kinase-inhibitor p21 may cause a delay of DNA synthesis in the course of the cell cycle (reviewed recently in Ref. [3]).

Different regulatory pathways are responsible for the control of p21 expression. The p53 tumour-suppressor gene product is a transcription factor for the p21 gene and p21 expression increases when the cellular levels of p53 increase. The regulation of cellular levels and transcriptional activity of p53 can occur via phosphorylation at multiple sites (recently reviewed in [4], [5]). In cells that are dividing normally, p53 is unstable, with a half-life measured in minutes. Complex mechanisms can increase p53 levels by influencing its degradation rate. MDM2 protein binds to the N-terminal region and targets p53 for ubiquitination and degradation. DNA damage stimulates the phosphorylation of p53 at Ser-15 and Ser-20, which prevents the interaction of MDM2 with p53. ARF protein also increases the half-life of p53 [6], [7]. The increased stability of p53 leads to the induction of p21 expression and consequently to the delay of DNA synthesis.

A p53-independent, Erk1/Erk2 MAP kinase cascade-dependent mechanism is also known to regulate p21 expression (reviewed in [8], [9]). High-intensity Raf signal exerted by a Raf-androgen receptor fusion protein causes cell cycle arrest mediated by p21 [10]. Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity in NIH 3T3 cells; Raf has been suggested to induce the expression of p21 in a p53-independent way [11]. Activation of Erk1/Erk2 has been found to mediate growth-factor-stimulated transcription of a p21 promoter–reporter construct [12]. Constitutively active components in the Erk1/Erk2 MAP kinase pathway activate p21 expression, and inhibitors or dominant negative constructs for the MEK pathway significantly decrease p21 induction in certain signalling systems [13]. Nevertheless, the molecular mechanism mediating the effects of the MAP kinase pathway on the expression of p21 gene has not been revealed.

On the other hand, the promoter of p21/Cip1/Waf1 contains binding sites for the Sp1 transcription factor and Sp1 has been found to play an important role in the transcriptional activity of the p21 gene [14], [15], [16] in different cell types including HepG2 cells [15]. Sp1 collaborates with the p300 coactivator in p21 promoter activation [17]. In hematopoietic cells that are deleted for p53, phorbol ester induces the transcription of p21 in a p53-independent manner. This phorbol ester-mediated transcriptional stimulation is controlled by a GC-rich Sp1-binding site located adjacent to the TATA box [14].

We have reported that the scattering of HepG2 cells induced by phorbol ester or HGF requires the activation of Erk1/Erk2 MAP kinases. Both phorbol ester and HGF stimulate the activation of the Erk1/Erk2 MAP kinase cascade for a significantly longer time than epidermal growth factor, which is not able to induce scattering in this cell type [18]. The aim of the present study was to investigate the time course of events, which are responsible for the phorbol-ester-induced transient growth inhibition of HepG2 cells. We found that growth inhibition correlated with the intensive activation of Raf. However, under this condition, the transient increase of p53 levels was also observed and the synergistic effects of Erk1/Erk2 activation and the transient increase of p53 levels might account for elevated p21 expression.

Section snippets

Chemicals

Phorbol myristate acetate (PMA) was purchased from Sigma (St. Louis, MO). HGF (human recombinant), the protein kinase C (PKC) inhibitor bisimdolylmaleimide I and the MEK inhibitor PD98059 were obtained from Calbiochem (San Diego, CA).

Cell culture

HepG2 human hepatoma cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) in a humidified atmosphere of 5% CO₂/95% air. The medium was supplemented with 10% (v/v) foetal

Activation of the Erk1/Erk2 MAP kinase cascade is essential to the increase of p21 levels

Treatment of HepG2 cells with phorbol ester induces a transient decrease of DNA synthesis under certain conditions [1]. In our experiments, the maximal growth inhibition (30–40%) was demonstrated after 6 h of incubation with 80 nM PMA. After 24 h of incubation, the rate of [³H]-thymidine incorporation was even higher in phorbol ester-treated cells than in control cells (Fig. 1A). We attempted to prevent the inhibitory effect of PMA with the PKC inhibitor BIM, but in cells growing in the

Acknowledgements

This work was supported by a Fogarty International Research Collaboration Award (NIH, USA) by the OTKA Grant T-032033, by the Hungarian Ministry of Welfare (30/2000), and by the Hungarian Ministry of Education (FKFP 0370/1999).

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¹: Present address: Lilly Research Laboratories, Lilly Corporation Center, Eli Lilly and Company, Indianapolis, IN, USA.

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Cellular Signalling

Activation of Erk1/Erk2 and transiently increased p53 levels together may account for p21 expression associated with phorbol ester-induced transient growth inhibition in HepG2 cells

Abstract

Introduction

Section snippets

Chemicals

Cell culture

Activation of the Erk1/Erk2 MAP kinase cascade is essential to the increase of p21 levels

Acknowledgements

Trends Cell Biol

J Biol Chem

J Biol Chem

J Biol Chem

Cell Signal

J Biol Chem

Cell Signal

J Cell Physiol

J Cell Physiol

Mol Pathol

Biochem J

Anthocyanins in Vascular Diseases

PMA increases M3 muscarinic receptor levels and decreases retinal cells proliferation through a change in the levels of cell-cycle regulatory proteins

Para-Phenylenediamine-induced autophagy in human uroepithelial cell line mediated mutant p53 and activation of ERK signaling pathway

Protein kinase C α trigger Ras and Raf-independent MEK/ERK activation for TPA-induced growth inhibition of human hepatoma cell HepG2

Regulation of de novo phosphatidylinositol synthesis

Protein kinase C modulates negatively the hepatocyte growth factor-induced migration, integrin expression and phosphatidylinositol 3-kinase activation