Prolonged activation of the mitogen-activated protein kinase pathway promotes DNA synthesis in primary hepatocytes from p21Cip-1/WAF1-null mice, but not in hepatocytes from p16INK4a-null mice

Abstract

In primary rat hepatocytes, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a decrease in DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor (CKI) proteins p21Cip-1/WAF1 and p16INK4a. To evaluate the relative importance of these CKIs in mediating this response, we determined the impact of prolonged MAPK activation on DNA synthesis in primary cultures of hepatocytes derived from mice embryonically deleted (null) for either p21Cip-1/WAF1 or p16INK4a. When MAPK was activated in wild-type mouse hepatocytes for 24 h, via infection with a construct to express an inducible oestrogen receptor-Raf-1 fusion protein (DeltaRaf:ER), the expression of p21Cip-1/WAF1 and p16INK4a CKI proteins increased, cyclin-dependent kinase 2 (cdk2) and cdk4 activities decreased, and DNA synthesis decreased. Inhibition of RhoA GTPase function increased the basal expression of p21Cip-1/WAF1 and p27Kip-1 but not p16INK4a, and enhanced the ability of MAPK signalling to decrease DNA synthesis. Ablation of the expression of CCAATT enhancer-binding protein alpha (C/EBPalpha), but not of the expression of C/EBPbeta, decreased the ability of MAPK signalling to induce p21Cip-1/WAF1. When MAPK was activated in p16INK4a-null hepatocytes for 24 h, the expression of p21Cip-1/WAF1 increased, cdk2 and cdk4 activities decreased and DNA synthesis decreased. In contrast with these findings, prolonged activation of the MAPK pathway in hepatocytes from p21Cip-1/WAF1-null mice enhanced cdk2 and cdk4 activities and caused a large increase in DNA synthesis, despite elevated expression of p16INK4a. Inhibition of RhoA GTPase activity in p21Cip-1/WAF1-null cells partly blunted both the basal levels of DNA synthesis and the ability of prolonged MAPK signalling to increase DNA synthesis. Expression of anti-sense p21Cip-1/WAF1 in either wild-type or p16INK4a-null hepatocytes decreased the ability of prolonged MAPK signalling to increase the expression of p21Cip-1/WAF1, and permitted MAPK signalling to increase both cdk2 and cdk4 activities and DNA synthesis. These results argue that the ability of prolonged MAPK signalling to inhibit DNA synthesis in hepatocytes requires the expression of p21Cip-1/WAF1, and that the increased expression of p16INK4a has a smaller role in the ability of this stimulus to mediate growth arrest. Our results also suggest that RhoA function can modulate DNA synthesis in primary hepatocytes via the expression of p21Cip-1/WAF1 and p27Kip-1.

Blunted DNA synthesis and delayed S-phase entry following inhibition of Cdk2 activity in the regenerating rat liver

“…Ras might also provide a link between the cyclin D/Cdk4 and the cyclin E/Cdk2 complex in cycling hepatocytes. 34,35 Therefore, the observation of similar upregulation of ras in rats administered the active compound compared to controls constitutes an additional argument for normal early cell cycle progression underscoring that CYC202 specifically inhibits late G1-S-phase transition, blunting cell proliferation.…”

Radiation-induced Release of Transforming Growth Factor α Activates the Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase Pathway in Carcinoma Cells, Leading to Increased Proliferation and Protection from Radiation-induced Cell Death

“…A short activation of the MAPK cascade by growth factors has been correlated with increased proliferation, via both increased cyclin D1 expression and an increased ability to progress through the G 2 -M transition. In contrast, prolonged elevation of MAPK activity has been demonstrated to inhibit DNA synthesis, via induction of the cyclin-dependent kinase inhibitor protein p21Cip-1/WAF1 (Auer et al, 1998b;Tombes et al, 1998). In addition to a role for MAPK signaling during G 1 -S phase, we and others have also argued that MAPK signaling is involved in the ability of cells to progress through G 2 -M, particularly in cells after irradiation (Warenius et al, 1996;Gokhale et al, 1997;Abbott and Holt, 1999;Vrana et al, 1999).…”

“…Increased signaling by EGFR and the MAPK pathway has been suggested in previous studies from our laboratory to be protective against both ionizing radiation and drug treatments, although the precise mechanism(s) by which this occurs are unclear (Balaban et al, 1996;Gokhale et al, 1997;Goldkorn et al, 1997;Schmidt-Ullrich et al, 1997;Carter et al, 1998;Kavanagh et al, 1998). Currently, the ability of the MAPK cascade to regulate differentiation and proliferative responses of cells is the focus of intense research (Sewing et al, 1997;Woods et al, 1997;Auer et al, 1998b;Dent et al, 1998;Tombes et al, 1998). The ability of MAPK signaling to regulate proliferation versus differentiation appears to depend on the cell type examined as well as on the amplitude and duration of MAPK activation.…”

CL100 expression is down-regulated in advanced epithelial ovarian cancer and its re-expression decreases its malignant potential

“…A speci®c antibody against b-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as loading control on the same membrane used to assay for the expression of CL100 or cyclin D1. ERK-2, JNK-1 and p38 kinase activities were measured as previously described with minor modi®cations (Auer et al, 1998).…”