Jean-Claude Chambard

ERK and cell death: Mechanisms of ERK‐induced cell death – apoptosis, autophagy and senescence

The Ras/Raf/extracellular signal‐regulated kinase (ERK) signaling pathway plays a crucial role in almost all cell functions and therefore requires exquisite control of its spatiotemporal activity. Depending on the cell type and stimulus, ERK activity will mediate different antiproliferative events, such as apoptosis, autophagy and senescence in vitro and in vivo. ERK activity can promote either intrinsic or extrinsic apoptotic pathways by induction of mitochondrial cytochrome c release or caspase‐8 activation, permanent cell cycle arrest or autophagic vacuolization. These unusual effects require sustained ERK activity in specific subcellular compartments and could depend on the presence of reactive oxygen species. We will summarize the mechanisms involved in Ras/Raf/ERK antiproliferative functions.

Intracellular pH controls growth factor-induced ribosomal protein S6 phosphorylation and protein synthesis in the G0→G1 transition of fibroblasts☆

Mitogen-induced intracellular alkalinization mediated by activation of a Na+/H+ antiporter is a common feature of eukaryotic cells stimulated to divide. A Chinese hamster fibroblast mutant (PS120) lacking Na+/H+ antiport activity (Pouysségur et al., Proc natl acad sci US 81 (1984) 4833) [42] possesses an intracellular pH (pHi) 0.2-0.3 units lower than the wild type (CCL39) and requires a more alkaline pHout (pHo) for growth. Here, we show that serum-stimulated ribosomal protein S6 phosphorylation, protein synthesis activation and DNA synthesis re-initiation are pH-regulated events that display a similar threshold pHo value (6.60) in CCL39 cells. pH-Dependencies for initiation of all three events are shifted toward higher pHo values in the mutant PS120, indicating that growth factor-induced alkalinization has a permissive effect on the pleiotypic response. However, cytoplasmic alkalinization per se is insufficient to trigger S6 phosphorylation, polysome formation, and subsequent DNA synthesis. Transient exposure to a non-permissive pHo (6.5) inhibits both the rate of leucine incorporation into proteins and the progression through the G1 phase of the cell cycle. In contrast, cells committed to DNA synthesis are unaltered by the acidic pHo. These observations suggest that pHi by controlling the rate of protein synthesis play a determinant role in the control of cell division.

Prolonged activation of ERK1,2 induces FADD-independent caspase 8 activation and cell death

Prolonged ERK/MAPK activation has been implicated in neuronal cell death in vitro and in vivo. We found that HEK293 cells, recently reported to express neuronal markers, are exquisitely sensitive to long term ERK stimulation. Activation of an inducible form of Raf-1 (Raf-1:ER) in HEK293 cells induced massive apoptosis characterized by DNA degradation, loss of plasma membrane integrity and PARP cleavage. Cell death required MEK activity and protein synthesis and occurred via the death receptor pathway independently of the mitochondrial pathway. Accordingly, prolonged ERK stimulation activated caspase 8 and strongly potentiated Fas signaling. The death receptor adaptator FADD was found to be rapidly induced upon ERK activation. However using RNA interference and ectopic expression, we demonstrated that neither FADD nor Fas were necessary for caspase 8 activation and cell death. These findings reveal that prolonged ERK/MAPK stimulation results in caspase 8 activation and cell death.

alpha-Thrombin-induced early mitogenic signalling events and G0 to S-phase transition of fibroblasts require continual external stimulation.

In resting Chinese hamster fibroblasts (CCL39) alpha‐thrombin rapidly stimulates several biochemical events implicated in the mitogenic response, including the breakdown of inositol phospholipids, activation of a plasma membrane Na+/H+ antiporter, phosphorylation of ribosomal protein S6 and increased expression of the proto‐oncogene c‐myc. Complete removal of the growth factor during cellular G0/G1 transit precludes the re‐initiation of DNA synthesis. The present study was designed to examine the fate of alpha‐thrombin‐activated early events following growth factor inactivation. In cells stimulated for 30 min with alpha‐thrombin, neutralization of the growth factor results in: (i) immediate arrest of inositol phosphate formation, (ii) rapid inactivation of Na+/H+ exchange, (iii) deactivation of the S6 phosphorylating system and (iv) strong reduction of c‐myc mRNA level. Our findings that commitment for DNA synthesis as well as persistent activation of ‘early’ cellular events requires continual growth factor stimulation suggest that: (i) growth factor‐induced transmembrane signals have a short life and (ii) the generation of these signals during the 8 h of the pre‐replicative phase is required for G0‐arrested cells to enter the S phase.

Thrombin-induced protein phosphorylation in resting platelets and fibroblasts: Evidence for common post-receptor molecular events

We have investigated the thrombin-stimulated protein phosphorylation associated with the activation of two cellular processes: 1) reinitiation of DNA synthesis in G0-arrested hamster fibroblasts and 2) stimulation of serotonin release in platelets. We found a rapid 4- to 6-fold increased phosphorylation of a peptide with apparent Mr = 27,000 in SDS-PAGE. In both cell systems, the 27,000 dalton phosphopeptide is cytosolic, is resolved by isoelectric focusing as multiple variants (pHi 5 to 6), and thrombin stimulation generates the most acidic phosphorylated forms. This result and the fact that the 27,000 dalton peptide is also stimulated by a variety of growth factors in fibroblasts, strongly suggests that thrombin action in platelets and growth factor-induced mitogenesis, share common post-receptor molecular events.

Growth-Promoting Activity of Thrombin in Fibroblasts: Temporal Action and Early Biochemical Events in the G0 → G1 Transition

Polypeptide growth factors and hormones have been shown to play an essential role in the regulation of cell proliferation and cell differentiation. However, the molecular mechanisms by which these extracellular signals act on quiescent cells to induce the G0→G1 transition, DNA replication and subsequent cell division or differentiation are unknown. The introduction by Sato and coworkers of cell culture in serum-free hormone-supplemented medium (1) has greatly facilitated the experimental analysis of the mechanisms of growth factor action. The addition of purified growth-promoting polypeptides to G0-arrested fibroblasts induces a multiple array of biochemical events leading to activation of protein synthesis and subsequent DNA synthesis. Changes in ionic membrane permeability and in protein phosphorylation occur rapidly after stimulation (2–8). These changes, which appear to be ubiquitous, raise the central question of how distinct growth factor receptors translate the variety of external signals into a common activation of cellular metabolism and cell proliferation. The purpose of this paper is to present the growth-promoting activity of α-thrombin in serum-free conditions We will analyze the temporal action of thrombin during the G0→G1 transition of the cell cycle and the post-receptor, early-stimulated biochemical events: activation of a Na+/H+ exchange and stimulation of protein phosphorylation.