P.J. Rijken

The epidermal growth factor.

Epidermal growth factor (EGF) is a single polypeptide of 53 amino acid residues which is involved in the regulation of cell proliferation. Egf exerts its effects in the target cells by binding to the plasma membrane located EGF receptor. The EGF receptor is a transmembrane protein tyrosine kinase. Binding of EGF to the receptor causes activation of the kinase and subsequently receptor autophosphorylation. The autophosphorylation is essential for the interaction of the receptor with its substrates. These bind to the receptor by the so‐called SH2 domains.

Nuclear responses to protein kinase C signal transduction are sensitive to gravity changes

A number of studies have suggested that gravity changes may influence mammalian cell growth and differentiation. To obtain insight in the molecular mechanisms underlying these effects, we have studied immediate early gene expression in response to activation of cytoplasmic signal transduction under microgravity conditions. In this paper we show that epidermal growth factor (EGF)- and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced expression of the c-fos and c-jun protooncogenes is decreased in microgravity, while no effect of gravity changes was observed on A23187- and forskolin-induced expression of these genes. These decrease in c-fos expression was not due to delayed kinetics under microgravity. These results demonstrate that gravity differentially modulates distinctive signal transduction pathways.

Chapter 7 Effects of Gravity on the Cellular Response to Epidermal Growth Factor

Publisher Summary Cells are exposed to a variety of physical forces, generated by the association with other cells and extracellular matrices, by polymerization/depolymerization reactions of cytoskeletal elements, and by the constant foxes placed on cells by gravity. Such forces can result in alterations in the cellular biochemistry. Illustrative in this respect is gravity perception in specialized plant root cells, the statocytes, which are localized in the mot tip. In statocytes, small cell organelles (amyloplasts) sediment according to the gravity vector toward the basal cell wall. Through an unknown mechanism, this triggers biochemical stimuli that lead to the induction of unidirectional cell proliferation along the gravity vector. This response is absent in microgravity. Changes in the gravitational environment may also lead to biochemical alterations in animal cells. Exposure of rats to microgravity, or immobilization of rats, leads to a change in calcium homeostasis, which results in the inhibition of bone formation. Increased gravity values also affect cellular processes. In human epithelial carcinoma cell line (HeLa cells), hrpergravity leads to alterations in the expression of specific nuclear genes. Hypergravity enhances the expression of the c-myc protooncogene, which is associated with a reduction of the duration of the G 1 cell cycle phase and results in a stimulation of proliferation of these cells. Although studies such as these have clearly demonstrated the translation of sometimes very subtle mechanical stimuli into biochemical signals, no clues have been provided as to the molecular mechanisms involved. The studies described in this chapter were performed in an attempt to unravel the molecular basis for microgravity effects on mammalian cells.

Identification of specific gravity sensitive signal transduction pathways in human A431 carcinoma cells.

Epidermal growth factor (EGF) activates a well characterized signal transduction cascade in human A431 epidermoid carcinoma cells. The influence of gravity on EGF-induced EGF-receptor clustering and early gene expression as well as on actin polymerization and actin organization have been investigated. Different signalling pathways induced by the agents TPA, forskolin and A23187 that activate gene expression were tested for sensitivity to gravity. EGF-induced c-fos and c-jun expression were decreased in microgravity. However, constitutive beta-2 microglobulin expression remained unaltered. Under simulated weightlessness conditions EGF- and TPA-induced c-fos expression was decreased, while forskolin- and A23187-induced c-fos expression was independent of the gravity conditions. These results suggest that gravity affects specific signalling pathways. Preliminary results indicate the EGF-induced EGF-receptor clustering remained unaltered irrespective of the gravity conditions. Furthermore, the relative filamentous actin content of steady state A431 cells was enhanced under microgravity conditions and actin filament organization was altered. Under simulated weightlessness actin filament organization in steady state cells as well as in EGF-treated cells was altered as compared to the 1 G reference experiment. Interestingly the microtubule and keratin organization in untreated cells showed no difference with the normal gravity samples. This indicates that gravity may affect specific components of the signal transduction circuitry.

Sulfhydryl Reagents Alter Epidermal Growth Factor Receptor Affinity and Association with the Cytoskeleton

Sulfhydryl (SH) reagents are known to influence the characteristics of many ligand-receptor systems. The SH reagent N-ethylmaleimide has been demonstrated to interact with EGF receptors, and to inhibit EGF receptor kinase activity. The data presented in this paper concern the effect of SH reagents on two intriguing features of the EGF receptor system, namely the presence of low and high affinity EGF binding sites, and the interaction of EGF receptors with the cytoskeleton. SH reagents were observed to induce a disappearance of high, but not low, affinity EGF receptors from the cell surface, and an increase in receptor-cytoskeleton interaction. Comparison of the effects of membrane-permeant and membrane-impermeant SH reagents on wild type and structurally modified EGF receptors suggested that sulfhydryl groups on the cytoplasmic, rather than the extracellular, receptor domain are involved. This indicates that the cytoplasmic domain of the EGF receptor plays a role in the high affinity binding of EGF, and in the interaction of EGF receptors with the cytoskeleton. Experiments with an anti-EGF receptor antibody that specifically blocks the binding of EGF to low affinity receptors indicated that EGF induces a shift in the EGF receptor from low to high affinity. SH reagents probably affect EGF binding by inhibiting this EGF-induced receptor conversion.