Epidermal growth factor: Modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport

Abstract

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose- and time-dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4-6 hours. EGF also stimulated the initial rate of putrescine transport in a dose- and time-dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 microM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 microM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF-treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 micrograms/ml) and actinomycin D (50 micrograms/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca++ while alpha-aminoisobutyric acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO-treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.

Indomethacin Interferes with EGF-Induced Activation of Ornithine Decarboxylase in Gastric Cancer Cells

“…Interestingly, both ODC and mRNA assays exhibited the biphasic activation after EGF stimulation. Such biphasic activation of ODC is a new finding, entirely different from other reports pertaining to rat liver, brain, and lung [25], cultured rat pancreatic AR42J cells [19], human epidermoid carcinoma A431 cells [20], and murine embryonic palate mesenchymal cells [21]. In all these cells, EGF induced only monophasic activation of ODC.…”

“…We focused this study on EGF-induced activation of ODC, because ODC is a key enzyme for the cell proliferation. EGF has been shown to activate ODC in some cultured cells [19][20][21][22], but not in other cells such as NIH 3T3 fibroblasts [23] and SW 403 human colon carcinoma cells [24].…”

The Spermidine Transport System Is Regulated by Ligand Inactivation, Endocytosis, and by the Npr1p Ser/Thr Protein Kinase in Saccharomyces cerevisiae

“…Polyamine-specific carriers are widely distributed in prokaryotes and eukaryotes (5-7) and can replenish polyamine pools upon inhibition of the biosynthetic enzymes (8 -10). Mammalian polyamine transport activity is also acutely controlled by cell cycle events (11,12) and hormonal stimulation (5,9,10,13,14). Polyamine transport is a saturable, carrier-mediated and energy-dependent process (5,15,16).…”

Analysis of the Presence of Cell Proliferation-Related Molecules in the <i>Tgf-</i>β_<i>3</i> Null Mutant Mouse Palate Reveals Misexpression of EGF and <i>Msx-1</i>

“…Both EGF and EGF-R were immunolocalized in the MEE and mesenchyme at all stages of palate development [Shiota et al, 1990;Dixon et al, 1991;Citterio and Gaillard, 1994;Jaskoll et al, 1996]. EGF has a recognized mitogenic activity in the MEE and mesenchyme of the developing palate [Abbott and Pratt, 1988;Gawel-Thompson and Greene, 1989;Yamamoto et al, 2003], while EGF-R -/-mice exhibit CP and delayed palatal shelf closure [Miettinen et al, 1999]. Furthermore, alterations of the EGF signaling pathway have been claimed to be the ultimate cause of some teratogenically induced CP [Abbott and Pratt, 1987;Bryant et al, 2001;Abbott et al, 2005].…”