Epidermal growth factor (EGF) receptor density controls mitogenic activation of normal rat kidney (NRK) cells by EGF

Abstract

Normal rat kidney (NRK) fibroblasts are immortalized cells that are strictly dependent on externally added growth factors for proliferation. When cultured in the presence of epidermal growth factor (EGF) as the only growth stimulating hormone, these cells have a normal phenotype and undergo density-dependent growth inhibition. It has been postulated that this density-arrest results from a decrease of EGF receptor levels below a threshold level which makes these cells unresponsive to stimulation by EGF. In the present study, we show that NRK cells, made quiescent by serum-deprivation at submaximum density, are mitogenically still responsive to EGF, but show enhanced mitogenic stimulation after 8 hr pre-treatment with either transforming growth factor beta (TGF beta) or retinoic acid (RA), while prostaglandin F2 alpha (PGF2 alpha) and bradykinin (BK) enhance the mitogenic stimulation by EGF only slightly under these conditions. Addition of TGF beta or RA results in an increase of both 125I-EGF-binding capacity and EGF receptor mRNA levels. Using flow cytometric analysis, we show that pre-treatment with TGF beta or RA increases the percentage of cells entering the cell cycle as a function of time. Furthermore, pre-treatment of the cells with TGF beta or RA increases the rate of mitogen-activated protein kinase (MAPK) phosphorylation by EGF. PGF2 alpha and BK also increase EGF receptor levels, but only with delayed kinetics. These results show that already in serum-deprived quiescent NRK cells, EGF receptor levels limit EGF-induced mitogenic stimulation. This observation provides further evidence for the regulating role of the EGF receptor in density-dependent growth control of NRK cells.

Epidermal growth factor receptor tyrosine kinase inhibition is not protective in PCK rats

“…Previous studies have shown that the mitogenic effect of EGF correlates with EGFR density [49]. The contrast in EGFR expression between the PCK rat and human ARPKD or murine models of human ARPKD remains unexplained.…”

Prostaglandin F2α induces unsynchronized intracellular calcium oscillations in monolayers of gap junctionally coupled NRK fibroblasts

“…Normal rat kidney (NRK) fibroblasts have been used widely as an in vitro model system for studying the mechanisms of density-dependent growth regulation and oncogenic cell transformation [25,26,50]. Previous studies have shown that the arachidonic acid metabolite prostaglandin F2a (PGF2a), which activates the Fprostanoid (FP)-subtype of prostanoid receptor [35], induces phosphoinositide turnover in NRK fibroblasts thereby increasing the intracellular calcium concentration in these cells [26].…”

Central role of epidermal growth factor (EGF) receptor density in anchorage‐independent growth of normal rat kidney cells

“…Optimal cycle numbers were determined as described [23]. Under these conditions competitive interference was absent and twofold changes in the abundance of PCR products were detected over a span of 4–5 cycles, making this method suitable to monitor EGF receptor mRNA expression [14].…”

“…When cultured in the presence of epidermal growth factor (EGF) as the only growth stimulating hormone, these immortalized cells have a normal phenotype and undergo DDGI. In the additional presence of modulating factors such as retinoic acid (RA) or transforming growth factor β (TGF‐β), which by themselves are not mitogenic for these cells, density arrested NRK cells become responsive again to the growth stimulatory effect of EGF, resulting in loss of DDGI [12–15]. It has been established that the number of EGF receptors in NRK cells decreases with increasing cell density.…”

“…It has been established that the number of EGF receptors in NRK cells decreases with increasing cell density. Therefore, we have postulated that EGF treated NRK fibroblasts become density arrested as a result of a reduction in EGF receptor levels below a critical level from which they can be released by addition of such factors as RA and TGF‐β that increase EGF receptor numbers [14, 15].…”