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

Epidermal growth factor (EGF) receptor levels are known to play a central role in density dependent growth regulation of normal rat kidney (NRK) fibroblasts. Here we show that EGF receptor expression is strongly decreased when NRK cells are cultured under anchorage independent conditions, and that expression is returned to original levels upon cell readherence. Agents that stimulate anchorage independent growth (AIG) of NRK cells in the presence of EGF are shown to upregulate both EGF receptor promoter activity and (125)I-EGF binding capacity. These data show that two aspects of phenotypic transformation of NRK cells, namely density arrest and AIG, can both directly be correlated to EGF receptor levels.

Phenotypic transformation of normal rat kidney fibroblasts by endothelin-1. Different mode of action from lysophosphatidic acid, bradykinin, and prostaglandin f2alpha.

In the present study, we compared the effects of endothelin (ET)-1 on cell proliferation and second messenger induction in normal rat kidney (NRK) fibroblasts, with those of other activators of G-protein-coupled receptors such as prostaglandin (PG)-F2alpha, bradykinin (BK), and lysophosphatidic acid (LPA). LPA is mitogenic by itself, while the other factors require the presence of EGF. In density-arrested NRK cells, ET-1 and LPA induce phenotypic transformation rapidly, with similar kinetics as retinoic acid (RA) and transforming growth factor (TGF)-beta, while BK and PGF2alpha only do so with delayed kinetics. ET-1 and PGF2alpha are strong inducers of anchorage-independent growth, with a similar level of induction as TGFbeta, in contrast to LPA and BK. When investigating the second messenger generation, we found that ET-1 is the strongest activator of arachidonic acid release and phosphatidylinositol diphosphate hydrolysis. Only in the case of ET-1 the cell depolarization is not reversible upon removal of the factor. Similarly, only the ET-1-induced transient enhancement of intracellular calcium concentration is paralleled by both homologous and heterologous desensitization. In conclusion, these data show that ET-1 is a potent inducer of second messengers and phenotypic transformation in NRK cells, with characteristics that clearly differ from those of other activators of G-protein-coupled receptors, most likely as a result of prolonged receptor activation.

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

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.

Effect of bradykinin on loss of density-dependent growth inhibition of normal rat kidney cells.

Normal rat kidney fibroblasts, density-arrested in the presence of epidermal growth factor (EGF), can be restimulated to proliferate in a synchronous way and acquire a transformed phenotype following treatment with additional growth factors like retinoic acid (RA) and transforming growth factor (TGF)-beta. It was found that bradykinin has a strong inhibitory effect on growth stimulation induced by these factors, an effect which cannot be mimicked by PGF2 alpha. The growth-inhibiting effect can be blocked by inhibitors of cyclo-oxygenase activity, indicating that the relevant second messenger is most likely a prostaglandin. Externally added PGJ2, at a concentration of 10 microM, can mimic the inhibitory effect of bradykinin on the loss of density-arrest induced by RA suggesting that PGJ2 is a possible candidate for being the bradykinin induced growth-inhibiting prostaglandin.