Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat liver epithelial cells. Evidence for protein kinase C-dependent and -independent pathways.

H S Earp,J R Hepler,L A Petch,A Miller,A R Berry,J Harris,V W Raymond,B K Mccune,L W Lee,J W Grisham

doi:10.1016/s0021-9258(18)68324-3

Abstract

Epidermal growth factor (EGF) stimulated the rapid accumulation of inositol trisphosphate in WB cells, a continuous line of rat hepatic epithelial cells. Since we previously had shown that EGF stimulates EGF receptor synthesis in these cells, we tested whether hormones that stimulate PtdIns(4,5)P2 hydrolysis would increase EGF receptor protein synthesis and mRNA levels. Epinephrine, angiotensin II, and [Arg8]vasopressin activate phospholipase C in WB cells as evidenced by the accumulation of the inositol phosphates, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. A 3-4-h treatment with each hormone also increased the rate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [35S]methionine-labeled cells. Northern blot analyses of WB cell EGF receptor mRNA levels revealed that agents linked to the phosphoinositide signaling system increased receptor mRNA content within 1-2 h. A maximal increase of 3-7-fold was observed after a 3-h exposure to EGF and hormones. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C also stimulated EGF receptor synthesis. Pretreatment of WB cells for 18 h with high concentrations of TPA "down-regulated" protein kinase C and blocked TPA-directed EGF receptor mRNA synthesis. In contrast, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Epinephrine-induced increases in EGF receptor mRNA were reduced from 4- to 2-fold. Similarly, 18 h TPA pretreatment abolished the effect of TPA on EGF receptor protein synthesis but did not affect EGF-dependent EGF receptor protein synthesis. The 18-h TPA pretreatment diminished by 30-50% the induction of receptor protein synthesis by epinephrine or angiotensin II. We conclude that in WB cells EGF receptor synthesis can be regulated by EGF and other hormones that stimulate PtdIns(4,5)P2 hydrolysis. In these cells, EGF receptor synthesis appears to be regulated by several mechanism: one pathway is dependent upon EGF receptor activation and can operate independently of protein kinase C activation; another pathway is correlated with PtdIns(4,5)P2 hydrolysis and is dependent, at least in part, upon protein kinase C activation.

Highlights

From the Lineberger Cancer Research Center “CellBiology and Chemical Carcinogenesis Programs, ‘Program inNeurobiology, the Departments of *Medicine, ‘Pharmacology,and hPathology, UniversityofNorth Carolina at Chapel Hill, Chapel Hill, North Carolina 27514
A 3-4-h treatment with each hormone increased therate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipi- Stimulation of cells by mitogenic peptides involves a comtation of EGF receptor from [36S]methionine-labeled plex series of events that begin within the first minute of cells
Increased EGF receptor protein synthesis is due at WB cells for 18 h with high concentrations of TPA “down-regulated” protein kinase C and blocked TPAdirected EGF receptor mRNA synthesis

Summary

RESULTS

The addition of purified moEuGseF t o WB cells prelabeled with [3H]inosit~lresulted in a time- and concentration-dependenthydrolysis of PtdIns[4,5]P2 as determined by the accumulation of inositolphosphates(InsP).TheEGF-induced increase in InsP levels was readily measurable within. The protein kinase C agonist TPA, an agent that does not stimulate InsP3 generation in WB cells, increased EGF receptor synthesis. Represents thecommon pathway by which all of these agents stimulated EGF receptor synthesiFso. llowing long term TPA treatment, the medium was changed and cells were treated withEGF,freshTPA,epinephrine,orangiotensin. The peak densitometric area for the control sample from each experiment was divided into thepeak area for each of the treated samples for that experiment This is reported in this table as the-fold change in immunoprecipitable EGF receptor for that treatment. Fresh TPA no longer increased receptor synthesis (Fig. 6, Table 11).Second, direct assay of protein kinase C in cytosol anddetergentextractedparticulatefractions prepared from WB cells was performed using an N-bromosuccinimide fragment of lysine-rich histone asasubstrate [38]. In control WB cells, a 15min exposure to 100 nM TPA completely blocked EGF- and hormone-dependent accumulation of InsPs (Fig. 7). Similar results were obtained with EGF and epinephrine in two other experiments

Increase above control

DISCUSSION

MultiPpalethways for Regulation of EGF Receptor Synthesis