DIFFERENTIAL MODULATION OF IMMEDIATE EARLY GENES IN MAMMARY EPITHELIAL CELLS

Immediate early genes (IEGs) represent a class of proteins, with different functions, with fast kinetics of upand down-regulation [1]. In this work we have studied the differential modulation of some IEGs at trascriptional and post-trascriptional levels, in mammary epithelial cells of different origin. We have examined trascription factors of the Egr, Fos and Jun family as well as amphiregulin (AREG), one of the seven ligands for the EGFR tyrosine kinase receptor, which has a fundamental role during pubertal mammary growth [3]. Primary and immortalized mammary epithelial cells (murine, feline, bovine) were used to study how these IEGs are regulated. We have analized the kinetic of expression, the factors and the pathways that affect IEGs modulation both at the mRNA and protein levels. Expression of the IEGs showed different patterns of modulation depending on the gene being analyzed. The Egr family (Egr-1/2/3) and the c-Fos trascrition factor were similarly regulated at the mRNA level, with fast upregulation that reached a significant peak after 45-60 minutes of stimulation with growth medium, followed by rapid downmodulation. Egr-1 protein levels followed exactly mRNA modulation with significant fast up- and down-regulation after growth medium stimulation. On the other hand c-Fos protein expression levels lasted longer being significantly upregulated after one hour but then slowly decreascing after more than 4 hours. Egr-1 and c-Fos depended on the Erk-1/2 pathway activation to sustain their expression both at the mRNA and protein levels. All factors that affected Erk1/2 phosphorylation, like epidermal growth factor or phorbol-mysistate-acetate, were also able to upregulate Egr-1 and c-Fos. mRNA levels of the c-Jun trascription factor were signifantly upregulated by growth medium, but at lower levels the Egr-1 or c-Fos. On the other side c-Jun protein levels were highly incresased by growth medium and remained sustained for 4-6 hours. Inhibiting the Erk1/2, the PI3K or the JUN kinases did not modify c-Jun protein or mRNA levels. AREG, at the mRNA levels, was stimulated but with slower kinetics, reaching a significant peak after 120 minutes of stimulation. A partial reduction of AREG mRNA was observed with Erk1/2, but not PI3K or JUN kinases inhibition. AREG protein levels were superimposable with c-Jun and, similarly, were not altered after Erk1/2, PI3K or JUN kinases inhibition. In conclusion, although IEGs share the common characteristic of fast up- and down-regulation the kinetics, the factors that stimulate them and the pathways that regulate them are different and deserve careful examination in order to unravel mammary gland biology during growth and function [3].