THE EFFECTS OF VARIOUS KINASE AND PHOSPHATASE INHIBITORS ON THE TRANSMISSION OF THE PROLACTIN AND EXTRACELLULAR-MATRIX SIGNALS TO RABBIT ALPHA-S-1-CASEIN AND TRANSFERRIN GENES

In all species, milk protein genes are specifically expressed in the m ammary gland under the control of lactogenic hormones and extracellula r matrix. In rabbit, casein gene expression is induced by prolactin al one and this induction is amplified by extracellular matrix. Transferr in gene expression is induced by extracellular matrix in the absence o f hormones. The transduction mechanisms of prolactin and extracellular matrix to milk protein genes is only partly known. The present study has been undertaken to determine if protein kinases and phosphatases a re involved in these mechanisms. Rabbit primary mammary cells were cul tured in three different conditions (i) directly on floating collagen I, (ii) on plastic after a trypsinization to remove endogenous extrace llular matrix, and (iii) on boating collagen I after a trypsinization to restore a functional extracellular matrix. In these culture conditi ons, prolactin and several protein kinase and phosphatase inhibitors w ere added to the medium. The expression of alpha S-1,-casein and trans ferrin genes was evaluated using Northern blotting analysis. In cells cultured directly on collagen 1, staurosporine, quercetin and 6-dimeth ylaminopurine strongly inhibited prolactin action of alpha S-1,-casein gene whereas herbimycin A was only partly inhibitory. An erbstatin an alogue, tyrosine phosphate, 1(5 isoquinolylsulphonyl) 2-methylpiperazi ne and GF 109 203 X did not alter prolactin action. The inhibitors whi ch inhibited prolactin action when cells were directly cult;red on col lagen I were also those which prevented the induction of alpha S-1,-ca sein gene expression when cells were cultured on plastic in the absenc e of extracellular matrix. The induction of transferrin gene by the ex tracellular matrix was inhibited slightly by quercetin. Okadaic acid, phenylarsine oxide and sodium pervanadate which inhibit Ser/Thr and Ty r phosphatase inhibitors were unable to mimic prolactin action on alph a S-1,-casein gene expression. On the contrary, these inhibitors preve nted prolactin action. These data suggest that a cascade including pro tein kinases and phosphatases for Ser/Thr and Tyr phosphate is involve d in the transduction of the prolactin message from its receptor to ca sein genes. The signal delivered to the mammary cells by the extracell ular matrix is quite different, possibly involving another cascade of protein kinases.