REPRESSION OF TRANSFORMING GROWTH-FACTOR BETA-1 PROTEIN BY ANTISENSE OLIGONUCLEOTIDE-INDUCED INCREASE OF ADRENAL-CELL DIFFERENTIATED FUNCTIONS

Transforming growth factor beta 1 (TGF beta 1) is a potent inhibitor o f several differentiated functions in bovine adrenal fasciculata cells (BAC). In addition, these cells express and secrete this factor. To d etermine whether this peptide plays an autocrine role in BAC, cells we re transfected with 10 mu M unmodified sense (SON) or antisense (AON) oligonucleotide complementary to the translation initiation region of the TGF beta 1 mRNA in an attempt to inhibit TGF beta 1 protein synthe sis. We investigated first, the cellular uptake, the stability, and th e intracellular distribution of P-32-labeled TGF beta 1 AON and SON; a nd second, the effects of both oligonucleotides on BAC specific functi ons. We have demonstrated that in BAC, the TGF beta 1 AON uptake reach ed a plateau after 8 h of transfection (16% of the radioactivity added ) and remained fairly constant for at least 24 h. In contrast, the upt ake of TGF beta 1 SON reached a plateau after 2 h of transfection (8% of the radioactivity added), remained stable for only 3 h, and then de clined. After 8 h of transfection, followed by 44 h of culture without oligonucleotides, the intracellular level of TGF beta 1 AON was still high with about 8% of the radioactivity added, whereas that of TGF be ta 1 SON represented only 1.2%. Moreover, AON was present in the cytop lasmic and nuclear fractions, and it was hybridized in both compartmen ts. However, TGF beta 1 SON was present mainly in the cytoplasmic frac tion where it was not hybridized. Neither TGF beta 1 AON nor SON modif ied TGF beta 1 mRNA levels; however, TGF beta 1 AON, but not SON, caus ed the disappearance of TGF beta 1 immunoreactivity inside the cells. Finally, the steroidogenic responsiveness of BAC transfected with TGF beta 1 AON increased about 2-fold, and this was associated with a 2-fo ld increase of the mRNA levels of both cytochrome P450 17 alpha-hydrox ylase and 3 beta-hydroxysteroid dehydrogenase. Neither TGF beta 1 SON nor a scrambled oligonucleotide containing the same number of G nucleo tides as TGF beta 1 AON had any effect on these parameters Thus, these studies demonstrate that TGF beta 1 has an autocrine inhibitory effec t on BAC differentiated functions, an effect that can be overcome by T GF beta 1 AON.