Transforming growth factor 1 (TGF1) is a potent inhibitor of several differentiated functions in bovine adrenal fasciculata cells (BAC). In addition, these cells express and secrete this factor. To determine whether this peptide plays an autocrine role in BAC, cells were transfected with 10 µM unmodified sense (SON) or antisense (AON) oligonucleotide complementary to the translation initiation region of the TGF1 mRNA in an attempt to inhibit TGF1 protein synthesis. We investigated first, the cellular uptake, the stability, and the intracellular distribution of P-labeled TGF1 AON and SON; and second, the effects of both oligonucleotides on BAC specific functions. We have demonstrated that in BAC, the TGF1 AON uptake reached a plateau after 8 h of transfection (16% of the radioactivity added) and remained fairly constant for at least 24 h. In contrast, the uptake of TGF1 SON reached a plateau after 2 h of transfection (8% of the radioactivity added), remained stable for only 3 h, and then declined. After 8 h of transfection, followed by 44 h of culture without oligonucleotides, the intracellular level of TGF1 AON was still high with about 8% of the radioactivity added, whereas that of TGF1 SON represented only 1.2%. Moreover, AON was present in the cytoplasmic and nuclear fractions, and it was hybridized in both compartments. However, TGF1 SON was present mainly in the cytoplasmic fraction where it was not hybridized. Neither TGF1 AON nor SON modified TGF1 mRNA levels; however, TGF1 AON, but not SON, caused the disappearance of TGF1 immunoreactivity inside the cells. Finally, the steroidogenic responsiveness of BAC transfected with TGF1 AON increased about 2-fold, and this was associated with a 2-fold increase of the mRNA levels of both cytochrome P450 17-hydroxylase and 3-hydroxysteroid dehydrogenase. Neither TGF1 SON nor a scrambled oligonucleotide containing the same number of G nucleotides as TGF1 AON had any effect on these parameters. Thus, these studies demonstrate that TGF1 has an autocrine inhibitory effect on BAC differentiated functions, an effect that can be overcome by TGF1 AON.

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