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Abstract
Transforming growth factor beta 1 (TGF beta 1) 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 microM 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 synthesis. We investigated first, the cellular uptake, the stability, and the intracellular distribution of 32P-labeled TGF beta 1 AON and SON; and second, the effects of both oligonucleotides on BAC specific functions. We have demonstrated that in BAC, the TGF beta 1 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 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 declined. 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 beta 1 SON represented only 1.2%. Moreover, AON was present in the cytoplasmic and nuclear fractions, and it was hybridized in both compartments. However, TGF beta 1 SON was present mainly in the cytoplasmic fraction where it was not hybridized. Neither TGF beta 1 AON nor SON modified TGF beta 1 mRNA levels; however, TGF beta 1 AON, but not SON, caused 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-fold increase of the mRNA levels of both cytochrome P450 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase. Neither TGF beta 1 SON nor a scrambled oligonucleotide containing the same number of G nucleotides as TGF beta 1 AON had any effect on these parameters. Thus, these studies demonstrate that TGF beta 1 has an autocrine inhibitory effect on BAC differentiated functions, an effect that can be overcome by TGF beta 1 AON.
Highlights
Consists of related disulfide-linked homodimers that have multifunctional regulatory activities in many cell types and are expressed in many normal and malignant tissues [1,2,3]
Effects of Transforming growth factor 1 (TGF1) AON and SON on bovine fasciculata adrenal cells (BAC) transforming growth factor  (TGF)1 mRNA and Protein Content—To assess whether the oligonucleotides were able to modify the transcription and/or the translation of TGF1, we investigated their effects on TGF1 mRNA by Northern blot and on cellular TGF1 protein content by immunocytochemistry
Effects of TGF1 AON and SON on BAC Functions—As described in the Introduction, exogenous TGF1 decreases the steroidogenic responsiveness of BAC; we investigated the effects of both oligonucleotides, TGF1 AON and SON, on the steroidogenic responsiveness to AngII of control and cyclosporine treated cells (Fig. 9)
Summary
TGF, transforming growth factor ; consists of related disulfide-linked homodimers that have multifunctional regulatory activities in many cell types and are expressed in many normal and malignant tissues [1,2,3]. In bovine fasciculata adrenal cells (BAC), the expression and the maintenance of specific differentiated functions are regulated by corticotropin (ACTH) and angiotensin-II (AngII), the two main hormones that control steroidogenesis, and by growth factors, which have been shown to have pleiotropic effects in addition to their mitogenic action. BAC, bovine adrenal fasciculata cell(s); ACTH, adrenocorticotropin hormone; AngII, angiotensin-II; 3 HSD, 3-hydroxysteroid dehydrogenase; AON, antisense oligonucleotide; SON, sense oligonucleotide; SCR, scrambled oligonucleotide; PBS, phosphate-buffered saline; PIPES, 1,4piperazinediethanesulfonic acid. TGF1 mRNA level, whereas AngII increases TGF1 mRNA and protein levels.2 All these data suggest that TGF1 local production could play an autocrine role on BAC differentiated functions. In the present study, using a TGF1 antisense oligodeoxynucleotide complementary to a sequence that includes the translation start site of the human TGF1 mRNA, we have inhibited TGF1 synthesis in BAC and demonstrated an autocrine role for TGF1 on BAC differentiated functions
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