ANTISENSE OLIGONUCLEOTIDE TARGETING THE TRANSFORMING-GROWTH-FACTOR BETA-1 INCREASES EXPRESSION OF SPECIFIC GENES AND FUNCTIONS OF LEYDIG-CELLS

Transforming growth factor beta 1 (TGF beta 1) has been reported to be a potent inhibitor of differentiated functions of many steroidogenic cells. Porcine Leydig cells (LC), as well as Sertoli cells (SC), expre ss TGF beta 1 mRNA and secrete this peptide, suggesting that it might play an autocrine role. Moreover, many studies have suggested a possib le paracrine regulation of LC by SC-secreted factors. To assess whethe r TGF beta 1 plays an autocrine/paracrine role on these steroidogenic cells, we attempted to inhibit TGF beta 1 protein synthesis by transfe cting LC, SC and LC+SC for 24 h with 10 mu M of an unmodified antisens e oligonucleotide (AON) complementary to the translation-initiation re gion of the TGF beta 1 mRNA and, as controls, with the corresponding s ense (SON) or scrambled (SCRON) oligonucleotides. First, we determined at which level, transcriptional or translational, the TGF beta 1 AON acts. Neither TGF beta 1 AON, SON nor SCRON modified TGF beta 1 mRNA l evels in LC, SC or LC+SC. However, TGF beta 1 AON caused the disappear ance of TGF beta 1 immunoreactivity in both cell types. In addition, T GF beta 1 AON reduced the attachment of TGF beta 1 mRNA in ribosomal a nd polyribosomal fractions. Then, we showed that the decrease of the T GF beta 1 protein induced by the AON results in an increase of the exp ression of LC specific genes and of LC steroidogenic capacity. In LC a nd LC + SC, TGF beta 1 AON increased the mRNA levels of both LH/hCG re ceptor (1.9-fold and 3.5-fold, respectively) and P450 c17 (5-fold and 8-fold, respectively). This was associated with an enhancement of hCG- induced testosterone production by both LC and LC+SC (1.6-fold and 3-. 2-fold, respectively) when compared with untransfected cells. The TGF beta 1 AON effects were always more pronounced on LC+SC than on LC. Th e present findings show that TGF beta 1 has an autocrine/paracrine inh ibitory effect on cultured porcine Leydig cells, an effect that can be overcome by TGF beta 1 AON.