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 Leyding cells (LC), as well as Sertoli cells (SC), express TGF beta 1 mRNA and secrete this peptide, suggesting that it might play an autocrine role. Moreover, many studies have suggested a possible paracrine regulation of LC by SC-secreted factors. To assess whether TGF beta 1 plays an autocrine/paracrine role on these steroidogenic cells, we attempted to inhibit TGF beta 1 protein synthesis by transfecting LC, SC and LC+SC for 24 h with 10 mu M of an unmodified antisense oligonucleotide (AON) complementary to the translation-initiation region of the TGF beta 1 mRNA and, as controls, with the corresponding sense (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 levels in LC, SC or LC+SC. However, TGF beta 1 AON caused the disappearance of TGF beta 1 immunoreactivity in both cell types. In addition, TGF beta 1 AON reduced the attachment of TGF beta 1 mRNA in ribosomal and polyribosomal fractions. Then, we showed that the decrease of the TGF beta 1 protein induced by the AON results in an increase of the expression of LC specific genes and of LC steroidogenic capacity. In LC and LC+SC, TGF beta 1 AON increased the mRNA levels of both LH/hCG receptor (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 2.2-fold, respectively) when compared with untransfected cells. The TGF beta 1 AON effects were always more pronounced on LC+SC than on LC. The present findings show that TGF beta 1 has an autocrine/paracrine inhibitory effect on cultured porcine Leydig cells, an effect that can be overcome by TGF beta 1 AON.