title: Antisense oligonucleotide targeting the transforming growth factor beta1 increases expression of specific genes and functions of Leydig cells.
abstract: Transforming growth factor beta1 (TGFbeta1) 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), express TGFbeta1 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 TGFbeta1 plays an autocrine/paracrine role on these steroidogenic cells, we attempted to inhibit TGFbeta1 protein synthesis by transfecting LC, SC and LC+SC for 24 h with 10 microM of an unmodified antisense oligonucleotide (AON) complementary to the translation-initiation region of the TGFbeta1 mRNA and, as controls, with the corresponding sense (SON) or scrambled (SCRON) oligonucleotides. First, we determined at which level, transcriptional or translational, the TGFbeta1 AON acts. Neither TGFbeta1 AON, SON nor SCRON modified TGFbeta1 mRNA levels in LC, SC or LC+SC. However, TGFbeta1 AON caused the disappearance of TGFbeta1 immunoreactivity in both cell types. In addition, TGFbeta1 AON reduced the attachment of TGFbeta1 mRNA in ribosomal and polyribosomal fractions. Then, we showed that the decrease of the TGFbeta1 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, TGFbeta1 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 TGFbeta1 AON effects were always more pronounced on LC+SC than on LC. The present findings show that TGFbeta1 has an autocrine/paracrine inhibitory effect on cultured porcine Leydig cells, an effect that can be overcome by TGFbeta1 AON.
authors.affiliation: INSERM-INRA U 418 and IFREL d'Endocrinologie, Hôpital Debrousse, Lyon, France.
authors.forename: C
authors.initials: C
authors.lastname: Le Roy
authors.forename: P
authors.initials: P
authors.lastname: Leduque
authors.forename: J
authors.initials: J
authors.lastname: Yuan Li
authors.forename: J M
authors.initials: JM
authors.lastname: Saez
authors.forename: D
authors.initials: D
authors.lastname: Langlois
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000235:genetics*; D007985:Leydig Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000201:enzymology / Q000378:metabolism; D012708:Sertoli Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics; D013552:Swine; D013739:Testosterone / Q000096:biosynthesis; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism
chemical_list: D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D013739:Testosterone; D013254:Steroid 17-alpha-Hydroxylase