Reducing betaglycan expression by RNA interference (RNAi) attenuates inhibin bioactivity in LbetaT2 gonadotropes.
Betaglycan is an inhibin-binding protein co-receptor, the forced expression of which confers inhibin responsiveness on cells previously non-responsive to inhibin. The present study determines whether removal of betaglycan expression in otherwise inhibin-responsive cells will render the cells insensitive to inhibin. Small interfering RNAs (siRNAs) designed to the betaglycan gene were transfected into LbetaT2 gonadotrope cells to 'knock-down' betaglycan expression. To control for non-specific effects, siRNAs corresponding to an unrelated sequence (BF-1) were used. Two activin-responsive promoter constructs were used to assess inhibin bioactivity; an ovine FSHbeta promoter (oFSHbeta-lux), and a construct containing three copies of the activin-responsive sequence from the GnRHR promoter (3XpGRAS-PRL-lux). Activin stimulated the activity of both promoters 5-8-fold. Inhibin suppressed these activin-stimulated promoter activities by 52+/-11% and 51+/-7%, respectively. Similar inhibin suppression was also seen for cells co-transfected with the control BF-1 siRNAs. In contrast, inhibin's ability to suppress activin-stimulated activity was significantly reduced (33+/-3%, p<0.005 and 24+/-4%, p<0.045, respectively) in cells co-transfected with betaglycan siRNAs. These results demonstrated that endocrine effects of inhibin as a negative feedback controller of FSH production in gonadotropes are dependent on betaglycan expression.
Escalona
Ruth M
RM
Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia.
Stenvers
Kaye L
KL
Farnworth
Paul G
PG
Findlay
Jock K
JK
Ooi
Guck T
GT
D000818:Animals; D055785:Gene Knockdown Techniques; D052681:Gonadotrophs / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D007265:Inhibins / Q000378:metabolism*; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D011509:Proteoglycans / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D011509:Proteoglycans; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; C071736:betaglycan; D007265:Inhibins