Cloning of Smad2, Smad3, Smad4, and Smad7 from the goldfish pituitary and evidence for their involvement in activin regulation of goldfish FSHbeta promoter activity.

Follicle-stimulating hormone (FSH), a glycoprotein consisting of an alpha subunit and a unique beta subunit, is essential for gonadal development and function in vertebrates including teleosts. FSH is regulated by a variety of neuroendocrine and endocrine factors, and its biosynthesis is primarily determined by the expression of the beta subunit. Although the regulation of FSH biosynthesis has been well documented in mammals, the molecular mechanisms underlying the regulation are poorly understood. Our previous studies demonstrated that activin stimulated goldfish FSHbeta expression in the primary pituitary cell culture and enhanced its promoter activity in the mouse gonadotrope cell line LbetaT-2 cells. However, little is known about the signal transduction pathway involved in the transcriptional activation of this gene by activin. To assess the involvement of intracellular signaling protein Smads in regulating goldfish FSHbeta promoter, we first cloned full-length cDNAs for goldfish Smad2, Smad3, Smad4, and Smad7 from the pituitary. All Smads cloned show high sequence conservation with their mammalian counterparts. The spatial expression of these Smads overlapped with that of activin subunits and its receptors in various tissues examined. In addition, we demonstrated that activin induced Smad3 and Smad7 expression, but not Smad2 and Smad4. Co-transfection of Smad2 or Smad3 cDNA into the LbetaT-2 cells with the reporter construct of goldfish FSHbeta promoter significantly enhanced basal and activin-stimulated reporter (SEAP, secreted alkaline phosphatase) expression, while Smad7 completely blocked basal and Smad2/3-stimulated FSHbeta activity. Interestingly, the effect of Smad3 was much higher than that of Smad2, suggesting that Smad3 is likely the principal signal transducing molecule involved in activin stimulation of FSHbeta expression in the goldfish. This work lays a foundation for further analysis of goldfish FSHbeta promoter for the cis-regulatory elements involved in activin signaling.