Signal Transduction Mediating Gene Expression of SP1, LHβ-Subunit and GH in Response to GnRH or GHRH in the Postnatal and Fetal Porcine Anterior Pituitary in Vitro

Abstract

To clarify signal transduction pathways mediating putative gene expression of transcription factor SP1 (selective promoter factor 1 or specificity protein 1) by GnRH or GHRH porcine anterior pituitary monolayer cultures were exposed for various time periods to GnRH, GHRH, activators of adenylate cyclase (AC) or proteinkinase C (PKC), and mRNA levels of SP1, LHbeta-subunit, and GH were determined by multiplex RT-PCR. In many experiments LH and GH release were measured as well for comparison. Another approach was to inactivate AC, PKC, or proteinkinase A (PKA) by specific inhibitors, MDL, GFX, and H89, respectively. Postnatally (4 weeks) SP1 mRNA level was maximally increased by GnRH, GHRH and both by activation of AC or PKC after 2 h of exposure. Two-hour stimulation of SP1 mRNA levels by dbcAMP was totally blocked by H89, while this inhibitor not clearly blocked GHRH stimulated SP1 mRNA levels. Stimulation of LHbeta mRNA by GnRH was suppressed by inactivation of AC or of PKC but not by inactivation of PKA. Inactivation of AC or PKA but not of PKC inhibited GHRH induced GH mRNA. Already at day 50 of fetal life (and likewise day 80) SP1 mRNA levels were stimulated by GHRH or activation of AC, but not by GnRH or activation of PKC. The results are consistent with the notion that SP1 plays an important role 1) in conferring GnRH responsiveness to the LHbeta-subunit gene by mediating the actions of both AC and PKC and 2) in conferring GHRH responsiveness to the GH gene through activation of the AC probably PKA pathway. Furthermore, the data are in line with the view that the GHRH/AC/SP1/GH pathway develops earlier during fetal life than the GnRH/PKC/SP1/LHbeta pathway.