Date of Award

Summer 2001

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

Program/Concentration

Chemistry

Committee Director

Ke-Wen Dong

Committee Member

Patricia A. Pleban

Committee Member

Michael J. Solhaug

Call Number for Print

Special Collections LD4331.C45 W46 2001

Abstract

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, we studied the mechanisms of the PKA positive regulation of the hGnRH gene expression in the human placenta. Studies in JEG3 cells showed that through the PKA catalytic subunit a, human GnRH upstream promoter activity was stimulated by PKA signaling pathway in a cAMP dependent mechanism. The sequence between —202 (Afl II) and —554 (BamH I) base pair in the human GnRH upstream promoter region appeared to be responsible for the PKA positive regulation of the gene expression. Furthermore, Western blot analysis demonstrated the involvement of phospho-GREB in the PKA regulation of hGnRH gene expression in JEG-3 cells, and GREB-binding protein (CBP) could further enhance the PKA stimulatory effect on the hGnRH upstream promoter activities.

Transient transfection studies showed that 10 μM forskolin, an activator of adenylate cyclase, stimulated the hGnRH upstream promoter activities in JEG-3 cells in a dose dependent fashion. The cAMP analogue, eight-bromo-cAMP(8- CPT-cAMP) also increased hGnRH gene expression in a dose- and time - dependent fashion. The 8-CPT-cAMP stimulatory effect on hGnRH gene expression was abolished by 10 μM of H-89, an antagonist of PKA. The role of PKA catalytic subunits in hGnRH gene expression was also determined: The a subunit stimulated the hGnRH promoter activity by two fold, while the γ subunit did not significantly change the activity.

When the fragment between sequences -202 and -554 bp was deleted away, the PKA positive stimulatory effect disappeared, suggesting that both cis- and trans- elements were involved. Western blot results showed an increased amount of phospho-GREB protein after treatment with 8-CPT-cAMP or co-transfection with PKA catalytic subunit a compared to the control cells without any treatment. Furthermore, transient transfection studies demonstrated that GREB-binding protein (CBP) enhanced the PKA stimulatory effect on hGnRH upstream promoter activities, suggesting that CBP may be also involved in the PKA regulation of hGnRH gene expression.

While PKA signaling pathway has been involved in many placental functions, we have demonstrated, for the first time, the PKA positive regulation on hGnRH gene expression which should help to further elucidate the mechanism of local hGnRH functions in the placenta.

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DOI

10.25777/zhcd-ak25

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