PARENT SESSION
PLATFORM SESSION 11: MOLECULAR MECHANISMS OF HORMONE ACTION
Chair: Nilson, John¹, ¹
Co-chair: Horvat, Regina¹, ¹
Grand Ballroom I-IV
2:00 PM-4:00 PM

(225) FSH REGULATES Akt AND FKHR IN GRANULOSA CELLS.

Cunningham, Melissa¹, Hammond, James¹, ¹ Pennsylvania State University College of Medicine, Hershey, PA

ABSTRACT- While the differentiative effects of FSH in granulosa cells are accomplished largely through a cAMP/PKA-dependent pathway, the signal transduction pathways that mediate the growth and survival effects of FSH on granulosa cells have not yet been established. Recent data suggest that PKA does not account for all of the intracellular actions of FSH. Apparently, FSH can stimulate the phosphorylation of Akt/PKB in a PI 3-kinase-dependent manner. Akt is known to promote cell survival in part via the phosphorylation and translocation of the transcription factor FKHR out of the nucleus, effectively inhibiting its role as a transactivator of apoptosis-inducing genes. However, these events have not been demonstrated in the ovary. We hypothesized that Akt activation and FKHR translocation is responsible for the survival effects of FSH on granulosa cells. We used serum-free cultures of porcine granulosa cells to investigate the effects of FSH on Akt and FKHR. By Western blot analysis, we found that FSH could stimulate the phosphorylation of Akt by 1.5h in addition to a more robust phosphorylation at 6h and 24h. Next we demonstrated that two FKHR family members, the 70kDa FKHR and 100kDa FKHRL1, are expressed in our cell system. We also examined expression of FKHR message by RT-PCR. We found no clear difference in the amount of FKHR between untreated and FSH-treated cells over 12h at either the message level or protein level in whole cell lysates. We next investigated the possibility that intracellular translocation of FKHR is an FSH-regulated step. In contrast to whole cell lysates, FKHR protein was decreased significantly in nuclear extracts 1h after FSH stimulation. There was a return to the nucleus by 6h. Changes in cytoplasmic levels of FKHR and FKHRL1 were more complex and may reflect either translocation and/or possibly targeted degradation. These data suggest that FSH can affect FKHR translocation out of the nucleus, perhaps mediated by FSH-activation of Akt. This is consistent with our hypothesis that FSH-regulated FKHR plays a role in granulosa cell survival. This work was supported by HD24565.

KEY WORDS: FKHR, Akt, Granulosa cell