Studies on the roles of gonadotropin releasing hormone : Involvement in the modulation of mitogenic responses

Abstract

GnRH-Mediated Modulation of Cellular Proliferation in Pituitary and Extra-Pituitary Tissues

Abstract and Synopsis Introduction This thesis explores the emerging role of gonadotropin-releasing hormone (GnRH) in modulating proliferative responses in pituitary and extra-pituitary tissues expressing GnRH receptors. GnRH is a hypothalamic decapeptide secreted in a pulsatile manner, binding to high-affinity receptors on pituitary gonadotropes to stimulate the release of LH and FSH, which regulate gonadal development and steroidogenesis.

While the pituitary is the primary site of GnRH action, receptors have also been identified in extrapituitary tissues such as placenta, brain, breast, ovary, testis, activated lymphocytes, and various tumors. Synthetic GnRH analogues have shown antiproliferative effects in ovarian, breast, endometrial, and prostate cancers, though the precise role of GnRH receptors in tumor biology remains unclear.

Pituitary Gonadotrope Studies Objectives:

Assess the effect of GnRH signal pattern on proliferation in aT3-1 cells.

Study GnRH receptor expression under different signal patterns using RT-PCR and immunofluorescence.

Monitor c-fos reporter activity in response to GnRH stimulation.

Extend findings to primary rat pituitary cultures.

Key Findings:

Pulsatile GnRH stimulation increased DNA synthesis two-fold, while continuous treatment had no effect.

Pulsatile GnRH promoted G0/G1-S phase transition in quiescent cells.

Continuous GnRH reduced receptor mRNA levels, whereas pulsatile stimulation enhanced receptor surface presentation.

Both continuous and pulsatile GnRH increased c-fos activity, but continuous treatment led to excessive activation, potentially abrogating long-term mitogenic signaling.

In primary cultures, pulsatile GnRH increased proliferation of LH-bearing cells, implicating GnRH in pituitary homeostasis.

Cancer Cell Line Studies Objectives:

Confirm GnRH receptor presence in T47D breast cancer cells.

Study effects of GnRH analogues on proliferation.

Measure cAMP accumulation and modulation by GnRH analogues.

Test effects on EGF-stimulated c-fos reporter activity.

Key Findings:

Receptors confirmed in T47D and OVCAR-3 cells by RT-PCR, Western blot, and FACS.

GnRH agonist (Buserelin) and antagonist (Cetrorelix) inhibited proliferation in T47D cells.

Cetrorelix inhibited forskolin-induced cAMP accumulation via Gi/o coupling, distinct from pituitary Gq/11 coupling.

GnRH analogues did not affect EGF-induced c-fos activity in T47D or MCF-7 cells, contradicting earlier reports of cross-talk with growth factor pathways.

Additional Work

Development of GnRH-specific monoclonal antibody (FID3C5) to study biologically active GnRH structure.

Validation of anti-receptor monoclonal antibody (FIG4) for identifying surface receptor expression.

Conclusions Pulsatile GnRH stimulation is essential for mitogenic responses in pituitary gonadotropes.

GnRH receptor regulation depends on signal pattern, with pulsatility maintaining receptor availability.

GnRH analogues exert direct antiproliferative effects on cancer cells, mediated by distinct intracellular pathways compared to pituitary cells.

Findings highlight GnRH’s dual role in pituitary homeostasis and tumor biology, with implications for therapeutic applications.