VEGF AND FGF-2 PARTICIPATION IN PITUITARY TUMORS OF DOPAMINERGIC D2 RECEPTOR (D2R) KNOCKOUT FEMALE MIC

CRISTINA,C., GÓNGORA,A., BALDI,A. DÍAZ-TORGA,G., LOW, M.J., BECÚ-VILLALOBOS, D

Miami U.S.A.

Simposio; The 2005 Miami Nature Biotechnology Winter Symposium "ANGIOGENESIS IN CANCER AND VASCULAR DISEASE"; 2006

SHORT REPORT (extended abstract) FOR  POSTER  PRESENTATION   VEGF AND FGF-2 PARTICIPATION IN PITUITARY TUMORS OF  DOPAMINERGIC D2 RECEPTOR (D2R) KNOCKOUT FEMALE MICE Cristina,C., Góngora,A., Baldi,A.  Díaz-Torga,G., Low, M.J. and Becú-Villalobos, D. Instituto de Biología y Medicina Experimental. CONICET. V. Obligado 2490. Buenos Aires, Argentina. * cristina@dna.uba.ar   INTRODUCTION: Prolactinomas constitute 30% on brain tumors. They are generally benign and can be effectively treated with dopaminergic agents. But 15% of these may become resistant to classical pharmacological therapy, are invasive and aggressive, and require extirpation. An alternative target would be desired in these circumstances. The D2R knockout (KO) female mouse provides an interesting model to study resistant prolactinomas, and pituitary tumor genesis. Females develop chronic hyperprolactinemia  and  pituitary hyperplasia.  Recently, we have shown that female KO mice have an overexpression of  vascular endothelial growth factor (VEGF) in pituitary folliculoestellate cells, suggesting a paracrine action of VEGF on pituitary endothelial cells (1) Numerous cytokines and growth factors modulate pituitary proliferation. Among them, the type-2 Fibroblast Growth Factor (FGF2), exerts its mitogenic action on a wide variety of cells, including endothelial and fibroblasts, and furthermore participates in angiogenic processes. We therefore studied the participation of FGF2 in comparison with VEGF in pituitary hyperplasia in KO female mice.   METHODS: Pituitary cells were cultured as previously described (2), and the effect of FGF-2 and VEGF on proliferation (MTS assay and 3H thymidine incorporation), prolactin (RIA) and ERK phosphorylation (Western blot) was measured. Human umbilical vein cord cells (HUVEC) were cultured as previously described (1) and the proliferative effect of conditioned media from pituitary cultures from KO and WT cells was tested. Specific antisera to FGF2 and VEGF were tested to abrogate the proliferative effect of CM on HUVECs.   RESULTS: We sought to determine whether FGF-2 had any differential action on pituitary cell proliferation or prolactin release between both genotypes.  In 3H-Thymidine captation assay 10 ng/ml FGF-2 induced the proliferation of cells from both genotypes (Percent increase: 138.5 + 4.5 and 128.1 +6.1 % for KO and WT, respectively), similar results were obtained using the MTS assay. When we studied the mechanism of signal transduction involved in the proliferative action of FGF-2, KO cells showed a higher phosphorylation of ERKs by FGF-2 than WT cells (at 5 min. 236+ 30% and 158+ 8%, respectively, p< 0.05). On the other hand, VEGF did not increase pituitary cell proliferation or ERK phosphorylation. Increased FGF-2 action on proliferation in KO pituitary cells correlates with enhanced FGF receptor expression in KO pituitaries as determined by Western blot. FGF-2 and VEGF increased prolactin secretion to the same extent in both genotypes. We observed that the conditioned media (CM) from pituitary cells of both genotypes cultured in vitro enhanced HUVECs growth, and an antibody against FGF2 blocked  the action of the CM from KO mice (48% reduction) while in the WT a 30% reduction was observed. This would indicate that FGF-2 was present in CM from KO cells, as we have previously determined for VEGF, and can induce the proliferation of endothelial cells.   DISCUSSION: These results suggest that while VEGF predominantly affects pituitary angiogenesis in prolactinomas of the D2R KO mice,  FGF2 may be involved not only in endothelial proliferation but also in  pituitary cell proliferation and secretion in our experimental model.       Reference List     1. Cristina C, Diaz-Torga G, Baldi A, Gongora A, Rubinstein M, Low MJ, Becu-Villalobos D 2005 Increased pituitary vascular endothelial growth factor-A in dopaminergic D2 receptor knockout female mice. Endocrinology   2. Diaz-Torga G, Feierstein C, Libertun C, Gelman D, Kelly MA, Low MJ, Rubinstein M, Becu-Villalobos D 2002 Disruption of the D2 dopamine receptor alters GH and IGF-I secretion and causes dwarfism in male mice. Endocrinology 143:1270-1279