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., BECÚ-VILLALOBOS, D.

Miami

Simposio; The Miami Nature Biotechnology Winter Symposium; 2006

University of Miami Miller School of Medicine

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., Becú-Villalobos, D. Instituto de Biología y Medicina Experimental. CONICET. V. Obligado 2490. Buenos Aires (1428), Argentina. * cristina@dna.uba.ar   INTRODUCTION: Prolactinomas constitute 30% on brain tumors. They are generally benign and can be effectively treated with dopaminergic agents. Only 15% of these may become resistant to classical pharmacological therapy and require extirpation. Furthermore, prolactinomas lack metastatic potential. 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. In the present work we wished to study the participation of basic Fibroblast Growth Factor (FGF2) in these tumors. FGF2 exerts its mitogenic action on a wide variety of cells and participates in angiogenic processes. It si is present in endocrine pituitary cells and upregulated by estrogen 2.   METHODS: Pituitary cells were cultured as previously described 3, and the effects of FGF2 and VEGF on proliferation (MTS assay and 3H thymidine incorporation), prolactin (RIA) and ERK phosphorylation (Western blot) were measured. Human umbilical vein cord cells (HUVEC) were cultured as previously described 1 and the proliferative effect of conditioned media (CM) from pituitary cultures from KO and wildtype (WT) cells was tested. Specific antisera to FGF2 and VEGF were tested to abrogate the proliferative effect of CM on HUVECs. FGF2 was determined by ELISA in pituitary homogenates from KO and WT female mice. Pituitary FGF2 Receptor (FGFR1) expression was determined by Western blot and immunohistochemistry.   RESULTS: We sought to determine whether FGF2 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 FGF2, 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 in either genotype. Increased FGF-2 action on proliferation in KO pituitary cells correlated with enhanced FGFR1 expression in KO pituitaries as determined by Western blot and immohistochemistry. FGF-2 and VEGF increased prolactin secretion to the same extent in both genotypes. We observed that CM from pituitary cells of both genotypes enhanced HUVECs proliferation, and an antibody against FGF2 blocked the action of the CM from KO mice to a similar extent in both genotypes (KO: 48+12%, WT 35+13 % reduction, NS).This would indicate that FGF-2 was present in CM from KO and WT cells. Finally we determined FGF2 in pituitary homogenates and found that the concentration of this growth factor was decreased in KO cells (915 + 152 vs. 2909 + 167 pg/ug protein, KO vs WT, p< 0.01).   DISCUSSION: The unexpected finding of decreased FGF2 in these tumors may be associated to the slow pace of pituitary tumor growth, the benign course of tumors and the rarity of metastases observed in animal and human prolactinomas. Present and previous1 results suggest that VEGF and not FGF2 affects pituitary angiogenesis and peliosis in prolactinomas of the D2R KO mice. Decreased pituitary FGF2 would be consistent with low estrogen levels found in female D2R KO and is probably related to the increased expression of FGFR1, which enhances the responses to exogenous FGF2. Correct therapeutic strategies will result from the knowledge of differential expression of angiogenic factors in each type of tumor.   REFERENCES    1.   Cristina,C., Diaz-Torga,G., Baldi,A., Gongora,A., Rubinstein,M., Low,M.J., and Becu-Villalobos,D. (2005) Endocrinology. 146, 2952-2962 .    2.   Heaney,A.P., Horwitz,G.A., Wang,Z., Singson,R., and Melmed,S. (1999) Nature Medicine 5, 1317-1321 .    3.   Diaz-Torga,G., Feierstein,C., Libertun,C., Gelman,D., Kelly,M.A., Low,M.J., Rubinstein,M., and Becu-Villalobos,D. (2002) Endocrinology. 143, 1270-1279.