Nerve growth factor induces vascular endothelial growth factor expression in granulosa cells via a trkA receptor/mitogen-activated protein kinase-extracellularly regulated kinase 2-dependent pathway - PubMed (original) (raw)

Nerve growth factor induces vascular endothelial growth factor expression in granulosa cells via a trkA receptor/mitogen-activated protein kinase-extracellularly regulated kinase 2-dependent pathway

Marcela Julio-Pieper et al. J Clin Endocrinol Metab. 2009 Aug.

Abstract

Context: Acquisition of ovulatory competence by antral follicles requires development of an adequate vascular supply. Although it is well established that ovarian angiogenesis is cyclically regulated by vascular endothelial growth factor (VEGF), the factors controlling VEGF production by ovarian follicles remain largely unknown. Nerve growth factor (NGF) may be one of these factors, because NGF promotes angiogenesis and synthesis of angiogenic factors in other tissues and is produced by human granulosa cells (hGCs).

Objective: The aim of the study was to determine whether NGF influences the production of VEGF by hGCs and to identify a potential signaling pathway underlying this effect.

Design: We conducted a prospective experimental study.

Patients: hGCs were obtained from 41 women participating in the in vitro fertilization program of our institution.

Methods: Changes in VEGF mRNA after exposure to NGF were evaluated in cultured hGCs by PCR and real-time PCR. The effect of NGF on VEGF secretion was determined by ELISA. The involvement of trkA, the high affinity NGF receptor, was examined by inhibiting the receptor's tyrosine kinase activity with K252a. The contribution of an ERK1/ERK2-mediated signaling pathway was identified by detecting NGF-dependent phosphorylation of these proteins and by blocking their activity with the inhibitor U0126.

Results: NGF promotes VEGF production in cultured hGCs. Blockade of trkA receptor tyrosine kinase activity blocks this effect. NGF induces MAPK-ERK2 phosphorylation, and blockade of this signaling pathway prevents the NGF-induced increase in VEGF production.

Conclusions: NGF promotes ovarian angiogenesis by enhancing the synthesis and secretion of VEGF from hGCs via a trkA- and ERK2-dependent mechanism.

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Figures

Figure 1

Figure 1

NGF increases mRNA expression in hGCs. A, Ethidium bromide-stained gel showing the presence of VEGF mRNA transcripts encoding the VEGF isoforms 121 and 165 in cultured hGCs, as measured by RT-PCR. MM, Molecular markers; (+), positive control (ovarian cancer); (−), negative control (PCR mix without cDNA); C, hGCs cultured under basal conditions; N, hGCs cultured for 8 h in the presence of 50 ng/ml NGF. This gel is representative of seven independent experiments. B, Effect of NGF on VEGF121 mRNA levels measured by semiquantitative PCR after different times of treatment (2, 4, 8, and 18 h). Each column represents the mean ±

sem

of seven independent experiments, each assayed in duplicate. C, Effect of NGF on VEGF165 mRNA levels. *, P < 0.05; **, P < 0.01 vs. untreated controls. AU, Arbitrary units.

Figure 2

Figure 2

NGF increases VEGF mRNA content in hGCs via activation of trk receptors. A, Inhibitory effect of the tyrosine kinase receptor inhibitor K252a (100 n

m

) on NGF-induced increase in VEGF165 mRNA abundance. The cells were pretreated with the inhibitor for 30 min before adding NGF (50 ng/ml), and the mRNA levels were measured after 8 h of NGF treatment. The inhibitor was maintained in the culture medium during this time. *, P < 0.05 vs. untreated control group; ≠, P < 0.05 vs. NGF-treated group. Each column (expressed in arbitrary units) represents the mean ±

sem

of five independent observations analyzed in triplicate by real-time PCR. B, K252a also abolishes the increase in VEGF secretion elicited by NGF, as measured by ELISA. Each value represents the mean ±

sem

(expressed as percentage of the mean value observed in untreated controls) of five independent observations measured in triplicate. *, P < 0.05 vs. control group; ≠, P < 0.05 vs. NGF-treated group.

Figure 3

Figure 3

Increase in immunoreactive VEGF levels in cultured hGCs treated with NGF (50 ng/ml) for 24 h. Representative microphotographs from five experiments are shown. A, hGCs contain VEGF immunoreactive material in the absence of NGF treatment. B, NGF treatment increases the content of VEGF in hGCs. C, Immunoneutralization of NGF actions with rabbit polyclonal antibodies blocks the effect of NGF on VEGF immunoreactive levels. D, Blockade of trk receptors with K252a also prevents the effect of NGF on VEGF immunoreactive levels. E, hGCs incubated in the absence of primary VEGF antibodies (ICC negative control). Scale bars, 50 μm.

Figure 4

Figure 4

Semiquantitative analysis of VEGF protein levels detected by ICC in cultured hGCs. VEGF protein levels are increased by NGF (***, P < 0.0001), and this effect was blocked by either neutralizing NGF antibodies (NGF-Ab; ***, P < 0.0001) or K252a, a tyrosine kinase receptor inhibitor (***, P < 0.0001). Each bar represents the mean ±

se

of five independent experiments.

Figure 5

Figure 5

NGF induces phosphorylation of MAPK ERK1/2 in hGCs. A, A representative Western blot showing that NGF induces a rapid (5 min), but transient, phosphorylation of ERK2, and to a lesser extent, ERK1, in cultured hGCs. B, Densitometric analysis of the blot shown in panel A. Each value represents the mean ±

sem

of eight experiments expressed as the ratio of phosphorylated ERK2/total ERK2 relative to untreated controls. *, P < 0.05 vs. t = 5 min and t = 10 min; **, P < 0.01 vs. t = 5 min and t = 1 min.

Figure 6

Figure 6

MAPK pathway activation mediates NGF-induced VEGF expression. Human GCs were treated with the MAPK inhibitor U 0126 for 8 h in the presence or absence of NGF (50 ng/ml), and VEGF165 mRNA levels were measured by semiquantitative PCR at the end of the treatment. Each column represents the mean ±

sem

of VEGF165 mRNA levels from four independent experiments, expressed as arbitrary units. **, P < 0.01 vs. NGF + U 0126. DMSO, Dimethylsulfoxide.

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