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.

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.

TrkA receptor activation by nerve growth factor induces shedding of the p75 neurotrophin receptor followed by endosomal gamma-secretase-mediated release of the p75 intracellular domain.

Neurotrophins are trophic factors that regulate important neuronal functions. They bind two unrelated receptors, the Trk family of receptor-tyrosine kinases and the p75 neurotrophin receptor (p75). p75 was recently identified as a new substrate for gamma-secretase-mediated intramembrane proteolysis, generating a p75-derived intracellular domain (p75-ICD) with signaling capabilities. Using PC12 cells as a model, we studied how neurotrophins activate p75 processing and where these events occur in the cell. We demonstrate that activation of the TrkA receptor upon binding of nerve growth factor (NGF) regulates the metalloprotease-mediated shedding of p75 leaving a membrane-bound p75 C-terminal fragment (p75-CTF). Using subcellular fractionation to isolate a highly purified endosomal fraction, we demonstrate that p75-CTF ends up in endosomes where gamma-secretase-mediated p75-CTF cleavage occurs, resulting in the release of a p75-ICD. Moreover, we show similar structural requirements for gamma-secretase processing of p75 and amyloid precursor protein-derived CTFs. Thus, NGF-induced endocytosis regulates both signaling and proteolytic processing of p75.

Nerve growth factor-dependent activation of trkA receptors in the human ovary results in synthesis of follicle-stimulating hormone receptors and estrogen secretion.

CONTEXT: Previous studies showed that nerve growth factor (NGF) induces the expression of functional FSH receptors (FSHR) in preantral follicles of the developing rat ovary. OBJECTIVE: The objective of this study was to determine whether NGF can affect granulosa cell (GC) function in human periovulatory follicles using intact human ovaries and isolated human GCs. PATIENTS AND INTERVENTIONS: Human GCs were obtained from in vitro fertilization patients and normal ovaries from women with elective pelvic surgery for nonovarian indications. RESULTS: In normal ovaries, NGF and trkA (NGF's high-affinity receptor) were detected by immunohistochemistry in GCs of preantral and antral follicles. NGF and trkA are also present in thecal cells of antral follicles. Both freshly collected and cultured GCs contained immunoreactive NGF and trkA in addition to their respective mRNAs. Human GCs respond to NGF with increased estradiol (E(2)) secretion and a reduction in progesterone output. Exposure of human GCs to NGF increased FSHR mRNA content within 18 h of treatment, and this effect was blocked by the trk tyrosine kinase blocker K-252a. Also, cells preexposed to NGF released significantly more E(2) in response to hFSH than cells not pretreated with the neurotropin, showing that the NGF-induced increase in FSHR gene expression results in the formation of functional FSHRs. CONCLUSIONS: These results suggest that one of the functions of NGF in the preovulatory human ovary is to increase the secretion of E(2) while preventing early luteinization via an inhibitory effect on progesterone secretion. NGF stimulates E(2) secretion both directly and by increasing the formation of FSHRs.

Plasticity in rat uterine sympathetic nerves: the role of TrkA and p75 nerve growth factor receptors.

Uterine sympathetic innervation undergoes profound remodelling in response to physiological and experimental changes in the circulating levels of sex hormones. It is not known, however, whether this plasticity results from changes in the innervating neurons, the neuritogenic properties of the target tissue or both. Using densitometric immunohistochemistry, we analysed the effects of prepubertal chronic oestrogen treatment (three subcutaneous injections of 20 microg of beta-oestradiol 17-cypionate on days 25, 27 and 29 after birth), natural peripubertal transition and late pregnancy (19-20 days post coitum) on the levels of TrkA and p75 nerve growth factor receptors in uterine-projecting sympathetic neurons of the thoraco-lumbar paravertebral sympathetic chain (T7-L2) identified using the retrograde tracer Fluorogold. For comparative purposes, levels of TrkA and p75 were assessed in the superior cervical ganglion (SCG) following prepubertal chronic oestrogen treatment. These studies showed that the vast majority of uterine-projecting neurons expressed both TrkA and p75. Both prepubertal chronic oestrogen treatment and the peripubertal transition increased the ratio p75 to TrkA in uterine-projecting neurons, whereas pregnancy elicited the opposite effect. Prepubertal chronic oestrogen treatment had no effects on levels of TrkA or p75 in sympathetic neurons of the SCG. Taken together, our data suggest that neurotrophin receptor-mediated events may contribute to regulate sex hormone-induced plasticity in uterine sympathetic nerves, and are in line with the idea that, in vivo, plasticity in uterine nerves involves changes in both the target and the innervating neurons.

Peroxisome proliferator-activated receptor gamma is a novel target of the nerve growth factor signaling pathway in PC12 cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, is subject to considerable interest because of its role in adipocyte differentiation, metabolic control, and anti-inflammatory action. PPARgamma research in brain cells is presently focused on glial PPARgamma because of its potential as a pharmacological target in the treatment of neurodegenerative diseases with an inflammatory component. In neurons PPARgamma function is far from clear, and PPARgamma agonist-dependent and -independent effects on cell survival or differentiation have been reported. We used PC12 cells, widely used to study neuronal signaling, such as nerve growth factor (NGF)-induced differentiation and survival or epidermal growth factor-dependent cell proliferation to dissect the possible involvement of PPARgamma in these pathways. We show that NGF but not epidermal growth factor increases the transcriptional activity of PPARgamma, and modulates the expression of this transcription factor. Because NGF signals through the tyrosine kinase (TrkA) NGF receptor and/or the p75NTR receptor, we used rescue experiments with a PC12 cell mutant lacking TrkA to show that NGF-induced PPARgamma activation is dependent on TrkA activation. Our results point out PPARgamma as a novel target of the TrkA-mediated neuronal cell survival and differentiating pathway and suggest a potential new inflammatory-independent therapeutic approach for pharmacological intervention in neurological disorders.

Low-density lipoprotein receptor-related protein mediates in PC12 cell cultures the inhibition of nerve growth factor-promoted neurite outgrowth by pregnancy zone protein and alpha2-macroglobulin.

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein closely related to human alpha(2)-macroglobulin (alpha(2)M). It has been demonstrated that monoamine-activated forms of human and rat alpha(2)M and rat alpha(1)M can bind to TrkA and, respectively, inhibit and stimulate NGF-promoted neurite outgrowth, Trk phosphorylation, and intracellular signal transduction in PC12 cells. However, the effect of PZP on neurons is unknown, and the molecular mechanism of neuroinhibition by monoamine-activated alpha(2)M is still unclear. In this report, we show that methylamine-activated PZP (MA-PZP), like MA-alpha(2)M, inhibits in a dose-dependent way the NGF-promoted neurite extension and TrkA phosphorylation in PC12 cells. On the other hand, normal PZP (N-PZP) had little or no effect. In addition, the inhibitory effect of activated alpha-macroglobulins (alphaMs) was reversible upon its removal from the cell culture. In addition, PZP, as well as alpha(2)M, is neuroinhibitory without being directly cytotoxic. It is known that the activated alphaMs bind to the multiligand receptor termed low-density lipoprotein receptor-related protein (LRP) and that the receptor-associated protein (RAP) specifically blocks uptake of all known LRP ligands. To investigate the potential role of LRP in neuromodulation by activated PZP/alpha(2)M, the effect of RAP on the neuroinhibitory activities of these alphaMs was also studied. Data presented here show that RAP blocked the neurite- and Trk-inhibitory activities of both MA-PZP and MA-alpha(2)M, whereas RAP itself had no neuromodulatory effect. Hence, we conclude that these data suggest that the LRP receptor and its alphaM ligands may play a role in regulating Trk receptors.