Structural characterization of NGF and its receptors

NGF is a prototype glycoprotein that belongs to the NT family. It is synthesized initially as pro-NGF (306 amino acids) that is cleaved and translocated to the rough endoplasmic reticulum, then cleaved by extracellular proteases to produce a biologically active protein (118 amino acids). The mature NGF is thus a covalent homodimer with a molecular weight of 130 kDa (Covaceuszach et al., Reference Covaceuszach, Cassetta, Cattaneo and Lamba2004; Mouri et al., Reference Mouri, Nomoto and Furukawa2007; Nomoto et al., Reference Nomoto, Takaiwa, Mouri and Furukawa2007).

The tridimensional structure of NGF was revealed by McDonald et al. (Reference McDonald, Lapatto, Murray-Rust, Gunning, Wlodawer and Blundell1991), who verified the structure using X-ray crystallography as having an elongated shape with a core formed by twisted beta sheets and bound by disulfide bridges. The molecule is made up of three subunits (α, β and γ complex). The β-NGF subunit is responsible for the biological activity and the γ-NGF subunit is a highly specific active protease (26-kDa serine protease of the kallikrein protease group) and is responsible for the conversion of the pro-NGF transcript into its active form. The function of the α-NGF subunit is not known and it appears to be inactive (Sofroniew et al., Reference Sofroniew, Howe and Mobley2001). During the activation of NGF, the γ-NGF subunit proteins are hydrolyzed to enable the conversion of pro-NGF synthesized from the gene into the active homodimer NGF. In this process, the protein remains stable and there is no segregation of α-NGF and γ-NGF subunits. It is believed that although this protein has not shown biological function, it may serve to protect NGF from proteolytic enzymes present in tissues (Levi-Montalcini & Calissano, Reference Levi-Montalcini and Calissano1986).

NGF begins its biological role by binding to two known receptors (Fig. 1). One of these shows high affinity to NGF and is called tyrosine kinase receptor A (TrkA) (Ibañez, Reference Ibañez1998; Terenghi, Reference Terenghi1999). The other is a low-affinity receptor called neurotrophin receptor p75 (p75^NTR or p75). NGF signalling occurs preferentially through the TrkA receptor (Kaplan et al., Reference Kaplan, Hempstead, Martin-Zanca, Chao and Parada1991), whereas p75 may potentiate or inhibit the biological responses mediated by TrkA (Kohn et al., Reference Kohn, Aloyz, Toma, Haak-Frendscho and Miller1999). Phylogenetic analysis of NGF and its TrkA receptor identified co-evolution between these loci, and increased the receptor–ligand specificity (Halböök et al., Reference Halböök, Lundin and Kullander1998).

Figure 1 Schematic model that illustrates high (TrkA) and low (p75) affinity receptors for NGF. Adapted from GrandPré et al. (Reference GrandPré, Li and Strittmatter2002).

The TrkA gene maps to chromosome band 1q22 (Valent & Bernheim, Reference Valent and Bernheim1997) and contains 17 exons that codify a transmembrane protein of 140 kDa, which has an extracellular binding domain (composed of three leucine-rich motifs flanked by two cysteine clusters, two immunoglobulin-like C2 type domains – Ig-C2), a single transmembrane domain and an intracellular domain that has tyrosine kinase activity (Greco et al., Reference Greco, Villa and Pierotti1996). NGF binding to the extracellular domain leads to receptor oligomerization and this rearrangement allows the domains of the closest kinases in the receptor chain to phosphorylate one another, in a process called autophosphorylation (Kaplan & Miller, Reference Kaplan and Miller2000; Alberts et al., Reference Alberts, Johnson, Lewis, Raff, Roberts and Walter2004). The activation of TrkA receptors begins the signalling cascades, including the intracellular pathways.

The receptor p75 is a member of the death-promoting tumour necrosis factor receptor (TNF-R) superfamily, which also includes the characteristic death receptors TNF-R apoptosis-inducing ligand (TRAIL)-R and Fas/CD95 (Haase et al., Reference Haase, Pettmann, Raoul and Henderson2008). p75 is a 75 kDa glycoprotein with four extracellular cysteine-rich repeats that are required for ligand binding (Chao, Reference Chao2003; Barker, Reference Barker2004). It is a single pass type I transmembrane receptor, with an intracellular domain that contains a juxtamembrane region and a type II consensus death domain (DD) sequence (Roux & Barker, Reference Roux and Barker2002). Although p75 binds dimeric neurotrophin ligands, there is some controversy over the oligomeric status of p75 and evidence indicates that it may also signal as a monomer or as a dimer (Vilar et al., Reference Vilar, Charalampopoulos, Kenchappa, Simi, Karaca, Reversi, Choi, Bothwell, Mingarro, Friedman, Schiavo, Bastiaens, Verveer, Carter and Ibanez2009). Recently, it was shown that p75 can form covalent homodimers through a disulphide bond in the transmembrane region. In this case, neurotrophin binding is understood to induce a conformational change in the receptor, such that it pivots on the disulfide bond at Cys257 to permit access of intracellular adaptor proteins to the intracellular domain in what is termed a ‘snail-tong model’ (Simi & Ibañez, Reference Simi and Ibañez2010). The receptor does not possess intrinsic enzymatic activity and instead transduces signals through recruitment of a variety of adaptor proteins to the intracellular domain, thus leading to proliferation, survival, or cell death (He & Garcia, Reference He and Garcia2004). Interestingly, p75 also serves as a receptor for immature pro-neurotrophins, which induce cell death in a manner that is dependent on binding to the co-receptor sortilin (Nykjaer et al., Reference Nykjaer, Lee, Teng, Jansen, Madsen, Nielsen, Jacobsen, Kliemannel, Schwarz, Willnow, Hempstead and Petersen2004).

Cellular signalling through NGF/receptor to regulate folliculogenesis

The effects of NGF are related to the activation of different biochemical pathways (Fig. 2), activation of the MAPK and PI3K pathway and inactivation of the apoptotic proteins Bad and Bax from the Bcl-2 family, both pathways are activated by the TrkA receptor. Previous researchers have also described the activation of the PLCγ pathway that produces DAG and IP3, which induce the release of calcium from the intracytoplasmic cellular stocks (Glebova & Ginty, Reference Glebova and Ginty2005). Finally, activation of the small GTPase Ras in response to NGF has been shown and linked to cell survival and differentiation (Caporali & Emanueli, Reference Caporali and Emanueli2009).

Figure 2 Schematic representation of signalling pathways between NGF and TrkA. NGF binds to its receptor TrkA, leading to dimerization and autophosphorylation. The Shc adaptor molecule binds to the phosphate in the receptor and to the Grb2–SOS complex. The Ras complex activates, exchanging the GDP for GTP. Activated Ras interacts directly with the Raf protein, which leads to the sequential activation of MEK and MAPK. MAPK translocate to the nucleus, where it phosphorylates transcriptional factors, promoting cellular differentiation. Ras also promotes PI3K activation, which activates substances responsible for follicular survival and growth. Activation of PLCγ1 results in the release of Ca²⁺ and PKC activation.

The details of activation performed by the p75 receptor are less well known, but it may result in the production of ceramides, or activation of nuclear factor-kappa B (NF-κB) or regulator kinases such as c-Jun N-terminal kinase (JNK; Lee et al., Reference Lee, Kermani, Teng and Hempstead2001; Chao, Reference Chao2003). p75 signalling can also lead to downstream activation of NF-κB, which promotes cell survival through upregulation of anti-apoptotic genes such as cFLIP and this event interferes with the activation of initiator caspase-8, the Bcl-2 family member Bcl-XL, and inhibitor of apoptosis proteins (IAPs) XIAP and cIAP1/2 (Karin, Reference Karin2006; Baud & Karin, Reference Baud and Karin2009). Together with the pro-apoptotic effects of p75, these findings show that p75 can act as a bifunctional switch to direct the cell down opposing pathways of cell death or survival (Molloy et al., Reference Molloy, Read and Gorman2011).

Thus, the p75 receptor shows an independent role in apoptosis that may be anti- or pro-apoptotic (Chao, Reference Chao2003). The anti-apoptotic stimulus begins with the activation of a ceramide-dependent signalling pathway (Shi et al., Reference Shi, Jin, Watanabe, Suzuki, Takahashi and Taya2004). In this case, NGF interacts with the p75 receptor to hydrolyze sphingomyelin, which results in the production of an apoptosis cascade signalling inhibitor called ceramide (Barrett, Reference Barrett2000). The pro-apoptotic stimulus induced by p75 follows the intrinsic apoptotic pathway, with the release of cytochrome c by mitochondria and activation of caspase-9 (Bhakar et al., Reference Bhakar, Howell, Paul, Salehi, Becker, Said, Bonni and Barker2003). However, cell apoptosis mediated by p75 does not occur when high affinity receptors (TrkA) are also expressed (Botchkarev et al., Reference Botchkarev, Botchkareva, Albers, Chen, Welker and Source2000).

Ras/MAPK pathway

Activation of the Ras protein occurs after NGF binds to its cellular receptor TrkA, which is needed for cell differentiation and survival. This process is mediated through an Shc adaptor molecule that binds to phosphorylated tyrosine 490, which is critical for the activation of the Ras signalling cascade. Shc is recruited to its recognized site in activated Trk by interactions with the Shc PTB domain (Nimnual et al., Reference Nimnual, Yatsula and Bar-Sagi1998). Shc phosphorylation produces a site on phosphotyrosine Shc that recruits other adaptor proteins that contain the SH2 domain, such as growth factor receptor-bound protein-2 (Grb2), which binds to the exchange factor of the nucleotide guanine (SOS – son of sevenless; Robinson et al., Reference Robinson, Manto, Buchsbaum, MacDonald and Meakin2005). The Grb2–SOS complex is translocated to the plasma membrane, where SOS activates a small G protein p21ras and promotes the transition from inactive Ras–GDP to active Ras–GTP (guanosine triphosphate; York et al., Reference York, Molliver, Grewal, Stenberg, McCleskey and Stork2000). The activated Ras protein stimulates signalling through several cytoplasmic kinase proteins, such as Raf (kinase specific for serine/threonine), MEK (MAPK kinase activator) and MAPK (Wood et al., Reference Wood, Sarnecki, Roberts and Blenis1992; Xing et al., Reference Xing, Kornhauser, Xia, Thiele and Greenberg1998). The activation of one or more kinases leads to phosphorylation and activation of MEK and isoforms of MAPK, ERK-1 and ERK-2 (kinases regulated by an extracellular signalling -1/-2) (Crews et al., Reference Crews, Alessandrini and Erikson1992; Wood et al., Reference Wood, Sarnecki, Roberts and Blenis1992). MAPK translocates to the cell nucleus, where it phosphorylates a group of molecules responsible for transcription, thus beginning cellular proliferation (Silva et al., Reference Silva, Horta, Alencastro and Pinto2009).

PI3K and PLCγ1 pathways

One of the main signalling mechanisms that involves lipids is the cleavage of the membrane phosphatidylinositol to form DAG and IP3 by PLCs. IP3 may bind to its receptors in the rough endoplasmic reticulum, releasing Ca²⁺ from these stocks, as shown previously, while DAG may activate several isoforms of protein kinase Cs (PKCs). Therefore, PI3K and PLCγ act as intracellular messengers, whose function is to transmit the membrane receptor signalling to several proteins that will make this sign noticeable by the cell (Lenz, Reference Lenz2000).

Considering that PI3Ks may be activated by Ras (Rodriguez-Viciana et al., Reference Rodriguez-Viciana, Warne, Dhand, Vanhaesebroeck, Gout, Fry, Waterfield and Downward1994) and by the subunits βγ of the protein G (Lopez-Ilasaca et al., Reference Lopez-Ilasaca, Crespo, Pellici, Gutkind and Wetzker1997), these enzymes represent an important connection point between the signalling activators of protein G, Ras and PKC and their numerous substrates (Lenz, Reference Lenz2000).

The Ras independent pathway occurs when the GAB1 adaptor proteins (GRB2-associated binding protein-1) and Shc are phosphorylated by TrkA. As soon as it is activated, they are associated with Grb2 to produce a complex that activates PI3K (Holgado-Madruga et al., Reference Holgado-Madruga, Moscatello, Emlet, Dieterich and Wong1997). However, Trk phosphorylation can also promote phosphorylation of the insulin 1 receptor, which recruits and activates PI3K (Yamada et al., Reference Yamada, Ohnishi, Sano, Nakatani, Ikeuchi and Hatanaka1997). Once activated, either by Ras or by its independent pathway, PI3K phosphorylates several proteins important for this process: Akt (also known as kinase B protein), the RAC-PK and the ribosomal protein p70 S6 kinase (p70S6K or p70) (Chung et al., Reference Chung, Grammer, Lemon, Kazlauskas and Blenis1994; Marte & Downward, Reference Marte and Downward1997). Akt is considered to be the most important protein phosphorylated by PI3K, and controls the activity of several other proteins that are important for cellular survival. This process occurs through the regulation of proteins that control the activity of certain transcriptional factors that promote apoptosis (Brunet et al., Reference Brunet, Datta and Greenberg2001), such as the BAD cascade (a pro-apoptotic member of the Bcl-2 family; Yuan et al., Reference Yuan, Hu and Xu2001).

PI3K has a role in cellular growth and differentiation through phosphorylated phosphatidylinositol that recruits the guanine exchange factors Cdc42/Rho/Rac (GEF), which act on the organization of actin filaments into cytoskeleton, and improves the orientation of mitotic fusion (Yuan et al., Reference Yuan, Lipinski and Degterev2003).

The activation of PLCγ by Trk promotes an increase in intracellular Ca²⁺ levels and the regulation of kinase C protein. These proteins activate numerous intracellular enzymes, which include all isoforms of kinase C protein and calcium-/calmodulin-dependent protein kinase C (CaMKII), which then activates other important proteins. Other such important proteins are MEK and ERK1/ERK2 (Corbit et al., Reference Corbit, Foster and Rosner1999).

Other Ras-independent pathways have been related to cellular survival and differentiation. Among these, the phosphorylation of the SNT molecule by NGF (Rabin et al., Reference Rabin, Cleghorn and Kaplan1993) can be highlighted. In this case, once phosphorylated, SNT is translocated into the nucleus, where it acts as a transcription factor by regulating the genes that control cell cycle (Rabin et al., Reference Rabin, Cleghorn and Kaplan1993).

Expression of NGF and its receptor in the ovary

The expression of NGF and its receptors (TrkA and p75) was verified in ovarian cells (oocyte, granulosa, theca and interstitial cells) of several species, including human (Spears et al., Reference Spears, Molinek, Robinson, Fulton, Cameron, Shimoda, Telfer, Anderson and Price2003; Salas et al., Reference Salas, Julio-Pieper, Valladares, Pommer, Veja, Mastronardi, Kerr, Ojeda, Lara and Romero2006), rodent (Romero et al., Reference Romero, Paredes, Dissen and Ojeda2002; Shi et al., Reference Shi, Jin, Watanabe, Suzuki, Takahashi and Taya2004), bovine (Dissen et al., Reference Dissen, Parrott, Skinner, Hill, Costa and Ojeda2000; Levanti et al., Reference Levanti, Germanà, Abbate, Montalbano, Veja and Germanà2005), ovine (Barboni et al., Reference Barboni, Mattioli, Giogia, Turriani, Capacchietti, Berardinelli and Bernabo2002) and caprine (Ren et al., Reference Ren, Medan, Weng, Jin, Li, Watanabe and Taya2005). Thus, NGF and its receptors regulate several functions in the ovary, such as sexual development (Lara et al., Reference Lara, McDonald and Ojeda1990), follicular development and ovulation (Dissen et al., Reference Dissen, Romero, Hirshfiel and Ojeda1996; Reference Dissen, Romero, Hirshfield and Ojeda2001) through autocrine and paracrine ways.

Levanti et al. (Reference Levanti, Germanà, Abbate, Montalbano, Veja and Germanà2005) verified a weak immunostaining for the protein p75 in the ovarian stromal in bovine by immunohistochemistry and a high immunoreactivity in the oocytes of preantral follicles (primordial, primary and secondary) and early antral follicles (tertiary). Immunoreactivity for the TrkA receptor was found in the oocyte, granulosa and theca cells, and appeared independent of the follicular maturation stage. In swine ovaries, the primordial and primary follicles showed positive immunostaining for p75 and TrkA only in granulosa cells, while in tertiary follicles, this same receptor was found both in follicular cells and oocytes. In addition, a weak immunoreactivity was found in stromal cells and high immunoreactivity in corpus luteum cells (Levanti et al., Reference Levanti, Germanà, Abbate, Montalbano, Veja and Germanà2005).

The TrkA receptor protein was localized in the oocytes of mouse primordial follicles and visualized using immunofluorescence, (Dissen et al., Reference Dissen, Romero, Hirshfield and Ojeda2001), while the NGF ligand was expressed in the theca and granulosa cells of preantral and antral follicles in rat ovaries (Dissen et al., Reference Dissen, Hill, Costa, Ma and Ojeda1991). The expression pathways found in the oocytes and somatic cells indicated that NGF is a potential factor that can regulate intra-oocyte activation of PI3K (Adhikari & Liu, Reference Adhikari and Liu2009). Noticeable amounts of NGF were also detected in the follicular fluid of rat antral follicles (Barboni et al., Reference Barboni, Mattioli, Giogia, Turriani, Capacchietti, Berardinelli and Bernabo2002), and probably originated from granulosa and theca cells (Dissen et al., Reference Dissen, Romero, Hirshfield and Ojeda2001).

In addition, other immunocytochemical studies revealed that the p75 receptor protein was expressed in the mesenchyme cells in the rat fetal ovary (Dissen et al., Reference Dissen, Hirshfield, Malamed and Ojeda1995; Ojeda et al., Reference Ojeda, Romero, Tapia and Dissen2000). In hamsters, the staining for NGF and its receptors (TrkA and p75) was detected in the oocyte, granulosa, theca, interstitial and luteinic cells of all follicular categories (Shi et al., Reference Shi, Jin, Watanabe, Suzuki, Takahashi and Taya2004). In mouse, TrkA was localized in the oocytes, granulosa and theca cells of primary and secondary follicles, while the p75 receptor was localized only in interstitial cells (Dissen et al., Reference Dissen, Romero, Hirshfield and Ojeda2001; Weng et al., Reference Weng, Shi, Tukada, Watanabe and Taya2009). In Rhesus monkey ovaries, detection of p75 receptor mRNA expression by immunocytochemistry indicates that this organ is able to synthesize this receptor (Dees et al., Reference Dees, Hiney, Schultea, Meyerhofer, Danilchik, Dissen and Ojeda1995).

Role of NGF on follicular development

Final consideration

NGF was found 50 years ago as a molecule that promoted the survival and differentiation of sympathetic and sensory neurons. Its role in neuronal development has been characterized extensively, but recent findings suggest an unexpected diversity of NGF actions in other organs, such as the ovary. Studies have demonstrated the essential role of NGF in mammal folliculogenesis both in a direct way, such as through the nervous stimulus in the ovary, or by its indirect role, through mesenchyme cells (theca cells).

The complete understanding of NGF actions within the ovaries is still unclear due to the great variety of kinases that may be expressed and also by the lack of studies on this growth factor in the ovary. Thus, the use of NGF in the media used for culture in vitro of ovarian follicles may be of fundamental importance to promote follicular development.