trkA Transcripts Research Articles

Expression of β-NGF and high-affinity NGF receptor (TrKA) in llama (Lama glama) male reproductive tract and spermatozoa.

β-Nerve growth factor (β-NGF) is a seminal plasma element, responsible for inducing ovulation in camelids. The main organ of β-NGF production remains nondescript. The aims of this study were to (a) characterize gene expression and protein localization of β-NGF and its main receptor tyrosine kinase receptor A (TrKA) in the llama male reproductive tract, and (b) determine whether the seminal β-NGF interacts with ejaculated sperm by localizing β-NGF and TrKA in epididymal, ejaculated, and acrosome-reacted (AR) sperms and, additionally, by identifying β-NGF presence in sperm-adsorbed proteins (SAP). Both β-NGF and TrkA transcripts are widely expressed along the male reproductive tract, with a higher expression level of β-NGF at prostate (p < 0.05). β-NGF immunolabeling was only positive for prostate, whereas TrKA label was present in epithelial and muscular cells of testis, prostate, bulbourethral glands, and epididymis. Using an immunofluorescent technique, β-NGF was colocalized with TrKA in the middle piece of ejaculated and AR sperm. However, only TrKA was observed in epididymal sperm indicating that β-NGF could have a seminal origin. This was also confirmed by the identification of four β-NGF isoforms in SAP. This study extends the knowledge about the participation of β-NGF/TrkA in llama reproduction, providing evidence that may have roles in the regulation of sperm physiology.

Expression of nerve growth factor and its receptor, tyrosine kinase receptor A, in rooster testes

Nerve growth factor (NGF), which is required for the survival and differentiation of the nervous system, is also thought to play an important role in the development of mammalian reproductive tissues. To explore the function of NGF in the male reproductive system of non-mammalian animals, we determined the presence of NGF and its receptor, tyrosine kinase receptor A (TrkA), in rooster testes and investigated the regulation of NGF and TrkA expression by follicle-stimulating hormone (FSH). The mRNA and protein levels of NGF and TrkA in 6-week-old rooster testes were lower than those in 12-, 16- or 20-week age groups; levels were highest in the 16-week group. Immunohistochemistry showed that NGF and TrkA were both detected in spermatogonia, spermatocytes and spermatids. NGF immunoreactivity was observed in Leydig cells and strong TrkA signals were present in Sertoli cells. Meanwhile, FSH increased TrkA transcript levels in rooster testes in a dose-dependent manner. We present novel evidence for the developmental and FSH-regulated expression of the NGF/TrkA system, and our findings suggest that the NGF/TrkA system may play a prominent role in chicken spermatogenesis.

Prognostic impact of MYCN, DDX1, TrkA, and TrkC gene transcripts expression in neuroblastoma

The aims of this study were to define the mRNA expression profiles of MYCN, DDX1, TrkA, and TrkC in biopsy tumor samples from 64 Brazilian patients with neuroblastomas of different risk stages and to correlate altered expression with prognostic values. Patients were retrospectively classified into low- (n = 11), intermediate- (n = 18), and high-risk (n = 35) groups using standard criteria. The mRNA levels of the above genes were measured by quantitative real-time polymerase chain reaction. Univariate analyses were performed and survival curves were plotted by the Kaplan-Meier method. Of the 64 patients, 53% were female and 62.5% were older than 18 months. The 5-year overall survival (OS) for the entire cohort was 40.3%, with inferior median OS in patients identified in the intermediate- and high-risk groups. A significant difference in OS with respect to TrkA mRNA expression was found for the high-risk group vs. either the low- or intermediate-risk groups (P < 0.01, log rank test). Within the intermediate-risk group, neuroblastoma patients with positive TrkA mRNA expression had better clinical outcomes than patients with no TrkA transcript expression (P = 0.004). Another difference in OS was only found between the intermediate- and high-risk groups (P < 0.027, log rank test). No significant correlation of mRNA expression and survival outcome could be detected for the MYCN, DDX1. Positive expression of TrkA mRNA may be a clinically useful addition to the current risk classification system, allowing the identification of NB tumors with favorable prognosis.

Detection of nerve growth factor (NGF) and its specific receptor (TrkA) in ejaculated bovine sperm, and the effects of NGF on sperm function

The objective was to confirm the presence of nerve growth factor (NGF) and its specific receptor, TrkA, in ejaculated bovine sperm, and to investigate the effects of NGF on specific aspects of bovine sperm function. Both TrkA transcripts and immunoreactivity typical of the translated protein were detected by RT-PCR and western blotting. However, only the NGF protein was detected in bovine sperm using western blotting, and there was no RT-PCR evidence for NGF transcripts in sperm. Using an immunofluorescent technique, NGF-immunoreactivity was localized to the sperm head and tail, whereas that of TrkA was detected in the acrosomal cap, nucleus, and tail regions When sperm were treated with exogenous NGF, both leptin secretion and sperm viability were increased (P < 0.05); moreover, the percentages of late apoptotic and dead sperm were increased (P < 0.05). However, NGF had no effects on insulin secretion, mitochondrial activity, intracellular calcium levels, or the acrosome reaction of sperm (P > 0.05). In conclusion, the presence of TrkA transcript, as well as NGF and TrkA immunoreactivity were confirmed in bovine sperm. Furthermore, exogenous NGF had significant effects on the secretion of leptin, cell viability, and sperm apoptosis. This study provided strong evidence that NGF/TrkA may have roles in regulation of sperm physiology and perhaps male fertility and infertility.

HAND transcription factors are required for neonatal sympathetic neuron survival

Expression of the basic helix-loop-helix transcription factor HAND2 begins early in sympathetic neuron development and is essential for the differentiation of noradrenergic neurons. Here, we show that the expression of HAND2 and related HAND1 are maintained in sympathetic neurons throughout fetal and postnatal development when these neurons depend on target-derived nerve growth factor (NGF) for survival. Short interfering RNA knockdown of endogenous HAND2 and, to a lesser extent, HAND1 in neonatal sympathetic neurons cultured with NGF, reduced the expression of the NGF receptor tyrosine kinase TrkA (tropomyosin-related kinase A), as well as neuronal survival. Chromatin immunoprecipitation analysis showed that NGF promotes HAND2 binding to the TrkA minimal enhancer and that transfection of sympathetic neurons with a TrkA expression plasmid rescued the neurons from HAND knockdown. These findings show that HAND transcription factors have a crucial function in sustaining the survival of neonatal sympathetic neurons with NGF by a feed-forward loop that maintains the expression of TrkA.

Zhangfei, a novel regulator of the human nerve growth factor receptor, trkA

The replication of herpes simplex virus (HSV) in epithelial cells, and during reactivation from latency in sensory neurons, depends on a ubiquitous cellular protein called host cell factor (HCF). The HSV transactivator, VP16, which initiates the viral replicative cycle, binds HCF as do some other cellular proteins. Of these, the neuronal transcription factor Zhangfei suppresses the ability of VP16 to initiate the replicative cycle. It also suppresses Luman, another cellular transcription factor that binds HCF. Interactions of nerve growth factor (NGF) and its receptor tropomyosin-related kinase (trkA) appear to be critical for maintaining HSV latency. Because the neuronal transcription factor Brn3a, which regulates trkA expression, has a motif for binding HCF, we investigated if Zhangfei had an effect on its activity. We found that Brn3a required HCF for activating the trkA promoter and Zhangfei suppressed its activity in non-neuronal cells. However, in neuron-like NGF-differentiated PC12 cells, both Brn3a and Zhangfei activated the trkA promoter and induced the expression of endogenous trkA. In addition, capsaicin, a stressor, which activates HSV in in vitro models of latency, decreased levels of Zhangfei and trkA transcripts in NGF-differentiated PC12 cells.

Novel Brn3a cis‐acting sequences mediate transcription of human trkA in neurons

TrkA, the receptor tropomyosin-related kinase for nerve growth factor, is critical not only for the correct spatial and temporal development of sensory neurons during embryogenesis but also for the survival of sensory neurons, the differentiation and apoptosis of neuronal tumors and suppression of latent herpes simplex virus genomes. While the regulation of the expression of trkA is a complex process, the transcription factor Brn3a is known to play an important role as an enhancer of trkA transcription during development in the mouse. Despite considerable information on the regulation of trkA during embryogenesis, the mechanisms by which the expression of trkA is regulated in differentiated neurons, or the factors that influence its expression in tumor cells, have not been identified. We initiated studies to determine whether Brn3a/trkA promoter interactions may be important in a model of differentiated neurons and in medulloblastoma cells. We constructed a plasmid that contains 1043 base pairs of genomic sequences that extend to 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a responsiveness in Vero cells. This region was also sufficient for Brn3a trans-activation in nerve growth factor-differentiated PC12 cells. At least two portions of the 190 bp fragment bind to Brn3a with an affinity high enough to be detected in electromobility shift assays. In addition, Brn3a increased levels of endogenous trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA.

Nerve growth factor and its receptor TrkA serve as potential markers for human corneal epithelial progenitor cells

Nerve growth factor (NGF), a member of the neurotrophin family, has been identified as an essential growth factor supporting stem cell self-renewal outside the nervous system and was previously shown to stimulate corneal epithelial proliferation both in vivo and in vitro. In this study, we evaluated the expression of NGF and its corresponding receptors in the human corneal and limbal tissues, as well as in primary limbal epithelial cultures by immunofluorescent staining and relatively quantitative real-time polymerase chain reaction. We found that NGF was uniquely expressed in the human limbal basal epithelium, together with its two corresponding receptors: the high-affinity receptor TrkA and the low-affinity receptor p75NTR. TrkA was shown to preferentially localize to limbal basal epithelial cells. NGF and TrkA were also found co-localized with stem cell-associated molecular markers (drug-resistance transporter ABCG2 and p63), but not with the differentiation marker cytokeratin 3 in the human limbal basal epithelial layer. In cultured limbal epithelial cells, NGF and TrkA were found to be preferentially expressed by a small population of limbal epithelial cells. The NGF and TrkA immuno-positive subpopulations were enriched for certain properties (including ABCG2 and p63 expression) of putative limbal epithelial stem cells ( P < 0.01, compared with the entire cell population). Levels of NGF and TrkA transcripts were found to be much more abundant in limbal than in corneal tissues, and in young cultured cells in the proliferative stage than in airlifted stratified cultures containing differentiated cells. The co-expression of NGF with its two corresponding receptors in limbal basal epithelial cells, but not in the cornea, suggests that NGF may function as a critical autocrine or paracrine factor supporting stem cell self-renewal in the limbal stem cell niche. The spatial expression of NGF and TrkA by small clusters of basal cells interspersed between negative cell patches suggests that they are potential markers for human corneal epithelial progenitor cells.

THIS ARTICLE HAS BEEN RETRACTED: Functional nerve growth factor and trkA autocrine/paracrine circuits in adult rat cortex are revealed by episodic ethanol exposure and withdrawal

AbstractThe hypothesis tested is that cortical neurotrophins communicate through an inducible autocrine/paracrine mechanism. As ethanol (Et) can induce cortical nerve growth factor (NGF) expression, adult rats were challenged with Et on three consecutive days per week for 6 weeks. The focus of the study was layer V, the chief repository of receptor‐expressing neuronal cell bodies. Brains were collected immediately after the sixth Et exposure or 72 h later [i.e., following withdrawal (WD)]. Double‐label in situ hybridization–immunohistochemistry studies showed that many neuronal somata co‐expressed NGF mRNA with NGF, trkA, or phosphorylated trk (p‐trk), essential components of an inducible autocrine system. The frequencies of co‐labeling were affected by neither Et nor WD. On the contrary, Et increased the number of NGF mRNA‐expressing neurons and the amount of NGF mRNA expressed per cell. Et also increased total cortical concentration of NGF protein, the number of layer V neurons expressing trkA transcript, the amount of trkA mRNA expressed per neuron, and trkA phosphorylation. Following WD, the frequency of NGF‐mRNA‐expressing cells increased, although transcript and protein content fell. WD induced an increase in trkA mRNA and protein expression, however, p‐trk expression was unaffected. Thus, Et treatment reveals that layer V has inducible autocrine/paracrine and anterograde neurotrophin systems. WD unveils the dynamism and recruitability of these systems.

Estrogen alters trkA and p75 neurotrophin receptor expression within sympathetic neurons

Survival and growth of sympathetic neurons is regulated by nerve growth factor acting through trkA and p75NTR receptors. Sympathetic neurons are also affected by gonadal steroid hormones, particularly estrogen. To determine if estrogen may influence sympathetic neurons via altered neurotrophin receptor expression, we investigated effects of acute or chronic estrogen administration on levels of trkA and p75NTR proteins, numbers of immunoreactive neurons, and numbers of neurons expressing trkA, p75NTR, and estrogen receptor-alpha transcripts. Superior cervical ganglia from ovariectomized or estradiol-treated rats were processed for in situ hybridization or immunohistochemistry, and percentages of stained neurons quantitated or processed for Western blot analysis. In ovariectomized rats, approximately 50% of sympathetic neurons expressed trkA mRNA and protein. Acute estrogen administration did not affect trkA transcript expression, but reduced trkA protein significantly. Chronic treatment did not alter neuronal trkA expression. Approximately 70% of sympathetic neurons in ovariectomized rats expressed p75NTR transcripts and about 50% showed p75NTR immunoreactivity. Acute estrogen did not affect p75NTR expression. However, chronic estrogen reduced p75NTR mRNA and protein expression significantly. Fifty to sixty percent of sympathetic neurons in ovariectomized rats displayed estrogen receptor-alpha mRNA. After acute estrogen administration, estrogen receptor-alpha transcript expression increased by 35%, although this was not maintained chronically. These findings indicate that estrogen can influence sympathetic neuronal neurotrophin receptor expression as well as estrogen receptor-alpha. Reduced trkA expression after acute estrogen may transiently predispose neurons to degenerative events, while diminished p75NTR expression by chronic estrogen administration may exert long-term effects on survival or axonal outgrowth in sympathetic neurons.

Ectopic expression of the TrkA receptor in adult dopaminergic mesencephalic neurons promotes retrograde axonal NGF transport and NGF-dependent neuroprotection

A recombinant adeno-associated virus (rAAV) was used to investigate the impact of an ectopic expression of the NGF high-affinity receptor in adult neurons. The rat TrkA cDNA cloned in a pCMX vector was first tagged with a human c-Myc sequence. The resulting vector was shown to encode a functional receptor which promoted the expression of TrkA immunoreactivity upon transfection of 293 fibroblasts or nnr5 cells, a TrkA-defective variant of PC12 cells. These cells also accumulate TrkA transcripts upon transfection and extended neurites in the presence of NGF. Therefore, the TrkA myc cassette was inserted into the pSSV9 plasmid. The new vectors shared properties similar to pCMX TrkA myc in 293 and nnr5 cells and enabled the preparation of rAAV TrkA myc viruses. Unilateral injection of this rAAV into the substantia nigra (SN) resulted in a protracted expression of TrkA (or c-Myc) immunoreactivity in numerous cell bodies, including tyrosine-hydroxylase (TH)-positive dopaminergic neurons. The presence of TrkA receptors in corresponding striatal dopaminergic endings was demonstrated by the advent of a striato-nigral retrograde axonal transport of 125I-NGF. Likewise, ectopic expression of TrkA in neurons of the parafascicular thalamic nucleus promoted a striatofuge transport of NGF toward this structure. To investigate whether ectopic expression of TrkA in SN neurons may confer neuroprotection, lesions were induced by 6-hydroxydopamine in striata located ipsilateral to the virus injection site. NGF or vehicle were next delivered dorsally to the virus-treated SN for 2 weeks, before sacrifice and processing of brains for TH-immunohistochemistry. NGF treatment, in contrast to treatment with vehicle, significantly enhanced the number of dopaminergic neurons counted in the lesioned SN. These data suggest that ectopic TrkA can mediate the trophic actions of NGF and influence neuronal plasticity in vivo.

Nerve Growth Factor Cooperates with p185 HER2 in Activating Growth of Human Breast Carcinoma Cells

Nerve growth factor (NGF) is known to exert a mitogenic effect on human breast cancer cells through proto-TrkA activation. Reverse transcriptase-PCR analysis of proto-TrkA expression in human breast carcinoma specimens and cell lines revealed trkA transcript in 12 of 14 human breast carcinoma specimens and in all of four cell lines tested. While cytofluorimetric and Western blot analysis indicated proto-TrkA expression in three of the four cell lines, NGF stimulated growth in only two of the three positive cell lines. Inhibition of NGF-induced MAPK activation by an antibody directed against the extracellular domain of TrkA but not by an inhibitor of TrkA phosphorylation demonstrated the requirement of NGF binding but not of proto-TrkA kinase activity for MAPK activation, suggesting the recruitment of another kinase for transmission of the mitogenic signaling. Indeed, NGF induced tyrosine phosphorylation and stimulated kinase activity of p185(HER2), a kinase receptor of the HER family. A TrkA phosphorylation inhibitor did not affect this activation. Moreover, the two receptors were coprecipitated by antibodies directed against proto-TrkA and p185(HER2). Down-modulation of p185(HER2) expression in a breast carcinoma line transfected with a construct containing an anti-p185(HER2) antibody sequence and expressing proto-TrkA impaired NGF-induced MAPK activation and proliferation. Together these data show that in cells expressing low levels of TrkA such as breast carcinoma cells, NGF must recruit other overexpressed receptors such as p185(HER2) in order to generate a biological signal that can induce breast cancer cell growth.

Regulation of NGF, BDNF and LNGFR gene expression in ROS [formula omitted] cells

The secosteroid hormone 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) has been recently shown to enhance the synthesis of NGF in mouse L929 fibroblasts. In view of the critical role of 1,25(OH) 2D 3 on bone metabolism, it has been investigated if ROS 17 2.8 osteoblastic cells were able to express the nerve growth factor (NGF) gene and if this process was responsive to 1,25(OH) 2D 3. Results indicate that these cells respond in a dose-dependent manner to the presence of 1,25(OH) 2D 3 by an increase in NGF mRNA levels. However, the phorbol ester PMA, previously reported to augment the synthesis of NGF via the recruitment of AP-1 complexes, depressed the expression of the NGF gene in ROS cells. In contrast, the mRNA levels of an NGF-related trophic factor, brain-derived neurotrophic factor (BDNF), was increased by PMA but not following 1,25(OH) 2D 3 treatment. Binding of 125I-NGF to ROS cells displayed the properties of a low affinity NGF receptor (dissociation constant K d ≈ 10 −9 M). In agreement with this result, the mRNA encoding the low affinity NGF receptor (LNGFR) was detected in ROS 17 2.8 cells, unlike trkA transcripts which encode the high affinity receptor. These data suggest that neurotrophins and their low affinity receptor could play an unsuspected role in bone tissue.

Coordinated expression and function of neurotrophins and their receptors in the rat inner ear during target innervation

We show that trkB and trkC mRNAs, encoding the high-affinity receptor tyrosine kinases for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), respectively, as well as low-affinity nerve growth factor receptor (p75 LNGFR) mRNA are expressed in the cochleovestibular ganglion (CVG) before and during innervation of the target fields. Correspondingly, from preinnervation stages onward, BDNF and NT-3, but neither nerve growth factor (NGF) nor neurotrophin-4 (NT-4) mRNAs are expressed in the sensory epithelium of the otic vesicle, the peripheral target field of CVG neurons. No neurotrophin transcripts were detected by in situ hybridization in the medullary central targets. In explant cultures, neuritogenesis from both the cochlear and vestibular part of the CVG was promoted by BDNF, while NT-3 evoked neuntes mainly from the cochlear neurons. Also NT-4 stimulated neurite outgrowth from the CVG in vitro. In dissociated neuron-enriched cultures, NT-3 and BDNF promoted survival of overlapping subsets of CVG neurons and, correspondingly, results from in situ hybridization showed that both trkC and trkB mRNAs were expressed in most neurons of this ganglion. The negligible effect of NGF seen in the bioassays agrees well with the expression of only a few trkA transcripts, encoding the high-affinity receptor for NGF, in the CVG. Based on the spatiotemporal expression patterns and biological effects in vitro, peripherally-synthesized BDNF and NT-3 regulate the survival of CVG neurons as well as the establishment of neuron-target cell contacts in the early-developing inner ear. In addition, the expression of trkB mRNA, more specifically its truncated form, and trkC as well as p75 LNGFR mRNAs in distinct non-neuronal structures indicates novel roles for these molecules during development.

Expression of mRNA for neurotrophic factors and their receptors in the rat dorsal root ganglion and sciatic nerve following nerve injury.

The adult rat dorsal root ganglion (DRG) produces mRNA for the neurotrophic factors nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and contains large populations of neurons responsive to these factors. We report that following a focal crush injury of the sciatic nerve, NGF mRNA expression increases threefold and BDNF mRNA two-fold, in the ipsilateral L4 and L5 DRGs. The mRNAs encoding the cognate neurotrophin receptors, p75NGFR, trkA, and trkB were expressed in the DRG throughout the post-injury time course, suggesting that DRG neurons remain responsive to both NGF and BDNF. p75NGFR mRNA levels became transiently depressed in the DRG during the first several days after the lesion but returned to normal within 1 week. trkB mRNA was expressed in the normal sciatic nerve and levels were not altered by nerve crush. RNase protection assays detected both full-length and truncated trkB transcripts in the DRG, but only truncated trkB mRNA, lacking the tyrosine kinase domain, was detected in the sciatic nerve. Likewise, trkA transcripts were not detected by RNase protection in normal sciatic nerve or in a segment of nerve distal to the crush site. These results are consistent with a model in which regenerating sensory neurons are supported by neurotrophic factors synthesized within the DRG.