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During adulthood, the neurotrophin Nerve Growth Factor (NGF) sensitizes nociceptors, thereby increasing the response to noxious stimuli. The relationship between NGF and pain is supported by genetic evidence: mutations in the NGF TrkA receptor in patients affected by an hereditary rare disease (Hereditary Sensory and Autonomic Neuropathy type IV, HSAN IV) determine a congenital form of severe pain insensitivity, with mental retardation, while a mutation in NGFB gene, leading to the aminoacid substitution R100W in mature NGF, determines a similar loss of pain perception, without overt cognitive neurological defects (HSAN V). The R100W mutation provokes a reduced processing of proNGF to mature NGF in cultured cells and a higher percentage of neurotrophin secreted is in the proNGF form. Moreover, using Surface Plasmon Resonance we showed that the R100W mutation does not affect NGF binding to TrkA, while it abolishes NGF binding to p75NTR receptors. However, it remains to be clarified whether the major impact of the mutation is on the biological function of proNGF or of mature NGF and to what extent the effects of the R100W mutation on the HSAN V clinical phenotype are developmental, or whether they reflect an impaired effectiveness of NGF to regulate and mediate nociceptive transmission in adult sensory neurons. Here we show that the R100 mutation selectively alters some of the signaling pathways activated downstream of TrkA NGF receptors. NGFR100 mutants maintain identical neurotrophic and neuroprotective properties in a variety of cell assays, while displaying a significantly reduced pain-inducing activity in vivo (n = 8–10 mice/group). We also show that proNGF has a significantly reduced nociceptive activity, with respect to NGF. Both sets of results jointly contribute to elucidating the mechanisms underlying the clinical HSAN V manifestations, and to clarifying which receptors and intracellular signaling cascades participate in the pain sensitizing action of NGF.

Background

Human bronchial smooth muscle cells (HBSMC) may regulate airway inflammation by secreting cytokines, chemokines and growth factors. The neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), have been shown to be elevated during airway inflammation and evoke airway hyperresponsiveness. We studied if HBSMC may be a source of NGF, BDNF and NT-3, and if so, how inflammatory cytokines may influence their production.

Methods

Basal and cytokine (IL-1β, IFN-γ, IL-4)-stimulated neurotrophin expression in HBSMC cultured in vitro was quantified. The mRNA expression was quantified by real-time RT-PCR and the protein secretion into the cell culture medium by ELISA.

Results

We observed a constitutive NGF, BDNF and NT-3 expression. IL-1β stimulated a transient increase of NGF, while the increase of BDNF had a later onset and was more sustained. COX-inhibitors (indomethacin and NS-398) markedly decreased IL-1β-stimulated secretion of BDNF, but not IL-1β-stimulated NGF secretion. IFN-γ increased NGF expression, down-regulated BDNF expression and synergistically enhanced IL-1β-stimulated NGF expression. In contrast, IL-4 had no effect on basal NGF and BDNF expression, but decreased IL-1β-stimulated NGF expression. NT-3 was not altered by the tested cytokines.

Conclusion

Taken together, our data indicate that, in addition to the contractile capacity, HBSMC can express NGF, BDNF and NT-3. The expression of these neurotrophins may be differently regulated by inflammatory cytokines, suggesting a dynamic interplay that might have a potential role in airway inflammation.

Nerve growth factor (NGF) is overexpressed not only in nervous system, but also in several types of cancers. However, the role of NGF in oesophageal squamous cell carcinoma (OESCC) remains unclear. Here, we show the first evidence of NGF-TrkA autocrine loop and clinical significance of NGF overexpression in OESCC. Immunohistochemical study of 109 OESCC specimens revealed that NGF overexpression, found in 63 out of 109 patients (57.8%), was associated with lymph node metastasis, distant metastasis, higher TNM stage, poorer tumour differentiation, and poorer survival. NGF overexpression was also associated with strong expression of TrkA and negative expression of low-affinity neurotrophin receptor (p75NTR). Semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) of 19 surgical specimens showed upregulation of NGF mRNA in 17 out of 19 (89%) patients. All five OESCC cell lines tested in vitro secreted detectable NGF in enzyme-linked immunosorbent assay, and expressed TrkA and p75NTR on RT–PCR and Western blot. The motility of HSA/c, one of the OESCC cell lines overexpressing NGF, was significantly decreased by either neutralising anti-NGF antibody, an inhibitor of TrkA, or NGF-small interfering RNA in transwell migration assay. Our findings suggest that NGF is of potential interest not only as a prognostic factor, but also as a novel therapeutic target in OESCC.

vgf is a neurotrophin response-specific, developmentally regulated gene that codes for a neurosecretory polypeptide. Its transcription in neuronal cells is selectively activated by the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3, which induce survival and differentiation, and not by epidermal growth factor. We studied a short region of the rat vgf promoter which is essential for its regulated expression. A cyclic AMP response element (CRE) within this region is necessary for NGF induction of vgf transcription. Two sites upstream of CRE, an E box and a CCAAT sequence, bind nuclear protein complexes and are involved in transcriptional control. The E box has a dual role. It acts as an inhibitor in NIH 3T3 fibroblasts, together with a second E box located downstream, and as a stimulator in the NGF-responsive cell line PC12. By expression screening, we have isolated the cDNA for a basic helix-loop-helix transcription factor, a homolog of the HTF4/HEB E protein, that specifically binds the vgf promoter E box. The E protein was present in various cell lines, including PC12 cells, and was a component of a multiprotein nuclear complex that binds the promoter in vitro. The E box and CRE cooperate in binding to this complex, which may be an important determinant for neural cell-specific expression.

The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-1 specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-1–induced NGF augmentation was mediated by the endothelin-A receptor, Giβγ, PKC, the Src family, EGFR, extracellular signal–regulated kinase, p38MAPK, activator protein-1, and the CCAAT/enhancer-binding protein δ element. Either conditioned medium or coculture with endothelin-1–stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-1–deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-1–deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late embryonic stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-1–deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-1 regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.

Two yeast artificial chromosomes containing the entire human nerve growth factor gene were isolated and mapped. By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to one of the YAC telomeres. Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts. In contrast to the plasmid-based reporter gene, expression and regulation of the YAC-based reporter gene was independent of the site of integration of the transgene. Basic fibroblast growth factor and okadaic acid stimulated expression of the YAC transgene, whereas transforming growth factor-beta and dexamethasone inhibited it. Although cyclic AMP strongly stimulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes. Downregulation of the secretion of endogenous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, but downregulation of the expression of NGF reporter genes occurred only at EC50 of 10 nM. This higher concentration was also required for upregulation of luciferase genes driven by the dexamethasone-inducible promoter of the mouse mammary tumor virus in L929 fibroblasts.

Introduction

The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been identified in the human intervertebral disc (IVD) and have been implicated in the mechanisms associated with nerve ingrowth and nociception in degeneration of the IVD. The aim of the current study was to investigate an association between neurotrophin expression in the IVD and the severity of disc degeneration, including the effect of disc-related proinflammatory cytokines on neurotrophin and neuropeptide expression in cells derived from the human IVD.

Methods

Immunohistochemical analysis was performed to examine the expression of NGF, BDNF and their high-affinity receptors Trk-A and Trk-B in human IVD samples, divided into three categories: non-degenerate, moderate degeneration and severe degeneration. In order to study the effect of disc-related cytokines on neurotrophin/neuropeptide gene expression, nucleus pulposus cells derived from non-degenerate and degenerate IVD samples were seeded in alginate and were stimulated with either IL-1β or TNFα for 48 hours. RNA was extracted, cDNA was synthesised and quantitative real-time PCR was performed to examine the expression of NGF, BDNF and substance P.

Results

Immunohistochemistry showed expression of NGF and BDNF in the native chondrocyte-like cells in all regions of the IVD and in all grades of degeneration. Interestingly only BDNF significantly increased with the severity of degeneration (P < 0.05). Similar expression was observed for Trk-A and Trk-B, although no association with disease severity was demonstrated. In cultured human nucleus pulposus cells, stimulation with IL-1β led to significant increases in NGF and BDNF gene expression (P < 0.05). Treatment with TNFα was associated with an upregulation of substance P expression only.

Conclusion

Our findings show that both the annulus fibrosus and nucleus pulposus cells of the IVD express the neurotrophins NGF and BDNF, factors that may influence and enhance innervation and pain in the degenerate IVD. Expression of Trk-A and Trk-B by cells of the nondegenerate and degenerate IVD suggests an autocrine role for neurotrophins in regulation of disc cell biology. Furthermore, modulation of neurotrophin expression by IL-1β and modulation of substance P expression by TNFα, coupled with their increased expression in the degenerate IVD, highlights novel roles for these cytokines in regulating nerve ingrowth in the degenerate IVD and associated back pain.

Nerve growth factor (NGF) is critical to the development and maintenance of the peripheral nervous system, but its possible roles in other organ systems are less well characterized. We have recently shown that human epidermal melanocytes, pigment cells derived from the neural crest, express the NGF receptor (p75 NGF-R) in vitro (Peacocke, M., M. Yaar, C. P. Mansur, M. V. Chao, and B. A. Gilchrest. 1988. Proc. Natl. Acad. Sci. USA. 85:5282-5286). Using cultured human skin-derived cells we now demonstrate that the melanocyte p75 NGF-R is functional, in that NGF stimulation modulates melanocyte gene expression; that exposure to an NGF gradient is chemotactic for melanocytes and enhances their dendricity; and that keratinocytes, the dominant epidermal cell type, express NGF messenger RNA and hence are a possible local source of NGF for epidermal melanocytes in the skin. These combined data suggest a paracrine role for NGF in human epidermis.

Background

The relationship between functional improvements in ischemic rats given a neural stem cell (NSC) transplant and the modulation of the class I major histocompatibility complex (MHC) mediated by NSC-derived neurotrophins was investigated.

Methods

The levels of gene expression of nerve growth factor (NGF), brain-derived neurotropic factor (BDNF) and neurotrophin-3 (NT-3) were assayed from cultures of cortical NSC from Sprague-Dawley rat E16 embryos. The levels of translated NGF in spent culture media from NSC cultures and the cerebral spinal fluid (CSF) of rats with and without NGF injection or NSC transplant were also measured.

Results

We found a significant increase of NGF, BDNF and NT-3 transcripts and NGF proteins in both the NSC cultures and the CSF of the rats. The immunochemical staining for MHC in brain sections and the enzyme-linked immunosorbent assay of CSF were carried out in sham-operated rats and rats with surgically induced focal cerebral ischemia. These groups were further divided into animals that did and did not receive NGF administration or NSC transplant into the cisterna magna. Our results show an up-regulation of class I MHC in the ischemic rats with NGF and NSC administration. The extent of caspase-III immunoreactivity was comparable among three arms in the ischemic rats.

Conclusion

Readouts of somatosensory evoked potential and the trap channel test illustrated improvements in the neurological function of ischemic rats treated with NGF administration and NSC transplant.

SUMMARY

Cell death in the developing retina is regulated, but so far little is known about what factors regulate the cell death. Several neurotrophic factors and receptors, including the neurotrophins and Trk receptors, are expressed during the critical time. We have studied the developing avian retina with respect to the role of nerve growth factor (NGF) in these processes. Our starting point for the work was that NGF and its receptor TrkA are expressed in a partially overlapping pattern in the inner nuclear layer of the developing retina. Our results show that TrkA and NGF-expressing cells are postmitotic. The first NGF-expressing cells were found on the vitreal side of the central region of E5.5–E6 retina. This pattern changed and NGF-expressing cells identified as horizontal cells were later confined to the external inner nuclear layer. We show that these horizontal cells co-express TrkA and NGF, unlike a subpopulation of amacrine cells that only expresses TrkA. In contrast to the horizontal cells, which survive, the majority of the TrkA-expressing amacrine cells die during a period of cell death in the inner nuclear layer. Intraocular injections of NGF protein rescued the dying amacrine cells and injection of antisense oligonucleotides for NGF that block its synthesis, caused death among the TrkA-expressing horizontal cells, which normally would survive. Our results suggest that NGF supports the survival of TrkA expressing avian horizontal cells in an autocrine mode of action in the retina of E10-E12 chicks. The cells co-express TrkA and NGF and the role for NGF is to maintain the TrkA-expressing horizontal cells. The TrkA-expressing amacrine cells are not supported by NGF and subsequently die. In addition to the effect on survival, our results suggest that NGF plays a role in horizontal cell plasticity.

Introduction

We previously described the presence of nerve growth factor receptors in the inflamed synovial compartment. Here we investigated the presence of the corresponding nerve growth factors, with special focus on nerve growth factor (NGF).

Methods

mRNA expression levels of four ligands (NGF, brain derived growth factor (BDNF), neurotrophin (NT)-3, NT-4) and their four corresponding receptors (tyrosine kinase (trk) A, trkB, trkC, NGFRp75) were determined in the synovial fluid (SF) cells of 9 patients with rheumatoid arthritis (RA) and 16 with spondyloarthritis (SpA) and compared with 7 osteoarthritis (OA) patients. NGF was also determined in synovial tissue (ST) biopsies of 10 RA and 10 SpA patients. The production of NGF by monocytes and lymphocytes was assessed by flow cytometry of SF cells, synovial tissue derived fibroblast-like synoviocytes (FLS) were assessed by ELISA on culture supernatant.

Results

SF cell analysis revealed a clear BDNF and NGF mRNA expression, with significantly higher NGF expression in RA and SpA patients than in the OA group. NGF expression was higher in ST samples of RA as compared to SpA. Using intracellular FACS analysis, we could demonstrate the presence of the NGF protein in the two inflammatory arthritis groups on both CD3+ T lymphocytes and CD14+ cells, i.e. monocytes/macrophages, whereas cultured FLS did not produce NGF in vitro.

Conclusions

Neurotrophins and especially NGF are expressed in the synovial fluid and tissue of patients with peripheral synovitis. The presence of neurotrophins as well as their receptors, in particular the NGF/trkA-p75 axis in peripheral synovitis warrants further functional investigation of their active involvement in chronic inflammatory arthritis.

Stathmin is a ubiquitous cytosolic protein which undergoes extensive phosphorylation in response to a variety of external signals. It is highly abundant in developing neurons. The use of antisense oligonucleotides which selectively block stathmin expression has allowed us to study directly its role in rat PC12 cells. We show that stathmin depletion prevents nerve growth factor (NGF)-stimulated differentiation of PC12 cells into sympathetic-like neurons although the expression of several NGF-inducible genes was not affected. Furthermore, we found that stathmin phosphorylation in PC12 cells which is induced by NGF depends on mitogen-activated protein kinase (MAPK) activity. We conclude that stathmin is an essential component of the NGF-induced MAPK signaling pathway and performs a key role during differentiation of developing neurons.

Background

Mechanisms underlying the malignant development in bladder cancer are still not well understood. Lipolysis stimulated lipoprotein receptor (LSR) has previously been found to be upregulated by P53. Furthermore, we have previously found LSR to be differentially expressed in bladder cancer. Here we investigated the role of LSR in bladder cancer.

Methods

A time course siRNA knock down experiment was performed to investigate the functional role of LSR in SW780 bladder cancer cells. Since LSR was previously shown to be regulated by P53, siRNA against TP53 was included in the experimental setup. We used Affymetrix GeneChips for measuring gene expression changes and we used Ingenuity Pathway Analysis to investigate the relationship among differentially expressed genes upon siRNA knockdown.

Results

By Ingenuity Pathway analysis of the microarray data from the different timepoints we identified six gene networks containing genes mainly related to the functional categories "cancer", "cell death", and "cellular movement". We determined that genes annotated to the functional category "cellular movement" including "invasion" and "cell motility" were highly significantly overrepresented. A matrigel assay showed that 24 h after transfection the invasion capacity was significantly increased 3-fold (p < 0.02) in LSR-siRNA transfected cells, and 2.7-fold (p < 0.02) in TP53-siRNA transfected cells compared to controls. After 48 h the motility capacity was significantly increased 3.5-fold (p < 0.004) in LSR-siRNA transfected cells, and 4.7-fold (p < 0.002) in TP53-siRNA transfected cells compared to controls.

Conclusion

We conclude that LSR may impair bladder cancer cells from gaining invasive properties.

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 air-lifted 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.

Familial dysautonomia is a hereditary disorder that affects autonomic and sensory neurons. Nerve growth factor (NGF) is required for the normal development of sympathetic and sensory neurons and it has been postulated that an abnormality involving NGF may be responsible for familial dysautonomia. Previous studies have shown that the beta-NGF gene is not linked to the disease. However, NGF appears to be abnormal by immunochemical assays; the putative altered form of NGF could result from a disturbance in the processing pathway. To study the processing of the 35-kD glycosylated NGF precursor and the secretion of NGF in familial dysautonomia, we have employed a recombinant vaccinia virus vector to express high levels of NGF mRNA in primary fibroblast cultures from patients with the disorder; the processing pathway was then studied directly. Cells from several unrelated patients all produce the same 35-kD NGF precursor, process this normally to NGF within the cell, and release NGF into the medium. There are no differences in the ability of cells from patients and from unaffected relatives to process and secrete NGF. The use of similar recombinant vaccinia virus vectors to express proteins at high level in primary cell lines should facilitate the detection of posttranslational processing defects in a variety of human disorders.

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Background

Nerve growth factor (NGF) is a potent growth factor that plays a key role in neuronal cell differentiation and may also play a role in hematopoietic differentiation. It has been shown that NGF induced synergistic action for the colony formation of CD34 positive hematopoietic progenitor cells treated with macrophage-colony stimulating factor (M-CSF or CSF-1), or stem cell factor (SCF). However, the exact role of NGF in hematopoietic system is unclear. It is also not clear whether NGF mediated signals in hematopoietic cells are identical to those in neuronal cells.

Results

To study the signal transduction pathways induced by NGF treatment in hematopoietic cells, we utilized the mastocytoma cell line HMC-1(V560G c-Kit) which expresses the NGF receptor, tropomyosin-receptor-kinase (Trk)A, as well as the constitutively activated SCF receptor, V560G c-Kit, which can be inhibited completely by treatment with the potent tyrosine kinase inhibitor imatinib mesylate (imatinib). NGF rescues HMC-1(V560G c-Kit) cells from imatinib mediated cell death and promotes proliferation. To examine the NGF mediated proliferation and survival in these cells, we compared the NGF mediated upregulated genes (30 and 120 min after stimulation) to the downregulated genes by imatinib treatment (downregulation of c-Kit activity for 4 h) by transcriptome analysis. The following conclusions can be drawn from the microarray data: Firstly, gene expression profiling reveals 50% overlap of genes induced by NGF-TrkA with genes expressed downstream of V560G c-Kit. Secondly, NGF treatment does not enhance expression of genes involved in immune related functions that were down regulated by imatinib treatment. Thirdly, more than 55% of common upregulated genes are involved in cell proliferation and survival. Fourthly, we found Kruppel-like factor (KLF) 2 and Smad family member 7 (SMAD7) as the NGF mediated novel downstream genes in hematopoietic cells. Finally, the downregulation of KLF2 gene enhanced imatinib induced apoptosis.

Conclusion

NGF does not induce genes which are involved in immune related functions, but induces proliferation and survival signals in HMC-1(V560G c-Kit) cells. Furthermore, the current data provide novel candidate genes, KLF2 and SMAD7 which are induced by NGF/TrkA activation in hematopoietic cells. Since the depletion of KLF2 causes enhanced apoptosis of HMC-1(V560G c-Kit), KLF2 may play a role in the NGF mediated survival signal.

The lower urinary tract (LUT) comprises a storage unit, the urinary bladder, and an outlet, the urethra. The coordination between the two structures is tightly controlled by the nervous system and, therefore, LUT function is highly susceptible to injuries to the neuronal pathways involved in micturition control. These injuries may include lesions to the spinal cord or to nerve fibres and result in micturition dysfunction. A common trait of micturition pathologies, irrespective of its origin, is an upregulation in synthesis and secretion of neurotrophins, most notably Nerve Growth Factor (NGF) and Brain Derived Neurotrophic Factor (BDNF). These neurotrophins are produced by neuronal and non-neuronal cells and exert their effects upon binding to their high-affinity receptors abundantly expressed in the neuronal circuits regulating LUT function. In addition, NGF and BDNF are present in detectable amounts in the urine of patients suffering from various LUT pathologies, suggesting that analysis of urinary NGF and BDNF may serve as likely biomarkers to be studied in tandem with other factors when diagnosing patients. Studies with experimental models of bladder dysfunction using antagonists of NGF and BDNF receptors as well as scavenging agents suggest that those NTs may be key elements in the pathophysiology of bladder dysfunctions. In addition, available data indicates that NGF and BDNF might constitute future targets for designing new drugs for better treatment of bladder dysfunction.

Rationale:

Nerve growth factor (NGF) promotes angiogenesis and cardiomyocyte survival, which are both desirable for postinfarction myocardial healing. Nonetheless, the NGF potential for cardiac repair has never been investigated.

Objective:

To define expression and localization of NGF and its high-affinity receptor TrkA (tropomyosin-related receptor A) in the human infarcted heart and to investigate the cardiac roles of both endogenous and engineered NGF using a mouse model of myocardial infarction (MI).

Methods and Results:

Immunostaining for NGF and TrkA was performed on heart samples from humans deceased of MI or unrelated pathologies. To study the post-MI functions of endogenous NGF, a NGF-neutralizing antibody (Ab-NGF) or nonimmune IgG (control) was given to MI mice. To investigate the NGF therapeutic potential, human NGF gene or control (empty vector) was delivered to the murine periinfarct myocardium. Results indicate that NGF is present in the infarcted human heart. Both cardiomyocytes and endothelial cells (ECs) possess TrkA, which suggests NGF cardiovascular actions in humans. In MI mice, Ab-NGF abrogated native reparative angiogenesis, increased EC and cardiomyocyte apoptosis and worsened cardiac function. Conversely, NGF gene transfer ameliorated EC and cardiomyocyte survival, promoted neovascularization and improved myocardial blood flow and cardiac function. The prosurvival/proangiogenic Akt/Foxo pathway mediated the therapeutic benefits of NGF transfer. Moreover, NGF overexpression increased stem cell factor (the c-kit receptor ligand) expression, which translated in higher myocardial abundance of c-kitpos progenitor cells in NGF-engineered hearts.

Conclusions:

NGF elicits pleiotropic beneficial actions in the post-MI heart. NGF should be considered as a candidate for therapeutic cardiac regeneration.

To explore the molecular mechanisms of nerve growth factor (NGF) action, we have attempted to identify proteins that immunoprecipitate with the NGF receptor. An anti-NGF receptor antibody was developed that immunoprecipitated the 75-Kd receptor in PC-12 cells. In [35S]methionine-labeled cells lysed with nonionic detergent, immunoprecipitation with this antireceptor antisera specifically brought down several associated proteins, although prior treatment of cells with NGF produced no apparent change in the distribution of these proteins. However, in vitro phosphorylation assays of the immunoprecipitated complex revealed the presence of a serine kinase that phosphorylated two predominant substrates with Mrs of 60 and 130 Kd. Prior treatment of cells produced no change in the appearance of the 60-Kd phosphoprotein, but NGF did stimulate the appearance of the 130-Kd protein. This effect was observed with as little as 0.1 nM NGF and was maximal at 5 min, but declined thereafter. Prior treatment of cells with NGF did not increase the phosphorylation of enolase added exogenously to the immunoprecipitates, suggesting that this action of NGF may have reflected the hormone-dependent association of the 130-Kd protein with the receptor, rather than activation of a receptor-associated kinase. Thus the association of the NGF 75-Kd receptor with a 130-Kd protein may be involved in signal transduction for the growth factor, although the role of this receptor in the NGF-dependent tyrosine phosphorylation remains unclear.

Images

We have recently shown that (a) human melanocytes express the p75 nerve growth factor (NGF) receptor in vitro; (b) that melanocyte dendricity and migration, among other behaviors, are regulated at least in part by NGF; and (c) that cultured human epidermal keratinocytes produce NGF. We now report that melanocyte stimulation with phorbol 12-tetra decanoate 13-acetate (TPA), previously reported to induce p75 NGF receptor, also induces trk in melanocytes, and TPA effect is further potentiated by the presence of keratinocytes in culture. Moreover, trk in melanocytes becomes phosphorylated within minutes after NGF stimulation. As well, cultures of dermal fibroblasts express neurotrophin-3 (NT-3) mRNA; NT-3 mRNA levels in cultured fibroblasts are modulated by mitogenic stimulation, UV irradiation, and exposure to melanocyte-conditioned medium. Moreover, melanocytes constitutively express low levels of trk-C, and its expression is downregulated after TPA stimulation. NT-3 supplementation to cultured melanocytes maintained in Medium 199 alone prevents cell death. These combined data suggest that melanocyte behavior in human skin may be influenced by neurotrophic factors, possibly of keratinocyte and fibroblast origin, which act through high affinity receptors.

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Background

Developing sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis after NGF withdrawal. This process requires de novo gene expression but only a small number of genes induced by NGF deprivation have been identified so far, either by a candidate gene approach or in mRNA differential display experiments. This is partly because it is difficult to obtain large numbers of sympathetic neurons for in vitro studies. Here, we describe for the first time, how advances in gene microarray technology have allowed us to investigate the expression of all known genes in sympathetic neurons cultured in the presence and absence of NGF.

Results

We have used Affymetrix Exon arrays to study the pattern of expression of all known genes in NGF-deprived sympathetic neurons. We identified 415 up- and 813 down-regulated genes, including most of the genes previously known to be regulated in this system. NGF withdrawal activates the mixed lineage kinase (MLK)-c-Jun N-terminal kinase (JNK)-c-Jun pathway which is required for NGF deprivation-induced death. By including a mixed lineage kinase (MLK) inhibitor, CEP-11004, in our experimental design we identified which of the genes induced after NGF withdrawal are potential targets of the MLK-JNK-c-Jun pathway. A detailed Gene Ontology and functional enrichment analysis also identified genetic pathways that are highly enriched and overrepresented amongst the genes expressed after NGF withdrawal. Five genes not previously studied in sympathetic neurons - trib3, ddit3, txnip, ndrg1 and mxi1 - were validated by real time-PCR. The proteins encoded by these genes also increased in level after NGF withdrawal and this increase was prevented by CEP-11004, suggesting that these genes are potential targets of the MLK-JNK-c-Jun pathway.

Conclusions

The sympathetic neuron model is one of the best studied models of neuronal apoptosis. Overall, our microarray data gives a comprehensive overview of, and provides new information about, signalling pathways and transcription factors that are regulated by NGF withdrawal.

Tibia fracture in rats results in chronic vascular and nociceptive changes in the injured limb resembling complex regional pain syndrome (CRPS) and up-regulates expression of interleukin 1β (IL-1β), interleukin IL-6 (IL-6), tumor necrosis factor-α(TNF-α), and nerve growth factor-β(NGF-β) in the hindpaw skin. When fracture rats are treated with cytokine or NGF inhibitors nociceptive sensitization is blocked. Because there is no leukocyte infiltration in the hindpaw skin we postulated that resident skin cells produce the inflammatory mediators causing nociceptive sensitization after fracture. To test this hypothesis rats underwent distal tibia fracture and hindlimb casting for 4 weeks, then the hindpaw skin was harvested and immunostained for keratin, cytokines and NGF. BrdU staining was used to evaluate cell proliferation. Hindpaw nociceptive thresholds, edema, and temperature were tested before and up to 96 hours after intraplantar injections of IL-6 and TNF-β. Tibia fracture caused keratinocyte activation, proliferation, and up-regulated IL-1, IL-6, TNF-α and NGF-β protein expression in the hindpaw keratinocytes. Local injections of IL-6 and TNF-α induced hindpaw mechanical allodynia lasting for several days and modest increases in temperature and edema. These data indicate that activated keratinocytes proliferate and express IL-1β, , IL-6, TNF-α, and NGF-β after fracture and that excess amounts of inflammatory mediators in the skin cause sustained nociceptive sensitization. This is the first study demonstrating in vivo keratinocyte expression of IL-6, TNF-α and NGF-β in a CRPS model and we postulate that the keratinocyte is the primary cellular source for the inflammatory signals mediating cutaneous nociceptive sensitization in early CRPS.

Due to the therapeutic potential of gene therapy for neuronal injury, many studies of neurotrophic factors, vectors, and animal models have been performed. The presumed dog β-nerve growth factor (pdβ-NGF) was generated and cloned and its expression was confirmed in CHO cells. The recombinant pdβ-NGF protein reacted with a human β-NGF antibody and showed bioactivity in PC12 cells. The pdβ-NGF was shown to have similar bioactivity to the dog β-NGF. The recombinant pdβ-NGF plasmid was administrated into the intrathecal space in the gene therapy group. Twenty-four hours after the vector inoculation, the gene therapy group and the positive control group were intoxicated with excess pyridoxine for seven days. Each morning throughout the test period, the dogs' body weight was taken and postural reaction assessments were made. Electrophysiological recordings were performed twice, once before the experiment and once after the test period. After the experimental period, histological analysis was performed. Dogs in the gene therapy group had no weight change and were normal in postural reaction assessments. Electrophysiological recordings were also normal for the gene therapy group. Histological analysis showed that neither the axons nor the myelin of the dorsal funiculus of L4 were severely damaged in the gene therapy group. In addition, the dorsal root ganglia of L4 and the peripheral nerves (sciatic nerve) did not experience severe degenerative changes in the gene therapy group. This study is the first to show the protective effect of NGF gene therapy in a dog model.

Summary

The product of the trk proto-oncogene encodes a receptor for nerve growth factor (NGF). Here we show that NGF is a powerful mitogen that can induce resting NIH 3T3 cells to enter S phase, grow in semisolid medium, and become morphologically transformed. These mitogenic effects are absolutely dependent on expression of gp140trk receptors, but do not require the presence of the previously described low affinity NGF receptor. gp140trk also serves as a receptor for the related factor neurotrophin-3 (NT-3), but not for brain-derived neurotrophic factor. Both NGF and NT-3 induce the rapid phosphorylation of gp140trk receptors and the transient expression of c-Fos proteins. However, NT-3 appears to elicit more limited mitogenic responses than NGF. These results indicate that the product of the trk proto-oncogene is sufficient to mediate signal transduction processes induced by NGF and NT-3, at least in proliferating cells.

Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.

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