Identification of dynamic proteome changes upon ligand activation of Trk-receptors using two-dimensional fluorescence difference gel electrophoresis and mass spectrometry

The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.     

Glucose regulates expression of the nerve growth factor (NGF) receptors TrkA and p75NTR in rat islets and INS-1E beta-cells

BACKGROUND: The function and survival of pancreatic beta-cells strongly depend on glucose concentration and on autocrine secretion of peptide growth factors. NGF and its specific receptors TrkA and p75NTR play a pivotal role in islet survival and glucose-dependent insulin secretion. We therefore investigated whether or not glucose concentration influences expression of TrkA and p75NTR in rat islets and in INS-1E beta-cells at the mRNA and protein level (INS-1E). METHODS: Gene expression of the NGF receptors TrkA and p75NTR but also of the metabolic gene liver-type pyruvate kinase (L-PK) and the neurotrophin receptors TrkB and TrkC was studied by semi-quantitative PCR and by real-time PCR in islets and INS-1E beta-cells. RESULTS: In rat islets, high glucose exposure (25 mmol/l) increased gene expression of TrkA, p75NTR and L-PK. Expression of TrkA, p75NTR and L-PK reflected insulin secretion at the respective glucose concentration. In rat INS-1E insulinoma cells, expression of L-PK and p75NTR was suppressed by low glucose as in the islets, while expression of TrkA was strongly increased by low glucose levels and thus was regulated differently than in islets. Expression of TrkB and TrkC was not regulated by glucose concentration at all. TrkA protein was regulated in the same fashion as its mRNA expression, while p75NTR protein was not significantly regulated within 24 h. CONCLUSION: Glucose interacts with gene expression of TrkA and p75NTR that are strongly involved in beta-cell growth and glucose-dependent insulin secretion. The fact that TrkA expression is regulated the opposite way in islets and in INS-1E beta-cells might reflect their specific grade of differentiation and tendency to proliferate.     

p53 is required for nerve growth factor-mediated differentiation of PC12 cells via regulation of TrkA levels

p53 is necessary for the elimination of neural cells inappropriately differentiated or in response to stimuli. However, the role of p53 in neuronal differentiation is not certain. Here, we showed that nerve growth factor (NGF)-mediated differentiation in PC12 cells is enhanced by overexpression of wild-type p53 but inhibited by mutant p53 or knockdown of endogenous wild-type p53, the latter of which can be rescued by expression of exogenous wild-type p53. Interestingly, p53 knockdown or overexpression of mutant p53 attenuates NGF-mediated activation of TrkA, the high-affinity receptor for NGF and a tyrosine kinase, and activation of the mitogen-activated protein kinase pathway. In addition, p53 knockdown reduces the constitutive levels of TrkA, which renders PC12 cells inert to NGF. And finally, we showed that both constitutive and stimuli-induced expressions of TrkA are regulated by p53 and that induction of TrkA by activated endogenous p53 enhances NGF-mediated differentiation. Taken together, our data demonstrate that p53 plays a critical role in NGF-mediated neuronal differentiation in PC12 cells at least in part via regulation of TrkA levels.     

P53/PUMA are potential targets that mediate the protection of brain-derived neurotrophic factor (BDNF)/TrkB from etoposide-induced cell death in neuroblastoma (NB)

The over-expressions of brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor TrkB have been reported to induce chemo-resistance in neuroblastoma (NB) cells. In this study, we investigated the roles of P53 and BCL2 family members in the protection of BDNF/TrkB from etoposide-induced NB cell death. TB3 and TB8, two tetracycline (TET)-regulated TrkB-expressing NB cell lines, were utilized. The expressions of P53 and BCL2 family members were detected by Western blot or RT-PCR. Transfection of siRNAs was used to knockdown P53 or PUMA. Activated lentiviral was used to over-express PUMA. Cell survival was performed by MTS assay, and the percentage of cell confluence was measured by IncuCyte ZOOM. Our results showed that etoposide treatment induced significant and time-dependent increase of P53, which could be blocked by pre-treatment with BDNF, and knockdown P53 by transfecting siRNA attenuated etoposide-induced TrkB-expressing NB cell death. PUMA was the most significantly changed BCL2 family member after treatment with etoposide, and pre-treatment with BDNF blocked the increased expression of PUMA. Transfection with siRNA inhibited etoposide-induced increased expression of PUMA, and attenuated etoposide-induced NB cell death. We also found that over-expression of PUMA by infection of activated lentiviral induced TrkB-expressing NB cell death in the absence of etoposide, and treatment of BDNF protected NB cells from PUMA-induced cell death. Our results suggested that P53 and PUMA may be potential targets that mediated the protection of BDNF/TrkB from etoposide-induced NB cell death.     

Expression of neurotrophin receptors in normal and malignant B lymphocytes

In order to define a cellular model suitable for studying, in vitro, the molecular properties and functions of neurotrophin receptors in human lymphocytes, TrkA, TrkB, TrkC and p75(NTR) expression was investigated in a panel of EBV immortalized lymphoblastoid (LCL) and Burkitt lymphoma-derived cell lines (BLs) compared to primary B lymphocytes by RT-PCR and flow cytometric analysis. Our data show that trkA and trkB are transcribed in most B cell lines of normal and malignant origin. For several of them, we also gained first evidence of trkC expression in B cells. All cell lines and primary B cells lack p75(NTR) expression. These data suggest that neurotrophin receptors expression in the B cell lines correlates to some extent with the phenotypic maturation stage and endogenous viral activity levels. Our data suggest that TrkA and TrkB, once activated, provide a partial rescue from apoptosis, whereas TrkC stimulates the progression through the cell cycle without affecting cell survival. Finally, the identification of a number of cell lines showing single expression of one of the Trk receptors has disclosed the availability of a cellular tool for further studies on their function, and mechanisms of signal transduction in the B cell moiety in the absence of p75(NTR).     

Expression of DeltaNp73 is a molecular marker for adverse outcome in neuroblastoma patients

The p73 gene is a p53 homologue which induces apoptosis and inhibits cell proliferation. Although p73 maps at 1p36.3 and is frequently deleted in neuroblastoma (NB), it does not act as a classic oncosuppressor gene. In developing sympathetic neurons of mice, p73 is predominantly expressed as a truncated anti-apoptotic isoform (DeltaNp73), which antagonizes both p53 and the full-length p73 protein (TAp73). This suggests that p73 may be part of a complex tumor-control mechanism. To determine the role of DeltaNp73 in NB we analyzed the pattern of expression of this gene in vivo and evaluated the prognostic significance of its expression. Our results indicate that DeltaNp73 expression is associated with reduced apoptosis in a NB tumor tissue. Expression of this variant in NB patients significantly correlates with age at diagnosis and VMA urinary excretion. Moreover it is strongly associated with reduced survival (HR=7.93; P<0.001) and progression-free survival (HR=5.3; P<0.001) and its role in predicting a poorer outcome is independent from age, primary tumor site, stage and MYCN amplification (OS: HR=5.24, P=0.012; PFS: HR=4.36, P=0.005). In conclusion our data seem to indicate that DeltaNp73 is a crucial gene in neuroblastoma pathogenesis.     

Romidepsin (FK228/depsipeptide) controls growth and induces apoptosis in neuroblastoma tumor cells

As histone deacetylase inhibitors such as romidepsin (depsipeptide, FK228) complete successful Phase I clinical trials in pediatric solid tumors, it is important that their mechanisms of action are delineated in order to inform the development of subsequent clinical trials as single agents or in combination therapies. In this study, we evaluate the effect of romidepsin as a single agent on a number of different neuroblastoma (NB) cell lines. We find that the growth of 6/6 human NB tumor cell lines but not an immortalized fibroblast cell line (NIH3T3) is inhibited by romidepsin (IC50 = 1-6.5 ng/ml) after 72 h of treatment. Romidepsin shows selective dose-dependent cytotoxicity in both single copy and N-myc amplified NB cell lines, in cell lines with wild-type or mutant p53 and those containing Alk mutations. The decrease in cell proliferation is accompanied by caspase-dependent apoptosis as shown by PARP cleavage, an accumulation of cells in the sub-G1 phase of the cell cycle and the ability of a pan-caspase inhibitor to reduce cell death. Romidepsin inhibits the growth of subcutaneous NB xenografts in a dose dependent manner in immunocompromised mice. Furthermore, romidepsin induces expression of genes such as p21 and expression of p75 and NTRK (TrkA) which are more highly expressed in the tumors from NB patients that have a good prognosis. These studies support continued investigations into the therapeutic activity of romidepsin in NB.     

Mechanisms of Neuroblastoma Cell Growth Inhibition by CARP-1 Functional Mimetics

Neuroblastomas (NBs) are a clinically heterogeneous group of extra cranial pediatric tumors. Patients with high-risk, metastatic NBs have a long-term survival rate of below 40%, and are often resistant to current therapeutic modalities. Due to toxic side effects associated with radiation and chemotherapies, development of new agents is warranted to overcome resistance and effectively treat this disease in clinic. CARP-1 functional mimetics (CFMs) are an emerging class of small molecule compounds that inhibit growth of diverse cancer cell types. Here we investigated NB inhibitory potential of CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited NB cell growth, in vitro, independent of their p53 and MYCN status. CFM-4 and -5 induced apoptosis in NB cells in part by activating pro-apoptotic stress-activated kinases (SAPKs) p38 and JNK, stimulating CARP-1 expression and cleavage of PARP1, while promoting loss of the oncogenes C and N-myc as well as mitotic cyclin B1. Treatments of NB cells with CFM-4 or -5 also resulted in loss of Inhibitory κB (IκB) α and β proteins. Micro-RNA profiling revealed upregulation of XIAP-targeting miR513a-3p in CFM-4-treated NB, mesothelioma, and breast cancer cells. Moreover, exposure of NB and breast cancer cells to CFM-4 or -5 resulted in diminished expression of anti-apoptotic XIAP1, cIAP1, and Survivin proteins. Expression of anti-miR513a-5p or miR513a-5p mimic, however, interfered with or enhanced, respectively, the breast cancer cell growth inhibition by CFM-4. CFMs also impacted biological properties of the NB cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Our studies indicate anti-NB properties of CFM-4 and 5, and suggest that these CFMs and/or their future analogs have potential as anti-NB agents.     

Cytoplasmically "sequestered" wild type p53 protein is resistant to Mdm2-mediated degradation.

The Mdm2 oncoprotein mediates p53 degradation at cytoplasmic proteasomes and is the principal regulator for maintaining low, often undetectable levels of p53 in unstressed cells. However, a subset of human tumors including neuroblastoma constitutively harbor high levels of wild type p53 protein localized to the cytoplasm. Here we show that the abnormal p53 accumulation in such cells is due to a profound resistance to Mdm2-mediated degradation. Overexpression of Mdm2 in neuroblastoma (NB)(1) cell lines failed to decrease the high steady state levels of endogenous p53. Moreover, exogenous p53, when introduced into these cells, was also resistant to Mdm2-directed degradation. This resistance is not due to a lack of Mdm2 expression in NB cells or a lack of p53-Mdm2 interaction, nor is it due to a deficiency in the ubiquitination state of p53 or proteasome dysfunction. Instead, Mdm2-resistant p53 from NB cells is associated with covalent modification of p53 and masking of the modification-sensitive PAb 421 epitope. This system provides evidence for an important level of regulation of Mdm2-directed p53 destruction in vivo that is linked to p53 modification.     

Expression of the TrkA or TrkB receptor tyrosine kinase alters the double-strand break (DSB) repair capacity of SY5Y neuroblastoma cells

In the childhood tumor neuroblastoma, high expression of the TrkA neurotrophin receptor is associated with a favorable prognosis and a lack of structural chromosomal changes, whereas TrkB is expressed in aggressive neuroblastomas demonstrating high genomic instability. The ability to repair DNA double-strand breaks (DSBs) is considered a central determinant of chromosomal stability with nonhomologous end joining (NHEJ) being the major pathway of DSB repair in vertebrates. Here, we used the SH-SY5Y human neuroblastoma cell line ectopically expressing either TrkA or TrkB as a model system to analyze the impact of Trk receptor expression on NHEJ-mediated DSB repair. In a cell-free NHEJ assay, SY5Y-TrkA cells displayed a significantly higher efficiency for NHEJ compared to SY5Y-TrkB cells. To detect possible underlying mechanisms, gene expression data (Affymetrix U95A microarray chips) obtained from the same SY5Y-TrkA/TrkB model system were reanalyzed focussing on genes involved in DNA repair. Expression of XRCC4, a central component of NHEJ, was significantly upregulated in SY5Y-TrkA compared to SY5Y-TrkB cells. Expression data were confirmed using real-time PCR and western blotting. Additionally, XRCC4 expression was enhanced in most primary neuroblastomas with high TrkA expression. The TrkA-induced increase in NHEJ activity could be reverted by XRCC4 knock-down, confirming the induction of XRCC4 by TrkA to be essential for the observed phenotype. Our data provide the first evidence for a functional relationship between tyrosine kinase receptor signaling and NHEJ-mediated DSB repair in cancer cells, potentially contributing to their genomic stability.     

Rotary bioreactor culture can discern specific behavior phenotypes in Trk-null and Trk-expressing neuroblastoma cell lines

Neuroblastoma is characterized by biological and genetic heterogeneity that leads to diverse, often unpredictable, clinical behavior. Differential expression of the Trk family of neurotrophin receptors strongly correlates with clinical behavior; TrkA expression is associated with favorable outcome, whereas TrkB with unfavorable outcome. Neuroblastoma cells cultured in a microgravity rotary bioreactor spontaneously aggregate into tumor-like structures, called organoids. We wanted to determine if the clinical heterogeneity of TrkA- or TrkB-expressing neuroblastomas was reflected in aggregation kinetics and organoid morphology. Trk-null SY5Y cells were stably transfected to express either TrkA or TrkB. Short-term aggregation kinetics were determined by counting the number of single (non-aggregated) viable cells in the supernatant over time. Organoids were harvested after 8 d of bioreactor culture, stained, and analyzed morphometrically. SY5Y-TrkA cells aggregated significantly slower than SY5Y and SY5Y-TrkB cells, as quantified by several measures of aggregation. SY5Y and TrkB cell lines formed irregularly shaped organoids, featuring stellate projections. In contrast, TrkA cells formed smooth (non-stellate) organoids. SY5Y organoids were slightly smaller on average, but had significantly larger average perimeter than TrkA or TrkB organoids. TrkA expression alone is sufficient to dramatically alter the behavior of neuroblastoma cells in three-dimensional, in vitro rotary bioreactor culture. This pattern is consistent with both clinical behavior and in vivo tumorigenicity, in that SY5Y-TrkA represents a more differentiated, less aggressive phenotype. The microgravity bioreactor is a useful in vitro tool to rapidly investigate the biological characteristics of neuroblastoma and potentially to assess the effect of cytotoxic as well as biologically targeted drugs.     

Caffeine induces G2/M arrest and apoptosis via a novel p53-dependent pathway in NB4 promyelocytic leukemia cells

Methylxantine derivative, caffeine, is known to prevent the p53-dependent apoptosis pathway via inhibition of ATM (ataxia telangiectasia mutated) kinase, which activates p53 by phosphorylation of the Ser-15 residue. In contrast, it has been reported that caffeine induces p53-mediated apoptosis through Bax protein in non-small-cell lung cancer cells. Therefore, the effects of caffeine on cellular growth in malignant cells are controversial. We investigated the effects of caffeine on cell proliferation, cell cycle progression, and induction of apoptosis in NB4 promyelocytic leukemia cells containing wild-type p53. Caffeine suppressed the cellular growth of NB4 cells in a dose- and time-dependent manner. Caffeine induced G(2)/M phase cell cycle arrest in NB4 cells in association with the induction of phosphorylation at the Ser-15 residue of p53 and induction of tyrosine phosphorylation of cdc2. Expression of Bax protein was increased in NB4 cells after treatment with caffeine. Interestingly, the antisense oligonucleotides for p53 significantly reduced p53 expression and caffeine-induced G(2)/M phase cell cycle arrest in NB4 cells. These results suggest that caffeine induces cell cycle arrest and apoptosis in association with activation of p53 by a novel pathway to phosphorylate the Ser-15 residue and induction of phosphorylation of cdc 2 in leukemic cells with normal p53.     

Two distinctly altered cellular responses to DNA double-strand breaks in human neuroblastoma

Neuroblastoma (NB), the most common extracranial solid tumors in children, presents with numerous genetic abnormalities that accumulate in a very short lifetime. To better understand this process, we have induced DNA double-strand breaks in NB cell lines and analyzed the activation of the ATM-H2AX/Chk2-p53 signaling pathway. We have found that NB cells could be classified into two distinct groups. The first group strongly expressed activated Chk2, displayed an important sub-G1 population, expressed very low levels of p21, and exhibited an attenuated G1 arrest. Conversely, the second group weakly expressed Chk2 pT68, displayed no sub-G1 cell population, strongly expressed p21, and exhibited a functional G1 arrest. These findings were independent of the MYCN amplification or p53 status of the NB cell lines tested. Interestingly, most p21 weakly expressing NB cells expressed neuron-specific enolase and Bcl2, two markers of N-type NB cells, but did not express vimentin, a marker of S-type NB cells. The expression profile was reversed in the p21 strongly expressing NB cells which highly expressed vimentin. Along with additional data, our findings lead us to propose that N-type-like NB cells would survive under stress conditions by antagonizing the Chk2-dependent apoptosis pathway, whereas S-type-like NB cells would survive by down-regulating Chk2 expression to facilitate the crossing of the senescence barrier.     

神经生长因子及其受体相关信号传导通路的研究进展

神经生长因子是神经营养因子家族成员之一,对不同时期神经元的存活、分化、生长及损伤后的修复和再生都有着十分重要的作用。不仅在神经系统中,随着人类的其他正常和肿瘤组织中同样也检测得到了NGF,神经生长因子在各方面的应用也得到了重视并均已得到了证实。NGF功能的发挥离不开与其受体的结合,根据NGF表面糖蛋白与凝集素结合能力的不同,其受体可被分为高亲和力受体酪氨酸激酶A和低亲和力受体p75。Trk A与NGF结合后所介导的信号通路主要有:1MAPK通路;2PLC-γ通路;3PI3K/PKB通路。而p75与NGF结合介导的信号传导通路主要包括:1NF-κB通路;2JNK-p53-Bax凋亡通路;3神经酰胺通路。Trk A一般介导的是正性信号,如促进神经细胞生长、维持神经细胞的存活等;而p75既可促进神经细胞存活,也可诱导神经细胞凋亡,但以后者为主。当Trk A与p75同时表达时,Trk A可抑制p75诱导细胞凋亡,使受损神经细胞大量增殖,所以其生物学总效应是促进神经细胞的生长和存活。     

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen interacts with multifunctional angiogenin to utilize its antiapoptotic functions

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with the angioproliferative Kaposi's sarcoma (KS). KSHV infection and the expression of latency-associated nuclear antigen (LANA-1) upregulates the angiogenic multifunctional 123-amino-acid, 14-kDa protein angiogenin (ANG), which is detected in KS lesions and in KSHV-associated primary effusion lymphoma (PEL) cells. ANG knockdown or the inhibition of ANG's nuclear translocation resulted in decreased LANA-1 gene expression and reduced KSHV-infected endothelial and PEL cell survival (Sadagopan et al., J. Virol. 83:3342-3364, 2009). Further studies here demonstrate that LANA-1 and ANG colocalize and coimmunoprecipitate in de novo infected endothelial cells and in latently infected PEL (BCBL-1 and BC-3) cells. LANA-1 and ANG interaction occurred in the absence of the KSHV genome and other viral proteins. In gel filtration chromatography analyses of BC-3 cell lysates, ANG coeluted with LANA-1, p53, and Mdm2 in high-molecular-weight fractions, and LANA-1, p53, and Mdm2 also coimmunoprecipitated with ANG. LANA-1, ANG, and p53 colocalized in KSHV-infected cells, and colocalization between ANG and p53 was also observed in LANA-1-negative cells. The deletion constructs of ANG suggested that the C-terminal region of amino acids 104 to 123 is involved in LANA-1 and p53 interactions. Silencing ANG or inhibiting its nuclear translocation resulted in decreased nuclear LANA-1 and ANG levels, decreased interactions between ANG-LANA-1, ANG-p53, and LANA-1-p53, the induction of p53, p21, and Bax proteins, the increased cytoplasmic localization of p53, the downregulation of Bcl-2, the increased cleavage of caspase-3, and the apoptosis of cells. No such effects were observed in KSHV-negative BJAB cells. The phosphorylation of p53 at serine 15, which is essential for p53 stabilization and for p53's apoptotic and cell cycle regulation functions, was increased in BCBL-1 cells transduced with short hairpin RNA targeting ANG. Together, these studies suggest that the antiapoptosis observed in KSHV-infected cells and the suppression of p53 functions are mediated in part by ANG, and KSHV has probably evolved to utilize angiogenin's multiple functions for the maintenance of its latency and cell survival. Thus, targeting ANG to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV infection and associated malignancies.     

Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line <Emphasis Type="Italic">in vitro</Emphasis>

Induction of p53 gene expression in cancer cells can lead to both cell cycle arrest and apoptosis. To clarify whether the level of p53 expression determines the apoptotic response of hepatocellullar carcinoma (HCC) cells, we assessed the effect of various levels of expression of p53 gene on a p53-deficient HCC cell line, Hep3B, utilizing a doxycycline (Dox)-regulated inducible p53 expression system. Our results showed that apoptosis was induced in HCC cells with high levels of p53 expression. However, lower level of p53 expression induced only cell cycle arrest but not apoptosis. Bax expression was up-regulated following high levels of p53 expression, while bcl-2 expression was not altered by the level of p53 expression. Moreover, p21 expression was observed in both high and low expression of p53. These results suggest the level of p53 expression could determine if the HCC cells would go into cell cycle arrest or apoptosis. Bax may participate, at least in part, in inducing p53-dependent apoptosis and the induction of p21 alone was able to cause cell cycle arrest but not apoptosis.     

Neurotrophins and Neurotrophin Receptors in Proliferative Diabetic Retinopathy

Neurotrophins (NTs) are emerging as important mediators of angiogenesis and fibrosis. We investigated the expression of the NTs nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) and their receptors TrkA, TrkB, and TrkC in proliferative diabetic retinopathy (PDR). As a comparison, we examined the expression of NTs and their receptors in the retinas of diabetic rats. Vitreous samples from 16 PDR and 15 nondiabetic patients were studied by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Epiretinal membranes from 17 patients with PDR were studied by immunohistochemistry. Rats were made diabetic with a single high dose of streptozotocin and retinas of rats were examined by Western blot analysis. Western blot analysis revealed a significant increase in the expression of NT-3 and NT-4 and the shedding of receptors TrkA and TrkB in vitreous samples from PDR patients compared to nondiabetic controls, whereas NGF and BDNF and the receptor TrkC were not detected with the use of Western blot analysis and ELISA. In epiretinal membranes, vascular endothelial cells and myofibroblasts expressed NT-3 and the receptors TrkA, TrkB and TrkC in situ, whereas NT-4 was not detected. The expression levels of NT-3 and NT-4 and the receptors TrkA and TrkB, both in intact and solubilized forms, were upregulated in the retinas of diabetic rats, whereas the receptor TrkC was not detected. Co-immunoprecipitation studies revealed binding between NT-3 and the receptors TrkA and TrkB in the retinas of diabetic rats. Our findings in diabetic eyes from humans and rats suggest that the increased expression levels within the NT-3 and NT-4/Trk axis are associated with the progression of PDR.