The Mechanisms Of TrKB Activation In A549 Cell

Background:Lung cancer continues to be the leading cause of death among the malignant tumors worldwide. Lung cancer can be separated into two major forms: non-small cell lung cancer (NSCLC) and small cell lung cancer, which account for 80 and 20% of all lung carcinomas, respectively. The incidence of non-small cell lung cancer (NSCLC) is still increasing. Given the insensitivity to cytotoxic agents, identifying molecules that drive lung cancer growth, survival, and metastasis is critical.Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily which has been indicated in the pathophysiology of the nervous system, and is important in a number of neurological and psychological conditions. Recently, several studies indicated that BDNF and/or its receptor, TrkB, might be involved in cancer growth and metastasis in several malignant tissues, such as neuroblastoma, pancreatic ductal carcinoma, prostate cancer, hepatocellular carcinoma, and lung cancer. While At present, little is known about the molecular mechanisms that elicit signalings downstream of TrkB in the progression of NSCLC.Members of the signal transducer and activator of transcription (STAT) family of transcription factors are potential targets for the treatment and prevention of cancers including non-small-cell lung cancer. Signal transducer and activator of transcription 3 (STAT3) has long been shown to regulate gene transcription in response to cytokines and growth factors through JAK1 or src-kinase.Researches indicated STAT3 as a downstream mediator of Trk signaling and functions in PC12 cells and in major pelvic ganglia (MPG) of rats.While whether STAT3 is also a mediator of BDNF/TrkB signaling in lung cancers, is still undown. In this study, we report, in A549 cells, BDNF stimuli increase the activation of STAT3, and the increased STAT3 activity further increase the synthesis of BDNF and that the release of BDNF can, in turn, activate prolonged TrkB signaling.Objective:1. To investigate whether STAT3 is also a mediator of BDNF/TrkB signaling in lung cancers,.2. To investigate whether BDNF stimuli increase the activation of STAT3.Methods:1. Cell culture and treatmentsThe human pulmonary epithelial cell line A549 was maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) and 5mM glutamine and cultured at 37° C in a humidified atmosphere of 5% CO2. The cells were seeded in six-well plates at 80% confluence.2. Western blotProtein level was measured by Western blot. Briefly, equal amounts of protein (10μg per lane) were separated by SDS-PAGE electrophoresis, and transferred to PVDF membrane (Bio-Rad Inc.). The membranes were incubated in blocking buffer (0.2 mM Tris,137 mM NaCI,5% no-fat milk, and 0.1% Tween-20) for an hour and then probed at 4℃ overnight with KL antibody. The membranes were rinsed with washing buffer (0.1% Tween 20,0.2 mM Tris, and 137 mM NaCl) and incubated with HRP-conjugated secondary antibody (1:5000) for an hour at room temperature, followed by chemiluminescent detection.3. Quantitative RT-PCR.Synthesis of BDNF was measured by quantitative RT-PCR. cells were washed with PBS followed by RNA extraction. Total RNA was extracted with TRNzol-A+ RNA isolation reagent (TIANGEN) according to the manufacturer’s instructions. Reverse transcription was performed with 1 u g of total RNA and RevertAid First Strand cDNA Synthesis Kit (Fermentas). Quantitative RT-PCR was performed in a cycler (MyiQ2, Bio-Rad) using SYBR green (Roche). The threshold cycle for each sample was chosen from the linear range and converted to a starting quantity by interpolation from a standard curve run on the same plate for each set of primers. The BDNF mRNA levels were normalized for each well to the β-actin mRNA levels using the 2-△△CT method. Each experiment was repeated three times.4. Statistical analysis.Statistical significance was assessed using Student’s t-test or analysis of variance (ANOVA). Data were presented as mean ± SEM, and p<0.05 was considered significant. All the experiments were repeated in three parallels.Results:TrkB is constitutively actived in human lung cancer cell line A549To characterize TrkB expression and activation status in vitro, A549 cells in different conditions was examined using Western blot analysis with pan-TrkB antibody to detect TrkB protein expression, or with phosphor-Trk antibody to detect activated TrkB, respectively. The cells pretreated with BDNF for 1h was used as a positive control. As shown in Fig1A, TrkB is commonly expressed and activated in A549 cells. The phospholation level of TrkB is decreased in A549 cells serumed starved for 1h, while the level of phosphorylated TrkB was increased in A549 cells serum starved for 24h. The sponataneous recovery of phosphorylation of TrkB in serum-deprived condition suggested the presence of an autocrine mechanism. To confirm this possibility, we detected the expression of BDNF, the ligand of TrkB receptors, in A549 cells (Fig.1B). The lysate of HEK293 transfected with BDNF overexpression construct was used as a positive control. The results indicated that there is endogenous expression of BDNF. This finding strongly suggests BDNF as the secretary factor that induced TrkB activation in A549 cells.STAT3 is the major medicator for BDNF expression in A549 cellsWhile what’s the medicator of BDNF expression in A549 cells? Signal transducer and activator of transcription 3 (STAT3) has long been shown to regulate gene transcription, and play roles in the progression of NSCLC. To study whether STAT3 is activated in serum-deprived condition, we performed western blot analysis to analysis the phosphorylation level of Stat3. The tyrosine phosphorylation of Stat3 in A549 cells was elevated after serum starvation for 24h, compared with control group. The result of tyrosine phosphorylation of Stat3 was similar (Fig.2A). The spontaneous recovery of Stat3 activation in A549 cells in serum-deprived condition suggested the presence of an unknown mechanism to ugregulateing STAT3 activity. Furthermore, levels of both BDNF mRNA and protein were reduced when cells were pretreated with specific STAT3 inhibitor——Sattic (Fig.2B). While under serum-deprived condition, Stat3 activity inhibition by Sattic could block A549 cell the response to serum starvation (Fig.2D).BDNF is a major regulate factor for Stat3 activation in A549 cellsSTAT3 was reported to be the downstream signaling target of BDNF/TrkB according to previous study. To study whether the activation of STAT3 is also regulated by BDNF/TrkB in A549 cells, we detected the phospholation level of STAT3 in cells with or without Trk activity inhibitor K252a. The results indicated that blocking TrkB activity could decrease the STAT3 phospholation; while more BDNF treatment could further active STAT3 (Fig.3A). The result indicated that BDNF works as regulator of STAT3. To confirm this possibility, the A549 cells were serum-deprived and applied to K252a in medium for 12 h. Then the phosphorylation of Stat3 was detected in serum-starved A549 cells under indicated medium. The results shown that, block TrkB activity could slowdown the spontaneous recovery of Stat3 activation in A549 cells in serum-deprived condition. This finding strongly suggests that BDNF works partially as a secretory factor that induced Stat3 activation in A549 cell.STAT3 induced BDNF expression is Involved in the Constitutive Activation of Akt.To examine if STAT3 induced BDNF expression was associated with the activation of Akt, a key event in the survival of cells exposed to different apoptotic stimuli such as serum deprivation; we used K252a and Sattic, a drug that inhibits STAT3 activity specifically to inhibit endogenous STAT3 activity in A549cells, and then we detected the phosphorylation of Akt. As shown, the phosphorylation of Akt was decreased by Sattic or K252a treatment. The results indicated that both BDNF/TrkB and STAT3 activity are involved in the Constitutive Activation of Akt. What is more, exogenous BDNF rescued the phosphorylation of Akt in cells treated with Sattic. These results strongly suggest that the STAT3 induced BDNF expression is Involved in the constitutive activation of Akt.DiscussionLung cancer continues to be the leading cause of death among the malignant tumors worldwide. The incidence of non-small cell lung cancer (NSCLC), the main form of lung cancer, is still increasing. Because of the insensitivity to cytotoxic agents, identifying molecules that drive lung cancer growth, survival, and metastasis is critical.BDNF is a member of the nerve growth factor family and is important to the phenotypic behavior of neurons or neural system cancers. BDNF mediates the cellular biological effects mainly through a tyrosine kinase receptor, tropo-myosin-related konase B (TrkB). The expression level and impact of TrkB has been reported in several types of carcinomas, although how this protein exerts its effects remains to be identified. The expression of TrkB and BDNF reported to be associated with poor prognosis in NSCLC patients. BDNF/TrkB signal is involved in malignant progression of invasiveness and tumorigenicity for large cell neuroendocrine carcinoma (LCNEC), and may be a potential target for LCNEC (12,13). While little is known about how the BDNF/TrkB signal is activated.In this study, we found a positive autocrine feedback loop existed between A549-derived BDNF and subsequent TrkB activation as well as the mechanisms underlying this positive feedback loop. Our suggest that Activation of STAT3 could drive BDNF expression; BDNF stimuli could further increase the synthesis of BDNF through activating STAT3 in A549 cells, and that the release of BDNF can, in turn, activate TrkB signaling and downstream signaling. A positive autocrine BDNF feedback loop from A549 cells may contribute to A549 biological function. Our results provide several new insights into the mechanism BDNF expression and signaling regulation.First, we reported that the activation TrkB in A549 cells was triggered by the autoctine BDNF; further, we indicated that this autocrine BDNF was dependent on STAT3 activity. According to previous report, there are overexpression and abnormal activation of TrkB in lung cancers, and exogenous BDNF could increase the TrkB activation, and affect the downstream function. But still we do not know, where is the source of BDNF in vivo. By using RT-PCR and WB blot, we results shown that there is endogenous expression of BDNF in A549 cells; and these engogenous expression of BDNF is involved in regulation of TrkB activity. Also, we found the BDNF expression is under control of STAT3 in A549 cells. Inhibiting STAT3 activity by Sattic, could decrease the BDNF expression. That is to say, STAT3 is a critical regulator of BDNF in A549 cells. Second, we found BDNF/TrkB signaling could further upregulate the activation of STAT3 in turn, these upregulation could further increase the expression of BDNF, and the activation of TrkB. In other word, not only STAT3 could regulate BDNF expression, BDNF could control STAT3 activaty reversely. Those BDNF controlled upregultation of STAT3 activation, will further strengthen the BDNF expression. So there is a Positive feedback loop of Autocrine BDNF from A549 cells, and that might be the reason of enhanced STAT3 and TrkB activation in lung cancers.Third, we found the STAT3 dependent positive feedback loop of autocrine BDNF is involved in activation of PI3K-AKT signaling. Our results shown, in A549 cells, block STAT3 activation by inhibitor, could cause the reduction of AKT activity; while this reduction could be rescued by exogenous BDNF. The result suggests that the STAT3 dependent positive feedback loop of autocrine BDNF is involved in AKT activity regulation in A549 cells.Significance:In this study, we report there is a STAT3 dependent positive feedback loop of autocrine BDNF:the STAT3 activity trigger the BDNF expression and activation of TrkB; this activation enhance the activity of STAT3, and further strengthen the BDNF expression, TrkB activation and downstream sigamling,such as PI3K-AKT. This positive autocrine BDNF feedback loop from A549 cells may contribute to A549 biological function.