The transcription factor hypoxia-inducible factor (HIF)-1 plays a central physiological role in oxygen and energy homeostasis, and is activated during hypoxia by stabilization of the subunit HIF-1α. Recent studies have demonstrated that non-hypoxic stimuli can also activate HIF-1α in a cell-specific manner. Here, we demonstrate that stimulation of BEAS-2B cells and primary human bronchial epithelial cells by proinflammatory cytokines TNFα/IL-4 strongly induced expression and transcriptional activity of HIF-1α under normoxic conditions and amplified hypoxic HIF-1α activation. TNFα/IL-4 stimulated de novo HIF-1α gene transcription and translation rather than affected HIF-1α protein degradation and mRNA decay process. The activation of HIF-1α by TNFα/IL-4 was countered by the phosphoinositol 3-kinase (PI3K) inhibitor LY-294002 and rapamycin, an antagonist of mammalian target of rapamycin (mTOR), but not by inhibition of the MAPK pathway. In line, TNFα/IL-4 also activated NF-κB, whereas blocking of NF-κB by an inhibitor or silencing NF-κB subunit p65 attenuated HIF-1α activation by TNFα/IL-4. We also found the collaborative induction of VEGF, a potent angiogenic factor required for airway remodeling, by TNFα/IL-4 and hypoxia partially via HIF-1α pathway in BEAS-2B cells. This study reports the previously unsuspected collaborative regulation of HIF-1α by TNFα/IL-4 and hypoxia in bronchial epithelial cells partially via PI3K-mTOR and NF-κB pathway, and thereby will lead to the elucidation of the importance of HIF-1 in integrating inflammatory and hypoxic response in the pathogenesis of airway diseases.
- hypoxia-inducible factor-1α
- airway epithelium
- tumor necrosis factor-α
hypoxia activates a number of genes that are important in the cellular adaptation to low oxygen conditions. Hypoxia inducible factor (HIF)-1 is a key regulator of hypoxia-inducible genes like erythropoietin and vascular endothelial growth factor (VEGF), as well as a growing number of glycolytic and metabolic enzymes (20). These genes are implicated in many different cellular functions such as cell survival, cell proliferation, apoptosis, glucose metabolism, and angiogenesis. The heterodimeric HIF-1 is composed of an oxygen-sensitive HIF-1α and a constitutive HIF-1β subunit, which both belong to the family of basic helix-loop-helix and PAS domain proteins (6, 34). HIF-1 activity is primarily regulated by the abundance of the HIF-1α subunit. Under normoxic conditions, HIF-1α is targeted for rapid degradation by hydroxylation of specific prolyl residues (Pro 402 and Pro 564), which are catalyzed by specific oxygenases, identified as prolyhydroxylase domain-containing proteins (PHDs) (43). Another factor in the degradation process is the Von Hippel-Lindau tumor suppressor protein, which facilitates degradation of HIF-1α through the ubiquitin-proteasome pathway(30). Under hypoxic conditions, HIF-1α is stabilized, translocates into the nucleus where it dimerizes with HIF-1β, and transactivates downstream target genes containing hypoxia-response elements (HRE) within their promoter or enhancer.
In addition to hypoxia, growth factors, lipopolysaccharides, and proinflammatory cytokines such as IL-1β and TNFα have been demonstrated to be critical regulators of HIF-1α activation(3, 13, 19, 50). There is growing evidence that HIF-1α is involved in the inflammatory process by regulating angiogenesis and functions of inflammatory cells (11, 53). Persistent inflammation of the respiratory tract in chronic pulmonary disease is mediated by increased expression of multiple inflammation mediators (1). The bronchial airway epithelium plays a critical role not only in the maintenance of physicochemical homoeostasis of the airways but also in the pathogenesis of airway diseases. During airway inflammation the epithelium is both a source of mediator production as well as a target of remodeling processes(42). Immunohistochemistry revealed induction of HIF-1α in bronchial epithelium cells by hypoxia (51). Studies showed that HIF-1α play an intergrative role in conditions of hypoxia and inflammation, and the microenvironments of bronchial epithelial cells are characterized by inflammatory and hypoxic conditions (39). Moreover, an orchestrated change among posttranscriptional mechanisms in human bronchial epithelial BEAS-2B cells following inflammatory stimulation has been documented, and treatment of BEAS-2B cells with IL-4/TNFα can increase the expression of a number of genes coding for cytokines and chemokines (52). Thus, in the present study, we investigated the potential activation of HIF-1α by IL-4/TNFα and hypoxia in bronchial airway epithelium cells and identified the signaling pathways involved in the process. We found that TNFα/IL-4 induced HIF-1α transcription and translation in BEAS-2B cells and primary human bronchial epithelial cells under normoxic conditions and amplified hypoxic HIF-1α activation. This response involves the activation of the phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway and of NF-κB. Furthermore, TNFα/IL-4 and hypoxia induced VEGF expression synergistically via the HIF-1α pathway. Because the microenvironments of bronchial epithelial cells are characterized by inflammatory and hypoxic conditions during the pathogenesis of inflammatory airway diseases, the cooperative regulation of HIF-1α by inflammatory cytokines and hypoxia provides important new insight into inflammatory airway diseases and identifies novel therapeutic targets.
MATERIALS AND METHODS
Cells and reagents.
The human bronchial epithelial cell line BEAS-2B cells were purchased from American Type Culture Collection (Rockville, MD). The cells were grown and maintained as previously described (5). Primary human bronchial epithelial cells (ScienCell, San Diego, CA) were maintained in bronchial epithelial cell medium by following the manufacturer's instructions. For TNFα/IL-4 stimulation, the medium was replaced, and the cells were cultured in medium containing 50 ng/ml each cytokine as described (52) or under hypoxic conditions for up to 24 h. None of the agents used significantly affected cell morphology or viability under these conditions. Unless indicated otherwise, all reagents used were obtained from Sigma-Aldrich.
Exposure to hypoxia.
Hypoxia and normoxia protocol was set up as we described previously (28). Briefly, on the day of the experiment, the medium was replaced with a thin layer of fresh medium (0.15 ml/cm2) with 10% fetal calf serum to decrease the diffusion distance of the ambient gas. Culture dishes were then placed in a humidified airtight incubator with inflow and outflow valves, and the hypoxic gas mixture (1% O2, 5% CO2, and balance N2) was delivered at 10 l/min for 25 min. The airtight incubator was kept at 37°C for preset time periods, whereas normoxic cells were placed at 37°C in a humidified incubator (5% CO2, 95% air) until harvest.
Reverse transcription and real-time quantitative PCR.
Real-time quantitative PCR estimation of mRNA levels was performed as described previously (28). Real-time quantitative PCR was performed by using SYBR green I as fluorescent dye (TaKaRa Biotechnology, Dalian, China). Primers were commercially obtained (Shanghai Sangon Biologic Engineering & Technology and Service, Shanghai, China), and the specific nucleotide sequences were as follows: HIF-1α, 5′-CCA GCA GAC TCA AAT ACA AGA ACC-3′ (sense) and 5′-TGT ATG TGG GTA GGA GAT GGA GAT-3′ (antisense); VEGF, 5′-TCT ACC TCC ACC ATG CCA AGT-3′ (sense) and 5′-GAT GAT TCT GCC CTC CTC CTT-3′ (antisense); β-actin, 5′-GTG GGC CGC CCT AGG CAC CA-3′ (sense) and 5′-GGT TGG CCT TAG GGT TCA GG-3′ (antisense). Amplification and detection were performed by using an ABI PRISM 7700 detection system (Applied Biosystems, Foster City, CA). Real-time PCR was performed in triplicate reactions with 20 ng of complementary DNA in a final volume of 10 μl containing 1× SYBR Green Master Mix and 200 nM of both primers. Agarose gel electrophoresis, purification, and DNA sequencing confirmed the identity of the PCR products. Reference gene β-actin, which was not altered by TNFα/IL-4 or any other treatment applied in this study, was used for normalization of the expression data.
Western blot analysis.
Whole cell or nuclear extracts were prepared as described previously (28). A total of 100 μg of protein was loaded onto a 10–15% SDS/polyacrylamide gel, and, after electrophoresis, it was blotted onto nitrocellulose membranes. The primary rabbit anti-human HIF-1α polyclonal antibodies (Upstate), mouse anti-human IL-8 monoclonal antibody (R&D Systems), polyclonal total Akt and phospho-Akt (Ser473, Cell Signaling Technology), total and phospho-4E-BP1, total and phosphor-p70s6k, NF-κB p65 antibody (Santa Cruz, CA), and anti-β-actin monoclonal antibody were used. The anti-rabbit IgG secondary antibody (KPL) was used at 1:2,000 dilution, and the signal was analyzed by enhanced chemiluminescence (Chemicon). The protein abundance was quantitated by means of densitometry with the values being calculated after normalization to the amount of β-actin using software Quantity One 4.2.3.
HIF-1α transcriptional activity assay.
The luciferase reporter plasmids (pGL3-luc) harboring HRE from human VEGF gene promoter region(−88/−54) and its HRE mutant were kindly provided by Dr. Amit Maity (Dept. of Radiation Oncology, Univ. of Pennsylvania School of Medicine, Philadelphia, PA). The Renilla luciferase expression plasmid pRL-SV40 was obtained from Promega (Madison, WI). BEAS-2B cells were plated 5 × 105 cells/well on the day before transfection. Each plate was transfected with a mixture of 200 ng of reporter gene plasmid and 50 ng of control plasmid pRL-SV40 for transfection efficiency, premixed with Lipofectamine 2000 and Opti-MEM reduced serum medium (Invitrogen, Shanghai, China). After 6 h, cells were recovered overnight and subsequently incubated with or without TNFα/IL-4 under normoxic conditions for another 18 h. The cells were harvested, and the luciferase activity of each well was measured in the same dosage of cell lysate with the use of Dual-Luciferase Reporter Assay System (Promega). The ratio of firefly:Renilla luciferase activity was determined and normalized to the values obtained from normoxic cells with the medium to obtain the relative luciferase activity.
RNA interference experiments.
The small interference RNA (siRNA) oligonucleotide for HIF-1α was designed as we described previously (29). Sense and antisense RNA strands for HIF-1α-siRNA: 5′-UGU GAG UUC GCA UCU UGA UTT-3′ and 5′-AUC AAG AUG CGA ACU CAC ATT-3′; for p65-siRNA: 5′-CUC AAG AUC UGC CGA GUA ATT -3′ and 5′-UUA CUC GGC AGA UCU UGA GTT-3′ were commercially synthesized (Genetimes Technology, Shanghai, China). Transfection of siRNA was performed at a concentration of 100 nM using Lipofectamine 2000. As a control for siRNA, we used a corresponding random siRNA sequence (scrambled siRNA: sense 5′-AGU UCA ACG ACC AGU AGU CTT-3′ and antisense 5′-GAC UAC UGG UCG UUG ATT-3′). Control cells were transfected without oligonucleotides under the same conditions. After transfection, the cells were split in aliquots, grown in six-well dishes for 24 h, and then exposed to normoxic or indicated condition for preset time periods. Cells were lysed, and total RNA or protein was extracted as described above.
Nuclear extracts were prepared from cells using a nuclear extraction kit according to the manufacturer's instructions (Pierce), and aliquots were incubated with γ-32P-ATP-labeled oligonucleotides encompassing the binding sites for NF-κB (5′-AGT TGA GGG ACT TTC CCA GGC-3′) (TaKaRa). EMSA was performed as previously described(12).
One-way analysis of variance was used to compare mean values across multiple treatment groups with a Tukey post hoc multiple comparison test. Statistical significance was defined as P <0.05. All data are expressed throughout the text as means ± SD of the indicated number of experiments.
Activation of HIF-1α by TNFα/IL-4 and hypoxia in BEAS-2B cells and primary human bronchial epithelial cells.
Previous investigation demonstrated that the response of BEAS-2B cells to TNFα/IL-4 was likely to be evident at 18 h of exposure (52). So we incubated cells under normoxic or hypoxic conditions for 0, 4, 8, 18, and 24 h with or without TNFα/IL-4 stimulation. Significant HIF-1α accumulation was first detected after 4 h of hypoxia. TNFα/IL-4 likewise induced HIF-1α accumulation even under normoxic conditions at 4 h, and peaking at 18 and 24 h of stimulation. At all time points, TNFα/IL-4 synergistically enhanced the hypoxia-induced HIF-1α accumulation (Fig. 1A). We then evaluated whether HIF-1α induced by TNFα/IL-4 is transcriptionally active. TNFα/IL-4 alone induced a moderate increase in the transcriptional activity of HIF-1α, which was significantly enhanced when cells were treated with TNFα/IL-4 under hypoxic conditions (Fig. 1B). To test whether TNFα/IL-4-induced HIF-1α transcriptional activity resulted in the expression of a HIF-1 target gene, VEGF expression was quantified. Treatment with TNFα/IL-4 for 18 h induced VEGF mRNA levels under normoxic and hypoxic conditions (Fig. 1C). To further confirm the activation of HIF-1α by TNFα/IL-4, we investigated the induction of HIF-1α in primary bronchial epithelial cells. Similar results were found in primary bronchial epithelial cells, with HIF-1α protein expression and transcriptional activity induction being found at 18 h of incubation with TNFα/IL-4 under normoxic and hypoxic conditions (Fig. 1, D and E). Collectively, these results illustrate that TNFα/IL-4 can induce HIF-1α activation in BEAS-2B cells and primary bronchial epithelial cells.
TNFα/IL-4 induces HIF-1α through mechanisms that are distinct from hypoxic induction.
We noticed a striking difference between the kinetics of HIF-1α protein accumulation induced by hypoxia and by TNFα/IL-4 (Fig. 1A). This indicates that different mechanisms are implicated in the activation of HIF-1α by hypoxia and by TNFα/IL-4 in these cells. It has been shown that increased transcription of HIF-1α gene was important for HIF-1α protein induction by nonhypoxic stimulations (3). Therefore, we evaluated whether TNFα/IL-4 could modify the levels of HIF-1α mRNA in BEAS-2B cells. As seen in Fig. 2A, TNFα/IL-4 stimulated the expression of HIF-1α mRNA under normoxic and hypoxic conditions. Hypoxia did not induce the expression of HIF-1α mRNA in BEAS-2B cells, as was the case in other cell models (3, 37). To determine whether mRNA stability contributes to the induction of HIF-1α mRNA in activated bronchial epithelial cells, we examined the decay of HIF-1α mRNA in nonstimulated and TNFα/IL-4 stimulated BEAS-2B cells. The cells were treated with the transcription inhibitor actinomycin D (Act D), and mRNA half-life was analyzed as we described previously (28). The level of the control β-actin transcript remained similar in the cells with or without stimulation (data not shown). The results showed that TNFα/IL-4 treatment did not affect the decay of HIF-1α mRNA (Fig. 2B). By contrast, hypoxia dramatically decreased the half-life of HIF-1α mRNA compared with normoxia (Fig. 2C). To clearly demonstrate that increased mRNA levels were important for HIF-1α protein induction, BEAS-2B cells were pretreated with Act D before stimulation with TNFα/IL-4 for 18 h. Real-time PCR revealed that the TNFα/IL-4-induced increase in HIF-1α expression was abolished by Act D under normoxic and hypoxic conditions (Fig. 3A). Respective changes in HIF-1α protein levels were observed with Act D treatment (Fig. 3B). The Act D inhibition was not due to a toxic effect because the ability of TNFα/IL-4 to increase IL-8, a protein known to be activated by TNFα/IL-4 in BEAS-2B cells (52), was not decreased (Fig. 3B). It is noteworthy to mention that HIF-1α protein levels induced during hypoxia were not decreased by treatment with Act D (Fig. 3B). These experiments suggest that increased transcription of HIF-1α mRNA is crucial to maintain elevated levels of HIF-1α in BEAS-2B cells by TNFα/IL-4. This pathway is distinct from hypoxic induction of HIF-1α in these cells, in which HIF-1α protein stability plays a decisive role (21). Nevertheless, these results also raised the question of whether increased amounts of HIF-1α protein were the result of reduced degradation of the protein or increased translation of the mRNA. To test whether TNFα/IL-4 increased the translation of HIF-1α mRNA, cycloheximide (CHX), an inhibitor of translation, was added 60 and 15 min before the end of 18-h incubation with TNFα/IL-4 under normoxic conditions. HIF-1α protein accumulation was clearly reduced by CHX (Fig. 3C), indicating that HIF-1α protein accumulation by TNFα/IL-4 is also the result of enhanced translation of HIF-1α mRNA. Very recently, it has been found that nonhypoxic stimulation stabilizes HIF-1α via the degradation pathway (33, 36). We then examined whether TNFα/IL-4 would stabilize HIF-1α protein. BEAS-2B cells were exposed to TNFα/IL-4 and hypoxia for 18 h and then returned to normoxia (reoxygenation). Although TNFα/IL-4 increased HIF-1α protein levels when incubated under hypoxic conditions, it did not affect HIF-1α degradation on reoxygenation (Fig. 3D). On the transition to reoxygenation, HIF-1α completely disappeared within 15 min in hypoxia and hypoxia plus TNFα/IL-4-treated cells. Moreover, inhibition of the proteasome pathway by MG132 led to a high level of HIF-1α, and TNFα/IL-4 induced HIF-1α in the presence of MG132 (Fig. 3E). Collectively, TNFα/IL-4 treatment seemed to increase HIF-1α protein levels by increasing HIF-1α gene transcription and translation rather than by inhibiting proline hydroxylase-dependent degradation pathway.
Implication of PI3K and NF-κB pathways in HIF-1α activation by TNFα/IL-4 in BEAS-2B cells.
Activation of PI3K pathway plays a major role in the induction of HIF-1α by different nonhypoxic stimuli (3, 14, 16, 25, 26, 31, 37, 46, 49). Studies suggest that activation of the PI3K pathway preferentially increases HIF-1α protein translation (26, 37, 49). Moreover, it has been shown that TNFα/IL-4 stimulation resulted in a global translation upregulation in BEAS-2B cells (52). To determine the signal transduction pathways mediating the effects of TNFα/IL-4 on HIF-1α activation, BEAS-2B cells were pretreated with LY-294002 or wortmannin (Calbiochem, San Diego, CA), which are selective pharmacological inhibitors of PI3K. In the BEAS-2B cells, TNFα/IL-4 activated the PI3K pathway, as seen by the phosphorylation of AKT (Fig. 4A). LY-294002 and wortmannin blocked the activation of AKT and also strongly inhibited the increase in HIF-1α protein expression by TNFα/IL-4 (Fig. 4A). Moreover, rapamycin, an inhibitor of mTOR kinase activity, also attenuated the induction of HIF-1α protein expression by TNFα/IL-4 (Fig. 4B). In contrast to their effects on the expression of HIF-1α induced by TNFα/IL-4 treatment, wortmannin had little inhibitory effect on the expression of HIF-1α in hypoxia-treated cells (Fig. 4C), providing further evidence that TNFα/IL-4 and hypoxia act by distinct molecular mechanisms. In contrast to the effects of these inhibitors, PD-98059, at concentrations that effectively inhibit the MAP kinase MEK1 (data not shown), evidenced only minimal effects on this induction of HIF-1α by hypoxia or TNFα/IL-4 in these cells (Fig. 4C). The inhibition of TNFα/IL-4-induced HIF-1α expression was achieved almost to the same degree by wortmannin alone and by the combined treatment with PD-98059 and wortmannin (Fig. 4C). Furthermore, the induction of HIF-1α mRNA, transcriptional activity, and VEGF mRNA expression was inhibited by these agents as seen for the inhibition of HIF-1α protein expression (Fig. 4, D–F). However, the contents of HIF-1α and VEGF in the cells preincubated with wortmannin or rapamycin were still higher than those in the control group, indicating that the inhibition of PI3K-AKT-mTOR pathway could not completely inhibit the induction effect of TNFα/IL-4 on expression of HIF-1α and VEGF. These results therefore demonstrate PI3K-AKT-mTOR pathway is partially involved in the activation of HIF-1α by TNFα/IL-4 in BEAS-2B cells. The signal transduction pathway involving PI3K-AKT-mTOR has been shown to regulate protein translation via phosphorylation of 4E-BP1 and p70s6k (15, 18, 38). In the present study, the phosphorylation of 4E-BP1 and p70s6k, which was induced by TNFα/IL-4 treatment for 4 or 18 h, could be blocked by wortmannin or rapamycin (Fig. 5), an effect consistent with its inhibition of TNFα/IL-4-induced HIF-1α and VEGF expression.
Recently, the involvement of NF-κB in cytokine-induced HIF-1α activation was proposed, especially the role of NF-κB in the transcription of HIF-1α gene (2, 13, 23, 50). To characterize in greater detail the downstream signaling pathways inherent to TNFα/IL-4-induced HIF-1α activation, we treated BEAS-2B cells with the NF-κB inhibitor pyrrolidinium dihthiocarbamate (PDTC) before incubation with TNFα/IL-4. By EMSA we found NF-κB activation by TNFα/IL-4 was reduced with PDTC (Fig. 6A). Accordingly, HIF-1α mRNA, protein, and transcriptional activity induced by TNFα/IL-4 was attenuated by PDTC (Fig. 6, B–D). To circumvent potential side effects of drugs, we substantiated the role of NF-κB by transfecting p65 siRNA into BEAS-2B cells, and then assessed the levels of HIF-1α expression. Transfection of cells with p65 siRNA significantly reduced the protein levels of p65 compared with cells transfected with a scrambled control siRNA (Fig. 6E). The knockdown of p65 also greatly reduced the effects of TNFα/IL-4 on HIF-1α activation (Fig. 6, E–G). Nevertheless, blocking of NF-κB activation cannot completely suppress the activation of HIF-1α by TNFα/IL-4, indicating other pathways involved in the activation effects of TNFα/IL-4 on HIF-1α in these cells.
Role of HIF-1α in TNFα/IL-4 and hypoxia-induced VEGF expression in BEAS-2B cells.
Finally, we aimed at elucidating the importance of HIF-1α in TNFα/IL-4-induced expression of the HIF-1 target gene VEGF. In the first step, we established a specific RNAi directed against HIF-1α. BEAS-2B cells were transfected with HIF-1α RNAi 24 h before the start of the experiments. After siRNA treatment, hypoxia and TNFα/IL-4 failed to induce the accumulation of HIF-1α protein in BEAS-2B cells (Fig. 7, A and B). Specificity of the selected sequence was confirmed by transfecting a mutant HIF-1α RNAi (RNAi scramble) that had neither an effect on hypoxia nor TNFα/IL-4-induced HIF-1α and VEGF accumulation (Fig. 7, A–D). Silencing of HIF-1α had no effect on the constitutive VEGF expression but significantly reduced the TNFα/IL-4-induced expression of VEGF under normoxic and hypoxic conditions (Fig. 7, C and D). Interestingly, however, although hypoxic and TNFα/IL-4-dependent HIF-1α induction was almost completely abolished by HIF-1α RNAi treatment, TNFα/IL-4 still induced VEGF expression, indicating that other signaling pathways could also be involved in this effect. Thus, HIF-1α seems to be partially involved in the TNFα/IL-4-induced VEGF expression in BEAS-2B cells.
The first report to relate HIF-1α with inflammation and inflammatory mediators is that IL-1β and TNFα stimulate the DNA binding of HIF-1 (19). Subsequent studies have demonstrated that inflammatory cytokines and inflammatory mediators such as IL-4 are critical regulators of the activation of HIF-1α (17, 41). Interestingly, some of these inductions have been shown to be equal or greater than the hypoxic induction of HIF-1α. In this study, we show that in bronchial epithelial cells: 1) TNFα/IL-4 and hypoxia can synergistically activate HIF-1α, 2) in contrast to hypoxia, TNFα/IL-4 does not regulate HIF-1α by the inhibition of its degradation but by de novo HIF-1α gene transcription and translation-dependent mechanisms, 3) the activation of HIF-1α by TNFα/IL-4 is mediated in part by the PI3K-AKT-mTOR and NF-κB pathways 4) HIF-1α pathway is involved in the stimulation of VEGF expression by TNFα/IL-4 and hypoxia.
Although hypoxia increases HIF-1α levels in all cell types, an increasing body of evidence indicates that this transcription factor may also be upregulated by a number of nonhypoxic stimuli in a cell type-specific manner (11). It is interesting that the mechanisms involved in activating HIF-1α under hypoxic and nonhypoxic conditions are strikingly different, insofar as unlike hypoxia, nonhypoxic stimuli do not induce HIF-1 by means of HIF-1α stabilization. Although very recently, it has been shown that nonhypoxia stimuli such as angiotensin II and transforming growth factor-β1 induced HIF-1α via protein stabilization (33, 36). Studies carried out by a number of laboratories have found that the predominant mechanism in this induction is an increase in HIF-1α protein translation via the PI3K pathway and mitogen-activated protein kinase pathway (32). Meanwhile, we recently have reported that volatile anesthetic isoflurane stimulates HIF-1α protein synthesis in the human hepatoma cell line Hep3B cells (29). So we focused first on the two pathways as a potential part of the intracellular signaling cascade activated by TNFα/IL-4 in BEAS-2B cells. In our current study, we found a significant activation of PI3K/AKT pathway after TNFα/IL-4 stimulation, which was followed by increased HIF-1α protein expression. The PI3K inhibitor LY-294002 and wortmannin induced a significant blockage of TNFα/IL-4-induced HIF-1α expression. Rapamycin, an mTOR inhibitor, abrogated the TNFα/IL-4-mediated induction of HIF-1α. However, our results indicate that the MEK inhibitor PD-98059 inhibited TNFα/IL-4-induced HIF-1α expression only partially. Our data demonstrate a striking correlation between the inhibition of TNFα/IL-4-induced HIF-1α protein and the inhibition of eIF-4E binding protein 1 (4E-BP1) and p70s6K phosphorylation by wortmannin and rapamycin in BEAS-2B cells. mTOR, a member of the PI3K-related kinase family, is an important signaling molecule downstream of PI3K and Akt (35, 44). In particular, mTOR activates p70s6K and 4E-BP1, thereby controlling the translation of specific subsets of mRNA (40, 45). These results demonstrate that activation of HIF-1α in response to TNFα/IL-4 stimulation is dependent on PI3K-AKT-mTOR kinase activity. Thus, by coordinately upregulating the levels of key factors necessary for translation, activated BEAS-2B cells can accommodate the elevated levels of HIF-1α mRNAs induced by TNFα/IL-4 treatment. However, inhibition of PI3K-AKT-mTOR pathway could not abrogate the activation effect of TNFα/IL-4 on HIF-1α completely, suggesting mechanisms other than this pathway involved in the process.
It has recently become apparent that HIF-1α and NF-κB, as well as acting independently in the regulation of gene expression in hypoxic inflammation, have a significant and profound level of cross talk (48). Moreover, a recent study shows short-term hypoxia activates NF-κB involving a PI3K/AKT-dependent pathway, resulting in a rapid and transient increase in HIF-1α mRNA and protein levels in pulmonary artery smooth muscle cells (2). In the present study, we show that TNFα/IL can stimulate NF-κB p65 subunit DNA binding activity. In addition, we demonstrate that inhibition or knockdown of NF-κB p65 subunits can attenuate HIF-1α mRNA, protein, and transcriptional activity levels induced by TNFα/IL-4. These results demonstrate that HIF-1α pathway is also partially reliant upon NF-κB signaling in response to TNFα/IL-4 stimulation in vitro. NF-κB has been previously shown to contribute to the regulation of HIF-1α protein, but not HIF-1α mRNA, by TNFα or colchicine in fibroblasts, and to HIF-1α translation by HGF in HepG2 cells (22, 24, 47, 54). Thus, the transcriptional regulation of HIF-1α by PI3K-AKT and NF-κB may act in concert with translation and stabilization of HIF-1α and appears to be cell type and/or stimulus specific. TNFα/IL-4 was also shown to induce VEGF production in BEAS-2B cells (52). However, the exact contribution of HIF-1α in TNFα/IL-4-induced VEGF production remained unexplored. Herein, we demonstrated that TNFα/IL-4-induced HIF-1α also plays a significant role in VEGF production in response to TNFα/IL-4 under hypoxic and normoxic conditions. VEGF, a potent angiogenic factor, is required for airway remodeling (27). Our findings are in line with recent results showing that HIF-1 may participate in airway epithelial remodeling under inflammatory conditions (4). This result provides an additional mechanistic link between inflammation and VEGF expression, and suggests that the same transcription factor HIF-1α partially mediates the production of VEGF triggered by both the hypoxic and the inflammatory zones of inflamed tissue. In addition, it was noticed that although the increased contents of VEGF induced by TNFα/IL-4 and hypoxia could be significantly inhibited by HIF-1α RNAi, they were still higher than those in the control group. This implied that some other pathways might also be involved in hypoxia and TNFα/IL-4-induced VEGF expression apart from the HIF-1α pathway.
The microenvironmental conditions found in areas of inflammation are characterized by low levels of oxygen (8). Several studies have shown the cellular responses in inflammation and hypoxia are closely linked (7, 9, 10). Our data underline the importance of HIF-1α in integrating the inflammatory and hypoxic response. During inflammation, low oxygen alone may not fully explain the activation of HIF-1α. Instead, the combination with cytokines may be important to assure fine-tuning of the system. Our data document evidence of a synergism between inflammatory cytokines and hypoxia on HIF-1α activation in bronchial epithelial cells, thus providing important new insight into inflammatory airway diseases and identifying novel therapeutic targets.
This study was supported by the Nature Science Foundation of Shanghai Science Committee (No. 074119626) and Shanghai Rising-Star Program (No. 08QA14044).
No conflicts of interest are declared by the author(s).
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