Microtubule Disruption Utilizes an NFkappa B-dependent Pathway to Stabilize HIF-1alpha Protein*

Yun-Jin JungDagger , Jennifer S. IsaacsDagger , Sunmin Lee§, Jane Trepel§, and Len NeckersDagger

From the Dagger  Cell and Cancer Biology Branch, Center for Cancer Research, NCI, National Institutes of Health, Rockville, Maryland 20850 and the § Medical Oncology Clinical Research Unit, CCR, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, September 24, 2002, and in revised form, December 11, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hypoxia-inducible factor (HIF)-1alpha levels are elevated in normoxic cells undergoing physiological processes involving large scale microtubule reorganization, such as embryonic development, wound healing, and tumor cell metastasis. Although alterations in microtubules affect numerous cellular responses, no data have yet implicated microtubule dynamics in HIF-1alpha regulation. To investigate the effect of microtubule change upon HIF-1alpha regulation, we treated cells with the microtubule-depolymerizing agents (MDAs) colchicine, vinblastine or nocodazole. We demonstrate that these agents are able to induce transcriptionally active HIF-1. MDA-mediated induction of HIF-1alpha required microtubule depolymerization, since HIF-1alpha levels were unchanged in cells treated with either the microtubule-stabilizing agent paclitaxel, or an inactive form of colchicine, or in colchicine-resistant cells. HIF-1 induction was dependent upon cellular transcription, as transcription inhibitors abrogated HIF-1alpha protein up-regulation. The ability of transcriptional inhibitors to interfere with HIF-1alpha accumulation was specific to the MDA-initiated pathway, as they were ineffective in preventing hypoxia-mediated HIF-1 induction, which occurs by a distinct post-translational pathway. Moreover, we provide evidence implicating a requirement for NFkappa B transcription in the HIF-1 induction mediated by MDAs. The ability of MDAs to induce HIF-1alpha is dependent upon activation of NFkappa B, since inhibition of NFkappa B either pharmacologically or by transfection of an NFkappa B super-repressor plasmid abrogated this induction. Collectively, these data support a model in which NFkappa B is a focal point for the convergence of MDA-mediated signaling events leading to HIF-1 induction, thus revealing a novel aspect of HIF-1alpha regulation and function.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hypoxia-inducible factor (HIF-1)1 is a heterodimeric transcription factor composed of the basis helix-loop-helix-PAS -domain containing proteins HIF-1alpha and aryl hydrocarbon receptor nuclear translocator (ARNT, HIF-1beta ) (1). HIF-1alpha and HIF-1beta mRNAs are constantly expressed under normoxic and hypoxic conditions (2). However, HIF-1alpha protein is significantly increased by hypoxia, whereas the HIF-1beta protein remains constant regardless of oxygen tension (3). Under normoxia, HIF-1alpha protein is remarkably unstable and its degradation by the proteasome is orchestrated by the ubiquitin protein ligase VHL (3-6). Under normoxia, VHL recognizes HIF-1alpha as a substrate due to the enzymatic modification of HIF-1alpha by prolyl hydroxylases, whose function is inhibited during hypoxia (7, 8). Hypoxic stabilization of HIF-1alpha is accompanied by its nuclear translocation, heterodimerization with HIF-1beta , and transcription of genes encoding proteins that function to increase O2 delivery, allow metabolic adaptation, and promote cell survival (9). HIF-transactivated genes such as iNOS, IGF, and VEGF play an important role in tumor metastasis and invasion (10, 11) and HIF-1alpha protein is overexpressed in a majority of nonhypoxic metastasic tumors and cell lines (12).

One of the major components of the cytoskeleton is the microtubule network. Because of the dynamic instability of tubulin dimers, microtubules are subject to constant remodeling (13). MDAs are potent anti-tumor agents that associate with microtubules and disrupt the microtubular system, thereby blocking cell division (14-16). The action of MDAs is thought to loosely mimic a wide range of cellular responses involving cytoskeletal rearrangement, such as wound healing, tumor cell metastasis, and invasion (17, 18). Microtubule reorganization has been also shown to correlate with changes in gene expression (19-21). For instance, it has been reported that microtubule disruption by MDAs modulates gene expression and activity of protein kinases and transcription factors such as NFkappa B (22-28).

NFkappa B is an ubiquitous transcription factor known to be activated by a wide variety of stimuli including infection, inflammation, oxidative stress, and the aforementioned microtubule disruption (29). NFkappa B transactivates a number of proinflammatory, apoptotic and oncogenic genes that collectively function to foster cellular adaptation to stress (29, 30). Although the mechanism of activation depends on the stimulus, most stimuli initiate various intracellular signaling cascades that result in the phosphorylation of inhibitory protein kappa B (Ikappa B) by Ikappa B kinases (IKKs) (31). NFkappa B is normally associated with Ikappa B in the cytoplasm, where it is kept in an inactive state (32). Stimulus-mediated phosphorylation and subsequent proteolytic degradation of Ikappa B (33, 34) allows the release and nuclear translocation of NFkappa B, where it transactivates a number of target genes.

The pathways involved in the nonhypoxic stabilization of HIF-1alpha remain unclear but are thought to be regulated by growth factor signaling cascades such as PI 3-kinase/AKT (35, 36). Interestingly, HIF-1alpha has been reported to be highly expressed in cells during physiological processes that entail massive microtubule reorganization (12, 37-39). However, there are no reports demonstrating a direct relationship between changes in microtubule dynamics and HIF-1alpha protein regulation. Therefore, in this study, we specifically investigated this connection. We show that reagents interfering with tubulin polymerization are able to induce NFkappa B transcription and we further show that this activation is necessary for the subsequent increase in HIF-1alpha protein expression. These results demonstrate a novel aspect of HIF-1alpha regulation and suggest that HIF-1alpha may play a broader role in sensing cytoskeletal change.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents-- The MDAs vinblastine, colchicine, and nocodazole, and the microtubule stabilizing agent paclitaxel were purchased from Sigma. The colchicine deriviative gamma -lumicolchicine and pyrrolidinedithiocarbamate and the transcriptional inhibitors actinomycin D (AcD) and 5,6-dichlorobenzimidazole riboside were also purchased from Sigma. Cobalt chloride and the iron chelator phenanthroline were obtained from the same supplier. The protein synthesis inhibitor cycloheximide (CHX) was from Sigma.

Cells and Transient Transfection-- A549 cells (human lung cancer cell line obtained from American Type Culture Collection) were cultured in F12-K (Kaighn's modification) medium (Invitrogen). Colchicine-resistant CHO (10193) and wild type CHO (10001) (a gift from Dr. M. M. Gottesman, National Institutes of Health) were grown in alpha -modified minimal essential medium (Sigma). Jurkat cells were cultured in RPMI 1640 medium (Biofluids). Unless specified, all other cell lines were grown in Dulbecco's modified Eagle's medium (DMEM, Biofluids). Media were supplemented with 10% fetal bovine serum, glutamine (for DMEM), Hepes, and penicillin/streptomycin. For transient transfections of NFkappa B super-repressor plasmid (40) or HIF-1alpha plasmids (41), cells were plated in 6-cm dishes and transfected with NFkappa B super-repressor plasmid (5 ug) or HIF-1alpha plasmids (3 ug) in the presence of FuGENE 6 (Roche Molecular Biochemicals). After 24 h, cells were subjected to the indicated drug treatments, lysates were harvested, and HIF-1alpha levels determined by Western blotting. HIF-1 protein stability was determined by treatment of cells with 200 µM CHX, followed by immunoblot and densitometric analysis. For transient transfection of reporter plasmids, cells were plated in 12-well plates and the following day cells were cotransfected with luciferase reporter plasmids containing either 3× NFkappa B binding sites (0.4 µg, a gift from Dr. M. Birrer, NCI) or 3× hypoxia response element (0.4 µg, a gift from Dr. G. Melillo, NCI), in combination with the internal control CMV Renilla luciferase plasmid (1:100 the amount of reporter plasmid, Promega). Luciferase activities of reporter plasmids were measured using the Dual-Luciferase Reporter Assay System (Promega). Transfection efficiency was evaluated using green fluorescence protein expression plasmid and determined to be 35-45% under these experimental conditions. Cell viability was determined by the trypan blue exclusion method. Cell viability was unchanged in each experimental condition.

Western Blotting-- Cells were lysed and nuclear and cytosolic extracts prepared as described (42). Cell lysates were electrophoretically separated using either 4-20 or 7.5% SDS-PAGE gels (BioRad). Proteins were transferred to nitrocellulose membrane (Protran, Schleicher & Schuell) and imunoblottted with either monoclonal anti-HIF-1alpha , polyclonal anti-iNOS antibodies (1:300 and 1:500, respectively, Transduction Laboratories), monoclonal anti-HA antibody (1:1000, Covance) or monoclonal anti-alpha -tubulin antibody (1:2000, Santa Cruz Biotechnology). HIF-1alpha in human cell lines was detected in 20 µg of nuclear extracts and HIF-1alpha in non-human cell lines was detected in 30-40 µg of nuclear extracts using monoclonal HIF-1alpha antibody (1:750, Novus). iNOS protein was detected in 40 µg of cytosolic extracts. All blots were developed with SuperSignal chemiluminescence substrate (Pierce) using anti-mouse horseradish peroxidase IgG (Amersham Biosciences).

Quantitative RT-PCR Analysis for HIF-1alpha Expression-- Cells were treated with MDAs for 3 h and lysed and total mRNA was extracted using Rneasy Mini Kit (Qiagen). The real-time quantification of HIF-1alpha mRNA was carried out using SYBR Green I dye (Applied Biosystems) with the following primer pairs: human HIF-1alpha forward, 5'-TCCAGTTACGTTCCTTCGATCA-3'; human HIF-1alpha reverse, 5'-TTTGAGGACTTGCGCTTTCA-3'. SYBR Green I, double-stranded DNA binding dye, was detected using the laser-based ABI Prism 7700 Sequence Detection System (Applied Biosystems). PCR amplification was performed using an optical 96-well reaction plate and caps. The final reaction mixture of 25 µl consisted of 200 nM each primer, 1× SYBR Green PCR Master Mix (Applied Biosystems) containing a reference dye, and cDNA at the following conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The cDNAs were prepared from each RNA sample using a TaqMan Reverse Transcription kit (Applied Biosystems).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MDAs Induce HIF-1alpha in Various Cell Lines under Normoxia-- Overexpression of HIF-1alpha and large scale changes in microtubule organization are common events associated with embryonic development, wound healing, and tumor cell invasion and metastasis. We therefore sought to investigate whether a correlation existed between microtubule disruption and HIF-1alpha expression. To examine this question, A549 cells were treated with the MDAs vinblastine, colchicine, or nocodazole, or with the microtubule-stabilizing agent paclitaxel, and HIF-1alpha protein expression was monitored. The concentrations used represent those required for maximal microtubule disruption (43). As shown in Fig. 1A, HIF-1alpha protein levels were similarly induced by all of the MDAs tested, while HIF-1alpha protein remained unchanged following treatment with paclitaxel, indicating that increased HIF-1alpha protein expression correlated with microtubule depolymerization, and not with stabilization. In Fig. 1B, the kinetics of vinblastine-mediated HIF-1alpha induction were investigated. The data indicate that the increase in HIF-1 levels is somewhat transient, with maximal induction occurring between 4-5 h and rapidly declining by 7 h.


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Fig. 1.   MDAs induce HIF-1alpha in various cell lines under normoxia. A, A549 cells were treated with vinblastine (vin, 0.1 µM), colchicine (col, 1 µM), nocodazole (noco, 10 µM), or paclitaxel (taxol, 1 µM) for 4 h. Following these treatments, nuclear extracts (20 µg) were prepared, and Western blotting was performed as described under "Materials and Methods." B, A549 cells were treated with vinblastine for the indicated times and HIF-1 levels were assessed in nuclear extracts. Topoisomerase II was used as an internal loading control. C, MCF-7, Jurkat, or NIH 3T3 cells were treated with vinblastine (0.1 µM; 1 µM for NIH 3T3 cells) or colchicine (1 µM; 5 µM for NIH-3T3 cells). Following these treatments, nuclear extracts were prepared, and Western blotting was performed as in A.

To determine whether the effect of MDAs on HIF-1alpha expression was a general phenomenon, we assessed the ability of MDAs to induce HIF-1alpha levels in a variety of cell lines. As shown in Fig. 1C, MDAs induced HIF-1alpha protein in cells derived from multiple lineages, irrespective of tumorigenicity or cell adherence, thereby demonstrating that this is a general signaling pathway shared by many, if not all, cell types.

MDAs Induce Both NFkappa B-dependent Transcription and Up-regulation of HIF-1alpha Protein-- We previously found that the inflammatory cytokines TNF-alpha and IL-1beta induce HIF-1alpha protein expression via NFkappa B activation.2 Coincidentally, MDAs are reported to activate NFkappa B gene transcription (24). Therefore, we wished to determine whether the HIF-1 induction following MDA treatment was potentially mediated by an NFkappa B-dependent pathway. First, we tested whether MDAs were capable of inducing NFkappa B activation in A549 cells, as assessed with transiently transfected NFkappa B-responsive luciferase constructs. As shown in Fig. 2A, the MDAs, but not paclitaxel, which promotes microtubule polymerization and stabilization, induced NFkappa B-responsive luciferase activity. Interestingly, MDA-induced NFkappa B activity correlated with the ability of these agents to induce HIF-1alpha protein levels (Fig. 1). We therefore explored the apparent correlation between MDA-induced NFkappa B activation and HIF-1alpha protein induction. To investigate this association, transiently transfected A549 cells were treated with increasing concentrations of either vinblastine or colchicine and NFkappa B activity was measured in parallel with HIF-1alpha protein expression. As shown in Fig. 2B, treatment of A549 cells with these agents resulted in a maximal level of NFkappa B activity, followed by a decline in activity at higher concentrations. When HIF-1alpha protein levels were examined from identically treated cells, the MDA-dependent increase in NFkappa B activity mirrored the increase in HIF-1alpha levels and maximal HIF-1alpha expression correlated with maximal NFkappa B activity. Similarly, at higher doses, HIF-1alpha protein levels declined in parallel with decreasing NFkappa B activity.


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Fig. 2.   MDAs induce both NFkB-dependent transcription and HIF-1alpha up-regulation. A, A549 cells were cotransfected with NFkappa B-dependent luciferase plasmid (0.4 ug) and CMV Renilla luciferase plasmid for 6 h. Subsequently, cells were treated with vinblastine (0.1 µM), colchicine (1 µM), nocodazole (10 µM), and paclitaxel (1 µM). Reporter activities were measured 6 h later and normalized to CMV Renilla luciferase activity. Data represent the mean of three separate experiments. B, a 549 cells were cotransfected as in A, and reporter activities were similarly measured at the indicated concentrations of vinblastine or colchicine. For detection of HIF-1alpha protein, cells were treated with varying concentrations of vinblastine or colchicine, as indicated, and nuclear extracts were immunoblotted for HIF-1alpha as in Fig 1.

MDA-dependent HIF-1 Induction Is Dependent upon New Transcription and NFkappa B Activation-- We sought to determine whether the NFkappa B activation induced by MDAs was responsible for mediating the increase in HIF-1alpha protein. First, we examined the requirement for general transcription in the MDA pathway. Cells were treated with MDAs in the presence of the transcription inhibitors AcD or 5,6-dichlorobenzimidazole riboside (DCB) and HIF-1alpha levels were examined. As shown in Fig. 3A (upper panel), HIF-1 induction by MDAs was completely inhibited by either of these agents. To confirm that the effect of transcriptional inhibitors upon HIF-1alpha levels was specific for the MDA-mediated pathway, these agents were added in combination with hypoxia mimetics. Cobalt chloride and phenanthroline (an iron chelator) are known to stabilize HIF-1alpha by preventing prolyl hydroxylases from modifying the protein, thereby rescuing HIF-1alpha from the destabilizing effects of VHL. As shown in Fig. 3A (lower panel), the transcriptional inhibitors did not interfere with the ability of hypoxia mimetics to induce HIF-1alpha protein. This result demonstrates a requirement for cellular transcription in MDA-mediated HIF-1 induction, thus defining this pathway as distinct from the hypoxia-mediated pathway.


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Fig. 3.   MDA-dependent HIF-1 induction is dependent upon new transcription and NFkB activation. A, upper panel, A549 cells were pretreated for 20 min with the transcription inhibitors actinomycin D (AcD,10 µM) or dichlorobenzimidazole riboside (DCB, 70 µM) before a 4-h treatment with MDAs, as indicated, and HIF-1alpha expression was monitored in nuclear extracts. Lower panel, A549 cells were pretreated with the same transcription inhibitor before treatment with either cobalt chloride (100 µM) or phenanthroline (phe, 200 µM) and HIF-1alpha levels were similarly detected. B, upper panel, A549 cells were transfected with NFkappa B super-repressor plasmid (NFRP, 5 ug) for 24 h followed by treatment with either vinblastine or colchicine and HIF-1alpha levels were detected. Lower panel, A549 cells were pretreated for 30 min with the NFkappa B inhibitor pyrrolidine dithiocarbamate (PDTC, 100 µM) followed by treatment with the hypoxia mimetic phenanthroline, or vinblastine or colchicine, as indicated. Cells were lysed 4 h later and HIF-1alpha immunoblots were performed using nuclear extract. C, A549 cells were treated with vinblastine for the indicated times. For the combination treatments of vinblastine and actinomycin D, cells were first pretreated with vinblastine for 3 h, followed by treatment with actinomycin D for either 1 or 2 additional hours (in the continued presence of vinblastine). D, wild type MCF-7 cells and MCF-7 cells stably transfected with dominant negative c-Jun construct (MCF-7/dn c-Jun) were treated with the indicated MDAs for 4 h and HIF-1alpha expression was examined.

We next examined whether the NFkappa B pathway was specifically implicated in the MDA-mediated induction of HIF-1alpha . To test this, A549 cells were transiently transfected with an NFkappa B super-repressor plasmid (expressing Ikappa B mutated to resist proteasome-mediated degradation) that effectively inhibits NFkappa B transcription (40), and these cells were subsequently subjected to treatment with MDAs. As shown in Fig. 3B (upper panel) MDA-mediated HIF-1 induction is severely impaired in the presence of NFkappa B repressor, thereby demonstrating a requirement for NFkappa B transcription in this pathway. To ensure that this result was not a nonspecific effect related to the transfections, the NFkappa B-inhibiting drug pyrrolidine dithiocarbamate (PDTC) was used to confirm these observations. As shown in Fig. 3B (lower panel), similar to the NFkappa B repressor effects, PDTC abrogated MDA-mediated HIF-1 induction. However, it had no effect upon the ability of hypoxia mimetics to induce HIF-1alpha , thereby validating the specificity of this pathway and emphasizing the crucial role of NFkappa B. To examine the effect of transcriptional inhibition upon MDA-stabilized HIF-1alpha protein, A549 cells were pretreated with vinblastine for 3 h, followed by either a 1 or 2 h treatment of actinomycin D (in the continued presence of vinblastine). As shown in Fig 3C, transcription inhibition was able to moderately reduce, but not eliminate, already stabilized HIF-1 protein. These data demonstrate that while constitutive transcription is needed for MDA-mediated up-regulation of HIF-1alpha , persistence of the stabilized protein is not dependent on transcription.

In addition to the NFkappa B pathway, it has been reported that MDAs can also activate the transcription factor AP-1 in a c-Jun-dependent manner (27). Therefore, we tested the potential contribution of AP-1 in the MDA-mediated HIF-1 induction using MCF-7 cells stably transfected with a dominant negative c-Jun construct (dn c-Jun) that inhibits c-Jun-dependent AP-1 activity (44). As shown in Fig. 3D, cells expressing dn c-Jun induced HIF-1alpha in response to MDAs to a degree comparable with wild type cells, thereby discounting an involvement of c-Jun-dependent AP-1 activity in this process.

MDA-induced HIF-1alpha Protein Is Transcriptionally Active-- It was of interest to determine whether the HIF-1alpha protein induced by MDAs was transcriptionally active. To test this, A549 cells were transfected with a HIF-1 responsive reporter plasmid that contains 3 hypoxia response elements of the iNOS gene (45). As shown in Fig. 4A, consistent with our Western results (Fig. 1), HIF-1-dependent luciferase activity was induced by treatment with MDAs. To confirm that HIF-1 reporter activity was induced in a HIF-1-specific manner, this experiment was repeated in hepa1c1c7 cells that contain wild type Arnt, and in matched hepa1c4 cells that are unable to transactivate HIF-1-dependent genes due to a genetic defect in Arnt (46, 47). First, we determined that MDAs induce over a 2-fold induction of HIF-1alpha protein in hepa1c1c7 (Fig. 4B). Furthermore, this increase in HIF-1alpha protein correlated with over a 2-fold increase in HIF-1 reporter activity. However, in Arnt-deficient hepa1c4 cells, MDA treatment failed to increase HIF-1-dependent luciferase activity, thereby demonstrating that MDA-mediated HIF-1 activation is dependent upon and accurately represents transcriptionally active HIF-1.


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Fig. 4.   MDAs induce HIF-dependent luciferase activity and upregulate iNOS protein in a HIF-1-dependent manner. A, A549 cells were cotransfected with a HIF-1-dependent iNOS luciferase reporter gene (0.4 µg) and CMV Renilla luciferase plasmid (4 ng) for 6 h. Subsequently, cells were treated with either vinblastine (0.1 µM), colchicine (1µM), or nocodazole (10µM). Reporter activities were measured 10 h later and were normalized to CMV Renilla luciferase activity. The data represent the mean of three separate experiments. B, upper panel, Hepa1c1c7 cells were treated for 4 h with either vinblastine (0.5 µM) or colchicine (5 µM), cells were lysed and HIF-1alpha was immunodetected. Lower panel, both hepa1c1c7 and hepa1c4 cells were cotransfected with a HIF-1-dependent iNOS luciferase reporter construct as in A prior to treatment with vinblastine (0.5 µM) or colchicine (5 µM). Reporter activities were measured 10 h later and were normalized to CMV Renilla luciferase activity. The data represent the mean of three separate experiments. C, both hepa1c1c7 and hepa1c1c4 cells were treated with either vinblastine (0.5 µM), or colchicine (5 µM) for 6 h. Following this treatment, cells were lysed and iNOS protein and alpha -tubulin (loading control) were immunoblotted using cytosolic extracts (40 µg).

We reasoned that the ability of MDAs to increase the population of transcriptionally active HIF-1 would result in the up-regulation of HIF-1 target proteins. Therefore, we examined whether the MDA-dependent increase in HIF-1 reporter activity correlated with an increase in endogenous iNOS protein, which is known to be a transcriptional target of HIF-1alpha (45). As shown in Fig. 4C, HIF-1alpha levels in hepa1c1c7 cells were induced following a 5-h treatment with the MDAs vinblastine or colchicine. However, these same agents were unable to elicit any iNOS induction in the Arnt-defective hepa1c4 cells. Therefore, the ability of MDAs to induce HIF-1 reporter activity in these cell lines reflects their ability to induce endogenous iNOS protein and is consistent with up-regulation of transcriptionally active HIF-1.

Microtubule Disruption Is Required for MDA-mediated HIF-1 Induction-- While the dependence for NFkappa B in MDA-mediated HIF-1 induction was definitive, it remained to be determined whether microtubule disruption itself was required for HIF-1 induction. To examine this issue, MDA-mediated HIF-1 induction was assessed in both wild type and colchicine-resistant Chinese Hamster Ovary (CHO) cells. The colchicine-resistant cells contain a mutation in tubulin that alters the association of MDAs with microtubules (48, 49). As shown in Fig. 5A, while the wild type CHO cells exhibited a marked increase in HIF-1 protein following MDA treatment, the colchicine-resistant cells failed to respond to this treatment. To further verify that microtubule disruption is a component of the signaling pathway of MDAs, A549 cells were treated with gamma -lumicolchicine, a structurally similar, but catalytically inactive analog of colchicine. As shown in Fig. 5B, this analog failed to induce HIF-1alpha protein, thereby demonstrating that MDA interaction with microtubules is required for MDAs to induce HIF-1alpha .


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Fig. 5.   Microtubule disruption is required for MDA-mediated HIF-1 induction. A, wild type and colchicine-resistant CHO cells were treated for 4 h with either colchicine (5 µM) or CoCl2 (100 µM), and HIF-1alpha expression was detected using nuclear extracts. B, A549 cells were treated for 4 h with colchicine (1 µM) or with gamma -lumicolchicine (gamma -lumicol, 1 µM), an inactive form of colchicine, and Western blotting was performed using nuclear extracts.

MDAs Up-regulate HIF-1alpha Protein at the Post-transcriptional Level-- Our data suggested that NFkappa B up-regulated HIF-1alpha at the transcriptional level, and we therefore examined whether HIF-1alpha mRNA levels were induced by MDAs. As shown in Fig. 6A (upper panel) RT-PCR analysis reveals that HIF-1alpha mRNA levels in A549 cells remain unchanged in response to a 3-h treatment of the indicated MDAs. The lower panel shows the RT-PCR of beta -actin, which was used as an internal control. This result indicates that MDAs up-regulate HIF-1alpha at the post-transcriptional level.


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Fig. 6.   MDAs upregulate HIF-1alpha at the posttranscriptional level and stabilize the protein in a VHL-dependent manner. A, A549 cells were treated with vinblastine (0.1 µM) or colchicine (1 µM) for 3 h. Total cellular mRNA was extracted and subjected to RT-PCR, as described. beta -actin mRNA levels were examined as an internal control. B, renal carcinoma cells that are deficient for VHL function (UMRC2), or a clonally selected line with VHL stably expressed (UMRC2/VHL), were treated for 4 h with vinblastine (vin, 0.1 µM), and HIF-1alpha protein was detected in nuclear extracts. C, A549 cells were transfected with either HA-tagged wild type HIF-1alpha or HA-tagged pmHIF-1alpha (HIF-1alpha proline-mutated at residues 402 and 564), a form resistant to VHL-dependent degradation. Following transfection, cells were treated as in B, and HIF-1alpha was immunodetected with an anti-HA antibody. D, A549 cells were either left untreated or were pretreated with vinblastine for 4 h, followed by addition of cycloheximide for the indicated times. Endogenous levels of HIF-1 were visualized from nuclear extracts (30 µg for vinblastine-treated cells and 80 µg for untreated cells). Blots were reprobed for ARNT expression as a control for equivalent loading within each group. Densitometric analysis of HIF expression was used to plot the decay rate of the protein.

One of the main regulators of HIF-1alpha protein under normoxia is VHL. Therefore, we examined whether VHL played a role in the MDA-mediated induction of HIF-1alpha . The effect of MDAs upon HIF-1alpha expression was examined in a matched pair of renal carcinoma lines either lacking VHL function, or with VHL replaced by stable transfection. As shown in Fig. 6B, vinblastine up-regulated HIF-1alpha protein in the cell line containing functional VHL protein (UMRC2/VHL). However, HIF-1alpha was not further up-regulated in the VHL-mutated parental line containing stable HIF-1alpha protein. To confirm these results, transfections were performed in the well-characterized A549 cell line. As shown in Fig. 6C, vinblastine up-regulated the levels of transfected wild type HIF-1alpha expressed in A549 cells, while this agent failed to upregulate a mutated form of HIF-1alpha that is VHL-resistant. These results suggested that MDA treatment stabilized HIF-1alpha protein to the effects of VHL. To test this hypothesis, the stability of endogenous HIF-1alpha protein in A549 cells was determined either in the presence or absence of vinblastine. As shown in Fig. 6D, while endogenous HIF-1alpha was extremely labile in normoxic cells, with a half-life of less than 4 min, vinblastine treatment significantly stabilized the protein and extended its half-life by more than 5-fold to 20 min.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, MDAs were used to simulate cellular responses activated by microtubule change. We demonstrate that cytoskeletal alteration mediated by a variety of microtubule-depolymerizing agents elevate protein levels of transcriptionally active HIF-1alpha in a pathway dependent upon NFkappa B activation in a variety of cell lines, suggesting that this is a basic mode of signaling universally employed by most, if not all, cell types. While MDA-mediated HIF-1 induction is not as pronounced as that elicited by hypoxia, it is significant enough to result in more than a 2-fold increase in HIF-1-regulated iNOS reporter activity and in a severalfold up-regulation of endogenous iNOS protein expression. By comparison, hypoxic stimulation of this same reporter was on the order of 3-4-fold (data not shown).

Our data conclusively demonstrate that transcription, likely mediated by NFkappa B activation, is a requirement for the ability of MDAs to induce HIF-1alpha . First, we show that transcription inhibitors interfere with the ability of MDAs to induce HIF-1alpha protein. Second, we demonstrate that MDAs induce NFkappa B activity, which correlates with the ability of these agents to induce HIF-1alpha levels. Third, and most compelling, treatment of cells with either a drug that inhibits NFkappa B or transfection with the NFkappa B super-repressor plasmid both abrogated the ability of MDAs to up-regulate HIF-1alpha protein. Finally, we demonstrate that the MDA signaling pathway for HIF-1 induction is distinct from the hypoxia-mediated pathway, in that NFkappa B inhibitors had no effect on reducing HIF-1alpha levels induced by a hypoxia mimetic, further emphasizing the unique transcriptional dependence of this mechanism.

Several reports demonstrate that drugs capable of microtubule disruption elevate NFkappa B activity (24, 50-51) and in agreement with these reports, we demonstrate that MDAs induced NFkappa B activity in A549 cells. However, the precise mechanism of MDA-induced NFkappa B activation remains unclear. In one model, MDAs are proposed to stimulate Ikappa B degradation, resulting in the nuclear accumulation and subsequent activation of NFkappa B (24, 52). In accordance with this model, we observed a slight increase in nuclear NFkappa B upon MDA treatment (data not shown). In a second model, mechanisms potentially independent of nuclear NFkappa B protein levels play a role. These mechanisms include posttranslational modification of the NFkappa B protein, such as phosphorylation or acetylation of p65 (53). Interestingly, although high doses of either vinblastine or colchicine resulted in decreased NFkappa B activity, no such corresponding decrease in nuclear protein levels was observed (data not shown), consistent with other reports (52). Therefore, although our data do not preclude the possibility that MDA-mediated nuclear translocation of NFkappa B is an important initial event, the down-regulation of NFkappa B activity without corresponding changes in nuclear p65 suggests that MDAs also initiate additional signaling events culminating in the post-translational modification of NFkappa B. Although a definitive signaling pathway remains elusive, it is notable that MDAs modulate the activities of a variety of kinases, such as protein kinases A and C, PI 3-kinase, and focal adhesion kinase (FAK), that regulate NFkappa B activity by phosphorylation of the p65 subunit (18, 54-55). Consistent with this hypothesis, MDAs at low, but not high concentrations, increase tyrosine phosphorylation of focal adhesion proteins such as FAK and paxillin (56). Finally, it has been demonstrated that microtubule-stabilizing agents such as paclitaxel are unable to promote activation of these same kinases (51, 57), correlating with their inability to activate NFkappa B and upregulate HIF-1alpha .

Our data demonstrate that the MDA-mediated pathway for HIF-1alpha induction is distinct from the hypoxia-mediated stabilization of HIF-1alpha . This is most clearly illustrated by data showing the dependence upon NFkB activation for the former, but not the latter pathway. However, similar to the hypoxia-mediated pathway, we find that MDAs induce HIF-1alpha protein at the posttranscriptional level. While we cannot absolutely rule out the possibility of increased translation of HIF-1alpha as an explanation of this phenomenon, in a manner similar to the effect of various growth factors (35, 58), we suggest that MDAs act by partially protecting HIF-1alpha protein from VHL-dependent degradation. MDA-mediated stabilization does not render the protein completely resistant to VHL, but rather appears to engender a less efficient degradation, resulting in a 5-fold increase in half-life. Compelling evidence for this notion is provided by our finding that HIF-1alpha levels in a cell line lacking VHL function remained unchanged upon exposure to vinblastine. However, in a matched line with stably expressed VHL, HIF-1alpha accumulated in response to vinblastine, suggesting that the MDA-stabilizing effect is dependent upon VHL expression. Similarly, vinblastine elevated the level of transiently expressed wild type HIF-1alpha protein in A549 cells, while these agents had no effect upon a transiently expressed proline-mutated, VHL-resistant HIF-1alpha protein. Finally, the ability of MDAs to activate NFkB was independent of VHL status (data not shown), supporting our hypothesis that NFkB activation by MDAs occurs prior to HIF-1 accumulation. Although the complete mechanism of MDA-induced HIF-1alpha accumulation remains unclear, the transcriptional dependence of this pathway suggests that the mediator(s) involved may be labile. Evidence for a labile mediator is further provided by our data (Fig. 3C) demonstrating that transcriptional inhibition of MDA-stabilized HIF-1 results in a moderate decrease in protein levels within the first hour. This labile mediator(s) may modify HIF-1alpha , or another protein involved VHL-HIF association, so as to render HIF-1alpha less susceptible to VHL-mediated degradation.

Tumor cell invasion and metastasis, hallmarks of the tumorigenic process, involve microtubule reorganization. We demonstrate that MDA-mediated activation of NFkappa B and subsequent induction of HIF-1alpha is initiated by and depends upon microtubule depolymerization. While the specific role NFkappa B may play in invasion and metastasis is unclear, several reports document overexpression and/or hyperactivity of NFkappa B in cancer lines (59) and tissues (60). Inhibition of NFkappa B correlates with suppression of metastasis and invasion (61, 62), down-regulation of VEGF mRNA (63), and suppression of angiogenesis (62), effects, which may be mediated through regulation of HIF-1alpha . Given that HIF-1alpha is overexpressed in a majority of tumors (12), the data in this study suggest that HIF-1alpha is among the pro-oncogenic factors induced by NFkappa B.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 301-402-3128, ext. 318; Fax: 301-402-4422; E-mail: len@helix.nih.gov.

Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M209804200

2 Y.-J. Jung, J. S. Isaacs, S. Lee, J. Trepel, Z.-G. Liu, and L. Neckers, submitted manuscript.

    ABBREVIATIONS

The abbreviations used are: HIF-1, hypoxia inducible factor-1; MDA, microtubule-depolymerizing agent; vin, vinblastine; col, colchicine; noco, nocodazole; taxol, paclitaxel; AcD, actinomycin-D; DCB, dichlorobenzimidazole riboside; phe, phenanthroline; gamma -lumicol, gamma -lumicolchicine; NFkappa B, nuclear factor kappa B; NFRP, NFkappa B super-repressor plasmid; iNOS, inducible nitric-oxide synthetase; ARNT, aryl hydrocarbon nuclear translocator; HA, hemagglutinin; PDTC, pyrrolidinedithiocarbamate; CHX, cycloheximide; PI, phosphatidylinositol; CMV, cytomegalovirus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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