Adenosine monophosphate-activated protein kinase (AMPK) is thought to inhibit cell proliferation or promote cell death, but the details remain unclear. In this study, we propose that AMPK inhibits the expression of anti-apoptotic B-cell lymphoma 2 (Bcl-2) by relying on the hypoxia-inducible factor 1 alpha (HIF-1α)-induced caveolin-1 (Cav-1) expression pathway in noninvasive human bladder tumor (RT4) cells.
In cells exposed to a hypoxic environment (0.5% oxygen), the levels of expression and phospho-activity of the relevant signaling enzymes were examined via Western blots and reverse transcription-polymerase chain reaction. Cell proliferation was assessed using a Cell Counting Kit-8 assay.
The level of expression of Cav-1 was very low or undetectable in RT4 cells. Hypoxia was associated with significantly decreased cell growth, along with marked induction of HIF-1α and Cav-1 expression; additionally, it suppressed the expression of the antiapoptotic marker Bcl-2 while leaving AMPK activity unchanged. Under hypoxic conditions, HIF-1α acts as a transcription factor for Cav-1 mRNA gene expression. The cell growth and Bcl-2 expression suppressed under hypoxia were reversed along with decreases in the induced HIF-1α and Cav-1 levels by AMPK activation with metformin (1mM) or phenformin (0.1mM). In addition, pretreatment with AMPK small interfering RNA not only increased the hypoxia-induced expression of HIF-1α and Cav-1, but also reversed the suppression of Bcl-2 expression. These results suggest that HIF-1α and Cav-1 expression in hypoxic environments is regulated by basal AMPK activity; therefore, the inhibition of Bcl-2 expression cannot be expected when AMPK activity is suppressed, even if Cav-1 expression is elevated.
For the first time, we find that AMPK activation can regulate HIF-1α induction as well as HIF-1α-induced Cav1 expression, and the hypoxia-induced inhibitory effect on the antiapoptotic pathway in RT4 cells is due to Cav-1-dependent AMPK activity.
- AMPK inhibits the expression of antiapoptotic Bcl-2 by relying on the HIF-1α-induced Cav-1 expression pathway in Cav-1-free RT4 bladder tumor cells, and the hypoxia-induced inhibitory effect on the anti-apoptotic pathway is due to Cav-1-dependent AMPK activity. These findings point to the interactions between AMPK and Cav-1 impacting Bcl-2 expression.
Hypoxia is known to affect various signals related to tumor aggression, prognosis, and metastasis [
Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that regulates cellular energy status and differentiation and responds to metabolic stress [
In addition to hypoxia-induced AMPK activation, hypoxia induces the differential expression of caveolin-1 (Cav-1), a cellular membrane scaffolding protein, in several cell types [
In this study, we demonstrated that Cav-1 expression was induced via a transcriptional pathway of HIF-1α in hypoxia-exposed RT4 cells, and hypoxia-induced inhibition of cell growth as indicated by a marked decrease in B-cell lymphoma 2 (Bcl-2) expression was induced by basal AMPK activity only in the presence of Cav-1 expression. Furthermore, we report that AMPK activation eliminated or reversed the hypoxia-related induction of HIF-1α and associated Cav-1 expression and also reversed the effect on Bcl-2 expression. These findings point to possible interactions between AMPK and Cav-1 impacting Bcl2 expression.
Phenformin hydrochloride, 1,1-dimethylbiguanide hydrochloride (metformin), echinomycin, and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for AMPKα, phospho-AMPKα (Thr172), phosphoAMPKα1 (Ser485), acetyl-CoA carboxylase, and phospho-acetyl-CoA carboxylase (Ser79) were purchased from Cell Signaling Technology (Beverly, MA, USA), and antibodies for Cav-1 and Bcl-2 as well as AMPKα1/2 small interfering RNA (siRNA) and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HIF-1α antibody was obtained from R&D Systems (Minneapolis, MN, USA).
RT4 cells (Korean Cell Line Bank, Seoul, Korea) were cultured in Dulbecco’s modified Eagle medium (Gibco, El Paso, TX, USA) containing 10% fetal bovine serum (genDEPOT, Barker, TX, USA) and 1% penicillin-streptomycin (Gibco), while T24 cells (Korean Cell Line Bank, Seoul, Korea) were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (genDEPOT), 1% penicillin-streptomycin (Gibco), and 20mM HEPES (Sigma-Aldrich). These cells were serially passaged in the laboratory using 0.25% trypsin-EDTA (Gibco). The cells were grown at 37°C in a humidified atmosphere of 5% carbon dioxide (CO2) and 95% air. For all experiments, cells were seeded into 60-mm plates overnight and then treated with relevant reagents or vehicle only for the indicated time.
Hypoxic conditions (0.5% O2 and 5% CO2 balanced with N2) were achieved in a humidified aerobic workstation (InvivO2 500 with I-CO2N2IC gas mixing system; The Baker Company, Sanford, ME, USA), which contained an oxygen sensor that continuously monitored the chamber oxygen tension.
For whole-cell extracts, cells were washed with ice-cold phosphate-buffered saline and collected by scraping in ice-cold NOS lysis buffer (320mM sucrose, 200mM HEPES, and 1mM EDTA; pH, 7.2) supplemented with Phos Stop (Roche, Mannheim, Germany) and protease inhibitor (Sigma-Aldrich). The harvested cells were sonicated 5 times for 5 seconds each at 3 W and centrifuged at 3,000 rpm, 10 minutes, and 4°C. The supernatant was quantified using the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA). An equal amount of protein for each blot was loaded on a 4%–20% Mini-PROTEAN TGX Precast Protein Gel (BIO-RAD, Hercules, CA, USA), transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), and then blocked with 5% nonfat milk in tris-buffered saline with 0.05% tween 20 (TBS-T) buffer. The membrane was incubated with the indicated antibody overnight at 4°C and was then washed 3 times in TBS-T buffer. After incubation with the secondary antibody, detection was performed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).
Cell viability and proliferation were measured with a Cell Counting Assay Kit-8 (Dojindo, Rockville, MD, USA). Trypsinized cells were resuspended in Dulbecco’s modified Eagle medium at 1×105 cells/mL, and 100 µL of cell suspension was distributed into each well of a 96-well flat-bottom microplate. Following incubation to allow adherent cell growth, the medium was removed, and 100 µL of fresh medium was distributed into each well and incubated in a CO2 incubator (37°C, with a humidified atmosphere of 5% CO2 and 95% air) or a hypoxia chamber (0.5% O2 and 5% CO2 balanced with N2) for 6, 12, 24, and 48 hours. After incubation, 10 µL of CCK-8 solution was added to each well of the plate and incubated for 2 hours in the incubator. The absorbance on the plate was then measured at 450 nm with a microplate reader.
Cells were washed with Opti-MEM medium (Gibco) and transfected with 10 pmol control or AMPK siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The medium was replaced with growth medium 24 hours after transfection, and the cells were incubated overnight for stabilization. After changing to fresh medium, the cells were incubated in a CO2 incubator (37°C, with a humidified atmosphere of 5% CO2 and 95% air) or a hypoxia chamber (0.5% O2 and 5% CO2 balanced with N2) for 24 and 48 hours. Protein expression was evaluated using Western blotting.
The total RNA was isolated using AccuZol Total RNA Extraction Reagent (Bioneer Corporation, Daejeon, South Korea) according to the manufacturer’s instructions. cDNA was synthesized using the AccuPower CycleScript RT Premix (dT20; Bioneer), and the synthesis was carried out as follows: 40 cycles for 1 minute at 15°C, 4 minutes at 50°C, 1 cycle for 5 minutes at 95°C, 60 minutes at 50°C, and 5 minutes at 95°C.
Reverse transcription-polymerase chain reaction (RT-PCR) was performed with Solg e-Taq DNA Polymerase (10X e-Taq Reaction Buffer, 10mM dNTP mix, Solg e-Taq), sense and antisense primers, and a cDNA template with the following protocol: 1 cycle for 2 minutes at 95°C, 35 cycles for 20 seconds at 95°C, 40 seconds at 55°C, 1 minute at 72°C, and 1 cycle for 5 minutes at 72°C. RT-PCR primers were used for Cav-1 (sense, 5ʹ-ACG TAG ACT CGG AGG GAC ATC TCT-3ʹ; antisense, 5ʹ-CTG CAA GTT GAT GCG GAC ATT GC-3ʹ) and glyceraldehyde 3-phosphate dehydrogenase (sense, 5ʹ-TCA TTG ACC TCA ACT ACA TGG T-3ʹ; antisense, 5ʹ-CTA AGC AGT TGG TGG TGC AG-3ʹ). The samples were then analyzed via 2% agarose gel electrophoresis with 0.5% tris-borate-EDTA buffer.
The statistical analysis was performed with GraphPad Prism software ver. 9 (GraphPad Software, San Diego, CA, USA). Data were represented as the mean±standard error of the mean of 3 or more independent experiments. Based on the Western blots, differences between groups (normoxia and hypoxia) and within groups (siRNA-treated and control for each condition) were assessed using the unpaired Student t-test. For the between-group comparisons, P-values of *P<0.05, **P<0.01, and ***P<0.001 were considered to indicate statistical significance, while P-values of #P<0.05 and ##P<0.01 were considered to do so for the within-group comparisons. Two-way analysis of variance, followed by the Tukey multiple comparison test, was used as the statistical test for cell proliferation levels (***P<0.001 between the normoxia and hypoxia groups).
AMPK is involved in the regulation of cell survival or proliferation. The purpose of this study was to examine the changes in the expression and activity of AMPK in urothelial cells after exposure to a hypoxic environment (0.5% O2). As an initial step, the level of expression of Cav-1 and the activity of Src kinase (a tyrosine kinase) was compared between noninvasive urothelial (RT4) cells and high-grade invasive urothelial carcinoma (T24) cells. In RT4 cells, the expression of Cav-1 was almost too low to be detected. In contrast, the activity of Src kinase (Tyr416) was much higher in RT4 cells than in T24 cells (data not shown).
In the present study, the levels of expression of HIF-1α (48 hours, **P=0.002) and Cav-1 (24 hours, *P=0.049; 48 hours, *P=0.038) were markedly greater in RT4 cells exposed to hypoxia than in the cells treated with normal oxygen concentrations (
To elucidate the impact of hypoxia on RT4 cell proliferation, the cells in the normoxic and hypoxic conditions were quantified over time with the CCK-8 assay. The doubling time of the cells was approximately 36 hours in normoxic conditions. At the early time point (~12 hours), no obvious difference was observed between normoxic and hypoxic conditions. In hypoxic conditions, the cell proliferation rate was significantly lower than in normoxic conditions at both 24 and 48 hours (~35% lower at 24 hours, ***P<0.001; ~70% lower at 48 hours, ***P<0.001;
Under hypoxic conditions, the expression of HIF-1α was significantly induced relative to normoxic conditions at both 24 and 48 hours (**P=0.005 and **P=0.007, respectively), while the expression of Cav-1 was significantly induced only at 48 hours (*P=0.027). The levels of AMPK activity, as well as the p-ACC/ACC ratio, were unchanged or slightly increased, with a decrease in AMPK expression, a decrease in p-AMPK-172 phospho-activity, and a significant decrease in p-AMPK-485 phospho-activity (*P=0.019) relative to normoxia. In contrast, the expression of Bcl-2 was reduced to approximately 60% of its level under normoxia (24 hours, **P=0.004; 48 hours, **P= 0.002) (
The levels of Cav-1 mRNA expression were assessed via RT-PCR at 6, 12, and 24 hours to determine whether hypoxia-induced Cav-1 expression was induced at the transcriptional level. As shown in
To demonstrate that HIF-1α acts as a transcription factor for Cav-1 induction, cell lines exposed to hypoxia were pretreated with echinomycin, a selective inhibitor of HIF-1α. As shown in
To confirm whether the HIF-1α transcription factor is involved in hypoxia-induced Cav-1 expression, we pretreated the cells under the hypoxic conditions with echinomycin (1nM and 2nM), a selective inhibitor of HIF-1α. The cytotoxicity associated with echinomycin was measured via the CCK-8 assay, and HIF-1α expression completely disappeared in the echinomycinpretreated hypoxic condition relative to the untreated hypoxic cells (#P=0.025 and #P=0.023) (
A transient knockdown with AMPK siRNA was applied to demonstrate that AMPK activity plays a role in the regulatory pathway of hypoxia-induced HIF-1α and Cav-1 expression. As shown in
In this study, we showed that HIF-1α-mediated Cav-1 expression was induced in Cav-1-free and noninvasive human bladder tumor (RT4) cells exposed to hypoxic conditions (<0.5% O2). Under these conditions, the activity of AMPK (as indicated by the p-ACC/ACC ratio), which has anti-cell proliferation or proapoptotic activity, was weakly increased or unchanged, while the expression of Bcl-2, an antiapoptotic signaling enzyme, was markedly decreased. That is, hypoxia appears to suppress the antiapoptotic Bcl-2 signaling pathway via an interactive mechanism between basal AMPK activity and Cav-1 expression.
The activation of AMPK under hypoxic conditions is well known [
More interestingly, in the present study, pretreatment with AMPK activators (metformin or phenformin) interfered with HIF-1α and Cav-1 induction, and this pretreatment suppressed the hypoxia-induced inhibition of cell growth or apoptosis while returning Bcl-2 expression to its normal level or increasing it. Therefore, in the RT4 cells exposed to hypoxic conditions, the triggering of Bcl-2 inhibition or the apoptosis pathway occurs only in the presence of AMPK activity and Cav-1 expression, and an excessive increase in AMPK activity suppresses hypoxia-induced Cav-1 expression (
Noninvasive bladder urothelial cells, such as RT4 cells, are free of Cav-1 expression, even though this protein is highly expressed in smooth muscle cells [
The expression of Cav-1 in bladder urothelial cells is an important marker in distinguishing cancer grade, squamous differentiation, and cancer progression [
Salani et al. [
In conclusion, we report a new finding that hypoxia-induced AMPK activation can regulate HIF-1α-induced Cav-1 expression in Cav-1-free noninvasive RT4 bladder tumor cells. Additionally, we found that the substantial AMPK activity in hypoxia-induced suppression of the anti-apoptotic signaling pathway is exhibited only in cases of inducible Cav-1 expression. Thus, this AMPK activity is Cav-1-dependent.
This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (NRF-2014R1A5A2009392 to C.-S. Park), as well as the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2020R1I1A1A01065009 to B.-H. Choi).
No potential conflict of interest relevant to this article was reported.
· Conceptualization:
· Data curation:
· Formal Analysis:
· Funding acquisition:
· Methodology:
· Project Administration:
· Writing – Original Draft:
· Writing – Review & Editing:
Basal and induced expression and/or phospho-activity of acetyl-CoA carboxylase (ACC), adenosine monophosphate-activated protein kinase (AMPK), hypoxia-inducible factor-1 (HIF-1α), caveolin-1 (Cav-1), and B-cell lymphoma 2 (Bcl-2) under normoxia and hypoxia in noninvasive superficial (RT4) and high-grade invasive (T24) human bladder tumor cells at 24 hours and 48 hours. pAMPK-172 and p-AMPK-485 refer to the stimulatory and inhibitory phosphorylation sites, respectively. In particular, substantial AMPK activity can be predicted based on the ratio of p-ACC/ACC. Black and grey bars indicate the normoxia and hypoxia groups, respectively. Values are expressed as the mean±standard error of the mean. *P<0.05 and **P<0.01 vs. normoxia (RT4 cells; statistical analysis was not performed for T24 cells).
ell proliferation over time under normoxic and hypoxic conditions. The cells were counted at baseline and at 4 time points (0, 6, 12, 24, and 48 hours). Each value represents the mean±standard error of the mean of 5 independent experiments. The difference between the normoxia and hypoxia groups was analyzed via 2-way analysis of variance with the Tukey multiple comparison test. ***P<0.001 vs. normoxia (24 hours and 48 hours).
Effects of the adenosine monophosphate-activated protein kinase (AMPK) activators metformin (Met) and phenformin (Phen) on the levels of expression and/or phospho-activity of acetyl-CoA carboxylase (ACC), AMPK, hypoxia-inducible factor-1 (HIF-1α), caveolin-1 (Cav-1), and B-cell lymphoma 2 (Bcl-2) under normoxia and hypoxia, at 24 hours and 48 hours. Hypoxia did not significantly affect either AMPK activity or the ratio of p-ACC/ACC, but it induced increases in both HIF-1α (**P<0.01) and Cav-1 (*P<0.05) and, inversely, a decrease in Bcl-2 (**P<0.01). These changes were reversed by pretreatment with Met or Phen, but nonsignificantly compared within the hypoxia group. The black and grey bars indicate the normoxia and hypoxia groups, respectively. The data presented are expressed as the mean±standard error of the mean.
Effects of the adenosine monophosphate-activated protein kinase (AMPK) activators metformin (Met) and phenformin (Phen) on caveolin-1 (Cav-1) mRNA induction under normoxic and hypoxic conditions at 6, 12, and 24 hours. Cav-1 mRNA expression was measured via real time-polymerase chain reaction. Time-dependent Cav-1 mRNA expression was induced by hypoxia (Con: control), but this was blocked by pretreatment with Met or Phen. The data presented are expressed as the mean±standard error of the mean of 2 independent experiments (% ratio of control [6 hours]) without a statistical analysis.
Effects of the hypoxia-inducible factor 1 (HIF-1α)-selective inhibitor echinomycin (Ech, 2nM) on caveolin-1 (Cav-1) mRNA induction under normoxia and hypoxia at 12, 24, and 48 hours. Cav-1 mRNA expression was measured via reverse transcriptasepolymerase chain reaction. Ech pretreatment decreased the time-dependent hypoxia-induced Cav-1 mRNA expression. The data presented are expressed as the mean±standard error of the mean of 2 independent experiments (% ratio of control [12 hours]) without a statistical analysis.
Effects of the hypoxia-inducible factor 1 (HIF-1α) selective inhibitor echinomycin (Ech, 2nM) on the expressions and/or phospho-activities of adenosine monophosphate-activated protein kinase (AMPK), HIF-1α, caveolin-1 (Cav-1), and B-cell lymphoma 2 (Bcl-2) under normoxic and hypoxic conditions at 24 hours and 48 hours. The black and grey bars indicate the normoxia and hypoxia groups, respectively. The data presented are expressed as the mean±standard error of the mean (*P<0.05, **P<0.01, vs. normoxia; #P<0.05 vs. control group of normoxic or hypoxic condition).
Effects of transient silencing of adenosine monophosphate-activated protein kinase (AMPK) on hypoxia-inducible factor 1 (HIF-1α), caveolin-1 (Cav-1), and B-cell lymphoma 2 (Bcl-2) expression. Despite Cav-1 overexpression, hypoxia did not induce the suppression of Bcl-2 in cells pretreated with AMPK siRNA due to the loss of AMPK activity. The black and grey bars indicate the normoxia and hypoxia groups, respectively. The data presented are expressed as the mean±standard error of the mean (*P<0.05, **P<0.01, ***P<0.001 vs. normoxia; #P<0.05, ##P<0.01 vs. the control group of normoxic or hypoxic conditions).