HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 19/8/1042 04-2841fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, H.-S. Articles by Walsh, K. Search for Related Content PubMed PubMed Citation Articles by Kim, H.-S. Articles by Walsh, K.

Akt/FOXO3a signaling modulates the endothelial stress response through regulation of heat shock protein 70 expression

Hyo-Soo Kim^*,,1, Carsten Skurk^*,1, Henrike Maatz^*, Ichiro Shiojima^*, Yuri Ivashchenko, Suk-Won Yoon, Young-Bae Park and Kenneth Walsh^*,2

^* Molecular Cardiology/Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, USA;
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea; and
AVENTIS Pharma, Cardiovascular Disease Group, Frankfurt, Germany

² Correspondence: Molecular Cardiology, Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany St., W611 Boston 02118, MA, USA. E-mail: kxwalsh{at}bu.edu

SPECIFIC AIMS

Apoptosis is an important regulatory feature of blood vessel remodeling. The Akt pathway is activated by stress to preserve cellular viability. To identify new antiapoptotic targets of the PI3K-Akt signaling pathway in endothelial cells, adenovirus-mediated Akt1 gene transfer and oligonucleotide microarrays were used to examine Akt-regulated transcripts and their functional significance in the endothelial stress response.

PRINCIPAL FINDINGS

1. Effects of myrAkt overexpression on gene expression in endothelial cells
Analysis of the gene expression profile in HUVEC transduced with Ad-myrAkt compared with those transduced with a control vector-expressing ß-galactosidase revealed that constitutive activation of Akt signaling by adenoviral gene transfer altered the expression of 462 transcripts of 12,532 sequences contained on the gene chip. A complete list of regulated transcripts can be found at www.kwalshlab.org. Up-regulation of HSP70 underwent the largest change in expression of any gene observed. We did not observe a difference of HSP70 gene expression in HUVEC transduced with ß-galactosidase compared with mock-infected endothelial cells, indicating a specific effect of myrAkt on HSP70 up-regulation.

2. Akt signaling regulates HSP70 expression in endothelial cells
To validate the microarray data, QRT-PCR analysis of HSP70 gene expression was performed. QRT-PCR analysis demonstrated a marked up-regulation of HSP70 gene expression after Ad-myrAkt gene transfer into HUVEC (P<0.01).

Subconfluent HUVEC were transduced with increasing concentrations of Ad-myrAkt. Ad-myrAkt-mediated gene transfer led to a dose-dependent increase in HSP70 protein levels as observed by Western immunoblot analysis (Fig. 1 ). Transduction with a viral vector expressing ß-galactosidase up to 50 MOI did not significantly increase HSP70 levels, indicating a specific effect of the Akt transgene. Conversely, preincubation of endothelial cells with a PI-3 kinase inhibitor (LY294002) for 12 h significantly down-regulated the expression of HSP70 protein in a dose-dependent manner.

View larger version (41K): [in this window] [in a new window]   Figure 1. The PI3-kinase/Akt signaling axis controls HSP70 protein expression in endothelial cells. Subconfluent HUVEC were transduced overnight with adenoviral vectors and whole cell lysates were collected for Western blot analysis. A) Representative Western blot indicating dose-dependent HSP70 up-regulation by myrAkt. Conversely, incubation of HUVEC with LY294002 (20 µM for 12 h) led to a significant down-regulation in HSP70 protein levels. Membranes were stripped and reprobed with HA indicating transgene expression. Tubulin indicates equal loading in each lane.

3. HSP70 regulation by FOXO3a
To test whether the Akt-regulated FOXO3a transcription factor participates in Hsp70 regulation, subconfluent HUVEC cultures were transduced with adenoviral vectors encoding wild-type (WT) and triple mutant (TM) FOXO3a, which has three Akt phosphorylation sites mutated to alanine residues. Transduction with WT-FOXO3a and TM-FOXO3a leads to a profound decrease in HSP70 expression in endothelial cells. In accordance with WT-FOXO3a phosphorylation by growth factor signaling via the PI3-kinase/Akt signaling axis, down-regulation of HSP70 expression could be partially rescued under high mitogen conditions (EGM+10% FBS), which lead to phosphorylation of the forkhead transcription factor and its exclusion from the nucleus.

4. FOXO3a-mediated suppression of HSP70 promotes apoptosis through activation of the intrinsic apoptotic pathway
HSP70 promotes cell viability through its ability to inhibit caspase-9 activation and the intrinsic apoptotic pathway. The contribution of caspase-9 to FOXO3a-induced cell death in this cell type has not been examined in detail. Adenovirus-mediated overexpression of TM-FOXO3a significantly reduced cell viability as assessed by a WST-1 assay, an indicator of disturbed mitochondrial function. FOXO3a increased DNA strand breaks as detected by TUNEL assay; these observations were corroborated by a quantitative analysis of hypodiploid DNA after FOXO3a gene transfer. Induction of apoptosis due to FOXO3a overexpression was significantly inhibited by cotransduction with an adenoviral vector expressing a dominant-negative form of caspase-9. Dominant-negative caspase-9 significantly protected endothelial cells from the FOXO3a-mediated reduction in mitochondrial function. Dominant-negative caspase-9 also decreased DNA strand breaks in FOXO3a-transduced cells as well as hypodiploid DNA content. These data provide evidence for the importance of caspase-9-mediated apoptosis in endothelial cells transduced with FOXO3a. To provide causal evidence that FOXO3a-mediated down-regulation of HSP70 is a feature of reduced endothelial cell viability, HUVEC were cotransduced with Ad-TM-FOXO3a and Ad-HSP70. Transduction with Ad-HSP70 rescued HUVEC from apoptotic cell death after FOXO3a overexpression as determined by quantitative FACS analysis of hypodiploid DNA. The rescue from apoptosis by Ad-HSP70 and Ad-dnCaspase- 9 was not complete, suggesting the participation of other mechanisms of FOXO3a toxicity. Similarly, cotransduction with CrmA, a caspase-8 inhibitor, partly rescues endothelial cells from FOXO3a-induced cytotoxicity. However, the effect of caspase-8 and caspase-9 blockade was additive, and cotransduction with both vectors blocked FOXO3a-induced apoptosis.

5. FOXO3a-induced down-regulation of HSP70 causes activation of caspase-3 and caspase-9
Caspase-9 activation was directly measured by a fluorescence assay. TM-FOXO3a gene transfer leads to a significant increase in caspase-9 activity that could be completely blocked by cotransduction with dominant-negative caspase-9. Cotransduction with HSP70 essentially reduced TM-FOXO3a-induced caspase-9 activity to baseline. To corroborate these data, activation of caspase-3 was determined by FACS analysis using red-DEVK-FMK. FOXO3a gene transfer caused activation of caspase-3. This cleavage could be significantly inhibited by cotransduction with dominant-negative caspase-9. Our data indicate that FOXO3a-induced down-regulation of HSP70 expression caused an activation of caspase-9 that is functionally significant.

6. FOXO3a inhibits the HSP70 stress response in endothelial cells
HSP70 is induced in response to a variety of cellular stresses. To elucidate the role of FOXO3a in regulating HSP70 during the stress response in endothelial cells, HUVEC were subjected to heat shock at 42°C, leading to a significant increase of HSP70 protein levels that was blocked by transduction of FOXO3a in a dose-dependent manner (Fig. 2 A). Heat shock caused a significant reduction in cell viability, exacerbated by the overexpression of FOXO3a. Transduction with an adenoviral vector encoding dominant-negative caspase-9 or Hsp70 significantly reduced apoptosis under these conditions (Fig. 2B ).

View larger version (21K): [in this window] [in a new window]   Figure 2. FOXO3a inhibits the cellular stress response to heat shock and anoxia/reoxygenation. A) Subconfluent cells were transduced with control vector (GFP) or TM-FOXO3a overnight and subjected to heat shock at 42°C for 4 h. Whole cell lysates were collected and Western blot analysis for HSP70 was performed. B) WST-1 assay of mitochondrial function after heat shock. The assay was performed 24 h after transduction. Data are presented as mean ± SE for 3 independent experiments (*P<0.01 vs. control 42°C, #P<0.01 vs. FOXO3a). C) Anoxia increases HSP70 protein expression in a time-dependent manner. The anoxic increase in HSP70 returned to baseline levels as cells were reoxygenated. A4/A15- anoxia for 4/15 h, R30'- reoxygenation for 30 min. D) FOXO3a prevents anoxia-induced up-regulation of HSP70. Adenovirus-transduced cells were subjected to anoxia for 24 h and Western blot analysis for HSP70 was performed on whole cell lysates. E) WST-1 assay illustrating cell viability after 24 h of anoxia. Data are presented as mean ± SE for 3 independent experiments in each group (*P<0.01 vs. GFP, #P<0.01 vs. FOXO3a).

In a second model of cellular stress, HUVEC were subjected to anoxia, an important feature of the angiogenic cellular response. When HUVEC were exposed to anoxic conditions, HSP70 protein expression increased significantly (Fig. 2C ). The anoxia-induced increase in HSP70 expression rapidly returned to baseline level when cells were reoxygenated. The increase of HSP70 protein expression in response to anoxia was almost completely blocked by overexpression of WT-FOXO3a or TM-FOXO3a (Fig. 2D ). Anoxic conditions led to a reduction in cell viability as assessed by WST-1 assay (Fig. 2E ). Cell viability was further reduced under these conditions by transduction with FOXO3a. Transduction with dominant-negative caspase-9 or Hsp70 significantly protected cells from FOXO3a-mediated death under these conditions.

CONCLUSIONS AND SIGNIFICANCE

In this study, we identify HSP70 as a new target for the Akt/FOXO signaling axis in endothelial cells. Microarray, QT-PCR, and Western immunoblot analysis showed that HSP70 mRNA and protein are positively regulated by Akt in endothelial cells. We extended these observations by showing that FOXO3a, a transcription factor downstream of Akt, plays a role in mediating this response. Akt/FOXO3a-mediated HSP70 regulation is functionally significant for endothelial cell survival under basal conditions as well as in response to anoxia or heat shock. This signaling axis is likely to augment the regulation of HSP70 by GSK-3ß (Fig. 3 ).

View larger version (17K): [in this window] [in a new window]   Figure 3. Proposed scheme for FOXO3a-mediated cytotoxicity in endothelial cells. Under conditions of growth factor stimulation, Akt is activated, causing phosphorylation and nuclear exclusion of FOXO3a. Under conditions of growth factor withdrawal, FOXO3a is dephosphorylated, leading to its activation and nuclear accumulation. FOXO3a activation leads to a suppression of HSP70 expression. Diminished HSP70 levels lead to activation of the intrinsic apoptotic cell death pathway via activation of the caspase-9 cascade. Phosphorylation/inactivation of GSK-3ß is another mechanism of Akt-mediated HSP70 regulation. FOXO3a promotes caspase-8 activation by suppressing intracellular FLIP levels. The regulatory axis identified by this study is indicated by bold lines.

The status of the FOXO3a/HSP70 signaling axis may participate in the balance between blood vessel growth and regression in ischemic tissues.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2841fje; doi: 10.1096/fj.04-2841fje

This article has been cited by other articles:

				H.-Y. Lee, H.-J. You, J.-Y. Won, S.-W. Youn, H.-J. Cho, K.-W. Park, W.-Y. Park, J.-S. Seo, Y.-B. Park, K. Walsh, et al. Forkhead Factor, FOXO3a, Induces Apoptosis of Endothelial Cells Through Activation of Matrix Metalloproteinases Arterioscler. Thromb. Vasc. Biol., February 1, 2008; 28(2): 302 - 308. [Abstract] [Full Text] [PDF]

				K. N. Papanicolaou, Y. Izumiya, and K. Walsh Forkhead Transcription Factors and Cardiovascular Biology Circ. Res., January 4, 2008; 102(1): 16 - 31. [Abstract] [Full Text] [PDF]

				H.-Y. Lee, S.-W. Youn, J.-Y. Kim, K.-W. Park, C.-I. Hwang, W.-Y. Park, B.-H. Oh, Y.-B. Park, K. Walsh, J.-S. Seo, et al. FOXO3a Turns the Tumor Necrosis Factor Receptor Signaling Towards Apoptosis Through Reciprocal Regulation of c-Jun N-Terminal Kinase and NF-{kappa}B Arterioscler. Thromb. Vasc. Biol., January 1, 2008; 28(1): 112 - 120. [Abstract] [Full Text] [PDF]

				H.-Y. Lee, J.-W. Chung, S.-W. Youn, J.-Y. Kim, K.-W. Park, B.-K. Koo, B.-H. Oh, Y.-B. Park, B. Chaqour, K. Walsh, et al. Forkhead Transcription Factor FOXO3a Is a Negative Regulator of Angiogenic Immediate Early Gene CYR61, Leading to Inhibition of Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Circ. Res., February 16, 2007; 100(3): 372 - 380. [Abstract] [Full Text] [PDF]

				P. Rafiee, M. E. Theriot, V. M. Nelson, J. Heidemann, Y. Kanaa, S. A. Horowitz, A. Rogaczewski, C. P. Johnson, I. Ali, R. Shaker, et al. Human esophageal microvascular endothelial cells respond to acidic pH stress by PI3K/AKT and p38 MAPK-regulated induction of Hsp70 and Hsp27 Am J Physiol Cell Physiol, November 1, 2006; 291(5): C931 - C945. [Abstract] [Full Text] [PDF]

				S. Schiekofer, I. Shiojima, K. Sato, G. Galasso, Y. Oshima, and K. Walsh Microarray analysis of Akt1 activation in transgenic mouse hearts reveals transcript expression profiles associated with compensatory hypertrophy and failure Physiol Genomics, October 11, 2006; 27(2): 156 - 170. [Abstract] [Full Text] [PDF]

				M. Liu, R. Aneja, C. Liu, L. Sun, J. Gao, H. Wang, J.-T. Dong, V. Sarli, A. Giannis, H. C. Joshi, et al. Inhibition of the Mitotic Kinesin Eg5 Up-regulates Hsp70 through the Phosphatidylinositol 3-Kinase/Akt Pathway in Multiple Myeloma Cells J. Biol. Chem., June 30, 2006; 281(26): 18090 - 18097. [Abstract] [Full Text] [PDF]

				C. Depre, L. Wang, X. Sui, H. Qiu, C. Hong, N. Hedhli, A. Ginion, A. Shah, M. Pelat, L. Bertrand, et al. H11 Kinase Prevents Myocardial Infarction by Preemptive Preconditioning of the Heart Circ. Res., February 3, 2006; 98(2): 280 - 288. [Abstract] [Full Text] [PDF]

				J. Magrane, K. M. Rosen, R. C. Smith, K. Walsh, G. K. Gouras, and H. W. Querfurth Intraneuronal {beta}-Amyloid Expression Downregulates the Akt Survival Pathway and Blunts the Stress Response J. Neurosci., November 23, 2005; 25(47): 10960 - 10969. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/8/1042 04-2841fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, H.-S. Articles by Walsh, K. Search for Related Content PubMed PubMed Citation Articles by Kim, H.-S. Articles by Walsh, K.