Angiotensin II–Inducible Smooth Muscle Cell Apoptosis Involves the Angiotensin II Type 2 Receptor, GATA-6 Activation, and FasL-Fas Engagement
Rationale: Fas ligand (FasL)-mediated smooth muscle cell (SMC) apoptosis within the vulnerable plaque may lead to plaque instability and rupture, events that underlie myocardial infarction and stroke.
Objective: The molecular mechanisms underlying FasL transcription and FasL-dependent SMC apoptosis were investigated in this study in vitro and in vivo.
Methods and Results: We demonstrate that GATA-6, the predominant GATA family member expressed in SMCs, stimulates SMC apoptosis in an extracellular FasL-dependent manner. Both GATA-6 and FasL were inducibly and transiently expressed following balloon injury to rat carotid arteries. We identified two potential GATA binding in the FasL promoter and demonstrated using DNA binding and chromatin immunoprecipitation assays that GATA-6 regulates FasL through one (−298TTATCA−303) but not both these elements. Angiotensin II (Ang II) stimulated expression of both GATA-6 and FasL. Ang II increased SMC apoptosis in an Ang II type 2 receptor–, caspase 8–, and FasL-dependent fashion. GATA-6 activation was MEK-ERK1/2– and JNK-dependent, and GATA-6 small interfering RNA blocked Ang II–inducible FasL expression and SMC apoptosis. Administration of Ang II to rats increased FasL expression and apoptosis in carotid artery SMCs in an Ang II type 2 receptor– and GATA-6–dependent manner.
Conclusions: This study provides new insights into the transcriptional events underpinning FasL-dependent SMC apoptosis after exposure to Ang II.
Fas ligand (FasL) is a 40-kDa cytotoxic type II transmembrane protein belonging to the tumor necrosis factor1 family. Although Fas (APO-1, CD95) is found in a variety of normal tissues, FasL is expressed mainly by cells of the immune system.2 In human atherosclerosis, FasL is expressed together with markers of apoptosis in inflammatory regions of plaques.3–5⇓⇓ FasL-mediated smooth muscle cell (SMC) apoptosis within the vulnerable plaque may lead to plaque instability and rupture, events well known to cause myocardial infarction and stroke.4,6⇓ Plasma levels of soluble FasL are elevated in patients with acute myocardial infarction and unstable angina pectoris.7 When FasL engages its receptor Fas, it triggers the formation of DISC (death-inducing signaling complex) by recruiting an adaptor molecule FADD (Fas-associating protein death domain).1 The N-terminal region of FADD when bound recruits procaspase 8.8 When procaspase 8 is proteolytically processed, its active form caspase 8 activates the extrinsic apoptotic pathway.9 Forced expression of FasL in rat carotid arteries by adenovirus leads to SMC apoptosis and inhibits neointima formation.10 FasL expression in SMCs is regulated at the level of transcription although this is poorly understood. Previous studies by our group and others11 have characterized transcription factors regulating FasL promoter activity in this cell type, which include Sp1,12,13⇓ c-Jun,14 and Ets-1.12 Roles for transcription factors such as Egr-1,15 NFAT,15 nuclear factor (NF)-κB,16,17⇓ and c-Myc18 have been reported in other cell types.
The GATA family of transcriptional regulatory proteins are comprised of 6 members. Though originally characterized in the context of hematopoiesis and heart development, GATA factors are expressed in a wide variety of tissues. GATA-6 is the predominant GATA family member expressed in vascular SMCs and controls the phenotype of these cells following vascular injury.19,20⇓ Forced expression of GATA-6, like FasL, in balloon-injured carotid arteries inhibits neointima formation.21 GATA-6 expression is downregulated in SMCs following mitogen stimulation or vascular injury.21 GATA-6 levels increase in endothelial cells subjected to laminar shear stress and urokinase plasminogen activator, a GATA-6–dependent gene is elevated in atherosclerotic lesions.22 Here, we report that Ang II stimulates GATA-6 and FasL expression. Ang II increases SMC apoptosis in a manner involving the Ang II type 2 (AT2) receptor, mitogen-activated protein kinase kinase (MEK)–extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), caspase 8, and extracellular FasL.
Details of materials and experimental procedures in respect of cell culture, plasmid constructs, transfection and luciferase analysis, polymerase chain reaction, nuclear protein extraction, electrophoretic mobility shift assay, flow cytometry using annexin V staining, Cell Death Detection ELISAPlus, TUNEL staining, real-time chromatin immunoprecipitation assay, Western blotting, rat carotid artery balloon injury, Ang II administration in rats, immunohistochemical staining, and statistical analysis are in the expanded Methods section in the Online Data Supplement, available at http://circres.ahajournals.org.13,23–30⇓⇓⇓⇓⇓⇓⇓⇓
FasL and GATA-6 Are Inducibly Expressed in Balloon-Injured Rat Carotid Arteries
Previous studies have reported that FasL is inducibly expressed in the carotid arteries of mice 7 days after balloon distension injury31 and can play a negative growth-regulatory role in the artery wall.10 For example, forced expression of FasL in rat carotid arteries by adenovirus leads to SMC apoptosis and inhibits neointima formation.10 However, it is not known whether endogenous FasL is expressed acutely after SMC injury (ie, within hours, rather than weeks), nor is the transcriptional basis for FasL induction in SMCs well understood. Immunohistochemical analysis of uninjured and balloon-injured rat carotid arteries revealed that FasL is inducibly expressed by medial SMCs within 4 hours of balloon injury (Figure 1A and 1B).
Analysis of the FasL promoter for putative transcription factor binding sites using TESS bioinformatics software revealed 2 putative GATA binding elements, −298TTATCA−303 and −176AGATAG−171 (Figure 1C). GATA-6 is the predominant GATA family member expressed in vascular SMCs and controls phenotype following vascular injury.19,20⇓ Like FasL, forced expression of GATA-6 in balloon-injured carotid arteries inhibits neointima formation.21 Thus far, no member of the GATA has been linked to FasL expression in any cell type (Table). GATA-6 staining was observed in medial SMCs 1.5 hour after injury but not after 4 hours (Figure 1A and 1B), indicating that this transcription factor is transiently expressed in response to injury, as observed previously with immediate-early genes Egr-132 and c-Jun.33 GATA-6 and FasL levels were at preinjury levels 24 hours after injury (Figure 1A and 1B). A previous study reported that GATA-6 levels were downregulated in rat carotid arteries after balloon injury, but GATA-6 expression was not examined earlier than 24 hours after injury.21
Ang II Increases FasL Expression in SMCs and Stimulates Apoptosis via the Extrinsic Pathway and the AT2 Receptor
Ang II, the effector peptide hormone of the renin-angiotensin system involved in the control of vascular tone, growth and apoptosis,34,35⇓ has been shown to stimulate FasL expression and stimulate apoptosis via the Ang II type 1 (AT1) receptor.36 The latter findings are surprising in light of other studies indicating a more dominant role for the AT2 receptor in Ang II–dependent SMC apoptosis.37 The transcriptional mechanisms underlying Ang II control of FasL is poorly understood, as is whether FasL plays a causal role in Ang II–dependent SMC apoptosis. Transient transfection analysis in cultured SMCs revealed that Ang II induced time-dependent activation of the human FasL promoter (Figure 2A). FasL promoter-dependent expression increased within 2 hours of exposure to Ang II, remained elevated for at least another 2 hours, and then returned to basal levels within 24 hours (Figure 2A). We used flow cytometry measuring annexin V staining to show that apoptosis induced by Ang II was abolished by 50 μg/mL Fas-Fc (Figure 2B) but not Fc alone. Fas-Fc binds to extracellular FasL and blocks Fas-dependent apoptosis.13 Moreover, Ang II–inducible SMC apoptosis was abrogated by the presence of Z-IETD-FMK, an inhibitor of caspase 8, but not by the caspase 9 inhibitor Z-LEHD-FMK (Figure 2C). SMC apoptosis stimulated by Ang II was blocked by the AT2 receptor PD123319, but not the AT1 receptor antagonist losartan (Figure 2C).
Moreover, in an assay measuring cytoplasmic histone-associated internucleosomal DNA fragmentation,13,27⇓ Ang II–inducible SMC apoptosis was inhibited by PD123319, Z-IETD-FMK, the MEK-ERK1/2 inhibitor PD98059, and JNK inhibitor II, whereas losartan, Z-LEHD-FMK, and the phosphatidylinositol 3-kinase inhibitor Ly294002 had no effect (Figure 2D). In the absence of Ang II, these inhibitors had negligible effects on apoptosis, except for Ly294002 and JNK inhibitor II, which produced a slight increase relative to untreated cells (Figure 2D). These findings demonstrate that Ang II stimulates SMC apoptosis via AT2, MEK-ERK1/2, JNK, and FasL-Fas engagement.
FasL-Fas Engagement Mediates GATA-6–Inducible SMC Apoptosis
To begin to define a direct relationship between GATA-6 and FasL, we cloned GATA-6 and GATA-4 cDNA into pcDNA3 and performed transient transfection analysis. Figure 3A demonstrates that GATA-6 activates the FasL promoter, whereas an identical amount of GATA-4, which is not or is poorly expressed in SMCs,19,20⇓ had no effect. GATA-6 but not GATA-4 increased annexin V staining assessed by flow cytometry (Figure 3B). GATA-6 induction of apoptosis was completely blocked when SMCs were incubated with Fas-Fc but unaffected by the presence of Fc alone (Figure 3C). These data indicate that FasL-Fas engagement mediates GATA-6–inducible SMC apoptosis.
We next determined whether Ang II regulates GATA-6 expression. Quantitative real-time PCR demonstrates that GATA-6 mRNA levels were increased by SMC exposure to Ang II (Figure 3D). Blockade of Ang II induction of GATA-6 expression with PD98059 and JNK inhibitor II revealed the requirement of MEK-ERK1/2 and JNK (Figure 3D). In contrast, phosphatidylinositol 3-kinase inhibition had no effect on GATA-6 expression stimulated by Ang II (Figure 3D). These inhibitors had no effect on GATA-6 expression in the absence of Ang II (Figure 3D). As with Ang II induction of apoptosis (Figure 2C), GATA-6 mRNA expression stimulated by Ang II was blocked by AT2 receptor inhibition (Figure 3D). The absence or presence of serum in the culture medium did not affect the extent of Ang II–inducible GATA-6 expression (Figure 3D).
GATA-6 Small Interfering RNA Blocks Ang II—Inducible FasL Expression and SMC Apoptosis
To more directly link GATA-6 with FasL and Ang II–inducible apoptosis, we used small interfering (si)RNA targeting GATA-6, which reduced GATA-6 mRNA levels in SMCs exposed to Ang II as compared to AllStars negative control siRNA (Qiagen) (Figure 4, upper left). GATA-6 knockdown also inhibited Ang II–inducible FasL expression (Figure 4, lower left) and blocked apoptosis stimulated by Ang II (Figure 4, right). These findings demonstrate that GATA-6 mediates Ang II induction of FasL expression and SMC apoptosis.
Ang II Stimulates GATA-6 Binding to a Region of the FasL Promoter Spanning the −298TTATCA−303 Element
Electrophoretic mobility-shift assay was performed with a 32P-labeled double-stranded oligonucleotide (32P-Oligo FasL−313/−277) spanning the −298TTATCA−303 element. An inducible nucleoprotein complex resulted that was eliminated by GATA-6 antibodies, but not Sp1 antibodies (Figure 5A, left). Mutation of 2 nucleotides in 32P-Oligo FasL−313/−277 (to −298TTTACA−303, producing 32P-Oligo mFasL−313/−277) did not support complex formation (Figure 5A, left). In contrast, an oligonucleotide (32P-Oligo FasL−202/−161) spanning the −176AGATAG−171 element did not produce an Ang II–inducible complex (Figure 5A, right). These findings were supported by real-time chromatin immunoprecipitation analysis. The FasL promoter amplicon−714/−207, spanning element −298TTATCA−303 and encompassing known Egr binding sites (Figure 1C),15 showed increased occupancy by GATA-6 in response to Ang II (Figure 5B). Egr-1, also bound the promoter (Figure 5B). The histone deacetylase (HDAC) inhibitor trichostatin A (TSA), which regulates transcription by promoting histone and transcription factor acetylation,38 suppressed Ang II–inducible enrichment of GATA-6 (and Egr-1) at the FasL promoter (Figure 5B). These findings, taken together, demonstrate that GATA-6 stimulated by Ang II binds the FasL promoter in an AT2 receptor– and HDAC-dependent manner and suggest that FasL induces SMC apoptosis via an autocrine loop involving caspase 8 activation.
Ang II Activates the FasL Promoter via the −298TTATCA−303 but Not −176AGATAG−171 Element
To demonstrate the requirement of one or both putative GATA binding elements in the FasL promoter for Ang II induction, we introduced mutations of −298TTATCA−303 or −176AGATAG−171 in the FasL promoter–reporter construct FasL-hsLuc.13 Ang II activation of the FasL promoter was blocked by mutation in −298TTATCA−303 (Figure 5C), whereas the −176AGATAG−171 element was redundant in this process (Figure 5C). Mutation of both elements, as with disruption of the −298TTATCA−303 alone, failed to support Ang II induction of FasL promoter activity (Figure 5C).
Ang II Increases FasL Expression and Apoptosis in SMCs of Rat Carotid Arteries in an AT2- and GATA-6–Dependent Fashion
To demonstrate the in vivo relevance of our findings, male Sprague–Dawley rats received IP injections of Ang II (1 mg/kg)39 in the absence or presence of losartan (200 μg/kg),40 PD123319 (200 μg/kg),40 GATA-6 siRNA (500 μg), or AllStars negative control siRNA (500 μg); after 1.5, 4, or 24 hours, the animals were euthanized and the carotid arteries were fixed in formalin and paraffin-embedded. Immunohistochemical analysis revealed that Ang II increased FasL (Figure 6A and 6B) and GATA-6 (Figure 6C and 6D) expression in medial SMCs. The inducible expression of each was abolished by the presence of PD123319 and GATA-6 siRNA (Figure 6A through 6D). In contrast, losartan and AllStars negative control siRNA had no effect (Figure 6A through 6D). These in vivo findings demonstrate that Ang II stimulates GATA-6 and FasL expression and that this is mediated via the AT2 but not the AT1 receptor. Moreover, these data show that siRNA knockdown of GATA-6 blocks Ang II induction of FasL expression in the SMCs. Absence of staining when an isotope-specific immunoglobulin was used as the primary antibody demonstrated specificity (Figure 6E). Ang II increased TUNEL+ cells in the media by 3.5-fold (Figure 6F and 6G). This induction was abolished by the presence of PD123319 and GATA-6 siRNA, but not losartan nor negative control siRNA (Figure 6F and 6G). Thus, consistent with our in vitro data, Ang II increases FasL expression and apoptosis in medial SMCs of rat carotid arteries in an AT2- and GATA-6–dependent manner.
Previous studies have shown that in human atherosclerotic plaques, FasL is expressed with active caspase in inflammatory regions.3–5⇓⇓ SMC apoptosis within the vulnerable plaque may lead to plaque instability and rupture, events well known to cause myocardial infarction and stroke.4 Interestingly, plasma levels of soluble FasL are elevated in patients with acute myocardial infarction and unstable angina pectoris.7 In this report, we report that GATA-6 stimulates SMC apoptosis in an extracellular FasL-dependent manner. GATA-6 regulates FasL transcription through −298TTATCA−303 but not a second putative element downstream. Moreover, Ang II increases GATA-6 and FasL expression and SMC apoptosis in an AT2 receptor–, caspase 8–, and extracellular FasL–dependent fashion. GATA-6 activation is dependent on MEK-ERK1/2 and JNK, whereas GATA-6 siRNA blocks Ang II–inducible FasL expression and SMC apoptosis. Administration of Ang II to rats increases FasL expression and apoptosis in medial SMCs of carotid arteries in an AT2- and GATA-6–dependent manner. This is the first study linking a GATA factor with FasL expression in any cell type, and provides key insights into the mechanisms underlying FasL-dependent apoptosis in SMCs exposed to Ang II (Figure 7).
Our results using PD123319 are in keeping with other studies indicating a more dominant role for the AT2 receptor in Ang II–dependent SMC apoptosis.37 Fas-Fc has proven to be a useful reagent in this context. For example, Alcouffe et al found that oxidized low-density lipoprotein triggers the early production of soluble FasL by T lymphocytes and that Fas-Fc inhibits oxidized low-density lipoprotein–mediated apoptosis.41 Our findings also indicate that TSA abolishes Ang II–mediated GATA-6 binding to the authentic FasL promoter. HDACs are enzymes that affect the acetylation status of histones and transcription factors. TSA, originally developed as an antifungal agent, is a potent HDAC inhibitor that can both stimulate and inhibit transcription. For example, we recently showed that TSA relieves repression of IGF1R following oxidative stress.42 TSA also upregulates the expression of angiostatic extracellular metalloproteinase ADAMTS1.43 Conversely, TSA can suppress tissue factor induction by tumor necrosis factor α, lipopolysaccharide, and interleukin-1β in vascular endothelial cells.44 Currently, there is only one report of HDAC regulation of GATA-6–dependent transcriptional activity. Yin et al used TSA to demonstrate that homeodomain protein Hop negatively regulates GATA-6/Nkx2.1/SRF activity in lung epithelial cells in a HDAC-dependent manner.45
The GATA family of transcription factors are so named because of their affinity for the consensus DNA binding motif 5′-(A/T)GATA(A/G)-3′. These transcription factors contain either one or two distinct zinc finger domains with an adjacent highly conserved basic region, which constitutes the DNA binding domain. Zinc finger proteins are thought to represent the largest class of DNA binding proteins in eukaryotes, and examples of other proteins of this class regulating vascular gene expression and cellular phenotype include Egr-1 and Sp1.24,46,47⇓⇓ GATA factors have been shown to play critical roles in development, such as cell fate specification, regulation of differentiation, and control of cell proliferation and migration.48 GATA factors play vital roles during development, and GATA-6 can act as a positive or negative regulator of SMC-specific gene expression.49 Microarray analysis demonstrates that GATA-6 regulates genes associated with synthetic SMC phenotype, involving cell-to-cell signaling and cell–matrix interactions.50 It will be interesting to determine the cofactors of GATA-6 involved in its regulation of FasL expression. Potential players in this process are NFAT51 and p300,52 which associate and cooperate with GATA-6. Similarly, it will be interesting to investigate the potential involvement of reactive oxygen species. Atherosclerosis leads to heightened oxidative stress in the vessel wall, and Ang II is well known to activate NADPH oxidases in SMCs. The resultant hydrogen peroxide is known to enhance DNA binding of redox sensitive zinc finger transcription factors, including GATA-4,53 and transcriptional activation of FasL.54
We thank Ryan Peden and Annie Au-Yeung for helpful comments and technical assistance.
Sources of Funding
This work was supported by grants from the National Health and Medical Research Council (Australia), Australian Research Council, and National Heart Foundation (Australia). L.M.K. is an Australia Fellow and R.S. is a Senior Principal Research Fellow of the National Health and Medical Research Council.
Original received December 18, 2008; resubmission received June 22, 2009; revised resubmission received July 13, 2009; accepted July 15, 2009.
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