Betacellulin and Amphiregulin Induce Upregulation of Cyclin D1 and DNA Synthesis Activity Through Differential Signaling Pathways in Vascular Smooth Muscle Cells
Activation of EGF receptors is closely involved in vascular proliferative diseases. The signaling mechanisms of EGF ligands, including betacellulin (BTC) and amphiregulin (AR), are poorly understood. We examined how BTC and AR induced DNA synthesis activity in primary cultures of human thoracic aortic smooth muscle cells (HTASMCs). BTC induced phosphorylation of all four EGF receptors present on HTASMCs: ErbB1, ErbB2, ErbB3, and ErbB4. BTC rapidly induced the phosphorylation of Akt, GSK3α/β, and two FoxO factors, FKHR and AFX, in a dose- and time-dependent manner. BTC increased nuclear β-catenin accumulation. BTC increased cyclin D1 mRNA, cyclin D1 protein, and DNA synthesis activity. Pretreatment with the phosphatidylinositol 3′-kinase (PI 3′-kinase) inhibitor wortmannin suppressed BTC-induced cyclin D1 mRNA and protein and DNA synthesis activity. In contrast, AR, a specific ErbB1 ligand, induced sustained ERK1/2 and Elk1 phosphorylation, increased cyclin D1 mRNA and protein, and increased DNA synthesis activity. AR did not produce any changes in Akt phosphorylation. Pretreatment with PD98059 suppressed AR-induced cyclin D1 mRNA and protein. Thus, the PI 3′-kinase/Akt/GSK/FoxO/β-catenin pathway could be the major signaling cascade for BTC-induced upregulation of cyclin D1 protein, whereas a sustained ERK/Elk1 activation could be the major signaling cascade for AR-induced upregulation of cyclin D1 protein in HTASMCs. Moreover, immunohistochemical staining revealed that that BTC, ErbB1, and ErbB4 are upregulated in the plaques of human atherosclerotic coronary arteries. Taken together, BTC and AR could be potent growth factors in proliferative vascular diseases.
- epidermal growth factor receptors
- phosphatidylinositol 3′-kinase
- extracellular signal-regulated kinase 1 and 2
Betacellulin (BTC) and amphiregulin (AR) are members of the EGF family. BTC was originally identified as a growth-promoting factor in the conditioned medium of a mouse pancreatic β-cell carcinoma (insulinoma) cell line,1 and it has since been identified in humans.2 AR was originally identified from conditioned medium of a phorbol 12-myristate 13-acetate–treated human breast carcinoma cell line.3 BTC has been shown to be a potent mitogen for fibroblasts, vascular smooth muscle cells (VSMCs), and retinal pigment epithelial cells, whereas AR is a cell type–dependent bifunctional modulator of cell growth.1,3 BTC and AR are synthesized in a wide range of adult tissues and in many cultured cells, including smooth muscle cells and epithelial cells.1–4
Four EGF receptors are currently known: ErbB1 (EGFR), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4).5–7 The receptors exist as inactive monomers that dimerize after ligand activation, either as homo- or heterodimers. After ligand binding, the tyrosine kinase intracellular domain of the receptor is activated, with autophosphorylation of the intracellular domain; the phosphorylated receptor then initiates a cascade of intracellular events.5–7 BTC is known to bind and stimulate homodimers of ErbB1 and ErbB4, and heterodimers of ErbB1/ErbB2, ErbB1/ErbB3, ErbB1/ErbB4, ErbB2/ErbB3, and, ErbB2/ErbB4.5–8 AR is known to preferentially bind and stimulate homodimers of ErbB1.6 Thus, BTC and AR produce physiological and pathological effects through ErbB receptors, although their binding and affinity vary among different cell types.
Recently, we showed that BTC induced angiogenesis in primary cultured endothelial cells through the activation of ErbB2, ErbB3, and ErbB4 receptors acting on two pathways: the intracellular phosphatidylinositol 3′-kinase (PI 3′-kinase)/Akt pathway and the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway.9 We also observed that BTC-immunopositive VSMCs are more abundant in the intimal region of atherosclerotic lesions than in the normal portions of human thoracic aortas.10 Notably, strong BTC-immunoreactivity in the migrating VSMCs and macrophages were observed around the core lesion of atherosclerotic plaques, suggesting that BTC may be closely involved in vascular proliferative diseases. Moreover, recent studies indicate that activation of PI 3′-kinase is the key signaling pathway for induction of cyclin D1 and cell cycle in VSMCs in animal models of vascular proliferation.11,12
Therefore, in this study, we examined how BTC and AR induced DNA synthesis activity in primary cultures of human thoracic aortic smooth muscle cells (HTASMCs). In addition, we reevaluated the localization of BTC and its receptors in atherosclerotic lesions of human coronary artery. Our results indicate that BTC and AR could be potent growth factors for proliferative vascular diseases.
Materials and Methods
Materials and Cell Culture
Recombinant human BTC and AR were purchased from Sigma-Aldrich. Recombinant human EGF was purchased from R&D Systems. PI 3′-kinase inhibitor wortmannin, Akt inhibitor, and mitogen-activated protein/extracellular signal-regulated kinase (MEK) 1/2 inhibitor PD98059 were purchased from Calbiochem. Antibodies for phosphospecific Akt (Ser473), GSK3α/β (Ser21/Ser9), FKHR (Ser256)/AFX, Elk1 (Ser383), and ERK1/2 (Thr202/Tyr204) were purchased from Cell Signaling Technology. Antibodies for ErbB1, ErbB2, ErbB3, ErbB4, and cyclin D1 were purchased from Santa Cruz Biotechnology. Antibodies for phospho-ErbB1, phospho-ErbB2, and phospho-tyrosine were purchased from Upstate Biotechnology. Antibody for β-catenin was purchased from BD Transduction Laboratories. HTASMCs were isolated from thoracic aortas of donors during heart transplant by the explant method and were cultured as described previously.13 Tissue collection was approved by the local Ethics Committee of Keimyung University Hospital. The primary cultured cells used in this study were between passage 3 and 5.
HTASMCs were incubated in DMEM with 1% fetal bovine serum for 16 hours before addition of the indicated growth factors. The phosphorylation of EGF receptors, Akt, GSK3α/β, FoxO factors, ERK1/2 and Elk1, and the protein levels of β-catenin and cyclin D1 were measured as previously described.9 All signals were visualized and analyzed by densitometric scanning (LAS-1000, Fuji Film).
HTASMCs were seeded onto glass coverslips and incubated for 24 hours before addition of BTC. Treated cells were washed with PBS, fixed with methanol/acetone (1:1) for 15 minutes, incubated with anti–β-catenin antibody (BD Transduction Laboratories), then incubated with FITC-conjugated secondary antibody, and counter-stained with Hoechst 33425. The slides were mounted and examined by confocal microscopy, and images were captured with an LSM 510 camera (Zeiss).
Ribonuclease Protection Assay (RPA)
RPA for human cyclin D1 and cyclophilin were performed on total RNAs using the Ambion RPA kit.
Assays of DNA Synthesis and Cell Cycle Progression
The DNA synthetic activity of HTASMCs was measured using PicoGreen fluorescent reagent (Molecular Probes) as previously described.9 Cell cycle progression was measured using propidium iodide staining with a FACSCalibur (BD Bioscience).
Sampling and Immunohistochemistry
Human atherosclerotic coronary arteries were obtained from autopsied cadavers with informed consent of the next of kin. Immunohistochemical detection of BTC and EGF receptors in the lesions was performed as described previously.10
Data are expressed as mean±SD. Statistical significance was tested using 1-way ANOVA followed by the Student-Newman-Keuls test. Statistical significance was set at P<0.05.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
BTC Induced Activation of All Four ErbB Receptors
Because BTC is known to exert biological effects through ErbB receptors,8 we examined the distribution of subtypes of EGF receptors in primary cultures of HTASMCs. Our Western blot analyses indicated that HTASMCs express all four ErbB receptors, ErbB1, ErbB2, ErbB3, and ErbB4, and that EGF phosphorylated all four receptors (data not shown). Furthermore, BTC phosphorylated all four receptors: ErbB1, ErbB2, ErbB3, and ErbB4 (see online Figure 1 in the online data supplement at http://www.circresaha.org).
BTC Induces Phosphorylation of Akt, GSK3α/β, FKHR, AFX, ERK1/2, and Elk1 and Induces Accumulation of Nuclear β-Catenin
Because growth factor receptors are known to mediate the activation of intracellular PI 3′-kinase/Akt and MEK/ERK cascades for upregulation of cyclin D1 and proliferation,14 we first examined the effect of BTC on phosphorylation of Akt (Ser473) and ERK1/2 in primary cultured HTASMCs. BTC (10 ng/mL) increased the phosphorylation of Akt and its downstream kinase, GSK3α/β (Ser21/Ser9), as early as 1 minute and produced a maximal effect at 1 minute (Figures 1A and 1B). The phosphorylation level declined but remained higher than control at 30 minutes. The maximum mean increases in Akt and GSK3β phosphorylation were 12.5-fold and 14.6-fold, respectively. BTC (0.03 to 10 ng/mL) increased the phosphorylation of both Akt and GSK3α/β in a dose-dependent manner (Figures 1C and 1D). In addition, pretreatment with PI 3′-kinase inhibitor, wortmannin (30 nmol/L), completely abolished the BTC-induced Akt and GSK3α/β phosphorylation (data not shown). Pretreatment with Akt inhibitor suppressed the BTC-induced GSK3α/β phosphorylation in a dose-dependent manner (Figure 1E). Both BTC (10 ng/mL at 1 minute) and EGF (10 ng/mL at 5 minutes) produced a similar level of Akt and GSK3α/β phosphorylation (Figure 1). Inactivation of GSK3α/β through dephosphorylation at two sites (Ser21/Ser9) is known to cause β-catenin to accumulate in the cytoplasm and then translocate into the nucleus.15 Accordingly, BTC (10 ng/mL) increased β-catenin protein level as early as 1 minute and produced a continued increase until 60 minutes (Figure 2A). Immunofluorescent staining revealed that more than 60% of BTC-treated cells have a marked increase of β-catenin in the nuclei (Figure 2B). Pretreatment with GSK inhibitor, LiCl (20 mmol/L), increased nuclear β-catenin protein level regardless of Akt inhibition (Figure 2C). In addition, pretreatment with Akt inhibitor markedly suppressed BTC-induced nuclear β-catenin protein level (Figure 2C). These data indicate that the BTC-induced increase in nuclear β-catenin protein level is mainly through the PI 3′-kinase/Akt/GSK3 cascade in VSMCs. Moreover, BTC increased the phosphorylation of Akt downstream transcription factors, FoxO transcriptional factors FKHR and AFX, in a time-dependent manner (Figure 2D). BTC (10 ng/mL) also increased the phosphorylation of ERK1/2 and its downstream transcription factor, Elk1, as early as 1 minute and produced a maximal effect at 10 minutes (Figures 3A and 3C). These effects declined but remained higher than control at 60 minutes. The maximum mean increases in ERK1/2 and Elk1 phosphorylation were 13.5-fold and 5.2-fold, respectively. BTC (0.03 to 10 ng/mL) increased ERK1/2 phosphorylation in a dose-dependent manner (Figure 3B). In addition, pretreatment with MEK inhibitor, PD98059 (50 nmol/L), completely abolished the BTC-induced ERK1/2 and Elk1 phosphorylation (data not shown). Both BTC (10 ng/mL at 10 minutes) and EGF (10 ng/mL at 10 minutes) produced a similar level of ERK1/2 phosphorylation (Figure 3). These data indicate that BTC is a strong activator of both the intracellular Akt/GSK3/FoxO/β-catenin cascade and the ERK/Elk1 cascade in VSMCs.
BTC Upregulates Cyclin D1 Protein Mainly Through the Intracellular PI 3′-Kinase/Akt Pathway
The phosphorylation of Akt, GSKα/β, and FoxO factors, and the accumulation of nuclear β-catenin play a crucial role in the upregulation of cyclin D1 protein level by suppressing cyclin D1 degradation and by increasing cyclin D1 transcription (16 to 19, MCB). Phosphorylation of ERK1/2 plays a crucial role in the upregulation of cyclin D1 protein level by increasing cyclin D1 transcription.14 Therefore, we next examined how BTC upregulated cyclin D1 protein levels in HTASMCs. BTC (10 ng/mL) increased cyclin D1 mRNA and protein in a time-dependent manner (Figures 4A and 4B). Interestingly, pretreatment with PI 3′-kinase inhibitor wortmannin (30 nmol/L) almost completely suppressed BTC-induced cyclin D1 mRNA and protein, whereas pretreatment with MEK inhibitor PD98059 (50 nmol/L) only weakly suppressed these increases (Figures 4C and 4D). Moreover, pretreatment with Akt inhibitor completely suppressed BTC-induced cyclin D1 protein. Thus, PI 3′-kinase/Akt/GSK/FoxO/β-catenin appears to be the major regulatory pathway for BTC-induced upregulation of cyclin D1 protein in VSMCs.
AR Upregulates Cyclin D1 Protein Mainly Through Sustained Activation of the Intracellular ERK/Elk1 Pathway
Because activation of different ErbB receptors is known to activate different intracellular signaling cascades, we examined the effect of specific ErbB1 ligand, AR, on signaling cascades and cyclin D1 levels in HTASMCs. Preliminary results indicated that 100 ng/mL of AR produced a maximal effect in ErbB1 and ERK1/2 phosphorylation (data not shown). Thus, we chose this concentration of AR for the subsequent experiments. As expected, AR (100 ng/mL) phosphorylated only ErbB1 among four ErbB receptors (Figures 5A through 5C). Notably, AR (100 ng/mL) did not produce any changes in Akt (Ser473) phosphorylation up to 60 minutes (Figure 5D). In contrast, AR (100 ng/mL) increased ERK1/2 and Elk1 phosphorylation as early as 1 minute and produced a maximal effect at 60 minutes (Figures 5E and 5F). These effects declined slightly but remained elevated up to 5 hours (data not shown). The maximum mean increases in ERK1/2 and Elk1 phosphorylation were 12.8-fold and 11.1-fold, respectively. Thus, AR-induced ERK/Elk1 activation was more sustained than BTC-induced ERK/Elk1 activation. AR (100 ng/mL) increased cyclin D1 mRNA in a time-dependent manner, whereas it increased cyclin D1 protein at 6 hours and maintained the same elevated levels of cyclin D1 protein at 12 and 24 hours after treatment (Figures 6A and 6B). In comparison, BTC (10 ng/mL) steadily increased cyclin D1 protein. Accordingly, pretreatment with PD98059 (50 nmol/L) markedly suppressed AR-induced cyclin D1 mRNA and protein, whereas pretreatment with wortmannin (30 nmol/L) slightly suppressed AR-induced cyclin D1 mRNA and protein (Figures 6C and 6D). Thus, AR-induced ErbB1 activation produced a sustained ERK/Elk1 activation, which could be a major regulator for AR-induced upregulation of cyclin D1 protein in VSMCs.
BTC and AR Increased DNA Synthetic Activity and Cell Cycle Progression Through Different Signaling Pathways
Because upregulation of cyclin D1 protein is a major regulatory step in the G1 phase of cell cycle,20 we examined the effects of BTC and AR on DNA synthesis in HTASMCs. BTC and AR increased DNA synthetic activity approximately 2.9-fold and 1.9-fold, respectively (see online Figure 2A). Moreover, BTC and AR increased cell cycle progression from G1 phase to S and G2/M phase (see online Figure 2). Pretreatment with wortmannin (30 nmol/L) almost completely suppressed BTC-induced DNA synthetic activity and cell cycle progression, whereas pretreatment with PD98059 (50 nmol/L) slightly suppressed BTC-induced DNA synthetic activity and cell cycle progression (see online Figure 2). In contrast, pretreatment with wortmannin (30 nmol/L) slightly suppressed AR-induced DNA synthetic activity and cell cycle progression, whereas PD98059 (50 nmol/L) almost completely suppressed AR-induced DNA synthetic activity and cell cycle progression (see online Figure 2). Thus, BTC-induced DNA synthetic activity and cell cycle progression is mainly dependent on PI 3′-kinase/Akt dependent upregulation of cyclin D1, whereas AR-induced DNA synthetic activity and cell cycle progression is mainly dependent on ERK dependent upregulation of cyclin D1.
BTC and ErbB Are Found in Atherosclerotic Plaques
Given that BTC is a potent factor for DNA synthesis and cell cycle progression, and the antibodies for ErbB1, ErbB2, and ErbB4 work well on paraffin-embedded human tissues, we used immunohistochemistry to evaluate the expression of BTC, ErbB1, ErbB2, and ErbB4 in human atherosclerotic coronary arteries. Consistent with our previous report,10 in the nonatherosclerotic portion of coronary artery, the intimal VSMCs form diffuse intimal thickening lesions. BTC immunostaining was detected weakly in the medial VSMCs and relatively intensely in the intimal VSMCs near the internal elastic lamina (data not shown). In the atherosclerotic portion of coronary artery, macrophages, macrophage-derived foam cells, lymphocytes, and VSMCs make up eccentric intimal thickening lesions, termed plaques. Strong BTC immunoreactivity was detected in macrophages and VSMCs (Figures 7A, 7B, and 7D). VSMCs, in particular, had BTC immunoreactivity near the inner elastic lamina of the plaque (Figures 7E and 7F). In these lesions, many ErbB1- (Figure 7I) or ErbB4- (Figure 7J) positive cells were observed, whereas ErbB2 immunoreactivities were hardly detected (data not shown). Macrophage-derived foam cells had no immunostaining of ErbB1 and ErbB4. VSMCs around a small vessel were positive for ErbB1 immunostaining (Figures 7H and 7I).
BTC is a ligand of multiple EGF receptors and is known to bind to homodimers of ErbB1 and ErbB4, and also to heterodimers of ErbB1/ErbB2, ErbB1/ErbB3, ErbB1/ErbB4, ErbB2/ErbB3, and ErbB2/ErbB4.8 Our biochemical results indicated that BTC induced phosphorylation of all four of EGF receptors in VSMCs. Subsequently, BTC-evoked dual signaling cascades (1) prompt phosphorylation of Akt, GSK3, and FoxO factors, and nuclear accumulation of β-catenin and (2) transient phosphorylation of ERK/Elk1. Thus, on BTC binding, all possible dimeric EGR receptors may be activated, and their signals mediated accordingly. In comparison, AR is a specific ErbB1 ligand and binds preferentially to homodimeric ErbB1.6 Accordingly, our biochemical results indicated that AR induced ErbB1 phosphorylation only. Subsequently, AR evoked relatively sustained ERK and Elk1 phosphorylation without any changes in Akt phosphorylation. Thus, BTC and AR evoke differential activation of EGF receptors and differential downstream signals in VSMCs (Figure 8).
The G1 phase of the cell cycle is controlled by D-type cyclins and cyclin-dependent kinases (CDKs).20 Progression of G1 phase is mainly effected through induction of cyclin D1 by growth factors including EGF ligands.20 Growth factor–induced activation of PI 3′-kinase/Akt and ERK has been linked to induction of cyclin D1 and cell cycle progression.14 In the present study, we noted that BTC rapidly activated Akt in a PI 3′-kinase–dependent manner. Akt activation is known to lead to inactivation of GSK3α and β through their phosphorylation at Ser21 and Ser9, respectively.21–23 GSK3 is a well-known regulator of cyclin D1 protein.16,21–23 Active GSK3β-induced phosphorylation of cyclin D1 at Thr286 causes increased association of cyclin D1 with a nuclear exportin (Crm-1) and promotes nuclear exit and subsequent proteasomal degradation of cyclinD1.16 Conversely, inactivated GSK3β results in suppression of cyclin D1 phosphorylation at Thr286 and thereby suppresses cyclin D1 degradation. Akt is also known to phosphorylate all three FoxO subfamily of forkhead transcriptional factors, specifically FKHR, AFX, and FKHR1. This phosphorylation results in nuclear exclusion and inhibition of FoxO factor–mediated gene expression.24 A recent report indicates that FoxO factors repress cyclin D1 expression at the transcriptional level.19 Our results suggest that BTC-induced phosphorylation of FKHR and AFX may contribute to the increase in cyclin D1 at the transcription level by releasing the repression by FoxO factors. Thus, BTC-induced Akt activation contributes to the increase in cyclin D1 at the level of degradation and transcription by inducing the phosphorylation of GSK3β and FoxO factors (Figure 8).
Another important target of GSK3 is β-catenin,22,25 a transcriptional coactivator that has important roles in early development. In unstimulated cells, active GSK3 constitutively phosphorylates β-catenin on N-terminal serine residues, targeting β-catenin for rapid ubiquitination and proteasome-mediated degradation.26 Inhibition of GSK3-dependent phosphorylation of β-catenin allows β-catenin to accumulate in the cytoplasm and then translocate into the nucleus.15 Once in the nucleus, β-catenin promotes transcription by binding to members of the lymphoid enhancer factor-1 (LEF-1)/T factor (TCF) family of DNA-binding proteins.27,28 In mammalian cells, β-catenin upregulates the transcription of cyclin D1.17,18 Thus, BTC-induced phosphorylated GSK3 can induce nuclear β-catenin accumulation, which increases induction of cyclin D1 mRNA transcription. In fact, our results showed that BTC rapidly phosphorylated GSK3 and induced nuclear β-catenin accumulation in a largely Akt-dependent manner in VSMCs. In addition, BTC induced cyclin D1 protein, DNA synthesis, and cell cycle progression in VSMCs. Accordingly, our results showed that pharmacological inhibition of PI 3′-kinase/Akt significantly suppressed BTC-induced cyclin D1 protein, DNA synthesis, and cell cycle progression, whereas MEK inhibition only slightly suppressed these BTC-induced processes. Taken together, our results indicate that PI 3′-kinase/Akt/GSK/FoxO/β-catenin cascades could be a major signaling mechanism by which BTC induces the upregulation of cyclin D1 protein and an increase in DNA synthesis and cell cycle progression (Figure 8).
Why was MEK inhibition unable to suppress BTC-induced cyclin D1, DNA synthesis, and cell cycle progression, even though BTC induced the MEK/ERK signaling cascade? Activation of the MEK/ERK pathway has been linked to the induction of cyclin D1. Sustained MEK/ERK activation is necessary for induction of cyclin D1 protein and DNA synthesis, whereas transient activation of the MEK/ERK signaling cascade may be insufficient to induce cyclin D1 protein and DNA synthesis.14,29,30 In fact, our results showed that BTC-induced ERK phosphorylation was relatively transient. Thus, BTC-induced ERK activation could not be a major signaling pathway for induction of cyclin D1 protein and DNA synthesis in VSMCs. In comparison, our results indicate that AR-induced ERK1/2 activation is more sustained than BTC-induced ERK activation, whereas AR did not produce any change in Akt activation. Previous reports suggested that sustained ERK1/2 activation is a major determinant of upregulation of cyclin D1 protein and DNA synthesis in several cell types.14,29,30 Likewise, our results suggest that sustained ERK/Elk1 activation could be a major signaling cascade for AR-induced upregulation of cyclin D1 protein and DNA synthesis in VSMCs. To support this idea, pharmacological inhibition of MEK significantly suppressed AR-induced cyclin D1 protein, DNA synthesis, and cell cycle progression, whereas PI 3′-kinase inhibition slightly suppressed AR-induced cyclin D1 protein, DNA synthesis, and cell cycle progression. Taken together, our results suggest that PI 3′-kinase/Akt/GSK3/FoxO/β-catenin cascades could be major signaling cascades for BTC-induced upregulation of cyclin D1 protein, DNA synthesis, and cell cycle progression, whereas a sustained ERK/Elk1 activation could be a major signaling cascade for AR-induced upregulation of cyclin D1 protein, DNA synthesis, and cell cycle progression (Figure 8).
Our immunolocalization study indicates that BTC, ErbB1, and ErbB4 are upregulated in the plaques of human atherosclerotic coronary artery. In these lesions, BTC is detected in macrophages and VSMCs, whereas ErbB1 receptor is detected in VSMCs. In the plaque shoulder, VSMCs are known to proliferate actively and to migrate from the media into the intima during the progression of atherosclerosis. BTC may contribute to the progression of atherosclerosis through autocrine and paracrine signaling. Several promising inhibitors for EGF receptors have recently been developed31; these inhibitors may prove useful for preventing the progression of atherosclerosis.
This work was supported by the Creative Research Initiatives of the Korean Ministry of Science and Technology and by the Brain Korea 21 Project. We thank Jennifer Macke for help in preparing the manuscript.
Original received March 6, 2003; revision received July 7, 2003; accepted July 8, 2003.
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