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Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, ChinaHepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, ChinaClinical Medical Research Center of Hepatic Surgery at Hubei Province, Wuhan, China
State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100021, China
Corresponding author. Departments of Gerontology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan, 430022, China.
Corresponding author. Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan, 430022, China.
TAK1 is activated in vascular smooth muscle cells (VSMCs) in the setting of aortic transplantation.
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TAK1 is involved in VSMCs proliferation and migration.
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TAK1 controls autophagy activation.
Abstract
Background and aims
The proliferation and migration of vascular smooth muscle cells (VSMCs) are fundamental hallmarks of vasculopathy. Transforming growth factor β-activated kinase-1 (TAK1) plays a crucial role in mediating cellular functions, including autophagy, which has been recently linked to the regulation of VSMC functions and the development of vasculopathy. This study aims to better dissect how TAK1 controls VSMC proliferation and migration.
Methods
A rat model of graft arteriosclerosis was employed to explore the influence of TAK1 signaling activation on VSMC proliferation, migration, autophagy, and neointima formation in vivo. Knockdown and pharmacological inhibition of TAK1 were utilized in cultured VSMCs to investigate the mechanisms underlying the progression of VSMC proliferation and migration.
Results
Increased phosphorylation of TAK1 (Thr-184/Thr-187) was examined in SMα-actin positive cells in the medial and neointimal lesions of aortic allografts. Lentivirus-mediated Tak1 shRNA transfection of aortic allografts robustly suppressed neointimal formation and lumen stenosis, as well as autophagy and cell proliferative responses. In cultured PDGF-BB-incubated VSMCs, genetic and pharmacological inhibition of TAK1 markedly attenuated autophagy activation, and blocked the progression of cell cycle, proliferation, and migration responses.
Conclusions
Activation of TAK1 in VSMCs in the setting of aortic transplantation is an early and critical event in VSMC proliferation and migration, as well as neointima formation, because it controls autophagy activation, constituting a potential molecular mechanism and target for preventing transplant vasculopathy.
Vascular smooth muscle cells (VSMCs) constitute the major cellular component of the vascular wall. In normal mature vessels, VSMCs predominantly reside in a quiescent contractile state with an extremely low rate of proliferation and function, principally maintaining vascular homeostasis. However, in response to vascular injury or inflammatory stimuli, VSMCs can dedifferentiate into a synthetic phenotype that is characterized by increased proliferation, migration, and matrix synthesis, subsequently leading to the formation of intimal lesions that can cause luminal narrowing and eventually complete occlusion [
]. It has been established that the aberrant proliferation and migration of VSMCs play central roles in neointima formation during the pathogenesis of a variety of vascular proliferative diseases, including atherosclerosis, postangioplasty restenosis, and transplant arteriosclerosis [
]. Therefore, a better understanding of the molecular mechanisms governing VSMC proliferation and migration is the key to developing novel therapeutic approaches for the treatment of these vascular disorders.
Transforming growth factor β-activated kinase-1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family and functions as a key signaling molecule in immune and pro-inflammatory signaling pathways activated by multiple extracellular stimuli, including interleukin-1 (IL-1), transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and platelet-derived growth factor-BB (PDGF-BB). TAK1 is a serine/threonine kinase whose activation is regulated by phosphorylation at certain activation segment residues, such as Thr-184 and Thr-187 [
Critical roles of threonine 187 phosphorylation in cellular stress-induced rapid and transient activation of transforming growth factor-beta-activated kinase 1 (TAK1) in a signaling complex containing TAK1-binding protein TAB1 and TAB2.
]. Activated TAK1 then phosphorylates and activates several downstream kinases, such as the IκB kinase (IKK) complex and MAPK kinases, leading to the activation of transcription factor nuclear factor-κB (NF-κB) and activating protein-1. Therefore, TAK1 plays a critical role in regulating cell survival, differentiation, apoptosis, oncogenesis, and inflammation. Recently, TAK1 has also been identified as a potent inducer of cellular autophagy through either AMPK/mTOR or IKK signaling [
]. Autophagy is a lysosome-dependent intracellular degradation process that maintains cellular metabolism under stress conditions, thereby extensively contributing to the dynamic regulation of cell survival, proliferation, and migration. Intriguingly, autophagy has been implicated in the regulation of VSMC functions and the development of neointimal lesions and vascular remodeling [
]. However, the potential roles of TAK1 in the regulation of autophagy and its contribution to VSMC proliferation and migration and transplant arteriosclerosis remain elusive.
In the present study, we used a rat aortic interposition allograft model to evaluate the effect of TAK1 knockdown on VSMC proliferation and neointimal lesion formation following transplantation. We demonstrate that TAK1 mediates cellular autophagy and the cell proliferative response after transplantation and that knockdown of TAK1 leads to impaired neointima formation in aortic allografts. We further provide evidence that autophagy is required for TAK1 signaling to control the proliferation and migration of VSMCs. These data suggest that targeting TAK1 may be a promising therapeutic strategy for the treatment of vascular proliferative diseases.
2. Materials and methods
2.1 Animals
Male Brown Norway (BN) and Lewis rats were obtained from HFK Bioscience Co. (Beijing, China). Rats were housed in a specific pathogen-free environment. The animal experimental protocol was approved by the Animal Care Committee of Tongji Medical College, Huazhong University of Science and Technology. The IACUC approval number is [2021] 2582.
2.2 Lentiviral vector construction
Recombinant lentiviral vectors (pGC-FU-Tak1-shRNA) with a specific Sm22α promoter (TAK1-KD) (GeneChem, Shanghai, China) and its corresponding negative control (NS-KD) were generated as described previously [
]. Briefly, a specific mouse Sm22α promoter (−441 to +41, Genbank accession no.U36589) and rat Tak1 shRNA sequence were co-cloned into a recombinant lentiviral vector pGC-FU utilizing a gene-recombinant method that drove the target gene (Tak1-shRNA) expression. The previously tested sequence of Tak1 gene shRNA is 5′-GCCCTAGTGTCAGAATGATTTCAAGAGAATCATTCTGACACTAGGGCTTTTTG-3’. The nonspecific sequence, as a negative control, is 5′-TTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTG-3’.
2.3 Aortic transplantation and tissue preparation
Abdominal aortic transplantation was performed using a modified protocol initially reported by Mennander et al. [
]. Rats weight 100–120g were used. Male Lewis rats were used as syngeneic donors and recipients as well as allogeneic recipients. Male BN rats served as allogeneic donors.
The rats were anesthetized with an inhaled anesthesia mixture of isoflurane (3%) and oxygen (1 L/min). A 1 cm-long abdominal aorta fragment was obtained from the donor and infected by incubating the corresponding lentivirus vector (2 × 107 TU/ml) 60 min at room temperature in Opti-MEM medium containing polybrene (5 μg/ml). The donor rats were sacrificed by bloodletting under deep anesthesia. Subsequently, the graft was transplanted into a heterotopic position below the renal artery and above the bifurcation in recipient rats, using an end-to-end interrupted anastomosis with 9-0 nylon suture. Metamizole (50 mg/100 ml in drinking water) was used for painkilling for 3 days post-transplantation. Immunosuppressants were not used in this study. No single graft was lost to thrombosis or technical failures.
The recipient rats were sacrificed by an overdose of pentobarbital sodium (180 mg/kg) 8 weeks post-transplantation. The graft segments were harvested for western blotting and quantitative RT-PCR, as well as histology and immunohistochemistry analyses.
2.4 Histology and morphometry
For histology and immunohistochemistry analyses, the graft segments were fixed with 4% paraformaldehyde and embedded in paraffin. Cross-sections (5 μm thick) were cut for hematoxylin-eosin (H&E) and elastic tissue fibers-Verhoeff's Van Gieson (EVG) staining. Two independent experimental design-blinded investigators measured lumen area, intimal area (the area within the internal elastic lamina minus the lumen area), medial area (the area within the external elastic lamina minus the area within the internal elastic lamina), and total vessel area (the area encompassed by the external elastic lamina) of digital images using Image-Pro Plus (Media Cybernetics, MD, USA). We calculated the mean of their results.
2.5 Immunohistochemistry
Cross-sections were deparaffinized with xylene and rehydrated in sequential gradients of ethanol. Antigen-retrieved and blocked sections were incubated with primary antibodies against SMα-actin (ab7817, mouse monoclonal, dilution 1:200, Abcam, Cambridge, UK), PCNA (ab92552, rabbit monoclonal, dilution 1:100, Abcam), Cyclin D1 (ab40754, rabbit monoclonal, dilution 1:250, Abcam), CDK4 (ab7955, rabbit polyclonal, dilution 1:100, Abcam), Beclin1 (ab62557, rabbit polyclonal, dilution 1:100, Abcam), and LC3B (3868, rabbit monoclonal, dilution 1:200, Cell Signaling Technology, Danvers, USA) overnight at 4 °C. Subsequently, we incubated the sections with biotinylated secondary antibodies, visualized them by 3, 3′- diaminobenzidine (DAB), and treated them with hematoxylin to counterstain the nucleus. We used a fluorescence microscope (Olympus, Tokyo, Japan) to acquire digital images.
2.6 Cell culture and treatment
Primary VSMCs were isolated using an explant method [
]. The media of BN rats thoracic aortas were dissected from intima and adventitia, cut into small pieces, placed onto a cell culture dish in DMEM/F12 (Life Technology, Grand Island, USA) with 10% fetal bovine serum (FBS) (Life Technology, Grand Island, USA), and incubated at 37 °C and 5% CO2 for 7 days. Outgrown VSMCs were passaged by trypsin and cultured with DMEM/F12 containing 10% FBS at 37 °C and 5% CO2. Passage third to sixth cells were used for cell experiments.
Cell quiescence was induced by serum starvation with 0.2% BSA supplement for 48 h before reagents treatment. Quiescent cells were pretreated with 5Z-7-oxozeanenol (0.3 μM, Sigma-Aldrich, Missouri, USA), bafilomycin A1 (50 nM, Selleck, Texas, USA) and spautin-1 (10 μM, Selleck), respectively, for 60 min before PDGF-BB (20 ng/ml, Peprotech, New Jersey, USA) stimulation.
For TAK1 knockdown, VSMCs were transfected with lentiviruses carrying the rat Tak1 shRNA driven by a specific Sm22α promoter (TAK1-KD) and its corresponding negative control shRNA (NS-KD) according to the manufacturer's protocol at a multiplicity of infection (MOI) of 100 in an Opti-MEM medium containing polybrene (5 μg/ml). Transfection efficiency was identified by western blotting 7 days later.
2.7 Quantitative RT-PCR
Total RNA was isolated using TRIzol reagent (Invitrogen, California, USA). Reverse transcription using PrimeScript RT Master Mix (Takara, Shiga, Japan) and quantitative real-time reverse-transcriptase(RT)-PCR using the SYBR Premix Ex Taq kit (Takara, Shiga, Japan) were performed in a thermocycler and an iCycler Real-Time PCR Detection System (Bio-Rad, California, USA), respectively. The relative mRNA expression level was normalized by GAPDH using the 2-ΔΔCt method. The sequences of primer were: Tak1, 5′-TATGCTGAA-GGAGGCTCGTTGT-3' (forward) and 5′-AGG-CTTGAGGTCCCTATGAATG-3' (reverse); Gapdh, 5′-GTTACCAGGGCTGCCTT-CTC-3' (forward) and 5′-GATGGTGATGGGTTTCC-CGT-3' (reverse).
2.8 Western blotting
Protein was extracted following a standard method with RIPA lysis buffer. Briefly, we separated VSMCs and aortic grafts lysates containing an equal amount of protein with SDS-PAGE and transferred them onto PVDF membranes. Blocked PVDF membranes were incubated with corresponding primary antibodies against phospho-TAK1 (Thr-184/Thr-187) (orb7050, rabbit polyclonal, dilution 1:1000, Biorbyt, Cambridge, UK), TAK1 (4505, rabbit polyclonal, dilution 1:1000, Cell Signaling Technology), GAPDH (ab9485, rabbit polyclonal, dilution 1:2500, Abcam), Cyclin D1 (ab40754, rabbit monoclonal, dilution 1:1000, Abcam), CDK4 (ab7955, rabbit polyclonal, dilution 1:1000, Abcam), Cyclin E1 (ab71535, rabbit polyclonal, dilution 1:2000, Abcam), CDK2 (ab32147, rabbit monoclonal, dilution 1:1000, Abcam), PCNA (rabbit monoclonal, dilution 1:100, Abcam), Beclin1 (ab62557, rabbit polyclonal, dilution 1:100, Abcam), and LC3B (3868, rabbit monoclonal, dilution 1:200, Cell Signaling Technology) followed by HRP-conjugated AffiniPure Goat Anti-rabbit IgG (BA1055, dilution 1:3000, Boster Biological Technology, Wuhan, China). A band of different proteins was visualized and recorded by the ChemiDoc imaging system (Bio-Rad, California, USA). GAPDH served as the loading control.
2.9 Immunofluorescence
Immunofluorescence staining was performed on cross-sections and cultured VSMCs according to our early research [
]. PDGF-BB-treated VSMCs were fixed onto cell slides with 4% paraformaldehyde. Cross-sections were deparaffinized with xylene and rehydrated in ethanol. Blocked sections and cell slides were incubated with primary antibodies against phospho-TAK1 (Thr-184/Thr-187) (orb7050, rabbit polyclonal, dilution 1:100, Biorbyt), SMα-actin (ab7817, mouse monoclonal, dilution 1:200, Abcam), and LC3B (3868, rabbit polyclonal, dilution 1:200, Cell Signaling Technology) overnight at 4 °C. Immunoreactions were visualized using CY3 Conjugated AffiniPure Goat Anti-rabbit IgG (BA1032, dilution 1:100, Boster Biological Technology) and FITC Conjugated AffiniPure Goat Anti-mouse IgG (BA1011, dilution 1:100, Boster Biological Technology). The nucleus was stained with Hoechst (Invitrogen, California, USA). A fluorescence microscope (Olympus, Tokyo, Japan) was used to acquire images.
2.10 Cell cycle analyses
Flow cytometry was performed to determine the distribution of cell cycle stages. After shRNA transfection and TAK1 inhibitor treatment, respectively, VSMCs were stimulated with PDGF-BB (20 ng/ml) for 24 h. We collected cells and washed them twice with PBS. The cells were then fixed in 70% ethanol overnight and stained with propidium iodide for 30 min before analyses. The samples were analyzed using a FACS machine (Becton, Dickinson and Company, New Jersey, USA).
2.11 Cell proliferation assay
VSMCs proliferation was evaluated by Cell Counting Kit-8 (CCK-8). Briefly, VSMCs were plated at 2 × 103 cells per well in 96-wells plates and stimulated with PDGF-BB (20 ng/ml) for the indicated time after transfection or inhibitor treatment. Then, the cells were incubated with the CCK-8 solution for 2 h in the dark. The absorbance of the samples was measured at 450 nm using a microplate reader (Thermo, Massachusetts, USA).
2.12 EdU incorporation assay
DNA synthesis was assessed by 5-Ethynyl-2′-deoxyuridine (EdU) incorporation. Cells were plated at 3 × 103 cells per well in 96-wells plates. Transfected or pretreated VSMCs were incubated with EdU-labeling solution (50 μM EdU) for 2 h in the dark. Then we fixed cells with 4% paraformaldehyde and stained them with Apollo® 567 for 30 min. The ensuing steps were carried out according to the manufacturer's protocol. The nucleus was stained with Hoechst (Invitrogen, California, USA). A fluorescence microscope (Olympus, Tokyo, Japan) was used to acquire images.
2.13 Cell migration
Cultured VSMCs migration was evaluated by wound-healing assay and Transwell. For the wound-healing assay, PDGF-BB (20 ng/ml) was introduced to starved VSMCs to induce cell migration. The migration rate was calculated as the ratio of the remaining wound area to the initial. For Transwell migration assay, VSMCs were seeded into the upper chambers of Transwell plates with an 8 μm pore size (Corning Inc., New York, USA), while the PDGF-BB (20 ng/ml) was added to the lower chambers. The plates were incubated for a further 6 h. Migrated cells on the filter bottom were stained with 0.1% crystal violet solution and imaged by a microscope.
2.14 Statistical analyses
At least three independent experiments were performed. Data were presented as mean ± standard error of mean (SEM). Firstly, the Shapiro-Wilk normality test was applied. Kruskal-Wallis test was used for nonnormally distributed data. For normally distributed data, the Levene test was performed to test the homogeneity of variance. For data with uneven variance, the Brown-Forsythe test and Tamhane's T2 test were applied. One-way ANOVA and Sidak multiple comparisons were performed to detect the statistical differences in data with normally distributed and homogeneity of variance. IBM SPSS Statistics 25.0 (IBM Co., New York, USA) was used for statistical analyses. p < 0.05 was considered a significant difference.
3. Results
3.1 TAK1 is required for PDGF-induced VSMC proliferation and migration
TAK1 has been identified as a key regulator of VSMC inflammatory activation in response to diverse extracellular stimuli [
]. We wondered whether TAK1 is involved in the regulation of VSMC proliferation and migration. Therefore, we initially performed Western blotting to explore the potential role of PDGF-BB, a potent inducer of VSMC proliferation, on TAK1 activation in cultured VSMCs. As expected, PDGF-BB induced TAK1 activation in VSMCs, as shown by a rapid apparent increase in TAK1 phosphorylation at residue Thr-184/Thr-187 (Fig. 1A). Next, 5Z-7-oxozeanenol, a selective TAK1 kinase inhibitor, was employed to investigate the effect of TAK1 activation on PDGF-BB-induced VSMC proliferation (Supplementary Fig. 1A). As measured by CCK-8 assay, pretreatment with 5Z-7-oxozeanenol resulted in significant suppression of VSMC proliferation in response to PDGF-BB stimulation (Supplementary Fig. 1B). To verify the effect of TAK1 signaling on VSMC proliferation, we performed shRNA-mediated knockdown of TAK1 in VSMCs, and the knockdown efficiency was validated by Western blotting (Fig. 1B). We found that knockdown of TAK1 markedly inhibited VSMC proliferation induced by PDGF-BB (Fig. 1C). Subsequently, VSMC proliferation was assessed by measuring of DNA synthesis based on the incorporation of EdU. We observed a proportion of 41.74% EdU-positive VSMCs following PDGF-BB stimulation, whereas this stimulatory effect was diminished to 13.8% after genetic knockdown of TAK1 and to 19.8% after 5Z-7-oxozeanenol-treatment (Fig. 1D and Supplementary Fig. 1C).
Fig. 1TAK1 is required for PDGF-induced VSMC proliferation and migration.
(A) VSMC were treated with PDGF-BB (20 ng/ml) for the indicated time. (B) NS-KD and TAK1-KD VSMC were stimulated with PDGF-BB (20 ng/ml) for 10 min. (C) VSMC proliferation was measured by CCK-8 assay in TAK1-KD and NS-KD VSMC exposed to PDGF-BB (20 ng/ml) for the indicated time (**p < 0.01 vs Control, ##p < 0.01 vs PDGF-BB). (D) EdU (red) incorporation assay was performed on NS-KD and TAK1-KD VSMC exposed to PDGF-BB (20 ng/ml) for 24 h. Cell nuclei were stained with Hoechst (blue). The bar graph shows the mean percentage of EdU positive cells to the overall cells. (E) VSMC migration was measured by Transwell assay in TAK1-KD and NS-KD VSMC. (F) The wound-healing assay was performed on TAK1-KD and NS-KD VSMC exposed to PDGF-BB (20 ng/ml) for 24 h. The bar graph shows the mean area change of migrated cells per field. *p < 0.05, **p < 0.01.
Additionally, we explored the functional effect of TAK1 activation on cell migration by performing wound-healing and Transwell assays. Consistent with the inhibitory effect of TAK1 blockade on cell proliferation, pretreatment with 5Z-7-oxozeanenol remarkably attenuated the migratory ability of VSMCs conferred by PDGF-BB (Supplementary Figs. 1D and E). We also confirmed these findings in TAK1-KD VSMCs, where knockdown of TAK1 led to impaired migration in response to PDGF-BB (Fig. 1E and F). Taken together, these results suggest that TAK1 plays a critical role in the regulation of VSMC proliferation and migration.
3.2 Knockdown of VSMC TAK1 attenuates transplant arteriosclerosis in rat aortic allografts
The proliferation and migration of VSMCs are known to be crucial events in the development of neointimal formation, a hallmark of vascular occlusive disorders such as in-stent restenosis and transplant vasculopathy. To determine the functional role of VSMC-derived TAK1 in the development of transplant vasculopathy in vivo, we established a rat model of BN in Lewis aortic transplantation following lentivirus-mediated transfer of shRNA targeting the TAK1 gene driven by the smooth muscle-specific Sm22α promoter. Quantitative real-time PCR and Western blotting were performed to measure the mRNA and protein expression levels of TAK1 in aortic grafts. Our results showed that the TAK1 phosphorylation rate in aortic allografts was 39.44%, which was higher than the 1.78% rate in aortic isografts (Fig. 2A and B). Moreover, immunofluorescence staining confirmed that marked expression of phospho-TAK1 led to its predominant colocalization with SMα-actin staining in the media and neointima of aortic allografts. Importantly, TAK1 activation induced by alloimmune injury was reduced from 28.31% to 5.39% by TAK1 shRNA transfection in aortic allografts (Fig. 2C and D), suggesting that lentivirus-mediated transfer of TAK1 shRNA is highly efficient in silencing TAK1 expression and activation in vivo.
Fig. 2Knockdown of VSMC TAK1 attenuates transplant arteriosclerosis in rat aortic allografts.
(A) Relative TAK1 mRNA in aortic grafts form abdominal aortic transplantation rats was detected by qRT-PCR. mRNA expression was normalized to GAPDH. n = 3 rats per group. (B) Aortic grafts total and phosphorylated (Thr184/187) TAK1 protein level was measured by Western blotting. The bar graph shows the quantification of phosphorylation presented as the ratio of p-TAK1 to total TAK1. (C and D) Representative aortic grafts cross-sections were immunostained for p-TAK1 (red) and SMα-actin (green). The bar graph shows the mean percentage of positively stained area to the medial and neointimal area of aortic grafts. n = 15 rats per group. (E) Representative H&E staining, EVG staining, and immunohistochemistry staining for SMα-actin of aortic grafts cross-sections from abdominal aortic transplantation rats were performed. The bar graphs show the quantitative analyses of intimal area (F), intima/media ratio (G), and lumen stenosis ratio (H). n = 15 rats per group. (I) Representative aortic grafts cross-sections from abdominal aortic transplantation rats were immunostained for PCNA. The bar graph shows the mean percentage of IHC positive cells within the medial and neointimal area. n = 15 rats per group. *p < 0.05, **p < 0.01.
Next, we performed histological and immunohistochemical analyses to evaluate the effect of TAK1 knockdown on neointimal formation in aortic grafts 8 weeks after transplantation. In aortic isografts, almost no neointimal lesions or morphological changes were detected, whereas neointima formation was pronounced and consisted mainly of SMα-actin-positive cells in aortic allografts (Fig. 2E), suggesting that accelerated neointima formation induced by alloimmune injury may be a result of excessive accumulation of VSMCs. Quantitative morphometric analyses showed that neointima formation was attenuated in TAK1-KD aortic allografts, as reflected by a significant 42.24% reduction in intima area, 54.70% reduction in lumen/media ratio and 63.13% reduction in lumen stenosis ratio compared with NS-KD aortic allografts (Fig. 2F–H). Furthermore, we conducted immunostaining for the proliferation marker PCNA to assess the degree of cell proliferation in the vascular wall. We found that the allogeneic immune response led to a substantial increase, 41.11%, in the number of PCNA-positive VSMCs in the media and neointima of aortic allografts, compared with the number in aortic isografts, but this number decreased to14.82% after TAK1 knockdown (Fig. 2I), confirming the critical role of TAK1 in controlling VSMC proliferation in vivo. Altogether, these findings suggest that VSMC-derived TAK1 critically contributes to the development of transplant arteriosclerosis by promoting VSMC proliferation and migration following transplantation.
3.3 TAK1 controls cell cycle progression into the S phase in VSMCs
Since cell proliferation is known to be controlled by progression through the cell cycle, we addressed whether TAK1 is functionally involved in regulating the cell cycle progression of VSMCs by flow cytometry analyses. After 48 h of serum starvation, the vast majority of VSMCs were arrested in the G0/G1 phase with a small population delayed in the S phase. Stimulation with PDGF-BB resulted in a marked S phase cell population increase to 12.48%, with a concomitant decrease in the cell population in the G0/G1 phase. In contrast, genetic knockdown or pharmacological inhibition of TAK1 remarkably reduced the proportion of cells in the S phase, to 4.31% and 9.89%, respectively, and enhanced the cell population in the G0/G1 phase in response to PDGF-BB (Fig. 3A and B). This outcome was similar to the influence of TAK1 loss-of-function on PDGF-BB-induced VSMC proliferation and DNA synthesis, as presented above. Therefore, these data indicate that TAK1 induces VSMC proliferation by promoting the G1-to-S phase transition of the cell cycle following PDGF-BB stimulation.
Fig. 3TAK1 controls cell cycle progression into S phase in VSMC.
(A) Flow cytometry was performed on VSMC pretreated with 5Z-7-Oxozeaenol (0.3 μM) for 1 h, followed by exposure to PDGF-BB (20 ng/ml) for 24 h. (B) Cell cycle distribution of NS-KD and TAK1-KD VSMC exposed to PDGF-BB (20 ng/ml) for 24 h was measured by flow cytometry. (C) Western blotting was performed to detect Cyclin D1, CDK4, Cyclin E1, CDK2, and PCNA protein expression in VSMC exposed to PDGF-BB (20 ng/ml) for 24 h after pretreated with 5Z-7-Oxozeaenol (0.3 μM) for 1 h. Densitometric analyses is shown as the relative ratio of the indicated protein to GAPDH. (D) NS-KD and TAK1-KD VSMC were exposed to PDGF-BB (20 ng/ml) for 24 h. (E) Representative aortic grafts cross-sections from abdominal aortic transplantation rats were immunostained for Cyclin D1 and CDK4. The bar graph shows the mean percentage of IHC positive cells within the medial and neointimal area. n = 15 rats per group. *p < 0.05, **p < 0.01.
Cell cycle progression is finely orchestrated by the activity of a plethora of cyclin/CDK complexes. Progression from G0/G1 to S phase is governed by cyclin D1/CDK4 and cyclin E/CDK2. To explore the underlying mechanism of TAK1-induced cell cycle progression, we measured the expression levels of cell cycle-related proteins in VSMCs. Consistent with the effect on cell cycle progression, PDGF-BB treatment dramatically enhanced the protein expression of cyclin D1, CDK4, cyclin E1, CDK2, and PCNA, by 191.01%, 172.56%, 87.28%, 93.33%, and 71.28%, respectively. However, PDGF-BB failed to induce the upregulation of those proteins in the presence of 5Z-7-oxozeanenol pretreatment (Fig. 3C). Similarly, TAK1 knockdown blunted the effect of PDGF-BB on the induction of these cell cycle-related proteins (Fig. 3D). Moreover, this negative influence of TAK1 silencing on the expression of cell cycle-related proteins was verified in vivo, as indicated by immunostaining in the tissue sections of aortic grafts (Fig. 3E). Collectively, these results suggest that TAK1 plays an essential role in controlling cell cycle progression and DNA synthesis via the regulation of cell cycle-related proteins in VSMCs.
3.4 TAK1 is necessary for the induction of autophagy in VSMCs
Autophagy is a crucial intracellular degradation process that regulates cellular metabolism under stress conditions, thereby contributing to a variety of biological functions, including cell survival, proliferation, and migration [
]. We wondered whether autophagy might be involved in the functional effect of TAK1 on VSMCs. Thus, we analyzed the changes in autophagic flux following TAK1 inactivation in VSMCs. Performing Western blotting, we found that PDGF-BB stimulation caused significant increases in the expression of autophagy genes, including Beclin1 and LC3B II, as well as low SQSTM1 accumulation, which can be markedly suppressed by 5Z-7-oxozeanenol or TAK1 knockdown (Fig. 4A and B). Immunofluorescence staining for LC3B revealed that PDGF-BB failed to trigger an increase in LC3B puncta formation in TAK1-KD VSMCs (Fig. 4C), suggesting an important role for TAK1 signaling in controlling autophagy activation in VSMCs. Accompanied by bafilomycin A1, a specific inhibitor that blocks autophagosome-lysosome fusion and inhibits acidification and protein degradation in lysosomes, PDGF-BB stimulation increased LC3B II expression, which was inhibited in TAK1-KD VSMCs (Fig. 4D). In line with these observations in vitro, we detected the increased accumulation of cells expressing autophagy markers Beclin1 and LC3B, mainly located in neointimal lesions and partially in the medial layer in aortic allografts, whereas selective knockdown of VSMC TAK1 reversed the increased expression of Beclin1 and LC3B in the aortic allografts (Fig. 4E). These observations suggest that TAK1 is necessary for the regulation and initiation of autophagy in VSMCs in response to growth factors and stresses.
Fig. 4TAK1 is necessary for the induction of autophagy in VSMC.
(A) Western blotting analyses of Beclin1, LC3B, and SQSTM1 protein expression in VSMC exposed to PDGF-BB (20 ng/ml) for 24 h after pretreated with 5Z-7-Oxozeaenol (0.3 μM) for 1 h. Densitometric analyses is shown as the relative ratio of the indicated protein to GAPDH. (B) NS-KD and TAK1-KD VSMC were exposed to PDGF-BB (20 ng/ml) for 24 h. (C) Immunofluorescence staining of LC3B (red) was performed in NS-KD and TAK1-KD VSMC stimulated with PDGF-BB (20 ng/ml) for 24 h. Cell nuclei were stained with Hoechst (blue). (D) NS-KD and TAK1-KD VSMC were stimulated with PDGF-BB (20 ng/ml) for 24 h. Bafilomycin A1 (50 nM) was used 1 h before harvest. (E) Representative aortic grafts cross-sections from abdominal aortic transplantation rats were immunostained for Beclin1 and LC3B. The bar graph shows the mean percentage of the positive areas within the medial and neointimal area. n = 15 rats per group. *p < 0.05, **p < 0.01.
3.5 Autophagy is critical for TAK1-induced VSMC proliferation and migration
To determine the role of autophagy in the regulation of VSMC proliferation, we used the autophagy inhibitor spautin-1 to block the initiation of autophagy in VSMCs. Consistent with the antiproliferative effect of TAK1 inhibition on VSMCs, pretreatment with spautin-1 profoundly suppressed cell proliferation stimulated by PDGF-BB, as measured by cell counting and EdU incorporation assays (Fig. 5A and B). However, the combination of Spautin-1 and TAK1 knockdown had no additive effect on VSMC proliferation (Fig. 5C and D), indicating that autophagy may act downstream of TAK1 signaling. Next, we asked whether autophagy might contribute to VSMC migration induced by TAK1 activation. Hence, we carried out Transwell and wound-healing assays to evaluate the migration ability of VSMCs after different treatments. In contrast, blocking autophagy with spautin-1 pretreatment attenuated the PDGF-BB-induced migration of VSMCs, while the combination of spautin-1 treatment and TAK1 knockdown led to no additive inhibitory effect on cell migration (Fig. 5E and F), suggesting that cell migration induced by TAK1 signaling may be dependent on autophagy induction. Overall, these data suggest that autophagy is critical for TAK1-induced VSMC proliferation and migration.
Fig. 5Autophagy is responsible for TAK1-induced VSMC proliferation and migration.
(A) CCK-8 assay was performed on VSMC pretreated with spautin-1 (10 μM) for 1 h before PDGF-BB (20 ng/ml) stimulation for the indicated time. (**p < 0.01 vs Control, #p < 0.05 vs PDGF-BB, ##p < 0.01 vs PDGF-BB.) (B) VSMC were pretreated with spautin-1 (10 μM) for 1 h, followed by PDGF-BB (20 ng/ml) for 24 h. EdU incorporation assay was performed. (C) VSMC were transfected with TAK1-KD shRNA or pretreated with spautin-1 (10 μM) for 1 h. CCK-8 assay was performed on VSMC stimulated with PDGF-BB (20 ng/ml) for 48 h. (D) EdU incorporation assay was performed on VSMC stimulated with PDGF-BB (20 ng/ml) for 24 h. (E) VSMC were transfected with TAK1-KD shRNA or pretreated with spautin-1 (10 μM) for 1 h, following by PDGF-BB (20 ng/ml) stimulation for 24 h. Transwell and the wound-healing assay (F) was performed. *p < 0.05, **p < 0.01.
The potential mechanism underlying the proliferation and migration of VSMCs and the mechanism contributing to vasculopathy remain unclear. In the present study, we successfully downregulated TAK1 expression in VSMCs and were thus able to experimentally characterize the key regulatory function of TAK1 in graft arteriosclerosis models. In addition to suppressing neointima formation, VSMC-derived TAK1 knockdown inhibited VSMC proliferation and autophagy. Consistently, blockade of TAK1 activity or expression in cultured VSMCs strikingly inhibited PDGF-BB-induced VSMC proliferation, migration, and cellular autophagy, indicating that TAK1 promoted VSMC proliferation and migration by inducing autophagy.
Although research on TAK1 in vascular diseases in vivo has been overshadowed by its pivotal role in vasculogenesis [
], the development of some approaches has led to exciting discoveries in this field. Our previous study using the 5Z-7-oxozeanenol showed that TAK1 is a key regulator of neointima formation [
], expanded our knowledge on the roles of TAK1 in VSMCs after abdominal aortic transplantation, demonstrating that VSMC-specific deletion of TAK1 attenuated neointima formation after transplantation.
The colocalization of phospho-TAK1 and SMα-actin in the neointimal area, together with the inhibition of neointima formation due to TAK1 knockdown, supported the idea that VSMC-derived TAK1 is critical for neointima formation. Traditionally, medial VSMCs have been treated as the predominant contributors to neointima. Recently, heterogeneous VSMCs have been introduced to participate in neointima formation [
MicroRNA-155 promotes the directional migration of resident smooth muscle progenitor cells by regulating monocyte chemoattractant protein 1 in transplant arteriosclerosis.
The origin of neointimal smooth muscle cells in transplant arteriosclerosis from recipient bone-marrow cells in rat aortic allograft.
Journal of Huazhong University of Science and Technology. Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban.2007; 27: 303-306
]. Regrettably, our study failed to provide direct evidence that TAK1 is phosphotylated/activated in medial VSMCs. Among further investigations, harvesting and testing grafts at multiple posttransplantation time points may help dynamically determine TAK1 activation in medial VSMCs and neointima formation.
We provided evidence, for the first time, on the activation of TAK1 by PDGF-BB in VSMCs. PDGF-BB triggered TAK1 phosphorylation rapidly and shortly. It had been previously reported that PDGF receptor (PDGFR) β, but not PDGFRα, was induced in SMα-actin-positive arterial intimal cells in allografts, but not in isografts [
Expression and localization of platelet-derived growth factor ligand and receptor protein during acute and chronic rejection of rat cardiac allografts.
Combined vascular endothelial growth factor and platelet-derived growth factor inhibition in rat cardiac allografts: beneficial effects on inflammation and smooth muscle cell proliferation.
]. However, there is no direct evidence showing that PDGF-BB induced TAK1 phosphorylation via PDGFR.
Conventionally regarded as a regulator of inflammation, TAK1 has also been linked to noninflammatory cell processes in VSMCs, including L-type calcium channel currents [
Toll-like receptor 9–dependent AMPKαActivation occurs via TAK1 and contributes to RhoA/ROCK signaling and actin polymerization in vascular smooth muscle cells.
], as well as cell proliferation, migration, and autophagic activation, as proven by our results. The contribution of VSMC proliferation and migration to the development of transplant arteriosclerosis has been established [
], suggesting that TAK1 may participate in neointima formation by regulating several different cellular processes.
Additionally, autophagy is required for TAK1 signaling to regulate the proliferation and migration of VSMCs. Autophagy has been recently shown to be regulated by TAK1 via the AMPK/mTORC1 axis [
]. Previous studies have indicated that PDGF induced NADPH oxidase 4 (Nox4) expression, while the NADPH inhibitor DPI and Nox4 siRNA inhibited PDGF-induced autophagy in VSMCs [
] induced autophagy exhibited a negative effect on VSMC proliferation. We provided supplementary evidence showing that PDGF induced VSMC proliferation via autophagy, speculating that reutilization of degraded cell components may meet the needs of essential macromolecule biosynthesis and energy production [
]. Nevertheless, the interaction between autophagy and VSMC proliferation remains to be elucidated.
Accumulating evidence has illustrated that autophagy is activated during neointima formation, exhibiting a complex effect that is both detrimental and protective. Our results revealed the induction of TAK1-mediated autophagy in the neointima. In addition, an autophagy accelerator, biolimus, and an autophagy inhibitor, 3-MA, both suppressed neointima formation [
]. To date, the possibility that distinct levels of autophagy are differentially involved in neointima formation and their potential clinical significance deserve further investigation.
In conclusion, this study has provided evidence that TAK1 acts as a major mediator of proliferation and autophagy in VSMCs, demonstrating the novel autophagy progress downstream of TAK1 in promoting VSMC proliferation and migration. Therefore, we provided potential prevention and therapeutic targets in VSMC proliferation-associated diseases such as transplant arteriosclerosis.
Financial support
This work was funded by grants from the National Natural Science Foundation of China to Z.S. (No. 81370582, 81670374, 81974040), W.L. (No.81974113), and D.S (No.81700425). All funding sources were used for purchasing experimental materials and data collection.
CRediT authorship contribution statement
Xichuan Zheng: Validation, Investigation, Writing – original draft. Qihong Yu: Validation, Investigation, Writing – original draft. Dan Shang: Validation, Investigation, Writing – original draft. Chuanzheng Yin: Visualization, Writing – review & editing. Dawei Xie: Resources, Data curation. Tong Huang: Resources, Data curation. Xiaolong Du: Resources, Data curation. Wenjie Wang: Resources, Data curation. Xueke Yan: Resources, Data curation. Chen Zhang: Visualization, Writing – review & editing. Wei Li: Conceptualization, Methodology. Zifang Song: Conceptualization, Methodology.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Appendix A. Supplementary data
The following is the Supplementary data to this article:
Critical roles of threonine 187 phosphorylation in cellular stress-induced rapid and transient activation of transforming growth factor-beta-activated kinase 1 (TAK1) in a signaling complex containing TAK1-binding protein TAB1 and TAB2.
MicroRNA-155 promotes the directional migration of resident smooth muscle progenitor cells by regulating monocyte chemoattractant protein 1 in transplant arteriosclerosis.
The origin of neointimal smooth muscle cells in transplant arteriosclerosis from recipient bone-marrow cells in rat aortic allograft.
Journal of Huazhong University of Science and Technology. Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban.2007; 27: 303-306
Expression and localization of platelet-derived growth factor ligand and receptor protein during acute and chronic rejection of rat cardiac allografts.
Combined vascular endothelial growth factor and platelet-derived growth factor inhibition in rat cardiac allografts: beneficial effects on inflammation and smooth muscle cell proliferation.
Toll-like receptor 9–dependent AMPKαActivation occurs via TAK1 and contributes to RhoA/ROCK signaling and actin polymerization in vascular smooth muscle cells.
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