CD 36 initiates the secretory phenotype during the establishment of cellular senescence
2018; Springer Nature; Volume: 19; Issue: 6 Linguagem: Inglês
10.15252/embr.201745274
ISSN1469-3178
AutoresMengyang Chong, Tao Yin, Rui Chen, Handan Xiang, Lifeng Yuan, Yi Ding, Christopher C. Pan, Zhen Tang, Peter B. Alexander, Qijing Li, Xiao‐Fan Wang,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoScientific Report18 May 2018free access Transparent process CD36 initiates the secretory phenotype during the establishment of cellular senescence Mengyang Chong Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Tao Yin Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Rui Chen Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Handan Xiang Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Lifeng Yuan Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Yi Ding Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Christopher C Pan Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Zhen Tang Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Peter B Alexander Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Qi-Jing Li Corresponding Author [email protected] Department of Immunology, Duke University, Durham, NC, USA Search for more papers by this author Xiao-Fan Wang Corresponding Author [email protected] Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Mengyang Chong Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Tao Yin Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Rui Chen Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Handan Xiang Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Lifeng Yuan Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Yi Ding Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Christopher C Pan Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Zhen Tang Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Peter B Alexander Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Qi-Jing Li Corresponding Author [email protected] Department of Immunology, Duke University, Durham, NC, USA Search for more papers by this author Xiao-Fan Wang Corresponding Author [email protected] Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Search for more papers by this author Author Information Mengyang Chong1,‡, Tao Yin1,‡, Rui Chen1, Handan Xiang1, Lifeng Yuan1, Yi Ding1, Christopher C Pan1, Zhen Tang1, Peter B Alexander1, Qi-Jing Li *,2 and Xiao-Fan Wang *,1 1Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA 2Department of Immunology, Duke University, Durham, NC, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 919 668 4070; E-mail: [email protected] *Corresponding author. Tel: +1 919 681 4861; E-mail: [email protected] EMBO Rep (2018)19:e45274https://doi.org/10.15252/embr.201745274 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cellular senescence is a unique cell fate characterized by stable proliferative arrest and the extensive production and secretion of various inflammatory proteins, a phenomenon known as the senescence-associated secretory phenotype (SASP). The molecular mechanisms responsible for generating a SASP in response to senescent stimuli remain largely obscure. Here, using unbiased gene expression profiling, we discover that the scavenger receptor CD36 is rapidly upregulated in multiple cell types in response to replicative, oncogenic, and chemical senescent stimuli. Moreover, ectopic CD36 expression in dividing mammalian cells is sufficient to initiate the production of a large subset of the known SASP components via activation of canonical Src–p38–NF-κB signaling, resulting in the onset of a full senescent state. The secretome is further shown to be ligand-dependent, as amyloid-beta (Aβ) is sufficient to drive CD36-dependent NF-κB and SASP activation. Finally, loss-of-function experiments revealed a strict requirement for CD36 in secretory molecule production during conventional senescence reprogramming. Taken together, these results uncover the Aβ–CD36–NF-κB signaling axis as an important regulator of the senescent cell fate via induction of the SASP. Synopsis In response to various senescence-inducing stimuli, normal mammalian cells rapidly upregulate the scavenger receptor CD36. Amyloid beta-dependent CD36 signaling then triggers NF-κB pathway activation, resulting in the production and secretion of numerous inflammatory proteins known to comprise the senescence-associated secretory phenotype. The multi-ligand receptor CD36 is induced in multiple senescence contexts. Amyloid beta activates CD36 to stimulate NF-κB-dependent cytokine and chemokine production. Sustained secretory molecule production leads to the onset of a comprehensive senescent cell fate. Introduction Cellular senescence, defined as a state of irreversible cell cycle arrest, was discovered in 1962 when Dr. Leonard Hayflick observed that upon prolonged cell culture, human diploid fibroblasts indefinitely lose their ability to proliferate 1, 2. In the ensuing decades, senescence has been increasingly appreciated for its physiological functions in vivo, with important roles during embryonic development and normal aging and in multiple pathological conditions including fibrosis and cancer 3-7. In addition to persistent replicative stress, various other types of stimuli, including DNA damage, oncogene activation, oxidative stress, and telomere dysfunction, are known to induce senescence in various cellular contexts 8. Moreover, the administration of specific chemical agents, such as doxorubicin and erlotinib, is sufficient to induce cellular senescence in certain cancer and normal epithelial cell types, respectively 9, 10. Across these various induction stimuli, senescent cells are currently thought to share two major molecular features. First, senescent cells have increased expression of at least one cyclin-dependent kinase (CDK) inhibitor, typically p16 or p21, which functions to activate the Rb tumor suppressor, resulting in cell cycle arrest 8. Second, senescent cells exhibit a unique secretory profile, termed the senescence-associated secretory phenotype (SASP). Upon senescence initiation, two transcription factors normally present in immune cells, NF-κB and CEBPβ, are activated to promote the transcription of a set of relatively conserved pro-inflammatory cytokines, chemokines, growth factors, and proteases 9-11. Some canonical signal transduction cascades, such as the mTOR and p38 MAPK pathways, have been shown to stimulate NF-κB and SASP formation during senescence 12, 13. However, the upstream inputs that trigger the activation of those pathways in order to produce the SASP remain largely unknown. CD36 is a multi-ligand scavenger receptor expressed in various mammalian cell types that functions in a context-dependent manner. Previous studies have identified diverse CD36 ligands including collagen, thrombospondin, and various lipoproteins and lipids that bind CD36 in order to regulate vascular and adipose homeostasis 14-17. In contrast, when expressed in macrophages and microglia cells, CD36 can generate a strong pro-inflammatory response through its interaction with secreted amyloid-beta 1–42 (Aβ) or oxidized low-density lipoprotein (oxLDL). Upon Aβ or oxLDL binding, CD36 stimulates MAPK signaling through Src family kinase activation, leading to the activation of NF-κB and subsequent cytokine and chemokine transcriptional initiation 18. However, the presence of this CD36-dependent pro-inflammatory signaling axis outside the immune system has not been previously described. In this study, we show that CD36 expression is rapidly and robustly induced in a variety of senescent states and cell types. Molecular analysis further revealed that the interaction of upregulated CD36 with its ligand Aβ is sufficient to promote Src–MAPK–NF-κB pathway activation and establishment of a SASP. Importantly, sustained exposure of CD36 to ligand drives pro-inflammatory cytokine production and cell cycle arrest in order to establish the overall senescent state in both epithelial cells and fibroblasts. Finally, loss-of-function experiments demonstrate that CD36 is strictly required for NF-κB phosphorylation and SASP initiation and maintenance during both oncogene- and chemical-induced senescence. Taken together, we identify CD36 as a novel SASP modulator involved in both senescence-associated secretome activation and the formation of a comprehensive senescent state. Results and Discussion CD36 is induced in multiple senescence contexts In previous work, we showed that targeted chemical inhibition of the epidermal growth factor receptor (EGFR) in primary human bronchial epithelial (HBE) cells is sufficient to trigger a comprehensive senescent phenotype within 3 days 9. Taking advantage of the efficiency of this method, we developed an unbiased gene expression profiling approach to compare senescent HBE cells with their proliferating counterparts in order to identify novel signaling molecules that function to regulate senescence initiation and the SASP. HBE cells, treated with either erlotinib or DMSO, were incubated with the fluorescent substrate C12FDG for the senescence-associated beta-galactosidase (SA-βGal), and senescent cells were purified using flow cytometry as previously described 19. The biological triplicated samples of senescent and proliferating cells were then screened by transcriptional profiling to identify genes differentially expressed during the early phase of senescence establishment (Fig 1A). This method revealed 331 genes with at least 1.5-fold transcriptional induction or suppression at 18 h after erlotinib-mediated senescence initiation (in press). Of these, 10 candidates were chosen for further investigation based on their plasma membrane localization (GeneCards confidence > 4), and time-dependent mRNA upregulation was validated by qPCR (Fig EV1A). As an indication that our method can detect bona fide senescence regulators, this subset of membrane proteins included the interleukin-1 receptor (IL-1R) and Notch3, both of which have been previously shown to modulate senescence but not SASP initiation per se 20, 21. However, among the candidates, the scavenger receptor CD36 stood out as the one with the strongest and most rapid induction (Fig EV1A). In fact, CD36 mRNA expression was upregulated approximately ten- and thirty-fold at the 6- and 48-h time points, respectively (Fig 1B). Normally at 6 h, most features of the senescent phenotype are not yet evident and signal transduction is still in its initiation phase. Thus, we hypothesized that CD36 might be functional in the senescence programming process. Figure 1. CD36 is induced in multiple senescence contexts Schematic of gene expression profiling approach to identify novel regulators of the SASP. Proliferating HBE cells were treated with DMSO or 1 μM erlotinib for 18 h, after which cell lysates were collected and analyzed using gene expression arrays. A total of 331 genes were significantly upregulated in senescent cells (1.5-fold cutoff threshold). Further protein localization analysis revealed a group of 10 surface proteins among this subset. * indicates proteins previously reported to regulate cellular senescence. *** indicates the candidate with the fastest and most robust upregulation. CD36 mRNA expression time-course analysis. Proliferating HBE cells were treated with DMSO or 1 μM erlotinib. Lysates were collected at 0, 4, 12, 18, 24, 36, and 48 h post-treatment. qPCR was performed to measure CD36 mRNA expression normalized to β-actin. Experiments were replicated three times independently (n = 3). Data are reported as the mean ± SEM. **P < 0.01 compared with control group, one-way ANOVA. CD36 mRNA and protein analysis during replicative senescence. IMR90 cells were collected at passages 27 (early) and 70 (late) for CD36 expression analysis by qPCR and immunoblotting. The immunoblot figures are a representative image of at least three independent experiments (n = 3). qPCR results are normalized to β-actin. Data are reported as the mean ± SEM. P-values were calculated based on at least three independent experiments (n = 3). **P < 0.01, Student's t-test. CD36 mRNA and protein analysis in erlotinib-induced senescence. Proliferating HBE cells were treated with either 1 μM erlotinib or vehicle (DMSO) for 48 h prior to sample collection for CD36 expression analysis by qPCR and immunoblotting. Immunoblot images are representative of five independent experiments (n = 5). qPCR results are normalized to β-actin (n = 5). Data are reported as the mean ± SEM. P-values were calculated based on five independent experiments. **P < 0.01, Student's t-test. CD36 mRNA and protein analysis during HRAS-induced senescence. Proliferating IMR90 cells were transfected with Tet-on HRAS or a control plasmid. After puromycin selection, cells were further treated with 1 μg/ml doxycycline for 7 days to fully establish senescence. Samples were then analyzed by qPCR and immunoblotting. Immunoblot figures are representative of three independent experiments (n = 3). qPCR results are normalized to β-actin (n = 3). Data are reported as the mean ± SEM. P-values were calculated based on three independent experiments. **P < 0.01, Student's t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. CD36 is rapidly upregulated upon senescence induction A. Candidate gene expression in senescent HBE cells. Proliferating HBE cells were treated with DMSO or 1 μM erlotinib, and lysates were collected at 0, 4, 12, 18, 24, 36 and 48 h post-treatment. Samples were analyzed by qPCR using primers specifically recognizing the indicated transcripts. Each measurement was normalized to β-actin (mean ± SEM, n = 3). B. CD36 expression analysis using GEO datasets. CD36 expression in control (proliferating) and senescent IMR90 fibroblasts was obtained from publicly available replicative (GSE53356) and oncogene-induced (GSE75207) senescence datasets, as indicated. Data are reported as means ± SEM. **P < 0.01, Student's t-test. C, D. IMR90 replicative senescence proliferation and SA-βGal staining analysis. IMR90 cells were cultured to 27 (early) and 70 (late) passages and collected. (C) Representative flow cytometry (left) and quantification (right) of cell proliferation. Cells were treated with EdU for 2 h prior to analysis by flow cytometry. (D) Representative SA-βGal staining images (left) and quantification (right) of passage 27 (early) and 70 (late) IMR90 cells. Scale bars, 50 μm. Data are reported as the mean ± SEM (n = 3). **P < 0.01, Student's t-test. E, F. Erlotinib-induced HBE cell senescence. (E) Representative flow cytometry (left) and quantification (right) of EdU incorporation. Proliferating HBE cells were treated with DMSO or 1 μM erlotinib for 3 days. Cells were then treated with EdU for 2 h and analyzed by flow cytometry. (F) Representative SA-βGal staining images (left) and quantification (right) of DMSO or erlotinib-treated HBE cells. Data are reported as the mean ± SEM (n = 3). **P < 0.01, Student's t-test. G, H. HRAS-induced IMR90 cell senescence. Proliferating IMR90 cells were infected with HRAS or control viruses for 9 days, and samples were collected for proliferation and SA-βGal staining assays. (G) Representative flow cytometry (left) and quantification (right) of cell proliferation. Cells were treated with EdU for 2 h prior to analysis. (H) Representative SA-βGal staining images (left) and quantification (right) of HRAS and control IMR90 cells. Scale bars, 50 μm. Data are reported as the mean ± SEM (n = 3). **P < 0.01, Student's t-test. I. CD36 expression in organs from aged (30 months old) and young (1 month old) mice. Lung, liver, and muscle tissues as indicated were collected and analyzed for CD36 expression by qPCR. Results were normalized to 18S rRNA. Data are reported as the mean ± S.D. (n = 3 technical replicates). **P < 0.01, Student's t-test Download figure Download PowerPoint To determine whether CD36 is also induced in other senescence models, we analyzed CD36 expression in replicative and oncogene-induced senescent fibroblasts using the publicly available Gene Expression Omnibus (GEO) database. In agreement with our chemical-induced senescence results, CD36 was found to also be significantly upregulated in both of these microarray datasets (Fig EV1B). To further validate our microarray results, we replicated and confirmed the models of erlotinib-induced, oncogene-induced, and replicative senescence using canonical senescence markers (Fig EV1C–H). We then measured CD36 expression in those senescent contexts. Consistently, a strong induction of CD36 mRNA and protein was observed in senescent cells induced by all three stimuli (Fig 1C–E). It is known that senescent cells accumulate within aged tissues 22, and this phenomenon is conserved across different species and proposed to be functionally responsible for the development of major aging-related phenotypes 23. To assess whether CD36 expression is correlated with senescent cell accumulation in aging organs, we measured CD36 expression in lung, liver, and muscle tissue of both young (1 month) and old (30 months) mice. Importantly, all tissue types tested contained elevated CD36 expression. Moreover, in lung tissue, which is the origin of HBE cells, CD36 had a ~100-fold induction in aged mice, indicating a strong correlation between CD36 induction and bronchial cell senescence and aging (Fig EV1I). Taken together, these results demonstrate that CD36 expression is consistently upregulated in a wide range of cell and tissue types by various senescent stimuli including replicative stress, oncogene activation, EGFR inhibition, and the natural aging process. Short-term CD36 expression initiates production of secretory molecules Based on its strong induction during the early stages of senescence initiation, we hypothesized that CD36 might have a functional impact on senescence programming. To investigate its possible sufficiency in inducing cellular senescence, we ectopically expressed CD36 in HBE cells using a Tet-on inducible expression system. Since there are no published reports describing a role for CD36 in cellular senescence, we first examined established features of the senescent phenotype including cell proliferation, CDK inhibitor expression (p16 and p21), SA-βGal activity, and the SASP. First, we performed SA-βGal staining (Fig 2A), 5-ethynyl-2A-deoxyuridine (EdU) incorporation (Fig 2B), and p16 and p21 Western blot assays (Fig 2C) using HBE cells engineered to overexpress CD36 for 7 days or control cells. However, these proliferation-related assays failed to reveal significant differences between CD36-expressing and control HBE cells at the 7-day time point, indicating that forced CD36 expression is insufficient to drive cell cycle arrest in the short term. Figure 2. Short-term CD36 upregulation initiates NF-κB signaling and secretome establishment in epithelial cells Representative images of SA-βGal staining (left) and quantification (right) of short-term CD36-expressing or control HBE cells. HBE cells were infected with Tet-on CD36 or a Tet-on control virus. Cells were then treated with 1 μg/ml doxycycline for 7 days. Subsequently, cells were fixed and stained for SA-βGal activity. Scale bars, 50 μm. Data are reported as the mean ± SEM; n = 3. N.S., not significant, Student's t-test. Flow cytometry (left) and quantification (right) of short-term CD36-overexpressing HBE cell proliferation. HBE cells overexpressing Tet-on CD36 or Tet-on control were treated with 1 μg/ml doxycycline for 7 days. Cells were then treated with EdU for 2 h and analyzed by flow cytometry. Data are reported as the mean ± SEM; n = 3. P-values were calculated based on at least three independent experiments. N.S., not significant, Student's t-test. Cyclin-dependent kinase inhibitors (p16 and p21) of short-term CD36-expressing HBE cells. Whole-cell lysates of control and CD36-overexpressing HBE cells (7 days) were collected and subsequently immunoblotted with the indicated antibodies. Blots are representative of three independent biological replicates (n = 3). Signal transduction analysis of short-term CD36-expressing HBE cells. Whole-cell lysates of control and CD36-overexpressing HBE cells (7 days) were collected and subsequently immunoblotted with the indicated antibodies. Blots are representative of four independent biological replicates (n = 4). NF-κB luciferase reporter assay of short-term CD36-expressing HBE cells. Luciferase reporters were transfected into control and CD36-overexpressing HBE cells (4 days). Luciferase reporter assays were then executed at day 7. Data are reported as the mean ± SEM; n = 3. P-values were calculated based on three independent experiments. **P < 0.01, Student's t-test. SASP transcriptional analysis of short-term CD36 expression in HBE cells. HBE cells from three independent donors were infected with Tet-on CD36 or Tet-on control and treated with 1 μg/ml doxycycline for 7 days. Samples were then collected and analyzed by qPCR. Results were normalized to control HBE cells. Red and blue colors represent up- and downregulated transcripts, respectively. P-values were calculated based on three cell donors analyzed in independent experiments. **P < 0.01; *P < 0.05; Student's t-test. Download figure Download PowerPoint Since CD36 is known to activate an inflammatory phenotype in certain immune system cell types 15, 16, 18, 24, we next considered the possibility that it might have a similar function during senescence reprogramming. SASP has been shown to be mediated through the activity of the NF-κB transcription factor complex, and a conventional indicator of NF-κB activation is the phosphorylation status of its functional subunit p65; we therefore measured p65 phosphorylation in CD36-overexpressing and control cells by Western blotting 25. Interestingly, these assays revealed increased phosphorylation of p65 along with activation of its upstream tyrosine kinase c-Src and MAP kinase p38 (Fig 2D). Luciferase reporter assays further verified the activation of NF-κB signaling in CD36-expressing HBE cells (Fig 2E). Since the conventional role of NF-κB is regulating cytokine production and secretion, the finding of CD36 driving NF-κB activation suggests that CD36 might promote the SASP through stimulating the Src–p38–NF-κB axis. To comprehensively explore a relationship between CD36 and the SASP, we performed quantitative PCR (qPCR)-based profiling analysis of 78 molecules previously reported as components of the SASP 26. Strikingly, across HBE cells from three independent human donors, 22 of these molecules showed a statistically significant (> 1.5-fold) upregulation upon ectopic CD36 expression (Fig 2F). Among these, many well-recognized conventional SASP components were found to be consistently produced upon CD36 expression, including interleukin 6 (IL-6) and interleukin 8 (IL-8) 11, 25, 27, 28. These results demonstrate that short-term expression of CD36 in HBE cells, while unable to trigger a cell cycle exit, can promote NF-κB signaling and production of a large set of SASP components. Long-term CD36 expression promotes a comprehensive senescent phenotype Some senescent stimuli, such as replicative exhaustion, require a period of weeks to yield cell cycle arrest 29. To further assess a possible effect of CD36 on the cell cycle, HBE cells expressing CD36 were maintained for a period of 14 days. Interestingly, prolonged CD36 expression led to a striking phenotype of cell cycle exit that was associated with increased levels of cyclin-dependent kinase inhibitors and SA-βGal activity (Fig 3A–C). Further signaling pathway analysis revealed a sustained activity of the Src–p38–NF-κB axis, indicating that long-term activation of CD36-dependent pro-inflammatory signaling might be responsible for proliferative arrest (Fig 3D–E). Overall, these results suggest that long-term CD36-dependent SASP signaling activation can trigger a comprehensive senescence phenotype in primary human epithelial cells. Figure 3. Long-term CD36 ectopic expression triggers a comprehensive senescent phenotype in epithelial cells Representative images of SA-βGal staining (left) and quantification (right) for long-term CD36-expressing and control HBE cells. HBE cells were infected with Tet-on CD36 or Tet-on control viruses and subsequently treated with 1 μg/ml doxycycline for 14 days. At day 14, cells were fixed and stained for SA-βGal activity. Scale bars, 50 μm. Data are reported as the mean ± SEM; n = 4. **P < 0.01, Student's t-test. Flow cytometry analysis (left) and quantification (right) of HBE cell proliferation after long-term CD36 overexpression. HBE cells were infected with Tet-on CD36 or Tet-on control and treated with 1 μg/ml doxycycline for 14 days. At day 14, cells were treated with EdU for 2 h and analyzed by flow cytometry. Data are reported as the mean ± SEM; n = 3. P-values were calculated based on three independent experiments. **P < 0.01, Student's t-test. Cyclin-dependent kinase inhibitor (p16 and p21) analysis of long-term CD36-expressing HBE cells. Whole-cell lysates from control or CD36-overexpressing HBE cells (14 days) were collected and subsequently immunoblotted with the indicated antibodies. Blots are representative of three independent biological replicates. Signal transduction analysis of long-term CD36-expressing HBE cells. Whole-cell lysates of control and CD36-overexpressing HBE cells (14 days) were collected and immunoblotted with the indicated antibodies. Blots are representative of three independent biological replicates. NF-κB luciferase reporter assay of long-term CD36-expressing HBE cells. Luciferase reporters were transfected into control or CD36-overexpressing HBE cells (11 days), and luciferase activity was measured at day 14. Data are reported as the mean ± SEM; n = 3. P-values were calculated based on three independent experiments. **P < 0.01, Student's t-test. Download figure Download PowerPoint Based on the fact that CD36 is broadly induced during epithelial cell and fibroblast senescence and is sufficient to promote cellular senescence in HBE cells, we next asked whether the effects of CD36 on NF-κB activation and cell cycle arrest are conserved across cell types. To investigate this, we ectopically expressed CD36 for 7 days in IMR90 human diploid fibroblasts. This short-term CD36 overexpression in IMR90 cells did not produce significant NF-κB activation (Fig 4A), cell cycle arrest (Fig 4C), or SA-βGal activity (Fig 4E). However, as in HBE cells, upon extended (17 days) CD36 expression, we observed a significantly decreased proliferative capability (Fig 4D) associated with increased SA-βGal staining (Fig 4F) and increased levels of p16, p21, and activated forms of NF-κB signaling pathway components (Fig 4B). Together, these findings suggest that the CD36–NF-κB–SASP signaling cascade exists in both human epithelial cells and diploid fibroblasts and prolonged exposure to this signaling is capable of inducing stable cell cycle arrest and appearance of a senescent phenotype. Figure 4. Long-term CD36 expression triggers a comprehensive senescence phenotype in human diploid fibroblasts A, B. Cyclin-dependent kinase inhibitor (p16 and p21) and signal transduction analysis of short-term (A) and long-term (B) CD36-expressing IMR90 cells. Whole-cell lysates of control and CD36-overexpressing IMR90 cells (7 or 17 days) were collected and immunoblotted with the indicated antibodies. Blots are representative of three independent biological replicates. C, D. Proliferation of IMR90 cells after short- (C) or long-term (D) CD36 expression. Representative flow cytometry (left) and quantification (right) of IMR90 cell proliferation. IMR90 cells were infected with Tet-on CD36 or Tet-on control viruses and treated with 1 μg/ml doxycycline for 7 or 17 days. Cells were then treated with EdU for 2 h and analyzed by flow cytometry. Data are reported as the mean ± SEM; n = 3. P-values were calculated based on three independent experiments. N.S., not significant; **P < 0.01; Student's t-test. E, F. SA-βGal staining of IMR90 cells after short- (E) or long-term (F) expression of CD36. Representative SA-βGal staining images (above) and quantification (below) are shown. IMR90 cells were infected with Tet-on CD36 or Tet-on control viruses and then treated with 1 μg/ml doxycycline for 7 or 17 days, after which cells were fixed and stained for SA-βGal activity. Scale bars, 50 μm. Data are reported as the mean ± SEM; n =3. N.S., not
Referência(s)