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. 2018 Apr;17(2):e12720.
doi: 10.1111/acel.12720. Epub 2018 Jan 24.

ATF3 represses PINK1 gene transcription in lung epithelial cells to control mitochondrial homeostasis

Marta Bueno  1   2 , Judith Brands  1   2 , Lauren Voltz  1 , Kaitlin Fiedler  1 , Brenton Mays  1 , Claudette St Croix  3 , John Sembrat  1   4 , Rama K Mallampalli  2   5 , Mauricio Rojas  1   4 , Ana L Mora  1   2
Affiliations

ATF3 represses PINK1 gene transcription in lung epithelial cells to control mitochondrial homeostasis

Marta Bueno et al. Aging Cell. 2018 Apr.

Abstract

PINK1 (PTEN-induced putative kinase 1) is a key regulator of mitochondrial homeostasis that is relatively depleted in aging lungs and in lung epithelial cells from patients with idiopathic pulmonary fibrosis (IPF), a disease linked with aging. Impaired PINK1 expression and accumulation of damaged mitochondria in lung epithelial cells from fibrotic lungs were associated with the presence of ER stress. Here, we show that ATF3 (activating transcription factor 3), a member of the integrated stress response (ISR), negatively regulates transcription of the PINK1 gene. An ATF3 binding site within the human PINK1 promoter is located in the first 150 bp upstream of the transcription start site. Induction of ER stress or overexpression of ATF3 inhibited the activity of the PINK1 promoter. Importantly, overexpression of ATF3 causes accumulation of depolarized mitochondria, increased production of mitochondrial ROS, and loss of cell viability. Furthermore, conditional deletion of ATF3 in type II lung epithelial cells protects mice from bleomycin-induced lung fibrosis. Finally, we observed that ATF3 expression increases in the lung with age and, specially, in lung epithelial cells from IPF lungs. These data provide a unique link between ATF3 and PINK1 expression suggesting that persistent stress, driven by ATF3, can dysregulate mitochondrial homeostasis by repression of PINK1 mRNA synthesis.

Keywords: ER stress; PTEN-induced putative kinase 1; activating transcription factor 3; aging; idiopathic pulmonary fibrosis; mitochondrial dysfunction.

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Figures

Figure 1
Figure 1
ER stress‐mediated transcriptional repression of PINK1. A549 cells show upregulation of ATF3 mRNA levels (a) after tunicamycin (TM) treatment. (b) PINK1 mRNA transcript levels are lower after TM treatment. (c) qRT‐PCR assay for PINK1 transcript stability after inhibition of transcription activity by actinomycin D does not display any differences. (d) Immunoblot analysis (see Figure S1B) of ATF3 and PINK1 protein levels at different time points after TM treatment confirmed upregulation of ATF3 and decreased PINK1. Primary human AECs exposed to low concentrations of TM show upregulation of ATF3 mRNA levels (e) and reduction in PINK1 transcript (f), concomitantly with upregulation of senescence markers (g). Data represent mean ± SEM of four (a–c) and three (d–g) independent experiments. *p < .01, two‐way ANOVA with multiple comparison test
Figure 2
Figure 2
Inactivation of ATF3 potentiates PINK1 transcription. (a) Representative immunoblot analysis of ATF3 and PINK1 in total cell lysates of A549 cells, transfected with GFP (transfection control) or ATF3. Cells overexpressing ATF3 for 48 hr show lower levels of PINK1 in whole cell lysates. A549 cells transfected with siRNA scramble control or ATF3 siRNA for a total of 48 hr and exposed to tunicamycin the last 24 hr (b–d). Less ATF3 mRNA after 24 hr TM treatment (b) and a recovery of the basal PINK1 transcript levels (c) were measured in knockdown ATF3 cells. (d) At 48 hr, protein levels of ATF3 also reflect these changes after TM treatment in the presence or absence of ATF3 silencing (see Figure S2F). Data represent mean ± SEM of four (a–c) and three (d) independent experiments. *p < .01, two‐way ANOVA with multiple comparison test
Figure 3
Figure 3
ATF3 binds to the PINK1 promoter to repress gene transcription. (a) A549 cells were treated with a low concentrations of DMSO (vehicle) or TM (1 μg/ml) for 5 hr. Chromatin immunoprecipitation (ChIP) assays on the PINK1 promoter were performed using antibodies against ATF3 and an IgG isotype control. Data represent mean ± SEM. *p < .01, two‐way ANOVA with multiple comparison test. (Negative locus, see Figure S3). (b) A549 cells transfected with a human PINK1 luciferase promoter reporter construct were treated with 10 μg/ml TM in the absence or presence of siATF3, and cotransfected with an ATF3 overexpressing plasmid. Luciferase and SEAP (secreted alkaline phosphatase) activities were measured after 24‐hr stimulation. (c) Schematic diagram of the 1.2 kb PINK1 promoter region in the 5′ flanking region upstream of the transcriptional starting site (TSS). (d) Deletion constructs of the 1.2 kb cloned PINK1 promoter luciferase reporter plasmid (pPINK‐A). Arrows represent the direction of transcription and the numbers detail the endpoint of each construct. The deletion plasmids were transfected in A549 cells, and after 24 hr of TM (10 μg/ml) treatment or overexpression of ATF3 plasmid (or GFP as control), promoter activity was measured. Luciferase activities were normalized to SEAP activities. Values obtained for the untreated (or GFP cotransfected) sample of each construct represent 100%. Data are reported as mean ± SEM of four independent experiments. One‐way ANOVA with multiple comparison test; *p < .05 vs. untreated, † p < .01 vs. TM 10 μg/ml (b). *p < .05 and **p < .01, unpaired, two‐tailed Student's t test vs. each corresponding untreated (or GFP cotransfected) sample (d)
Figure 4
Figure 4
ATF3 modulates mitochondrial homeostasis and cell viability. ATF3 was silenced in A549, and experiments were performed after 24 hr of tunicamycin treatment. (a) Hoechst 33342 staining was used to assess cell viability. Cell transfected with siATF3 did not lost cell viability under TM exposure. Mitochondrial mass (b) and depolarization (c) were assessed by MitoTracker and JC‐1 staining, respectively. ATF3 silencing was beneficial to the injured cells, showing complete recovery from the ER stress‐induced accumulation of depolarized mitochondria. (d) Mitochondrial ROS generation, by MitoSOX staining, was also rescue in the knockdown ATF3 cells. A549 cells were transfected with different quantities of ATF3 plasmid for 48 hr, and cell viability and mitochondrial health were measured. (e) Transfected cells showed a dose‐dependent cell death after exposure to high amounts of ectopically expressed ATF3. ATF3 overexpression was detrimental to the cells, showing a dose‐dependent accumulation (f) of depolarized mitochondria (g). Mitochondrial ROS generation (h) was also elevated in the cell overexpressing ATF3. All measurements were taken 48 hr after transfection. Data represent mean ± SEM of 24 replicates per condition, in three independent experiments. *p < .01 vs. untreated, one‐way ANOVA with multiple comparison test
Figure 5
Figure 5
Upregulation of ATF3 in bleomycin‐induced fibrotic lung and in the lung of the aging. Young mice (3 months old) were treated with 1.5 U/kg of bleomycin intratracheally and their lungs were harvested at different time points. In total lung lysate, mRNA transcript levels of ATF3 (a) are upregulated while levels of PINK1 are downregulated (b). When comparing young (3 months old) vs. old (20 months old) mice, the old mice present elevated levels of ATF3 (c) concomitantly with lower PINK1 transcript (d). After treating young mice with tunicamycin (TM) intratracheally, their lungs show similar mRNA levels of ATF3 (c) and PINK1 (d) as the old mice. Data represent mean ± SEM of n = 6. (a–c) *p < .01 vs. day 0 and # p < .01 as indicated, one‐way ANOVA with multiple comparison test. (c–d) *p < .01 vs. young and # p < .01 as indicated, two‐way ANOVA with multiple comparison test
Figure 6
Figure 6
ATF3 increases susceptibility to lung fibrosis. (a) Representative Masson trichrome staining in lung sections from ATF3 WT and ATF3 spc‐KO (conditional type II lung epithelial cells ATF3 knockout mice) showing decreased collagen deposition (blue) at day 15 postbleomycin instillation. (b) Decreased collagen deposition in lungs of ATF3 spc‐KO mice after bleomycin determined by hydroxyproline levels. (c) Bleomycin‐mediated upregulation of ATF3 mRNA in the lung of the WT mice is reduced in the ATF3 spc‐KO mice, hand‐in hand, with an improvement in PINK1 transcript levels (d). Higher fibronectin (e) and collagen I (f) transcript levels in lungs of the WT mice compared to the AECII‐specific KO littermates. (g) Relative change in the levels of TGF‐β transcripts. Relative change in (h) TNF‐α, (i) IL‐10, and (j) IL‐6 mRNA levels after bleomycin treatment in ATF3 WT and ATF3 spc‐KO mice. (k) ATF3 spc‐KO mice present lower levels of senescence markers in total lung lysate. Data represent mean ± SEM of n = 6–8. (b–k) *p < .01 vs. ATF3 WT PBS and # p < .01 as indicated, two‐way ANOVA with multiple comparison test
Figure 7
Figure 7
Upregulation of ATF3 with aging and in idiopathic pulmonary fibrosis (IPF) lungs. Analysis of ATF3 expression in total lung lysates of young donor (<50 years old), old donor (more than 50 years old), and IPF lungs (n = 17 patients per group). Significantly increased ATF3 expression in old donor and IPF tissue was detected by mRNA (a) transcript levels and protein densitometry (b). Data represent mean ± SEM. *p < .01, **p < .01, and *** p < .001 vs. young donor, one‐way ANOVA with multiple comparison test. (c) Representative images (n = 3) of immunohistochemistry analyses from young and old donors and IPF lungs using ATF3 antibody. Arrows denote epithelial cells showing nuclear staining. (d) Representative immunofluorescence using anti‐ABCA3 (type II epithelial cell marker; red) and anti‐ATF3 (green) antibodies plus DAPI to stain nuclei blue, showing high ATF3 expression in the nuclei of hyperplasic AECIIs from honeycombs in IPF lung
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References

    1. Armanios, M. Y. , Chen, J. J. , Cogan, J. D. , Alder, J. K. , Ingersoll, R. G. , Markin, C. , … Loyd, J. E. (2007). Telomerase mutations in families with idiopathic pulmonary fibrosis. The New England Journal of Medicine, 356, 1317–1326. https://doi.org/10.1056/NEJMoa066157 - DOI - PubMed
    1. Bueno, M. , Lai, Y. C. , Romero, Y. , Brands, J. , St Croix, C. M. , Kamga, C. , … Mora, A. L. (2015). PINK1 deficiency impairs mitochondrial homeostasis and promotes lung fibrosis. The Journal of Clinical Investigation, 125, 521–538. https://doi.org/10.1172/JCI74942 - DOI - PMC - PubMed
    1. Chambers, R. C. , Leoni, P. , Kaminski, N. , Laurent, G. J. , & Heller, R. A. (2003). Global expression profiling of fibroblast responses to transforming growth factor‐β1 reveals the induction of inhibitor of differentiation‐1 and provides evidence of smooth muscle cell phenotypic switching. American Journal of Pathology, 162, 533–546. https://doi.org/10.1016/S0002-9440(10)63847-3 - DOI - PMC - PubMed
    1. Chen, B. P. , Wolfgang, C. D. , & Hai, T. (1996). Analysis of ATF3, a transcription factor induced by physiological stresses and modulated by gadd153/Chop10. Molecular and Cellular Biology, 16, 1157–1168. https://doi.org/10.1128/MCB.16.3.1157 - DOI - PMC - PubMed
    1. Duan, X. , Tong, J. , Xu, Q. , Wu, Y. , Cai, F. , Li, T. , & Song, W. (2014). Upregulation of human PINK1 gene expression by NFkappaB signalling. Molecular Brain, 7, 57 https://doi.org/10.1186/s13041-014-0057-y - DOI - PMC - PubMed

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