"V体育官网入口" Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official VSports app下载. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2014 Jun 3;111(22):E2261-70.
doi: 10.1073/pnas.1320843111. Epub 2014 May 19.

Mechanisms controlling the smooth muscle cell death in progeria via down-regulation of poly(ADP-ribose) polymerase 1

Haoyue Zhang  1 , Zheng-Mei Xiong  1 , Kan Cao  2
Affiliations

"V体育平台登录" Mechanisms controlling the smooth muscle cell death in progeria via down-regulation of poly(ADP-ribose) polymerase 1

Haoyue Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a severe human premature aging disorder caused by a lamin A mutant named progerin. Death occurs at a mean age of 13 y from cardiovascular problems. Previous studies revealed loss of vascular smooth muscle cells (SMCs) in the media of large arteries in a patient with HGPS and two mouse models, suggesting a causal connection between the SMC loss and cardiovascular malfunction. However, the mechanisms of how progerin leads to massive SMC loss are unknown. In this study, using SMCs differentiated from HGPS induced pluripotent stem cells, we show that HGPS SMCs exhibit a profound proliferative defect, which is primarily caused by caspase-independent cell death. Importantly, progerin accumulation stimulates a powerful suppression of PARP1 and consequently triggers an activation of the error-prone nonhomologous end joining response. As a result, most HGPS SMCs exhibit prolonged mitosis and die of mitotic catastrophe. This study demonstrates a critical role of PARP1 in mediating SMC loss in patients with HGPS and elucidates a molecular pathway underlying the progressive SMC loss in progeria VSports手机版. .

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Characterization of SMCs differentiated from normal and HGPS iPS cells. (A) Schematic diagram of SMC differentiation timeline and representative phase contrast images underneath each denoted stage. EB, embryoid body. (Scale bar, 400 μm.) (B) Western blot with anti-SMA, calponin, and GAPDH antibodies on the cell samples at various differentiation stages. (C) Percentages of SMA and calponin positive population in normal and HGPS SMCs at day 14. Results were generated from two biological replicates. (D) Representative phase control images showing the contraction of normal and HGPS SMCs at days 14 and 21 upon angiotensin II treatment. Cell contraction at 30 min after stimulation was indicated by red arrows in the enlarged images. (Scale bar, 400 μm.) (E) Quantification of D, showing the percentage of contracted SMCs upon stimulation. Results were generated from three independent experiments.
Fig. 2.
Fig. 2.
Increased caspase-independent cell death in HGPS SMCs. (A) Growth curve of normal and HGPS SMCs during differentiation. Results were generated from three biological replicates. *P < 0.05, **P < 0.01. Blue and green arrows referred to the images at days 7 and 21 in B, respectively. (B) Representative phase contrast images of normal and HGPS SMCs at days 7 and 21 during SMC differentiation. (Scale bar, 200 μm.) (C) Cell cycle analysis of normal and HGPS SMCs at day 14, illustrating the distribution of G0/G1, S, and G2/M phases. Results were generated from three biological replicates. *P < 0.05. (D) PI-annexin V flow cytometry analysis on normal and HGPS SMCs at various time points during SMC differentiation. The gates were set according to the positive and negative controls as suggested by the manufacturer. The cells in the Lower Right quadrant were scored as the dying cell population. (E) Quantification of D, showing the percentage of dying population based on the PI-annexin V assay. Results were generated from three biological replicates. *P < 0.05, **P < 0.01. (F) RT-qPCR analysis with Bax-specific primers on normal and HGPS SMCs at different time points during SMC differentiation. (G) Basal caspase 3 activity of normal and HGPS SMCs at day 14. Results were generated from two independent biological replicates. (H) Western blot with anti-PARP1 and GAPDH antibodies on normal and HGPS SMCs at indicated time points during differentiation. (I) Percentage of the sub-G1 population in normal and HGPS SMCs at days 14, 21, and 28.
Fig. 3.
Fig. 3.
Progerin down-regulates PARP1. (A, Upper) Western blot with anti-PARP1 and GAPDH antibodies on two HGPS SMC lines (HGADFN167 and HGADFN164) and a normal control SMC line at day 14. (Lower) Quantification of PARP1 in each sample. The intensity of PARP1 was first normalized to corresponding GAPDH and then normalized to the control SMC. (B) Representative images of immunofluorescence staining with anti-PAPR1 antibody in normal and HGPS SMCs at day 14. (Scale bar, 50 μm.) (C) Quantification of B, showing the PARP1 red fluorescence intensity. ***P < 0.001. (D) Representative images of immunofluorescence staining with anti-PARP1 in normal SMCs transfected with pEGFP-C1-LA or pEGFP-C1-HG plasmids. The transfected and untransfected cells are indicated by green and white arrows, respectively. The nuclei were outlined by white dashed lines. (Scale bar, 20 μm.) (E) Quantification of D, showing the correlation between EGFP-HG or EGFP-LA fluorescence intensity (x axis) and PARP1 fluorescent intensity (y axis). More than 100 cells were randomly picked for each group. (F) Western blot with anti-PARP1 and GAPDH antibodies on normal SMCs transfected with different amounts of pEGFP-C1-HG plasmid. (G) Quantification of F, showing a negative-linear relationship between PARP1 (y axis) and progerin (x axis) protein amounts. Signals were normalized to the untransfected control. (H) Survival curve of early passage normal and HGPS fibroblasts upon a PARP1 inhibitor (Olaparib) treatment. Results were generated from two independent experiments. *P < 0.05.
Fig. 4.
Fig. 4.
Progerin induces PARP1 mislocalization and Ran gradient disruption. (A) Representative images showing normal SMCs coexpressing PARP1-GFP with DsRed, DsRed-LA, DsRed-HG (with or without 2 μM FTI), or DsRed-HG-SSIM. Nuclei was outlined by white dashed lines. (Scale bar, 10 μm.) (B) Quantification of A, showing the percentage of PARP1-GFP–mislocalized cells under indicated conditions. More than 100 cells were counted for each group. Results were generated from three independent experiments. *P < 0.05, **P < 0.01. (C) Representative images of immunofluorescence staining with anti-Ran antibody on HGPS SMCs transfected with PARP1-GFP. Cells with both normally or mislocalized PARP1-GFP were shown. The nuclei were outlined by white dashed lines. (Scale bar, 20 μm.) (D) Quantification of C, showing the Ran gradient in HGPS SMCs transfected with PARP1-GFP. ***P < 0.001. (E) Representative images of immunofluorescence staining with anti-Ran antibody in normal SMCs transfected with DsRed, DsRed-LA, DsRed-HG (with or without 2 μM FTI) or DsRed-HG-SSIM. The transfected and untransfected cells were indicated by green and white arrows, respectively. The nuclei were outlined by white dashed lines. (Scale bar, 20 μm.) (F) Quantification of E, showing the correlation between DsRed fluorescent intensity and Ran gradient (Ran N/C) in N and C refer to the nucleus and cytoplasm, respectively. More than 50 cells were randomly picked for each group.
Fig. 5.
Fig. 5.
HGPS SMCs show impaired HR and activated NHEJ. (A) Representative image showing immunofluorescence staining with anti-γH2AX and Rad51 antibodies on normal and HGPS SMCs recovered for various amounts of time after 100 nM CPT treatment. (Scale bar, 10 μm.) (B) Quantification of A, showing of the number of γH2AX (x axis) and colocalized Rad51 (y axis) foci in normal and HGPS SMCs at different time points. More than 100 cells were counted for each case. The trend lines of normal (in gray) and HGPS (in red) SMCs were also presented. (C) The percentage of nuclei containing more than five γH2AX foci in normal and HGPS SMCs at each indicated time point after 100 nM CPT treatment. (D) Representative images of immunofluorescence staining with anti-γH2AX and 53BP1 antibodies on normal and HGPS SMCs at 10 min after 100 nM CPT treatment. (Scale bar, 10 μm.) (E) Quantification of D, showing the number of γH2AX (x axis) and colocalized 53BP1 (y axis) foci in normal and HGPS SMCs at different time points. Over 50 cells were analyzed.
Fig. 6.
Fig. 6.
Mitotic catastrophe in HGPS SMCs. (A) Cell cycle analysis of the normal and HGPS SMCs at denoted time points. Results were generated from two biological replicates. (B) DIC live cell imaging of normal and HGPS SMCs at day 21. (Top) Normal mitotic division of a normal SMC; (Middle) mitotic catastrophe of an HGPS SMC; and (Bottom) failed mitosis of an HGPS SMC. (C) Percentage of normal and defective mitosis in normal and HGPS SMCs. n = 4 and 7 for normal and HGPS SMCs, respectively. (D) Quantification of the percentages of BrdU-positive nuclei at 48 h and 96 h after BrdU pulse in normal and HGPS SMCs at day 14. Results were generated from two independent experiments. *P < 0.05 and **P < 0.01.
Fig. 7.
Fig. 7.
Working model. The accumulation of membrane-associated progerin not only causes HR deficiency via an unknown mechanism, but also causes PARP1 down-regulation, likely through interfering with PARP1 nuclear import. This PARP1 reduction results in the activation of the NHEJ pathway in the HR-deficient HGPS SMCs, leading to error-prone DNA repair, chromosome aberration, and mitotic catastrophe in HGPS SMCs.
See this image and copyright information in PMC

References

    1. Rothkamm K, Krüger I, Thompson LH, Löbrich M. Pathways of DNA double-strand break repair during the mammalian cell cycle. Mol Cell Biol. 2003;23(16):5706–5715. - PMC - PubMed
    1. Chen JH, Hales CN, Ozanne SE. DNA damage, cellular senescence and organismal ageing: Causal or correlative? Nucleic Acids Res. 2007;35(22):7417–7428. - PMC - PubMed
    1. Kraemer KH, et al. Xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome: A complex genotype-phenotype relationship. Neuroscience. 2007;145(4):1388–1396. - PMC - PubMed
    1. Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature. 2006;443(7113):787–795. - PubMed (VSports)
    1. Vakifahmetoglu H, Olsson M, Zhivotovsky B. Death through a tragedy: Mitotic catastrophe. Cell Death Differ. 2008;15(7):1153–1162. - PubMed

Publication types

MeSH terms (VSports在线直播)

Substances (VSports注册入口)