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. 2017 Mar 7;18(10):2480-2493.
doi: 10.1016/j.celrep.2017.02.012.

Integrin Beta 3 Regulates Cellular Senescence by Activating the TGF-β Pathway

Valentina Rapisarda  1 , Michela Borghesan  1 , Veronica Miguela  2 , Vesela Encheva  3 , Ambrosius P Snijders  3 , Amaia Lujambio  2 , Ana O'Loghlen  4
Affiliations

Integrin Beta 3 Regulates Cellular Senescence by Activating the TGF-β Pathway

Valentina Rapisarda et al. Cell Rep. .

Abstract

Cellular senescence is an important in vivo mechanism that prevents the propagation of damaged cells. However, the precise mechanisms regulating senescence are not well characterized. Here, we find that ITGB3 (integrin beta 3 or β3) is regulated by the Polycomb protein CBX7. β3 expression accelerates the onset of senescence in human primary fibroblasts by activating the transforming growth factor β (TGF-β) pathway in a cell-autonomous and non-cell-autonomous manner. β3 levels are dynamically increased during oncogene-induced senescence (OIS) through CBX7 Polycomb regulation, and downregulation of β3 levels overrides OIS and therapy-induced senescence (TIS), independently of its ligand-binding activity. Moreover, cilengitide, an αvβ3 antagonist, has the ability to block the senescence-associated secretory phenotype (SASP) without affecting proliferation. Finally, we show an increase in β3 levels in a subset of tissues during aging. Altogether, our data show that integrin β3 subunit is a marker and regulator of senescence.

Keywords: CBX7; ITGB3; Palbociclib; SASP; TGFβ; aging; cilengitide; integrin; senescence; β3.

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Figures

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Graphical abstract
Figure 1
Figure 1
SILAC Screen Identifies Putative Regulators of Senescence (A) Left panel: schematic representation of the senescence model used in the SILAC screen. Human primary breast fibroblasts (BFs) were transduced with a lentivirus harboring an shRNA targeting CBX7 (shCBX7). Right panel: immunoblot showing CBX7 knockdown efficiency and an increase in p16INK4A protein levels. β-tubulin is used as loading control. (B) Senescence induced upon shCBX7 is shown by an increase in the percentage of cells staining positive for SA-β-galactosidase (SA-β-Gal) activity. Quantification of two to three independent experiments is shown. (C) Scatterplot of mass spectrometry (MS) results from both forward and reverse SILAC experiments. A 2-fold difference in expression upon shCBX7 is indicated with orange circles, outlining CDKN2A and ITGB3 in blue. Gray circles represent unchanged proteins. (D) Pathway analyses (KEGG) show that proteins with a 2-fold difference in expression fall within the categories of the extracellular matrix (ECM)-interacting and FA pathways. (E) Comparison of the proteins significantly deregulated in the SILAC experiment with a published dataset for genes regulated by CBX proteins in human diploid fibroblasts (Pemberton et al., 2014). (F) List of 20 proteins in the SILAC screen whose genes could be regulated by CBX proteins, highlighting CDKN2A (red) and ITGB3 (green).
Figure 2
Figure 2
The Genes Encoding the Proteins Found in the SILAC Screen Are Regulated by CBX7 (A) qPCR analyses show the relative mRNA levels of the selected genes upon shCBX7. INK4A is highlighted in red as a known CBX7-regulated gene, and ITGB3 is highlighted in green as a potentially new gene regulated by CBX7. Data are normalized to the control, shown as a gray shade, and represent the mean ± SD of two independent experiments. (B) Overexpression of Cbx7 reduces the expression of its target genes. Relative mRNA levels are shown by qPCR. Data are normalized to the control, shown as a gray shade, and represent the mean ± SD of two independent experiments. (C) ChIP for endogenous CBX7 (black bars) shows enrichment at the transcription start site (TSS) of its target genes, in comparison with immunoglobulin G (IgG) control (white bars). There is no CBX7 enrichment at the TSS of non-PRC1 target genes (Controls): ARF (encoding p14ARF) and ACTB (β-actin). Data represent the mean ± SD of a representative experiment. (D) ChIP for other PRC1 proteins (CBX8 and RING1B) show enrichment at the TSS of ITGB3, in comparison with IgG (white bars). A representative experiment is shown. (E) Representative images showing integrin αvβ3 (green) and F-actin (red) staining in fibroblasts expressing empty vector or shCBX7. The formation of αvβ3-stained FA complexes can be observed only in cells harboring shCBX7 (white arrows). The quantification indicates the percentage of cells positive for αvβ3 staining ± SD (three to five independent experiments). Scale bar, 20 μm. (F) A representative blot for BFs overexpressing Cbx7 shows reduced levels of endogenous β3 subunit and mouse Cbx7 overexpression levels. β-actin is used as loading control. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Figure 3
Figure 3
ITGB3 Ectopic Expression Induces Senescence via p21CIP/p53 Pathway (A–F) Overexpression of a retroviral construct encoding ITGB3 in BFs induces senescence. H-RasG12V (RAS) is used as a positive control for inducing senescence. (A) We show a reduction in proliferation in BFs expressing ITGB3 by measuring the percentage of cells incorporating BrdU (left panel: quantification levels; right panel: representative pictures). Proliferation was assessed 4–5 days after plating. BFs expressing ITGB3 show (B) an increase in p21CIP and p53 protein levels by IF 4–5 days after plating (left panel: percentage of cells stained positive for p21CIP and p53; right panel: representative pictures for p21CIP staining) and (C) an increase in CDKN2B (encoding p15INK4B) mRNA levels by qPCR. No changes were observed in CDKN1B (encoding p27KIP1) or CDKN2A (p16INK4A). (D) Expression of ITGB3 also induced an increase in senescence-associated β-galactosidase activity (SA-β-Gal). Data represent the percentage of cells staining positive for SA-β-Gal ± SD. Staining was performed 7–10 days after plating; (E) an increase in the levels of ROS, measured by 8-oxoG staining, and (F) a mild increase of the mRNA levels of different SASP by qPCR. Cells were subjected to analysis (either by IF or qPCR) 4–5 days after plating. (G) An shRNA against p53 (shp53) prevents the activation of senescence induced by ITGB3 ectopic expression, as shown by the reversion in the percentage of BFs incorporating BrdU induced by ITGB3 (left graph) and the decrease in SA-β-Gal activity (right graph). BrdU was added 24 hr prior to fixing the cells for IF. Data represent the mean ± SD of more than two independent experiments. Scale bars, 100 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Figure 4
Figure 4
ITGB3 Regulates Senescence Independently of Its Binding Activity (A) Endogenous αvβ3 expression increases during OIS upon RAS expression in BFs. Representative pictures for αvβ3 (green) and F-actin (red) staining by IF in vector and RAS cells are shown. αvβ3-stained FA complexes are indicated with white arrows. Data represent the percentage of cells positive for αvβ3 staining. Scale bar, 20 μm. (B) ITGB3 is endogenously upregulated during DNA-damage-induced senescence (DDIS). BFs were treated with 100 μM etoposide (Etop) for 2 days and replaced with fresh media for 5 days. (C) MCF7 breast cancer cells were treated with 200 nM palbociclib (Palbo) for 7 days, after which cells were lysed for immunobloting. An increase in β3 subunit and p53 can be observed after Palbo treatment. (D) mRNA analyses for ITGB subunits 1–8 during RAS-induced senescence in BFs. (E) CBX7 binding to ITGB3 TSS is reduced during OIS. ChIP for CBX7 enrichment (black bars) versus IgG control (white bars) at an ITGB3 TSS and a coding region in BFs expressing vector or RAS. (F) Schematic representation of the timings used to determine the role for ITGB3 overcoming OIS (top panel). Lower left panel: relative cell number in RAS-expressing BFs transduced with a vector or an shRNA targeting ITGB3 (shITGB3). Lower right panel: representative immunoblot showing β3 subunit knockdown efficiency. (G) Top panel shows the experimental planning. Senescence was induced by Palbo treatment, after which siITGB3 was transfected (green bar). Two independent siRNAs targeting ITGB3 (siITGB3) overcame the senescence arrest induced by treating MCF7 cells with Palbo for 7 days. BrdU was added 24 hr before the end of the experiment. (H) Cells expressing RAS were treated with DMSO or αvβ3 inhibitor (cilengitide) for 48 hr, and the relative cell number was calculated. Increasing concentrations of cilengitide (10, 25, and 50 nM) show no reversion of the proliferation arrest induced by RAS. An inhibitor for TGF-β-receptor 1 (TGFBR1, 4 μM) was used as positive control. (I) Immunoblot for the conditioned media (CM) from cells expressing either vector or RAS treated with or without 50 nM of αvβ3 inhibitor (cilengitide) for 48 hr, followed by a 72-hr incubation in fresh media. Coomassie staining is shown as loading control. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Figure 5
Figure 5
ITGB3 Induces Senescence by Activating the TGF-β Pathway in a Cell-Autonomous and Non-Cell-Autonomous Fashion (A and B) Shown here are (A) a schematic representation and (B) timings of a miniscreen for small molecule inhibitors (Inh) used to determine the pathway activating senescence induced by β3. (C) BFs expressing vector or ITGB3 were treated for 48 hr with a variety of drugs inhibiting different signaling pathways. Drugs were renewed every 24 hr, and BrdU was added 16 hr prior to fixing the cells. The percentage of BrdU-positive cells with or without the inhibitors is shown. The graph indicates the inhibitor’s targets: 40 μM PD98059 (targeting MEK1/2), 20 μM SB202190 (p38MAPK), 100 nM TORIN2 (mammalian target of rapamycin; mTOR), 4 μM TGF-β-R1 (TGFBR1), 8 μM Vegfr-2/Flt3/C-Kit (VEGFR), 150 nM GSK429286A (ROCK1/2, Rho-associated kinase), 50 nM Cpd22 (ILK, integrin-linked kinase), and 50 nM cilengitide (αvβ3). Except when indicated, asterisks represent the statistical differences for cells expressing ITGB3 treated with DMSO or the different small molecule inhibitors. (D) Knockdown of TGFBR2 overcomes senescence induced by β3. We measured the proliferation (left graph) and p21CIP levels (right graph) in BF fibroblasts transiently transfected with a scramble (Scr) or two independent siRNAs against TGFBR2 (siTR2_2 and 7) for 4–5 days. (E) qPCR analyses of different regulators of the TGF-β pathway: ligands (TGFB1, -2, and -3), effectors (SMAD2, -3, and -4), receptors (TGFBR1 or ALK5, TGFBR2), and ECM proteins (LTBP1, -3, or latent TGF binding proteins). (F) Representative IF pictures for SMAD2/3 staining and quantification of the percentage of cells positive for nuclear SMAD2/3. Scale bar, 50 μm. (G) MMP1–MMP9 mRNA levels upon ITGB3 expression. (H and I) Normal BFs were treated with conditioned media (CM) from BFs expressing vector or ITGB3. (H) Pan-specific neutralizing anti-TGF-β1-3 antibodies inhibit the stabilization of p53 induced by the CM taken from ITGB3 cells. A species matching IgG was used as negative control. Bottom panel: a diagram showing the experimental planning. CM was collected after 7 days and transferred to normal BFs with or without different treatments. (I) A TGFBR1 inhibitor (4 μM) blocks the nuclear translocation of SMAD2/3 induced by the CM taken from ITGB3 cells. Data represent mean ± SD of 2–4 independent experiments. ∗p < 0.05; ∗∗∗p < 0.001.
Figure 6
Figure 6
β3 Subunit Is Upregulated during Replicative Senescence and Aging in Human and Mouse (A) Immunoblot for β3 subunit in BFs expressing empty vector, RAS or the dominant-negative telomeric repeat binding factor 2 (TRF2ΔBΔM), mimicking replicative senescence. β-actin is used as loading control. (B) Immunoblot for β3 subunit (left panel) and qPCR analysis for Itgb3 mRNA levels (right panel) in mouse hepatic stellate cells (mHSCs) upon different days of doxycycline (Dox) withdrawal. (C) mRNA levels are shown for Ink4a and Itgb3 in livers taken from C57BL/6J female mice aged 4, 19, and 25 months old. (D) Kidneys from young (4 months) and old (25 months) C57BL/6J mice were subjected to qPCR to determine Ink4a and Itgb3 mRNA expression levels. (E) Representative immunoblot for β3 subunit in human primary fibroblasts derived from young and old donors. We observed similar results with other young and old samples. β-actin is the loading control. (F) Quantification of the percentage of cells stained positive for αvβ3 in FA complexes by IF in young and old human fibroblasts. Data represent the mean ± SD of fibroblasts derived from young and old donors. (G) qPCR analyses for TGF-β receptors 1 and 2 and SMAD3 and 4 in human fibroblasts from young (Y) and old (O) donors. In (C) and (D), data represent the mean ± SD of 4–5 mice per condition. In (F) and (G), data represent the mean ± SD from fibroblasts from 2–4 young and 6–7 old donors.
Figure 7
Figure 7
Changes in the Expression Levels of ITGB3 Affect Aging and Senescence Cellular Features (A and B) Ectopic expression of a construct encoding either RAS or ITGB3 induces a senescence-like arrest in human fibroblasts derived from two independent young donors. (A) Percentage of BrdU-positive cells and (B) p21CIP protein levels quantified by IF are shown. (C and D) RNAi targeting ITGB3 in fibroblasts derived from old donors averts cellular features of aging and senescence. Cells derived from two young donors were used as controls (white filling). Fibroblasts from old donors (black filling) were transiently transfected with a scramble (Scr) or two independent siRNAs against ITGB3 (siITGB3_3 and 4) for 4–5 days. sip53 was used as a control. (C) The percentage of cells staining positive for BrdU and (D) p21CIP were quantified by IF. (E and F) Treatment of cells derived from old donors with cilengitide does not affect senescence/aging. Cells from two independent old donors (black filling) were treated with 50 nM cilengitide αvβ3 inhibitor for 2 days, and (E) the percentage of BrdU- and (F) p21CIP-positive cells was assessed. inh., inhibitor. All data represent the mean ± SD from cells derived from two independent young (A and B) or old (C–F) individuals. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
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