Integrin-mediated adhesions in regulation of cellular senescence

Abstract

Bioinformatic and functional data link integrin-mediated cell adhesion to cellular senescence; however, the significance of and molecular mechanisms behind these connections are unknown. We now report that the focal adhesion-localized βPAK-interacting exchange factor (βPIX)-G protein-coupled receptor kinase interacting protein (GIT) complex controls cellular senescence in vitro and in vivo. βPIX and GIT levels decline with age. βPIX knockdown induces cellular senescence, which was prevented by reexpression. Loss of βPIX induced calpain cleavage of the endocytic adapter amphiphysin 1 to suppress clathrin-mediated endocytosis (CME); direct competition of GIT1/2 for the calpain-binding site on paxillin mediates this effect. Decreased CME and thus integrin endocytosis induced abnormal integrin signaling, with elevated reactive oxygen species production. Blocking integrin signaling inhibited senescence in human fibroblasts and mouse lungs in vivo. These results reveal a central role for integrin signaling in cellular senescence, potentially identifying a new therapeutic direction.

Fig. 1. Decreased βPIX levels in aged tissues and cells.

(A) Expression of βPIX and related adhesion molecules in the lung, kidney, spleen, heart, or skin from 1-, 15-, or 24-month-old mice. Protein levels were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are representative of three experiments. (B) Immunohistochemical staining with anti-βPIX (top) or anti-p16 (bottom) antibody in lung from 3-month-old (n = 4) and 24-month-old (n = 4) mice (five bronchioles per mouse). Blue arrows indicate positive staining. Scale bars, 10 μm. (C) Quantification for (B). *P < 0.001, Student’s t test (t test). (D) Immunohistochemical staining of βPIX (top) and p16 (bottom) in bronchioles from young (12 to 16 years old; n = 5) or old (64 to 75 years old; n = 5) human lung (three bronchioles per lung). M, male. Blue arrows indicate positive staining. Scale bar, 10 μm. (E) Quantification for (D). *P < 0.001, t test. (F) Expression of βPIX and p16 was analyzed by immunoblotting with anti-βPIX or anti-p16 in young or old HDF cells. Protein levels were normalized by GAPDH. (G) SA-β-Gal staining in young or old passage HDF cells. Scale bar, 100 μm. (H) Quantification for (G). n > 200 cells per group from three independent experiments. *P < 0.001, Mann-Whitney rank sum test. For all panels, quantified values are means ± SEM.

Fig. 2. βPIX knockdown induces cellular senescence in vitro and in vivo.

(A) Co-staining for SA-β-Gal (blue) and pγH2AX (brown; inset) in siRNA-treated HDFs. (B and D) Quantification of SA-β-Gal staining. n > 100 cells per group from three independent experiments. *P < 0.001, Mann-Whitney rank sum test. (C and E) Immunoblotting. (F and I) Experimental scheme for (G) and (H) and (J) and (K), respectively. IHC, immunohistochemistry. (G) SA-β-Gal staining and immunostaining for senescence markers in siRNA-treated mice bronchioles. (H) Quantification of senescence markers. n = 3 mice per group (five bronchioles per mouse). *P < 0.001, t test. siPIXm, mouse-specific siPIX. (J) Staining for senescence markers in bronchioles after lentivirus-mediated expression of green fluorescent protein (GFP) or GFP-βPIX. (K) Quantification of senescence markers. n = 3 mice per group (five bronchioles per mouse). *P < 0.001, t test. Blue arrows in (G) and (J) indicate positive staining. Scale bars, 20 μm (A) and 10 μm (G and J). For all panels, quantified values are means ± SEM.

Fig. 3. βPIX knockdown activates integrin signaling.

(A) Phosphorylation of FAK and paxillin over a 4-day period after siPIX transfection. siRNA-treated cell lysates were immunoblotted. Data are representative of three independent experiments. (B) Representative images for FAs and actin bundles (red). siRNA-treated cells were incubated with 100 μM control or RGD peptides for 3 days. (C) Quantification of number of FAs. n ≥ 50 cells per group from three independent experiments. *P < 0.001, Mann-Whitney rank sum test. (D) Quantification of intensity of actin bundles. n ≥ 50 cells per group from three independent experiments. *P < 0.001, t test. (E) Representative images for SA-β-Gal staining. HDFs treated with siRNA were incubated with 100 μM control or RGD peptides for 3 days. (F and G) Effect of 100 μM RGD peptides (F) and 20 nM PF573028 (G) on senescence. n ≥ 200 cells per group from three independent experiments. *P < 0.001, t test. (H) Experimental scheme for (I) and (J). (I) Immunohistochemical staining for indicated proteins in bronchioles of siRNA-treated mice. Blue arrows indicate positive staining. (J) Quantification of indicated proteins (I). n = 3 mice per group (five bronchioles per mouse). *P < 0.001, t test. Scale bars, 20 μm (B), 100 μm (E), and 10 μm (I). For all panels, quantified values are means ± SEM.

Fig. 4. βPIX knockdown blocks CME.

(A) β₁ Integrin uptake in βPIX-depleted HDFs. siRNA-treated cells were pretreated with nocodazole and incubated with anti–active β₁ integrin antibody. Cells were then subject to acid rinse to remove unbound antibody and nocodazole and chased in medium containing 10% fetal bovine serum (FBS) for 60 min. Integrin uptake was visualized by staining for the β₁ integrin antibody. (B) Quantification of internalized β₁ integrin. n ≥ 100 cells per group from three independent experiments. *P < 0.001, t test. (C) Transferrin endocytosis in siRNA-treated HDFs. At 3 days after siRNA transfection, transferrin uptake was measured. DAPI, 4′,6-diamidino-2-phenylindole. (D) Quantification of endocytosed transferrin. n ≥ 100 cells per group from three independent experiments. *P < 0.001, t test. (E) Human βPIX siRNAs show no off-target effects for transferrin endocytosis. (F) Quantification of internalized transferrin. n = 10 fields (cells per group, >100). *P < 0.001. Mann-Whitney rank sum test. A.U., arbitrary units. (G) Transferrin endocytosis in the bronchi of siRNA-treated mice. Transferrin was applied by the intratracheal delivery technique. Internalized transferrin was observed with confocal microscope. (H) Quantification. n = 3 mice per group (five bronchioles per mouse). *P < 0.001, t test. Scale bars, 20 μm (A, C, and E) and 10 μm (G). For all panels, quantified values are means ± SEM.

Fig. 5. Calpain-resistant amphiphysin (V392G) prevents βPIX knockdown–induced events in vivo.

(A) Experimental scheme to analyze calpain-resistant amphiphysin I. (B) Uptake of Alexa Fluor 594–transferrin was visualized in bronchioles of siRNA-treated mouse lung expressing GFP (control) and amphiphysin (AMPH-WT or AMPH-V392G). Scale bar, 20 μm. siPIXm1, mouse specific siRNA for silencing βPIX. (C) Quantification of transferrin uptake. n = 3 mice per group (five bronchioles per mouse). *P < 0.001, t test. (D) SA-β-Gal and immunohistochemical staining for senescence markers were performed in the bronchioles of siRNA-treated mice expressing GFP (control) and amphiphysin (AMPH-WT or AMPH-V392G). Red arrows indicate positive staining. Scale bars, 10 μm. (E) Quantification of senescence markers. n = 3 mice per group (five bronchioles per mouse). *P < 0.001, t test. For all panels, quantified values are means ± SEM.

Fig. 6. GIT-CT prevents βPIX knockdown–induced events in vivo.

(A) Competition of GIT1 and calpain-2 binding to paxillin in vitro. Ni²⁺ beads–bound paxillin was incubated with GST–calpain-2, and then the indicated concentrations of GST–GIT-CT (left) or GST (negative control; right) were added. Protein complexes were washed and analyzed by immunoblotting. Data are representative of two independent experiments. (B) Experimental scheme for (C) and (E). (C) Visualization of transferrin uptake in bronchioles of siRNA-treated mouse lung expressing GFP (control) or GIT-CT. Scale bars, 20 or 10 μm (enlarged). (D) Quantification of transferrin uptake. n = 4 mice per group (five bronchioles per mouse). *P < 0.001, t test. (E) SA-β-Gal and immunohistochemical staining for senescence markers in bronchioles of siRNA-treated mice. Scale bars, 10 μm. (F) Quantification of senescence markers. n = 4 mice per group (five bronchioles per mouse). *P < 0.001, t test. (G) Model for the role of βPIX-GIT in CME and senescence. Left: When βPIX-GIT complex is sufficient, CME would occur at physiological levels. Middle: When βPIX and/or GIT is depleted, calpain-2 binds paxillin and cleaves amphiphysin, which would lead to defective CME. Right: Defective endocytosis of active β₁ integrin results in persistent activation of integrin signaling, which leads to senescence. For all panels, quantified values are means ± SEM.