Reactive oxygen species control senescence-associated matrix metalloproteinase-1 through c-Jun-N-terminal kinase

Abstract

The lifetime exposure of organisms to oxidative stress influences many aging processes which involve the turnover of the extracellular matrix. In this study, we identify the redox-responsive molecular signals that drive senescence-associated (SA) matrix metalloproteinase-1 (MMP-1) expression. Precise biochemical monitoring revealed that senescent fibroblasts increase steady-state (H(2)O(2)) 3.5-fold (13.7-48.6 pM) relative to young cells. Restricting H(2)O(2) production through low O(2) exposure or by antioxidant treatments prevented SA increases in MMP-1 expression. The H(2)O(2)-dependent control of SA MMP-1 is attributed to sustained JNK activation and c-jun recruitment to the MMP-1 promoter. SA JNK activation corresponds to increases and decreases in the levels of its activating kinase (MKK-4) and inhibitory phosphatase (MKP-1), respectively. Enforced MKP-1 expression negates SA increases in JNK phosphorylation and MMP-1 production. Overall, these studies define redox-sensitive signaling networks regulating SA MMP-1 expression and link the free radical theory of aging to initiation of aberrant matrix turnover.

Fig. 1

Oxygen-dependent control of senescence associated MMP-1 expression. (A) Morphologic differences between young and old cells. (B) Immunoblot analysis of MMP-1 and senescence markers, p21 and p16 in cells cultured at 21% O₂ verses 3% O₂. (C) MMP-1 protein and mRNA levels from young (passage 12-14) and old (passage 24-26) IMR90 cells. Protein levels are reported as relative densitometric intensity measure by NIH ImageJ and error bars represent ± S.E. of mean (n=7). RNA levels were measured by real-time PCR which were normalized to β-actin and reported as cDNA units ± S.E. of mean (n=4). (D) Immunoblot analysis of MMP-1 from aged cells in 3% O₂ or acutely transitioned to 21% O₂. Representative blot shown (n=3).

Fig. 2

Oxygen tension controls steady-state oxidant production. Intracellular reactive oxygen species (ROS) were determined using an aminotriazole inhibition of catalase assay and a redox-sensitive green fluorescent protein (RoGFP) as described in materials and methods. (A-C) Exponential decline of catalase activity following treatment with ATZ in IMR-90 fibroblasts cultured at 3% and 21% O₂. (D) Steady-state intracellular [H₂O₂] in pM in IMR-90 cells at 3% and 21% O₂ and cells that were transitioned from low to high O₂ or vice versa. The levels were determined based on the rate of catalase inhibition. Values are reported ± S.E. of mean (n=3). (E) Excitation ratio at λ₄₁₀/λ₄₇₀ was determined in senescent cells under 3% and 21% O₂ or following transfer of cells from 3% to 21% O₂ and vice-versa. Data reported as a ratio of excitation at λ₄₁₀/λ₄₇₀ with a higher ratio 410/470 indicating an increased oxidized state and represented as ± S.E. of mean. A minimum of 10 cells were measured for roGFP1 oxidation.

Fig. 3

Inhibition of JNK signaling impairs senescence associated MMP-1 production. Old IMR90 cells at passage 25 under 21% O₂ were treated with various pharmacological inhibitors (50μM LY294002 for PI3K, 25μM PD98059 for MEK/Erk, 10μM SB203580 for p38 and 10μM SP600125 for JNK) overnight. MMP-1 protein and RNA levels were analyzed as described above. p<0.05, using one-tailed unpaired t-test of protein (A) and RNA (B). Data represented as ± S.E. of means, n=3 and normalized to cell number for protein and to β-actin for RNA. (C) Senescent IMR90 cells under 21% O₂ were infected with adenovirus expressing GFP or a dominant/negative JNK-1-GFP fusion protein and MMP-1 production determined by immunoblotting (n=3). A representative immunoblot and quantification (mean ± S.D.) are shown in inset. Adenoviral infection was confirmed by monitoring GFP as shown in the representative transmitted light and fluorescent images.

Fig. 4

The activation of the c-Jun N-terminal kinase pathway is both age and redox-responsive. (A) Immunblot analysis of JNK-1/2 phosphorylation in young and old cells cultured at 3% or 21% O₂. p-JNK levels were normalized to total JNK. Data reported as relative densitometric intensity measured by NIH ImageJ and errors bars represent ± S.E. of mean, n=3. (B) ChIP analysis of c-Jun binding to the MMP-1 promoter (n=6). Data was normalized to input as well as a no antibody control. (C) Immunoblot anlaysis of Akt and ERK phosphorylation in young and old IMR90 cells cultured in low and high oxygen. Representative blot shown, n=3.

Fig. 5

Antioxidants restrict senescence associated MMP-1 expression. (A) MMP-1 immunoblot analysis from IMR-90 cells maintained at 21% and 3% O₂ after treatment with 2mM dose of antioxidant, n-Acetyl cysteine (NAC) for 8hrs and 24hrs (B) MMP-1 protein levels determined by densitometry. Data reported as relative densitometric intensity measured by NIH ImageJ and errors bars represent ± S.E. of mean, n=3. MMP-1 levels were normalized to cell number. (C) Phospho-JNK levels in IMR-90 fibroblasts determined by cytometric bead array following treatment with 10mM NAC for 8hrs. Final levels of phospho-JNK were normalized to total JNK and GAPDH. Levels were reported as U/ml ± S.E. of mean, n=5. (D) Analysis of young and old cells cultured at 3 or 21% oxygen following treatment with 20 μM of, the Sod-Catalase mimetic, FeTBAP. A representative immunoblot is shown. (E) MMP-1 analysis of aged cells treated with FeTBAP or its inactive analogue MesoTBAP. Data reported as relative densitometric intensity measure by NIH ImageJ and represented as mean ± S.E, n=3.

Fig. 6

SA increases in MMP-1 are associated with oxidative-depletion of MKP-1. Immunoblotting for (A) phospho-MKK-4 (n=3), and (B) total MKP-1 in young and old IMR-90 fibroblasts at 21% and 3% O₂ (n=2). Human GAPDH used as a loading control. Data reported as relative densitometric intensity measured by NIH ImageJ and error bars represent +/− S.E. of mean. (C) Young and old cells under 21% O₂ and (D) senescent cells grown at 21% and 3% O₂ were treated with or without 10μM of proteasome inhibitor MG132 overnight followed by immunoprecipitation for MKP-1. Samples were electrophoresed and immunoblotted for anti-ubiquitin using an antibody that detects both mono- and polyubiquitinated forms of the protein.

Fig. 7

Lentiviral mediated over-expression of MKP-1 suppresses senescence associated MMP-1 expression. (A) Immunoblotting for MMP-1 and MKP-1 in young and old IMR-90 fibroblasts at 21% O₂ following transient infection with lentiviruses expressing MKP-1. Cells were also infected with lentivirus expressing the phosphatase dead MKP-1 that has a single amino-acid mutation C258S (MKP-1 C→S ). As control, cells were infected with viruses expressing LacZ (empty vector). Human GAPDH was used as loading control for MKP-1. MMP-1 monitored as described in Fig 1. Representative blot shown (n=3). (B) Real Time RT-PCR of MMP-1 in IMR-90 cells following infection with lentiviruses expressing MKP-1 and MKP-1 C→S. Data was normalized to β-actin RNA levels and reported as DNA units ± S.E. of mean (n=3). (C) Phospho-JNK levels determined by cytometric bead array in IMR-90 fibroblasts following infection with lentiviruses expressing MKP-1 and mutant MKP-1. Phospho-JNK levels were normalised to total JNK and GAPDH. Levels were reported as U/ml ± S.E. of mean, n=2. (D) Schematic for the redox and senescence associated regulation of MMP-1 expression.