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, 284 (26), 17595-606

Enhanced Endothelial Cell Senescence by Lithium-Induced Matrix metalloproteinase-1 Expression

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Enhanced Endothelial Cell Senescence by Lithium-Induced Matrix metalloproteinase-1 Expression

Ian T Struewing et al. J Biol Chem.

Abstract

Endothelial cell (EC) senescence and dysfunction occurring after chronic injury and inflammation are highly associated with the development and progression of cardiovascular diseases. However, the factors involved in the establishment of EC senescence remain poorly understood. We have previously shown that lithium, an inhibitor of glycogen synthase kinase (GSK)-3beta and activator of the Wnt/beta-catenin signaling pathway, induces an EC senescent-like phenotype. Herein, we show that lithium induces a rapid and pronounced up-regulation of the matrix metalloproteinase (MMP)-1, an inflammation and senescent cell marker, at the mRNA and protein levels, whereas the induction of two other senescent cell markers is either weak (interleukin-8) or delayed (plasminogen activator inhibitor-1). Lithium effect on MMP-1 expression is also specific among other MMPs and not mediated by GSK3beta inhibition. Lithium affects MMP-1 expression mainly at the transcriptional level but neither the AP1/Ets regulatory sites nor the redox sensitive (-1607/2G) site in MMP-1 promoter are involved in lithium-dependent MMP-1 regulation. However, down-regulation of p53, a target of lithium in EC, dampens both basal and lithium-induced MMP-1 expression, which further links MMP-1 up-regulation with the establishment of cell senescence. Although increased MMP-1 levels are usually associated with angiogenesis in enabled proliferative EC, the exogenous addition of activated MMP-1 on lithium- arrested EC increases the number of EC positive for the senescent-associated-beta-galactosidase marker. Conversely, down-regulation of MMP-1 expression by small interfering RNAs blunts the lithium-dependent increase in senescent-associated-beta-galactosidase positive cells. Altogether our data indicate that lithium-induced MMP-1 may participate in the reinforcement of EC senescence and reveal a novel mechanism for lithium-induced tissue remodeling.

Figures

FIGURE 1.
FIGURE 1.
Lithium induces specifically in a time- and dose-dependent manner the expression of MMP-1 mRNA. In A, BAEC were plated 24 h prior to being treated with either 10 mm NaCl as control or 10 mm LiCl for 24 h. In B, BAEC were treated with either 10 mm NaCl or 10 mm LiCl for the indicated times. In all cases after cell harvest, the total RNAs were extracted and reverse transcribed. The levels of the target mRNAs were determined by real-time PCR as described under “Experimental Procedures” using rpL30 mRNAs for normalization. The results are expressed as the fold-change in target mRNA levels with NaCl-treated cells being equal to 1 (mean ± S. E. (error bars), n = 2–5 independent experiments). *, results were considered statistically different from sodium-treated controls at p < 0.05 (t test).
FIGURE 2.
FIGURE 2.
Lithium increases collagenase activities and MMP-1 protein levels in EC. A, BAECs were grown on DQ-FITC-collagen type I-coated slides for 24 h prior to being treated with either 10 mm NaCl or 10 mm LiCl for an additional 24-h period. The cells were then fixed and mounted in the presence of 4′,6-diamidino-2-phenylindole (DAPI) to stain nuclei. Representative immunofluorescence microscopy images are shown. B, BAECs were plated 24 h prior to being treated with either 10 mm NaCl or 10 mm LiCl for 36 h. The levels of MMP-1 were assessed in the conditioned media (CM) and whole cell extracts (WCE) by Western blotting. For the whole cell extracts, β-actin was used as internal control. The purified (Calbiochem-EMD) and recombinant (Chemicon) human MMP-1 were used as positive controls.
FIGURE 3.
FIGURE 3.
Lithium-induced MMP1 expression is independent of inositol depletion and GSK3β inhibition. A, BAEC were pretreated with or without 1 mm myo-inositol for 1 h prior to being treated with either 10 mm NaCl or 10 mm LiCl for 36 h. B, BAEC were treated with different inhibitors of GSK3β: 10 mm LiCl (Li), 1 mm sodium-valproate, or 5 μm indirubin for 36 h. C, BAEC were transfected with pCR3 as control, wild type-GSK3β-His (wt-GSK), the constitutively active S9A-GSK3β-His (S9A-GSK), kinase-dead K85R-GSK3β-His (K85R-GSK), or S37A-β-catenin-hemagglutinin (S37A-bcat) constructs for 36 h. D and E, 24 h after plating, BAEC were transfected with 35 nm of either siRNA-CT-1 or siRNA-GSK3β in standard culture medium without antibiotics for a total 48-h period. For the last 24 h the cells were treated with either 10 mm NaCl (−) or 10 mm LiCl (+) prior to being harvested either for whole cell lysis (D) or RNA extraction (E). In all cases (A–C and E), the total RNAs were extracted and reverse transcribed. The levels of the target mRNAs were determined by real-time PCR as described under “Experimental Procedures” using rpL30 mRNAs for normalization. The results are expressed as the fold-change in target mRNA levels with NaCl-treated cells being equal to 1 (mean ± S. E. (error bars), n = 3–5 independent experiments). *, results were considered statistically different from controls at p < 0.05 (t test). D, the levels of phospho-GSK3β and GSK3β were analyzed by Western blotting using β-actin as control. CT, control.
FIGURE 4.
FIGURE 4.
Lithium does not affect specifically MMP-1 mRNA stability. BAECs were transfected with the indicated 3′-UTR Renilla luciferase reporters in the presence of 5 ng of CMV-β-galactosidase vector for normalization purposes. Twenty-four hours after transfection, BAECs were treated with either 10 mm NaCl or 10 mm LiCl for an additional 24-h period prior to quantification of the Renilla luciferase and β-galactosidase activities. The graph represents the relative ratio of the normalized Renilla luciferase activity in lithium-treated cells versus sodium-treated cells (mean ± S. E. (error bars), n = 4–5 independent experiments).
FIGURE 5.
FIGURE 5.
Lithium-dependent transcriptional activation of MMP1 expression in EC does not involve proximal AP1 and distal AP1/Ets1 redox sensitive binding sites. A, BAECs and HEK293 cells were transfected with 5 ng of CMV-β-galactosidase vector for normalization purposes and with 125 ng of pGL3-basic as control, −600/+60-MMP1P-Luc, (1G)−2235/+60-MMP1P-Luc, or (2G)−2235/+60-MMP1P-Luc reporter constructs for 24 h prior to being treated with 10 mm NaCl, 10 mm LiCl, or 100 nm phorbol myristate acetate (PMA) as indicated for an additional 24-h period. The firefly luciferase and β-galactosidase activities were determined and the results are expressed as the fold-induction of the normalized luciferase activity in treated cells as compared with NaCl-treated control cells (mean ± S. E. (error bars), n = 4–5 independent experiments). B, BAECs and HEK293 cells were treated with either 10 mm NaCl or 10 mm LiCl for 36 h prior to being lysed for the analysis of Jun activation (phospho-S63-Jun) and Jun levels (c-Jun) by Western blotting. A representative experiment is shown using β-actin as normalization control. C, BAEC were treated with either NaCl or LiCl (10 mm) for the indicated times prior to staining with 2 μm CM-H2DCFDA for 30 min. BAEC were also challenged for the indicated times with 1 μg/ml lipopolysacchride as positive control. The fluorescence intensity was determined at λexc = 485 nm/λem = 535 nm (Fusion, PerkinElmer Life Sciences) and normalized for variations in cell number. The results are expressed as the relative levels of the normalized fluorescence intensity in treated versus control cells (mean ± S. E. (error bars), n = 6). *, results were considered statistically different from NaCl-treated control cells at p < 0.05 (t test)).
FIGURE 6.
FIGURE 6.
Down-regulation of p53 abrogates basal and lithium-induced MMP-1 expression. A and B, BAEC were transfected with 35 nm of either siRNA-CT-1 or siRNA-p53 for 24 h prior to being treated with either 10 mm NaCl (−) or 10 mm LiCl (+) for a 24-h period. A, after cell lysis, the levels of p53 and Bax, a p53 target, were analyzed by Western blotting using β-actin as control (CT). B, after RNA extraction and reverse transcription, the levels of the target mRNAs were determined by real-time PCR using rpL30 mRNAs for normalization. The results are expressed as the fold-change in target mRNA levels with NaCl-treated cells being equal to 1 (mean ± S. E. (error bars), n = 3–4 independent experiments). C, BAEC were co-transfected with either control (CT) or p53 siRNA duplexes (35 nm) and the indicated MMP-1-promoter luciferase reporters for 48 h prior to cell lysis and determination of the firefly luciferase and β-galactosidase activities. Treatments with either 10 mm NaCl or 10 mm LiCl were performed in the last 24 h of transfection. The graph represents the relative levels of normalized luciferase activity with si-Control equal to 1 for each of the reporters (mean ± S. E. (error bars), n = 3 independent experiments performed in duplicates). *, results were considered statistically different from NaCl-treated siRNA control-transfected cells at p < 0.05 (t test).
FIGURE 7.
FIGURE 7.
Activated MMP-1 increases lithium-induced SA-β-galactosidase marker and p21Cip expression. A, 24 h after plating, BAEC were treated with either 10 mm NaCl or 10 mm LiCl in the absence or presence of 5 nm activated MMP-1. Five days later, the cells were fixed and stained for SA-β-galactosidase activity. Representative phase-contrast color images are shown. The numbers of SA-β-galactosidase positive cells were counted as well as the total number of cells for each treatment and the results are presented in the graph (mean ± S. E. (error bars), n = 4–5 independent experiments performed in duplicates). *, results were considered statistically different from NaCl-treated control cells at p < 0.05 (one-way analysis of variance-Bonferoni post test). B, BAEC were treated as described in A for 36 h prior to RNA extraction and gene expression analysis by real-time PCR. *, results were considered statistically different from NaCl-treated control cells at p < 0.05 (t test).
FIGURE 8.
FIGURE 8.
Down-regulation of MMP-1 expression affects lithium-induced senescent cell markers. A–C, BAECs were transfected with 35 nm of the indicated siRNA duplexes for 24 h prior to being treated with either 10 mm NaCl or 10 mm LiCl for an additional 24-h period. Then, the conditioned media were collected for Western blot analysis of MMP-1 levels (B) and the cells were harvested for RNA extraction and real-time PCR analysis of the indicated target genes (A and C). D, for determining the SA-β-galactosidase activity, BAECs were transfected with the indicated siRNAs and treated with either 10 mm NaCl or 10 mm LiCl for 4 days. The graph represents the number of SA-β-galactosidase positive cells per 104 cells (mean ± S. E. (error bars), n = 4–5 independent experiments performed in duplicates). *, results were considered statistically different at p < 0.05 (t test).

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