Activation of heat shock transcription factor 1 in atherosclerosis

Abstract

Previous work established that increased expression of heat shock proteins (HSPs) in the vessel wall might evoke proinflammatory and autoimmune reactions in the pathogenesis of atherosclerosis. The present study was designed to further scrutinize the molecular mechanisms of HSP expression involving activation of heat shock transcription factors (HSFs) in atherosclerotic lesions in animal models. Severe atherosclerotic lesions developed in the aortas of rabbits 16 weeks after feeding a 0.2% cholesterol diet. When protein extracts from the aortas were subjected to Western blot analysis, the level of HSF1 in proteins from atherosclerotic lesions of hypercholesterolemic rabbits were significantly higher than those of normal vessels. Gel mobility shift assays revealed the formation of protein-heat shock element complexes containing HSF1 in protein extracts from atherosclerotic lesion. Furthermore, triglyceride-rich lipoprotein, oxidized-triglyceride-rich lipoprotein, low-density lipoprotein, and oxidized low-density lipoprotein did not activate HSF1 in cultured smooth muscle cells, whereas HSF1 was highly activated in cells treated with tumor necrosis factor-alpha. Interestingly, mechanical stretching of smooth muscle cells resulted in HSF1 translocation from the cytoplasm to the nucleus and hyperphosphorylation followed by increased HSP70 expression. Thus, our findings provide the first evidence that HSF1 is activated and highly expressed in atherosclerotic lesions and that cytokine stimulation and disturbed mechanical stress to the vessel wall may be responsible for such activation.

Figure 1.

Blood cholesterol levels and atherosclerotic lesions in rabbits. a: The values of serum cholesterol were measured every 2 or 4 weeks using an enzymatic procedure. Note that serum cholesterol levels in rabbits fed a 0.2% cholesterol diet were significantly higher than in those receiving the chow diet. b and c: H&E-stained sections from animals receiving chow (b) or cholesterol-enriched diets (c) for 16 weeks. Arrows indicate internal elastic lamina.

Figure 2.

Elevated HSF1 proteins in atherosclerotic lesions. Intima and media (I/M) from chow-fed rabbits and media (M) and atherosclerotic intimal lesions (AS) from cholesterol-fed rabbits (16-week diet) were dissected on ice from the remaining adventitia with tweezers and scissors. Tissues were frozen in liquid nitrogen and homogenized with a polytron homogenizer. a: Protein extracts (50 μg/lane) were separated on 7% sodium dodecyl sulfate-polyacrylamide gel, transferred onto membranes, and probed with the antibody against HSF1 (top) and anti-actin antibodies after stripping (bottom). Immunocomplexes were visualized by a Western blot detection kit. Each lane represents an individual animal. The molecular weight of HSF1 and actin is ∼90 and 39 kd, respectively. Graphic data show mean ± SD obtained from five rabbits per group. *, Significant difference from the intima/media, P < 0.001. b: Protein extracts (10 μg/lane) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel, transferred onto membranes, and probed with the antibody against HSF1. Immunocomplexes were visualized by a Western blot detection kit.

Figure 3.

Gel mobility shift assays for protein extracts from atherosclerotic lesions. Intima and media (I/M) from chow-fed rabbits and media (M) and atherosclerotic intimal lesions (AS) from cholesterol-fed rabbits (16-week diet) were dissected and protein extracts were prepared as described in the Materials and Methods. a: Gel mobility shift assay performed using protein extracts (20 μg protein per lane) from I/M of control animals, atherosclerotic lesions (AS), or media (M). Arrowhead indicates protein-DNA-binding complexes; NS, nonspecific binding. b: Extracts obtained from atherosclerotic lesions were incubated with a radiolabeled oligonucleotide containing a HSF-binding site (HSE) with no addition (−), in the presence of unlabeled NF-κB, or unlabeled HSE, oligonucleotides (50:1). c: Protein extracts obtained from the media (M) or atherosclerotic lesions (AS) were incubated with a radiolabeled oligonucleotide containing a HSF-binding site in the presence of antibodies (1:20, Ab) specific to HSF1. Supershifted DNA-binding complexes indicative of HSF1 protein-containing complexes are indicated by the arrow. Each lane on the blot (a) represents an individual animal.

Figure 4.

TRLP, remnant lipoprotein, oxidized TRLP, LDL, and oxidized LDL do not activate HSF-DNA binding. a: Gel mobility shift assay for protein extracts from cultured SMCs. Rat SMCs were treated with TRLP, remnant lipoprotein, oxidized TRLP (100 μg/ml), LDL, and oxidized LDL (100 μg/ml) at 37°C for 1 hour. The cells were washed and harvested with cold Tris-buffered saline. Nuclear proteins were prepared as described in Materials and Methods and protein concentration was determined with a Bio-Rad assay. Protein extracts (5 μg per lane) were incubated with a radiolabeled HSE oligonucleotide on ice for 20 minutes. The gel mobility shift assay was performed in 4% gel. Arrowhead indicates specific HSE-binding complexes. b: Western blot analysis. SMCs were treated with TRLP, remnant lipoprotein, oxidized TRLP, LDL, and oxidized LDL (μg/ml) at 37°C for 12 hours. Protein extracts (50 μg/lane) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel, transferred onto a membrane, and probed with the antibody against HSF1. Immunocomplexes were visualized by a Western blot detection kit. c: Western blot analysis for ERK1/2. Protein extracts from cultured SMCs treated with lipoproteins were separated on 15% sodium dodecyl sulfate-polyacrylamide gel, transferred onto a membrane, and probed with the antibody against phospho-ERK1/2 and pan-ERK1/2. Blots were stripped between antibody incubations. Immunocomplexes were visualized by a Western blot detection kit. Ctl⁻, negative control without treatment; CtL⁺, SMCs treated at 42°C for 30 minutes.

Figure 5.

TNF-α- and mechanical stretch-activated HSF1 in SMCs. a: Gel mobility shift assay of protein extracts from SMCs treated with TNF-α (50 ng/ml) or H₂O₂ (200 μmol/L) at 37°C for 1 hour. b: Gel mobility shift assay of protein extracts from SMCs treated with mechanical stretch (1 Hz) for the indicated times. c: Western blot analysis for HSF1 using nuclear proteins (left) and cell proteins (right) of SMCs treated with mechanical stretch (1 Hz) 37°C for 1 hour. d: Western blot analysis for HSP70 of SMCs treated with mechanical stretch (1 Hz) 37°C for the indicated times. Ctl⁻, negative control without treatment; CtL⁺, SMCs treated at 42°C for 30 minutes.