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Cardiovascular Research 2006 69(1):66-75; doi:10.1016/j.cardiores.2005.07.004
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Copyright © 2005, European Society of Cardiology

Small interfering RNA knocks down heat shock factor-1 (HSF-1) and exacerbates pro-inflammatory activation of NF-{kappa}B and AP-1 in vascular smooth muscle cells

Yu Chen and R. William Currie*

Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5

* Corresponding author. Tel.: +1 902 494 3343; fax: +1 902 494 1212. Email address: wcurrie{at}dal.ca

Received 29 March 2005; revised 15 June 2005; accepted 5 July 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: Heat shock and elevated expression of heat shock proteins suppress activation of the pro-inflammatory transcription factor NF-{kappa}B. We hypothesized that knocking down the expression of heat shock factor-1 (HSF-1) with RNAi technology would exacerbate angiotensin (Ang) II-induced inflammatory injury in vascular smooth muscle cells (VSMC).

Methods: Rat aorta A10 cells and human intestinal smooth muscle cells were grown without transfection or with transfection with HSF-1 small interfering RNA (siRNA), or negative control siRNA. Cells were stimulated with Ang II (100 nM) to activate the NF-{kappa}B signaling pathway.

Results: HSF-1 siRNA significantly knocked down HSF-1 expression, and one of the downstream heat shock proteins (Hsp), Hsp27, in both cells lines. HSF-1 siRNA also affected cells stressed with heat shock or Ang II treatment. Ang II induced activation of NF-{kappa}B and AP-1 in untransfected VSMCs, however, Ang II induced significantly higher activities of these pro-inflammatory transcription factors in HSF-1 siRNA transfected cells. Control siRNA had no apparent effect on HSF-1 and Hsp27 expression and Ang II-induced NF-{kappa}B and AP-1 activation.

Conclusions: These data indicate that the knock down of HSF-1 exacerbates Ang II-induced inflammation in VSMCs, and suggests that heat shock proteins protect against inflammatory injury by suppression of pro-inflammatory transcription factors such as NF-{kappa}B and AP-1.

KEYWORDS HSF-1; Heat shock proteins; Angiotensin II; NF-{kappa}B; AP-1; Inflammation

See Editorial by A.A. Knowlton (pages 7–8) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Heat shock proteins (Hsps) are a family of highly conserved molecular chaperones that maintain protein solubility, cytoskeletal function and cellular homeostasis, and protect cells against environmental stress and pathophysiological injuries [1–3]. In rats, whole body heat shock (42 °C for 15 min) induces significantly increased levels of Hsp70 in heart, and this is associated with improved cardiac functional recovery after global ischemia [4]. Transgenic mice expressing high levels of the human inducible Hsp70 demonstrated the direct role of Hsp70 in the protection of the myocardium and hippocampal neurons from ischemia injury [5,6]. Recently, heat shock treatment, with induced high levels of Hsp70 and Hsp27, was shown to protect against angiotensin (Ang) II-induced hypertension and inflammation in aorta and heart [7,8]. However, whether the elevated levels of Hsps or other changes in gene expression are directly regulating the inflammatory intracellular injury pathways is still an open question.

The various heat shock genes are regulated by heat shock transcript factors (HSFs) that are constitutively expressed [9]. At present, four different HSFs have been identified, i.e., HSF-1, HSF-2, HSF-3, and HSF-4, each with distinct characteristics. HSF-1 regulates the transcription of Hsp genes after environmental or pathophysiological stress that denature or precipitate cellular proteins. HSF-2 and HSF-3 regulate the transcription of Hsp genes in specific tissues, undergoing processes of differentiation and development, and HSF-3 has only been described in chicken [9,10]. HSF-4 is structurally related to the other three HSFs, however, it is functionally distinct and does not respond to stresses like heat [11]. Furthermore, the transcription of Hsp70, Hsp90, and Hsp27 genes are reduced when HSF-4 is overexpressed [11]. It has been suggested that HSF-4 is not a transcription factor because it lacks the carboxy-terminal repeat shared by HSF-1, HSF-2 and HSF-3 [11]. HSF-1 is the major stress responsive HSF mediating the heat shock response in mammalian cells [12]. HSF-1 is constitutively localized in the cytosol and nucleus and is maintained in an inactivated form by binding with the constitutive 70 kDa heat shock protein (Hsc70) or Hsp90. Upon various forms of cellular stress such as heat, hypoxia, ethanol, and sodium arsenite that denature proteins, the constitutive Hsps are recruited to the denatured proteins, freeing HSF-1. Free HSF-1 becomes phosphorylated and shifts from the cytosolic to nuclear compartment and becomes organized in active DNA-binding homotrimers [9].

In the present study, we used RNA interference (RNAi) technology to knock down the expression of the HSF-1 gene in vascular smooth muscle cells (VSMC). We hypothesized that (1) the knock down of HSF-1 would also disrupt the downstream gene expression of Hsps, and (2) the knock down of HSF-1 would exacerbate Ang II-induced inflammatory injury in VSMCs. Such evidence would support the notion that heat shock proteins play a direct role in suppressing Ang II-induced inflammatory signaling pathways and subsequent inflammation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture
VSMC cell line derived from fetal rat aorta (A10 cells) was obtained from the American Type Culture Collection (ATCC, Rockville, MD, Cat # CRL 1476). Human intestinal smooth muscle cells (HISM cells) were also obtained from ATCC (Cat # CRL 1692), and are thought to be a VSMC cell line [13]. The cells were cultured routinely in Dulbecco's modified Eagle's medium (DMEM) containing 10% of fetal bovine serum (FBS), penicillin (50 U/ml) and streptomycin (50 µg/ml) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.

2.2 RNAi production and transfection
HSF-1 siRNA was chemically synthesized by the Ambion company (Austin, TX). The method of Elbashir et al. [14] was used to determine the target sequence, as following: 5'-AAGAGAAAGATCCCTCTGATG-3'. The targeted sequence resides within the open reading frame of the rat HSF-1 gene (accession: xm_343270), 613 nucleotides downstream of the start codon. Negative control siRNA, a 21-nucleotide RNA duplex with no known sequence homology, was also purchased from Ambion (Cat # 4611).

Transfection of siRNA into cells was achieved by using oligofectamine reagent (Invitrogen, Burlington, ON). Briefly, 2 x 104 cells/well were plated into 24-well plates overnight to achieve 50–70% confluent monolayers. On the day of transfection, cultured cells were washed with DMEM without serum or antibiotics. Twenty µM stock oligonucleotide and oligofectamine were diluted and mixed gently with DMEM to achieve the final concentration of oligofectamine (manufacturer recommendation, 2 µl/well) and 1 x and 2 x for siRNA (1 x concentration was 133 nM). The oligonucleotide–oligofectamine complexes were then added to each well and the cells were incubated at 37 °C for 4 h. FBS was then added to the cells to achieve the final concentration to 10% in DMEM and incubated for 44 h at 37 °C in the CO2 incubator.

2.3 MTT assay for determining cell viability
After siRNA transfection or sham treatment, culture medium was changed to normal growth medium with 450 µl DMEM with 10% FBS. The tetrazolium salt MTT (Sigma, St. Louis, MI, Cat # M2128) 50 µl, 5 mg/ml in PBS, was added to the cells at a final concentration of 0.5 mg/ml and cells were incubated at 37 °C for 4 h. MTT was converted to formazan crystals by viable cells. Formazan crystals were dissolved in 500 µl solubilizing buffer (0.1 M HCl and 10% SDS) by incubating cells at 37 °C for 1 h. The absorbance was read at 562 nm in a microplate reader, FLx800 (Bio-Tek instrument Inc., Winooski, VT). The viability of the cells was expressed as a percentage of sham-treated cells.

2.4 Angiotensin II and heat shock treatment
After 48 h of siRNA transfection, both transfected and non-transfected (sham) cells were cultured for 1 day in serum-free DMEM media to arrest growth before Ang II treatment or HS treatment. Human Angiotensin II (Sigma, Cat # A9525) was added at a concentration of 100 nmol/L. For heat shock treatment, after the growth arrest, cells were incubated at 44 °C for 20 min in an incubator and recovered at 37 °C. Cells were collected for Hsp27, NF-{kappa}B and AP-1 examination at different time points after angiotensin II or HS treatment.

2.5 Immunocytochemistry
For immunocytochemistry, cells were incubated with rabbit polyclonal anti-HSF-1 antibody (1:2000, StressGen, Victoria, Canada, Cat # SPA-901) or rabbit polyclonal anti-Hsp27 antibody (1:2000, StressGen, Cat # SPA-801). The primary HSF-1 and Hsp27 antibodies were reacted with a secondary antibody, goat anti-rabbit conjugated to alexa-546 (1:500, Molecular Probes, Eugene, OR). Digital images were obtained with a Zeiss Axiovert 200 and AxioCam HRc camera and image capture system.

2.6 Preparation of protein extracts
Proteins from the various conditions described above were harvested and pooled from 6 wells of 24-well plates and all protein extraction procedures were performed on ice. Cells were rinsed with cold 0.1 M PBS and lysed with 50 µl ice-cold buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 1 mM 1,4-dithiothreitol (DTT), 0.5 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1% Triton X-100]. After a 10-min incubation on ice, the homogenates were centrifuged at 4000 g for 4 min at 4 °C. The supernatants were stored as cytoplasmic extracts at –70 °C. The pellets were resuspended in 40 µl buffer B [20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin]. The suspension was incubated for 30 min on ice. After centrifuging at 20,000 g for 15 min at 4 °C, the supernatants were transferred in aliquots to new tubes and stored at –70 °C until analyzed. Protein concentrations were determined by the method of Lowry et al. [15].

2.7 Western analysis
Protein samples (10 µg) were used for Western analysis following the method by Chen et al. [7,8]. Blots were reacted with either anti-HSF-1 antibody (1:5000), anti-Hsp27 antibody (1:5000), or rat monoclonal anti-HSF-2 antibody (1:5000, StressGen, Cat # SPA-960) followed by appropriate peroxidase-conjugated secondary antibody (1:2000, Vector Laboratories, Burlingame, CA) incubation. The level of HSF-1, HSF-2, and Hsp27 was determined by enhanced chemiluminescence system (Amersham Pharmacia) according to the manufacturer's protocol and then exposed to films or detected using the Storm system (Storm 840, Amersham Biosciences, Sunnyvale, CA). Protein levels were quantified by scanning densitometry using image-analysis systems (Scion Corp, Frederick, ML). Equal amounts of protein loading/lane were checked by amido black staining or Western blotting with rabbit polyclonal anti-actin antibody (1:200, Sigma, Cat # A2066).

2.8 Electrophoretic mobility shift assay (EMSA)
NF-{kappa}B and AP-1 consensus oligonucleotide sequences (5'-AGTGAGGGACTTTCCCAGGC-3' and 5'-CGCTTGATGAGTCAGCCGGAA-3', respectively) (Promega, Madison, WI) were used for EMSA following the protocol previously described by Chen et al. [7,8]. Briefly, binding reactions, each containing 10 µg of nuclear extracts, were separated on a nondenaturing 4% acrylamide gel. Gels were dried onto Whatman 3 MM paper and exposed to X-ray film at –70 °C overnight. To establish the specificity of the reaction, competition assays with 50 x excess of unlabeled specific competitor oligonucleotides and non-specific competitor oligonucleotide, SP-1 (5'-ATTCGATCGGGGCGGGGCGAGC-3') (Promega), were performed by adding unlabeled probes 10 min before the addition of the labeled probe. Negative controls contained no nuclear extracts. For supershift assays, 2 µg of anti-p65 (sc-109 X, Santa Cruz Biotechnology, Santa Cruz, CA), anti-p50 (sc-114 X, Santa Cruz Biotechnology), and anti-c-Fos (sc-52, Santa Cruz Biotechnology), antibodies were added to nuclear protein extracts and incubated for 1 h after the addition of the labeled probe. The DNA-binding activities were quantified by scanning densitometry using image-analysis systems (Scion Corp).

2.9 Statistical analysis
Data are expressed as mean ± SEM. The significance of differences was determined by ANOVA and post hoc multiple comparison test by using SPSS 11.5 software (SPSS, Chicago, IL). P<0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 RNAi knocks down the expression of HSF-1 and Hsp27 in unstressed A10 cells
Immunocytochemistry to detect the expression of HSF-1 in A10 VSMCs (Fig. 1A–E) revealed that there was a significant knock down effect of HSF-1 siRNA. In untransfected (sham) A10 cells, HSF-1 was mainly localized in nuclei and diffusely distributed in cytoplasm (Fig. 1A). To rule out the effect of the transfection itself, cells were transfected with a control siRNA at 1 x and 2 x dosage. There was no apparent effect of the control siRNA transfection on HSF-1 expression (Fig. 1B, C). With the HSF-1 siRNA transfection, the expression of HSF-1 decreased in a dose-dependent manner (Fig. 1D, E). The transfection efficiency was ~90%. Treatment of cells with siRNA transfection suppressed cell viability to about 70% of that of the sham-treated cells (P<0.05) (Fig. 1F). However, there was no significant difference between the control siRNA and HSF-1 siRNA treatment at both 1 x and 2 x dosages.


Figure 1
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  		Fig. 1 HSF-1 siRNA knocks down the expression of HSF-1 and down stream Hsp27 in A10 cells. (A) Sham-treated cells had mainly nuclear and diffused cytoplasm expression of HSF-1. (B, C) Cells treated with control siRNA at 1 x and 2 x dosages, respectively, had no apparent difference compared to sham-treated cells. (D and E) Cells treated with HSF-1 siRNA at 1 x and 2 x dosages, respectively. The HSF-1 expression appeared to be knocked down in a dose-dependent manner. (F) Cell viability determined with MTT assay showed that siRNA transfection suppressed cell viability, however, there was no significant difference between transfection treatments. (G) Western analysis confirmed HSF-1 RNAi knock down effect on expression of HSF-1, and also revealed that HSF-1 siRNA (2 x dosage) suppressed the expression of Hsp27. (H) Semi-quantification of Western analysis. Relative levels of Hsp90, Hsp70, and Hsp27 were normalized to actin. Data are mean ± SEM and are representative of 3 separate experiments. *P<0.05, **P<0.01 vs. sham. Scale bar in E=100 µm in panels (A, B, C, D and E).

 
The Western analysis also confirmed that the level of HSF-1 was decreased with the RNAi treatment. The anti-HSF-1 antibody recognized one 85 kDa protein in cytoplasm and both 85 kDa and 95 kDa proteins in the nucleus, corresponding to the molecular masses of the inactive and activated forms of HSF-1, respectively, on SDS–PAGE immunoblots (Fig. 1G). HSF-1 siRNA with 2 x dosage decreased HSF-1 levels in both the cytoplasm and nucleus, however, HSF-1 siRNA with 1 x dosage only decreased the nuclear level of HSF-1 (Fig. 1G, H). The effect of HSF-1 RNAi on one of the HSF-1 down stream heat shock proteins, Hsp27, was also examined. HSF-1 siRNA knocked down the expression of Hsp27 in A10 cells at 2 x dosage (Fig. 1G, H). HSF-1 siRNA had no effect on either HSF-2 or actin levels (Fig. 1G).

3.2 RNAi knocks down the expression of HSF-1 and Hsp27 in unstressed HISM cells
HSF-1 was mainly localized in nuclei in unstressed HISM cells. Sham and control siRNA treatment resulted in no apparent difference on the HSF-1 expression (Fig. 2A, B). Both dosages of HSF-1 siRNA knocked down the expression of HSF-1 in HISM cells in both nuclei and cytoplasm (Fig. 2C, D). In normal HISM cells, Hsp27 was localized mainly in the nucleoplasm and lightly in the cytoplasm (Fig. 2E). The control siRNA had no apparent effect on Hsp27 expression in HISM cells (Fig. 2F). Interestingly, HSF-1 siRNA treatment at 1 x dosage decreased the nuclear expression of Hsp27 (Fig. 2G), and 2 x dosage decreased both the nuclear and cytoplasmic expression of Hsp27 (Fig. 2H).


Figure 2
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  		Fig. 2 HSF-1 siRNA knocks down the expression of HSF-1 and Hsp27 in HISM cells. (A) Sham-treated cells show positive nuclear-cytoplasm expression of HSF-1. (B) Control siRNA 1 x -treated cells show no apparent difference compared to sham-treated cells. (C, D) Cells treated with HSF-1 siRNA at 1 x and 2 x dosage show cytoplasm and nuclear expression of HSF-1 has been knocked down in a dose-dependent manner. (E) Sham-treated cells show positive nuclear-cytoplasm expression of Hsp27. (F) Control siRNA 1 x -treated cells show no apparent difference compared to sham-treated cells. (G, H) Cells treated with HSF-1 siRNA at 1 x and 2 x dosage show nuclear and cytoplasm expression of Hsp27 has been knocked down in a dose-dependent manner. Scale bar in panels (D and H)=100 µm in all.

 
3.3 RNAi knocks down the expression of Hsp27 in heat shock or Ang II-treated A10 cells
To investigate the effect of RNAi in stressed cells, HSF-1 siRNA transfected A10 VSMCs were treated with heat shock or Ang II. HSF-1 siRNA significantly knocked down Hsp27 expression (P<0.05) in non-HS (0) and HS-treated cells (Fig. 3A). In untransfected (sham) A10 cells, HS treatment had only minimal effect on Hsp27 expression, i.e., Hsp27 was increased only at 12 h after HS treatment when compare to the non-HS (0)-treated cells. Compared to untransfected (sham) cells, control siRNA transfection had no apparent effect on Hsp27 expression.


Figure 3
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  		Fig. 3 Western blot analysis of Hsp27 in (A) heat shock or (B) angiotensin II-treated A10 cells. (A) A10 cells were untransfected (sham) or transfected with control siRNA 1 x, or HSF-1 siRNA 2 x for 2 days followed by 1 day of serum-free incubation before the HS treatment. Cells were harvested for analysis without HS treatment (0) or at 6, 12, 24, or 48 h after HS treatment. HS treatment resulted in a modest increase in Hsp27 expression only at 12 h after HS treatment. HSF-1 siRNA transfection significantly suppressed Hsp27 expression in non-HS-treated (0) and HS-treated cells with 6 to 48 h of recovery. Compared to sham, control siRNA transfection had no apparent effect on the expression of Hsp27. (B) A10 cells were untransfected (sham), or transfected with control siRNA 1 x or HSF-1 siRNA 2 x for 2 days followed by 1 day of serum-free incubation before Ang II treatment. Cells were harvested for analysis without Ang II treatment (0) or after 0.25, 1, 2, 6, or 24 h of Ang II treatment. In untransfected cells (sham), Ang II treatment increased Hsp27 expression at 1 to 6 h. HSF-1 siRNA transfection significantly suppressed Hsp27 expression in non-Ang II-treated (0) and cells-treated Ang II for 0.25 to 24 h. Compared to sham, control siRNA transfection had no apparent effect on Hsp27 expression. *, **P<0.05 and 0.01 vs. sham without HS nor Ang II treatment. #, ##P<0.05 and 0.01 HSF-1 siRNA treated vs. sham or control siRNA treated at each time point. The expression of Hsp27 was normalized to actin levels and the experiments were repeated three times.

 
HSF-1 siRNA significantly knocked down Hsp27 expression (P<0.05) in non-Ang II and Ang II-treated cells (Fig. 3B). In untransfected (sham) A10 cells, Ang II treatment for 1 to 6 h significantly increased Hsp27 expression. Compared to untransfected (sham) cells, control siRNA transfection had no apparent effect on Hsp27 expression.

3.4 The effect of HSF-1 expression knock down on Ang II-induced activation of NF-{kappa}B
In untransfected (sham) cells, Ang II treatment induced significant activation of NF-{kappa}B by 6 and 24 h of treatment (P<0.05) in both A10 and HISM cells (Fig. 4). In HSF-1 siRNA transfected cells, the DNA-binding activity of NF-{kappa}B induced by Ang II was significantly higher (P<0.05) at 6 h and 24 h of Ang II treatment compared to that of untransfected (sham) cells (Fig. 4). There was no apparent difference in the control siRNA transfected cells compared to the untransfected (sham) cells without Ang II treatment or with Ang II treatment for 6 h.


Figure 4
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  		Fig. 4 Effect of knock down of HSF-1 expression on Ang II-induced nuclear factor (NF)-{kappa}B activation. (A) A10 cells. (B) HISM cells. Ang II induced activation of NF-{kappa}B in both A10 and HISM cells after Ang II treatment for 6 and 24 h. Ang II induced significantly higher activation of NF-{kappa}B in HSF-1 siRNA 2 x knocked down cells. Control siRNA 1 x had no apparent effect on Ang II-induced NF-{kappa}B activation. Competition assay confirmed the specificities of the complexes for NF-{kappa}B. The complexes of NF-{kappa}B were compromised by adding an antibody against p65 and p50 in A10 and were supershifted in HISM cells. *, **P<0.05 and 0.01 vs. sham without Ang II treatment, respectively. #, ##P<0.05 and 0.01 HSF-1 siRNA treated vs. sham or control siRNA treated, respectively. Data are representative of 3 separate experiments.

 
3.5 The effect of HSF-1 expression knocked down on Ang II-induced activation of AP-1
In untransfected (sham) A10 cells, Ang II treatment for 6 and 24 h also induced activation of AP-1 (Fig. 5A). Although RNAi treatment did not cause significantly higher activity of AP-1 at 6 h compared to that of the sham, HSF-1 siRNA treatment doubled Ang II-induced AP-1 activation at 24 h. In untransfected (sham) HISM cells, Ang II only induced activation of AP-1 after 24 h of treatment (Fig. 5B). In contrast, in HSF-1 siRNA transfected cells, Ang II induced significantly elevated activation of AP-1 at both 6 and 24 h of treatment (P<0.01). In addition, HSF-1 siRNA treatment significantly increased (P<0.01) AP-1 activation after 6 and 24 h on Ang II treatment compared to the untransfected (sham) cells. There was no apparent difference in the control siRNA transfected cells compared to the untransfected (sham) cells without Ang II treatment or with Ang II treatment for 6 h.


Figure 5
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  		Fig. 5 Effect of knock down of HSF-1 expression on Ang II-induced AP-1 activation. (A) A10 cells. Ang II induced activation of AP-1 after treatment for 6 and 24 h. Ang II induced significantly higher activation of AP-1 in HSF-1 siRNA 2 x knocked down cells at 24 h of treatment (P<0.05). (B) Ang II only activated AP-1 at 24 h in sham-treated HISM cells, however, Ang II activated AP-1 at significantly higher level at both 6 and 24 h of treatment in HSF-1 siRNA 2 x -treated cells (P<0.05). Control siRNA 1 x had no apparent effect on Ang II-induced AP-1 activation. Competition assay confirmed the specificity of the complexes for AP-1. *, **P<0.05 and 0.01 vs. sham without Ang II treatment, respectively. ##P<0.01 HSF-1 siRNA treated vs. sham or control siRNA treated. Data are representative of 3 separate experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study, we found that HSF-1 siRNA specifically knocked down HSF-1 expression in two cell lines, A10 and HISM. The suppression of HSF-1 expression by HSF-1 RNAi subsequently suppressed the constitutive and inducible expression of one heat shock protein, Hsp27. This suppression of HSF-1 and Hsp27 expression caused increased activation of NF-{kappa}B and AP-1 in VSMCs after Ang II stimulation of inflammatory signaling pathways.

This study used HSF-1 siRNA to knock down the expression of HSF-1 in A10 and HISM cells. This siRNA had no apparent effect on HSF-2 or actin expression and on cell morphology. The negative control siRNA had no effect on HSF-1 or Hsp27 expression. This suggests that RNAi is a specific and effective approach to suppress expression of HSF-1. Because the amounts of oligofectamine were the same (2 µl/well) and there was no apparent difference between negative control siRNA treatments at 1 x and 2 x dosages on HSF-1 expression and cell viability (Fig. 1), as well as HSF-2 expression (data not shown), control siRNA at 1 x dosage were used as controls in the rest of the experiments. In this study, Hsp27 expression was suppressed in the HSF-1 siRNA-treated cells, however, the possibility of other Hsps being decreased has not been ruled out.

In this study, HSF-1 siRNA not only suppressed Hsp27 in unstressed cells, it also suppressed Hsp27 levels in HS or Ang II-treated cells (Fig. 3). Consistent with van de Klundert et al. [16], HS treatment had only minimal effect on Hsp27 expression in untransfected A10 cells. However, in HSF-1 siRNA transfected cells, HS treatment increased Hsp27 levels at 12 and 24 h of recovery. Hsp27 levels declined after 48 h of recovery from HS treatment in HSF-1 siRNA transfected cells. The HS treatment appears to temporarily counteract the HSF-1 siRNA effect at 12 and 24 h of recovery, and the HS effect appears to dissipate at 48 h.

How heat shock proteins are regulating inflammation is not entirely clear. Heat shock proteins are highly conserved stress proteins that protect cells and organisms from stress. Their induction is rapid and correlates with the induction of tolerance to high temperature and other stresses in a wide variety of cells and organism [17]. One function of heat shock proteins is to protect cells from damaged and non-functional proteins. Heat shock proteins identify stress-damaged polypeptides and target them for degradation through intrinsic proteolytic pathways [17] or reactivate stress-damaged proteins through the molecular chaperone role of Hsps such as Hsp70, Hsp60, Hsp90 and Hsp27 [17]. Under stressful conditions, constitutive Hsps dissociate from HSFs, are recruited to damaged or denatured proteins, and facilitate the refolding, assembly and stabilization of the denatured proteins [1,18].

Evidence is also accumulating that Hsps can interact with and regulate cell signaling pathways. For example, Hsp70 inhibits stress-induced apoptosis by suppressing c-Jun NH2-terminal kinase (JNK) [19] and procaspase-9 [20]. Hsp27 is cytoprotective against apoptosis by regulating caspase-3 [21] and cytochrome c-dependent activation of procaspase-9 [22].

In addition to regulating apoptotic signaling pathways, Hsps also appear to regulate inflammatory signaling transduction pathways. The Hsp60 family has been found to induce macrophages to secrete pro-inflammatory mediators such as TNF-{alpha}, IL-6, IL-12 and nitric oxide [23,24]. In contrast, other studies have demonstrated an anti-inflammatory role of Hsps. Overexpression of Hsp70 accelerates mouse recovery after endotoxic challenge [25]. In HSF-1 deficient (HSF–/–) mice expressing low levels of constitutive Hsps, greater amounts of pro-inflammatory cytokines are expressed and the mice are more susceptible to endotoxin-mediated lethality compared with wild-type mice [26]. In autoimmune diseases, nasal administration of Hsp60 180–188 peptides induces highly effective protection against adjuvant-induced arthritis through generation of regulatory T cells [27]. Mycobacterial Hsp10 [28] and Hsp70 [29] have also been found to suppress adjuvant-induced arthritis. Transgenic overexpression of Hsp70 protects against ischemia/reperfusion injury in heart [5,30] and brain [6,31].

Heat shock treatment with the concomitant high levels of Hsp70 and Hsp27 and their phosphorylation also protects against Ang II-induced cardiovascular inflammation by suppressing the pro-inflammatory NF-{kappa}B pathway [7,8]. Our work [7,8] and others [32,33] have found that HS treatment suppresses NF-{kappa}B activation by inhibiting the phosphorylation and degradation of its inhibitor, I{kappa}B-{alpha}. HS treatment also inhibits I{kappa}B kinase (IKK) activation [33] and decreases the cytoplasmic level of IKK-{alpha} [8]. HS treatment also leads to IKK complex insolubilization [34]. Interestingly, Hsp70 and Hsp27 have been found to interact directly with NF-{kappa}B, I{kappa}B-{alpha}, IKK-{alpha}, and IKK-β in down-regulation of stimuli-induced NF-{kappa}B activation [35–37]. In addition, Hsp70 induced by HS treatment has been shown to suppress other pro-inflammatory pathways such as JNK and p38 [38,39]. While Hsps suppress pro-inflammatory pathways, their role may be more general, if they are found to also regulate anti-inflammatory pathways. Hsps could be differentially regulating pro-inflammatory and anti-inflammatory signaling pathways [40].

Here, we have shown that knocking down HSF-1 suppresses expression of Hsp27 and this exacerbates Ang II-induced inflammation by causing significantly higher activation of NF-{kappa}B and AP-1 in VSMCs. This provides strong evidence for a role of Hsps, and specifically Hsp27, suppressing Ang II-induced inflammatory injury.

In conclusion, the knock down of HSF-1 with subsequent suppression of Hsp27 exacerbates Ang II-induced inflammation in VSMCs, which suggests a new role for heat shock proteins in regulation of inflammation.


   Acknowledgements
 
The authors thank Cindee Leopold, Wei Jiang and Kay Murphy for their excellent technical assistance. We also thank Dr. Yongde Peng (Shanghai Jiaotong University Affiliated First People's Hospital) for his help on siRNA design. This work was funded by a grant from the Heart and Stroke Foundation of New Brunswick to R. William Currie. Yu Chen was supported by scholarships from the Killam Trusts and from Nova Scotia Health Research Foundation.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Hartl F.U. Molecular chaperones in cellular protein folding. Nature (1996) 381:571–579.[CrossRef][Medline]
  2. Polla B.S., Bachelet M. Stress proteins in inflammation. Ann N Y Acad Sci (1998) 851:75–85.[Free Full Text]
  3. Beere H.M. Stressed to death: regulation of apoptotic signaling pathways by the heat shock proteins. Sci STKE (2001) 31:RE1.
  4. Currie R.W., Karmazyn M., Kloc M., Mailer K. Heat-shock response is associated with enhanced postischemic ventricular recovery. Circ Res (1988) 63:543–549.[Abstract/Free Full Text]
  5. Plumier J.C., Ross B.M., Currie R.W., Angelidis C.E., Kazlaris H., Kollias G., et al. Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest (1995) 95:1854–1860.[ISI][Medline]
  6. Plumier J.C., Krueger A.M., Currie R.W., Kontoyiannis D., Kollias G., Pagoulatos G.N. Transgenic mice expressing the human inducible Hsp70 have hippocampal neurons resistant to ischemic injury. Cell Stress Chaperones (1997) 2:162–167.[CrossRef][ISI][Medline]
  7. Chen Y., Ross B.M., Currie R.W. Heat shock treatment protects against Angiotensin II-induced hypertension and inflammation in aorta. Cell Stress Chaperones (2004) 9:99–107.[CrossRef][ISI][Medline]
  8. Chen Y., Arrigo A.-P., Currie R.W. Heat shock treatment suppresses Angiotensin II-induced activation of NF-{kappa}B pathway and heart inflammation: a role for IKK depletion by heat shock? Am J Physiol (2004) 287:H1104–H1114.[ISI]
  9. Snoeckx L.H., Cornelussen R.N., Van Nieuwenhoven F.A., Reneman R.S., Van Der Vusse G.J. Heat shock proteins and cardiovascular pathophysiology. Physiol Rev (2001) 81:1461–1497.[Abstract/Free Full Text]
  10. Nakai A., Morimoto R.I. Characterization of a novel chicken heat shock transcription factor, heat shock factor 3, suggests a new regulatory pathway. Mol Cell Biol (1993) 13:1983–1997.[Abstract/Free Full Text]
  11. Nakai A., Tanabe M., Kawazoe Y., Inazawa J., Morimoto R.I., Nagata K. HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator. Mol Cell Biol (1997) 17:469–481.[Abstract]
  12. Rabindran S.K., Giorgi G., Clos J., Wu C. Molecular cloning and expression of a human heat shock factor, HSF1. Proc Natl Acad Sci U S A (1991) 88:6906–6910.[Abstract/Free Full Text]
  13. Mandal A.K., Puchalski J.T., Lemley-Gillespie S., Taylor C.A., Kohno M. Effect of insulin and heparin on glucose-induced vascular damage in cell culture. Kidney Int (2000) 57:2492–2501.[CrossRef][ISI][Medline]
  14. Elbashir S.M., Harborth J., Weber K., Tuschl T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods (2002) 26:199–213.[CrossRef][ISI][Medline]
  15. Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the folin phenol reagent. J Biol Chem (1951) 193:265–275.[Free Full Text]
  16. van de Klundert F.A., Gijsen M.L., van den IJssel P.R., Snoeckx L.H., de Jong W.W. Alpha B-crystallin and hsp25 in neonatal cardiac cells-differences in cellular localization under stress conditions. Eur J Cell Biol (1998) 75:38–45.[ISI][Medline]
  17. Parsell D.A., Lindquist S. The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet (1993) 27:437–496.[CrossRef][ISI][Medline]
  18. Rogalla T., Ehrnsperger M., Preville X., Kotlyarov A., Lutsch G., Ducasse C., et al. Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation. J Biol Chem (1999) 274:18947–18956.[Abstract/Free Full Text]
  19. Gabai V.L., Mabuchi K., Mosser D.D., Sherman M.Y. Hsp72 and stress kinase c-jun N-terminal kinase regulate the bid-dependent pathway in tumor necrosis factor-induced apoptosis. Mol Cell Biol (2002) 22:3415–3424.[Abstract/Free Full Text]
  20. Beere H.M., Wolf B.B., Cain K., Mosser D.D., Mahboubi A., Kuwana T., et al. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol (2000) 2:469–475.[CrossRef][ISI][Medline]
  21. Rocchi P., So A., Kojima S., Signaevsky M., Beraldi E., Fazli L., et al. Heat shock protein 27 increases after androgen ablation and plays a cytoprotective role in hormone-refractory prostate cancer. Cancer Res (2004) 64:6595–6602.[Abstract/Free Full Text]
  22. Garrido C., Bruey J.-M., Fromentin A., Hammann A., Arrigo A.-P., Solary E. HSP27 inhibits cytochrome c-dependent activation of procaspase-9. FASEB J (1999) 13:2061–2070.[Abstract/Free Full Text]
  23. Ohashi K., Burkart V., Flohe S., Kolb H. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol (2000) 164:558–561.[Abstract/Free Full Text]
  24. Flohe S.B., Bruggemann J., Lendemans S., Nikulina M., Meierhoff G., Flohe S., et al. Human heat shock protein 60 induces maturation of dendritic cells versus a Th1-promoting phenotype. J Immunol (2003) 170:2340–2348.[Abstract/Free Full Text]
  25. Paidas C.N., Mooney M.L., Theodorakis N.G., De Maio A. Accelerated recovery after endotoxic challenge in heat shock-pretreated mice. Am J Physiol (2002) 282:R1374–R1381.[ISI]
  26. Xiao X., Zuo X., Davis A.A., McMillan D.R., Curry B.B., Richardson J.A., et al. HSF1 is required for extra-embryonic development, postnatal growth and protection during inflammatory responses in mice. EMBO J (1999) 18:5943–5952.[CrossRef][ISI][Medline]
  27. Prakken B.J., Roord S., van Kooten P.J., Wagenaar J.P., van Eden W., Albani S., et al. Inhibition of adjuvant-induced arthritis by interleukin-10-driven regulatory cells induced via nasal administration of a peptide analog of an arthritis-related heat-shock protein 60 T cell epitope. Arthritis Rheum (2002) 46:1937–1946.[CrossRef][ISI][Medline]
  28. Agnello D., Scanziani E., Di G.M., Leoni F., Modena D., Mascagni P., et al. Preventive administration of Mycobacterium tuberculosis 10-kDa heat shock protein (hsp10) suppresses adjuvant arthritis in Lewis rats. Int Immunopharmacol (2002) 2:463–474.[CrossRef][ISI][Medline]
  29. Wendling U., Paul L., van der Zee R., Prakken B., Singh M., van Eden W. A conserved mycobacterial heat shock protein (hsp) 70 sequence prevents adjuvant arthritis upon nasal administration and induces IL-10-producing T cells that cross-react with the mammalian self-hsp70 homologue. J Immunol (2000) 164:2711–2717.[Abstract/Free Full Text]
  30. Marber M.S., Mestril R., Chi S.H., Sayen M.R., Yellon D.M., Dillmann W.H. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest (1995) 95:1446–1456.[ISI][Medline]
  31. Kelly S., Bieneman A., Horsburgh K., Hughes D., Sofroniew M.V., McCulloch J., et al. Targeting expression of hsp70i to discrete neuronal populations using the Lmo-1 promoter: assessment of the neuroprotective effects of hsp70i in vivo and in vitro. J Cereb Blood Flow Metab (2001) 21:972–981.[CrossRef][ISI][Medline]
  32. Pritts T.A., Wang Q., Sun X., Moon M.R., Fischer D.R., Fischer J.E., et al. Induction of the stress response in vivo decreases nuclear factor-kappa B activity in jejunal mucosa of endotoxemic mice. Arch Surg (2000) 135:860–866.[Abstract/Free Full Text]
  33. Yoo C.G., Lee S., Lee C.T., Kim Y.W., Han S.K., Shim Y.S. Anti-inflammatory effect of heat shock protein induction is related to stabilization of I kappa B alpha through preventing I kappa B kinase activation in respiratory epithelial cells. J Immunol (2000) 164:5416–5423.[Abstract/Free Full Text]
  34. Pittet J.F., Lee H., Pespeni M., O'Mahony A., Roux J., Welch W.J. Stress-induced inhibition of the NF-{kappa}B signaling pathway results from the insolubilization of the I{kappa}B kinase complex following its dissociation from heat shock protein 90. J Immunol (2005) 174:384–394.[Abstract/Free Full Text]
  35. Shimizu M., Tamamori-Adachi M., Arai H., Tabuchi N., Tanaka H., Sunamori M. Lipopolysaccharide pretreatment attenuates myocardial infarct size: A possible mechanism involving heat shock protein 70-inhibitory kappaBalpha complex and attenuation of nuclear factor kappaB. J Thorac Cardiovasc Surg (2002) 124:933–941.[Abstract/Free Full Text]
  36. Guzhova I.V., Darieva Z.A., Melo A.R., Margulis B.A. Major stress protein Hsp70 interacts with NF-{kappa}B regulatory complex in human T-lymphoma cells. Cell Stress Chaperones (1997) 2:132–139.[CrossRef][ISI][Medline]
  37. Park K.J., Gaynor R.B., Kwak Y.T. Heat shock protein 27 association with the I kappa B kinase complex regulates tumor necrosis factor alpha-induced NF-kappa B activation. J Biol Chem (2003) 278:35272–35278.[Abstract/Free Full Text]
  38. He H., Chen C., Xie Y., Asea A., Calderwood S.K. HSP70 and heat shock factor 1 cooperate to repress Ras-induced transcriptional activation of the c-fos gene. Cell Stress Chaperones (2000) 5:406–411.[CrossRef][ISI][Medline]
  39. Wang Y., Li C., Wang X., Zhang J., Chang Z. Heat shock response inhibits IL-18 expression through the JNK pathway in murine peritoneal macrophages. Biochem Biophys Res Commun (2002) 296:742–748.[CrossRef][ISI][Medline]
  40. Chen Y., Currie R.W. Heat shock treatment suppresses angiotensin II-induced SP-1 and AP-1 and stimulates Oct-1 DNA-binding activity in heart. Inflamm Res (2005) 54. [in press].

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