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Cardiovascular Research 2007 74(2):262-269; doi:10.1016/j.cardiores.2007.01.011
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Copyright © 2007, European Society of Cardiology

Downregulation of smooth muscle {alpha}-actin expression by bacterial lipopolysaccharide

Nathan Sandbo, Sebastien Taurin, Douglas M. Yau, Steven Kregel, Richard Mitchell and Nickolai O. Dulin*

Department of Medicine, The University of Chicago, Chicago, IL 60637, United States

* Corresponding author. Section of Pulmonary and Critical Care Medicine, the University of Chicago Department of Medicine, 5841 S. Maryland Ave., MC 6076, Chicago, IL 60637, United States. Tel.: +1 773 702 5198. Email address: ndulin{at}medicine.bsd.uchicago.edu

Received 10 December 2006; revised 12 January 2007; accepted 15 January 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Smooth muscle {alpha}-actin (SMA) is a cytoskeletal protein characteristic to vascular smooth muscle cells (VSMC), and it serves to facilitate cell contraction and migration. Bacterial lipopolysaccharide (LPS), a major mediator of septic shock secondary to infection, is known to directly affect VSMC. The objective of this study was to investigate the effect of LPS on the expression levels of SMA in VSMC.

Methods: This study was performed on cultured VSMC derived from human aorta, human coronary artery, or rat aorta.

Results: We show that SMA expression in VSMC, induced by endothelin-1 (ET1) or transforming growth factor-β (TGF-β), is potently inhibited by a LPS. This parallels a decreased migration of VSMC after LPS treatment. Downregulation of SMA by LPS is not a result of altered signaling of ET1 or TGF-β receptors, and it is not mediated by canonical (for LPS) mechanisms, such as production of prostaglandins or nitric oxide, or secretion of other endocrine factors. On a molecular level, downregulation of SMA expression by LPS occurs at the level of transcription, as both SMA mRNA levels and SMA promoter activity are inhibited by LPS. The SMA promoter is controlled largely by two major regulatory elements–CArG boxes activated by serum response factor (SRF), and TGF-β control elements (TCE). LPS does not affect the activity of SRF, but it potently inhibits both basal and inducible TCE activation.

Conclusion: We show for the first time that LPS attenuates SMA transcription and protein expression in VSMC likely through inhibition of a TCE element on the SMA promoter.

KEYWORDS Endotoxins; Endothelins; Gene expression; Signal transduction; Smooth muscle

Abbreviations: COX, cylcooxygenase • ET1, endothelin-1 • ERK, extracellular signal regulated protein kinase • HASMC, human aortic smooth muscle cells • HCASMC, human coronary artery smooth muscle cells • iNOS, inducible nitric oxide synthase • KLF4, Kruppel-like factor 4 • L-NAME, NG-nitro-L-arginine methyl ester • LPS, lipopolysaccharide • PKA, protein kinase A • RPA, ribonuclease protection assay • SMA, smooth muscle {alpha}-actin • SRF, serum response factor • TCE, transforming growth factor β control element • TGF-β, transforming growth factor β • TLR4, toll-like receptor 4 • VSMC, vascular smooth muscle cells • WKY, Wistar–Kyoto • YY1, Yin Yang 1


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Vascular smooth muscle cells (VSMC) maintain elevated levels of contractile proteins, such as smooth muscle {alpha}-actin (SMA) due, in part, to the effects of local mediators, such as endothelin-1 (ET1)[1] and transforming growth factor β1 (TGF-β1)[2]. Increased levels of smooth muscle {alpha}-actin and other contractile proteins serve to facilitate normal cellular functions in smooth muscle cells, such as contraction and migration. Various stimuli have been shown disrupt contractile protein expression in vivo and in vitro, with disruption of normal vascular smooth muscle function [3]. Altered vascular smooth muscle contractile gene expression is thought to play a role in the pathogenesis of atherosclerosis, and may be important in other vascular diseases as well.

Bacterial lipopolysaccharride (LPS) is an essential component of the bacterial wall of pathogenic gram negative bacteria and is known to be an important mediator in septic shock secondary to infection [4]. It is known that LPS mediates its vasodilator effects, in part, by altering the contractile function of smooth muscle via upregulation of inducible nitric oxide sythase [5,6] and prostaglandins E2/I2 production [7,8]. Recently, several studies have suggested that low level endotoxemia may also play a role in mediating the vascular injury seen in atherosclerosis[9]. Coincident with this information, other investigators have demonstrated the ability of vascular smooth muscle cells to express TLR4 and CD14, and show signaling and gene expression responses to very low levels of LPS [10]. Given the importance of lipopolysaccharide in mediating septic shock and atherosclerosis, we asked whether LPS may affect the expression of contractile genes, such as smooth muscle actin (SMA).

SMA expression is typically controlled at the level of transcription through two major regulatory elements–CArG boxes activated by serum response factor (SRF), and transforming growth factor TGF-β-control elements (TCE) [11,12]. In this study, we show for the first time that LPS potently and dose-dependently inhibits SMA expression at the level of transcription by a mechanism involving inhibition of TCE but not SRF.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture
The rat aortic smooth muscle cells (RASMC) derived from Wistar–Kyoto (WKY) rat aortas were characterized previously [15]. The cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml streptomycin, 250 ng/ml amphotericin B, and 100 U/ml penicillin. The cells were serum deprived using DMEM containing 0.2% calf serum and 2 mM L-glutamine. Primary cultures of human aortic smooth muscle cells (HASMC) and human coronary artery smooth muscle cells (HCASMC) were purchased from Clonetics, and grown per the manufacture's protocol, all experiments were carried out on culture cells between passage 5–10. Serum starvation and all stimulations were performed in DMEM containing 0.1% bovine serum albumin (BSA).

The research protocol of this study was approved by the Institutional Animal Care and Use Committee of the University of Chicago. The investigation also conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 DNA and reagents
The luciferase reporter plasmids for smooth muscle {alpha}-actin promoter (–125 base pairs) and for SRF (SRE.L [13]) were used previously [14,15]. The TCE-reporter plasmid (pTARE) was from Stratagene. The antibodies were from the following sources: anti-SM-{alpha}-actin and anti-β-actin, were from Sigma; anti-SMAD2, anti-phospho SMAD2 (Ser-465/467), anti-ERK1/2, anti-phospho ERK1/2 from Cell Signaling and anti-SRF were from Santa Cruz Biotechnology. Endothelin-1, human recombinant transforming growth factor β-1 (TGF-β), NG-nitro-L-arginine methyl ester (L-NAME), NS-398, SC-560, and bacterial lipopolysaccharide (E. coli O111:B4) were from EMD biosciences. Indomethacin was from Sigma.

2.3 Transient transfection of DNA
Transient transfections were performed using LipofectAMINE-PLUS reagent (Invitrogen) following the manufacturer's recommendations. Cells were incubated with DNA–liposome complexes in FBS-free and antibiotic-free DMEM for 3 h, followed by FBS supplementation to a final concentration of 10% and incubation for additional 6 h. The cells were then starved in DMEM containing 0.1% BSA overnight. Stimulations with desired agonists were carried out in DMEM containing 0.1% BSA.

2.4 Western blotting
Following stimulation of quiescent cells with desired agonists, cells were lysed in RIPA buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 1 mM NaF, 200 µM Na-orthovanadate and protease inhibitors (1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM PMSF). The lysates were cleared from insoluble material by centrifugation at 20,000xg for 10 min, boiled in Laemmli buffer, subjected to polyacrylamide gel electrophoresis and analyzed by Western blotting with desired primary antibodies followed by HRP-conjugated secondary antibodies (Calbiochem), and developed by enhanced chemiluminescence reaction (Pierce).

2.5 Luciferase assay
Cells seeded at equal density and grown in 24-well plates were co-transfected with 100 ng/well desired luciferase reporter plasmid and 10 ng/well promoterless renilla luciferase plasmid. Cells were serum starved overnight following transfection, and then treated with lipopolysaccharide the following day. After 24 h of incubation in LPS, cells were stimulated with the desired agonists for 6–24 h, washed with PBS, and lysed in protein extraction reagent. The lysates were assayed for firefly and renilla luciferase activity using the Promega Dual luciferase assay kit (Promega, Madison, WI). In order to account for differences in transfection efficiency, firefly luciferase activity of each sample was normalized to renilla luciferase activity and expressed as fold of control (unstimulated cells).

2.6 Non-radioactive in vitro assay for PKA activity [14]
Following stimulation with desired agonists, the cells (grown in 12-well plates) were lysed in 0.15 ml/well lysis buffer containing 25 mM HEPES (pH 7.5), 0.5% NP-40, protease inhibitors (1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM PMSF), and phosphatase inhibitors (1 mM NaF, 200 µM Na-orthovanadate). The lysates were cleared from insoluble material by centrifugation at 20,000xg for 10 min, and 5 µl cleared lysates were subjected to a kinase reaction with the fluorescence-labeled PKA substrate, kemptide (Promega), following the manufacturer's protocol. The reaction was stopped by boiling the samples for 10 min. The phosphorylated kemptide was separated from non-phosphorylated kemptide by 0.8% agarose electrophoresis. The fluorescent images were taken by Luminescent Image Analyzer LAS-3000 (Fujifilm).

2.7 RNase protection assay (RPA)
Cellular RNA was prepared by an acid guanidinium–thiocyanate–phenol–chloroform extraction method using RNA-STAT-60 (Tel-Test "B" Inc., Friendswood, TX) from rat aortic smooth muscle cell cultures. Samples were quantified for RNA content. Ribonuclease protection assay was performed with [32P]uridine triphosphate-labeled antisense RNA synthesized from rat SMA template using the RiboquantTM transcription and RPA kits (BD Biosciences). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) hybridization signals were used for normalization control between different RNA samples.

2.8 Chromatin immunoprecipitation assay
The chromatin immunoprecipitation (ChIP) assay was performed using the corresponding kit (Upstate Biotechnology Inc.). Immunoprecipitation of SRF was performed using anti-SRF antibodies (sc-335X, Santa Cruz Biotechnology Inc.). The DNA bound to SRF was eluted from the immune complex and amplified by PCR using the primers flanking CArG-B and CArG-A elements of the rat smooth muscle {alpha}-actin promoter (5'-AGCAGAGCAGAGGAATGCAGTGGAAGAGACCC-3' and 5'-CCCTCCCACTCGCTTCCCAAACAAGGAGC-3'). PCR products were separated by a 1.5% agarose gel electrophoresis and visualized by SYBR safe DNA staining (Invitrogen).

2.9 Wound migration assay
Details of this method [16] have been previously published. Briefly, small circular wounds were made in the confluent monolayer with a rubber stylet, and the wound closure was measured 36 h after wound creation. Microscope images were photographed using a digital camera attached to a Nikon Diaphot inverted-stage microscope, and images were assembled using Photoshop 7.0 program. Analysis of perimeter length and area of the remaining wound in each image was performed using ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD). Values were normalized to time 0 values. Intra-operator variance was <1% for wounds of 1.5 mm2, and inter-operator variance was <3%.

2.10 Statistical analysis
Quantitative data were analyzed by the Student's t test and values of p<0.05 were considered as statistically significant. The data represent the results of at least three experiments and are expressed as the mean±SD.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 LPS inhibits smooth muscle {alpha}-actin expression and migration of VSMC
We have consistently observed that the expression of smooth muscle (SM) {alpha}-actin (SMA) induced by a commercial "SM-differentiation" media (Clonetics) is attenuated by pretreatment with bacterial lipopolysaccharides (LPS), both in primary cultured human aortic smooth muscle cells (HASMC), or human coronary artery smooth muscle cells (HCASMC) (Fig. 1A, B). To understand the molecular mechanism by which LPS inhibits the SMA expression, as well as the functional significance of this effect, we then used the rat aortic smooth muscle cells (RASMC) that we have previously characterized {Davis, 2003 #12}, as a model for a dynamic and profound SMA expression response to a single agonist (as opposed to a non-defined "SM-differentiation" media). Fig. 1C shows a profound induction of SMA expression in RASMC by endothelin-1 (ET1) that is inhibited by LPS in a dose-dependent manner. Downregulation of SMA by LPS was accompanied by reduced migration of cells as assessed by wound assay (Fig. 1D). By contrast, proliferation of cells was not affected by LPS (Fig. 1E).


Figure 1
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  		Fig. 1 LPS inhibits smooth muscle {alpha}-actin expression and migration of VSMC. (A–C) Serum starved primary cultured human aortic smooth muscle cells (A), human coronary artery smooth muscle cells (B) or rat aortic smooth muscle cells RASMC-WKY (C) were incubated with indicated concentrations of LPS for 24 h and then stimulated with commercial "SM-differentiation media" (A, B) or with 100 nM ET1 (C) for an additional 24 h. Cells were lysed, and equal amounts of protein were analyzed by Western blotting with antibodies against smooth muscle {alpha}-actin (SMA) and β-actin. Shown are the representative images from at least three independent experiments. (D) Serum starved RASMC-WKY cells were incubated with LPS (1 µg/ml) or vehicle for 24 h, a circular wound was then introduced, followed by stimulation with serum. Wound area measurements were obtained at 0 and 36 h as described in Methods. Shown are means±SD for migration distances of 4 independent wounds per condition (*p<0.05). (E) Serum starved RASMC-WKY cells were incubated with or without LPS (1 µg/ml) for 24 h followed by stimulation with serum. 3H-thymidine thymidine incorporation assay was then performed as described in Methods.

 
3.2 Downregulation of SMA by LPS is not mediated by altered ET1 receptor signaling, or by cyclooxygenases, or by nitric oxide
It is known that downregulation of receptors to vasoactive agonists can occur in response to LPS [17,18] resulting in reduced downstream signaling. We addressed this possibility by measuring ERK1/2 and AKT phosphorylation–established VSMC responses to ET1 [15]. As shown in Fig. 2A, ET1-induced phosphorylation of either ERK1/2 or AKT was unaffected by LPS pretreatment, suggesting that the ET1 signaling is preserved. Likewise, since common mechanisms of LPS action include the induction of nitric oxide synthase (iNOS), and/or cyclooxygenase-2 (COX2), we examined their possible role by using pharmacological inhibitors. If these enzymes mediate downregulation of SMA by LPS, then the corresponding inhibitor should reverse this effect. Fig. 2B shows that pretreatment of cells with specific COX-1 or COX-2 inhibitors, sc-560 and ns-398, respectively, or with the non-selective COX1/2 inhibitor, indomethacin, did not restore the induction of SMA expression by ET1 in the presence of LPS. The efficacy of these inhibitors was confirmed by their inhibition of ET1-induced PKA activity (Fig. 2B, lower panel), which is stimulated via an endocrine mechanism involving the synthesis of prostaglandins (our unpublished data). Similarly, pre-treatment with the iNOS inhibitor L-NAME, also did not reverse the effect of LPS (Fig. 2C). This suggests that neither NO, nor the prostaglandin pathways are involved in downregulation of SMA by LPS. Finally, we addressed the possibility of other endocrine factors (secreted upon LPS stimulation) that may affect SMA expression. After a 24-h treatment, LPS was thoroughly washed out, followed by stimulation with ET1. As shown in Fig. 2D, ET1 failed to stimulate SMA expression even when LPS was washed out. This suggests that downregulation of SMA by LPS occurs not through an endocrine mechanism, but via intracellular changes in the cells.


Figure 2
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  		Fig. 2 Downregulation of SMA by LPS is not mediated by altered ET1 receptor signaling, by cyclooxygenases, or by nitric oxide. (A) Serum starved RASMC-WKY cells were incubated with LPS (1 µg/ml) or vehicle for 24 h followed by stimulation with 100 nM ET1 for 5 min. Cells were then lysed and the cell lysates were analyzed by Western blotting with phospho-specific or total antibodies against ERK1/2 or AKT as indicated. (B, C) Serum starved RASMC-WKY cells were pretreated with the cyclooxygenase inhibitors SC-560 (300 nM), NS-398 (10 µM), and indomethacin (20 µM) or with NOS inhibitor L-NAME (100 µM) for 30 min, followed by 24 h treatment with LPS (1 µg/ml) and subsequent stimulation of cells with ET1 for 24 h (except in panel B, bottom blot, where stimulation with ET1 was performed for 5 min). Cells were then lysed and cell lysates were analyzed for SMA or β-actin expression by western blotting with corresponding antibodies, or for PKA activity using kemptide as a substrate (B, bottom blot). (D) Serum starved cells were pretreated with LPS for 24 h, then the media were either washed out or left in place. Cells were then stimulated with 100 nM ET1 for an additional 24 h. Lysates were analyzed for SMA and β-actin. Shown are the representative images from at least three independent experiments.

 
3.3 LPS inhibits SMA gene transcription
To examine whether the downregulation of SMA by LPS occurs at transcriptional level, we first measured SMA mRNA levels by RNAase protection assay (RPA). As shown in Fig. 3A, ET1-induced SMA transcript levels reach a peak at 6 h, which is in accord with previously published reports of SMA transcriptional responses to other vasoactive agonists. Pretreatment with 1 µg/ml LPS abolishes SMA transcript induction, without affecting the levels of GAPDH (Fig. 3). We then examined the effect of LPS on the activity of a minimal SMA promoter (–125 base pairs), which is sufficient for induction by ET1 [15,19]. As shown in Fig. 3B, LPS pretreatment attenuated –125-SMA promoter activity. Together, these data indicate that LPS downregulates SMA expression at a transcriptional level through inhibition of SMA promoter activity.


Figure 3
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  		Fig. 3 LPS inhibits SMA gene transcription. (A) Serum starved RASMC-WKY cells were pretreated with LPS (1 µg/ml) or vehicle for 24 h, stimulated with ET1 or vehicle for 1, 3, and 6 h and lysed followed by subsequent RNA extraction. Cellular RNA was probed for the expression of SMA and GAPDH using ribonuclease protection assay (RPA). (B) VSMC were transfected with luciferase reporter constructs for –125 base pairs of SM-{alpha}-actin promoter (–125 SMA-Luc) together with promoterless renilla luciferase plasmid. Cells were serum starved for 24 h, preincubated with LPS (1 µg/ml) for 24 h, followed by stimulation with 100 nM ET1 for 6 h. Luciferase activity in cell lysates was then measured, normalized to renilla luciferase activity and expressed as fold of control (mean±SD from three independent experiments performed in quadruplicates, *p<0.05).

 
3.4 LPS does not affect SRF activity
It is well established that serum response factor (SRF) is a key transcription factor regulating smooth muscle specific gene expression, and that its transactivation and binding to its consensus cis-elements, the CArG boxes, are sufficient to convey induction of genes which contain these elements. The –125 SMA promoter contains two CArG boxes that are required for a promoter induction by vasoactive agonists [12]. To determine whether regulation of SMA promoter by LPS occurs through inhibition of SRF, we first utilized an SRF luciferase reporter, SRE.L [13]. Fig. 4A shows a profound induction of SRF activity by ET1 after 6 h of stimulation. This effect was not inhibited (but rather slightly potentiated) by preincubation with LPS. To confirm this, we examined the SRF binding to SMA promoter in the context of the chromatin microenvironment, by using a chromatin immunoprecipitation (ChIP) assay. As shown in Fig. 4B, SRF binding to a 151 bp region of the proximal SMA promoter (containing both CArG boxes) was significantly increased upon a 30-min ET1 stimulation, and LPS pretreatment had no effect. Together, these data convincingly demonstrate that LPS downregulates SMA expression, but not via inhibition of SRF.


Figure 4
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  		Fig. 4 LPS does not affect SRF activity. (A) RASMC-WKY cells were transfected with cDNA for SRF-luciferase reporter (SRE.L) together with promoterless renilla luciferase plasmid, Cells were serum starved for 24 h, preincubated with LPS (1 µg/ml) for 24 h, followed by stimulation with 100 nM ET1 for 6 h. Luciferase activity in cell lysates was then measured, normalized to renilla luciferase activity and expressed as fold of control (mean±SD from three independent experiments performed in quadruplicates). (B) RASMC-WKY cells were grown to confluence, serum starved, and treated with LPS 1 µg/ml or vehicle for 24 h, followed by stimulation with 100 nM ET1 or vehicle for 30 min. Cells were then cross-linked with formaldehyde and chromatin immunoprecipation (ChIP) assay was performed with antibodies against SRF or with normal IgG and primers against smooth muscle {alpha}-actin promoter as described in Methods. Shown are the representative data from three independent experiments.

 
3.5 LPS inhibits the activity of TGF-β-control element (TCE) without affecting TGF-β signaling
In addition to two CArG boxes, –125 SMA promoter contains TGF-β-control element (TCE), that is also required for inducible SMA gene transcription [11]. Therefore, we examined if LPS affects TCE activity, using TGF-β as an agonist. As shown in Fig. 5, LPS greatly inhibited both SMA protein expression (Fig. 5A), and –125 SMA promoter activation (Fig. 5B) in response to TGF-β. To assess the effect of LPS on TCE activity, we used the TCE-luciferase reporter (pTARE, Stratagene). As shown in Fig. 5C, LPS pretreatment drastically reduced both basal and TGF-β-induced activity of TCE reporter. Importantly, TGF-β-induced SRF activation was not inhibited by LPS (Fig. 5D), as in case ET1 stimulation (Fig. 4A). Finally, LPS had no effect on TGF-β-induced SMAD2 phosphorylation (Fig. 5E), suggesting that TGF-β receptors and immediate signaling are preserved upon LPS pretreatment. Together, these results suggest that downregulation of SMA by LPS may occur through inhibition of the TCE within the SMA promoter, at the level downstream or distinct from SMAD phosphorylation.


Figure 5
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  		Fig. 5 LPS inhibits the activity of TGF-β-control elements (TCE) without affecting TGF-β signaling. (A) VSMC were starved and incubated with 1 µg/ml LPS for 24 h, followed by stimulation with TGF-β (3 ng/ml) or vehicle for an additional 24 h, with subsequent analysis of cell lysates for SM-{alpha}-actin and β-actin expression by Western blotting. (B–D) VSMC were transfected with luciferase reporter constructs for the activities of –125 SM-{alpha}-actin promoter (B), of TCE (C), or of SRE.L (D) together with promoterless renilla luciferase plasmid, as described in Methods. Cells were serum starved for 24 h, preincubated with LPS (1 µg/ml) for 24 h, followed by stimulation with 3 ng/ml TGF-β for 24 h. Luciferase activity in cell lysates was then measured, normalized to renilla luciferase activity and expressed as fold of control (mean±SD from three independent experiments performed in quadruplicates, *p<0.05). (E) Serum starved VSMC were incubated with LPS (1 µg/ml) or vehicle for 24 h followed by stimulation with 3 ng/ml TGF-β for varying time intervals, and cell lysates were analyzed by Western blotting with antibodies against SMAD2 or phospho-SMAD2. Shown are the representative images from at least three independent experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We show for the first time that bacterial lipopolysaccharide attenuates SMA expression in vascular smooth muscle cells. SMA is regarded as a critical marker for smooth muscle cell differentiation, and is a component of the contractile apparatus. Decreased levels of SMA expression correlate with diminished cellular contractile function, as SMA knockout mice have hypocontractile aortas [20]. Thus, it is possible that decreased actin expression may contribute to the hypocontractility seen with exposure to LPS ex vivo [21], or in disease such as that seen in septic shock [4]. Other investigators have shown the ability of vascular smooth muscle cells to respond to LPS with cytokine production [9], which likely contributes to the local vascular dysfunction seen in atherosclerosis and sepsis. However, no one to date has shown alteration in contractile gene expression, which is the hallmark of normal differentiated vascular smooth muscle cells.

Interestingly, this effect of LPS is not due to its immediate signaling, as short term preincubation (1 h) with LPS does not appear to inhibit SMA expression induced by ET1 or TGF-β (data not shown). Rather, a delay, likely due to LPS-induced expression of some intracellular factors, is required. Also, downregulation of SMA by LPS is probably not mediated by canonical (for LPS) mechanisms, such as production of prostaglandins or nitric oxide, or secretion of other endocrine factors (Fig. 2). Similarly, LPS preconditioning does not alter immediate receptor mediated signaling by ET1 (Fig. 2A).

More importantly, we found that LPS inhibits SMA gene transcription (Fig. 3) without altering the activity of SRF (Fig. 4), but rather through inhibition of TGF-β-control elements, TCE (Fig. 5). TGF-β has emerged as a powerful inducer of SM-gene transcription through activation of both TCE and CArG elements, as mutation of either of them abrogates the effect of TGF-β [11]. Our data (i) confirm that TGF-β stimulates both TCE and CArG elements, and (ii) suggest that TGF-β-induced activation of TCE, but not of SRF is inhibited by LPS pretreatment (Fig. 5C, D). Importantly, TGF-β-induced SMAD2 phosphorylation was not affected by LPS, suggesting that TGF-β signaling is preserved after LPS pretreatment. This is in accord with our notion (see above) that the increased (or decreased) expression of intracellular factors (perhaps regulators of transcription) is required for this effect of LPS. SMA transcription may be regulated by Yin Yang 1 (YY1) through repression of either SRF [22] or of SMAD [23]; or by Kruppel-like factor GKLF/KLF4 through regulation of TCE [24]. Identification of the TCE-regulatory cofactors that are modulated by LPS is the goal of our future studies.


   Acknowledgements
 
This study was supported by NIH grant HL071755 (NOD) and American Heart Association postdoctoral fellowship awards (NS, ST).


   Notes
 
Time for primary review 28 days


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

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S. Taurin, N. Sandbo, D. M. Yau, N. Sethakorn, and N. O. Dulin
Phosphorylation of {beta}-catenin by PKA promotes ATP-induced proliferation of vascular smooth muscle cells
Am J Physiol Cell Physiol, May 1, 2008; 294(5): C1169 - C1174.
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