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Effect of Salusin-ß on Peroxisome Proliferator-Activated Receptor Gamma Gene Expression in Vascular Smooth Muscle Cells and its Possible Mechanism

Background/Aims: salusin-ß is considered to be a potential pro-atherosclerotic factor. Regulation and function of vascular smooth muscle cells (VSMCs) are important in the progression of atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARγ) exerts a vascular protective role beyond its metabolic effects. Salusin-ß has direct effects on VSMCs. The aim of the present study was to assess the effect of salusin-ß on PPARγ gene expression in primary cultured rat VSMCs. Methods: Western blotting analysis, real-time PCR and transient transfection approach were used to determine expression of target proteins. Specific protein knockdown was performed with siRNA transfection. Cell proliferation was determined by 5-bromo-2'-deoxyuridine incorporation. The levels of inflammation indicators interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) were determined using enzyme-linked immunosorbent assay. Results: Salusin-ß negatively regulated PPARγ gene expression at protein, mRNA and gene promoter level in VSMCs. The inhibitory effect of salusin-ß on PPARγ gene expression contributed to salusin-ß-induced VSMCs proliferation and inflammation in vitro. IγBa-NF-γB activation, but not NF-γB p50 or p65, mediated the salusin-ß-induced inhibition of PPARγ gene expression. Salusin-ß induced nuclear translocation of histone deacetylase 3 (HDAC3). HDAC3 siRNA prevented salusin-ß-induced PPARγ reduction. Nuclear translocation of HDAC3 in response to salusin-ß was significantly reversed by an IγBa inhibitor BAY 11-7085. Furthermore, IγBa-HDAC3 complex was present in the cytosol of VSMCs but interrupted after salusin-ß treatment. Conclusion: IγBa-HDAC3 pathway may contribute to salusin-ß-induced inhibition of PPARγ gene expression in VSMCs.


Western blot and Immunoprecipitation
The whole cell lysate, nuclear, and cytoplasmic extracts were prepared by using commercial kits (Beyotime Co., Jiangsu, China) and protein concentration was determined using a BCA Protein Assay Reagent (Beyotime Co., Jiangsu, China) according to the manufacturer's instructions. Equal amounts of protein were separated and then electro-transferred to nitrocellulose membranes (Bio-Rad, USA). Membranes were incubated with the appropriate primary antibodies, and incubated in the dark with the appropriate IRDye 680RD secondary antibodies (1:15000). The blot was imaged and quantitated using the Odyssey infrared imaging system (LI-COR Biosciences, Inc.). To detect multiple signals from one membrane, the membrane was treated with a stripping buffer (Beyotime Co., Jiangsu, China) for 20 min at 37 °C before the next blotting.
For immunoprecipitation, cellular extracts were precleared with protein A/G-agarose (Santa Cruz, CA, USA) for 2h at 4°C. After preclearing, the lysate was incubated with antibody overnight at 4°C. The immune complexes were washed with immunoprecipitation buffer and resolved by SDS-PAGE. Proteins were detected by western blot using specific antibodies.

RNA isolation and real-time PCR
Quantitative real time PCR analysis was used to measure mRNA expression. Total RNA was extracted using Trizol reagent (Takara, Otsu, Shiga, Japan). RNA was added as a template to reverse-transcriptase reactions carried out using the PrimeScript RT Master Mix Kit (Takara, Otsu, Shiga, Japan). PCRs were carried out with the resulting cDNAs using the SYBR Green Premix (Takara, Otsu, Shiga, Japan) with ABI steponeplus real time pcr system (Applied Biosystems, Foster City, CA). The primers are quoted as follows: PPARγ 5'-ATT CTG GCC CAC CAA CTT CGG -3' (forward), 5'-TGG AAG CCT GAT GCT TTA TCC CCA -3' (reverse); 18S 5'-AGT CCC TGC CCT TTG TAC ACA-3' (forward), 5'-GAT CCG AGG GCC TCA CTA AAC-3' (reverse). Experimental cycle threshold (Ct) values were normalized to housekeeping gene 18S and relative mRNA expression was calculated versus a reference sample. Each sample was run and analyzed in triplicate.

Plasmid and transient transfection assays
The PPARγ promoter luciferase reporter plasmid pPPARγ-Luc contains the 5'-flanking region (-2333 bp) of the PPARγ gene promoter in a luciferase reporter plasmid, and was a gift from Yajun Zhou

Enzyme-linked immunosorbent assay (ELISA)
The levels of IL-6 and TNF-α in the VSMCs culture supernatants were measured with ELISA kits according to the manufacturer's instructions (Shanghai Westang Biotechnology Co. Ltd, Shanghai, China). The OD value of each well was measured at a 450 nm wavelength, and their concentrations were determined by interpolation of a standard calibration curve.
Cell proliferation was measured by 5-bromodeoxyuridine (BrdU)-incorporation assay using a cell proliferation ELISA kit (Roche Diagnostics, Mannheim, Germany).Briefly, after the incubation period, the cells were incubated with a monoclonal anti-BrdU antibody during the last 4 h incubation period, followed by a peroxidase-conjugated secondary antibody for 1.5 h and substrates for detection. BrdU incorporation into DNA was determined by a standard elisa reader.

Statistical analysis
Data were reported as mean ± SD. All values were analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison test using Graphpad Prism 5 software; P<0.05 was taken to indicate a significant difference between groups.

Salusin-β reduced PPARγ gene expression in VSMCs
To examine whether salusin-β regulates PPARγ gene expression in VSMCs, the cultured VSMCs were stimulated with or without salusin-β. Western blot analysis showed that 10 -7 mol/L salusin-β significantly reduced PPARγ protein expression in a timedependent manner (Fig. 1A). VSMCs were then stimulated with salusin-β at different doses (10 -9 -10 -7 mol/L) for 24h. A dose-dependent inhibitory effect of salusin-β on PPARγ expression at protein and mRNA levels was demonstrated in Fig. 1B, C. To further verify the role of salusin-β in PPARγ gene expression, VSMCs were transfected with a PPARγ promoter luciferase reporter plasmid pPPARγ-Luc and incubated with salusin-β for 24h. Measurement of luciferase activities demonstrated that salusin-β caused a dose-dependent reduction in PPARγ promoter activity (Fig. 1D). This result was consistent with that shown in Fig. 1B, C. Taken together, these results strongly suggested that salusin-β might inhibit PPARγ gene expression in primary cultured rat VSMCs.
IκBα-NF-κB activation, but not NF-κB p50 or p65, mediated salusin-β-induced inhibition of PPARγ gene expression Our previous study has shown that mitogen-activated protein kinases (ERK1/2, JNK, and p38) and AKT pathways can be activated by salusin-β in VSMCs [11] and other researchers reported that salusin-β facilitated NF-κB signals in aortic endothelial cells [8,9]. To determine which pathway is required for the salusin-β-induced inhibition of PPARγ gene expression, kinase-specific chemical inhibitors were used to evaluate each pathway. The result showed that pretreatment of VSMCs with a NF-κB inhibitor, BAY 11-7085 which suppresses IκBα phosphorylation and thus stabilizes NF-κB, attenuated salusin-β-induced reductions in PPARγ protein, mRNA expression and PPARγ promoter activity, whereas pretreatment with inhibitors of ERK1/2, JNK, p38 or AKT had no significant effect ( Fig Cellular Physiology and Biochemistry B, C). These findings suggested that it is possible that NF-κB pathway is involved in salusinβ-induced inhibition of PPARγ gene expression. We next confirmed that NF-κB pathway could be activated by salusin-β in VSMCs. Salusin-β-treatment of VSMCs resulted in robust phosphorylation and loss of cytoplasmic IkBa at early time period, between 15min and 60min, with robust accumulation of p50 and p65 in the nucleus (Fig. 2D). These results indicate that the salusin-β could activate NF-κB by classical pathway of phosphorylation and degradation of IκBα.
On the basis of the above observations, the role of the NF-κB pathway was further examined. We used RNA interference to knock down NF-κB p50 and NF-κB p65 gene expression in VSMCs. However, not as expected, silencing neither NF-κB p50 nor p65 impacted salusin-β-induced reduction in PPARγ protein (Fig. 3), mRNA expression and PPARγ promoter activity (data not shown).

HDAC3 was required for salusin-β-induced inhibition of PPARγ gene expression
HDACs are repressive transcriptional regulators and involved in various signaling pathways. Because class I members HDAC1, HDAC2 and HDAC3 are associated with both PPARγ and IkBα, we further analyzed the role of them in salusin-β-mediated reduction in PPARγ in VSMCs. Western blot analysis revealed salusin-β treatment had no effect on HDAC1,2 and 3 protein level in whole cell lysates (Fig. 4A). However, with salusin-β treatment, the HDAC3 level was significantly decreased in the cytoplasm, but increased in nuclear IκBα may mediate nuclear translocation of HDAC3 in response to salusin-β in VSMCs We next investigated whether BAY 11-7085 influences nuclear translocation of HDAC3 in response to salusin-β. VSMCs were pretreated with BAY 11-7085 for 1h before incubation with salusin-β for an additional 30min. As shown in Fig. 6A, salusin-β-induced IκBα phosphorylation, degradation and nuclear translocation of p50 and p65 were significantly reversed by BAY 11-7085. Furthermore, Bay11-7085 pretreatment caused evidently increased protein expression of HDAC3 in the cytoplasm and accordingly decreased protein expression of HDAC3 in the nuclear compartment (Fig. 6B). Thus, the nuclear translocation of HDAC3 may be a consequence of IκBα degradation. To test this possibility, the association of HDAC3 with IκBα was investigated using coimmunoprecipitation. Cytoplasmic extract of VSMCs were immunoprecipitated with either anti-IκBα or HDAC3 antibody. As expected, HDAC3 was found in the immunoprecipitation product of IκBα antibody, and IκBα was identified in the immunoprecipitation product of HDAC3 antibody strongly indicating that IκBα and HDAC3 coexist in the same protein complex in cells (Fig. 6C). However, treatment with salusin-β for 30min, IκBα was little detected in the immunoprecipitation product of HDAC3 (Fig. 6D), consisting with the observation that IκBα was degraded by salusin-β stimulation at early time (Fig. 2D). Taken together, these data suggest that IκBα may mediate nuclear translocation of HDAC3 in response to salusin-β in VSMCs.  5. Inhibition of salusin-β activity by HDAC inhibitor and HDAC siRNA. A-C, VSMCs were pretreated with HDAC Inhibitor TSA (1μmol/L) for 30min and further treated with or without 10 -7 mol/L salusin-β for 24h to detect PPARγ expression. A, Nuclear extracts were obtained and protein levels were measured by western blot analysis (n=3). B, mRNA levels were examined by Real-time PCR analysis (n=6). C, Transfection assay for analysis of PPARγ promoter activity (n=6). Data are expressed as means ± SD. *P< 0.05 versus the control with no treatment; # P< 0.05 versus 10 -7 mol/L salusin-β treatment alone. D, VSMCs were transfected with no siRNA (con), control siRNA (con siRNA), HDAC1 siRNA, HDAC2 siRNA and HDAC3 siRNA. HDAC1, HDAC2 and HDAC3 protein level were assessed 24h after transfection by western blot. Representatives were shown here from three independent experiments. E-G, VSMCs were transfected with control siRNA (con siRNA), HDAC1 siRNA, HDAC2 siRNA or HDAC3 siRNA for 24h, serum-starved for 24h, and treated with or without 10 -7 mol/L salusin-β for 24h to detect PPARγ expression. E, Nuclear extracts were obtained and protein levels were measured by western blot analysis (n=3). F, mRNA levels were examined by Real-time PCR analysis (n=6). G, Transfection assay for analysis of PPARγ promoter activity (n=6). Data are expressed as means ± SD. *P< 0.05 versus the con siRNA without salusin-β (the first column on the left); # P< 0.05 versus con siRNA with 10 -7 mol/L salusin-β treatment (the second column on the left).

Increased cellular levels of PPARγ protein or PPARγ ligands down-regulated salusin-βinduced VSMCs proliferation and inflammation
To directly assess the functional significance of salusin-β-induced PPARγ suppression, we tested whether increasing PPARγ expression antagonizes the proliferative and inflammatory effects of salusin-β in VSMCs. Transfection of VSMCs with 0.4μg of PPARγexpression plasmid significantly increased PPARγ protein expression (Fig. 7A). The result showed that increasing amounts of transfected PPARγ expression plasmid reduced salusinβ-induced VSMCs proliferation as indicated by BrdU incorporation into DNA in a dosedependent manner (Fig. 7B). Moreover, salusin-β could promote the release of inflammation indicators interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) from VSMCs, while this release was also inhibited in VSMCs transfected with the PPARγ expression plasmid (Fig. 7C  and D).

Discussion
In the present study, we examined PPARγ regulation by salusin-β in primary cultured rat VSMCs and investigated its possible mechanism. The findings of current study provided the first demonstration that salusin-β directly inhibited PPARγ gene expression at protein level, mRNA level and PPARγ gene promoter activity level. Furthermore, salusin-β activated IκBα may mediate nuclear translocation of HDAC3 in response to salusin-β in VSMCs. A and B, VSMCs were pretreated with BAY 11-7085 (BAY, 10μmol/L) for 1h and further treated with or without 10 -7 mol/L salusin-β for 30min. A, Cytoplasmic extracts (Cy) were obtained and the phosphorylated IκBα, IκBα and HDCA3 protein levels were measured by western blot analysis. B, nuclear extracts (Nu) were obtained and the nuclear p50, p65 and HDAC3 protein levels were measured by western blot analysis. Data are expressed as means ± SD (n=3). *P< 0.05 versus the control with no treatment; # P< 0.05 versus 10 -7 mol/L salusin-β treatment alone. C, association of IκBα and HDAC3 in coimmunoprecipitation. The cytoplasmic extract was made from VSMCs and used in the coimmunoprecipitation. IgG was a control for nonspecific signal. Representatives were shown here from three independent experiments. D, Serum-starved VSMCs were treated with 10 -7 mol/L salusin-β for 30min, The amount of IκBα associated with the HDAC3 protein in cytoplasmic extract was assessed by immunoprecipitation. Input samples were loaded as 10% of whole cell lysates used for the immunoprecipitation. Representatives were shown here from three independent experiments. Fig. 7. Increased cellular levels of PPARγ protein down-regulated salusin-β-induced VSMCs proliferation and inflammation. VSMCs were transfected with increased amount of pCMX-PPARγ expression plasmid or pCMX empty vector for 24h. The empty vector was used to ensure the equal amount of total DNA in transfection assay. After transfection and recovery, cells were serum-starved for 24 h before the stimulation with or without 10 -7 mol/L salusin-β for an additional 24 h. A, Nuclear extracts were obtained and PPARγ protein levels were measured by westernblot analysis. Representatives were shown here from three independent experiments. B, Cell proliferation was determined by the Brdu assay. C and D, the levels of IL-6 and TNF-a were determined using an ELISA assay. Data are expressed as means ± SD (n=6). *P< 0.05 versus the 0.4μg pCMX empty vector without salusin-β (the first column on the left); # P< 0.05 versus 0.4μg pCMX empty vector with 10 -7 mol/L salusin-β treatment (the second column on the left).

Cellular Physiology and Biochemistry
IκBα-NF-κB, but decreased PPARγ gene expression through HDAC3 without involving NF-κB p50 and p65. In addition, the inhibitory effect of salusin-β on PPARγ gene expression contributes to salusin-β-induced VSMCs proliferation and inflammation in vitro. PPARγ has been recognized as an important regulator in the vasculature and maintaining an appropriate expression level in vascular wall cells is essential for assuring that vascular structure and function adapt to atherogenic factors acting on the vascular wall [4]. VSMCs are the main constitutive stromal cells of the vascular wall, continually exposed to mechanical signals and biochemical components generated in the blood compartment [22]. Various genes have been examined with respect to their abilities to prevent atherosclerosis, and of these, PPARγ is a good candidate because it is present in all cells composing blood vessels including VSMCs [4]. Many studies suggest that PPARγ ligands such as the thiazolidinediones not only ameliorate insulin sensitivity, but also have pleiotropic effects on the pathophysiology of vascular disease including atherosclerosis [18,19]. Activation of PPARγ suppresses production of inflammatory cytokines, and there is accumulating data that PPARγ ligands exert anti-inflammatory, antioxidative, and antiproliferative effects on VSMCs [2 ,4]. In addition to the atheroprotective effects of PPARγ ligands identifing a potential role for PPARγ as a regulator of VSMCs biology, many genetic evidences further support a normal role for PPARγ in inhibiting VSMCs proliferation and inflammation in the context of restenosis or atherosclerosis. For example, dominant-negative loss-of-function PPARγ mutations have also been introduced into in vivo and in vitro experiments. It is shown that transfer of a PPARγ-dominant-negative gene promoted neointima formation in balloon injured animal arteries and enhances VSMCs proliferation, inflammation, and vascular remodeling [15][16][17]; in contrast transfer of the PPARγ-wild-type gene was found to inhibit smooth muscle proliferation and reduce neointima formation after balloon injury [15,17]. In addition, VSMCs-specific PPARγ deficiency augmented angiotensin II-induced atherosclerosis [23,24]. It should be noted that many growth factors and cytokines, such as platelet-derived growth factor (PDGF), TGF-β, or angiotensin II-induced atherosclerosis through interactions with PPARγ expressed in VSMCs [25,26]. As a new potential proinflammatory and proatherosclerotic factor, salusin-β has direct effects on VSMCs [5,11,13]. The present results showed that salusin-β caused a marked reduction in PPARγ gene expression at protein and mRNA levels and reduced PPARγ gene promoter activity in VSMCs. Since vascular PPARγ plays a pivotal role in protection from vascular disease, we speculate that the inhibitory action of salusin-β on PPARγ gene expression contributes to its proliferative and inflammatory effects on VSMCs. This postulate is confirmed by the result that increased cellular levels of PPARγ protein down-regulated salusin-β-induced VSMCs proliferation and inflammation in the present study.
PPARγ has also been shown in vitro to function at the nodal point of various cellsignaling pathways [17,27]. Our previous study revealed that salusin-β stimulation in VSMCs invokes mitogen-activated protein kinases (MAPKs) and AKT signaling pathways which are key components of the signals that transduct extracellular stimuli into cells [11]. However, the present study showed that they were not involved in salusin-β inhibition of PPARγ gene expression. Other studies have demonstrated that salusin-β is a powerful activator of the NF-κB pathway in both cultured endothelial cells and in intact arteries [8,9,10]. NF-κB is a transcription factor that participates in vascular damage through the regulation of several genes involved in proliferation and immune responses. The prototypical NF-kB heterodimer consists of RelA (p65) and p50 that contain the transcriptional activation domain. The classical NF-kB activation model involves cytoplasmic sequestration of NF-kB by inhibitory protein IκBα, preventing NF-kB from migrating to the nucleus. When IκBα is phosphorylated, it is degraded, allowing NF-kB to translocate to the nucleus, followed by the activation of specific target genes [28,29]. The present study showed that BAY 11-7085, a known pharmacologic inhibitor of IκBα phosphorylation [30], markedly suppressed salusinβ-induced reduction in PPARγ gene expression. Furthermore, to our knowledge, the present study is the first to report that like in endothelial cells, salusin-β could also activate NF-κB by classical pathway of phosphorylation and degradation of IkBa with parallel induction of nuclear translocation of NF-kB p65 and p50 in VSMCs. However, silencing with NF-κB p50 or p65 did not impact salusin-β-induced down-regulation of PPARγ gene expression. These findings suggest that IκBα may have a new and NF-kB-independent role in salusinβ-induced PPARγ reduction.
HDACs are enzymes that remove acetylation from lysine residues within histones, resulting in a closed chromatin structure and an overall reduction in transcriptional potential. The interplay between histone acetyltransferases and HDACs permits rapid changes in gene expression in response to extrinsic or intrinsic signals [31,32]. There- fore, although originally shown to be involved in cancer and neurological disease, HDACs have been found to be able to modulate most steps of atherosclerosis in the traditional view, e.g. inflammation, proliferation and apoptosis of both ECs and VSMCs [31]. There are 18 characterized members of HDACs, which have been classified into four groups. Class I members HDAC1, HDAC2 and HDAC3 are considered as mitogen-responsive genes in VSMCs [33]. Moreover, HDAC1 and HDAC3 are well known to be transcriptional corepressors of PPARγ. For example, in adipose cells, recruitment of HDAC1 and HDAC3 are involved in the regulation of PPARγ function [34,35], leading to the inhibition of adipocyte differentiation. siRNA-mediated inhibition of HDAC3 attenuated angiotensin II mediated reductions in PPARγ abundance in VSMCs [26]. TGF-β1 down-regulates PPARγ gene expression through recruitment of HDAC1 at the PPARγ promoter in cardiac fibroblasts [36]. Importantly, it has been reported that cytoplasmic IκBα increases NF-κB-independent transcription through binding to HDAC1 and HDAC3 [37]. Thus, we next examined the effects of salusin-β on HDAC1, HDAC2 and HDAC3 and the relationship between them and PPARγ. Our results showed that salusin-β induced nuclear translocation HDAC3, which was required for salusin-β-induced inhibition of PPARγ gene expression. HDAC3 is found to shuttle between nucleus and cytoplasm [31]. Since it is generally believed that HDACs could suppress gene expression only when they are in the nucleus [31], modulation of cellular localization is considered to be critical in the effect of HDAC3 on PPARγ in the present study. It has been reported that nuclear translocation of HDAC3 is controlled by IκBα in the IκBα-HDAC3 model. IκBα binds HDAC3 in the cytosol through the ankyrin repeats [37], and IκBα degradation leads to nuclear translocation of HDAC3 [38]. In the present study, the same mechanism is shown to be responsible for the PPARγ inhibition by salusin-β in VSMCs by four lines of evidence: (a) Either IκBα phosphorylation inhibitor Bay11-7085 or siRNA knockdown targeting HDAC3 can significantly inhibit salusin-β-induced reduction in PPARγ gene expression; (b) salusin-β-induced nuclear translocation of HDAC3 was coupled with IκBα phosphorylation and IκBα degradation; (c) nuclear translocation of HDAC3 in response to salusin-β was significantly reversed by (d) The present study firstly demonstrated that IκBα-HDAC3 complex was also present in the cytosol of VSMCs but interrupted after salusin-β treatment in parallel with IκBα degradation and HDAC3 translocation.
Conclusion: The present study indicated that salusin-β negatively regulated PPARγ gene expression at protein, mRNA and gene promoter level in VSMCs and IκBα-HDAC3 pathway may contribute to salusin-β-induced inhibition PPARγ gene expression (Fig. 9). Given that serum salusin-β levels are significantly higher in patients undergoing coronary artery disease than those in healthy controls [6], the results in the present study might have potential implications for clarifying the mechanisms of presence and severity of coronary artery disease associated with elevated salusin-β level.