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Am J Physiol Cell Physiol 293: C1226-C1238, 2007. First published July 11, 2007; doi:10.1152/ajpcell.00152.2007
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VASCULAR BIOLOGY

Lacking cytokine production in ES cells and ES-cell-derived vascular cells stimulated by TNF-{alpha} is rescued by HDAC inhibitor trichostatin A

Anna Zampetaki,* Lingfang Zeng,* Qingzhong Xiao, Andriani Margariti, Yanhua Hu, and Qingbo Xu

Cardiovascular Division, King's College London, London, United Kingdom

Submitted 12 April 2007 ; accepted in final form 11 July 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Inflammation and TNF-{alpha} signaling play a central role in most of the pathological conditions where cell transplantation could be applied. As shown by initial experiments, embryonic stem (ES) cells and ES-cell derived vascular cells express very low levels of TNF-{alpha} receptor I (TNFRp55) and thus do not induce cytokine expression in response to TNF-{alpha} stimulation. Transient transfection analysis of wild-type or deletion variants of the TNFRp55 gene promoter showed a strong activity for a 250-bp fragment in the upstream region of the gene. This activity was abolished by mutations targeting the Sp1/Sp3 or AP1 binding sites. Moreover, treatment with trichostatin A (TSA) led to a pronounced increase in TNFRp55 mRNA and promoter activity. Overexpression of Sp1 or c-fos further enhanced the TSA-induced luciferase activity, and this response was attenuated by Sp3 or c-jun coexpression. Additional experiments revealed that TSA did not affect the Sp1/Sp3 ratio but caused transcriptional activation of the c-fos gene. Thus, we provide the first evidence that ES and ES-cell-derived vascular cells lack cytokine expression in response to TNF-{alpha} stimulation due to low levels of c-fos and transcriptional activation of Sp1 that can be regulated by inhibition of histone deacetylase activity.

inflammation; TNFRp55; c-fos; Sp1

TNF-{alpha} IS A PLEIOTROPHIC CYTOKINE that possesses strong proinflammatory and immunostimulatory activities and can induce cellular responses that vary from cell proliferation to apoptosis, depending on tissue type, precise cellular context, timing, and duration of TNF-{alpha} action (43). Previous studies have shown that it plays a crucial role in the pathophysiology of cardiovascular diseases. Extremely elevated levels of this mediator are observed after heart failure (27, 40), and TNF-{alpha} signaling is associated with carotid atherosclerosis, while its inhibition reduces neointima formation in APoE null mice (4, 7).

The biological activities of TNF-{alpha} are induced through binding to two cell surface receptors, tumor necrosis factor receptor 1 (TNFRp55) and tumor necrosis factor receptor 2 (TNFRp75). Both receptors have significant homologies in their extracellular domains with repeat cysteine-rich sequences, but they are functionally distinct. TNFRp75 appears to play a major role in the lymphoid system, whereas TNFRp55 is the main mediator of the inflammatory responses exerted by TNF-{alpha}. Mice lacking TNFRp55 are resistant to endotoxic shock and TNF-{alpha}-mediated cytotoxicity, although highly susceptible to infection (25, 32, 34, 36, 38). Although both receptors are expressed in most cell types, knockout mice lacking the TNFRp55 and TNFRp75 are normal, indicating that TNF-{alpha} signaling is not required for normal mouse development and homeostasis under basal conditions (31).

TNF-{alpha} signaling can also be an important concern in tissue regenerative and engineering approaches. Generation of living tissue replacements or transplantation of differentiated progenitors capable of rescuing organ function represents a promising therapeutic potential for cardiovascular and chronic diseases (23, 33). For instance, vascular progenitors and endothelial cells derived from stem cells can effectively repair damaged endothelial monolayer in an animal model of femoral artery injury (47, 51). However, the transplanted cells will have to survive in a very hostile environment that is usually deprived of nutrients, blood supply, and highly enriched in inflammatory mediators like cytokines. Even though it is still unclear whether transient secretion of factors from the circulating progenitor cells or replacement of the injured tissue from the newly differentiated cells is required, characterization of the inflammatory response of these progenitors will provide useful insight into the efficiency of such interventions.

ES cells have the ability to differentiate to a wide variety of cell types and thus represent an attractive target for cell-based repair therapies (9, 17, 26, 28). In the present study, we investigated TNF-{alpha} signaling in mouse embryonic stem cells (ES), ES cell-derived endothelial progenitors (esEC), and ES cell-derived smooth muscle cells (esSMC). Our experiments show that ES and ES cell-derived vascular cells do not induce cytokine expression in response to TNF-{alpha} stimulation. This is mainly due to low levels of TNFRp55. Further experiments revealed that Sp1/Sp3 and AP-1 binding site on the 5'-flanking region of TNFRp55 regulated its expression. Treatment with trichostatin A (TSA) led to a significant increase in TNFRp55 mRNA and promoter activity. Additional experiments indicated that TSA did not affect the Sp1/Sp3 ratio in ES cells but increased the binding of Sp1 and AP1 transcription factors to the TNFRp55 promoter. Moreover, TSA treatment led to a transcriptional activation of the c-fos gene. Finally, we reveal a cross talk between Sp1 and c-fos was detected as coexpression of c-fos and Sp1 that plays a key role in the TNFRp55 expression.


   METHODS
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 METHODS
RESULTS
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Cell culture. SMCs were isolated by enzymatic digestion of mouse aortas, as described elsewhere (13, 22) and were cultured in DMEM supplemented with 15% FCS, 2 mM L-glutamine and 100 mg/l gentamicin. Mouse ES cells (D3) were obtained from American Type Culture Collection (Manassas, VA, USA) and grown on gelatin-coated flasks. Cell passages 3–15 were used for experiments. To maintain the ES cells in an undifferentiated state, leukemia inhibitory factor (LIF; 1,000 U/ml) was added to the culture medium (DMEM, ATCC) supplemented with 10% FCS, 2 mM L-glutamine, 100 mg/l gentamicin and 10–4 M 2-mercaptoethanol (2-ME).

Endothelial progenitors derived from ES cells (esEC) were obtained as described previously (51); ES were plated on collagen IV slides and cultured in alpha-MEM supplemented with 10% FBS, 2 mM L-glutamine, 100 mg/l gentamicin, and 5 x 10–5 M 2-ME for 4 days. The cells were subsequently subjected to shear stress at 12 dyn/cm2for 24 h. Expression of EC markers was detected by RT-PCR and confirmed by Fluorescence-Activated Cell Sorter Analysis (FACS).

esSMC were derived from ES cells cultured on type IV mouse collagen-coated flasks grown as described previously (46). In brief, Sca-1+cells were sorted from the cell culture by magnetic labeling cell sorting (MACS) with anti-Sca-1 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Sca-1+cells were resuspended and cultured in fresh ES cell growth medium. For SMC differentiation, Sca-1+cells were plated on collagen IV-coated dishes or flasks, and cultured in {alpha}MEM supplemented with 10% FCS and 5 x 10–5 M 2-ME and 10 ng/ml PDGF-BB (Sigma). esSMC passage 5 was used for all experiments. Expression of SMC markers was detected by RT-PCR and confirmed by FACS.

RNase protection assay. Total RNA was extracted using the Qiagen kit according to the manufacturer's instructions. To estimate the expression of cytokines, RNase Protection Assay (RPA) was performed, using mCK2b, mCR4, and mCK5c multiprobe template sets (RiboQuant, Pharmingen, San Diego, CA) and [{alpha}-32P] UTP (Amersham Biosciences, Piscataway, NJ), according to the manufacturer's recommendations. The "RNase-protected" fragments were purified and resolved on a 5% sequencing gel and autoradiographed. The housekeeping gene L32 was used as a loading control.

RT-PCR. Total RNA was extracted using the Qiagen kit according to the manufacturer's instructions, and any potential contaminating chromosomal DNA was digested using the DNA-free kit (Ambion, Austin, TX). The procedure used for RT-PCR was similar to that described elsewhere (1). In brief, 2 µg of RNA was converted to cDNA using Promega Reverse Transcription System (Promega, Madison WI). cDNA products were amplified by PCR using gene-specific primers. The primers used were TNFRp55 forward: ACC AAG TGC CAC AAA GGA AC; TNFRp55 reverse: CAC GCA CTG GAA GTG TGT CT; TNF-{alpha} forward: AGC CCC CAG TCT GTA TCC TT, TNF-{alpha} reverse: CTC CCT TTG CAG AAC TCA GG; IL-6 forward: CGA TGA TGC ACT TGC AGA AA; IL-6 reverse: GGA AAT TGG GGT AGG AAG GA; Sp1 forward: ACA GCA GGT GGA GAA GGA GA; Sp1 reverse: TGA GGC TCT TCC CAC ACT GT", Sp3 forward: GCT CCA CCT TTT GTG TTT CC; Sp3 reverse: TCT TGT TTC ACG GGC TTT TC; c-fos forward: GGG GCA AAG TAG AGC AGC TA; c-fos reverse: GGC TGC CAA AAT AAA CTC CA; GAPDH forward: CGG AGT CAA CGG ATT TGG TCG TAT; and GAPDH reverse: AGC CTT CTC CAT GGT GGT GAA GAC. PCR conditions were as follows: 94°C for 3 min and then 30 cycles for TNFRp55, TNF-{alpha}, IL-6, Sp1, Sp3, c-fos, or 26 cycles for GAPDH at 94°C for 30 s, 58°C for 1 min and 72°C for 1 min, followed by 72°C for 10 min. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.

Real-time PCR experiments were performed with the Brilliant SYBR Green quantitative PCR core reagent kit (Stratagene, Cedar Creek, TX), using the Mx4000 (Stratagene) real-time thermocycler, according to the company's instructions. Amplification was performed with 40 cycles, and an annealing temperature of 58°C. Copy numbers were calculated by the instrument's software from standard curves. The specificity of the amplification reaction was determined by a melting curve analysis. For quantification TNFRp55, TNF-{alpha} and IL-6 mRNA expression were normalized to the house keeping gene GAPDH.

To determine the half-life of TNFRp55 or c-fos, cells were treated with 5 µg/ml actinomycin D for the indicated time, and total RNA was harvested. Levels of TNFRp55 mRNA were analyzed by quantitative real-time PCR. Target mRNA levels were normalized to GAPDH mRNA expression. Gene expression levels at time 0 were set as 100. Values are means ± SD. Data presented are the sum of four independent experiments.

Plasmid construct. Expression vector harboring sequences of the mouse TNFRp55 or c-fos promoters were created using genomic DNA from SMCs. The 5'-flanking region of the respective gene was amplified by PCR. The PCR product was then digested, gel purified, and cloned into the pGL3-basic vector. Mutants pGL3p55(Sp1-MUT12), pGL3p55(Sp1-MUT3), and pGL3p55(AP1-MUT) were generated by PCR using pGL3p55(-373, -129) vector as a template and mutant pGL3p55(Sp1-MUT123) using pGL3p55(Sp1-MUT12) as a template. PCR products were gel purified and ligated. The expression vectors pShuttle(Sp1), pShuttle(Sp3), pShuttle(c-fos), and pShuttle(c-jun) encoding the mouse Sp1, Sp3, c-fos, and c-Jun proteins, respectively, were generated by PCR using mouse cDNA as a template. The PCR product was then digested, gel purified, and cloned into the pShuttle vector. All primers and cloning sites are shown in Table 1. All vectors were verified by sequencing.


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  		Table 1. Primers used for cloning

 
Transient transfection. For transfection experiments, 5 x 104 ES cells per well were seeded in 12-well plates and left to adhere overnight. The cells were then transfected with the reporter with or without expression plasmid vectors using Fugene-6-Reagent (Roche Molecular Biochemicals, Indianapolis, IN), according to the manufacturer's instructions. pCMV-beta-galactosidase was included as an internal control. After 24 h, fresh medium was added to the transfected cells, and the plates were returned to the incubator and harvested 48 h later. The cells were then washed twice with ice cold PBS and lysed in the reporter lysis buffer (Promega). Luciferase and the beta-galactosidase activity were determined using luciferase and beta-galactosidase enzyme assay systems, respectively (Promega), and the latter was used to calculate transfection efficiency in each experiment. At least, three independent transfections were performed in triplicate.

siRNA knockdown. The siRNA for control, Sp1, and c-fos were purchased from Ambion. siRNA experiments were performed using the siIMPORTER (Millipore, Billerica, MA), according to the company's recommendations. In brief, ES cells were plated on gelatin-coated 12-well plates and 24 h later, siRNA, together with pGL3p55(-1263, -129) and pCMV-beta-galactosidase were introduced into the cells with siIMPORTER. The untransfected and control siRNA-transfected cells were included as a control. The transfected cells were further cultured for 72 h. Luciferase and the beta-galactosidase activity were determined using luciferase and beta-galactosidase enzyme assay systems, respectively (Promega), and the latter was used to calculate transfection efficiency in each experiment. Samples were run in triplicate, and the data presented are the sum of at least three independent experiments. Western blot analysis was performed to confirm Sp1 and c-fos gene knockdown.

Western Blot analysis. The procedure used was similar to that described previously (49). Antibodies against TNFRp55, beta-actin, Sp1, Sp3, and c-fos were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Specific antibody-antigen complexes were detected by using the ECL Western Blot Detection Kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK).

Bisulfite modification. DNA bisulfite modification and purification was performed using the EZ-DNA Methylation kit (Zymo Research, Orange, CA) according to manufacturer's recommendations. The modified DNA was amplified using primers for bisulfite sequencing PCR that were designed using the program "Meth Primer: designing primers for Methylation PCRs". The primers used were: Part TA, TNFRp55 BIS1 L: AGT GAT TTT GGG TTA GGA TTT AGG TA and TNFRp55 BIS1 R: AAA AAC CAA CCC TCC TAT CTC AC; and Part TB: TNFRp55 BIS2 L AAT AGG TTT AGA GGG GTT AGT TTA T and TNFRp55 BIS2 R: AAA AAA AAC CAA AAT TCT TTA AC. PCR conditions were as follows: 94°C for 3 min and then 35 cycles at 94°C for 30 s, 58°C for 1 min and 72°C for 1 min, followed by 72°C for 10 min. PCR products were gel purified and cloned into pGEM-T vector (Promega), according to the company's instructions, and 20 colonies were purified as minipreps and screened for correct insertion. In each case, 10 independent clones were sequenced.

Chromatin immunoprecipitation. The chromatin immunoprecipitation (ChIP) assays were performed as described previously (50) with minor modifications. In brief, cells were treated with 1% (vol/vol) formaldehyde at room temperature for 10 min and then quenched with glycine at room temperature. The medium was removed, and cells were harvested for sonication. The sheared samples were diluted into 1 ml of immunoprecipitation buffer containing 25 mM Tris·HCl, pH 7.2, 0.1% NP-40, 150 mM NaCl, and 1 mM EDTA; immunoprecipitation was conducted with rabbit anti-acetyl-histone H4, anti-acetyl-histone H3, together with single-strand salmon sperm DNA saturated with Protein G-Sepharose beads. Normal IgG was used as a control. Immunoprecipitates were pelleted by centrifugation. The immunoprecipitates were eluted from the beads using 100 µl elution buffer (50 mM NaHCO3, 1% SDS). A total of 200-µl proteinase K solution was added to a total elution volume of 300 µl and incubated at 60°C overnight. DNA was extracted, purified, and then used to amplify target sequences by PCR. The primers used to amplify the TNFRp55 promoter were ChIP TNFRp55 forward: CTT GGC CCT CTC CTC ACT CC and ChIP TNFRp55 reverse: GAG AAG CTG AAA GTC AGA GG. Aliquots of chromatin were also analyzed before immunoprecipitation and served as an input control.

Statistics. Statistical analyses were performed by one-way ANOVA, and P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ES cells and ES-cell-derived vascular cells do not upregulate cytokine expression after TNF-{alpha} challenge due to low levels of TNF-{alpha} receptors. To assess the inflammatory response of embryonic stem cells (ES) and ES-cell-derived smooth muscle cells (esSMC), we treated these cells for 1 or 2 h with TNF-{alpha} and analyzed the mRNA levels of various cytokines using RPA. Surprisingly, except macrophage migration inhibitory factor, no significant cytokine mRNA levels were observed even after 2 h of treatment (Fig. 1). Mature smooth muscle cells (SMC) were used as a positive control. Very low levels of cytokines were detected after prolonged exposure of the film, but these levels were dramatically lower compared with those observed in mature cells at baseline, and no cytokine upregulation in response to TNF-{alpha} was identified. Interestingly, esSMC constitutively expressed elevated levels of IP-10 and TCA-3 that were not affected by the TNF-{alpha} treatment (Fig. 1C). Of note, this short-term TNF-{alpha} treatment does not affect cell proliferation rate.


Figure 1
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  		Fig. 1. ES and ES-cell-derived smooth muscle cells do not upregulate cytokine expression in response to TNF-{alpha}. Cytokine expression after TNF{alpha} stimulation. Total RNA was isolated from cells after TNF-{alpha} treatment (20 ng/ml) for the indicated times. Samples were analyzed by RNase protection assay (RPA). L32, a ribosomal protein mRNA, was used as a loading control. Left lanes indicate the unprotected probe sizes.

 
TNF-{alpha} elicits its biological activities through two structurally related but functionally distinct receptors, TNFRp55 and TNFRp75. Previous studies have shown that TNFRp55 mediates most of the inflammatory responses induced by TNF-{alpha}, and thus, our studies focused on this receptor (8, 34). To determine whether ES cells and ES-cell-derived vascular cells do not upregulate inflammatory cytokine expression due to the low abundance of this receptor, we studied its expression levels. TNFRp55 mRNA (Fig. 2, A and B) and protein (Fig. 2C) was extremely low in ES cells compared with mature SMC. Similar results were also obtained for esSMC (Fig. 2D), and endothelial progenitors derived from ES cells after shear stress (Fig. 2E).


Figure 2
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  		Fig. 2. Expression of TNFRp55 in embryonic stem (ES) and ES-cell-derived vascular cells. A: TNF-{alpha} receptor expression levels in ES and mature smooth muscle cell (SMC) were analyzed by RPA. Different lanes indicate independent experiments. B: quantitative real-time PCR analysis of the TNFRp55 mRNA levels. Data were normalized to GAPDH mRNA expression. Values are means ± SD. C: Western blot analysis of TNFRp55 protein expression in ES and mature SMC. beta-actin was included as a loading control. D: TNF-{alpha} receptor expression levels in ES-cell-derived smooth muscle cells (esSMC) and in ES-cell-derived endothelial progenitor cells obtained by shear stress (E) were analyzed by RPA. Left lanes indicate the unprotected probe sizes.

 
Regulation of TNFRp55 expression in ES cells. Regulation of the mRNA decay rate is an important process in determining the abundance of cellular transcripts. Aiming to identify any differences in the TNFRp55 mRNA stability in the two cell lines, we estimated the half-life of the transcript. As shown in Fig. 3A, no differences in the mRNA degradation were detected.


Figure 3
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  		Fig. 3. Transcriptional regulation of TNFRp55 expression in ES cells. A: turnover of TNFRp55 mRNA in SMC and ES cells. Cells were treated with 5 µg/ml actinomycin D for the indicated time, and total RNA was harvested and levels of TNFRp55 mRNA were analyzed by quantitative real-time PCR. Data were normalized to GAPDH mRNA expression. Values are means ± SD. Data presented are the sum of four independent experiments. B: TNFRp55 promoter is not methylated in ES cells. Schematic diagram of the TNFRp55 upstream region. CpG dinucleotides are depicted as blue bars below the promoter. DNA methylation profile of the individual CpG elements at Part TA and TB in TNFRp55 5'-flanking region in SMCs and ES, as identified by bisulfite sequence analysis of 10 separate clones. Unmethylated CpGs are represented with open circles and methylated CpGs with solid circles.

 
In mammals, reversible methylation of cytosine is an epigenetic modification known to suppress gene expression (3, 39). To explore the possibility that DNA methylation of the TNFRp55 promoter inhibits its expression in ES, we did a sequence analysis of the 5'-flanking region. Two regions enriched in CpG dinucleotides were identified. To study the methylation state, bisulfite modification of isolated genomic DNA from embryonic stem cells and the mature SMCs and PCR amplification of Part TA and Part TB was performed. As shown in Fig. 3B, only some random methylation events were observed at low frequency, and the TNFRp55 promoter was largely unmethylated (11). These results suggest that downregulation of TNFRp55 expression in ES cells involves the control of the transcription rate.

To define the minimal region in the mouse TNFRp55 promoter that is necessary and sufficient for gene expression, deletions ranging from -3794 to -177 upstream of the translation initiation site of TNFRp55 were cloned into pGL3-basic vector, and reporter activity was measured after transient transfection. A 244-bp fragment spanning residues -373 to -129 was identified to show extremely strong promoter activity, comparable to that observed from the 3.6-kb promoter fragment (Fig. 4A).


Figure 4
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  		Fig. 4. Regulatory elements on the mouse TNFRp55 promoter. A: schematic illustration of different deletion variants of the TNFRp55 promoter. Luciferase activity of various promoter deletions cloned into pGL3-basic vector was measured and normalized to the expression of cotransfected pCMV-beta-galactosidase control plasmid. Values are means ± SD. Samples were run in triplicates. Data presented are the sum of at least three independent experiments. B: mutation analysis of the TNFRp55 promoter. *Significant difference from unmutated sample, P < 0.05. C: expression levels of Sp1/Sp3 and AP-1 transcription factors as assessed by RT-PCR (left) and Western blot analysis (right).

 
Sequence analysis of this fragment revealed the presence of putative binding sites for transcription factors Sp1/Sp3 and AP-1. Mutation of Sp1/Sp3 or AP-1 binding sites significantly attenuates TNFRp55 promoter activity (Fig. 4B), indicating that both transcription factors are involved in the expression of TNFRp55. In line with these findings, in pGL3(-278, -129) reporter where all Sp1/Sp3 sites have been deleted, significantly lower levels of luciferase activity were observed (Fig. 4B). Interestingly, although no significant differences in Sp1/Sp3 expression were observed between SMC and ES cells, c-fos expression levels were remarkably reduced in ES cells (Fig. 4C).

Trichostatin A upregulates TNFRp55 expression. Histone deacetylases have been shown to play a crucial role in ES cell differentiation (21). To investigate whether histone deacetylase (HDAC) can also regulate the inflammatory response of ES cells, we studied the effect of HDAC inhibition on TNFRp55 expression. As shown in Fig. 5, A and B, TSA, an inhibitor of histone deacetylases induced a significant upregulation in TNFRp55 mRNA and could slightly induce TNFRp55 protein. Of note, the concentrations of the reagent used did not affect cell proliferation and did not induce apoptosis. Moreover, even very low levels of TSA could induce TNFRp55 expression in ES cells (Fig. 5C). To verify whether the effect of this reagent on TNFRp55 mRNA levels was due to transcriptional regulation, transient transfection assays were performed. As shown in Fig. 5D, significantly elevated promoter activity was observed in TSA-treated cells. Additionally, the major TSA response element seems to be located within the 244-bp fragment of the minimal promoter of TNFRp55, as the 3.6-kb promoter fragment displayed luciferase activity comparable to that observed with the 244-bp fragment (data not shown). Further analysis using mutation of Sp1 and AP1 putative binding sites revealed that both Sp1 and AP1 binding is essential for the upregulation of TNFRp55 promoter activity by TSA, as disruption of either site ablated the increase in luciferase activity observed after TSA treatment. To further test whether this upregulation of TNFRp55 expression in ES cells was sufficient to restore cytokine expression, we assessed TNF-{alpha} and IL-6 mRNA levels after 1 h of TNF-{alpha} challenge using quantitative real-time PCR. Indeed, significantly higher levels of cytokine mRNA were detected in TSA-pretreated cells in response to TNF-{alpha} challenge (Fig. 5, E and F).


Figure 5
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  		Fig. 5. Trichostatin A upregulates TNFRp55 expression. A: TNF-{alpha} receptors mRNA levels in ES cells after TSA treatment, as analysed by RPA.L32, a ribosomal protein mRNA was used as a loading control. B: effect of TSA on TNFRp55 protein. C: effect of various concentrations of Trichlostatin A (TSA) on TNFRp55 mRNA as assessed by RTPCR. D: effect of TSA on various mutants of the TNFRp55 promoter. *Significant difference from the untreated mutated sample. Values are means ± SD. Samples were run in triplicate. E and F: quantitative real-time PCR of TNF-{alpha} and IL-6 mRNA levels in ES cells incubated with TSA and then stimulated with TNF-{alpha} (20 ng/ml) for 1 h. Data were normalized to GAPDH mRNA expression. Values are means ± SD. *Significant difference from the unstimulated sample incubated with the inhibitor, P < 0.05.

 
The transcription factor AP-1 has been implicated in a large variety of biological processes, including cell differentiation, proliferation, and apoptosis. It is a dimer composed of members of the Fos, Jun, and activation transcription factor families of proteins. c-fos/c-jun heterodimer is known to bind to the sequence element TGA(G/C)TCA, as the one detected in the TNFRp55 minimal promoter (15, 37). As mutation of the above site could abolish promoter activity, we performed a chromatin immunoprecipitation assay to identify possible binding to the upstream regulatory regions of the gene. As shown in Fig. 6A, a strong binding of the c-fos transcription factor was identified in TSA-treated ES but not in the untreated cells. To study the functional role of c-fos, c-jun in regulating TNFRp55 gene expression, we cotransfected ES cells with a TNFRp55 promoter luciferase construct, together with c-fos and c-jun or c-fos and c-jun alone. Coexpression of c-fos and c-jun resulted in significantly elevated levels of TNFRp55 promoter activity (Fig. 6B) in ES cells; however, this response was abrogated when Sp1 or AP1 binding sites were mutated (Fig. 8A). Interestingly, in the presence of TSA coexpression of c-fos and c-jun or overexpression of c-fos or c-jun alone did not further increase the reporter activity (Fig. 6B, lanes 5–8).


Figure 6
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  		Fig. 6. TSA induces TNFRp55 partly through transcriptional activation of c-fos in ES cells. A: chromatin immunoprecipitation assays were performed using antibodies against c-fos. Normal rabbit IgG was used as a negative control. Aliquots of chromatin before immunoprecipitation served as an input control. B: ES cells were transiently transfected with a pGL3p55(-1263,-129) reporter construct, pShuttle(c-fos), pShuttle(c-jun), or the empty vector pShuttle. After 24 h treatment with TSA (50 nM) or DMSO, the luciferase activity was assessed. Values are means ± SD. Samples were run in triplicate. Data presented are the sum of at least three independent experiments. *Significant difference from empty vector, P < 0.05. C: mRNA levels as assessed by quantitative PCR (Q-PCR). *Significant difference from untreated ES cells. Right: mRNA levels of c-fos in ES cells treated with various concentrations of TSA as assessed by RT-PCR. D: mRNA levels of c-fos in ES cells treated with TSA (50 nM) for the indicated times as assessed by RT-PCR. E: ES cells were pretreated with actinomycin D (5 µg/ml) for 1 h and then stimulated with TSA (50 nM) for 4 h. mRNA levels of c-fos were assessed by RT-PCR. F: ES cells were pretreated with cycloheximide (50 µg/ml) for 2 h and then stimulated with TSA (50 nM) for 4 h. mRNA levels of c-fos were assessed by RT-PCR. Data were normalized to GAPDH expression. G: turnover of c-fos mRNA in ES cells and TSA-treated cells ES. Cells were stimulated with TSA (50 nM) for 4 h and then treated with 5 µg/ml actinomycin D for the indicated time, and total RNA was harvested and levels of c-fos mRNA were analyzed by quantitative real-time PCR. Data were normalized to GAPDH mRNA expression. Values are means ± SD. Data presented are the sum of four independent experiments. H: ES cells were transfected with the pGL3(c-fos) reporter construct. After 24 h of treatment 50 nM of TSA, the luciferase activity was assessed. Values are means ± SD. Samples were run in triplicate. Data presented are the sum of at least three independent experiments. *Significant difference from untreated sample, P < 0.05. I: ES cells were treated with TNF-{alpha} for various time points and c-fos and TNFRp55 expression was assessed by RT-PCR. Data were normalized to GAPDH mRNA expression.

 

Figure 8
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  		Fig. 8. Sp1 and c-fos synergistically control the expression of TNFRp55 in TSA treated ES cells. A: ES cells were transiently transfected with a pGL3p55(-1263,-129), pGL3(SP1-MUT3), pGL3(AP1-MUT) reporter constructs, pShuttle(c-fos), pShuttle(c-jun), pShuttle(Sp1) or the empty vector pShuttle. After 24 h treatment with TSA (50 nM) or DMSO, the luciferase activity was assessed. Results were normalized to the expression of beta-galactosidase. Values are means ± SD. Samples were run in triplicate. Data presented are the sum of at least three independent experiments. *Significant difference from empty vector, #Significant difference from TSA-treated empty vector, P < 0.05. B: effect of knocking down Sp1 or c-fos on TNFRp55 promoter activity. ES cells were transfected with siRNA for the respective factors and pGL3p55(-1263,-129) reporter construct. Untransfected and control siRNA were included as control. Data are presented are the sum of three independent experiments. *Significant difference from TSA ctrsiRNA and untreated sample, P < 0.05.

 
To study whether increased binding of c-fos by TSA was due to upregulation of c-fos expression, we studied the c-fos mRNA levels. As shown in Fig. 6C, significantly higher levels of c-fos were detected. More importantly, increased mRNA levels were observed after TSA treatment at concentrations as low as 10 nM (Fig. 6D, right). Interestingly, this induction occurred very early, after only 2 h of treatment. Levels peaked at 4 h and remained significantly elevated even after 24 h (Fig. 6D). c-fos mRNA is known to be rapidly degraded (35). To determine whether this upregulation involved mRNA stabilization rather than transcriptional process, ES cells were pretreated with the transcription inhibitor actinomycin D for 1 h and then treated with TSA for 4 h. As indicated in Fig. 6E, this pretreatment completely abrogated c-fos mRNA induction by TSA. Additional experiments were performed using cycloheximide that has been previously shown to block c-fos mRNA turnover. Combined treatment of TSA and the protein synthesis inhibitor resulted in significantly higher levels of c-fos mRNA than treatment with cycloheximide, suggesting that TSA leads to induction rather than inhibition of degradation of c-fos mRNA (Fig. 6F). Aiming to identify any differences in the c-fos mRNA stability in TSA-treated and untreated cells, we estimated the half-life of the transcript. As shown in Fig. 6G, no differences in the c-fos mRNA degradation were detected. To further determine the effect of TSA on c-fos transcriptional regulation, we studied the effect of TSA on the c-fos promoter activity. Our results reveal a robust upregulation of the luciferase activity (Fig. 6H) in response to TSA and provide additional support for the transcriptional regulation of c-fos by TSA. However, elevated levels of c-fos are not sufficient to drive transcription from the TNFRp55 promoter. As shown in Fig. 6I, c-fos expression was upregulated in TNF-{alpha}-stimulated cells, but TNFRp55 mRNA was not affected, indicating that high levels of c-fos are not adequate to induce TNFRp55 expression and activation of Sp1, as observed after inhibition of deacetylases is also required.

Sp1 and Sp3 are also involved in the regulation of TNFRp55. They are two ubiquitously expressed transcription factors with similar structural features and highly conserved DNA binding domains. Sp1 and Sp3 recognize the same DNA element and have similar binding affinities. However, whereas Sp1 is known as a transcriptional activator, Sp3 is considered to be a repressor, as it has been shown to repress Sp1-mediated gene transactivation (2, 10, 18). Aiming to delineate the molecular mechanisms involved in the induction of TNFRp55 by the TSA, we studied the Sp1 and Sp3 mRNA and protein levels after TSA treatment in ES cells. As shown in Fig. 7, A and B the expression of the two transcription factors is not affected by inhibition of deacetylases. Interestingly, TSA treatment resulted in enhanced binding of Sp1 to the promoter of TNFRp55 as estimated by chromatin immunoprecipitation assay, although the detected levels were still lower than those observed in mature SMCs (Fig. 7C).


Figure 7
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  		Fig. 7. Role of Sp1 and Sp3 in the expression of TNFRp55. A: mRNA levels of Sp1 and Sp3 in ES cells treated with TSA as assessed by RT-PCR. B: Western blot analysis of Sp1 and Sp3 protein expression in ES in the presence or absence of TSA. C: chromatin immunoprecipitation assays were performed using antibodies against Sp1 and Sp3. Normal rabbit IgG was used as a negative control. Aliquots of chromatin before immunoprecipitation served as an input control. D: ES cells were transiently transfected with a pGL3p55(-1263,-129) reporter construct, pShuttle(Sp1), pShuttle(Sp3), or the empty vector pShuttle. After 24 h of treatment with 50 nM of TSA or DMSO, the luciferase activity was assessed. Values are means ± SD. Samples were run in triplicate. Data presented are the sum of at least three independent experiments. *Significant difference from empty vector. #Significant difference from TSA-treated empty vector, P < 0.05.

 
Moreover, overexpression of Sp1 led to a robust increase in the promoter activity of TNFRp55 in TSA-treated ES cells, as indicated by a reporter gene assay (Fig. 7D), while coexpression of Sp1 and Sp3 attenuated this response. The above data identify Sp1 as a positive modulator of TNFRp55 and suggest a negative regulatory role for Sp3. Interestingly, when the Sp1 binding site was mutated, Sp1 overexpression could no longer enhance the luciferase activity, indicating that binding of Sp1 to the TNFRp55 promoter is necessary. Similar results were also obtained when the AP1 binding site was mutated, suggesting that both Sp1 and AP1 elements synergistically control the expression of TNFRp55 (Fig. 8A).

Sp1 and c-fos synergistically control the expression of TNFRp55 in TSA-treated ES cells. To evaluate the putative cross talk between c-fos and Sp1, we coexpressed the two factors and assessed the TNFRp55 promoter activity. As shown in Fig. 8A, in untreated ES cells, c-fos and Sp1 coexpression induced significantly higher luciferase activity than Sp1 overexpression, whereas in TSA-treated ES cells, coexpression of c-fos and Sp1 resulted in similar levels of TNFRp55 promoter activity as overexpression of Sp1 alone. Importantly, coexpression of Sp1 and c-jun could abrogate this response and bring reporter activity to the levels detected with the empty vector. These data reveal a positive regulatory role for c-fos, while c-jun emerges as a negative modulator of TNFRp55. Importantly, reporter gene assays using constructs harboring mutated sites for Sp1 or AP1 highlighted the requirement for intact binding sites for both transcription factors, providing further evidence for their synergistic function in TNFRp55 expression (Fig. 8A).

Knockdown experiments of Sp1 and c-fos also support a synergistic role for the two factors in the TSA induction of TNFRp55. Although in untreated ES cells, siRNA for Sp1 and c-fos has no effect on the reporter activity, a strong downregulation in luciferase levels was observed when deacetylase activity is inhibited by TSA treatment of ES cells (Fig. 8B).


   DISCUSSION
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 ABSTRACT
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Tissue repair is a complex process that involves degeneration, inflammation, regeneration, and fibrosis. Healing is a continuous sequence of inflammation and repair in which a variety of immune cells invade and interact with the damaged tissue (29). ES cells derived from the inner cell mass of blastocysts have the potential to differentiate to any cell type and thus provide transplantable cells for treating failing organs, blood disorders, cardiovascular, neurological, and chronic diseases. The beneficial outcome of stem cell infusion very much depends on the growth and differentiation factors within the tissue, cell to cell interactions, the degree of injury, and inflammation (19, 24, 30, 41).

TNF-{alpha} is an important mediator of the innate immunity. It is involved both in tissue regeneration and destruction. This dual function makes TNF-{alpha} signaling an important determinant of the efficiency of stem cell-based therapeutic approaches. In the present study, we investigated the TNF-{alpha} signaling in mouse ES cells and ES-cell-derived vascular cells and demonstrated the mechanism by which TSA, an inhibitor of HDAC induces TNFRp55 gene expression. Here, we provide the first evidence that in ES cells and ES cell-derived vascular cells, transcriptional regulation of TNFRp55 results in very low levels of expression and thus renders these cells unable to upregulate cytokines in response to TNF-{alpha} stimulation. Previous studies from our group revealed that ES and ES cell-derived vascular progenitors are also unresponsive to endotoxin challenge (48), suggesting that these cells have a diminished inflammatory response and would not further augment the damage in the injured area when used in tissue regeneration approaches. This response is clearly distinct from previous observations in mesenchymal stem cells. Recent reports showed that these cells upregulate the expression of TNF-{alpha}, IL-6, and a variety of growth factors after stimulation, indicating that they represent a significant sources of paracrine factors (5, 44).

Histone deacetylase activity is extremely important for the differentiation of ES cells (21). Here, we demonstrate that it is also important for their inflammatory response to TNF-{alpha}. Our experiments showed that histone deacetylase inhibitor, TSA, could sufficiently induce functional TNFRp55 expression and confer cytokine expression after stimulation with TNF-{alpha}, even at concentrations as levels as low as 10 nM. Additionally, we identified two major response elements to the histone deacetylase inhibitor, TSA. Further studies revealed that TSA treatment led to upregulation of c-fos and increased binding of both c-fos and Sp1 to the TNFRp55 promoter. These two factors synergistically affect the transcription rate from the TNFRp55 promoter.

AP-1 is involved in diverse cellular functions ranging from proliferation to transformation or cell death. It can induce or prevent apoptosis, and the outcome is highly tissue- and developmental stage-specific (12, 37). Mutational analysis and knockdown experiments identified the dimeric transcription factor AP-1 as a critical regulator of TNFRp55. Our experiments showed that direct binding of AP-1 to the promoter region is required for the transcriptional activation of TNFRp55 as mutation of the putative binding site totally attenuated the reporter activity. Additionally, ChIP assays indicated that in TSA-treated ES cells the high levels of receptor coincided with strong binding of the c-fos on the promoter of TNFRp55, while coexpression of c-fos and c-jun significantly upregulated the reporter activity at baseline.

A robust elevation of c-fos was observed even at very low levels of TSA. After 2 h of treatment, ES cells upregulated c-fos expression in a dose-dependent manner. Previous studies have shown that c-fos transcription is suppressed by repressive factors that can bind to the fos intragenic regulatory element (FIRE) in the first exon. This poses a blockade to the transcription. However, once protein synthesis is inhibited, rapid degradation without replacement of these factors occurs, leading to increased stability of c-fos mRNA. This is the mechanism by which cycloheximide, a protein synthesis inhibitor, stabilizes c-fos transcript (20). Our experiments show that upregulation of c-fos by TSA does not involve stabilization of the transcript but rather requires de novo synthesis. Pretreatment with the transcription inhibitor Actinomycin D completely suppressed the c-fos induction by TSA, while the estimated half-life of the transcript was not affected by HDAC inhibition. Furthermore, the fact that TSA and cycloheximide treatment has an additive effect on the levels of c-fos mRNA argues against a mechanism of induction due to depletion of repressive factors and points to the direction of increased transcriptional activity of the promoter. These findings are also supported by reporter gene assays that indicate high luciferase levels after TSA stimulation.

Although elevated levels of c-fos are important, they are not sufficient to upregulate TNFRp55 transcription. Overexpression of c-fos in ES did not increase the activity of the TNFRp55 reporter, either at baseline or after TSA treatment. Additionally, in TSA-treated ES cells, siRNA experiments demonstrated that knockdown of c-fos can reduce TNFRp55 promoter activity but cannot bring it back to baseline levels, indicating that other transcription factors are also involved. Interestingly, coexpression of c-fos and Sp1 led to significantly elevated reporter activity, suggesting that there is a cross talk between the two factors. Our experiments clearly indicate that TSA induction of TNFRp55 expression requires intact binding sites for Sp1 and AP1 transcription factors. Overexpression of either transcription factor cannot lead to enhanced promoter activity if any of the two elements is mutated. Additionally, our chromatin immunoprecipitation assays reveal enhanced binding of both Sp1 and c-fos on the promoter of TNFRp55 in TSA-treated ES cells. The above data suggest that both elements are crucial in the induction of TNFRp55 by TSA.

The Sp family consists of four members, Sp1, Sp2, Sp3, and Sp4. Of these, Sp1 and Sp3 are ubiquitously expressed, while Sp2 and Sp4 are restricted to specific tissues and cell types. All four Sp-family members have similar domain structure. They contain three zinc fingers close to the C-terminus and glutamine-rich domains adjacent to serine/threonine stretches in their N-terminal region. Their DNA binding domain is the most conserved part of the proteins. In fact, because of conserved amino acids within the third zinc finger, Sp1, Sp3, and Sp4 recognize and act through the classical Sp1 binding site with identical affinity. These GC-rich elements are highly significant for the activity of TATA-less promoters. Sp1 and Sp3 differ in their capacity to activate or repress transcription and are thought to compete for similar binding sites. The relative rate of transcription is affected by the outcome of this competition (16, 42), alteration in the Sp1/Sp3 ratio is a well-known mechanism of transcriptional regulation (6, 10, 45).

Our experiments indicate that Sp1 signaling, at least in part, mediates TNFRp55 expression. Mutation analysis showed that binding of Sp1 to GC boxes in the minimal promoter area is crucial for the transcription of the gene, while Sp1 knockdown significantly attenuated the promoter activity. Additional data from ChIP assays also support this notion. The higher levels of Sp1 bound to the promoter of TNFRp55 in mature SMC compared with ES cells correlate with higher gene expression. Noteworthy, Sp1 and Sp3 levels do not differ in mature SMC and ES cells. In accordance with this observation, overexpression of Sp1 or Sp3 does not affect TNFRp55 promoter activity at baseline. Interestingly, inhibition of HDAC seems to play a major role in Sp1 signaling. In the presence of TSA, although no difference in the Sp1/Sp3 ratio was observed, ChIP assays indicated that higher levels of Sp1 bind to the TNFRp55 upstream region. This coincides with elevated TNFRp55 promoter activity that is further enhanced by overexpression of Sp1. Importantly, in the presence of this inhibitor, a negative regulatory role for Sp3 is revealed. Coexpression of Sp1 and Sp3 completely abrogated the additive effect of Sp1 overexpression on the reporter. This suggests that inhibition of HDAC facilitates the functional interaction between Sp1 and the rest of the transcription factors and coactivators that are required for the TNFRp55 promoter activation.

Interestingly, a recent report has shown that TSA treatment in cancer cells leads to downregulation of TNFRp55 expression (14). Although cell-type specific responses between embryonic stem cells and cancer cells may account for the differences observed, the extremely high levels of TSA (2–4 µM) used by Imre et al. (14) are in sharp contrast to the low concentration of TSA (10–50 nM) used in the present study.

In conclusion, we have shown here for the first time that ES cells do not induce cytokine expression after TNF-{alpha} stimulation due to low levels of TNFRp55. This occurs through regulation of the transcription rate of the gene rather than decreased half-life of the transcript or methylation of the promoter region. Coordinated de novo synthesis of c-fos and increased binding of c-fos and Sp1 to the TNFRp55 promoter after treatment with the HDAC inhibitor TSA can induce the expression of functional TNFRp55 and restore the ability to upregulate the proinflammatory cytokine in TNF-{alpha}-stimulated ES cells.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the British Heart Foundation and the Oak Foundation.


   ACKNOWLEDGMENTS
 
We thank Dr. Benjamin Adams for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Zampetaki, Cardiovascular Div., School of Medicine, King's College London, James Black Centre, 125 Coldharbour Lane, London, SE5 9NU, UK (e-mail: anna.zampetaki{at}kcl.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* These authors contributed equally to this study. Back


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Allen RD, Staley TA, Sidman CL. Differential cytokine expression in acute and chronic murine graft-versus-host-disease. Eur J Immunol 23: 333–337, 1993.[Web of Science][Medline]

2. Ammanamanchi S, Brattain MG. Sp3 is a transcriptional repressor of transforming growth factor-beta receptors. J Biol Chem 276: 3348–3352, 2001.[Abstract/Free Full Text]

3. Bird AP, Wolffe AP. Methylation-induced repression—belts, braces, and chromatin. Cell 99: 451–454, 1999.[CrossRef][Web of Science][Medline]

4. Branen L, Hovgaard L, Nitulescu M, Bengtsson E, Nilsson J, Jovinge S. Inhibition of tumor necrosis factor-alpha reduces atherosclerosis in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 24: 2137–2142, 2004.[Abstract/Free Full Text]

5. Crisostomo PR, Wang M, Herring CM, Markel TA, Meldrum KK, LillemoeKD, Meldrum DR. Gender differences in injury induced mesenchymal stem cell apoptosis and VEGF, TNF, IL-6 expression: role of the 55 kDa TNF receptor (TNFR1). J Mol Cell Cardiol 42: 142–149, 2007.[CrossRef][Web of Science][Medline]

6. Discher DJ, Bishopric NH, Wu X, Peterson CA, Webster KA. Hypoxia regulates beta-enolase and pyruvate kinase-M promoters by modulating Sp1/Sp3 binding to a conserved GC element. J Biol Chem 273: 26087–26093, 1998.[Abstract/Free Full Text]

7. Elkind MS, Cheng J, Boden-Albala B, Rundek T, Thomas J, Chen H, Rabbani LE, Sacco RL. Tumor necrosis factor receptor levels are associated with carotid atherosclerosis. Stroke 33: 31–37, 2002.[Abstract/Free Full Text]

8. Erickson SL, de Sauvage FJ, Kikly K, Carver-Moore K, Pitts-Meek S, Gillett N, Sheehan KC, Schreiber RD, Goeddel DV, Moore MW. Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice. Nature 372: 560–563, 1994.[CrossRef][Medline]

9. Fraidenraich D, Stillwell E, Romero E, Wilkes D, Manova K, Basson CT, Benezra R. Rescue of cardiac defects in id knockout embryos by injection of embryonic stem cells. Science 306: 247–252, 2004.[Abstract/Free Full Text]

10. Hagen G, Muller S, Beato M, Suske G. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J 13: 3843–3851, 1994.[Web of Science][Medline]

11. Hattori N, Nishino K, Ko YG, Ohgane J, Tanaka S, Shiota K. Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells. J Biol Chem 279: 17063–17069, 2004.[Abstract/Free Full Text]

12. Hess J, Angel P, Schorpp-Kistner M. AP-1 subunits: quarrel and harmony among siblings. J Cell Sci 117: 5965–5973, 2004.[Abstract/Free Full Text]

13. Hu Y, Zou Y, Dietrich H, Wick G, Xu Q. Inhibition of neointima hyperplasia of mouse vein grafts by locally applied suramin. Circulation 100: 861–868, 1999.[Abstract/Free Full Text]

14. Imre G, Gekeler V, Leja A, Beckers T, Boehm M. Histone deacetylase inhibitors suppress the inducibility of nuclear factor-kappaB by tumor necrosis factor-alpha receptor-1 down-regulation. Cancer Res 66: 5409–5418, 2006.[Abstract/Free Full Text]

15. Jochum W, Passegue E, Wagner EF. AP-1 in mouse development and tumorigenesis. Oncogene 20: 2401–2412, 2001.[CrossRef][Web of Science][Medline]

16. Kadonaga JT, Carner KR, Masiarz FR, Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 51: 1079–1090, 1987.[CrossRef][Web of Science][Medline]

17. Kehat I, Khimovich L, Caspi O, Gepstein A, Shofti R, Arbel G, Huber I, Satin J, Itskovitz-Eldor J, Gepstein L. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol 22: 1282–1289, 2004.[CrossRef][Web of Science][Medline]

18. Kumar AP, Butler AP. Transcription factor Sp3 antagonizes activation of the ornithine decarboxylase promoter by Sp1. Nucleic Acids Res 25: 2012–2019, 1997.[Abstract/Free Full Text]

19. Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol 23: 845–856, 2005.[CrossRef][Web of Science][Medline]

20. Lamb NJ, Fernandez A, Tourkine N, Jeanteur P, Blanchard JM. Demonstration in living cells of an intragenic negative regulatory element within the rodent c-fos gene. Cell 61: 485–496, 1990.[CrossRef][Web of Science][Medline]

21. Lee JH, Hart SR, Skalnik DG. Histone deacetylase activity is required for embryonic stem cell differentiation. Genesis 38: 32–38, 2004.[CrossRef][Web of Science][Medline]

22. Leitges M, Mayr M, Braun U, Mayr U, Li C, Pfister G, Ghaffari-Tabrizi N, Baier G, Hu Y, Xu Q. Exacerbated vein graft arteriosclerosis in protein kinase Cdelta-null mice. J Clin Invest 108: 1505–1512, 2001.[CrossRef][Web of Science][Medline]

23. Levenberg S. Engineering blood vessels from stem cells: recent advances and applications. Curr Opin Biotechnol 16: 516–523, 2005.[CrossRef][Web of Science][Medline]

24. Lindvall O, Kokaia Z. Stem cells for the treatment of neurological disorders. Nature 441: 1094–1096, 2006.[CrossRef][Medline]

25. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104: 487–501, 2001.[CrossRef][Web of Science][Medline]

26. McCloskey KE, Gilroy ME, Nerem RM. Use of embryonic stem cell-derived endothelial cells as a cell source to generate vessel structures in vitro. Tissue Eng 11: 497–505, 2005.[CrossRef][Web of Science][Medline]

27. Meldrum DR, Cleveland JC Jr, Cain BS, Meng X, Harken AH. Increased myocardial tumor necrosis factor-alpha in a crystalloid-perfused model of cardiac ischemia-reperfusion injury. Ann Thorac Surg 65: 439–443, 1998.[Abstract/Free Full Text]

28. Menard C, Hagege AA, Agbulut O, Barro M, Morichetti MC, Brasselet C, Bel A, Messas E, Bissery A, Bruneval P, Desnos M, Puceat M, Menasche P. Transplantation of cardiac-committed mouse embryonic stem cells to infarcted sheep myocardium: a preclinical study. Lancet 366: 1005–1012, 2005.[CrossRef][Web of Science][Medline]

29. Mourkioti F, Rosenthal N. IGF-1, inflammation and stem cells: interactions during muscle regeneration. Trends Immunol 26: 535–542, 2005.[CrossRef][Web of Science][Medline]

30. Olsen AL, Stachura DL, Weiss MJ. Designer blood: creating hematopoietic lineages from embryonic stem cells. Blood 107: 1265–1275, 2006.[Abstract/Free Full Text]

31. Peschon JJ, Torrance DS, Stocking KL, Glaccum MB, Otten C, Willis CR, Charrier K, Morrissey PJ, Ware CB, Mohler KM. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J Immunol 160: 943–952, 1998.[Abstract/Free Full Text]

32. Pfeffer K, Matsuyama T, Kundig TM, Wakeham A, Kishihara K, ShahinianA, Wiegmann K, Ohashi PS, Kronke M, Mak TW. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell 73: 457–467, 1993.[CrossRef][Web of Science][Medline]

33. Riha GM, Lin PH, Lumsden AB, Yao Q, Chen C. Review: application of stem cells for vascular tissue engineering. Tissue Eng 11: 1535–1552, 2005.[CrossRef][Web of Science][Medline]

34. Rothe J, Lesslauer W, Lotscher H, Lang Y, Koebel P, Kontgen F, Althage A, Zinkernagel R, Steinmetz M, Bluethmann H. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 364: 798–802, 1993.[CrossRef][Medline]

35. Roy N, Laflamme G, Raymond V. 5' untranslated sequences modulate rapid mRNA degradation mediated by 3' AU-rich element in v-/c-fos recombinants. Nucleic Acids Res 20: 5753–5762, 1992.[Abstract/Free Full Text]

36. Shalaby MR, Sundan A, Loetscher H, Brockhaus M, Lesslauer W, Espevik T. Binding and regulation of cellular functions by monoclonal antibodies against human tumor necrosis factor receptors. J Exp Med 172: 1517–1520, 1990.[Abstract/Free Full Text]

37. Shaulian E, Karin M. AP-1 as a regulator of cell life and death. Nat Cell Biol 4: E131–E136, 2002.[CrossRef][Web of Science][Medline]

38. Sheehan KC, Pinckard JK, Arthur CD, Dehner LP, Goeddel DV, Schreiber RD. Monoclonal antibodies specific for murine p55 and p75 tumor necrosis factor receptors: identification of a novel in vivo role for p75. J Exp Med 181: 607–617, 1995.[Abstract/Free Full Text]

39. Siegfried Z, Eden S, Mendelsohn M, Feng X, Tsuberi BZ, Cedar H. DNA methylation represses transcription in vivo. Nat Genet 22: 203–206, 1999.[CrossRef][Web of Science][Medline]

40. Smith SC, Allen PM. Neutralization of endogenous tumor necrosis factor ameliorates the severity of myosin-induced myocarditis. Circ Res 70: 856–863, 1992.[Abstract/Free Full Text]

41. Srivastava D, Ivey KN. Potential of stem-cell-based therapies for heart disease. Nature 441: 1097–1099, 2006.[CrossRef][Medline]

42. Suske G. The Sp-family of transcription factors. Gene 238: 291–300, 1999.[CrossRef][Web of Science][Medline]

43. Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ 10: 45–65, 2003.[CrossRef][Web of Science][Medline]

44. Wang M, Crisostomo PR, Herring C, Meldrum KK, Meldrum DR. Human progenitor cells from bone marrow or adipose tissue produce VEGF, HGF, and IGF-I in response to TNF by a p38 MAPK-dependent mechanism. Am J Physiol Regul Integr Comp Physiol 291: R880–R884, 2006.[Abstract/Free Full Text]

45. Wooten LG, Ogretmen B. Sp1/Sp3-dependent regulation of human telomerase reverse transcriptase promoter activity by the bioactive sphingolipid ceramide. J Biol Chem 280: 28867–28876, 2005.[Abstract/Free Full Text]

46. Xiao Q, Zeng L, Zhang Z, Hu Y, Xu Q. Stem cell-derived Sca-1+ progenitors differentiate into smooth muscle cells, which is mediated by collagen IV, which is mediated by collagen IV-integrin {alpha}1/beta1/{alpha}v and PDGF receptor pathways. Am J Physiol Cell Physiol 292: C342–C352, 2007.[Abstract/Free Full Text]

47. Xiao Q, Zeng L, Zhang Z, Margariti A, Ali ZA, Channon KM, Hu Y, Xu Q. Sca-1+ progenitors derived from embryonic stem cells differentiate into endothelial cells capable of vascular repair after arterial injury [Online]. Arterioscler Thromb Vasc Biol 26: 2244, 2006.[Abstract/Free Full Text]

48. Zampetaki A, Xiao Q, Zeng L, Hu Y, Xu Q. TLR4 expression in mouse embryonic stem cells and in stem cell-derived vascular cells is regulated by epigenetic modifications. Biochem Biophys Res Commun 347: 89–99, 2006.[CrossRef][Web of Science][Medline]

49. Zampetaki A, Zhang Z, Hu Y, Xu Q. Biomechanical stress induces IL-6 expression in smooth muscle cells via Ras/Rac1-p38 MAPK-NF-{kappa}B signaling pathways. Am J Physiol Heart Circ Physiol 288: H2946–H2954, 2005.[Abstract/Free Full Text]

50. Zeng L, Lu M, Mori K, Luo S, Lee AS, Zhu Y, Shyy JY. ATF6 modulates SREBP2-mediated lipogenesis. EMBO J 23: 950–958, 2004.[CrossRef][Web of Science][Medline]

51. Zeng L, Xiao Q, Margariti A, Zhang Z, Zampetaki A, Patel S, Capogrossi MC, Hu Y, Xu Q. HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells. J Cell Biol 174: 1059–1069, 2006.[Abstract/Free Full Text]




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