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(Circulation Research. 1997;80:305-311.)
© 1997 American Heart Association, Inc.

Articles

E-Selectin Gene Expression in Vascular Smooth Muscle Cells

Evidence for a Tissue-Specific Repressor Protein

Xi-Lin L. Chen, Pradyumna E. Tummala, Lyn Olliff, Russell M. Medford

the Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga.

Correspondence to Russell M. Medford, MD, PhD, Emory University, Division of Cardiology, 1639 Pierce Drive, WMB 319, Atlanta, GA 30322.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
E-Selectin is an inducible, endothelium-specific, cell surface adhesion molecule that mediates inflammatory responses in the vasculature. Nonendothelial cell types such as cultured human aortic smooth muscle cells (HASMCs) lack the ability to express E-selectin. We tested the hypothesis that HASMCs express a negative regulatory factor that inhibits E-selectin gene expression. E-Selectin mRNA and gene transcription were not detected in HASMCs after treatment with tumor necrosis factor- (TNF-) by Northern and nuclear runoff analyses, respectively. However, both E-selectin mRNA and gene transcription were dramatically induced by TNF- in the same cells pretreated with the protein synthesis inhibitor cycloheximide. Lipopolysaccharide demonstrated similar effects. Furthermore, E-selectin was detected on the cell surface of HASMCs after washing out of cycloheximide. Cycloheximide pretreatment enabled immortalized human dermal microvascular endothelial cells that have lost the ability to express E-selectin to induce both E-selectin mRNA and gene transcription in response to TNF-. Induction of E-selectin mRNA by lipopolysaccharide or TNF- in cycloheximide-treated HASMCs was inhibited by the antioxidant pyrrolidinedithiocarbamate and the serine protease inhibitor N-L-tosyl-L-phenylalanine chloromethyl ketone, suggesting that a nuclear factor-B–like mechanism may play an important role in E-selectin gene expression in HASMCs. These data strongly suggest that a labile repressor protein(s) plays an important role in inhibiting E-selectin gene expression in HASMCs likely at the level of gene transcription. Except for this repressor, HASMCs and endothelial cells may share similar regulatory mechanisms for controlling E-selectin expression.

Key Words: E-selectin • vascular smooth muscle cell • gene expression

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
E-selectin is a member of the selectin family of endothelial cell adhesion proteins that recognize carbohydrate ligands on circulating leukocytes.^{1 2} E-Selectin plays a central role in the binding and extravasation of leukocytes from the blood stream into sites of inflammation.^{1 2} Expression of E-selectin is both tissue specific and stimulus specific, as it is expressed only on endothelial cells in response to induction by cytokines such as interleukin-1ß, TNF-, and lipopolysaccharides.^{3 4} E-Selectin expression is restricted to venular and capillary endothelial cells and is observed only at sites of acute and chronic inflammation.⁵ E-Selectin gene expression is primarily, but not exclusively, regulated at the level of transcription.^{4 6}

The NF-B and CRE/ATF families are two principal transcription factors involved in E-selectin promoter regulation. There are three NF-B binding sites in the E-selectin proximal promoter region.^{6 7 8 9} NF-B is involved in the control of cytokine-induced expression of many immune and inflammatory-response genes. In endothelial cells, NF-B is retained in the cytoplasm in an inactive complex with IB. Inducing agents such as cytokines cause the dissociation of IB from NF-B. NF-B is then translocated to the nucleus, where it binds to its recognition sites on the E-selectin promoter.^{10 11} A second element for regulation of the E-selectin gene is the CRE/ATF-like binding site. In contrast to NF-B, DNA binding of ATFs to the E-selectin promoter elements is constitutive.¹²

E-Selectin exhibits a highly restricted pattern of tissue-specific expression. E-Selectin is expressed only in endothelial cells activated by inflammatory stimuli.^{4 6 13 14} To date, E-selectin has not been reported to be expressed by HASMCs.^{15 16} The molecular mechanisms mediating this tissue-specific pattern of expression remain unclear. Endothelial cells, but not HASMCs, may express a positive regulatory factor that induces E-selectin expression. Conversely, HASMCs, but not endothelial cells, may express a negative regulatory factor that inhibits E-selectin expression. To test this hypothesis, we studied expression of the E-selectin gene in HASMCs by exposing HASMCs to inflammatory stimuli in the presence of the protein synthesis inhibitor cycloheximide. Our results suggest that a labile repressor protein(s) plays an important role in repressing E-selectin gene expression in HASMCs. Furthermore, these results suggest that, except for this repressor protein, HASMCs and endothelial cells may share similar regulatory mechanisms for controlling E-selectin expression.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
HASMCs obtained from Clonetics, Inc, were cultured in Smooth muscle Basal medium (Clonetics, Inc) supplemented with SmGm SingleQuots (Clonetics, Inc) and 5% fetal bovine serum. Experiments were performed with HASMCs at passages 5 to 9. Immortalized HMECs were obtained from the Department of Dermatology, Emory University School of Medicine. HMECs were cultured in MCDB 131 medium supplemented with 10% fetal bovine serum, hydrocortisone (1 ng/mL), epidermal growth factor (10 ng/mL), and bovine brain extracts containing heparin, gentamycin, and amphotericin (Clonetics, Inc). HeLa cells were obtained from American Type Tissue Culture Collection and maintained in minimal essential medium supplemented with 10% fetal bovine serum.

Determination of Cell Surface Expression of E-Selectin by ELISA
HASMCs were plated in 96-well plates and incubated with TNF- (100 U/mL), the protein synthesis inhibitor cycloheximide (10 µg/mL), or both for 4 hours. Cells were then washed three times with PBS and replenished with fresh medium. Cell surface expression of E-selectin was determined by ELISA after 4 hours of incubation. Primary antibodies for E-selectin were obtained from Dr Barry Wolitsky (Hoffman-LaRoche, Inc, Nutley, NJ). Cell surface expression of E-selectin was determined by primary binding with specific mouse antibodies, followed by secondary binding with a horseradish peroxidase–conjugated goat anti-mouse IgG antibody. Quantification was performed by determination of colorimetric conversion at 450 nm of 3,3',5,5'-tetramethylbenzidine.

Preparation of RNA and Northern Blot Analysis
Total cellular RNA was isolated by a single extraction using TriPure reagent (Boehringer Mannheim). Total cellular RNA (15 µg) was size-fractionated using 1% agarose-formaldehyde gels in the presence of 1 µg/mL ethidium bromide. The RNA was transferred to a nitrocellulose filter and covalently linked by ultraviolet irradiation using a Stratalinker UV cross-linker. Hybridizations were performed at 68°C for 1 hour in QuickHyb solution (Stratagene). Approximately 1 to 2x10⁶ cpm/mL of ³²P-labeled probes were used per hybridization. After hybridization, filters were washed with a final stringency of 0.2x SSC at 60°C for 30 minutes. The cDNAs used were a 1.35-kb Eco RI fragment of human E-selectin cDNA, a 1.9-kb HindIII–Xho I fragment of human VCAM-1 cDNA, an Xba I fragment of human ICAM-1 cDNA, and a 1.2-kb Pst I fragment of human GAPDH cDNA.¹⁷ Autoradiography was performed with an intensifying screen at -70°C. Laser densitometry and digital analysis of scanned images were used for quantification of autoradiographs.

Nuclear Run-on Transcription Assays
HASMCs and HMECs were pretreated with or without the protein synthesis inhibitor cycloheximide (10 µg/mL) for 15 minutes, followed by exposure to TNF- (100 U/mL). Nuclei were isolated 3 hours after induction. Nuclear run-on assay was performed as previously described,¹⁸ with modification. Briefly, nuclei (3x10⁷ per treatment for HMECs and 1x10⁷ per treatment for HASMCs) were resuspended in 300 µL of transcription buffer containing 100 µCi of [-³²P]UTP and incubated for 30 minutes at 30°C. The labeled RNA was purified by single extraction using TriPure reagent and hybridized to a nylon membrane filter that contained alkali denatured target cDNA. The filters were prepared by slot blotting of 5 µg of target DNA and covalently linked by UV irradiation using a Stratalinker UV cross-linker. The cDNAs used were a 1.35-kb EcoRI fragment of human E-selectin cDNA and a 1.2-kb Pst I fragment of human GAPDH cDNA.

DNA Transfection and CAT Analysis
HASMCs were split at the ratio to give 60% confluence in 60-mm tissue culture plates. HASMCs were transfected with 10 µg of p3418E-selectin/CAT plasmid (coordinates -3418 to +49; a gift of Dr Tucker Collins, Harvard University, Boston, Mass) by the calcium phosphate coprecipitation technique¹⁸ for 16 hours. After an 8-hour recovery period, HMECs were treated with or without TNF- (100 U/mL). After 18 hours, cell extracts were prepared by rapid freeze/thaw in 0.25 mol/L Tris, pH 8.0. Protein was assayed for CAT activity as previously described by Ahmad et al.¹⁹ Acetylated and unacetylated forms of chloramphenicol were separated by thin-layer chromatography. Imaging and quantification were performed using the PhosphorImager 445Si (Molecular Dynamics).

Nuclear Extract Preparation and Gel-Shift Analysis
HASMC and HMEC nuclear extracts were prepared as described by Ahmad et al.¹⁹ The oligonucleotide containing the E-selectin NF-B consensus sequence was synthesized. Its sequence is as follows: 5'GCCATTGGGGATTTCCTCTTTACTGGGCTCGAGATCTATG-3'. The sequences of the NF-B consensus binding site are underlined. The underlined italicized sequences represent an unrelated tail sequence added to serve as a template for synthesis of the double-stranded DNA. Preparation and labeling of the double-stranded DNA probe was performed as previously described.¹⁹ The DNA binding reaction was performed at 30°C for 15 minutes in a volume of 20 µL, containing 5 µg of nuclear extract, 225 µg/mL bovine serum albumin, 1.0x10⁵ cpm of ³²P-labeled probe, 0.1 µg/mL poly(dI-dC), and 15 µL of binding buffer (12 mmol/L HEPES, pH 7.9, 4 mmol/L Tris, 60 mmol/L KCl, 1 mmol/L EDTA, 12% glycerol, 1 mmol/L dithiothreitol, and 1 mmol/L phenylmethylsulfonyl fluoride). After the binding reaction, the samples were subjected to electrophoresis in 1x Tris-glycine buffer using 4% native polyacrylamide gels.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Protein Synthesis Inhibitors Enable Cytokine-Stimulated HASMCs and HMECs to Express E-Selectin mRNA
To investigate whether E-selectin gene expression can be induced by cytokines in cultured HASMCs, HASMCs were treated with LPS (100 ng/mL) or TNF- (100 U/mL). After 4 hours, total RNA was isolated and analyzed for the expression of adhesion molecule mRNA by Northern analysis. As expected, treatment of HASMCs with LPS or TNF- did not induce E-selectin mRNA accumulation (Fig 1A, lanes 1 to 3) but markedly induced ICAM-1 and VCAM-1 mRNA accumulation (lanes 2 and 3). To determine whether a regulatory protein inhibits E-selectin expression in HASMCs, we pretreated HASMCs with three different protein synthesis inhibitors. As shown in Fig 1A, the protein synthesis inhibitor cycloheximide (10 µg/mL) alone did not induce E-selectin mRNA accumulation in HASMCs (lane 4). However, when HASMCs were pretreated with cycloheximide 15 minutes before and throughout a 4-hour exposure to either LPS or TNF-, E-selectin mRNA was dramatically induced 4 hours after exposure to LPS (lane 5) or TNF- (lane 6) to levels comparable to ICAM-1 (lanes 4 and 5) and VCAM-1 (lanes 4 and 5). Similarly, pretreatment with two other protein synthesis inhibitors, puromycin (100 µmol/L) or anisomycin (100 µmol/L) alone did not induce E-selectin mRNA expression (Fig 1B, lane 3 or 5) in HASMCs. In the presence of puromycin or anisomycin, TNF- induced a dramatic increase in E-selectin mRNA accumulation (Fig 1B, lane 4 or 6). The kinetics of induction of E-selectin mRNA in HASMCs by TNF- in the presence of cycloheximide were very similar to those reported in cultured human endothelial cells.^{4 6} In HASMCs, TNF- (100 U/mL) in the presence of cycloheximide (10 µg/mL) induced E-selectin mRNA accumulation within 2 hours, with peak induction occurring at 4 to 8 hours (Fig 2). Cycloheximide alone did not induce E-selectin mRNA accumulation at any time points. These findings suggest that a labile repressor protein inhibits cytokine-induced E-selectin gene expression in HASMCs.

View larger version (37K): [in this window] [in a new window]   Figure 1. A, Effects of protein synthesis inhibitor cycloheximide (CHX) on the induction of E-selectin, VCAM-1, and ICAM-1 mRNA by LPS or TNF- in HASMCs. HASMCs were either pretreated (lanes 4 to 6) or not (lanes 1 to 3) with CHX (10 µg/mL) for 15 minutes and then exposed to either LPS (100 ng/mL, lanes 2 and 5) or TNF- (100 U/mL, lanes 3 and 6) for a 4-hour period. Total RNA was isolated, and 15 µg was size-fractionated by denaturing agarose–formaldehyde gel electrophoresis, transferred to a nitrocellulose membrane, hybridized with 32P-labeled human E-selectin, VCAM-1, ICAM-1, or GAPDH-specific cDNA, and visualized by autoradiography. CTL indicates control (lane 1). B, Effects of protein synthesis inhibitor, puromycin (Puro) or anisomycin (Aniso), on the induction of E-selectin mRNA by TNF- in HASMCs. HASMCs were either pretreated or not with Puro (100 µmol/L, lanes 3 and 4) or Aniso (100 µmol/L, lanes 5 and 6) for 15 minutes and then exposed to TNF- (100 U/mL; lanes 2, 4, and 6) for a 4-hour period. E-Selectin and GAPDH mRNA levels were determined by Northern analysis as described in panel A.

View larger version (37K): [in this window] [in a new window]   Figure 2. Kinetics of induction of E-selectin mRNA by TNF- in cycloheximide (CHX)–treated HASMCs. Total RNA was isolated from untreated or CHX (10 µg/mL)– and TNF- (100 U/mL)–treated HASMCs at the indicated times. E-Selectin and GAPDH mRNA levels were determined by Northern analysis as described in Fig 1A.

HMECs are an immortalized human dermal microvascular endothelial cell line that has lost the ability to express E-selectin.²⁰ To determine whether this is due to a repressor protein similar to that in HASMCs, we treated HMECs with TNF- (100 U/mL) in the presence or absence of cycloheximide (10 µg/mL) 15 minutes before and throughout a 4-hour exposure. As in HASMCs, TNF- alone did not induce E-selectin mRNA accumulation (Fig 3, lane 3) but dramatically induced VCAM-1 mRNA accumulation (Fig 3, lane 3). However, in the presence of cycloheximide, TNF- markedly induced E-selectin mRNA accumulation at 4 hours (Fig 3, lane 4). These results suggest that HMECs express a labile repressor protein, functionally analogous to that found in HASMCs, that inhibits the expression of the E-selectin gene.

View larger version (58K): [in this window] [in a new window]   Figure 3. Effects of cycloheximide (CHX) on the induction of E-selectin and VCAM-1 mRNA accumulation by TNF- in immortalized HMECs. HMECs were either pretreated (lanes 2 and 4) or not (lanes 1 and 3) with CHX (10 µg/mL) for 15 minutes and then exposed to TNF- (100 U/mL, lanes 3 and 4) for a 4-hour period. Total RNA was isolated, and E-selectin, VCAM-1, and GAPDH mRNA levels were determined by Northern analysis as described in Fig 1A.

To investigate whether nonvascular cells can express the E-selectin gene, human epithelial HeLa cells were exposed to LPS (100 ng/mL) or TNF- (100 U/mL) in the presence or absence of cycloheximide (10 µg/mL). Cycloheximide alone did not induce E-selectin gene expression in HeLa cells. Both LPS and TNF- induced ICAM-1 mRNA accumulation in HeLa cells. However, LPS or TNF-, alone or in the presence of cycloheximide, did not induce E-selectin gene expression in HeLa cells (data not shown). These data suggest that HeLa cells might lack tissue-specific positive regulatory factors for E-selectin gene expression found in HASMCs and HMECs.

Induction of E-Selectin Cell Surface Protein Expression in HASMCs
To investigate whether the translational and posttranslational apparatus for E-selectin gene expression is functional in HASMCs, a washout experiment was performed. To induce a high level of E-selectin mRNA, HASMCs were treated with TNF- (100 U/mL) in the presence of cycloheximide (10 µg/mL, 15 minutes before and throughout the experiment) for 4 hours. Cells were then washed three times with PBS to remove cycloheximide and incubated for another 4 hours, thus allowing translation of the mRNA encoding the E-selectin protein.²¹ Surface expression of E-selectin was determined by ELISA. As shown in Fig 4, neither TNF- nor cycloheximide alone induced E-selectin expression on the surface of HASMCs. However, in the presence of cycloheximide, TNF- induced E-selectin protein expression on the surface of HASMCs. These data suggest that HASMCs have an intact translational and posttranslational apparatus for cell surface E-selectin expression.

View larger version (25K): [in this window] [in a new window]   Figure 4. Expression of cell surface E-selectin in HASMCs. HASMCs in 96-well plates were pretreated with cycloheximide (CHX, 10 µg/mL) for 15 minutes and exposed to TNF- for 4 hours. Then the cells were washed with PBS three times and further incubated for 4 hours. Cell surface expression of E-selectin was determined by primary binding with specific mouse antibodies, followed by secondary binding with horseradish peroxidase–tagged goat anti-mouse (IgG) antibody. Quantification was performed by determination of colorimetric conversion at 450 nm of 3,3',5,5'-tetramethylbenzidine. Values represent mean±SD (n=12). *P<.05 compared with control group (CTL).

Cycloheximide Enables TNF- to Activate E-Selectin Gene Transcription in HASMCs and HMECs
To define the molecular mechanism through which this labile repressor protein(s) inhibits E-selectin gene expression, we performed nuclear run-on experiments to assess E-selectin gene transcription. HASMCs and HMECs were treated with or without cycloheximide (10 µg/mL) before and throughout a 3-hour exposure to TNF- (100 U/mL). Nuclei were isolated, and radiolabeled RNA generated by these nuclear preparations was hybridized to E-selectin cDNA or GAPDH cDNA. As shown in Fig 5A, when HASMCs were treated with cycloheximide or TNF- alone, there was no detectable E-selectin transcription (lanes 2 and 3). However, in TNF-–treated and cycloheximide-treated HASMCs and HMECs, E-selectin gene transcription was readily detected (lane 4). These data demonstrate that by blocking synthesis of a labile repressor protein, cycloheximide enables TNF- to activate E-selectin gene transcription in both HASMCs and HMECs.

View larger version (24K): [in this window] [in a new window]   Figure 5. Nuclear run-on analysis of E-selectin gene transcription after exposure to TNF-, cycloheximide (CHX), or both in HASMCs or immortalized HMECs. A, HASMC (top) or HMEC (bottom) cells were either pretreated (lanes 2 and 4) or not (lanes 1 and 3) with CHX (10 µg/mL) for 15 minutes and then exposed to TNF- (100 U/mL, lanes 3 and 4) for a 4-hour period. Nuclei were isolated, and 32P-labeled transcripts were purified. The RNA was hybridized to specific cDNA inserts of E-selectin and GAPDH immobilized on a nylon filter. CTL indicates control (lane 1). B, Graphic presentation of the E-selectin gene transcription rate after normalization with GAPDH. Filters were imaged using the PhosphorImager 445Si (Molecular Dynamics) and NIH Image 1.55 computer software.

The Antioxidant PDTC and the Serine Protease Inhibitor TPCK Inhibit TNF-–Induced andCycloheximide-Induced E-Selectin mRNA Accumulation in HASMCs
To investigate the potential role of NF-B in TNF-–induced E-selectin gene expression in cycloheximide-pretreated HASMCs, two inhibitors of NF-B activation were used: TPCK, an alkylating agent and a chymotrypsin-like serine protease inhibitor, and the thiol antioxidant PDTC.^{17 22 23} HASMCs were treated with or without TPCK (10 µmol/L) or PDTC (100 µmol/L) for 1 hour before and throughout a 4-hour exposure to TNF- in the presence of cycloheximide. As shown in Fig 6, in the presence of cycloheximide, TNF-–induced or LPS-induced E-selectin mRNA accumulation (lanes 2 and 3) was abolished by TPCK treatment (lanes 4 and 5), suggesting that induction of E-selectin mRNA by LPS or TNF- in the presence of cycloheximide in HASMCs depends on proteolysis-sensitive activity. Pretreatment of HASMCs with PDTC also abolished LPS-induced or TNF-–induced accumulation of E-selectin mRNA in cycloheximide-treated HASMCs (lanes 6 and 7). These data demonstrate that induction of E-selectin mRNA by LPS or TNF- in the presence of cycloheximide depends on proteolytic activity and is thiol antioxidant sensitive. These results are consistent with an NF-B–like mechanism playing a role in LPS-induced or TNF-–induced E-selectin gene expression in cycloheximide-treated HASMCs.

View larger version (38K): [in this window] [in a new window]   Figure 6. The serine protease inhibitor TPCK and the thiol antioxidant PDTC inhibit cycloheximide (CHX)– and TNF-–induced E-selectin mRNA accumulation in HASMCs. HASMCs were pretreated with CHX and either TPCK (10 µmol/L, lanes 4 and 5) or PDTC (100 µmol/L, lanes 6 and 7) for 1 hour and then exposed to LPS (100 ng/mL; lanes 2, 4, and 6) or TNF- (100 U/mL; lanes 3, 5, and 7) for an additional 4-hour period. CTL indicates control (lane 1). RNA was isolated, and E-selectin and GAPDH mRNA levels were determined by Northern analysis as described in Fig 1A.

Labile Repressor Protein Does Not Inhibit Activation of NF-B or E-Selectin Promoter Transactivation by TNF-
To further characterize the role of NF-B, nuclear extracts were prepared from HASMCs 1 hour after exposure to TNF- (100 U/mL) in the presence or absence of cycloheximide (10 µg/mL, 15 minutes before and throughout the experiment) and tested by electrophoretic mobility shift assay for NF-B binding activity to the E-selectin NF-B consensus sequence elements. As shown in Fig 7, there was a small basal nuclear NF-B binding activity in nonstimulated cells (lane 1). Cycloheximide alone did not increase NF-B binding activity compared with nonstimulated cells (lane 2). Treatment with TNF- led to a large increase in NF-B binding (lane 3). Cotreatment with cycloheximide and TNF- produced a small superinduction of NF-B binding (lane 4). Specificity of the NF-B–like binding activity was confirmed by inhibition of complex formation by 100-fold excess molar of unlabeled probe, but not by an unrelated probe. There is a second nonspecific binding band that is not inhibited by unlabeled probe. To determine whether NF-B activation is sufficient to transactivate the E-selectin promoter, HASMCs were transfected with p3418E-selectin/CAT, a deletion construct of the human E-selectin promoter (coordinates -3418 to +49). As shown in Fig 8, there was some basal activity in nonstimulated cells; treatment with TNF- (100 U/mL) markedly induced p3418E-selectin/CAT activity in HASMCs. These data demonstrate that accumulation of functional nuclear NF-B is not sufficient to activate E-selectin gene expression but is sufficient to transactivate the E-selectin promoter. These data further suggest that the elements from -3418 to +49 of the E-selectin promoter are not sufficient to direct cell-specific labile repressor–mediated expression of E-selectin.

View larger version (40K): [in this window] [in a new window]   Figure 7. Effects of cycloheximide (CHX), TNF-, or both on induction of nuclear NF-B–like binding activity in HASMCs. Nuclear extracts from unstimulated HASMCs (CTL, lane 1) and HASMCs treated with CHX (10 µg/mL, lane 2), TNF- (100 U/mL, lane 3), or TNF-+CHX (lane 4) for 1 hour were incubated with E-selectin wild-type probe containing the NF-B consensus sequence. NF-B indicates NF-B–like binding activity; Non-spe, nonspecific binding activity.

View larger version (35K): [in this window] [in a new window]   Figure 8. TNF- transactivates the human E-selectin promoter HASMCs. A, HASMCs were transfected with p3418E-selectin/CAT by the calcium phosphate coprecipitation technique. After an 18-hour exposure to TNF- (100 U/mL), cytoplasmic extracts were prepared, and CAT activity was determined. Ac indicates acetylated [14C]chloramphenicol, which correlates with promoter transcription activity; Non-Ac, nonacetylated [14C]chloramphenicol; and CTL, control. B, Graph shows CAT activity for the same experiment expressed by percent conversion of Non-Ac to Ac.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Until the present report, E-selectin gene expression has been observed exclusively on activated endothelial cells.^{4 6 13 14} The molecular mechanisms underlying this endothelium-specific expression of the E-selectin gene are not clear. In the present study, we have established that E-selectin mRNA accumulation can be induced in cultured HASMCs by LPS or TNF- in the presence of the protein synthesis inhibitor cycloheximide. To our knowledge, this is the first report of E-selectin gene expression in nonendothelial cells. From the present study and published data, we have established three different types of tissue-specific regulation of E-selectin gene expression: (1) In endothelial cells, TNF- or LPS can directly induce E-selectin gene expression. (2) In vascular smooth muscle cells, TNF- or LPS can only stimulate E-selectin gene expression in the presence of a protein synthesis inhibitor. (3) In HeLa cells, E-selectin gene expression could not be induced regardless of the presence of a protein synthesis inhibitor. These data suggest that vascular nonendothelial cells, such as HASMCs, express the necessary regulatory factors required for E-selectin gene expression, yet their ability to express the E-selectin gene is suppressed by a labile repressor protein; in contrast, nonvascular cells, such as HeLa cells, do not possess the essential regulatory factors required for E-selectin gene expression.

Vascular smooth muscle cells may participate in inflammatory responses in the vascular wall by expression of leukocyte adhesion molecules. It is reported that the VCAM-1 and ICAM-1 genes are expressed in vascular smooth muscle cells in vivo in atherosclerotic plaques in humans and rabbits.^{24 25} We reported previously that TNF- can induce VCAM-1 and ICAM-1 but not E-selectin gene expression in HASMCs.¹⁵ Similar findings have been reported by Couffinhal et al.¹⁶ To explore whether negative regulatory proteins suppress E-selectin gene expression in HASMCs, we found that LPS or TNF- can induce E-selectin mRNA accumulation in protein synthesis inhibitor–pretreated HASMCs. We have also demonstrated that E-selectin mRNA transcribed in the presence of cycloheximide is functional. When cycloheximide was washed out of the cultures, HASMCs expressed E-selectin on the cell surface, as measured by ELISA. These data suggest that HASMCs possess the intact transcriptional, translational, and posttranslational apparatus for E-selectin gene expression. However, HASMCs express an additional labile negative regulatory element(s) that inhibits E-selectin expression.

We extended these observations to HMECs, an immortalized human dermal microvascular endothelial cell line that has lost the ability to express E-selectin while retaining the ability to express two other inducible adhesion molecules, VCAM-1 and ICAM-1.²⁰ In the present study, we found that E-selectin mRNA can be induced in HMECs by TNF- in the presence of the protein synthesis inhibitor cycloheximide. These data suggest that rather than loss of positive regulatory factors for E-selectin gene expression, HMECs express a specific labile negative regulatory protein that inhibits E-selectin gene expression. This labile repressor protein in HMECs is functionally analogous to that which we described in HASMCs.

The labile E-selectin repressor protein(s) may function through two regulatory mechanisms. First, it may function as a transcriptional repressor and inhibit E-selectin gene transcription. Second, it may function as a specific RNase and selectively degrade newly synthesized E-selectin mRNA. There are numerous reports on the selective degradation of hormone- or cytokine-induced mRNAs.^{26 27 28 29} E-Selectin mRNA has a short half-life.²¹ Consistent with this, the 3'-untranslated region of this mRNA carries multiple AUUUA elements characteristic of highly unstable mRNA.^{28 30} Protein synthesis inhibitors, such as cycloheximide, superinduce E-selectin mRNA in human umbilical vein endothelial cells as a result of both increased E-selectin mRNA stability and increased E-selectin gene transcription initiation.²¹ However, according to our nuclear run-on transcription analysis, the E-selectin repressor protein functions as a transcriptional repressor, although an additional role in control of mRNA stability cannot be ruled out.

Activation of the transcriptional factor NF-B is necessary but not sufficient for the induction of E-selectin gene expression in endothelial cells.^{6 7 8 9} TPCK and PDTC have been reported to block NF-B activation.^{17 22 23} Both TPCK and PDTC inhibited LPS-induced or TNF-–induced E-selectin gene expression in cycloheximide-treated HASMCs. These results suggest that an NF-B–like transcriptional mechanism may play an important role in cytokine-induced E-selectin gene expression in cycloheximide-treated HASMCs.

Several mechanisms have been proposed for negative regulation of gene transcription in eukaryotes.^{31 32 33} One of the earliest steps at which a repressor could interfere with the activity of a transcriptional factor is inhibition of transport of the activator from the cytoplasm into the nucleus. A second mechanism is competition between the repressor and the activating factor. A third mechanism is blocking DNA-bound activating factor to the transcription initiation complex. A fourth mechanism for negative control of gene transcription is silencing, wherein repressor binding blocks transcription irrespective of the location of the operator relative to the enhancer and the promoter.

Three lines of evidence suggest that this labile repressor protein does not interfere with NF-B signaling. First, two other adhesion molecules, VCAM-1 and ICAM-1, whose induction is also mediated by NF-B,^{34 35} are induced by TNF- and LPS alone, suggesting functional activation of NF-B. Second, TNF- alone induces nuclear NF-B binding activity to the E-selectin promoter/enhancer in HASMCs and HMECs. Third, TNF- alone can transactivate transiently transfected p839- or p3418-E-selectin/CAT promoter constructs in HMECs.

Whelan and colleagues^{6 36} have reported that the pattern of interleukin-1ß–induced expression of E-selectin/reporter gene chimeras (coordinates -2500 to +80) in HeLa cells is very similar to that observed in endothelial cells.^{6 36} We reported in the present study that transcriptional regulatory elements between -3418 and +49 are not sufficient to confer cell-specific regulation of E-selectin gene expression. Lack of cell specificity for the E-selectin promoter further suggests that this labile E-selectin repressor does not interfere with NF-B binding or the ability of bound NF-B to transactivate the E-selectin promoter in HMECs, since this requires the repressor to act in the immediate vicinity of, or exactly at, the activator DNA binding site for its action. Tissue-specific regulation of gene expression can be conferred as far as 10 kb upstream from the transcription start site or at downstream sequences.^{37 38 39}

The CRE/ATF element of the E-selectin promoter is necessary for full cytokine responsiveness.^{12 40} Recently, Pescini et al⁴¹ have identified a variant of the ATF/CREB transcriptional factor ATF-a0, which can form a dominant transcription inhibitor in an ATF-a heterodimer. It has been reported⁴⁰ that increases in cAMP levels decrease the E-selectin promoter response to TNF- in endothelial cells and that cAMP-mediated inhibition maps to the ATF element.^{41 42} However, CRE/ATF elements are unlikely to be involved in inhibiting E-selectin expression in HMECs, as the E-selectin promoter used in transfection contains the CRE-ATF binding site.

Although by transient transfection study the E-selectin promoter/enhancer does not have tissue specificity, the transiently transfected promoter does not integrate into genomic DNA. The interaction of the transcription factor with the promoter may be different in episomal conditions compared with a genomic DNA environment. We cannot rule out the possibility that the repressor protein may interact with the proximal E-selectin promoter when it resides in its natural genomic DNA environment. Alternatively, alteration in chromosome structure^{31 32 33} or DNA methylation³⁶ could mediate this effect. However, there is no report that this mechanism is mediated through repressor proteins.

In summary, the E-selectin gene is not expressed by cytokine-activated HASMCs. However, in the presence of the protein synthesis inhibitor cycloheximide, LPS or TNF- markedly induces E-selectin gene expression. Similar to endothelial cells, an NF-B–like transcription mechanism is involved in TNF-–induced expression of E-selectin in cycloheximide-treated HASMCs. These data demonstrate that a labile repressor protein plays an important role in E-selectin expression in HASMCs. These results suggest that except for this repressor protein, vascular smooth muscle cells and endothelial cells share similar regulatory mechanisms for controlling E-selectin expression.

	Selected Abbreviations and Acronyms

 

ATF = activating transcription factor CAT = chloramphenicol acetyltransferase CRE = cAMP-responsive element ELISA = enzyme-linked immunosorbent assay GAPDH = glyceraldehyde phosphate dehydrogenase HASMC = human aortic smooth muscle cell HMEC = human dermal microvascular endothelial cell IB = inhibitor-B ICAM-1 = intercellular adhesion molecule-1 LPS = lipopolysaccharide NF-B = nuclear factor-B PDTC = pyrrolidine dithiocarbamate TNF- = tumor necrosis factor- TPCK = N-L-tosylphenylalanine chloromethyl ketone VCAM-1 = vascular cell adhesion molecule-1

	Acknowledgments

 
This study was supported by National Institutes of Health research grant PO1-HL-48667 (Dr Medford) and by an American Heart Association, Georgia Affiliate, Grant-in-Aid (Drs Chen and Medford). Dr Medford is an Established Investigator of the American Heart Association. We are very thankful to Dr Margaret Offermann (Winship Cancer Center, Emory University) for critical reading of the manuscript and to Kate W. Harris for editing the manuscript.

Received July 15, 1996; accepted November 27, 1996.

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