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Am J Physiol Gastrointest Liver Physiol 291: G26-G34, 2006. First published February 23, 2006; doi:10.1152/ajpgi.00467.2005
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LIVER AND BILIARY TRACT

Shear stress induces hepatocyte PAI-1 gene expression through cooperative Sp1/Ets-1 activation of transcription

Hideki Nakatsuka,1,* Takaaki Sokabe,2,* Kimiko Yamamoto,2 Yoshinobu Sato,1 Katsuyoshi Hatakeyama,1 Akira Kamiya,2 and Joji Ando2

1Department of Digestive and General Surgery, Graduate School of Medicine, Niigata University, Niigata; and 2Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

Submitted 7 October 2005 ; accepted in final form 15 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Partial hepatectomy causes hemodynamic changes that increase portal blood flow in the remaining lobe, where the expression of immediate-early genes, including plasminogen activator inhibitor-1 (PAI-1), is induced. We hypothesized that a hyperdynamic circulatory state occurring in the remaining lobe induces immediate-early gene expression. In this study, we investigated whether the mechanical force generated by flowing blood, shear stress, induces PAI-1 expression in hepatocytes. When cultured rat hepatocytes were exposed to flow, PAI-1 mRNA levels began to increase within 3 h, peaked at levels significantly higher than the static control levels, and then gradually decreased. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent and accompanied by increased hepatocyte production of PAI-1 protein. Shear stress increased PAI-1 transcription but did not affect PAI-1 mRNA stability. Functional analysis of the 2.1-kb PAI-1 5'-promoter indicated that a 278-bp segment containing transcription factor Sp1 and Ets-1 consensus sequences was critical to the shear stress-dependent increase of PAI-1 transcription. Mutations of both the Sp1 and Ets-1 consensus sequences, but not of either one alone, markedly prevented basal PAI-1 transcription and abolished the response of the PAI-1 promoter to shear stress. EMSA and chromatin immunoprecipitation assays showed binding of Sp1 and Ets-1 to each consensus sequence under static conditions, which increased in response to shear stress. In conclusion, hepatocyte PAI-1 expression is flow sensitive and transcriptionally regulated by shear stress via cooperative interactions between Sp1 and Ets-1.

portal flow; hemodynamic force; immediate-early gene; partial hepatectomy; liver regeneration

THE PROGNOSIS FOR RECOVERY after partial hepatectomy, portal branch ligation, and liver transplantation depends on how well the liver regenerates after the operation. Thus understanding the mechanisms of liver regeneration is of fundamental importance to the prevention of postoperative liver failure and improving the outcome. The role of a variety of cytokines and growth factors has been extensively studied as triggers of liver regeneration (5, 16), but priming by hepatocytes themselves rather than cytokines or growth factors is now considered necessary for the initiation of liver regeneration, because liver regeneration fails to occur when a large dose of hepatocyte growth factor (HGF), a complete mitogen for hepatocytes, is administered (6, 32). The results of recent studies, including our own, have suggested that hemodynamic forces arising from changes in portal blood flow and pressure after partial hepatectomy or portal branch ligation may be involved in a priming mechanism for liver regeneration (18, 21, 23, 31); however, how hemodynamic forces affect liver cells has never been elucidated.

Because hepatocytes face the Disse spaces, into which portal blood flows through the sieve plate of the hepatic sinusoids, they are directly exposed to the mechanical force of shear stress exerted by flowing blood. When portal blood flow increases in intact liver tissue after partial hepatectomy or portal branch ligation, the shear stress to which hepatocytes are exposed increases and may affect hepatocyte functions. Alterations of the morphology, function, and gene expression of various types of cells, including vascular endothelial cells, vascular smooth muscle cells, and bone cells, have been shown to occur in response to shear stress (1, 2, 19), but few studies have investigated the effect of shear stress on hepatocytes. Kan et al. (9) observed that shear stress enhanced the rates of ammonia removal and urea synthesis by hepatocytes in a hepatocyte/nonparenchymal cell coculture system. Tilles et al. (30) investigated the function of rat hepatocytes cocultured with fibroblasts exposed to flow in a bioreactor and showed that shear stress affects hepatocyte function, including the synthesis of albumin and urea. Recent in vivo studies (17, 22, 27, 29) have revealed that the expression of a number of genes, including immediate-early genes such as early growth response factor-1 (Egr-1), plasminogen activator inhibitor-1 (PAI-1), and phosphatase of regenerating liver-1 (PRL-1), is induced in hyperperfused lobes during the first few hours after partial hepatectomy or portal branch ligation. An in situ hybridization analysis of PAI-1 mRNA in the liver after partial hepatectomy showed that the majority of the positive cells were hepatocytes, although PAI-1 mRNA was also localized in venous endothelial cells, capsular mesothelial cells, and sinusoidal cells (22, 29). Nevertheless, it remained unclear whether the induction of these genes in hepatocytes was attributable to shear stress.

To investigate whether shear stress affects gene expression by hepatocytes, we exposed cultured hepatocytes to controlled levels of shear stress in a flow-loading apparatus and examined them for changes in gene expression. We focused on PAI-1 rather than other immediate-early genes, such as a transcription factor Egr-1 and growth-related tyrosine kinase PRL-1, because PAI-1 is involved in fibrinolysis and in a number of biological processes, including extracellular matrix degradation, cell migration, and angiogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. All protocols were approved by the Institutional Animal Care and Use Committee of the University of Tokyo. Rat hepatocytes were isolated from male Wistar strain rats by a method in which the liver was perfused with collagenase (type I, Sigma), as previously described (24). Isolated cells were plated on a collagen type I (0.3 mg/ml)-coated culture dishes and cultured in Williams' medium E (Sigma) supplemented with 10% FBS. Rat hepatocytes transformed with SV40 [RTH33 (28)] were obtained from the RIKEN Cell Bank (Tsukuba, Japan) and grown in DMEM supplemented with 10% FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin on collagen type I (0.3 mg/ml)-coated tissue culture dishes. Human hepatoma cells (C3A) were obtained from the American Type Culture Collection (Manassas, VA) and cultured in MEM containing 10% FBS, 1 mM sodium pyruvate, 50 U/ml penicillin, and 50 µg/ml streptomycin on collagen type I (0.3 mg/ml)-coated dishes. Murine hepatocytes transformed with SV40 [TLR-2 (33)] were obtained from the RIKEN Cell Bank and grown in MEM{alpha} supplemented with 2% FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin on collagen type I (0.3 mg/ml)-coated dishes.

Flow-loading experiments. We applied controlled levels of shear stress to cultured cells by using the same parallel plate-type flow chamber as described previously (14). One side of the chamber was formed by the coverslip on which the hepatocytes were cultured, the base and walls were machined from a polymethacrylate plate, and the two flat surfaces of the coverslip and base were held ~200 µm apart by a Silicone rubber gasket. The chamber had an entrance and an exit for the medium, and the entrance was connected to a reservoir with a Silicone tube. The medium was perfused through the chamber by a roller/tube pump (ATTO; Tokyo, Japan). The entire circuit was placed in an automated CO2 incubator, and the flow-loading experiments were performed at 37°C in an atmosphere of 95% room air and 5% CO2. The intensity of shear stress ({tau}; in dyn/cm2) on the cell layer was calculated by the formula {tau} = 6µQ/a2b, where µ is the viscosity of the perfusate (in Poise), Q is flow volume (in ml/s), and a and b are cross-sectional dimensions of the flow path. Because the maximum Reynolds number corresponding to the highest flow rate used in this study was around 40, we assumed that the flow was laminar.

Real-time PCR analysis. Total RNA samples were prepared from cells with ISOGEN (Nippon Gene; Tokyo, Japan), and first-strand cDNAs were generated using Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL) and RNA primed with oligo-dT primer. After reverse transcription of the RNA into cDNA, real-time PCR was used to monitor gene expression with a Smart Cycler (Cepheid) according to standard procedures. PCR was performed with a Takara EX Taq R-PCR kit (Takara) and primer pairs for each PAI-1 of the rat (5'-GAGCCAGATTCATCATCAACG-3' and 5'-CTGCAATGAACATGCTGAGG-3'), human (5'- GTGTTTCAGCAGGTGGCGC-3' and 5'-CCGGAACAGCCTGAAGAAGTG-3'), and mouse (5'-AGGGCTTCATGCCCCACTTCTTCA-3' and 5'-AGTAGAGGGGCATTCACCAGCACCA-3'), respectively. The temperature profile consisted of initial denaturation for 30 s at 95°C, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and elongation at 72°C, and fluorescence monitoring at 85°C. The specificity of the amplification reaction was determined by performing a melting curve analysis. Relative signal quantification was achieved by normalizing the signals of the different genes to the signals of the beta-actin gene.

ELISA. The amount of PAI-1 excreted by the hepatocytes was assayed using commercially available ELISA kits (Biopool). Briefly, 50 µl of perfusate were incubated for 2 h on a microplate coated with anti-rat monoclonal antibody against PAI-1. After a wash with detergent, PAI-1 conjugate was added, and incubation was performed for 1 h. The color reagent was then added, and incubation was continued for 20 min. Absorbance at 492 nm was measured with a microplate reader (Bio-Rad), and the concentration of PAI-1 in each sample was determined by comparison with the standard curve.

Nuclear runon assay. A nuclear runon assay was performed by the method described previously (14). Briefly, nuclear extracts obtained from hepatocytes were reacted in reaction buffer containing [{alpha}-32P]dCTP, and 32P-labeled RNA was extracted. Plasmids containing cDNA inserts for PAI-1 or beta-actin (Clontech) were spotted onto nylon membranes, and the DNA was hybridized to the radiolabeled RNA. Autoradiograms were obtained with the GS363 Molecular Imager System (Bio-Rad).

Luciferase assay. Reporter plasmids containing the human PAI-1 promoter (a kind gift of Dr. M. Kurabayashi, School of Medicine, Gunma University, Gunma, Japan) and the rat PAI-1 promoter (a kind gift of Dr. T. Kietzmann, Institute of Biochemistry and Molecular Cell Biology, Georg-August-Universitat, Göttingen, Germany) were used for the transcription assay. The following constructs were generated: –2071 luc, –766 luc, –278 luc, and –414 luc. For –2071 luc, PCR was performed to amplify –2074 to +50 of the rat PAI-1 promoter upstream region using a rat genome as a template, two synthetic oligonucleotide primers (5'-CTGGTTGCCCTGGTATCTGTTTAC-3' and 5'-TTCCTCCTTCACAAAGCTCTCG-3'), and pyrobest DNA polymerase. The 2,071-bp fragment was cloned into pBluescript II KS vector with EcoRV, digested with SacI and XhoI, and cloned into pGL2-basic. For –766 luc, 779 bp (–753 to +26) of the rat PAI-1 upstream region was cloned into pGL2-basic with SacI and HindIII. For –278 luc, 307 bp (–281 to +26) of the rat PAI-1 upstream region was cloned into pGL2-basic with KpnI and HindIII. For –414 luc, 657 bp (–575 to +82) of the human PAI-1 upstream region was cloned into pGL2-basic with KpnI and HindIII.

The constructs were transfected into hepatocytes with FuGENE (Roche Diagnostics), and the phRL-TK vector (Promega) was cotransfected to normalize transfection efficiency. After 24 h, cells were either incubated under static conditions or exposed to shear stress for 6 h, and luciferase activity was determined with a dual-luciferase reporter assay system (Promega) and a luminometer (Berthold).

Site-directed mutagenesis. A QuikChange Site-Directed Mutagenesis Kit (Stratagene) was used to generate mutations at a consensus binding site (5'-TGGGTGGGGCT-3' in humans and rats) for the known transcription factor Sp1 and a consensus binding site (5'-TATTTCCTGC-3' in humans and 5'-TATTTCCGGC-3' in rats) for the known transcription factor Ets-1. Briefly, PCR was performed using templates (–278 luc or –414 luc) and primers containing specific mutations (indicated by underlines) for rat Sp1 (5'-GAGTTAGAAGGTGTTGTGGGCTGGAACATG-3' and 5'-CATGTTCCAGCCCACAACACCTTCTAACTC-3'), rat Ets-1 (5'-TCATCTATTCTTGGCCCACATCTGGTATAA-3' and 5'-TTATACCAGATGTGGGCCAAGAATAGATGA-3'), human Sp1 (5'-GAGCCAGTGAGTGTTTTGGGCTGGAACATG-3' and 5'-CATGTTCCAGCCCAAAACACTCACTGGCTC-3'), and human Ets-1 (5'-TCATCTATTCTTTGCCCACATCTGGTATAA-3' and 5'-TTATACCAGATGTGGGCAAAGAATAGATGA-3') using PfuTurbo DNA polymerase.

EMSA. EMSA was performed using nuclear extracts obtained from hepatocytes as previously described (26). An oligonucleotide containing a rat or human Sp1 or Ets-1 consensus element (Table 1) was labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP. Binding reactions between the radiolabeled oligonucleotides and 2.5 µg of nuclear extracts protein were allowed to proceed at 22°C in a total volume of 10 µl binding buffer [10 mM Tris·HCl, 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 4% glycerol, 0.5 mM DTT, and 0.05 mg/ml poly(dI-dC)], and the reaction mixtures were separated by 4.7% PAGE in 0.5x Tris·HCl-boric acid-EDTA buffer (45 mM Tris·HCl, 45 mM boric acid, and 1 mM EDTA; pH 8.3) for 70 min at 350 V at 4°C. The protein-DNA complexes were analyzed with a GS363 molecular imager system (Bio-Rad). In a supershift assay, antibodies were added to the binding reaction, and the mixture was incubated for 30 min before the labeled oligonucleotide was added. Antibodies against Sp1 and Ets-1 were purchased from Santa Cruz Biotechnology.


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  		Table 1. Sequences of DNA probes used for EMSA

 
Chromatin immunoprecipitation assay. After 1 x 107 cells were cross linked with 1% formaldehyde at room temperature for 15 min, the cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 M. Cells were washed with ice-cold PBS, centrifuged, and resuspended in lysis buffer (1% SDS, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 50 mM Tris·HCl; pH 8.1), and then sonicated for 15 s three times. Supernatants were then recovered by centrifugation and diluted 1:10 in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 16.7 mM Tris·HCl; pH 8.1). Nonspecific background was removed by incubating the chromatin resuspension with 2 µg of salmon sperm DNA and 20 µl of protein A-Sepharose (Amersham) for 30 min at 4°C. A 10-µl sample of each chromatin supernatant was saved before the chromatin immunoprecipitation (ChIP) assay as "Input" (positive control). Immunoprecipitation was performed overnight with specific antibodies against Sp1 and Ets-1 (Santa Cruz Biotechnology), respectively, at 4°C with rotation. Then, 2 µg of sheared salmon sperm DNA and 20 µl of protein A-Sepharose (of 50% slurry) were added and allowed to react for 2 h at 4°C. The beads were pelleted by centrifugation and washed sequentially with the following buffers: low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris·HCl; pH 8.1), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, and 20 mM Tris·HCl; pH 8.1), and LiCl wash buffer (0.25 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris·HCl; pH 8.1). Bead precipitates were washed twice with Tris·HCl-EDTA buffer (1 mM EDTA and 10 mM Tris·HCl; pH 8.0) and eluted with 1% SDS and 0.1 M NaHCO3, and the eluates were pooled and heated at 65°C overnight to reverse the formaldehyde cross linking. Supernatants were incubated for 1 h at 45°C with 40 µg/ml proteinase K, and genomic DNA fragments were recovered.

PCR was performed with Ex Taq polymerase (Takara) and the following primers that amplify the part of rat and human PAI-1 promoters that contain the Sp-1 binding sites: 5'-GCAAGTTACTGGGAGGGAGG-3' and 5'-GATGAACTCATGTTCCAGCC-3' or 5'-TCAGCAAGTCCCAGAGAGGG-3' and 5'-GATGAACTCATGTTCCAGCC-3', respectively; and the part that contains the Ets-1 binding sites: 5'-GAACATGAGTTCATCTATTTC-3' and 5'-GATCTGCAGCAGCCTGATCC-3' or 5'-GAACATGAGTTCATCTATTTC-3' and 5'-CAGCGCTCTTGGCCCTGCAG-3', respectively. "Mock" samples contained dilution buffer instead of chromatin (negative control). Amplified DNA fragments were cloned into the pCR-II-Topo vector (Invitrogen), and DNA sequences showed complete homology to the parts of each gene (Applied Biosystems).

Statistical analysis. All results are expressed as means ± SD. Statistical significance was evaluated by ANOVA and a Bonferonni adjustment applied to the results of a t-test performed with SPSS software. P values of <0.05 were regarded as statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Flow-induced changes in expression of the PAI-1 gene in hepatocytes. Cultured hepatocytes were exposed for 3, 6, 12, 24, 48, and 72 h to flow exerting a shear stress of 10 dyn/cm2, and changes in PAI-1 mRNA levels were analyzed by real-time PCR. The PAI-1 mRNA level of primary cultures of rat hepatocytes began to increase 3 h after the start of exposure to flow, peaked at around threefold the static control level at 12 h, and then gradually decreased, remaining at around twofold the control level after exposure to flow for 72 h (Fig. 1A). A flow-induced increase in PAI-1 expression was also observed in cultured rat hepatocytes transformed with SV40 (RTH33; Fig. 1B), human hepatoma cells (C3A; Fig. 1C), and murine hepatocytes transformed with SV40 (TLR-2; Fig. 1D), although the temporal pattern of the PAI-1 response to flow varied from cell line to cell line.


Figure 1
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  		Fig. 1. Effect of flow on plasminogen activator inhibitor (PAI)-1 mRNA levels in hepatocytes. A: primary cultures of rat hepatocytes. B: rat hepatocytes transformed with SV40 (RTH33). C: human hepatoma cells (C3A). D: murine hepatocytes transformed with SV40 (TLR-2). Total RNA was isolated from hepatocytes that had been exposed to flow (laminar shear stress: 10 dyn/cm2) for the periods indicated or maintained as a static control, indicated by time 0. Samples of mRNA were then analyzed using real-time PCR. Flow increased the PAI-1 mRNA level in primary cultures of rat hepatocytes, rat RT33 cells, human C3A cells, and murine TLR-2 cells. All values are means ± SD of 3 separate experiments. *P < 0.01 vs. static control.

 
Flow-induced changes in the release of PAI-1 by hepatocytes. Rat hepatocytes (RTH33) were exposed to flow exerting a shear stress of 10 dyn/cm2 for 3, 6, 12, 24, 48, and 72 h, and the concentration of PAI-1 protein in the perfusion medium was determined by ELISA. The hepatocytes released very little PAI-1 under static conditions, but release of PAI-1 increased markedly in response to flow (Fig. 2). PAI-1 release began to increase at 3 h, peaked at ~14-fold the control level at 12 h, then gradually decreased, remaining at ~2-fold the control level at 72 h. The temporal patterns suggested that the increase in PAI-1 protein release was based on the increase in PAI-1 expression.


Figure 2
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  		Fig. 2. Effect of flow on the release of PAI-1 protein by hepatocytes. Amounts of PAI-1 in perfusates of rat RTH33 cells cultured under static conditions or exposed to flow (shear stress: 10 dyn/cm2) for 3, 6, 12, 24, 48, or 72 h were measured by ELISA. bullet, Time course of the increases in PAI-1 release in response to flow; {circ}, basal release under static conditions. Results are presented as means ± SD of 3 separate samples. *P < 0.01 vs. static control.

 
Shear stress dependency of flow-induced PAI-1 expression. To determine whether the flow-induced PAI-1 expression was dependent on shear stress or shear rate, rat hepatocytes (RTH33) were subjected to flow by two perfusates having different viscosities. PAI-1 mRNA levels increased as the shear rate increased, but at the same shear rate they increased even more when the viscosity or shear stress was higher, and the data yielded two separate curves (Fig. 3, top). When PAI-1 mRNA levels were plotted against shear stress, on the other hand, the data yielded a single curve (Fig. 3, bottom). These findings indicate that flow-induced PAI-1 expression is shear stress dependent rather than shear rate dependent.


Figure 3
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  		Fig. 3. Shear stress dependency of flow-induced PAI-1 expression. Rat RTH33 cells were exposed to flow by two perfusates having different viscosities at various shear rates for 6 h, and changes in PAI-1 mRNA levels were determined by real-time PCR. The low-viscosity perfusate consisted of medium alone, and the high-viscosity perfusate consisted of medium plus 5% dextran (mol wt 162,000; Sigma). The low- and high-viscosity perfusates had viscosities of 0.0095 and 0.0378 Poise, respectively, specific gravities of 1.005 and 1.025, respectively, and osmolarities of 289 and 292 mosmol/l, respectively. The PAI-1 mRNA level increased with the shear rate, but at the same shear rate, the increase was always greater at the higher viscosity or at the higher shear stress (top). When plotted against shear stress, the mRNA data almost formed a single curve (bottom), suggesting that the flow-induced increase in the PAI-1 mRNA level is shear stress rather than shear rate dependent. The curves were drawn by curvilinear regression with Microsoft Excel software (Microsoft). Data are means ± SD of 3 samples.

 
Shear stress activates PAI-1 transcription in hepatocytes. A nuclear runon assay was performed to investigate whether shear stress affects the transcription of PAI-1. Exposure to shear stress (10 dyn/cm2, 6 h) significantly increased PAI-1 transcription in rat hepatocytes (RTH33) and human hepatoma cells (C3A) compared with the static control but had no effect on the level of beta-actin transcription (Fig. 4, A and B).


Figure 4
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  		Fig. 4. Effects of shear stress on PAI-1 transcription. A: rat hepatocytes (RTH33). B: human hepatoma cells (C3A). Left, nuclear runon assay. Nuclei were harvested from hepatocytes (1 x 107 cells) that had been incubated under static conditions or exposed to shear stress (10 dyn/cm2) for 6 h, and transcription was allowed to continue in the presence of [{alpha}-32P]UTP. Labeled RNA was extracted and used as a probe against immobilized plasmids containing cDNA inserts for rat or human PAI-1 and beta-actin (positive control) and the plasmid pBlue KS+ (negative control). Shear stress markedly enhanced PAI-1 transcription in both rat hepatocytes and human hepatoma cells. Shear stress had no effect on beta-actin transcription. Right, quantitative densitometry analysis. The ratio of the density of PAI-1 in shear-stressed cells was significantly higher than that in the static control cells. All values are means ± SD of 3 separate experiments. *P < 0.01 vs. static control.

 
Actinomycin D chase experiments were performed to investigate whether shear stress affects the stability of PAI-1 mRNA in rat hepatocytes. The amount of PAI-1 mRNA decreased with exposure time to actimomycin D, and no difference was observed between static control cells and shear stressed cells (Fig. 5). These findings indicate that shear stress increases PAI-1 expression by activating transcription, not by stabilizing mRNA.


Figure 5
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  		Fig. 5. Effect of shear stress on PAI-1 mRNA stability. Rat RTH33 cells were incubated under static conditions or subjected to shear stress (10 dyn/cm2) for 6 h and then exposed to actinomycin D for 1, 2, 3, 4, or 5 h. Competitive PCR was performed to measure the changes in PAI-1 mRNA levels. No differences in the time course of degradation of PAI-1 mRNA were seen between static control and shear-stressed cells. All values are means ± SD of 4 separate experiments.

 
A luciferase assay was conducted to confirm the positive effect of shear stress on PAI-1 transcription. The luciferase activity of hepatocytes exposed to shear stress was significantly higher than that of static control cells (Fig. 6A, rat RTH33 cells, –2071 luc; Fig. 6B, human C3A cells, –2895 luc), confirming that shear stress activates PAI-1 transcription.


Figure 6
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  		Fig. 6. Deletion and mutation analysis of the PAI-1 promoter. A: rat hepatocytes (RTH33). B: human hepatoma cells (C3A). Hepatocytes transfected with reporter plasmids containing the rat or human PAI-1 promoter linked to the luciferase (Luc) gene were exposed to shear stress (10 dyn/cm2) for 6 h, and changes in luciferase activity were measured with a luminometer. The horizontal axis represents the percentage of the luciferase activity of –2071 luc (top) or –2895 luc (bottom) under static conditions. Shear stress significantly increased the luciferase activity of rat hepatocytes transfected with –2071 luc, –766 luc, and –278 luc and significantly increased the luciferase activity of human hepatoma cells transfected with –2895 luc and –414 luc. Mutation of the Sp1 or Ets-1 binding motif alone (rat, –278 luc Sp1 mut and –278 luc Ets-1 mut; human, –414 luc Sp1 mut and –414 luc Ets-1 mut) only partially suppressed basal PAI-1 transcription and the PAI-1 response to shear stress, whereas mutation of both motifs (rat, –278 luc Sp1/Ets-1 mut; human, –414 luc Sp1/Ets-1 mut) significantly inhibited basal transcription of PAI-1 and completely abolished the shear stress responsiveness. Values are means ± SD of data from 5 separate coverslips. **P < 0.01; *P < 0.05.

 
Sp1 and Ets-1 binding motifs are essential for shear stress activation of PAI-1 transcription. A deletion analysis was performed to localize the cis-acting regions in the PAI-1 promoter that are responsible for the shear stress activation of PAI-1 transcription. An increase in PAI-1 transcription occurred in response to shear stress even when the promotor length of rat PAI-1 was reduced to –278 bp and the promoter length of human PAI-1 was reduced to –414 bp, suggesting that the shear stress-responsive elements lie within these regions of the PAI-1 promoter (Fig. 6). Inspection of the rat and human PAI-1 promotor revealed a Sp1 binding motif located at base pairs –78/–68 and –75/–65 and an Ets-1 binding motif located at base pairs –51/–42 and –48/–39, respectively, relative to the transcription start site. To determine whether these elements are involved in the PAI-1 response to shear stress, chimeric genes generated by site-specific mutagenesis at the Sp1 and/or Ets-1 binding motif were constructed and tested by a luciferase assay. Mutation of either the Sp1 or Ets-1 binding motif only partially suppressed PAI-1 basal transcription and the PAI-1 response to shear stress, whereas mutation of both motifs significantly inhibited the basal transcription of PAI-1 and completely abolished the shear stress responsiveness. These findings indicate that the Sp1 and Ets-1 binding motifs cooperatively function as shear stress-responsive elements of PAI-1.

Transcription factors Sp1 and Ets-1 are involved in shear stress activation of PAI-1 gene transcription. EMSA was performed to identify the nuclear proteins that bind to the Sp1 and Ets-1 consensus element. Incubation of a 30-mer oligonucleotide containing the Sp1 or Ets-1 binding motif with nuclear extracts obtained from rat RTH33 cells and human C3A cells cultured under static conditions or subjected to a shear stress of 10 dyn/cm2 resulted in the formation of a protein-DNA complex, indicating the presence of nuclear proteins that bind to the Sp1 and Ets-1 elements (Fig. 7, A and B). The protein-DNA complexes in the nuclear extracts from shear stressed cells were larger than in the nuclear extracts from static control cells. Antibody to Sp1 or Ets-1 markedly inhibited the formation of the protein-DNA complexes, and, when an EMSA was performed with oligonucleotides bearing the mutated Sp1 or Ets-1 binding motif as the labeled probe, no protein-DNA complex bands appeared.


Figure 7
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  		Fig. 7. Effects of shear stress on binding of the transcription factors to the Sp1 or Ets-1 consensus element. A: rat hepatocytes (RTH33). B: human hepatoma cells (C3A). EMSA was performed to investigate the complexes formed by oligonucleotides bearing the Sp1 or Ets-1 consensus element (Sp1, lanes 1–6; Ets-1, lanes 12–17) or mutated elements (Sp1, lanes 7–11; Ets-1, lanes 18–22) and nuclear extracts from static cells (Sp1, lanes 2, 3, 8, and 9; Ets-1, lanes 13, 14, 19, and 20) or shear-stressed cells (Sp1, lanes 4, 5, 10, and 11; Ets-1, lanes 15, 16, 21, and 22). Lanes 1, 7, 12, and 18, no nuclear extracts; lanes 3, 5, 9, 11, 14, 16, 20, and 22, addition of relevant unlabeled oligonucleotides as a competitor in 500-fold excess; lanes 6 and 17, addition of antibody to Sp1 and Ets-1, respectively. The arrow points to the shifted band representing the protein-DNA complex. Shear stress enhanced the band of the protein-DNA complex (Sp1, lanes 2 vs. 4; Ets-1, lanes 13 vs. 15). The addition of antibody to Sp1 or Ets-1 caused a band shift (lane 6) or abolished the band (lane 17) seen in static control cells (Sp1, lane 2; Ets-1, lane 13) and shear-stressed cells (Sp1, lane 4; Ets-1, lane 15). The mutation in the Sp1 or the Ets-1 consensus sequence also abolished the band for the protein-DNA complex (Sp1, lanes 8 and 10; Ets-1, lanes 19 and 21). All experiments were repeated 3 times, and the results were similar.

 
ChIP assays were performed to further study the interactions between Sp1 and Ets-1 and the PAI-1 promoter. After static and shear-stressed rat RTH33 cells and human C3A cells were cross linked with formaldehyde, immunoprecipitatres of Sp1 or Ets-1 were subjected to PCR using primers directed against the PAI-1 promoter region containing the Sp1 or Ets-1 consensus sequence. The PAI-1 promoter interacted with Sp1 and Ets-1 (Fig. 8), and Sp1 and Ets-1 binding to the PAI-1 promoter increased in response to shear stress. The identity of the amplified immunoprecipitated DNA fragment was confirmed by gel elution and subsequent sequencing (data not shown).


Figure 8
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  		Fig. 8. Sp1 and Ets-1 bind to the consensus element in the rat and human PAI-1 promoter. Chromatin extracts were prepared from rat hepatocytes (RTH33) and human hepatoma (C3A) cells that had been cultured under static conditions or subjected to shear stress (10 dyn/cm2) for 6 h and immunoprecipitated (ChIP) with antibodies as indicated. Final DNA extracts were amplified with a pair of primers that included the rat or human PAI-1 promoter containing the Sp1 or Ets-1 consensus site. Input represents the positive control and Mock represents the negative control, as described in MATERIALS AND METHODS. All experiments were performed 3 times, and the results were similar.

 
These results of the EMSA and ChIP assays suggested that cooperative interaction by Sp1 and Ets-1 with the PAI-1 promoter is involved in the shear stress activation of PAI-1 transcription in hepatocytes.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study demonstrated a marked increase in PAI-1 expression and PAI-1 protein release when rat cultured hepatocytes were subjected to flow. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent. Shear stress in the 0–40 dyn/cm2 range increased PAI-1 expression in a dose-dependent manner, indicating that hepatocytes are responsive to this range of shear stress, although the actual physiological levels of shear stress acting on hepatocytes are unknown. The increase in PAI-1 expression in response to shear stress was observed in human hepatoma cells and murine hepatocytes as well as rat hepatocytes. These findings suggest that hepatocytes are sensitive to flow and that their function and gene expression are regulated not only by chemical mediators, such as hormones, growth factors, and cytokines, but by the mechanical force generated by flowing blood, shear stress.

The shear stress-dependent PAI-1 activation in hepatocytes occurred at the transcription level, not at the posttranscription level. Deletion analysis of the rat PAI-1 promoter revealed that Sp1 and Ets-1 binding sites function as the cis-element for shear stress responsiveness. EMSA and ChIP assays showed that Sp1 and Ets-1 are actually involved in the shear stress-dependent activation of PAI-1 transcription. Cooperative interactions between Sp1 and Ets-1 have also been shown to play a critical role in regulating the transcription of genes, such as the genes encoding Fas ligand and the platelet-derived growth factor A-chain, in vascular smooth muscle cells (10, 20). Nevertheless, how shear stress activates Sp1 and Ets-1, including whether shear stress increases their gene expression or activates them through posttranscriptional modifications, such as by glycosylation and phosphorylation, has not yet been determined.

A considerable amount of research on shear stress signaling has been done in vascular endothelial cells. Although the shear stress receptors have not been identified, it has become apparent that shear stress activates multiple signaling pathways in which Ca2+, inositiol (1,4,5)-trisphosphate, small G proteins, and many protein kinases, such as extracellular signal-regulated kinase and focal adhesion kinase, are involved, and many transcription factors, including activator protein-1, NF-{kappa}B, Egr-1, Sp1, and GATA6, are activated downstream of the shear stress signaling (11–13, 25, 26). In contrast to hepatocytes, however, laminar shear stress has no effect on PAI-1 production in endothelial cells (4). Thus it seems that it will be necessary to investigate the signaling pathways involved in the shear stress-dependent increase in PAI-1 transcription in hepatocytes rather than in endothelial cells.

Induction of PAI-1 expression has been shown to occur in hepatocytes in the regenerating liver a few hours after partial hepatectomy. PAI-1 induction has also been observed in vitro in hepatoma cells stimulated with glucocorticoids, transforming growth factor-beta, epidermal growth factor, and interleukin-1 (3, 7, 8, 15), and thus systemic or local release of such chemical mediators after partial hepatectomy may account for the PAI-1 induction. Partial hepatectomy increases portal pressure in the remaining lobes (17, 22, 29), and the compression or stretching tension generated by the increased portal pressure may be involved in the induction of PAI-1. A recent study demonstrated that stretching tension stimulates endothelial cells to increase PAI-1 production. The results of our study suggest that the rise in shear stress generated by the increase in portal blood flow is capable of increasing PAI-1 expression in hepatocytes. However, further study is needed to clarify the extent of the role played by chemical mediators and hemodynamic forces in the PAI-1 induction after partial hepatectomy and the role of PAI-1 in the initiation of liver regeneration.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was partly supported in part by grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science and by a research grant for cardiovascular diseases from the Japanese Ministry of Health, Labour and Welfare.


   ACKNOWLEDGMENTS
 
The authors thank Yuko Sawada for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Ando, Dept. of Biomedical Engineering, Graduate School of Medicine, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (e-mail: joji{at}m.u-tokyo.ac.jp)

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.

* H. Nakatsuka and T. Sokabe contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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