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Circulation Research. 2006;98:913-922
Published online before print March 9, 2006, doi: 10.1161/01.RES.0000216954.55724.a2
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(Circulation Research. 2006;98:913.)
© 2006 American Heart Association, Inc.

Molecular Medicine

Novel Role for STAT-5B in the Regulation of Hsp27–FGF-2 Axis Facilitating Thrombin-Induced Vascular Smooth Muscle Cell Growth and Motility

Huiqing Cao, Nagadhara Dronadula, Farhan Rizvi, Quanyi Li, Kalyan Srivastava, William T. Gerthoffer, Gadiparthi N. Rao

From the Department of Physiology (H.C., N.D., F.R., Q.L., K.S., G.N.R.), University of Tennessee Health Science Center, Memphis; and Department of Pharmacology (W.T.G.), University of Nevada School of Medicine, Reno.

Correspondence to Gadiparthi N. Rao, PhD, Department of Physiology, University of Tennessee Health Science Center, 894 Union Ave, Memphis, TN 38163. E-mail grao{at}physio1.utmem.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Previously, we have demonstrated that STAT-3 plays a role in thrombin-induced VSMC motility. To learn more about the role of STATs in the mitogenic and chemotactic signaling events of thrombin, here we have studied the role of STAT-5. Thrombin activated STAT-5 as measured by its tyrosine phosphorylation, DNA binding, and reporter gene activity. Inhibition of STAT-5B, but not STAT-5A, by adenovirus-mediated expression of its respective dominant-negative mutants suppressed thrombin-induced VSMC growth and motility. Thrombin induced the expression of Hsp27 and FGF-2 in a time- and STAT-5B-dependent manner in VSMC. In addition, small interfering RNA-directed depletion of Hsp27 levels or adenovirus-mediated expression of its dominant-negative mutant attenuated thrombin-induced FGF-2 expression, growth, and motility of VSMC. An increased association of STAT-5B with STAT-3 occurred in response to thrombin and adenovirus-mediated expression of dnSTAT-3 suppressed thrombin-induced Hsp27 and FGF-2 induction, DNA synthesis and motility in VSMC. Together, these results indicate that thrombin-induced VSMC growth and motility require STAT-5B/STAT-3–dependent expression of Hsp27 and FGF-2. These observations also suggest that STAT-5B/STAT-3/Hsp27/FGF-2 signaling via its involvement in the regulation of VSMC growth and motility may play an important role in the pathogenesis of vascular diseases such as restenosis after angioplasty.


Key Words: fibroblast growth factor-2 • heat shock protein 27 • G protein-coupled receptor • signal transducer and activator of transcription • vascular smooth muscle cell


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Vascular smooth muscle cell (VSMC) growth and motility play a major role in the pathogenesis of vessel wall diseases such as atherosclerosis and restenosis following angioplasty.1,2 Thrombin, a serine protease and a G protein-coupled receptor (GPCR) agonist, is a potent mitogen and chemoattractant for many cell types, including VSMC and has been reported to be involved in the development of the above vascular lesions.3–6 Thrombin exerts its mitogenic and chemotactic effects to a major extent via activation of protease-activated receptor-1 (PAR-1).3,7,8 In addition, thrombin-induced mitogenic and chemotactic effects exhibit a requirement for transactivation of receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR), fibroblast growth factor receptor-1 (FGFR-1), and insulin-like growth factor-I receptor (IGF-IR) in various cell types, including VSMC.3,9–13 In addition to transactivation of RTKs, thrombin has been shown to stimulate mitogen-activated protein kinases such as extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 mitogen-activated protein kinase (p38MAPK) and transcriptional factors such as activator protein-1 (AP-1) and activating transcription factor-1 (ATF-1) in facilitating its mitogenic and chemotactic effects in these cells.14–17 Although a large body of data accumulated thus far clearly demonstrate the capacity of thrombin to influence growth and motility in many cell types, including VSMC, and activation of some of the biochemical events associated with these processes, the underlying signaling mechanisms are still far from clear.

The signal transducers and activators of transcription (STATs) have been shown to play an important role in the regulation of cell growth and differentiation.18–20 In recent years, work from various laboratories including ours has demonstrated that STATs, particularly STAT-3, also play a role in the regulation of VSMC growth and motility.21–25 To understand the signal transduction mechanisms of the mitogenic and chemotactic effect of thrombin, and thereby its involvement in vessel wall remodeling further, here we have studied the role of STAT-5. Our results suggest that thrombin-induced VSMC growth and motility require activation of STAT-5B. In addition, we found that STAT-5B in crosstalk with STAT-3 targets the induction of expression of Hsp27 and FGF-2 in triggering the thrombin-induced VSMC growth and motility. Based on these novel findings, it is likely that the STAT-5B/STAT-3/Hsp27/FGF-2 axis plays a critical role in vessel wall diseases such as restenosis following angioplasty.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Reagents
Thrombin was purchased from Sigma-Aldrich (St Louis, Mo). Anti-phospho-Hsp27 (S78/S82) antibodies, anti-STAT-5A antibodies (AF2168), and human FGF-2 ELISA kit (DFB50) were from R&D Systems (Minneapolis, Minn). Anti-Hsp27 (SC-1049) antibodies, anti-STAT-3 antibodies (SC-482), anti-STAT-5B antibodies (SC-836 and SC-1656), anti-PY20 antibodies (SC-508), STAT-5 consensus binding oligonucleotide (5'-AGA TTT CTA GGA ATT CAA TCC-3') (SC-2565) and STAT-5 mutant oligonucleotide (5'-AGA TTT AGT TTA ATT CAA TCC-3') (SC-2566) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Anti-STAT-5A/B antibodies (06–588) were obtained from Upstate Biotechnology (Lake Placid, NY). Phosphospecific anti-STAT-5 (9351S) antibodies were from Cell Signaling Technology (Beverly, Mass). Rat Hsp27 small interfering RNA (siRNA) duplexes (sense, 5'-GCGUGUCCCUGGACGUCAAdTdT-3'; antisense, 5'-UUGA-CGUCCAGGGACACGCdGdC-3') were made by QIAGEN (Valencia, Calif). Scrambled control siRNA (5'-UAGCGACUAAACACA-UCAA-3') was obtained from Dharmacon (Lafayette, Co). T4 polynucleotide kinase was obtained from Promega (Madison, Wis). Super Script First Strand Synthesis System for RT-PCR and TRIzol reagent were bought from Invitrogen (Carlsbad, Calif). [{gamma}32P]-ATP (3000 Ci/mmol) and [14C]-chloramphenicol (59 mCi/mmol) were from Amersham Biosciences (Piscataway, NJ). All the primers were obtained from Integrated DNA Technologies (Coralville, Iowa).

Cell Culture
Rat VSMC were isolated and subcultured as described previously.24

Construction of Recombinant dnSTAT-3, dnSTAT-5A, and dnSTAT-5B Adenoviral Vectors
C-terminally truncated STAT-5A (deletion at C terminus from 713 to 793 amino acid [AA] residues) and STAT-5B (deletion at C terminus from 718 to 793 AA residues) cDNAs26 were released from pmSTAT-5A-713-Prk5 and pmSTAT-5B-718-Prk5 plasmids by digestion with EcoRI and EcoRV and cloned into the same sites of entry vector pENTR3C. In the case of dnSTAT-3, its cDNA fragment was released from pFS3DM vector18 by digestion with EcoRI and SalI and cloned into EcoRI and XhoI sites of entry vector pENTR3C. The final constructs pAd-dnSTAT-5A, pAd-dnSTAT-5B, and pAd-dnSTAT-3 were generated by specific recombination of pdnSTAT-5A-ENTR3C, pdnSTAT-5B-ENTR3C, and pdnSTAT-3-ENTR3C with pAdCMV/V5DEST (Invitrogen). Adenovirus (Ad-dnSTAT-5A, Ad-dnSTAT-5B, and Ad-dnSTAT-3) expressing dnSTAT-5A, dnSTAT-5B, or dnSTAT-3 were produced by transfecting HEK293 cells with pAd-dnSTAT-5A, pAd-dnSTAT-5B, and pAd-dnSTAT-3 plasmids, respectively. Construction of pAd-GFP and pAd-3A (pAd-dnHsp27) have been described previously.27,28 Adenovirus was purified with cesium chloride centrifugation and the titer was determined by plaque assay as described previously.27

Cell Motility
VSMC motility was measured by cell-wounding assay, as described previously.24

Cell Number
VSMC at 72 hours of appropriate treatments were trypsinized, rinsed with and suspended in PBS, and counted using a hemacytometer.

DNA Synthesis
VSMC DNA synthesis was measured by labeling cells with 1 µCi/mL [3H]-thymidine, as described previously.16

Electrophoretic Mobility-Shift Assay
After appropriate treatments, VSMC nuclear extracts were made and analyzed for DNA binding activity, as described previously.24

Enzyme-Linked Immunosorbent Assay
After appropriate treatments, FGF-2 released into the culture medium of VSMC was measured using human FGF-2 ELISA kit following the instructions of the supplier.

Immunoprecipitation
After appropriate treatments, cell extracts were made and an equal amount of protein from each condition was subjected to pull-down assay followed by Western blot analysis.

RNA Isolation, cDNA Synthesis, and RT-PCR
RNA was isolated from cells using TRIzol reagent as per the guidelines of the manufacture. Reverse transcription was performed with Superscript First Strand Synthesis System for RT-PCR based on the protocol of the supplier. The cDNA was then used as template for PCR using specific primers for rat FGF-2 (forward, 5'-CAGCT-CCAAGCAGAAGAGAG-3'; reverse, 5'-ACAGTATGGCCTTCT-GTCCA-3'), Hsp27 (forward, 5'-TGTCAGAGATCCGACAGACG-3'; reverse, 5'-GACAGGGAAGAGGACACCAA-3'), and ß-actin (forward, 5'-CGTTGACATCCGTAAAGACC-3'; reverse 5'-GATAGAGCC ACCAATCCACA-3'). The amplification was performed on Gene Amp PCR System 2400 (Applied Biosystems, Calif) using the following amplification systems for the abovementioned genes as follows: for FGF-2, at 94°C for 5 minutes followed by 27 cycles at 94°C for 1 minute, 58°C for 1 minute and 72°C for 1 minute, with final extension at 72°C for 5 minutes; Hsp27, at 94°C for 5 minutes followed by 30 cycles at 94°C for 45 seconds, 56°C for 1 minute and 72°C for 45 seconds, with final extension at 72°C for 5 minutes; for ß-actin, at 94°C for 5 minutes followed by 25 cycles at 94°C for 45 seconds, 55°C for 1 minute and 72°C for 45 seconds, with final extension at 72°C for 5 minutes. The amplified RT-PCR products were separated on 2% (wt/vol) agarose gel containing ethidium bromide and the band intensities were quantified using NIH ImageJ software.

Western Blot Analysis
After appropriate treatments, VSMC extracts were made and analyzed by Western blotting for the protein of interest using its specific antibodies as described previously.16

CAT Assay
VSMC were transfected with pSIE-CAT plasmid in serum- and antibiotic-free DMEM using FuGENE 6 reagent (Invitrogen). Cells were then quiesced and treated with and without thrombin (0.5 U/mL) for the indicated times, and cell extracts were prepared. In the case of testing the effect of Ad-STAT-5A/B, cells were infected first with the respective virus followed by transfection with pSIE-CAT plasmid DNA. Cell extracts normalized for protein were assayed for CAT activity using [14C]-chloramphenicol and acetyl coenzyme A as substrates as described previously.24

Statistics
All the experiments were repeated three times with similar results. Data are presented as mean±SD. The treatment effects were analyzed by Student’s t test. Values of P<0.05 were considered to be statistically significant. In the case of CAT activity, electrophoretic mobility-shift assay (EMSA), RT-PCR, and Western blotting, 1 representative set of data are shown.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Thrombin Activates STAT-5–Dependent Transcription in VSMC
To understand the role of STATs in GPCR agonist-induced VSMC growth and motility, here we have studied the effect of thrombin on STAT-5 activation. Thrombin (0.5 U/mL) stimulated tyrosine phosphorylation of STAT-5 in a time-dependent manner (Figure 1A). On phosphorylation, STATs undergo either homo- or heterodimerization and translocate to the nucleus, where they influence their target gene transcription.29 To find whether the increases in STAT-5 phosphorylation correlate with its transcriptional transactivation, the STAT-5/DNA-binding activity was measured. Consistent with its effect on phosphorylation, thrombin (0.5 U/mL) also stimulated STAT-5/DNA-binding activity in a time-dependent manner (Figure 1B). No STAT-5/DNA-binding activity was observed with a mutant probe. Furthermore, preincubation of nuclear protein with anti-STAT-5 antibodies (1 µg) that recognize both A and B isoforms reduced the protein/DNA-binding activity, suggesting the presence of STAT-5 in these complexes (Figure 1C). To find whether STAT-5 influences the transcription of genes containing SIE (sis-inducing element) or SIE-like elements in their promoter regions, cells were transfected with pSIE-CAT plasmid DNA, quiesced, and treated with and without thrombin (0.5 U/mL) for the indicated times, and the CAT activity was measured. Consistent with its effect on DNA-binding activity, thrombin induced SIE-dependent CAT activity in a time-dependent manner (Figure 1D). To confirm these results further, we tested the effect of dnSTAT-5A/B. VSMC that were first infected with adenovirus expressing either GFP, dnSTAT-5A, or dnSTAT-5B at a multiplicity of infection (moi) of 80 were transfected with pSIE-CAT plasmid DNA, quiesced, and treated with and without thrombin (0.5 U/mL) for 8 hours, and the CAT activity was measured. As shown in Figure 1E, thrombin induced CAT activity by approximately 3-fold as compared with green fluorescence protein (GFP) control, and this response was blocked by both dnSTAT-5A and dnSTAT-5B. These findings suggest that thrombin activates STAT-5A/B in VSMC.


Figure 1
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  		Figure 1. Thrombin activates STAT-5 in VSMC. Quiescent VSMC were treated with and without thrombin (0.5 U/mL) for the indicated times, and either cell or nuclear extracts were prepared. A, Cell extracts containing an equal amount of protein from control and each treatment were immunoprecipitated with anti-STAT-5A/B antibodies, and the immunocomplexes were analyzed by Western blotting using anti-PY20 antibodies. The blot was reprobed with anti-STAT-5A/B antibodies for normalization. B and C, Nuclear extracts containing an equal amount of protein from control and each treatment were assayed for protein/DNA-binding activity using [32P]-labeled STAT-5 consensus and mutant oligonucleotides as probes. Wherever indicated, anti-STAT-5A/B antibodies were added to nuclear extracts 30 minutes before the addition of the [32P]-labeled STAT-5 consensus oligonucleotide probe. D, VSMC were transfected with pSIE-CAT plasmid DNA, quiesced, and treated with and without thrombin (0.5 U/mL) for the indicated times, and cell extracts were prepared. Cell extracts normalized for protein were assayed for CAT activity using [14C]-chloramphenicol and acetyl coenzyme A as substrates. E, VSMC were transduced first with Ad-GFP (control), Ad-dnSTAT-5A, or Ad-dnSTAT-5B at a moi of 80, and, 24 hours later, they were transfected with pSIE-CAT plasmid DNA and quiesced. Cells were then treated with and without thrombin (0.5 U/mL) for 8 hours, and cell extracts were prepared and assayed for CAT activity as described in D. To show the expression of dnSTAT-5A and -B, an equal amount of protein from each sample was analyzed by Western blotting using their specific antibodies. The bar graph represents quantitative analysis of 3 independent experiments. *P<0.01 vs control.

STAT-5B Mediates Thrombin-Induced VSMC Growth and Motility
To understand the role of STAT-5 in thrombin-induced VSMC growth and motility, we next tested the effect of dnSTAT-5A and dnSTAT-5B. Adenovirus-mediated expression of dnSTAT-5B but not dnSTAT-5A attenuated thrombin-induced VSMC DNA synthesis, cell number, and motility (Figure 2A through 2C). Because only STAT-5B appears to be involved in thrombin-induced VSMC growth and motility, we focused next on identification of its downstream effector molecules.


Figure 2
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  		Figure 2. Thrombin-induced VSMC growth and motility require activation of STAT-5B but not STAT-5A. VSMC that were transduced with Ad-GFP (control), Ad-dnSTAT-5A, or Ad-dnSTAT-5B with a moi of 80 were quiesced and treated with and without thrombin (0.5 U/mL) for appropriate time periods, and DNA synthesis, cell number, and motility were measured. A, DNA synthesis was measured by [3H]-thymidine incorporation into TCA-precipitable material. B, Cell number was measured by Trypan Blue dye exclusion assay. C, Cell motility was measured by cell-wounding assay. *P<0.01 vs control, **P<0.01 vs thrombin treatment alone.

Thrombin Induces the Expression of Hsp27 via STAT-5B Activation
Some reports have shown that Hsps, particularly Hsp27, play a role in the regulation of cell motility.30,31 To understand the mechanisms by which STAT-5B is involved in thrombin-induced VSMC growth and motility, we studied the time-course effect of thrombin on Hsp27 expression. Thrombin (0.5 U/mL) induced the expression of Hsp27 both at mRNA and protein levels in a time-dependent manner, with a maximum of 2-fold increase at 4 hours and sustaining these levels at least for 16 hours (Figure 3A and 3B). DnSTAT-5B inhibited thrombin-induced Hsp27 expression both at mRNA and protein levels (Figure 4A and 4B).


Figure 3
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  		Figure 3. Thrombin induces the expression of Hsp27 in a time-dependent manner in VSMC. Quiescent VSMC were treated with and without thrombin (0.5 U/mL) for the indicated times, and either total cellular RNA or protein were isolated. A, An equal amount of RNA from control and each treatment was subjected to 2-step RT-PCR using rat specific Hsp27 and ß-actin primers. B, An equal amount of protein from control and each treatment was analyzed by Western blotting for Hsp27 using its specific antibodies. The blot was reprobed with anti-STAT-5B antibodies for normalization. The bar graphs represent the quantitative analysis of 3 independent experiments. *P<0.05 vs control.


Figure 4
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  		Figure 4. STAT-5B mediates thrombin-induced Hsp27 expression in VSMC. VSMC that were transduced with Ad-GFP (control) or Ad-dnSTAT-5B with a moi of 80 were quiesced and treated with and without thrombin (0.5 U/mL) for 8 hours and 16 hours in the case of RNA extraction and 16 hours in the case of protein isolation. A, An equal amount of RNA from control and each treatment was subjected to 2-step RT-PCR using rat specific Hsp27 and ß-actin primers. B, An equal amount of protein from control and each treatment was analyzed by Western blotting for Hsp27 using its specific antibodies. The blot was reprobed with anti-STAT-5B antibodies for normalization. To show the overexpression of dnSTAT-5B, the blot was reprobed with anti-STAT-5A/B antibodies as they recognize the N-terminal region of STAT-5B. The bar graphs represent the quantitative analysis of three independent experiments. *P<0.01 vs control, *P<0.05 vs thrombin treatment alone.

A Requirement for Hsp27 in Thrombin-Induced VSMC Growth and Motility
To understand the functional significance of STAT-5B-Hsp27 signaling, we next studied the role of Hsp27 in thrombin-induced VSMC growth and motility using siRNA approach. Hsp27 siRNA but not control siRNA depleted thrombin-induced Hsp27 levels (Figure 5A). In addition, Hsp27 siRNA blocked thrombin-induced VSMC DNA synthesis, cell number, and motility (Figure 5B through 5D). To confirm these findings, we tested the effect of dnHsp27. DnHsp27 is a phosphorylation-null mutant.28,30 Thrombin induced the Ser86 phosphorylation of Hsp27 (equivalent of Ser82 in human Hsp27) in a time-dependent manner (Figure 6A). Adenovirus-mediated expression of dnHsp27 inhibited thrombin-induced Hsp27 Ser86 phosphorylation (Figure 6B). In addition, dnHsp27 inhibited thrombin-induced increases in VSMC DNA synthesis, cell number, and motility (Figure 6C through 6E).


Figure 5
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  		Figure 5. Hsp27 siRNA attenuates thrombin-induced VSMC growth and motility. A, VSMC were transfected with scrambled control or Hsp27 siRNA, quiesced, and treated with and without thrombin (0.5 U/mL) for 16 hours, and cell extracts were prepared. An equal amount of protein from control and each treatment was analyzed by Western blotting for Hsp27 using its specific antibodies. The blot was reprobed with anti-STAT-5B antibodies for normalization. B through D, VSMC that were mock or Hsp27 siRNA transfected were quiesced and subjected to thrombin-induced VSMC DNA synthesis, cell number, or motility, as described in the legend of Figure 2. The bar graph represents the quantitative analysis of 3 independent experiments. *P<0.01 vs control, **P<0.01 vs thrombin treatment alone.


Figure 6
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  		Figure 6. DnHsp27 inhibits thrombin-induced VSMC growth and motility. A, Quiescent VSMC were treated with and without thrombin (0.5 U/mL) for the indicated times, and cell extracts were prepared. An equal amount of protein from control and each treatment was analyzed by Western blotting for pHsp27 using its phosphospecific antibodies. B, VSMC were transduced with Ad-GFP (control) or Ad-dnHsp27 at a moi of 80, quiesced, and treated with and without thrombin (0.5 U/mL) for 10 minutes, and cell extracts were prepared and analyzed for pHsp27 as described in A. The blots in A and B were reprobed with anti-STAT-5B antibodies for normalization. C through E, Conditions were the same as in B, except that cells after quiescence were treated with and without thrombin (0.5 U/mL) for appropriate time periods and DNA synthesis, cell number, and motility were measured as described in the legend of Figure 2. *P<0.01 vs control, **P<0.01 vs thrombin treatment alone.

Thrombin Induces FGF-2 Expression via STAT-5B Activation
We have previously shown that thrombin via activation of phosphatidylinositol 3-kinase/Akt/Fra-1 signaling induces the expression of FGF-2 in VSMC and that neutralizing anti-FGF-2 antibodies inhibit thrombin-induced VSMC DNA synthesis and motility.32 In addition, we found that inhibition of Jak-2 suppresses 5(S)-hydroxyeicosatetraenoic acid-induced FGF-2 expression in human dermal microvascular endothelial cells.33 Based on these results, we hypothesized that FGF-2 may be an effector molecule of STAT-5B in thrombin-induced VSMC growth and motility. To test this, we studied the effect of dnSTAT-5B on thrombin-induced FGF-2 expression. Adenovirus-mediated expression of dnSTAT-5B completely blocked thrombin-induced FGF-2 expression both at mRNA and protein levels (Figure 7A and 7B).


Figure 7
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  		Figure 7. Thrombin induces FGF-2 expression in STAT-5B- and Hsp27-dependent manner in VSMC. VSMC that were transduced with Ad-GFP (control), Ad-dnSTAT-5B, or Ad-dnHsp27 at a moi of 80, or transfected with control or Hsp27 siRNA, were quiesced and treated with and without thrombin (0.5 U/mL) for the indicated times or 16 hours, and RNA and protein were isolated. A, C, and E, An equal amount of RNA from control and each treatment was subjected to 2-step RT-PCR using rat-specific FGF-2 and ß-actin primers. B, D, and F, An equal amount of protein from control and each treatment was analyzed by Western blotting for FGF-2 using its specific antibodies. The blots were reprobed with anti-STAT-5B antibodies for normalization. To show the expression of dnSTAT-5B, the blot in B was reprobed with anti-STAT-5A/B antibodies that recognize the N-terminal region of STAT-5B. The bar graphs represent the quantitative analysis of 3 independent experiments. *P<0.01 vs control, **P<0.01 vs thrombin treatment alone.

Thrombin-Induced FGF-2 Expression Requires Hsp27
Because STAT-5B was also involved in thrombin-induced Hsp27 expression, we wanted to learn whether there is any crosstalk between Hsp27 and FGF-2. To address this, we tested the role of Hsp27 in thrombin-induced FGF-2 expression. Hsp27 siRNA inhibited thrombin-induced FGF-2 expression both at mRNA and protein levels (Figure 7C and 7D). To confirm these findings, we also studied the effect of dnHsp27 on thrombin-induced FGF-2 expression. Consistent with the effect of Hsp27 siRNA, dnHsp27 also blocked thrombin-induced FGF-2 expression both at mRNA and protein levels (Figure 7E and 7F). To find whether thrombin-induced increases in the expression of FGF-2, in turn, leads to its release, VSMC that were transduced with Ad-GFP (control), Ad-dnSTAT-5B, or Ad-dnHsp27 or transfected with control or Hsp27 siRNA were quiesced and treated with and without thrombin (0.5 U/mL) for 16 hours, and the FGF-2 release into the medium was measured by ELISA. As shown in Figure 8A and 8B, thrombin induced the release of FGF-2 by approximately 3-fold as compared with control and this effect was substantially blocked by dnSTAT-5B, dnHsp27, and Hsp27 siRNA. Furthermore, neutralizing anti-FGF-2 antibodies blocked thrombin-induced VSMC growth and motility.32


Figure 8
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  		Figure 8. Thrombin-induced Hsp27 and FGF-2 expression requires activation of STAT-3. VSMC that were transduced with Ad-GFP (control), Ad-dnSTAT-5B, or Ad-dnHsp27 at a moi of 80 (A) or transfected with either scrambled control or Hsp27 siRNA (B) were quiesced and treated with and without thrombin (0.5 U/mL) for 16 hours, and FGF-2 released into the medium was measured using an ELISA kit. C, An equal amount of protein from control and thrombin-treated VSMC was immunoprecipitated with anti-STAT-3 antibodies and the resulting immunocomplexes were analyzed by Western blotting for STAT-5B (top) or STAT-3 (bottom) using their specific antibodies. D, VSMC that were transduced with Ad-GFP (control) or Ad-dnSTAT-3 at a moi of 80 were quiesced and treated with and without thrombin (0.5 U/mL) for 16 hours, and an equal amount of protein from each condition was analyzed by Western blotting for Hsp27 and FGF-2 levels using their specific antibodies. To show the expression of dnSTAT-3, the same blots were reprobed with anti-STAT-3 antibodies. E and F, Conditions were the same as in D, except that after quiescence, cells were treated with and without thrombin (0.5 U/mL) for 24 hours and DNA synthesis and motility were measured as in the legend of Figure 2. *P<0.01 vs control, **P<0.01 vs thrombin treatment alone.

Thrombin-Induced Hsp27 and FGF-2 Expression Exhibits a Crosstalk Between STAT-5B and STAT-3
Previously, we have reported that STAT-3 mediates thrombin-induced VSMC motility.24 To learn whether there is any crosstalk between STAT-5B and STAT-3 in the induction of expression of Hsp27 and FGF-2 toward mediating the mitogenic and chemotactic effects of thrombin, we first analyzed their interactions by pull-down assay. As shown in Figure 8C, increased association of STAT-5B with STAT-3 was observed in response to thrombin as compared with control. Using a dominant-negative mutant approach, we next tested the role of STAT-3 in thrombin-induced Hsp27 and FGF-2 expression. Adenovirus-mediated expression of dnSTAT-3 suppressed thrombin-induced induction of Hsp27 and FGF-2 (Figure 8D). Adenovirus-mediated expression of dnSTAT-3 also blocked thrombin-induced VSMC DNA synthesis and motility (Figure 8E and 8F).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The important findings of the present study are as follows. (1) Thrombin activates STAT-5 in VSMC. (2) Thrombin induces the expression of Hsp27 and FGF-2 in a time- and STAT-5B-dependent manner. (3) Thrombin-induced FGF-2 expression is also dependent on Hsp27. (4) Thrombin-induced VSMC growth and motility exhibited a requirement for STAT-5B-dependent Hsp27 and FGF-2 expression. (5) STAT-5B forms a complex with STAT-3 in response to thrombin and blockade of STAT-3 activation via adenovirus-mediated expression of its dominant-negative mutant inhibited thrombin-induced Hsp27 and FGF-2 induction in VSMC and the growth and motility of these cells. Earlier studies have shown that STAT-5 plays a role in the regulation of epithelial cell survival and proliferation.26,34 STAT-5 has also been shown to be involved in cytokine-induced hematopoietic cell growth.35,36 The findings that STAT-5 regulates the expression of cyclin D1 and Bcl(XL) further support its role in the regulation of cell proliferation and survival.37,38 The present results reveal that STAT-5B also targets both Hsp27 and FGF-2 in influencing the cellular growth and motility, at least in VSMC in response to thrombin. It was demonstrated that thrombin-induced VSMC DNA synthesis and motility require FGF-2 release.4,12 However, the mechanism(s) by which thrombin influences FGF-2 expression in VSMC have not been explored previously. In this regard, we have shown earlier that Akt plays a role in thrombin-induced FGF-2 expression in VSMC.32 In addition, our present results show that FGF-2 is also a target molecule of STAT-5B in thrombin-induced VSMC growth and motility. In view of these results, it appears that several mechanisms are involved in thrombin-induced FGF-2 expression and that FGF-2 may be a converging molecule for signaling events that target cell proliferation and motility in VSMC.

The involvement of Hsp27 in cell motility and tumor progression has been reported previously.30,39–41 Hsp27 interacts with actin and modulates actin cytoskeleton dynamics.42 However, the present observations reveal that Hsp27 is required for thrombin-induced FGF-2 expression. Earlier studies by other investigators have shown that Hsp27 colocalizes with FGF-2 and facilitates its release from endothelial cells in response to estradiol.43 Based on these results, it is likely that besides its capacity to regulate actin cytoskeleton dynamics, Hsp27 plays a role in the regulation of expression of FGF-2 toward mediating cell proliferation and motility. FGF-2 is a potent mitogen and chemoattractant for many cell types including VSMC.44–46 Because both Hsp27 and FGF-2 expression are regulated by STAT-5B and interference with the function of Hsp27 suppressed thrombin-induced FGF-2 induction, it is possible that Hsp27 acts downstream to STAT-5B in the induction of expression of FGF-2. It was demonstrated that Hsp27 via enhancing the degradation of ubiqitinated I{kappa}B{alpha} is involved in the activation of nuclear factor {kappa}B.47 A mechanism similar to this is quite possible for Hsp27 involvement in thrombin-induced FGF-2 expression as well.

We have previously shown that thrombin-induced VSMC motility requires STAT-3 activation.24 In addition to these observations, the present results reveal that STAT-3 interacts with STAT-5B in thrombin-induced expression of Hsp27 and FGF-2 and thereby in the regulation of VSMC growth and motility. These observations, along with our previous findings,32 suggest that FGF-2 is a downstream effector molecule of several signaling events that influence VSMC growth and motility. Based on these findings, we envision a potential role for STAT-5B/STAT-3/Hsp27/FGF-2 signaling in the vessel wall remodeling, particularly in vascular injury settings.


*    Acknowledgments
 
This work was supported by NIH grant HL64165 (to G.N.R.).


*    Footnotes
 
Original received October 20, 2005; resubmission received February 1, 2006; revised resubmission received February 23, 2006; accepted February 28, 2006.


*    References
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  1. Schwartz SM, deBlois D, O’Brien ER. The intima. Soil for atherosclerosis and restenosis. Circ Res. 1995; 77: 445–465.[Free Full Text]
  2. Hansson GK, Libby P, Schonbeck U, Yan ZQ. Innate and adaptive immunity in the pathogenesis of atherosclerosis. Circ Res. 2002; 91: 281–291.[Abstract/Free Full Text]
  3. Sabri A, Short J, Guo J, Steinberg SF. Protease-activated receptor-1-mediated DNA synthesis in cardiac fibroblast is via epidermal growth factor receptor transactivation: distinct PAR-1 signaling pathways in cardiac fibroblasts and cardiomyocytes. Circ Res. 2002; 91: 532–539.[Abstract/Free Full Text]
  4. Rauch BH, Millette E, Kenagy RD, Daum G, Fischer JW, Clowes AW. Syndecan-4 is required for thrombin-induced migration and proliferation in human vascular smooth muscle cells. J Biol Chem. 2005; 280: 17507–17511.[Abstract/Free Full Text]
  5. Rao GN, Runge MS. Cyclic AMP inhibition of thrombin-induced growth in vascular smooth muscle cells correlates with decreased JNK1 activity and c-Jun expression. J Biol Chem. 1996; 271: 20805–20810.[Abstract/Free Full Text]
  6. Rade JJ, Schulick AH, Virmani R, Dichek DA. Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury. Nat Med. 1996; 2: 293–298.[CrossRef][Medline] [Order article via Infotrieve]
  7. Chaikof EL, Caban R, Yan CN, Rao GN, Runge MS. Growth-related responses in arterial smooth muscle cells are arrested by thrombin receptor antisense sequences. J Biol Chem. 1995; 270: 7431–7436.[Abstract/Free Full Text]
  8. Darmoul D, Gratio V, Devaud H, Lehy T, Laburthe M. Aberrant expression and activation of the thrombin receptor protease-activated receptor-1 induces cell proliferation and motility in human colon cancer cells. Am J Pathol. 2003; 162: 1503–1513.[Abstract/Free Full Text]
  9. Daub H, Weiss FU, Wallasch C, Ullrich A. Role of transactivation of the EGF receptor in signaling by G protein-coupled receptors. Nature. 1996; 379: 557–560.[CrossRef][Medline] [Order article via Infotrieve]
  10. Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C, Ullrich A. EGF receptor transactivation by G protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature. 1999; 402: 884–888.[Medline] [Order article via Infotrieve]
  11. Kalmes A, Vesti BR, Daum G, Abraham JA, Clowes AW. Heparin blockade of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor (EGF) receptor transactivation by heparin-binding EGF-like growth factor. Circ Res. 2000; 87: 92–98.[Abstract/Free Full Text]
  12. Rauch BH, Millette E, Kenagy RD, Daum G, Clowes AW. Thrombin and factor Xa-induced DNA synthesis is mediated by transactivation of fibroblast growth factor receptor-1 in human vascular smooth muscle cells. Circ Res. 2004; 94: 340–335.[Abstract/Free Full Text]
  13. Rao GN, Delafontaine P, Runge MS. Thrombin stimulates phosphorylation of insulin-like growth factor-1 receptor, insulin receptor substrate-1, and phospholipase C-{gamma}1 in rat aortic smooth muscle cells. J Biol Chem. 1995; 270: 27871–27875.[Abstract/Free Full Text]
  14. Marinissen MJ, Servitja JM, Offermanns S, Simon MI, Gutkind JS. Thrombin protease-activated receptor-1 signals through GQ- and G13-initiated MAPK cascades regulating c-Jun expression to induce cell transformation. J Biol Chem. 2003; 278: 46814–46825.[Abstract/Free Full Text]
  15. DellaRocca GJ, Maudsley S, Daaka Y, Lefkowitz RJ, Luttrell LM. Pleiotropic coupling of G protein-coupled receptors to the mitogen-activated protein kinase cascade. Role of focal adhesions and receptor tyrosine kinases. J Biol Chem. 1999; 274: 13978–13984.[Abstract/Free Full Text]
  16. Rao GN, Katki KA, Madamanchi NR, Wu Y, Birrer MJ. JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells. J Biol Chem. 1999; 274: 6003–6010.[Abstract/Free Full Text]
  17. Ghosh SK, Gadiparthi L, Zeng ZZ, Bhanoori M, Tellez C, Bar-Eli M, Rao GN. ATF-1 mediates protease-activated receptor-1 but not receptor tyrosine kinase-induced DNA synthesis in vascular smooth muscle cells. J Biol Chem. 2002; 277: 21325–21331.[Abstract/Free Full Text]
  18. Wu YY, Bradshaw RA. Activation of the Stat3 signaling pathway is required for differentiation by interleukin-6 in PC12–E2 cells. J Biol Chem. 2000; 275: 2147–2156.[Abstract/Free Full Text]
  19. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C, Darnell JE Jr. Stat3 as an oncogene. Cell. 1999; 98: 295–303.[CrossRef][Medline] [Order article via Infotrieve]
  20. Klein C, Wustefeld T, Assmus U, Roskams T, Rose-John S, Muller M, Manns MP, Ernst M, Trautwein C. The IL-6-gp130-STAT3 pathway in hepatocytes triggers liver protection in T cell-mediated liver injury. J Clin Invest. 2005; 115: 860–869.[CrossRef][Medline] [Order article via Infotrieve]
  21. Madamanchi NR, Li S, Patterson C, Runge MS. Thrombin regulates vascular smooth muscle cell growth and heat shock proteins via the JAK-STAT pathway. J Biol Chem. 2001; 276: 18915–18924.[Abstract/Free Full Text]
  22. Yellaturu CR, Rao GN. Cytosolic phospholipase A2 is an effector of Jak/STAT signal transducers and activators of transcription signaling and is involved in platelet-derived growth factor BB-induced growth in vascular smooth muscle cells. J Biol Chem. 2003; 278: 9986–9992.[Abstract/Free Full Text]
  23. Neeli I, Liu Z, Dronadula N, Ma ZA, Rao GN. An essential role of Jak-2/STAT-3/cPLA2 axis in platelet-derived growth factor BB-induced vascular smooth muscle cell motility. J Biol Chem. 2004; 279: 46122–46128.[Abstract/Free Full Text]
  24. Dronadula N, Liu Z, Wang C, Cao H, and Rao GN. STAT-3-dependent expression of cPLA2 is required for thrombin-induced vascular smooth muscle cell motility. J Biol Chem. 2005; 280: 3112–3120.[Abstract/Free Full Text]
  25. Seki Y, Kai H, Shibata R, Nagata T, Yasukawa H, Yoshimura A, Imaizumi T. Role of the Jak-STAT pathway in rat carotid artery remodeling after vascular injury. Circ Res. 2000; 87: 12–18.[Abstract/Free Full Text]
  26. Wang D, Stravopodis D, Teglund S, Kitazawa J, Ihle JN. Naturally occurring dominant negative variants of Stat5. Mol Cell Biol. 1996; 16: 6141–6148.[Abstract]
  27. Liu Z, Zhang C, Dronadula N, Li Q, Rao GN. Blockade of nuclear factor of activated T cells activation signaling suppresses balloon injury-induced neointima formation in a rat carotid artery model. J Biol Chem. 2005; 280: 14700–14708.[Abstract/Free Full Text]
  28. Martin JL, Mestril R, Hilal-Dandan R, Brunton LL, Dillmann WH. Small heat shock proteins and protection against ischemic injury in cardiac myocytes. Circulation. 1997; 96: 4343–4348.[Abstract/Free Full Text]
  29. Horvath CM, Wen Z, Darnell JE Jr. A STAT protein domain that determines DNA sequence recognition suggests a novel DNA-binding domain. Genes Dev. 1995; 9: 984–994.[Abstract/Free Full Text]
  30. Hedges JC, Dechert MA, Yamboliev IA, Martin JL, Hickey E, Weber LA, Gerthoffer WT. A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem. 1999; 274: 24211–24219.[Abstract/Free Full Text]
  31. McMullen ME, Bryant PW, Glembotski CC, Vincent PA, Pumiglia KM. Activation of p38 has opposing effects on the proliferation and migration of endothelial cells. J Biol Chem. 2005; 280: 20995–21003.[Abstract/Free Full Text]
  32. Cao H, Dronadula N, Rao GN. Thrombin induces the expression of FGF-2 via activation of PI3K-Akt-Fra-1 signaling axis leading to DNA synthesis and motility in vascular smooth muscle cells. Am J Physiol. 2006; 290: C172–C182.
  33. Zeng ZZ, Yellaturu CR, Neeli I, Rao GN. 5(S)-hydroxyeicosatetraenoic acid stimulates DNA synthesis in human microvascular endothelial cells via activation of Jak/STAT and phosphatidylinositol 3-kinase/Akt signaling, leading to induction of expression of basic fibroblast growth factor 2. J Biol Chem. 2002; 277: 41213–41219.[Abstract/Free Full Text]
  34. Cui Y, Riedlinger G, Miyoshi K, Tang W, Li C, Deng CX, Robinson GW, Hennighausen L. Inactivation of Stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation. Mol Cell Biol. 2004; 24: 8037–8047.[Abstract/Free Full Text]
  35. Udy GB, Towers RP, Snell RG, Wilkins RJ, Park SH, Ram PA, Waxman DJ, Davey HW. Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc Natl Acad Sci U S A. 1997; 94: 7239–7244.[Abstract/Free Full Text]
  36. Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D, Wang D, Brown M, Bodner S, Grosveld G, Ihle JN. Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell. 1998; 93: 841–850.[CrossRef][Medline] [Order article via Infotrieve]
  37. Matsumura I, Kitamura T, Wakao H, Tanaka H, Hashimoto K, Albanese C, Downward J, Pestell RG, Kanakura Y. Transcriptional regulation of the cyclin D1 promoter by STAT5: its involvement in cytokine-dependent growth of hematopoietic cells. EMBO J. 1999; 18: 1367–1377.[CrossRef][Medline] [Order article via Infotrieve]
  38. Versteeg HH, Spek CA, Slofstra SH, Diks SH, Richel DJ, Peppelenbosch MP. FVIIa: TF induces cell survival via G12/G13-dependent Jak/STAT activation and BclXL production. Circ Res. 2004; 94: 1032–1040.[Abstract/Free Full Text]
  39. Piotrowicz RS, Hickey E, Levin EG. Heat shock protein 27 kDa expression and phosphorylation regulates endothelial cell migration. FASEB J. 1998; 12: 1481–1490.[Abstract/Free Full Text]
  40. Oesterreich S, Weng CN, Qiu M, Hilsenbeck SG, Osborne CK, Fuqua SA. The small heat shock protein hsp27 is correlated with growth and drug resistance in human breast cancer cell lines. Cancer Res. 1993; 53: 4443–4448.[Abstract/Free Full Text]
  41. Rocchi P, So A, Kojima S, Signaevsky M, Beraldi E, Fazli L, Hurtado-Coll A, Yamanaka K, Gleave M. Heat shock protein 27 increases after androgen ablation and plays a cytoprotective role in hormone-refractory prostate cancer. Cancer Res. 2004; 64: 6595–6602.[Abstract/Free Full Text]
  42. de Graauw M, Tijdens I, Cramer R, Corless S, Timms JF, van de Water B. Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis. J Biol Chem. 2005; 280: 29885–29898.[Abstract/Free Full Text]
  43. Piotrowicz RS, Martin JL, Dillman WH, Levin EG. The 27-kDa heat shock protein facilitates basic fibroblast growth factor release from endothelial cells. J Biol Chem. 1997; 272: 7042–7047.[Abstract/Free Full Text]
  44. Garmy-Susini B, Delmas E, Gourdy P, Zhou M, Bossard C, Bugler B, Bayard F, Krust A, Prats AC, Doetschman T, Prats H, Arnal JF. Role of fibroblast growth factor-2 isoforms in the effect of estradiol on endothelial cell migration and proliferation. Circ Res. 2004; 94: 1301–1309.[Abstract/Free Full Text]
  45. Nugent MA, Karnovsky MJ, Edelman ER. Vascular cell-derived heparan sulfate shows coupled inhibition of basic fibroblast growth factor binding and mitogenesis in vascular smooth muscle cells. Circ Res. 1993; 73: 1051–1060.[Abstract/Free Full Text]
  46. Bilato C, Pauly RR, Melillo G, Monticone R, Gorelick-Feldman D, Gluzband YA, Sollott SJ, Ziman B, Lakatta EG, Crow MT. Intracellular signaling pathways required for rat vascular smooth muscle cell migration. Interactions between basic fibroblast growth factor and platelet-derived growth factor. J Clin Invest. 1995; 96: 1905–1915.[Medline] [Order article via Infotrieve]
  47. Parcellier A, Schmitt E, Gurbuxani S, Seigneurin-Berny D, Pance A, Chantome A, Plenchette S, Khochbin S, Solary E, Garrido C. HSP27 is a ubiquitin-binding protein involved in I-kappaBalpha proteasomal degradation. Mol Cell Biol. 2003; 23: 5790–5802.[Abstract/Free Full Text]



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