help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on January 10, 2003, doi:10.1165/rcmb.2002-0206OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2002-0206OCv1
29/1/39    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, H. W.
Right arrow Articles by Solway, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, H. W.
Right arrow Articles by Solway, J.
American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 39-47, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0206OC

The RhoA/Rho Kinase Pathway Regulates Nuclear Localization of Serum Response Factor

Hong Wei Liu*, Andrew J. Halayko*, Darren J. Fernandes, Gregory S. Harmon, Joel A. McCauley, Pawel Kocieniewski, John McConville, Yiping Fu, Sean M. Forsythe, Paul Kogut, Shashi Bellam, Maria Dowell, Jason Churchill, Heinte Lesso, Kamrouz Kassiri, Richard W. Mitchell, Marc B. Hershenson, Blanca Camoretti-Mercado and Julian Solway

Departments of Medicine and Pediatrics, University of Chicago, Chicago, Illinois; Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; and Loyola University Medical Center, Maywood, Illinois

Address correspondence to: Julian Solway, M.D., Professor of Medicine and Pediatrics, University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637. E-mail: jsolway{at}medicine.bsd.uchicago.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
RhoA and its downstream target Rho kinase regulate serum response factor (SRF)-dependent skeletal and smooth muscle gene expression. We previously reported that long-term serum deprivation reduces transcription of smooth muscle contractile apparatus encoding genes, by redistributing SRF out of the nucleus. Because serum components stimulate RhoA activity, these observations suggest the hypothesis that the RhoA/Rho kinase pathway regulates SRF-dependent smooth muscle gene transcription in part by controlling SRF subcellular localization. Our present results support this hypothesis: cotransfection of cultured airway myocytes with a plasmid expressing constitutively active RhoAV14 selectively enhanced transcription from the SM22 and smooth muscle myosin heavy chain promoters and from a purely SRF-dependent promoter, but had no effect on transcription from the MSV-LTR promoter or from an AP2-dependent promoter. Conversely, inhibition of the RhoA/Rho kinase pathway by cotransfection with a plasmid expressing dominant negative RhoAN19, by cotransfection with a plasmid expressing Clostridial C3 toxin, or by incubation with the Rho kinase inhibitor, Y-27632, all selectively reduced SRF-dependent smooth muscle promoter activity. Furthermore, treatment with Y-27632 selectively reduced binding of SRF from nuclear extracts to its consensus DNA target, selectively reduced nuclear SRF protein content, and partially redistributed SRF from nucleus to cytoplasm, as revealed by quantitative immunocytochemistry. Treatment of cultured airway myocytes with latrunculin B, which reduces actin polymerization, also caused partial redistribution of SRF into the cytoplasm. Together, these results demonstrate for the first time that the RhoA/Rho kinase pathway controls smooth muscle gene transcription in differentiated smooth muscle cells, in part by regulating the subcellular localization of SRF. It is conceivable that the RhoA/Rho kinase pathway influences SRF localization through its effect on actin polymerization dynamics.

Abbreviations: ethylenediamine tetraacetic acid, EDTA • fluorescein isothiocyanate, FITC • nuclear localization sequence, NLS • smooth muscle myosin heavy chain, smMHC • serum response factor, SRF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Rho GTPases, including RhoA, Rac1, and Cdc42, activate downstream targets that regulate cell morphology, cytoskeletal microfilaments and focal adhesion formation, cell motility, and smooth muscle contraction (1–8). Rho GTPases can also influence transcription of immediate early genes (9–12), of skeletal muscle actin (13), and of smooth muscle contractile apparatus–encoding genes (14), through pathways that lead to activation of serum response factor (SRF). SRF is a nuclear protein that binds to its DNA target sequence (the "CArG box," CC[A/T]6GG) in the regulatory regions of these and a number of other striated and smooth muscle–specific contractile apparatus encoding genes, and so activates their transcriptional expression. Previously, we found that the transcription-promoting activity of SRF can be controlled physiologically through regulated nuclear localization (15). In long-term serum-deprived airway myocytes, SRF was redistributed out of the nucleus and into the cytoplasm, resulting in selective reduction of SRF-dependent smooth muscle gene transcription. The mechanisms that regulate SRF nuclear trafficking remain largely unknown (16–18).

In the present study, we tested the hypothesis that RhoA and its downstream target, Rho kinase, control smooth muscle contractile apparatus gene transcription in part by regulating the subcellular localization of SRF. We formulated this hypothesis by reasoning that: (i) RhoA and Rho kinase regulate SRF-dependent gene transcription in vascular smooth muscle (14) and in other systems (9–13, 19–22), through a mechanism that depends upon actin cytoskeletal dynamics (23, 24); (ii) long-term serum deprivation redistributes SRF out of the nucleus of cultured airway myocytes (15); and (iii) because serum components activate RhoA activity, perhaps changes in RhoA/Rho kinase pathway activity also regulate SRF subcellular localization and so also influence SRF-dependent smooth muscle contractile apparatus gene expression. Our results confirm that this mechanism does operate in airway myocytes, and thereby disclose a previously unknown role for this important signaling pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Canine tracheal myocytes were grown on uncoated plastic dishes or glass coverslips (15, 25), and were studied at passage 1 or 2. Serum-fed myocytes were maintained in Dulbecco's modified Eagle's medium:F-12 (1:1) plus 10% fetal bovine serum, 0.1 mM nonessential amino acids, 50 U/ml penicillin, and 50 µg/ml streptomycin. Serum-deprived cells were grown to confluence, then maintained for 1–2 d in serum-free Dulbecco's modified Eagle's medium:F-12 containing 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium, as well as nonessential amino acids and antibiotics as above. In some experiments, the specific Rho kinase inhibitor, Y-27632 (10 µM; kindly provided by Welfide Corporation) (26, 27), was added to culture medium continuously within 24 h after plating at low density, as cells grew to near confluence, and during 1–2 d subsequent serum deprivation.

Plasmids
Promoter reporters. In pSM22luc, transcription of the luciferase cDNA in pGL2basic (Promega Corporation, Madison, WI) is directed by bp -445 to +41 of the mouse SM22 gene (28). In p5xCArGluc, luciferase expression is directed by an artificial promoter containing five copies of the SRF binding site (CC[A/T]6GG) upstream of a minimal TATA box (Stratagene, La Jolla, CA). In psmMHCluc, 3.3 kb of human smooth muscle myosin heavy chain (smMHC) promoter drive luciferase expression (15). pMSVluc and pMSVßgal, in which the viral MSV-LTR promoter controls luciferase or ß-galactosidase expression, have been described previously (28, 29). In p4xAP2luc, four copies of the human keratin K14 promoter sequence, which contains an AP-2 binding site, were cloned upstream of the minimal K14 promoter in the K14mpLuc vector (15).

RhoA pathway modulators. Plasmids pEVX-RhoAV14 and pEVX-RhoAN19 (13), which express the constitutively active V14 and dominant-negative N19 mutants of RhoA, respectively, were kindly provided by Dr. A. Hall, as was pRK5-myc, which expresses the C3 exotoxin of Clostridium botulinum (30). All plasmids were purified on CsCl gradients before transfection.

Transfections
Transient transfection of plasmids was accomplished with cationic lipids (Lipofectamine; Invitrogen Corporation, Carlsbad, CA). Subconfluent myocytes in 6-well plates were transfected for 5 h in Optimem (Invitrogen Corporation) with 12 µg Lipofectamine and 1.2 µg DNA, including 0.6 µg luciferase reporter plasmid, 0.6 µg pMSVßgal (used to normalize transfection efficiency), and 0–25 ng plasmid encoding modulators of the RhoA pathway (total DNA was held constant with empty pEVX vector). Myocytes were refed with 10% fetal bovine serum containing medium for another 19 h after transfection, and thereafter were placed in serum-free medium for an additional 24 h. In some experiments, the specific Rho kinase inhibitor, Y-27632 (10 µM) was added to serum-free medium; in these and in corresponding untreated wells, 50 ng pcDNA3.1 were also included in the transfection. In the above experiments, cells were harvested 48 h after transfection for measurement of luciferase and ß-galactosidase activities, and luciferase activity was normalized to ß-galactosidase activity for each well (28, 29). In additional experiments, latrunculin B (0.5 µM) in serum-containing medium was added after 6 h transfection, and cells were harvested after 12 h latrunculin exposure. In all experiments, normalized luciferase activities from triplicate wells were averaged to provide the datum; experiments were repeated three to seven times, and the average (± SEM) shown.

Preparation of Nuclear, Cytoplasmic, and Whole Cell Extracts
Canine tracheal myocytes were plated at 15–30% confluence, grown to confluence in serum-containing medium, then serum-deprived for 2 d as described above. Whole cell extracts were prepared from similarly treated cultures using the methods previously reported (31). In some studies requiring isolation of nuclear lysates, 10 µM Y-27632 was included throughout growth and serum-deprivation periods. In other experiments, nuclear extracts were prepared from serum-fed myocytes pretreated with latrunculin B (0.5 µM) for 0, 6, or 12 h, and soluble cytoplasmic lysates were also collected and stored.

Nuclear and cytoplasmic extracts were prepared from myocytes after 2 d serum deprivation using a method modified from that described by Dignam and coworkers (32). Cells were harvested from cell culture using 0.5% trypsin–0.53 mM ethylenediamine tetraacetic acid (EDTA) and scraping. All subsequent steps were performed at 4°C using ice-cold reagents. Cells were pelleted by centrifugation (10 min, 800 x g), resuspended again in 5 vols of ice cold phosphate-buffered saline, and re-centrifuged. The resulting pellet was gently resuspended in five packed cell volumes of hypotonic Buffer A (in mM: 10 HEPES pH 7.9, 1.5 MgCl2, 10 KCl, 0.5 DTT, 0.5 PMSF) and incubated for 10 min. Ruptured cells were collected by centrifugation (10 min, 800 x g); the supernatant was carefully aspirated and retained as a crude cytosolic lysate for subsequent analysis of soluble protein content; protease inhibitors (leupeptin, antipain, chymostatin, and pepstatin A, 5 µg/ml each; Sigma, St. Louis, MO) were added, and aliquots were frozen -80°C until use. The remaining pellet was resuspended in two packed cell volumes of Buffer A, then nuclei were further separated from adherent cytosolic components using 10 gentle strokes of a glass Dounce homogenizer (#885303–0007 with Type B pestle; Kontes, Vineland, NJ). Cell disruption and nuclear integrity was confirmed by visual inspection under a phase contrast microscope, then nuclei were collected by centrifugation (10 min, 800 x g). The pellet was carefully transferred to an Oakridge centrifuge tube and centrifuged (20 min, 25,000 x g). Supernatant was carefully removed and the remaining nuclear pellet was resuspended in two packed cell volumes of Buffer C (in mM: 20 HEPES pH 7.9, 1.5 MgCl2, 420 NaCl, 0.5 DTT, 0.5 PMSF, and 25% glycerol), then further disrupted using a glass Dounce homogenizer with Type B pestle and gently shaken for 30 min. The crude nuclear lysate was centrifuged (30 min, 25,000 x g) and the supernatant was dialyzed with Buffer D (in mM: 20 HEPES pH 7.9, 100 KCl, 0.2 EDTA, 0.5 DTT, 0.5 PMSF, and 20% glycerol) overnight using dialysis membrane (12–14 kD exclusion; Invitrogen). Protease inhibitors (leupeptin, antipain, chymostatin, and pepstatin A, 5 µg/ml each; Sigma) were added to the nuclear extract, and aliquots were frozen -80°C until use.

Electrophoretic Mobility Shift Assay
Double-stranded DNA fragments harboring the sequences of interest were prepared by annealing complementary synthetic oligonucleotides, and were end-labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP. CArG-box-containing oligonucleotides encompassed the 5' (5'-GCTGCCCATAAAAGGTTTTTG-3') or 3' (5'-CTTTCCCCAAATATGGAGCCTG-3') CArG boxes (underlined) of the mouse SM22 promoter. An oligonucleotide harboring an AP2 binding site (5'-TCGAACTGACCGCCCGCGGCCCGT-3') was also used. 20,000 dpm (1–5 fmol) radiolabeled oligonucleotide were pre-incubated for 15 min with 1.5 µl binding buffer (50 mM Tris HCl pH 7.5, 20% Ficoll, 375 mM KCl, 5 mM EDTA, 5 mM DTT) and 1 µg poly (dI-dC). When indicated, 200-fold molar excess of unlabeled competitor oligonucleotide (identical to probe, or containing a myb binding site, 5'-TACAGGCATAACGGTTCCGTAGTG-3') was added. For supershift experiments, 3 µg of antibody were added to the incubation mixture; supershift antibodies included anti-SRF, anti-AP2{alpha}, anti-AP2ß, and anti-AP2{gamma} (all from Santa Cruz Biotechnology, Santa Cruz, CA) or anti-IL5 (provided by Searle Laboratories), used as a negative control. Binding reactions (3–6 µg nuclear extract protein) were performed at room temperature in 15 µl for 30 min. DNA–protein complex formation was analyzed by electrophoresis on 5% nondenaturing polyacrylamide gels in TBE buffer and autoradiography.

Western Analysis
Nuclear extracts or total cell lysates from 2 d serum-deprived myocytes with or without Y-27632 treatment were resolved using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. SRF and AP2 protein level were detected as ~ 67 kD and ~ 50 kD bands using anti-SRF (Santa Cruz Biotechnology) and anti–AP-2{alpha} primary antibodies (Santa Cruz Biotechnology), respectively, horseradish peroxidase–conjugated anti-rabbit IgG secondary antibody, and enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ). Western analysis of SRF and AP2 protein levels was also performed on nuclear extracts and cytoplasmic proteins obtained from serum-fed myocytes exposed to latrunculin B for 0–12 h.

Quantitative Immunocytochemistry
Cellular localization of SRF was identified by immunocytochemistry performed as previously described (15), and the subcellular distribution of SRF was quantified as follows. Airway myocytes were cultured from two different dogs. Cells from each dog were passaged onto four coverslips; two coverslips from each dog were treated with Y-27632 as above and two coverslips were untreated, but otherwise handled identically. Coverslips were immunostained for SRF (Santa Cruz primary antibody; fluorescein isothiocyanate [FITC]-conjugated secondary antibody) and nuclei were counterstained with propidium iodide, then two randomly chosen fields on each coverslip were imaged by laser scanning confocal microscopy (Olympus, Melville, NY). Stacks of multiple planar images (x100) in each field were collected at 0.5 µm spacing; we found that the entire cell height for every cell in each field was encompassed in 4–7 stacked images, which were analyzed for SRF subcellular distribution. Nuclear compartments of all the cells in each image of each field were identified by positive propidium iodide staining, and regions of interest were drawn to identify these nuclear compartments (there were 7–25 nuclear regions in each image). SRF fluorescence intensity in the cytoplasmic and nuclear compartments was measured as follows. For each image in the stack of images comprising a field, the areas (in square microns) and average FITC fluorescence intensities of the entire image and of individual nuclear compartments were measured using the UltraView Version 3.0 software package (Olympus LSR). Background fluorescence intensity (arbitrary units) was measured as the average of values from 3 acellular regions on each image, and was subtracted from all fluorescence intensity measurements to yield the corresponding SRF fluorescence intensity. Total cellular SRF content in each field was calculated by summing SRF fluorescence intensity times area (arbitrary units x sq. microns) across all images in the field, and the corresponding nuclear SRF content was calculated from the sum of products of SRF fluorescence times nuclear compartment area, including all nuclear compartments within each image. The percent nuclear SRF for each field was then calculated as nuclear SRF content/total cellular SRF content x 100. Percent cytoplasmic SRF for each field was calculated as 100 minus percent nuclear SRF. Eight fields were analyzed for each condition (2 dogs x 2 coverslips/dog x 2 fields/coverslip for each condition); results from each field were treated as independent data points.

In additional experiments, we determined the ratio of filamentous (F) to globular (G) actin ratio using quantitative fluorescence imaging of cultured airway myocytes obtained from three dogs, grown on glass coverslips. Cells were untreated or treated with 0.5 uM latrunculin B, for 12 h, then fixed. We stained F-actin using Texas Red-X phalloidin (5 U/ml or 165 nM) and stained G-actin with FITC-DNase I (0.45 µg/ml or 165 nM), both for 20 min at 4°C. Average fluorescence intensity in each field was calculated for each dye in each field, subtracting the background intensity as determined from acellular regions of the glass coverslip. A total of 12 fields were evaluated for each treatment group (2 fields/coverslip x 2 coverslips/dog x 3 dogs).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SRF-Dependent Smooth Muscle Gene Transcription Varies Directly with RhoA Activity in Airway Myocytes
Figure 1 (top panel) shows the activities of the mouse SM22, human smMHC, SRF-dependent 5xCArG, MSV-LTR, and AP2-dependent 4xAP2 promoters during transient cotransfection with expression plasmid encoding the constitutively active RhoAV14. RhoAV14 more than doubled the transcriptional activities of the smooth muscle–specific SM22 and smMHC promoters in a dose-dependent fashion. Because of the central importance of SRF in activating transcription from these promoters (33–40) and because RhoA has been implicated in the regulation of SRF-dependent transcription in nonmuscle cells and in vascular muscle cells, we also tested the effect of RhoA activation on transcription from the purely SRF-dependent 5xCArG promoter in airway smooth muscle. Like its effect on the SM22 and smMHC promoters, RhoAV14 did activate the 5xCArG reporter, but it had no effect on transcription from the MSV-LTR or 4xAP2 promoters. Conversely, inhibition of endogenous RhoA activity by cotransfection with plasmid encoding the dominant negative RhoAN19 selectively reduced transcription from the SM22, smMHC, and 5xCArG promoters, but did not alter MSV-LTR or 4xAP2 activities (Figure 1, bottom panel). Inhibition of endogenous RhoA activity by transient expression of C3 exotoxin (2 ng plasmid/well) also disproportionately reduced activities of the smooth muscle–specific and SRF-dependent promoters (Figure 2, top panel). In light of the known requirement for SRF activity in SM22 and smMHC transcription, these data demonstrate that RhoA activity directly determines the activation of smooth muscle–specific gene transcription in cultured airway myocytes, and that it mediates this effect at least in part through changes in SRF transcription-promoting activity.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 1. Overexpression of constitutively active RhoAV14 increases transcription from the SRF-dependent SM22, smMHC, and 5xCArG promoters in a dose-dependent fashion, whereas dominant-negative RhoAN19 has the opposite effect (P < 0.03 each, Kruskal-Wallis one-way ANOVA). In contrast, RhoA activity does not influence transcription from the SRF-independent MSV-LTR or 4xAP2 promoters (P > 0.48 each, Kruskal-Wallis one-way ANOVA). Amounts of RhoAV14 or RhoAN19 expression plasmids are shown in nanograms. Open bars, 0 ng; shaded bars, 10 ng; black bars, 25 ng.

 


View larger version (35K):
[in this window]
[in a new window]
  		Figure 2. Both C3 toxin from C. botulinum, which inhibits RhoA (upper panel), and Y-27632, which inhibits Rho kinase (lower panel), selectively suppress transcription from SRF-dependent promoters. Each bar shows promoter activity in the presence of C3 toxin or Y-27632 as a fraction of activity without C3 toxin or Y-27632, respectively. In this presentation, relative promoter activities smaller than 1.0 reflect inhibition of promoter activities by either C3 toxin or Y-27632. ANOVA disclosed that both C3 toxin and Y-27632 reduced transcription from the SRF-dependent SM22, smooth muscle myosin heavy chain (smMHC), and artificial 5xCArG promoters to a significantly greater extent than they reduced transcription from the non–SRF-dependent MSV and artificial 4xAP2 promoters (included as controls for any nonspecific influence of either agent on transcription in this assay).

 
Rho Kinase in Part Mediates the Influence of RhoA on SRF-Dependent Smooth Muscle Gene Transcription
We used the selective Rho kinase inhibitor, Y-27632, to test the requirement for p160ROCK in RhoA-mediated activation of smooth muscle–specific gene transcription. As shown in Figure 2 (bottom panel), Y-27632 (10 µM) decreases transcription from the SM22, smMHC, and SRF-dependent promoters (14, 41, 42). In marked contrast, inhibition of Rho kinase activity did not alter transcription from the MSV-LTR or AP2-dependent promoters (Figure 2, bottom panel). Together, these data demonstrate that RhoA selectively activates SRF-dependent gene transcription in airway smooth muscle at least in part through activation of Rho kinase.

Rho Kinase Regulates SRF Subcellular Localization
Previously, we demonstrated that long-term serum deprivation of cultured airway myocytes reduces transcription from the SM22 and smMHC promoters, in part by redistributing SRF from the nucleus into the cytoplasm. Because long-term serum deprivation likely reduces activity of the RhoA/ROCK pathway, we hypothesized that these signaling intermediates may exert their influence on SRF transcription promoting activity in part by altering its subcellular localization. We tested this hypothesis using Y-27632 to inhibit Rho kinase; treated cells were exposed to 10 µM Y-27632 as they grew from 15–30% confluence through confluence, and during two subsequent days of serum deprivation. As revealed by electrophoretic mobility shift assay analysis, the binding of SRF to the 3' CArG box of the mouse SM22 promoter was markedly reduced in nuclear extracts from Y-27632–treated airway myocytes (Figure 3A; similar results were obtained for SRF binding to the 5' CArG box, data not shown). In contrast, Rho kinase inhibition had no effect upon or tended to increase binding of AP2 to its consensus target sequence (Figure 3A). To discern whether the decreased binding of SRF stems from diminished nuclear SRF protein abundance, we performed Western analysis to quantify SRF and AP2 in nuclear extracts from untreated and Y-27632–treated airway myocytes (Figure 3B). Y-27632 treatment reduced SRF band intensity by 39.2 ± 15.2 (SEM)% (n = 7, P < 0.05, paired t test), even though it simultaneously tended to increase nuclear AP2 band intensity, by 26.8 ± 25.8% (n = 7, P = NS, paired t test). The SRF gene promoter is itself activated by SRF (43, 44). Therefore, to test the possibility that the decrease in nuclear SRF caused by Y-27632 stems from whole-cell depletion of SRF, we directly measured total cellular SRF by Western analysis of whole cell lysates. In Y-27632–treated myocytes, there was a small and nonsignificant increase (7.0 ± 17.7%; n = 4, P = NS, paired t test) in SRF content compared with paired, untreated myocyte cultures; this finding therefore excludes the stated possibility. Finally, we used laser scanning confocal microscopy to quantify SRF throughout the entire nuclear or cytoplasmic compartments of Y-27632–treated or untreated airway myocytes (Figure 4); note that use of such volumetric quantification of nuclear SRF and of cytoplasmic SRF negates any potential influence of the cell shape change per se that accompanies Rho kinase inhibition (Figure 4). These studies demonstrated that 51.5 ± 2.3% of total cellular SRF resides within the nucleus of untreated cells, whereas only 27.9 ± 1.0% of total cellular SRF is found in the nucleus of Y-27632–treated myocytes (n = 8 random fields/condition; P < 0.001, unpaired t test). Conversely, the cytoplasmic fraction of SRF increased from 48.5% in untreated cells to 72.1% in Y-27632–treated myocytes. Note that the effect of Y-27632 treatment on change in nuclear SRF content appeared roughly similar when assessed using quantitative immunocytochemistry (45.8%) or Western analysis of nuclear extracts (39.2%). Together, these data suggest that the RhoA/Rho kinase pathway alters SRF-dependent gene transcription in airway smooth muscle in part by regulating SRF subcellular localization.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 3. Inhibition of Rho kinase promotes extranuclear redistribution of SRF. (A) EMSA demonstrating reduced SRF binding activity in nuclear extracts from canine tracheal myocytes treated with Y-27632. In contrast, AP2 containing complexes are unchanged or increased in Y-27632 treated cells. Comp/Ab denotes cold oligonucleotide competitor (self, same as radiolabeled probe; myb, contains the consensus binding site for c-myb) or antibody ({alpha}SRF, anti-SRF; {alpha}IL-5, anti–interleukin-5; {alpha}AP2{alpha},ß,{gamma}, anti-AP2{alpha},ß,{gamma}) added to nuclear extract/probe binding reaction. (B) Typical immunoblot and summary graph demonstrating selectively reduced nuclear SRF content in Y-27632–treated canine tracheal myocytes. Y-27632 treatment reduced SRF band intensity on Western blots by 39.2 ± 15.2% (n = 7, P < 0.05, paired t test), even though it simultaneously tended to increase nuclear AP2{alpha} band intensity, by 26.8 ± 25.8% (n = 7, P = NS, paired t test).

 


View larger version (94K):
[in this window]
[in a new window]
  		Figure 4. Confocal analysis of the effects of 10 µM Y-27632 on SRF distribution in canine tracheal myocytes. A–D and E–H show two representative, partial stacks of confocal images of myocytes grown to subconfluence, then serum starved for 24 h. Images were obtained at x100 magnification and 0.5 µm vertical spacing, and are shown with acellular background intensity subtracted from entire image; A and E are closest to the glass coverslips on which cells were grown. Myocytes in E–H were treated with Y-27632 during growth and serum deprivation, whereas the control myocytes in A–D were untreated throughout. Immunoreactive SRF is shown in green; DNA staining is shown in red; merged image in each panel shows SRF and DNA staining superimposed. Note that SRF staining is prominent in the nucleus of control cells, but is much more obvious in the cytoplasm of Y-27632–treated myocytes.

 
Latrunculin B Also Partially Redistributes SRF out of the Nucleus
The RhoA/ROCK pathway is a prominent regulator of actin cytoskeletal dynamics; inhibiting RhoA/ROCK activity leads to diminished actin polymerization. Other interventions that oppose actin polymerization (but which act independent of the RhoA/ROCK pathway) similarly inhibit SRF-dependent gene transcription; among these has been treatment with latrunculin, which binds to and stabilizes depolymerized actin molecules, preventing their incorporation into F-actin. Together with our results above, these findings suggested the possibility that the RhoA/ROCK pathway might influence its effect on SRF subcellular localization through its effect on actin dynamics. To test this possibility, we first confirmed that latrunculin B treatment leads to actin filament depolymerization in our cultured airway myocytes, using quantitative fluorescence microscopy. As anticipated, we found that latrunculin B significantly reduced F/G actin ratio from 31.2 ± 24.7 (SD) (arbitrary units) to 11.4 ± 6.1 (SD) (P < 0.001, Mann-Whitney rank sum test). Next, we performed Western analysis of nuclear and cytoplasmic fractions of serum-fed confluent myocytes treated with latrunculin B. As shown in Figure 5, latrunculin B exposure lowered nuclear SRF content, whereas it increased cytoplasmic SRF content. Thus, inhibition of actin polymerization appears sufficient to influence SRF subcellular localization. Finally, we compared the influence of latrunculin B treatment of airway myocytes from three different dogs on transcription from artificial SRF-dependent (5xCArG), AP2-dependent (4xAP2), and viral MSV promoters. Similar to Y-27632, latrunculin treatment decreased transcription from all three promoters, but its influence on SRF-dependent transcription (78 ± 4.1 [SD]% reduction) was significantly more marked (P = 0.015, ANOVA for repeated measures) than on AP2-dependent (56.9 ± 4.2% reduction) or MSV-LTR–dependent (49.4 ± 9.6% reduction) transcription.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 5. Treatment with latrunculin B (0.5 µM) causes extranuclear redistribution of SRF in cultured serum-fed airway myocytes. Top panel shows representative Western blots from cytoplasmic and nuclear compartments of airway myocytes in a single experiment. Protein loading and exposure times are shown for each panel. Bottom panel shows the average (± SEM) fractional change in cytoplasmic or nuclear SRF content at 0, 6, and 12 h after addition of latrunculin B to cultures (n = 3 experiments); data are log transformed to facilitate comparison of fractional increases and decreases on the same plot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results of this study disclose a novel mechanism through which the RhoA/Rho kinase intracellular signaling pathway may in part control gene transcription in differentiated smooth muscle cells: by regulating the subcellular localization of serum response factor, a critical activator of a variety of muscle-specific and immediate early genes. We found that increasing the intracellular activity of RhoA in cultured airway myocytes, by cotransfection with an expression plasmid encoding a constitutively active mutant of RhoA (RhoAV14), selectively enhanced transcription from the SM22 and smMHC promoters and from a purely SRF-dependent promoter (5xCArG), but had no effect on transcription from the MSV-LTR promoter or from an AP2-dependent promoter (4xAP2) (Figure 1). Conversely, inhibition of RhoA or of its downstream effector, Rho kinase, by cotransfection with a plasmid expressing a dominant-negative mutant of RhoA (RhoAN19), by cotransfection with a plasmid expressing C3 toxin from C. botulinum, or by incubation with the Rho kinase inhibitor, Y-27632, all selectively reduced SRF-dependent smooth muscle promoter activity (Figures 1 and 2). Furthermore, treatment with Y-27632 selectively reduced binding of SRF from nuclear extracts to its consensus DNA target (Figure 3), selectively reduced nuclear SRF protein content (Figure 3), and partially redistributed SRF from nucleus to cytoplasm, as revealed by quantitative immunocytochemistry. Together, these results demonstrate for the first time that the RhoA/Rho kinase pathway controls smooth muscle gene transcription at least in part by regulating the subcellular localization of SRF. Prior studies implicated Rho GTPases in the transcriptional regulation of skeletal muscle {alpha}-actin (13), and Rho kinase has been shown to regulate SRF activity in fibroblasts (19). In addition, a more recent study by Mack and colleagues using vascular myocytes (14) reports generally similar activation of smooth muscle–specific gene promoters by RhoA and Rho kinase as demonstrated here. Our study extends those prior results, by disclosing a cellular mechanism through which the RhoA/Rho kinase pathway may regulate SRF-dependent gene transcription, and by confirming its relevance in living airway smooth muscle cells. It is conceivable that reduced RhoA/Rho kinase pathway activity in long-term serum-deprived airway myocytes explains their extranuclear SRF relocalization and reduced smooth muscle gene transcription (15).

Very little is known about the nuclear trafficking of SRF. Rech and coworkers (16) and Gauthier-Rouvier and colleagues (17) identified a classical basic nuclear localization sequence (NLS) within AA 95–100 of human SRF (91-LGAERRGLKRSLSE-104; NLS boxed, basic residues bold) that is required for SRF nuclear localization (as proved through mutational analysis), and which confers nuclear localization to IgG when expressed as an SRF-NLS-IgG fusion protein. Interestingly, none of three other potential basic NLS sequences within SRF were functional. Basal protein kinase A activity was required for SRF nuclear entry, but this did not depend upon PKA phosphorylation of SRF, because mutation to alanine of the Ser101 or Ser103 residues near the NLS (within the sequence shown above) did not prevent its ability to confer nuclear import to heterologous fusion proteins. Indeed, PKA activity was also required for nuclear import mediated by the SV40 large-T NLS (which cannot be phosphorylated by PKA), suggesting that PKA exerts its influence on nuclear import in a cargo-nonspecific fashion.

No previous studies have specifically addressed the nuclear export of SRF, but it is now certain that SRF is not always localized to the nucleus. Ding and coworkers (18) recently observed cytoplasmic redistribution of SRF when cultured, nontransformed NIH3T3T cells underwent differentiation into adipocytes. In addition, those differentiated adipocytes lost the ability to increase transcription from the c-fos promoter (which relies critically on SRF binding) upon serum stimulation. Thus, exclusion of SRF from the nucleus also suppressed SRF-dependent gene transcription in NIH3T3T cells. We independently gathered strong evidence that prolonged serum deprivation of cultured airway myocytes redistributes SRF out of the nucleus, and that this redistribution suppresses SRF-dependent transcription of smooth muscle contractile apparatus-encoding genes (15). Furthermore, Gauthier-Rouviere and colleagues (17) demonstrated that hemaglutinin-tagged SRF disappeared from the nucleus of cultured fibroblasts within 15 min of inhibiting SRF nuclear import, suggesting that active nuclear export of SRF occurs. Ultimately, it is the balance of nuclear import and nuclear export that determines the net SRF subcellular localization.

We do not yet know how the RhoA/Rho kinase pathway promotes SRF nuclear localization in differentiated airway smooth muscle cells, but recent studies implicate three potential mechanisms: 1) Enhanced nuclear import or impaired nuclear export due to protein kinase C (PKC){alpha} and/or PKC{epsilon} dependent phosphorylation of SRF. Multiple studies demonstrate that RhoA functions as an upstream activator of various PKC isoforms to transduce a variety of external signals (45–47), and PKC{alpha}, RhoA, and Rho kinase all translocate to the cell membrane simultaneously on muscarinic stimulation of vascular smooth muscle cells (48), suggesting their interrelated co-activation. Recently, Soh and coworkers (49) demonstrated that PKC{alpha} and PKC{epsilon} are required for RhoA-induced transcription from a mutated c-fos promoter whose serum response element bound SRF, but not Ets transcription factors. SRF contains six consensus PKC phosphorylation sites (27-TGR-29, 162-SKR-164, 199-TRK-201, 210-TGK-212, 262-TLK-264, and 504-STK-506), and so it is conceivable that PKC{alpha} and/or PKC{epsilon} phosphorylate SRF directly, thereby modifying its NLS or NES function. 2) Enhanced nuclear retention attributable to increased complexing with p300/CBP. Prior reports indicate that p300 interacts with SRF (50, 51), as it does with many other transcription factors, including HIF-1{alpha} (52) and GATA proteins (53–55). In addition, p300 sequesters interferon response factor-3 (IRF-3) in the nucleus through high affinity binding (56). Together, these findings suggest the as yet untested possibility that RhoA promotes nuclear retention by increasing affinity of SRF for p300. 3) Altered SRF nuclear import or export due to changes in actin polymerization. Recent studies each implicate actin polymerization in the activation by RhoA of SRF-dependent transcription in nonmuscle cells (23, 24) or rat vascular smooth muscle cells (14). Indeed, our experiments using latrunculin B to modulate actin polymerization dynamics (Figure 5) show that inhibition of actin polymerization is sufficient to cause partial redistribution of SRF from nucleus to cytoplasm. Thus, it seems likely that RhoA/Rho kinase–induced actin polymerization, or its consequent effects on G-actin, similarly alters the nuclear import or export of SRF, or its sequestration in cytoplasmic or nuclear compartments. The extent to which any of these mechanisms accounts for the influence of the RhoA/Rho kinase pathway on SRF subcellular localization observed here remains to be evaluated more fully.

Two recent reports confirm that entry of SRF into the nucleus is required for differentiation of smooth muscle cells, and that the RhoA/ROCK pathway influences SRF relocalization during differentiation. Lu and colleagues (57) showed that inhibition of Rho kinase prevented the epithelial to mesenchymal transformation and expression and nuclear localization of SRF that occur during development of coronary arterial myocytes, whereas Beqaj and coworkers (58) noted that high RhoA activity promotes the exclusion of SRF from the nucleus of undifferentiated mesenchymal precursors of smooth muscle myocytes. The results of our present study extend those reports, by demonstrating that the RhoA/Rho kinase pathway controls the subcellular distribution of SRF in differentiated smooth muscle cells, in a way that could at least in part explain the influence of this pathway on SRF-dependent transcription.

Results of the present study could have potential medical implications, because they suggest a possible strategy for inhibiting contractile apparatus protein accumulation in smooth muscle: by suppressing SRF-dependent smooth muscle gene transcription through inhibition of the RhoA/Rho kinase pathway. Several important diseases involve aberrant contraction of smooth muscle, including asthma and pulmonary hypertension. Conceivably, suppressing contractile apparatus accumulation in airway or pulmonary vascular smooth muscle could inhibit contraction of smooth muscle in these structures, and so inhibit the obstruction to airflow or pulmonary blood flow that characterizes these diseases. The potential for this therapeutic approach may be of interest for future study.


   Acknowledgments
 
This work was supported by SCOR HL56399, HL64095, Inspiraplex, CIHR 15562, Manitoba Health Research Council, Glaxo Smith Kline, and the Australia-New Zealand Thoracic Society. The authors are indebted to Dr. William T. Gerthoffer for providing canine tracheal tissue for smooth muscle cell isolation and culture.


   Footnotes
 
* These authors contributed equally to the work presented in this article. Back

Received in original form October 4, 2002

Received in final form December 25, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Van Aelst, L., and C. D'Souza-Schorey. 1997. Rho GTPases and signaling networks. Genes Dev. 11:2295–2322.[Free Full Text]
  2. Uehata, M., T. Ishizaki, H. Satoh, T. Ono, T. Kawahara, T. Morishita, H. Tamakawa, K. Yamagami, J. Inui, M. Maekawa, and S. Narumiya. 1997. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389:990–994.[CrossRef][Medline]
  3. Lamb, R. F., C. Roy, T. J. Diefenbach, H. V. Vinters, M. W. Johnson, D. G. Jay, and A. Hall. 2000. The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho. Nat. Cell Biol. 2:281–287.[CrossRef][Medline]
  4. Nobes, C. D., and A. Hall. 1999. Rho GTPases control polarity, protrusion, and adhesion during cell movement. J. Cell Biol. 144:1235–1244.[Abstract/Free Full Text]
  5. Togashi, H., C. W. Emala, I. P. Hall, and C. A. Hirshman. 1998. Carbachol-induced actin reorganization involves Gi activation of Rho in human airway smooth muscle cells. Am. J. Physiol. 274:L803–L809.
  6. Hall, A. 1998. Rho GTPases and the actin cytoskeleton. Science 279:509–514.[Abstract/Free Full Text]
  7. Hotchin, N. A., and A. Hall. 1995. The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. J. Cell Biol. 131:1857–1865.[Abstract/Free Full Text]
  8. Nobes, C. D., and A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81:53–62.[CrossRef][Medline]
  9. Hill, C. S., J. Wynne, and R. Treisman. 1994. Serum-regulated transcription by serum response factor (SRF): a novel role for the DNA binding domain. EMBO J. 13:5421–5432.[Medline]
  10. Price, M. A., C. Hill, and R. Treisman. 1996. Integration of growth factor signals at the c-fos serum response element. Philos. Trans. R. Soc. Lond. B Biol. Sci. 351:551–559.[Medline]
  11. Zohar, M., H. Teramoto, B. Z. Katz, K. M. Yamada, and J. S. Gutkind. 1998. Effector domain mutants of Rho dissociate cytoskeletal changes from nuclear signaling and cellular transformation. Oncogene 17:991–998.[CrossRef][Medline]
  12. Hill, C. S., J. Wynne, and R. Treisman. 1995. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 81:1159–1170.[CrossRef][Medline]
  13. Wei, L., W. Zhou, J. D. Croissant, F. E. Johansen, R. Prywes, A. Balasubramanyam, and R. J. Schwartz. 1998. RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation. J. Biol. Chem. 273:30287–30294.[Abstract/Free Full Text]
  14. Mack, C. P., A. V. Somlyo, M. Hautmann, A. P. Somlyo, and G. K. Owens. 2001. Smooth muscle differentiation marker gene expression is regulated by RhoA-mediated actin polymerization. J. Biol. Chem. 276:341–347.[Abstract/Free Full Text]
  15. Camoretti-Mercado, B., H. W. Liu, A. J. Halayko, S. M. Forsythe, J. W. Kyle, B. Li, Y. Fu, J. McConville, P. Kogut, J. E. Vieira, N. M. Patel, M. B. Hershenson, E. Fuchs, S. Sinha, J. M. Miano, M. S. Parmacek, J. K. Burkhardt, and J. Solway. 2000. Physiological control of smooth muscle-specific gene expression through regulated nuclear translocation of serum response factor. J. Biol. Chem. 275:30387–30393.[Abstract/Free Full Text]
  16. Rech, J., I. Barlat, J. L. Veyrune, A. Vie, and J. M. Blanchard. 1994. Nuclear import of serum response factor (SRF) requires a short amino-terminal nuclear localization sequence and is independent of the casein kinase II phosphorylation site. J. Cell Sci. 107:3029–3036.[Abstract]
  17. Gauthier-Rouviere, C., M. Vandromme, N. Lautredou, Q. Q. Cai, F. Girard, A. Fernandez, and N. Lamb. 1995. The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation. Mol. Cell. Biol. 15:433–444.[Abstract]
  18. Ding, W., M. M. Witte, and R. E. Scott. 1999. Transformation blocks differentiation-induced inhibition of serum response factor interactions with serum response elements. Cancer Res. 59:3795–3802.[Abstract/Free Full Text]
  19. Chihara, K., M. Amano, N. Nakamura, T. Yano, M. Shibata, T. Tokui, H. Ichikawa, R. Ikebe, M. Ikebe, and K. Kaibuchi. 1997. Cytoskeletal rearrangements and transcriptional activation of c-fos serum response element by Rho-kinase. J. Biol. Chem. 272:25121–25127.[Abstract/Free Full Text]
  20. Alberts, A. S., O. Geneste, and R. Treisman. 1998. Activation of SRF-regulated chromosomal templates by Rho-family GTPases requires a signal that also induces H4 hyperacetylation. Cell 92:475–487.[CrossRef][Medline]
  21. Sahai, E., A. S. Alberts, and R. Treisman. 1998. RhoA effector mutants reveal distinct effector pathways for cytoskeletal reorganization, SRF activation and transformation. EMBO J. 17:1350–1361.[CrossRef][Medline]
  22. Treisman, R., A. S. Alberts, and E. Sahai. 1998. Regulation of SRF activity by Rho family GTPases. Cold Spring Harb. Symp. Quant. Biol. 63:643–651.[CrossRef][Medline]
  23. Sotiropoulos, A., D. Gineitis, J. Copeland, and R. Treisman. 1999. Signal-regulated activation of serum response factor is mediated by changes in actin dynamics. Cell 98:159–169.[CrossRef][Medline]
  24. Geneste, O., J. W. Copeland, and R. Treisman. 2002. LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics. J. Cell Biol. 157:831–838.[Abstract/Free Full Text]
  25. Mitchell, R. W., A. J. Halayko, S. Kahraman, J. Solway, and M. E. Wylam. 2000. Selective restoration of calcium coupling to muscarinic M(3) receptors in contractile cultured airway myocytes. Am. J. Physiol. Lung Cell. Mol. Physiol. 278:L1091–L1100.[Abstract/Free Full Text]
  26. Ishizaki, T., M. Uehata, I. Tamechika, J. Keel, K. Nonomura, M. Maekawa, and S. Narumiya. 2000. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol. Pharmacol. 57:976–983.[Abstract/Free Full Text]
  27. Fujisawa, K., P. Madaule, T. Ishizaki, G. Watanabe, H. Bito, Y. Saito, A. Hall, and S. Narumiya. 1998. Different regions of Rho determine Rho-selective binding of different classes of Rho target molecules. J. Biol. Chem. 273:18943–18949.[Abstract/Free Full Text]
  28. Solway, J., J. Seltzer, F. F. Samaha, S. Kim, L. E. Alger, Q. Niu, E. E. Morrisey, H. S. Ip, and M. S. Parmacek. 1995. Structure and expression of a smooth muscle cell-specific gene, SM22 alpha. J. Biol. Chem. 270:13460–13469.[Abstract/Free Full Text]
  29. Camoretti-Mercado, B., S. M. Forsythe, M. M. LeBeau, R. Espinosa iii, J. E. Vieira, A. J. Halayko, S. Willadsen, B. Kurtz, C. Ober, G. A. Evans, R. Thweatt, S. Shapiro, Q. Niu, Y. Qin, P. A. Padrid, and J. Solway. 1998. Expression and cytogenetic localization of the human SM22 gene (TAGLN). Genomics 49:452–457.[CrossRef][Medline]
  30. Lamarche, N., N. Tapon, L. Stowers, P. D. Burbelo, P. Aspenstrom, T. Bridges, J. Chant, and A. Hall. 1996. Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade. Cell 87:519–529.[CrossRef][Medline]
  31. Halayko, A. J., B. Camoretti-Mercado, S. M. Forsythe, J. E. Vieira, R. W. Mitchell, M. E. Wylam, M. B. Hershenson, and J. Solway. 1999. Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes. Am. J. Physiol. 276:L197–L206.
  32. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475–1489.[Abstract/Free Full Text]
  33. Li, L., Z. Liu, B. Mercer, P. Overbeek, and E. N. Olson. 1997. Evidence for serum response factor-mediated regulatory networks governing SM22alpha transcription in smooth, skeletal, and cardiac muscle cells. Dev. Biol. 187:311–321.[CrossRef][Medline]
  34. Li, L., J. M. Miano, P. Cserjesi, and E. N. Olson. 1996. SM22 alpha, a marker of adult smooth muscle, is expressed in multiple myogenic lineages during embryogenesis. Circ. Res. 78:188–195.[Abstract/Free Full Text]
  35. Li, L., J. M. Miano, B. Mercer, and E. N. Olson. 1996. Expression of the SM22alpha promoter in transgenic mice provides evidence for distinct transcriptional regulatory programs in vascular and visceral smooth muscle cells. J. Cell Biol. 132:849–859.[Abstract/Free Full Text]
  36. Kim, S., H. S. Ip, M. M. Lu, C. Clendenin, and M. S. Parmacek. 1997. A serum response factor-dependent transcriptional regulatory program identifies distinct smooth muscle cell sublineages. Mol. Cell. Biol. 17:2266–2278.[Abstract]
  37. Madsen, C. S., C. P. Regan, and G. K. Owens. 1997. Interaction of CArG elements and a GC-rich repressor element in transcriptional regulation of the smooth muscle myosin heavy chain gene in vascular smooth muscle cells. J. Biol. Chem. 272:29842–29851.[Abstract/Free Full Text]
  38. Madsen, C. S., J. C. Hershey, M. B. Hautmann, S. L. White, and G. K. Owens. 1997. Expression of the smooth muscle myosin heavy chain gene is regulated by a negative-acting GC-rich element located between two positive-acting serum response factor-binding elements. J. Biol. Chem. 272:6332–6340.[Abstract/Free Full Text]
  39. Katoh, Y., E. Loukianov, E. Kopras, A. Zilberman, and M. Periasamy. 1994. Identification of functional promoter elements in the rabbit smooth muscle myosin heavy chain gene. J. Biol. Chem. 269:30538–30545.[Abstract/Free Full Text]
  40. Zilberman, A., V. Dave, J. Miano, E. N. Olson, and M. Periasamy. 1998. Evolutionarily conserved promoter region containing CArG*-like elements is crucial for smooth muscle myosin heavy chain gene expression. Circ. Res. 82:566–575.[Abstract/Free Full Text]
  41. Ishizaki, T., M. Naito, K. Fujisawa, M. Maekawa, N. Watanabe, Y. Saito, and S. Narumiya. 1997. p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions. FEBS Lett. 404:118–124.[CrossRef][Medline]
  42. Nakano, K., K. Takaishi, A. Kodama, A. Mammoto, H. Shiozaki, M. Monden, and Y. Takai. 1999. Distinct actions and cooperative roles of ROCK and mDia in Rho small G protein-induced reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells. Mol. Biol. Cell 10:2481–2491.[Abstract/Free Full Text]
  43. Spencer, J. A., and R. P. Misra. 1999. Expression of the SRF gene occurs through a Ras/Sp/SRF-mediated mechanism in response to serum growth signals. Oncogene 18:7319–7327.[CrossRef][Medline]
  44. Spencer, J. A., and R. P. Misra. 1996. Expression of the serum response factor gene is regulated by serum response factor binding sites. J. Biol. Chem. 271:16535–16543.[Abstract/Free Full Text]
  45. Strassheim, D., L. G. May, K. A. Varker, H. L. Puhl, S. H. Phelps, R. A. Porter, R. S. Aronstam, J. D. Noti, and C. L. Williams. 1999. M3 muscarinic acetylcholine receptors regulate cytoplasmic myosin by a process involving RhoA and requiring conventional protein kinase C isoforms. J. Biol. Chem. 274:18675–18685.[Abstract/Free Full Text]
  46. Hippenstiel, S., T. Kratz, M. Krull, J. Seybold, C. von Eichel-Streiber, and N. Suttorp. 1998. Rho protein inhibition blocks protein kinase C translocation and activation. Biochem. Biophys. Res. Commun. 245:830–834.[CrossRef][Medline]
  47. Nozu, F., Y. Tsunoda, A. I. Ibitayo, K. N. Bitar, and C. Owyang. 1999. Involvement of RhoA and its interaction with protein kinase C and Src in CCK-stimulated pancreatic acini. Am. J. Physiol. 276:G915–G923.
  48. Taggart, M. J., Y. H. Lee, and K. G. Morgan. 1999. Cellular redistribution of PKCalpha, rhoA, and ROKalpha following smooth muscle agonist stimulation. Exp. Cell Res. 251:92–101.[CrossRef][Medline]
  49. Soh, J. W., E. H. Lee, R. Prywes, and I. B. Weinstein. 1999. Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol. Cell. Biol. 19:1313–1324.[Abstract/Free Full Text]
  50. Kim, H. J., J. H. Kim, and J. W. Lee. 1998. Steroid receptor coactivator-1 interacts with serum response factor and coactivates serum response element-mediated transactivations. J. Biol. Chem. 273:28564–28567.[Abstract/Free Full Text]
  51. Ramirez, S., S. Ait-Si-Ali, P. Robin, D. Trouche, A. Harel-Bellan, and S. Ait Si Ali. 1997. The CREB-binding protein (CBP) cooperates with the serum response factor for transactivation of the c-fos serum response element. J. Biol. Chem. 272:31016–31021.[Abstract/Free Full Text]
  52. Kallio, P. J., K. Okamoto, S. O'Brien, P. Carrero, Y. Makino, H. Tanaka, and L. Poellinger. 1998. Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha. EMBO J. 17:6573–6586.[CrossRef][Medline]
  53. Blobel, G. A., T. Nakajima, R. Eckner, M. Montminy, and S. H. Orkin. 1998. CREB-binding protein cooperates with transcription factor GATA-1 and is required for erythroid differentiation. Proc. Natl. Acad. Sci. USA 95:2061–2066.[Abstract/Free Full Text]
  54. Kakita, T., K. Hasegawa, T. Morimoto, S. Kaburagi, H. Wada, and S. Sasayama. 1999. p300 protein as a coactivator of GATA-5 in the transcription of cardiac-restricted atrial natriuretic factor gene. J. Biol. Chem. 274:34096–34102.[Abstract/Free Full Text]
  55. Wada, H., K. Hasegawa, T. Morimoto, T. Kakita, T. Yanazume, and S. Sasayama. 2000. A p300 protein as a coactivator of GATA-6 in the transcription of the smooth muscle-myosin heavy chain gene. J. Biol. Chem. 275:25330–25335.[Abstract/Free Full Text]
  56. Kumar, K. P., K. M. McBride, B. K. Weaver, C. Dingwall, and N. C. Reich. 2000. Regulated nuclear-cytoplasmic localization of interferon regulatory factor 3, a subunit of double-stranded RNA-activated factor 1. Mol. Cell. Biol. 20:4159–4168.[Abstract/Free Full Text]
  57. Lu, J., T. E. Landerholm, J. S. Wei, X. R. Dong, S. P. Wu, X. Liu, K. Nagata, M. Inagaki, and M. W. Majesky. 2001. Coronary smooth muscle differentiation from proepicardial cells requires rhoA-mediated actin reorganization and p160 rho-kinase activity. Dev. Biol. 240:404–418.[CrossRef][Medline]
  58. Beqaj, S., S. Jakkaraju, R. R. Mattingly, D. Pan, and L. Schuger. 2002. High RhoA activity maintains the undifferentiated mesenchymal cell phenotype, whereas RhoA down-regulation by laminin-2 induces smooth muscle myogenesis. J. Cell Biol. 156:893–903.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
G. Elberg, L. Chen, D. Elberg, M. D. Chan, C. J. Logan, and M. A. Turman
MKL1 mediates TGF-{beta}1-induced {alpha}-smooth muscle actin expression in human renal epithelial cells
Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1116 - F1128.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
A. J. Halayko, T. Tran, and R. Gosens
Phenotype and Functional Plasticity of Airway Smooth Muscle: Role of Caveolae and Caveolins
Proceedings of the ATS, January 1, 2008; 5(1): 80 - 88.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
T. Tran, K. Ens-Blackie, E. S. Rector, G. L. Stelmack, K. D. McNeill, G. Tarone, W. T. Gerthoffer, H. Unruh, and A. J. Halayko
Laminin-Binding Integrin {alpha}7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes
Am. J. Respir. Cell Mol. Biol., December 1, 2007; 37(6): 668 - 680.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
D. A. Kass, H. C. Champion, and J. A. Beavo
Phosphodiesterase Type 5: Expanding Roles in Cardiovascular Regulation
Circ. Res., November 26, 2007; 101(11): 1084 - 1095.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
D. Schaafsma, K. D. McNeill, G. L. Stelmack, R. Gosens, H. A. Baarsma, B. G. J. Dekkers, E. Frohwerk, J.-M. Penninks, P. Sharma, K. M. Ens, et al.
Insulin increases the expression of contractile phenotypic markers in airway smooth muscle
Am J Physiol Cell Physiol, July 1, 2007; 293(1): C429 - C439.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
W. Zhou, C. Dasgupta, S. Negash, and J. U. Raj
Modulation of pulmonary vascular smooth muscle cell phenotype in hypoxia: role of cGMP-dependent protein kinase
Am J Physiol Lung Cell Mol Physiol, June 1, 2007; 292(6): L1459 - L1466.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
H. Kajimoto, K. Hashimoto, S. N. Bonnet, A. Haromy, G. Harry, R. Moudgil, T. Nakanishi, I. Rebeyka, B. Thebaud, E. D. Michelakis, et al.
Oxygen Activates the Rho/Rho-Kinase Pathway and Induces RhoB and ROCK-1 Expression in Human and Rabbit Ductus Arteriosus by Increasing Mitochondria-Derived Reactive Oxygen Species: A Newly Recognized Mechanism for Sustaining Ductal Constriction
Circulation, April 3, 2007; 115(13): 1777 - 1788.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
L. Fan, A. Sebe, Z. Peterfi, A. Masszi, A. C.P. Thirone, O. D. Rotstein, H. Nakano, C. A. McCulloch, K. Szaszi, I. Mucsi, et al.
Cell Contact-dependent Regulation of Epithelial-Myofibroblast Transition via the Rho-Rho Kinase-Phospho-Myosin Pathway
Mol. Biol. Cell, March 1, 2007; 18(3): 1083 - 1097.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. M. Miano, X. Long, and K. Fujiwara
Serum response factor: master regulator of the actin cytoskeleton and contractile apparatus
Am J Physiol Cell Physiol, January 1, 2007; 292(1): C70 - C81.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
Y. Goueffic, C. Guilluy, P. Guerin, P. Patra, P. Pacaud, and G. Loirand
Hyaluronan induces vascular smooth muscle cell migration through RHAMM-mediated PI3K-dependent Rac activation
Cardiovasc Res, November 1, 2006; 72(2): 339 - 348.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. Camoretti-Mercado, D. J. Fernandes, S. Dewundara, J. Churchill, L. Ma, P. C. Kogut, J. F. McConville, M. S. Parmacek, and J. Solway
Inhibition of Transforming Growth Factor beta-enhanced Serum Response Factor-dependent Transcription by SMAD7
J. Biol. Chem., July 21, 2006; 281(29): 20383 - 20392.
[Abstract] [Full Text] [PDF]