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J. Biol. Chem., Vol. 277, Issue 25, 22889-22895, June 21, 2002

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Transcriptional Regulation of a Contractile Gene by Mechanical Forces Applied through Integrins in Osteoblasts^*

Jiaxu Wang, Ming Su, Jennie Fan, Arun Seth, and Christopher A. McCulloch

From the Canadian Institutes for Health Research (CIHR) Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E8, Canada

Received for publication, April 1, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We examined mechanotranscriptional regulation of the contractile gene, -smooth muscle actin (SMA), in osteoblastic cells. Tensile forces were applied through collagen-coated magnetite beads to ROS17/2.8 cells. These cells were desmin, vimentin+ and expressed low levels of SMA. After force application (480 piconewton/cell), SMA protein and mRNA were increased but -actin was unchanged. Beads coated with bovine serum albumin or poly-L-lysine produced no change of SMA. In cells transiently transfected with plasmids containing the SMA promoter fused to -galactosidase or green fluorescent protein coding sequences, SMA promoter activity was increased by ~60% after 4 h of force, whereas control (Rous sarcoma virus) promoter activity was unaffected. Transfections with -galactosidase or green fluorescent protein reporter constructs showed that force-loaded cells exhibited higher -galactosidase activity than cells without force. Cytochalasin D and latrunculin B inhibited force-induced increases of SMA promoter activity. Deletion analyses showed that SMA promoter activity was increased ~70% after force with a minimal construct containing 155 bp upstream of the translation start site. The force effect on the SMA promoter was abrogated in cells transfected with CArG-B box mutants. Gel mobility shift analyses of nuclear extracts showed strong binding to the CArG-B motif after force. We conclude that the CArG-B box is a force-responsive element in the SMA promoter.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In fibroblasts (1), osteoblasts (2), and chondrocytes (3) the expression of -smooth muscle actin (SMA)¹ is associated with increased generation of contractile forces. Currently, the mechanisms by which applied forces regulate SMA expression are undefined. As SMA is an important example of a contractile force protein in non-muscle cells that is important in remodeling of extracellular matrices, a detailed definition of the force-sensitive, promoter-regulatory elements is important for a comprehensive understanding of how physical signals regulate cytoskeletal gene expression.

The regulation of SMA gene expression involves a complex interaction of multiple positive and negative cis elements that act in a cell type-specific fashion (4). Studies of the chicken, mouse, rat, and human SMA genes have demonstrated the importance of cell context in the characterization of promoter elements that function in transcriptional regulation (5-8). The SMA promoter contains a number of highly conserved regulatory elements. Some of the important elements include CArG boxes, which have the general sequence motif CC(A/T)₆GG (9). The SMA promoter contains three CArG elements, designated CArG A (at 62), CArG B (at 112), and an intronic CArG (at +1001). These elements are completely conserved in all species in which the promoter has been examined and are required for generation of full promoter activity in cultured smooth muscle cells (9, 10). The CArG box was first described as the core sequence of the serum response element (SRE) in early response genes such as c-fos (11). CArG elements can direct developmental and tissue-specific expression of many muscle-specific genes (12-15) and are required for the transient transcriptional responses of immediate-early response genes such as c-fos, -skeletal actin, and SMA following serum or growth factor stimulation (11, 16, 18, 19, 39).

Force application can induce expression of fetal type genes such as SMA (20), a process that could involve conserved CArG elements (8) or other upstream domains. However, presently, these processes are not defined. The CArG box-binding factor regulates the expression of SMA by binding to the CArG box, which overlaps an ETS1 binding site in the SRE of the SMA promoter (21). However, the force-induced activation of the SRE in the SMA promoter in connective tissue cells such as osteoblasts may be different from the mechanisms involved in c-Fos activation and may also be cell type-specific. Notably, applied mechanical force can regulate SMA gene expression by the p38 kinase pathway in cardiac fibroblasts (22), a process that in turn may regulate the binding of the serum response factor (SRF) to two critical CArG boxes in the SMA promoter (23). Cognizant of this background we have examined the responsiveness of the SMA promoter to applied forces in an in vitro model system (24, 25). As SMA is expressed in osteoblasts during contractile force generation (2), we used ROS17/2.8 cells, a rat osteoblastic cell line that exhibits phenotypic characteristics of differentiated osteoblasts. The data show that the SMA promoter is regulated by force-responsive elements associated with the CArG-B box.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Mouse monoclonal antibodies to SMA (clone no. 1A4), -actin (clone no. AC-15), collagenase (C5138), rhodamine phalloidin, BSA, fibronectin, and poly-L-lysine were purchased from Sigma. Goat anti-mouse IgG2a and goat anti-mouse IgG1 were purchased from Caltag (Burlingame, CA). Collagen was obtained from Celltrix (Palo Alto, CA).

Cell Culture-- Rat osteosarcoma cells (ROS 17/2.8; hereafter ROS cells) were incubated in complete -MEM medium at 37 °C. The medium contained 10% fetal bovine serum and a 1:10 dilution of an antibiotic solution (0.17% w/v penicillin V, 0.1% gentamicin sulfate, and 0.01 µg/ml amphotericin; Sigma). Cells were maintained in a humidified incubator gassed with 95% O₂-5% CO₂ and were passaged with 0.01% trypsin (Invitrogen).

Bead Preparations-- As described earlier (26, 27), 0.4 g of magnetite beads (Sigma-Aldrich) were incubated for 1 h with 1 ml of an acidic bovine collagen solution (>95% type I collagen) at 37 °C and neutralized to pH 7.4 with 100 µl of 1 N NaOH. Under these conditions collagen polymerizes and forms fibrils around the beads within 30 min. The beads were sonicated to eliminate clumps. BSA or poly-L-lysine beads were prepared in a similar fashion by incubating beads in solutions of 1 mg/ml BSA or 1 mg/ml poly-L-lysine and then dispersed. Analysis of bead size was performed by electronic particle counting (Coulter Channelyzer, Coulter Electronics, Hialeah, FL). Particles tended to exhibit a heterogeneous size distribution with a pronounced modal peak at 5 µm, although there were many particles with smaller diameters. Beads were rinsed in PBS, washed three times, and resuspended in Ca²⁺- and Mg²⁺-free PBS.

Force Generation-- A ceramic permanent magnet (grade 8, 2.2 × 9.6 × 11 cm; Jobmaster, Mississauga, Ontario, Canada) was used to generate perpendicular, tensile forces on beads attached to the dorsal surface of cells. For all experiments the pole face was parallel with and 2 cm from the cell culture dish surface. At this distance the force on a single osteoblast with ~750 µm² area of dorsal bead coverage was 480 piconewton/cell or 0.64 piconewton/µm². As the surface area of the magnet was larger than the culture dishes, and as bead covering was relatively uniform for all cells, the forces applied to cells across the width of the culture dish were relatively uniform (27). A constant force of varying duration was used for all experiments. Before incubating cells, beads were rinsed in PBS, washed three times, resuspended in calcium-free buffer and added to attached cells in full medium for 10 min. Cells were washed three times to remove unbound beads and exposed to force in PBS (pH 7.4) containing calcium and magnesium ions.

Immunofluorescence and Immunoblotting-- We assessed SMA content in cells that were fixed, immunostained for SMA, and counterstained with fluorescein isothiocyanate-conjugated goat anti-mouse IgG. Cells were examined in an epifluorescence microscope and photographed. For immunoblotting, bead-associated proteins or cell lysates prepared from cell cultures (60-mm dishes) that had been subjected to an applied force for discrete time intervals were analyzed. Cells were rinsed with PBS, lysed with 200 µl of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% w/v bromphenol blue), and transferred to a Microfuge tube. The samples were kept on ice and boiled for 5 min. Protein concentration was assessed by the BioRad assay, and equal amounts of protein were loaded in each lane. Isolated proteins were separated by SDS-PAGE (10% acrylamide) and transferred to nitrocellulose membranes. SMA and -actin were identified by immunoblotting. Blots were blocked for 1 h with 5% skim milk in Tween-TBS and incubated with the indicated antibody (diluted 1:1000 in 0.5% Tween-TBS) for 1 h at room temperature. Blots were washed with buffer for 10 min, incubated with appropriate second antibodies for 1 h, washed 4× in Tween-TBS, and developed by chemiluminescence (ECL; Amersham Biosciences). X-OMAT Kodak films were exposed to the blots, and the density of the bands was analysis by IP Lab Gel Scientific Image Processing (Signal Analytics Corporation, Vienna, VA).

Northern Analysis-- Total RNA was isolated from cells with the Qiagen RNAeasy total RNA kit according to the manufacturer's instructions and quantified by spectrophotometry (Ultrospec 3000; Amersham Biosciences). RNA samples (10 µg) were separated in 1.2% denaturing agarose gels containing 2.2 M formaldehyde in MOPS running buffer, transferred to nitrocellulose membranes (Optitran; Schleicher & Schuell), cross-linked by UV, and hybridized with ³²P-labeled oligonucleotide probes. These probes were designed from portions of the sequences of the rat -SMA mRNA 5'-untranslated region: (5'-GAAAAGAACTGAAGGCGCTGATCCACAAAACATTCACAGTTG-3') and from the rat -actin mRNA 3'-untranslated region (5'-CGCCTTCACCGTTCCAGTTTTTAAATCCTTGAGTCAAAAGCGCCA-3'). The oligonucleotides were synthesized by the Biotechnology Service Centre (Hospital for Sick Children, Toronto, Ontario). Probes were labeled with [³²P]ATP (PerkinElmer Life Sciences) using 3' end labeling. The blots were washed 4× with 0.5% SSC + 0.5% SDS at room temperature for 10 min and twice for 40 min at 50 °C and exposed to Kodak X-OMAT films at 70 °C.

Transient Transfections, Reporter Gene Assays, and DNA Constructs-- ROS cells were grown and maintained in complete -modified Eagle's medium with 10% fetal calf serum. Prior to transfection, ROS17/2.8 cells were plated at 1 × 10⁴ cells/well in 6-well plates and incubated in complete -Dulbecco's modified Eagle's medium without antibiotics for 24 h. Cells were transiently transfected with reporter plasmids containing p547/LacZ constructs obtained from Dr. G. Owens in which the whole promoter is present. Cells were co-transfected with a Rous sarcoma virus (RSV) expression plasmid to normalize for equal loading using LipofectAMINE 2000 reagent according to the supplier's instructions (Invitrogen). Following transfection (20 h), collagen beads were incubated with cells and washed, and force was applied for 4 h. Cell extracts were prepared after force application with a detergent lysis method. -galactosidase reporter enzyme activity was measured with an enzyme assay system (Roche). -galactosidase activities were normalized to RSV luciferase activity as a transfection control. The RSV (124 to +34)/luciferase construct was provided by H. P. Elsholtz (University of Toronto) and was used as described earlier (24). RSV luciferase assays were conducted using a luciferase assay system (Promega) according to the manufacturer's instructions. RSV luciferase activity (reflecting promoter activity) was unaffected by all treatments at all experimental time periods.

The p547/ -galactosidase construct obtained from Dr. G. Owens comprises the promoter region of the SMA gene fused to -galactosidase coding sequences. Constructs P371, P155, P92, and P56 were made by PCR amplification using P547 as the template and the following specific PCR primers: P371 = 5'-NNCCCAAGCTTTTAGCTAATGGACC-3', P155 = 5'-NNCCCAAGCTTTGGCCACCCAGATT-3', P92 = 5'-NNCCCAAGCTTCAGCTTCAGCCTGT-3', P56 = 5'-NNCCCAAGCTTGAGTGGGAGGGGATCAGACCAG-3', and reverse primer 5'-GGGGTACCCCTGATGGCGACTG-3'. Inserts of interest in the rat SMA promoter were excised with HindIII and KpnI, and the inserts were religated into the pUC19/AUG -galactosidase vector. The CArG-B mutation in the rat SMA promoter (CCCTATATCA; mutations underlined) was constructed by PCR amplification using the p2600Int/Laz CArG-B mutation SMA promoter obtained from Dr. Owens as a template (10). A SMA "core promoter" construct (p155GFP) was made using the proximal 155 bp of the SMA promoter, which was inserted into the promoterless pEGFP-1 vector (CLONTECH) directly proximal to the EGFP reporter gene. P155EGFP promoter constructs were synthesized by digesting the p547 plasmid with HindIII and KpnI for 4 h at 37 °C. The resultant promoter-containing restriction fragments were purified and ligated into the HindIII/KpnI-digested pEGFP-1 vector. The ligated product was transformed into competent DH5-' cells (Stratagene), and individual colonies were cultured overnight in 5 ml of LB + kanamycin (30 µg/µl). Plasmid DNA was isolated from these cultures (Qiagen), and the correct orientation of the insert was verified through diagnostic restriction digests with HindIII and KpnI. Subsequently, the authenticity of the inserts was established by sequencing (Centre of Applied Genomics, Hospital for Sick Children, Toronto, Ontario).

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSAs)-- Nuclear extracts were prepared according to a modified Digman's method (28). The oligonucleotides used for EMSAs were synthesized and purified commercially (Invitrogen). The following double-stranded oligonucleotides (with only the sense strands shown) were used as probes: CArG-A, 5'-TTGCTCCTTGTTGGGGAAGC-3', CArG-B, 5'-GAGGTCCCTATAGGTTTGTG-3', and CArG-B mutation, 5'-GAGGTCCCTATATCATTGTG-3'. EMSA probes were generated by end labeling single-stranded oligonucleotides (20 µM) with 150 µCi of [³²P]ATP (3000 Ci/mm; Mandel) using T4 polynucleotide kinase. Labeled single-stranded oligonucleotides were annealed and purified from unincorporated nucleotides using ProbeQuant TM G-50 Micro columns (Amersham Biosciences). EMSAs were incubated for 30 min at room temperature in 1× binding buffer (10 mmol/liter Tris-HCl, pH 7.5, 100 mmol/liter KCl, 50 mmol/liter NaCl, 1 mmol/liter dithiothreitol, 1 mmol/liter EDTA, and 5% glycerol) and performed in a binding reaction (20 µl) containing ~50 pg (50,000 cpm) of labeled probe, 10 µg of nuclear extract, and 0.25 µg of poly(dA-dT) in 1× binding buffer (12 mM HEPES, pH 7.9, 100 mM KCl, 5 mM MgCl₂, 4 mM Tris-HCl, pH 7.5, 0.6 mM EDTA, 0.6 mM dithiothreitol, and 10% glycerol). Samples were separated by electrophoresis on 4.5% polyacrylamide gels at 150 V in 45 mM Tris borate and 1 mM EDTA. Serum response factor protein was produced in vitro using the Promega rabbit reticulocyte system.

In Situ -Galactosidase Staining of Transfected Cells-- To monitor the efficiency of transfection after force application, we used in situ staining for -galactosidase. After transfection and force application, cells were washed with PBS (containing calcium and magnesium), fixed with 2% formaldehyde and 0.05% glutaraldehyde for 5 min at room temperature, and washed with PBS. Cells were stained (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl₂ 0.2% 5-bromo-4-chloro-3 indolyl--D-galactopyranoside (X-gal)), and plates were incubated overnight at room temperature prior to rinsing and observation in an inverted microscope.

Statistical Analysis-- For all assays, three or more separate experiments were performed. Means ± S.E. were calculated for continuous variables, and when appropriate, comparisons between two groups were analyzed by unpaired t-tests.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

SMA Is Expressed in ROS17/2.8 Cells after Force Application-- To determine whether force regulates SMA expression in osteoblastic cells in vitro, we used collagen-coated magnetite beads and permanent magnets to deliver precisely regulated tensile forces to the actin cytoskeleton through integrins (27). ROS cells are well differentiated osteoblastic cells that we have used to study regulation of smooth muscle actin promoter function by mechanical forces in vitro. Immunohistochemistry of ROS cells showed positive staining for vimentin (Fig. 1A), an intermediate filament protein that is a hallmark of connective tissue cells. Conversely, cells were not stained for desmin (Fig. 1, B and C), an intermediate filament protein marker that is expressed by smooth muscle cells. ROS cells showed very weak staining for SMA prior to force application (Fig. 1D). After only 4 h of force application, cells exhibited increased SMA staining intensity that was notably less than cardiac myofibroblasts (Fig. 1, E and F), cells which strongly express SMA in vitro (22).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Characterization of ROS cells. Immunohistochemical staining shows that ROS cells are vimentin+ (A) and desmin (B). Same cells in B are stained with 4',6-diamidino-2-phenylindole to show nuclei (C). SMA staining is weak in control ROS cells with collagen beads alone (D) but is much stronger after force treatment (4 h; E). Cardiac fibroblasts constitutively exhibit strong SMA staining (F).

We examined whether ROS cells, following incubation with collagen, BSA, or poly-L-lysine-coated beads, would exhibit variations of SMA protein content after force application. After tensile force application for 4 h, immunoblotting of whole cell lysates showed that forces applied through collagen-coated beads increased SMA content but did not affect -actin content (Fig. 2A). In contrast, cultured cardiac fibroblasts, which express very high basal levels of SMA, showed reduced SMA after force application (Fig. 2B), an effect that has been observed previously (22). The force-induced increase of SMA in ROS cells was apparently mediated through integrins because cells incubated with either BSA or poly-L-lysine beads showed no increase of SMA content after force application (Fig. 2, C and D).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   SMA expression after tensile force application through magnetite beads. Immunoblots of SMA and -actin in ROS cells (A) or cardiac fibroblasts (B) incubated with collagen-coated beads show differential effects of SMA content after force application in the two cell types. In ROS cells, force strongly increases SMA expression. Following application of force to ROS cells for 4 h, immunoblotting of whole cell lysates showed no change of SMA in cells incubated with BSA-coated (C) or poly-L-lysine-coated beads (D). E, Northern blotting to measure mRNA content of ROS cells or cardiac fibroblasts. Equivalent amounts of total RNA (10 µg) were loaded on each lane and probed with oligonucleotides specific for the rat SMA mRNA 5'-untranslated region: (5'-GAAAAGAACTGAAGGCGCTGATCCACAAAACATTCACAGTTG-3') and from the rat -actin mRNA 3'-untranslated region: (5'-CGCCTTCACCGTTCCAGTTTTTAAATCCTTGAGTCAAAAGCGCCA-3'). Probes were labeled with [32P]ATP using 3' end labeling. SMA and -actin mRNA content were measured after force application. -actin mRNA was unchanged by force application, but SMA was induced by force. Note that the basal level of SMA in control cells (cardiac fibroblasts) was high. In F, the ratio of SMA to -actin mRNA as determined by Northern blotting was increased 6-fold at 4 h after force (p < 0.05; n = 3).

The increase of SMA protein content by applied force suggested that force might affect selectively SMA mRNA content. Northern blotting of SMA and -actin mRNA content showed that compared with cardiac fibroblasts, which express very high basal levels of SMA mRNA in culture (22, 26), the basal SMA mRNA content of ROS cells was virtually undetectable (Fig. 2E). After only 4 h of force application, although -actin mRNA content was unchanged, the ratio of SMA to -actin mRNA was increased 6-fold (p < 0.05; n = 3; Fig. 2F). Thus ROS osteoblastic cells provide a convenient, rapidly responsive model for studying how SMA gene expression is regulated by mechanical forces in vitro.

SMA Promoter Activity Is Regulated by Force-- To determine whether force regulates SMA promoter activity, we transfected ROS cells with -galactosidase SMA reporter constructs (P547/LacZ) in which the whole SMA promoter (9) was present. As an internal transfection control, ROS cells were co-transfected with a pEGFP-1 vector containing an SV40 promoter that constitutively drives expression of the EGFP reporter gene (Fig. 3). To directly examine SMA promoter function in response to force application, transfected ROS cells were stained for -galactosidase in situ. Cells transfected with a promoter-less construct (Fig. 3A) or transfected with the SMA construct but without force showed minimal staining (Fig. 3B); but after force application cells with bound beads exhibited much more intense staining than cells without force (Fig. 3C). Fluorescence attributable to EGFP driven by an SV-40 promoter in parallel cultures of co-transfected cells was unaffected by force application (Fig. 3, D-F). This experiment indicated that in combination with force applied through collagen-coated beads, ROS cells provide a sensitive and specific model for studying SMA promoter regulation by mechanical forces in vitro.

View larger version (70K): [in this window] [in a new window]   Fig. 3.   Force effect on SMA promoter activity. ROS cells transfected with -galactosidase reporter constructs driven by the SMA promoter (P547) in which the whole promoter is present. Cells were co-transfected with a GFP construct in which GFP expression is under the control of a SV-40 promoter as an internal transfection control. After transfection, the proportion of cells exhibiting -galactosidase staining as a measure of SMA promoter activity was increased in cells subjected to 4 h of force compared with no force. A, promoterless -galactosidase construct. B, no force in cells transfected with SMA P547 construct and loaded with beads. C, same as B but with force for 4 h. Arrows point to -galactosidase-stained cells. D-F, parallel cultures of ROS cells co-transfected with EGFP construct under the control of an SV-40 promoter and the same -galactosidase constructs as A-C show no change in proportion of fluorescent cells after force application.

Force-mediated Activation of SMA Gene Expression Is Mediated by Specific Promoter Domains-- As a first step in determining the contributions of specific promoter regions to transcriptional regulation by force, cells were transfected with constructs containing specific domains of the SMA promoter (P547, P371, P208, P155, P92, and P56; Fig. 4A). These constructs were designed to include known regulatory domains of the SMA promoter including the TATA, CArG-A, CArG-B, GArC (GGAAGAGACC), and E boxes (9). The SMA constructs were made by PCR amplification using P547/-galactosidase (9) as the template and PCR primers that facilitated excision of the appropriate segment in the rat SMA promoter. The inserts were ligated into the pUC19/AUG -galactosidase vector. Cells were co-transfected with the RSV-luciferase construct as a loading control. Transfection efficiencies for all experiments were 40-50%. The -galactosidase activity normalized to RSV luciferase activity (Fig. 4B) gave an estimate of promoter activity that compensated for variations of transfection efficiency. In cells transfected with P547, P371, and P155 constructs, the adjusted -galactosidase activity was increased by ~60% after force application compared with no force application (p < 0.05). In contrast, cells transfected with the P92 and P56 constructs showed no significant change of -galactosidase activity after force. As SMA promoter activity was increased maximally with the P155 construct and not at all with the P92 or P56 constructs, it appeared likely that the CArG-B box (but not the CArG-A box) is an important force-responsive element in the SMA promoter.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   A, structure of rat SMA promoter--galactosidase reporter constructs. Promoter sequences were made by PCR amplification using P547 as the template and PCR primers for P371, P155, P92, P56, and reverse primer described under "Experimental Procedures." Appropriate segments in the rat SMA promoter were excised with HindIII and KpnI. The inserts were religated into the pUC19/AUG -galactosidase vector. B, cells were co-transfected with the SMA promoter constructs and with RSV luciferase as loading control. Cells transfected with P547, P371, and P155 showed >50% increase of -galactosidase normalized to RSV luciferase (p < 0.05) after force application compared with no force. SMA promoter activity was increased maximally after transfection with the P155 construct. The P547, P371, and P155 constructs all included the CArG-A and CArG-B boxes, while the P92 and P56 constructs contain only the CArG-A box. There was no significant force effect in cells transfected with the P92 and P56 constructs.

The 122-bp core promoter of the chicken SMA gene in myoblasts and fibroblasts has been identified previously (5). As the increased promoter activity of the P155 construct after force application was equivalent to that of longer promoter constructs, we considered that the 122-bp core promoter contained within the P155 construct was important for the SMA response to force. We also considered that the P155 construct would provide a useful reagent for examining potential cellular factors that regulate force-induced SMA promoter activity. Accordingly, the first 155 bp of the SMA promoter was cloned into the promoterless pEGFP-1 vector (CLONTECH) proximal to the EGFP reporter gene. After cells were transfected with this construct, the number of fluorescent cells and the total number of cells in microscopic fields were counted (×25 objective), and the proportion of EGFP to total cell counts was computed in each field. A total of 1674 cells were counted in 50 fields. Analogous to the data shown above for the P155 -galactosidase construct, the proportion of EGFP-expressing cells was increased 1.8-fold after force treatment compared with the proportion of fluorescent cells without force (Fig. 5). Parallel cultures that were transfected with the pEGFP construct under the control of the SV40 promoter showed no change of the proportion of EGFP fluorescent cells after force application. As previous studies have indicated that actin filaments are essential for mechanotranscriptional coupling of cytoskeletal genes including filamin A (25), we determined if force-mediated induction of SMA required intact actin filaments. Cells were loaded with beads and treated with cytochalasin D (0.5 µm) or latrunculin B (1 µm) to depolymerize actin filaments. Cells were then subjected to force (or not), and the proportion of EGFP fluorescent cells was computed. Although the proportion of untreated (baseline) EGFP fluorescent cells was lower than untreated cells and was different between the two experiments in which latrunculin or cytochalasin were used, both agents abrogated the force-induced increase of SMA promoter activity above their respective baseline levels (Fig. 5).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Requirement for actin filaments in force-induced activation of SMA core promoter. Cultures transfected with a pEGFP construct under the control of the SV40 promoter showed no change of the proportion of EGFP fluorescent cells after force application. For studies of SMA promoter function, the first 155 bp of the SMA promoter was purified and ligated into a HindIII/KpnI-digested pEGFP-1 vector. Cultures transfected with a promoterless construct showed virtually no fluorescent cells. Transfections with the pEGFP-p155 construct demonstrated a 1.8-fold increase of the proportions of fluorescent cells after force application compared with no force controls (p < 0.01). Cytochalasin D (0.5 µm) and latrunculin B (1 µm) abrogated the force effect on SMA promoter activity. A total of 1674 cells were counted in 50 fields for these experiments.

The CArG-B Box Is a Force-responsive Element-- Our data showed that the proximal 155 bp of the SMA promoter mediated a high level of transcriptional expression in ROS cells in response to force. Within this region are two CArG boxes designated CArG-A and CArG-B. These elements are 100% conserved between the four species in which the SMA promoter has been cloned (9), suggesting that they may be important regulators of SMA expression. Indeed, CArG box elements have been shown to direct developmental and tissue-specific transcriptional expression for both the skeletal and cardiac -actin genes (12, 15, 29). As the P547, P371, and P155 constructs all include the CArG-B box, we determined whether the CArG-B box is involved in the regulation of the SMA promoter by force using a mutational approach (10). A two-point mutation was introduced in the CArG-B box in the P155 and P371 constructs, changes that were verified by sequencing. Cells transfected with either construct in which the CArG-B box mutants were present showed complete abrogation of the force effect compared with wild type controls (Fig. 6). These data suggest that the CArG-B box is an important regulatory domain for mediating force-induced expression of SMA.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Effects of CArG-B box flanking mutations on SMA promoter activity after force application. Two point mutations were introduced (5'-CCCTATATGG-3' to 5'-CCCTATATCA-3') in the CArG-B box. The mutation was confirmed by sequencing the P371 and P155 constructs. The SMA constructs and RSV-LUC were co-transfected into ROS cells followed 20 h later by 4 h of force application. -galactosidase activity was normalized to RSV-luciferase activity (mean ± S.E.). Transfection with CArG-B box mutants (P155 and P 371) eliminated force-induced SMA promoter activity.

Electrophoretic mobility studies have demonstrated that CArG elements in the SMA promoter, like the SRE, bind the SRF (9) and that muscle-derived cells express higher levels of SRF than non-muscle-derived cells (31). EMSA analyses were performed to confirm that the CArG-B box is specifically involved in mediating force responses. We compared CArG-A box, CArG-B, and CArG-B-mutation probes in binding nuclear extracts from ROS cells subjected to force or no force for 4 h. The CArG-A probe (Fig. 7, lanes 1-4) did not bind to nuclear extracts with or without force application. The CArG-B probe (Fig. 7, lanes 5-8) bound to the nuclear extract more strongly after force application than without force. The CArG-B mutation probe showed no binding (Fig. 7, lanes 9-12). The gel shift produced by binding of the CArG-B probe to nuclear extracts in force-treated cells was similar to the shift observed with an SRF-specific probe. Competitor studies indicated that the force-induced binding of CArG-B-labeled probes to nuclear extracts was progressively inhibited by co-incubation with increasing concentrations of unlabeled probes (Fig. 8). Collectively, these results indicated that force application through collagen-coated beads stimulates SMA expression by binding to the CArG-B box in the SMA promoter.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   EMSA analyses of nuclear extracts and CArG boxes. A comparison of the binding of CArG-A box, CArG-B box, and CArG-B box mutation probes to nuclear extracts (10 µg) from ROS cells subjected to 4 h of tensile force is shown. CArG-A box, CArG-B box, and CArG-B mutation probes were synthesized as described under "Experimental Procedures." SRF protein was expressed in vitro using the Promega rabbit reticulocyte system. The CArG-A probe (lanes 1-4) did not bind to nuclear extracts with or without force application. The CArG-B probe (lanes 5-8) bound to nuclear extracts after force application, but the CArG-B mutation (lanes 9-12) blocked binding. Purified SRF shifted binding similar to the CArG-B probe. Lanes: 1, probe only; 2, no force; 3, force; 4, SRF; 5, probe; 6, no force; 7, force; 8, SRF; 9, probe; 10, no force; 11, force; 12, SRF.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Competition analysis of nuclear extracts for CArG-B box binding. Nuclear extracts (10 µg) from ROS cells were subjected to 4 h of tensile force. Radiolabeled wild type 20-bp CArG-B oligonucleotide duplex was incubated with nuclear extracts. Competition reactions were performed with unlabeled CArG-B oligonucleotides. Competitor oligonucleotides were added at 100-400-fold molar excess relative to the radiolabeled DNA. Lanes: 1, labeled CArG-B DNA probe only; 2, radiolabeled wild type + force; 3, radiolabeled wild type + 100× unlabeled wild type + force; 4, radiolabeled wild type + 200× unlabeled wild type + force; 5, radiolabeled wild type + 400× unlabeled wild type + force; 6, radiolabeled wild type + no force; 7, radiolabeled wild type + 100× unlabeled wild type + no force; 8, radiolabeled wild type + 200× unlabeled wild type + no force; 9, radiolabeled wild type + 400× unlabeled wild type + no force.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

SMA is a prominent actin isoform that in vivo is expressed by fibroblasts (1), osteoblasts (2), and chondrocytes (3) in association with increased generation of contractile forces or during wound contraction (32). In cultured connective tissue cells, SMA is a phenotypic marker of contractile cells (1). Previous studies have suggested that the actin cytoskeleton may also link transmission of external applied forces to specific patterns of gene expression and cellular organization (33, 34). The major novel findings of this study are that contractile forces applied through integrins in osteoblastic cells induce SMA expression, and that the CArG box in the SMA promoter is a critical, force-sensitive element in this transcriptional response. To assess regulation of SMA promoter function by mechanical forces in vitro, the nature and the authenticity of the in vitro model system are of critical importance.

ROS Cells as a Model for Studying Regulation of SMA by Force-- We used ROS cells because they are well differentiated osteoblastic cells that provide a culture model for the increased SMA that is seen in force-loaded osteoblasts in vivo (2). We found that SMA is expressed at very low levels in untreated ROS cells but is increased significantly after force application, similar to what is observed in vivo (2). Second, ROS cells abundantly express collagen receptors (35) that provide the attachment for collagen-coated beads and the linkage to the actin cytoskeleton (22, 26). Indeed, compared with NIH-3T3 and Chinese hamster ovary cells, ROS cells exhibited stronger binding to collagen beads and were not dislodged from the culture dish after force application (data not shown). Further, ROS cells showed specific responses of SMA to forces applied through collagen but not BSA or poly-L-lysine-coated beads, supporting the view that the force effect was indeed mediated by specific collagen receptors (i.e. _1,2,31 integrins). Third, ROS cells were more easily transfected than other fibroblastic and osteoblastic cell lines that were examined (e.g. NIH-3T3 cells; data not shown) and consistently exhibited higher transfection efficiencies. Fourth, it was important to choose a cell line that would exhibit force-induced increases of endogenous SMA as we found for ROS cells. Notably, Owens and co-workers have demonstrated that the regulatory elements responsible for transcriptional expression of the SMA gene are highly dependent upon the cell type in which the promoter is studied (9). They found that the P547 construct was transcriptionally active in smooth muscle cells and skeletal myotubes but was inactive in endothelial cells or skeletal myoblasts, cell types that do not express SMA. In this context, ROS cells provide a permissive model system in which force-induced expression of SMA is induced from very low baseline levels. Finally, as cytochalasin D or latrunculin B strongly inhibited the force-induced increase of SMA, the model fulfills the concept that force mediates transcriptional responses through intact actin filaments (24, 25).

Force-responsive Elements in the SMA Promoter-- Gene expression induced by mechanical loading can be divided temporally into immediate-early genes and late response genes, of which the SMA gene is apparently an example. Immediate-early genes such as the transcription factor c-Fos can couple trophic signals to changes in actin gene expression (36), and c-Fos transcription is critical for the regulation of actin expression both in vivo (37) and in vitro (38). In turn, stretch-induced activation of the c-Fos promoter is dependent on the serum response element and on a signaling complex that includes a SRF and the p62^TCF-ternary complex (30). Although there are regulatory elements in the SMA promoter upstream of the conserved CArG elements (8), previous studies of the chicken SMA gene demonstrated that the first 122 bp of the promoter were sufficient to confer a moderate to high level of transcriptional activity in chicken smooth muscle cells, skeletal myoblasts, and fibroblasts (5, 8). Consistent with these data, our deletion analyses showed little difference in the force-induced activation of the SMA promoter between constructs containing either 547 or 155 bp upstream of the translation start site. The force-induced effect was surprisingly rapid because within 4 h of force after force application there was a large increase of promoter activity in experiments that used the 155-bp construct.

More detailed analyses using mutant constructs and EMSA showed that the force effect was mediated by the CArG-B box, while the TATA and CArG-A boxes were apparently not involved in the regulation of transcriptional responses to force application. We suggest that tensile force induces SRF to form complexes with as yet unidentified transcription factors. This SRF complex may bind to the CArG-B box in the SMA promoter and then stimulate SMA transcription. Although the identity of the proteins in this putative force-sensitive complex is unknown, our data do show that the CArG-B box is a force-responsive element in the SMA promoter and are consistent with the general notion that actin isoforms are subject to mechanotranscriptional regulation (17).

	ACKNOWLEDGEMENTS

We thank G. K. Owens for the SMA constructs and for valuable advice, Caroline Chu for assistance with preparation of the manuscript, the Heart and Stroke Foundation of Canada, and the Canadian Institutes of Health Research for financial support.

	FOOTNOTES

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

To whom correspondence should be addressed: Rm. 244, Fitzgerald Building, University of Toronto, 150 College St., Toronto, Ontario M5S 3E8, Canada. Tel.: 416-978-1258; Fax: 416-978-5956; E-mail: christopher.mcculloch@utoronto.ca.

Published, JBC Papers in Press, April 12, 2002, DOI 10.1074/jbc.M203130200

	ABBREVIATIONS

The abbreviations used are: SMA, smooth muscle actin; SRE, serum response element; SRF, serum response factor; BSA, bovine serum albumin; ROS, rat osteosarcoma cells; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; RSV, Rous sarcoma virus; EGFP, enhanced green fluorescence protein; EMSA, electrophoretic mobility shift assay; GFP, green fluorescence protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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