Dec 15, 2003. We have previously shown that the pro-collagen type I alpha 2 gene contains an enhancer which. Paper, we delineate a specific vascular smooth muscle cell (vSMC) element: a 100-bp sequence around 1 Bengali Serial Shubho Bibaho. 6.6. Mouse lymphoblast cell line grown in suspension (European Collection of Ani. Jan 19, 2007. Vascular smooth muscle cells (VSMCs) are an important component of atherosclerotic plaques, responsible for prom. Of approximately 20% of the medial VSMCs is cleared within a week of stopping treatment, and without any obvious release of pro-inflammatory cellular contents or secondary necrosis.

Tissue preparation. Male Sprague-Dawley rats (300–350 g) were killed by decapitation, and the anal canal with an adjacent region of the rectum was quickly removed and transferred to oxygenated (95% O 2-5% CO 2) Krebs physiological solution (KPS) of the following composition (in mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl 2, 1.16 MgSO 4, 1.01 NaH2PO 4, 25 NaHCO 3, and 11.10 glucose (37°C). Adventitious structures connected to the IAS and the RSM were removed carefully by sharp dissection. After this, the mucosa was removed using sharp dissection, and circular SM strips (∼1 × 7 mm) of the IAS and RSM were prepared as explained previously (, ).

The experimental protocols were approved by the institutional animal care and use committee of Thomas Jefferson University in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care. Measurement of isometric tension. The SM strips were transferred to a 2-ml muscle bath containing oxygenated KPS at 37°C. One end of the strips was anchored at the bottom of the muscle bath, whereas the other end was connected to a force transducer (model FT03; Grass Instruments, Quincy, MA). Isometric tension was measured by the Powerlab/8SP data-acquisition system (AD Instruments, Castle Hill, Australia) and recorded using chart 4.1.2 (AD Instruments). Each SM strip was initially stretched to a tension of 0.7 g. The muscle strips were then given at least 1 h to equilibrate during which they were washed with KPS every 20 min.

For IAS only the SM strips that developed spontaneous tone and responded by relaxation to electrical field stimulation were used for the study (, ). The RSM was characterized by the presence of a low-grade tone with the superimposed phasic contractions. After the equilibration period, the SM strips were treated with different concentrations of inhibitors. Preparation of dispersed IAS and RSM SMC. SMCs were isolated from IAS and RSM by sequential enzymatic digestion, filtration, and centrifugation as described previously (, ).

Briefly, the SM tissues were cut into 0.2 × 0.2-mm blocks and incubated in KPS containing 0.1% collagenase and 0.01% trypsin inhibitor. The partly digested tissues were washed, and SMC were allowed to disperse spontaneously for 30 min. Cells were harvested by filtration through 500 μM Nitex mesh and centrifuged two times at 350 g for 10 min. Sample preparation.

IAS and RSM SMC were homogenized with tissue homogenizer in homogenization buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, and 0.5% sodium deoxycholate) on ice. Smooth muscle actin was precipitated using agarose-bound actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Briefly, 2,000 μg of lysate were incubated with 200 μg of agarose-bound antibody for 4 h at 4°C. At the end of incubation, actin was precipitated with agarose via centrifugation, and supernatant was collected. Protein samples were purified using a two-dimensional clean-up kit (GE Healthcare). Protein concentrations were determined using the GE Healthcare Quant Kit (Piscataway, NJ).

Samples were stored at −80°C until further processing. Fluorescent tagging: 2D-DIGE labeling (minimal labeling) and electrophoresis. The protein samples were brought to pH of 8–8.5 with 1 M NaOH to optimize minimal labeling.

To overrule any dye-based artifacts in quantitation, samples were randomly labeled with Cy3 or Cy5 from each group (IAS or RSM SMC). Each sample aliquot of 50 μg of protein was labeled with Cy3 or Cy5 (400 pmol). Equal amounts of protein from every sample were mixed to create a normalization pool, and then an aliquot (50 μg) of the pool was labeled with Cy2 (400 pmol). The labeling reaction was stopped by addition of 1 μl of 10 mM lysine and incubated on ice for 15 min. Equal amounts (50 μg) of Cy3-labeled sample, Cy5-labeled sample, and Cy2-labeled pool sample were combined and applied to each gel. Use of a normalization pool (which serves as an internal standardization) nearly abolishes the possibility of erroneous results due to different concentration loads and other related issues (, ). An equal volume of 2× sample buffer [2 M thiourea, 7 M urea, 2% IPG buffer (pH 3–10; nonlinear and 1.2% DeStreak reagent)] was added to all samples to give a final volume of 150 μl.

The 18-cm pH 3–10 nonlinear gradient Immobiline DryStrips (GE Healthcare) were rehydrated for 12 h with 350 μl of protein sample in rehydration buffer [DeStreak Rehydration Solution containing 0.5% IPG buffer (pH 3–10) using an IPG-phor (GE Healthcare)] following the manufacturer's instructions. Proteins were focused by using the following steps: 500 V for 3 h (step and hold), 1,000 V for 6 h (gradient), and finally 8,000 V for 6 h (step and hold). After isoelectric focusing the IPG strips were incubated for 15 min in equilibration buffer I (0.375 M TrisHCl, pH 8.8, 6 M urea, 2% SDS, 20% glycerol, and 13 mM dithiothreitol) to eliminate disulfide bonds in the focused proteins in preparation for the second dimension.

The IPG strips were then soaked in equilibration buffer II [0.375 M TrisHCl (pH 8.8), 6 M urea, 2% SDS, 20% glycerol, and 2.5% iodoacetamide] for an additional 15 min to alkylate the sulfhydryl groups. Next, isoelectric focusing strips were applied to 12.5% polyacrylamide gels (26 cm width × 20 cm height × 1 mm thick), sealed with 0.7% low-melting-point agarose containing bromophenol blue in a buffer of 1× Tris/glycine/SDS buffer [25 mM Tris, 192 mM glycine, and 0.1% (wt/vol) SDS, pH 8.3]. This was run for 30 min at 2 W/gel and then for 6–7 h at 20 W/gel at 20°C using the Ettan DALTtwelve system (GE Healthcare) for separation of proteins on the basis of molecular weight.

For preparative (picking) gels, an aliquot of 350 μg of sample was diluted with an equal volume of 2× sample buffer [2 M thiourea, 7 M urea, 2% IPG buffer (pH 3–10), and 1.2% DeStreak reagent]. The total volume was brought up to 450 μl with rehydration buffer [DeStreak Rehydration Solution and 0.5% IPG buffer (pH 3–10)].

Proteins were focused as described above for other gels. Each of these strips was equilibrated in equilibration solutions as mentioned above. For the preparative picking gel, a single plate for each gel plate sandwich was treated with Bind-Silane solution (80% ethanol, 0.02% glacial acetic acid, and 0.001% Bind-Silane) and had reference markers placed on them. After the completion of electrophoresis, the plates that had not been silane treated were removed from the sandwich, and the gels were fixed overnight with 30% ethanol and 7.5% glacial acetic acid. The preparative picking gels were then stained with Deep Purple Total Protein Stain (GE Healthcare) for 2 h. Gel scanning and image analysis. All two-dimensional gels were imaged on a Typhoon 9400 fluorescent imager (GE Healthcare) at a resolution of 100 μm.

Photomultiplier tube voltages were individually set for each of the three colored lasers to ensure maximum linear signals. The same voltages were used for all the gels. The DIGE gels were imaged at three different wavelengths (Cy2: 520 nm; Cy3: 580 nm; Cy5: 670 nm), and the Deep Purple Total Protein Stain-stained gels were imaged at 100 μm with a separate filter (610 nm).

Gel images were imported into the DeCyder software 5.01 (GE Healthcare) for analysis. Gel alignment was conducted automatically and then checked manually to ensure correct alignment.

A reference gel with minimum distortion and streaks was then selected from the Cy2 gels. Spot detection and spot matching across all the gels were conducted automatically, and then spot matching was checked and manually edited to ensure correct matching, merging, and splitting of spots. All of the spots included in analysis were transported to the DIA module of DeCyder using a value of 1,000 as the initial estimate of protein spots present. Quantitation of spots was accomplished by comparing the ratio of each Cy3 and Cy5 value with the values obtained from the normalization pool/Cy2 channel present on each gel. DIA analyses were collected in a single analysis using the BVA module of DeCyder, and final values for the expression ratio of specific protein spots between IAS and RSM were calculated.

Statistical analysis was performed by t-test to confirm the level of significance among various groups. For identified proteins having multiple isoforms, the normalized volumes of all isoforms of a given protein were added together, and statistical analysis was repeated on the totals. Protein identification with ProteomeX electrospray ion-trap. For identification of spots, protein spots were picked from picking gels using a robot-directed spot picker (Ettan Spot Picker; GE Healthcare). The spots selected for picking were determined on the basis of differential expression from the 2D-DIGE analyses. The picker head was calibrated using the reference stickers placed on the preparative picking gel, and gel plugs were picked and placed in a bar-coded 96-well plate.

The picked spots were then identified by using ProteomeX Electrospray Ion-Trap (ThermoElectron) MS where protein primary sequence information was determined using Peptide Mass Fingerprinting techniques (). The data were submitted to the MASCOT search engine using the NCBI nonredundant and SwissProt databases against mammals for identification.

MASCOT confidence interval scores of >95% were considered as positive protein identification. Measurement of SM22 phosphorylation. Levels of phosphorylated SM22 were determined from IAS and RSM in the basal state and following Y-27632 treatments. The SM were homogenized on ice in lysis buffer (composed of 50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 2 mM NaVO 4, and 25 mM NaF), and SM22 was immunoprecipitated using a Roche Diagnostics immunoprecipitation kit (Protein G) (Fisher). Lysate (200 μg) in 250 μl were precleared with 25 μl protein G agarose beads. Precleared lysate was incubated with 2 μg of SM22 rabbit polyclonal antibody (H-75, sc-50446; Santa Cruz Biotechnology) for 1 h.

Later, 25 μl of protein G agarose beads were added and further incubated for 4 h to immobilize antigen-antibody complexes. Agarose beads were centrifuged for 20 s at 10,000 g and washed three times with wash buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, and 0.5% sodium deoxycholate) to remove nonspecific bindings. Later 50 μl of low-salt buffer (LSB) were added to the beads and placed in a boiling water bath for 5 min. Protein samples were separated by 15% SDS-PAGE, and Western blot was performed using anti-phospho-serine monoclonal antibody (Calbiochem). Coimmunoprecipitation of SM22-actin complexes.

Actin-SM22 complexes were coimmunoprecipitated using agarose-conjugated actin polyclonal antibody (Santa Cruz). Briefly, 200 μg of lysate in 250 μl were incubated with 20 μl of anti-actin antibody-agarose for 4 h. Agarose beads were centrifuged for 20 s at 10,000 g and washed repeatedly with wash buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, and 0.5% sodium deoxycholate) to remove nonspecific binding. Later 50 μl of LSB were added to the beads and placed in a boiling water bath for 5 min. Protein samples were separated by 15% SDS-PAGE, and Western blot analysis was performed using rabbit anti-SM22 antibody (Santa Cruz).

Immunoblot analysis. Protein samples (20 μg in SDS loading buffer) were separated by electrophoresis on 15% SDS-polyacrylamide gel, transferred to polyvinylidene difluoride membrane, and probed with the specific primary antibodies (listed in Chemicals and drugs) diluted in TBS-T containing 1% milk for 1 h at room temperature (RT). After being washed three times, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 for each bovine anti-rabbit for SM22 and bovine anti-mouse for phospho-serine antibody). After the blot was washed three times, the corresponding bands were visualized with enhanced chemiluminescence substrate using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and Hyperfilm MP (Amersham Bioscience, Piscataway, NJ). The membranes were stripped of secondary and primary antibodies by incubating with Restore Western blot stripping buffer (Pierce) for 15 min at RT. The membranes were again reprobed with α-actin antibody.

Bands corresponding to different proteins on X-ray films were scanned with a scanner (model SNAPSCAN 310; Agfa, Ridgefield Park, NJ), and their relative densities were determined using Image-Pro Plus 4.0 software (Media Cybernetics). SMC transfections and measurement of SMC lengths. Isolated SMC from IAS and RSM were cultured in Dulbecco's modified Eagle's medium with 5% penicillin-streptomycin, 50 μg/ml gentamycin, and 2 μg/ml amphotericin B at 37°C with 5% CO 2 for 24 h. Later, IAS and RSM SMC were transfected with pFLAG-SM22 () using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) at a final concentration of 0.5, 1, and 2 μg/ml plasmid DNA. Control SMCs were similarly transfected with empty vector. SMC were further incubated at 37°C with 5% CO 2 for 36 h. Likewise, SMC were also transfected with different concentrations of SM22 siRNA (50–200 nM).

Control SMC were similarly transfected with scrambled (control) siRNA. At the end of incubation period, culture medium was removed from the plates, and SMCs were incubated for 5–10 min with 1 ml of 0.05% trypsin-EDTA. SMC were then centrifuged, washed two times with fresh KPS, and then resuspended in KPS, and 10 4 SMC were suspended in 100 μl of KPS.

Lengths of the individual cells were measured by computerized image microscopy. Average length of SMC from different groups was obtained from 50 cells encountered randomly in successive microscopic fields. Confocal microscopy. SMC were cultured in culture medium as described above on Lab-Tek II chamber slides (Nulge Nunc, Naperville, IL) at 37°C with 5% CO 2 for 24 h. Later, culture medium was removed, and SMC were fixed in 4% paraformaldehyde solution in Dulbecco's phosphate-buffered saline (DPBS) at RT for 15 min. SMCs were washed three times with DPBS and incubated overnight at RT in a humid environment with 1:100 dilution of SM22 primary antibody (raised in rabbit; Santa Cruz Biotechnology) in DPBS containing 0.2% Triton X-100 and 0.5% bovine serum albumin.

Later, SMC were washed three times with DPBS and further incubated with Texas red-conjugated anti-rabbit secondary antibody (1:200 dilution in DPBS; Santa Cruz Biotechnology) and fluorescein isothiocyanate-conjugated α-actin antibody (1:800; Sigma Chemical, St. Louis, MO) in DPBS with 0.3% Triton X-100 and 2% donkey serum for 1 h. SMC were then washed three times with DPBS, and chambers were removed from slides. The slides were air-dried, and coverslips were mounted on the slides using Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Fluorescence was analyzed with a Bio-Rad MRC 600 laser-scanning confocal microscope (Zeiss Anxiovert 100; Zeiss, Overkochen, Germany), and images were generated automatically by the imaging software ().

RESULTS 2D-DIGE analysis of the IAS vs. RSM SMC protein samples demonstrated decreased expression of SM22 in the tonic IAS SMC. As shown in, studies have identified three spots (nos.

1262, 1380, and 1381) having accession number 62653429, molecular mass 22.6 kDa, isoelectric point 9.4 and that were downregulated in the IAS (tonic) vs. RSM (phasic). Quantitative differences between IAS vs. RSM revealed an average significant decrease (−1.87) in all three spots in their expression in the IAS SMC. ProteomeX Electrospray Ion-Trap MS analysis of tryptic digests of the three downregulated proteins against NCBI database identified all of these spots to be SM22 as discussed below.

Overlaid representative images for 2-dimensional gels (pH 3–10) used in separations of proteins extracted from the internal anal sphincter (IAS) smooth muscle cells (SMC, labeled with Cy5 in red) and rectal smooth muscle (RSM) cells (labeled with. We analyzed the changes in these proteins and determined the role of SM22 in the functionality of the phenotypic tonic vs. Phasic SMs, especially how higher expression of SM22 in the RSM keeps it from becoming completely tonic. (In this regard data in have identified three spots that specifically were downregulated in the IAS vs. Effects of SM22 overexpression and SM22 silencing on IAS SMC.

As a follow up of proteomic analysis showing discrete differences in the phasic vs. Tonic SM with regard to the role of SM22, to gain further insights into the role of SM22 in these phenotypic SMCs, we overexpressed SM22 (using pFLAG-SM22) and silenced SM22 expression (using SM22 siRNA) in these cells and monitored changes in their length for the functional (contraction/relaxation) role in the IAS vs. Transfection of pFLAG-SM22 (0.5–2 μg/ml) concentration dependently and significantly ( P. Confocal microscopic studies showing the effects of SM22 siRNA on SM22 expression in IAS vs.

To further confirm the significance of SM22 expression differences in IAS and RSM SMCs, we performed confocal microscopy studies under the treatment of SM22 siRNA. Such studies using SM22 antibody confirmed the success of SM22 siRNA by the decreased expression of SM22 in the IAS and RSM SMC transfected with 200 nM of SM22 siRNA (). Also, in the basal state, data revealed higher immunofluorescence intensity of SM22 in the RSM SMC vs.

IAS SMC (), in agreement with the proteomic data for the expression differences. In these studies, α-actin antibody was used as a marker for SMC. Effects of ROCK inhibitor Y-27632 on phosphorylation of SM22 in the IAS. Because RhoA/ROCK and PKC pathways have been suggested to be the major determinant of the tonic and phasic SM contractions, respectively (), it was considered important to determine the effect of inhibitions of these pathways on phospho-SM22.

In that regard, we first proceeded to determine the effect of ROCK inhibitor Y-27632. SM22 was precipitated via immunoprecipitation using SM22 antibody. Precipitates were further analyzed via Western blot analysis using antiphosphoserine antibody to detect phosphorylation of SM22 in vivo. As described in, data revealed higher levels of phospho-SM22 in the IAS vs. In sharp contrast, levels of total SM22 were higher in the RSM vs. Y-27632 concentration dependently (10 −8 to 10 −5 M) and significantly ( P. Effects of Y-27632 on binding of SM22 to actin in the IAS.

Because SM22 is transgelin or actin-binding protein, it was important to determine the effect of ROCK inhibition in relation to SM22 biding to actin. For that, SM22-actin complexes were coimmunoprecipitated using agarose-bound actin antibody. Complexes were analyzed via Western blot using SM22-specific antibody to determine the levels of actin-bound SM22. Y-27632 concentration dependently (10 −8 to 10 −5 M) and significantly ( P. DISCUSSION Preliminary studies from our laboratory (for details, see materials and methods) revealed downregulation of SM22 (transgelin/calponin) protein expression in the tonic IAS vs. Phasic RSM SMC.

Because SM22 plays a significant role in the SM contraction with its role as actin-binding protein (), in the present investigation, we focused on its mechanistic differential role in developing basal tone within IAS and RSM SMC. SM22-actin binding data reveal lower levels in the IAS vs. Further studies show that overexpression of wild-type SM22 causes relaxation of IAS SMC. Additionally, silencing of SM22 by SM22 siRNA contracts IAS SMC.

Data further show higher levels of SM22 phosphorylation in the IAS vs. Additional data suggest that SM22 phosphorylation may be ROCK mediated as in the ROCK inhibitor decreases phosphorylation of SM22 and increases its binding to actin in the IAS. In the form of a cartoon illustrates the role of SM22 and p-SM22 in the IAS vs.

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