Phosphorylated HSP27 essential for acetylcholine-induced association of RhoA with PKC{alpha}

Suresh B. Patil, Mercy D. Pawar, and Khalil N. Bitar

Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109

Submitted 16 June 2003 ; accepted in final form 23 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reorganization of the cytoskeleton and association of contractile proteins are important steps in modulating smooth muscle contraction. Heat shock protein (HSP) 27 has significant effects on actin cytoskeletal reorganization during smooth muscle contraction. We investigated the role of phosphorylated HSP27 in modulating acetylcholine-induced sustained contraction of smooth muscle cells from the rabbit colon by transfecting smooth muscle cells with phosphomimic (3D) or nonphosphomimic (3G) HSP27. In 3G cells, the initial peak contractile response at 30 s was inhibited by 25% (24.0 ± 4.5% decrease in cell length, n = 4). The sustained contraction was greatly inhibited by 75% [9.3 ± .9% decreases in cell length (n = 4)]. Furthermore, in 3D cells, translocation of both PKC{alpha} and of RhoA was greatly enhanced and resulted in a greater association of PKC{alpha}-RhoA in the membrane fraction. In 3G transfected cells, PKC{alpha} and RhoA failed to translocate in response to stimulation with acetylcholine, resulting in an inhibition of association of PKC{alpha}-RhoA in the membrane fraction. Studies using GST-RhoA fusion protein indicate that there is a direct association of RhoA with PKC{alpha} and with HSP27. The results suggest that phosphorylated HSP27 plays a crucial role in the maintenance of association of PKC{alpha}-RhoA in the membrane fraction and in the maintenance of acetylcholine-induced sustained contraction.

phosphorylation; cytoskeleton


ON AGONIST STIMULATION and triggering of signaling pathways, smooth muscle cells adjust their functional properties by modulating the association of contractile proteins and reorganizing their cytoskeleton. The low-molecular-weight heat shock protein (HSP) 27 has significant effects on actin cytoskeletal reorganization (10, 21). HSP27 has been implicated in the regulation of the contraction and relaxation of smooth muscle (4, 7). Preincubation of smooth muscle cells from the rabbit rectosigmoid with a monoclonal antibody to HSP27 inhibits PKC-induced contraction (4). On stimulation of freshly isolated intestinal smooth muscle cells with contractile agonists, HSP27 colocalizes and coimmunoprecipitates with the contractile proteins such as actin, tropomyosin, and caldesmon (18), and it also associates with translocated PKC{alpha} and with translocated RhoA in the membrane fraction (3).

HSP27 is a member of the mammalian small HSP family. It is expressed in a variety of tissues including smooth muscle, both in the presence or absence of stress, and has been shown to exhibit chaperone activity in vitro and modulate actin filament microdynamics. HSP27 is phosphorylated in response to heat shock and in response to different stimuli such as cytokines, growth factors, and peptide hormones (2, 17, 22, 27). Landry et al. (22) have mapped the phosphorylation sites in human (h) HSP27 and showed that MAPK-activated protein kinase-2 (MAPKAP2 kinase) phosphorylates hHSP27 on Ser-15, Ser-78, and Ser-82. Ser-82 appears to be the major site of in vivo phosphorylation (30). It has been reported that phosphorylation results in the preponderance of smaller oligomers (14).

Phosphorylation of HSP27 changes the actin cytoskeleton and modulates actin-associated events (23), including modulation of smooth muscle contraction. Recently, Bitar (2) reported that agonist-induced contraction of colonic smooth muscle cells is associated with phosphorylation of HSP27 and that transfection of smooth muscle cells with the nonphosphomimic (3G) mutant of HSP27 results in inhibition of agonist-induced association of actin-myosin.

Evidence suggests that the ras-related small GTP binding protein RhoA is an important signaling protein that mediates various actin-dependent cytoskeletal functions including smooth muscle contraction (15, 29, 34). RhoA modulates agonist-induced signal-transduction cascades in smooth muscle contraction, and it colocalizes on the membrane with the actin-binding protein HSP27, as observed under confocal microscopy, and thus may regulate smooth muscle contraction through cytoskeletal reorganization of HSP27 (36).

We have previously shown that PKC{alpha} and HSP27 are translocated to the membrane during agonist-induced smooth muscle contraction (3). However, it is not known whether phosphorylation of HSP27 modulates the translocation and association of contractile and signaling proteins. We hypothesized that HSP27 may affect smooth muscle thin-filament regulation through its phosphorylation and may play an active role in the contractile machinery through its association with contractile proteins in smooth muscle. We have investigated the role of phosphorylated HSP27 in modulating the translocation and association of contractile and signaling proteins during agonist-induced smooth muscle contraction. We have used recombinant GST-RhoA fusion protein, recombinant hHSP27, and recombinant hPKC{alpha} to study in vitro protein-protein interactions and examined the direct interaction of RhoA with HSP27 and with PKC{alpha}. We have investigated whether phosphorylation of HSP27 is required for translocation of PKC{alpha} to the membrane fraction and whether the translocation of PKC{alpha} was primordial to the assocition in the membrane fraction of RhoA with PKC{alpha} and with HSP27. We have studied gain or loss of function of HSP27 phosphorylation. We used constructs of constitutively phosphorylated or nonphosphorylatable hHSP27 stably transfected to cultured rabbit colon smooth muscle cells. The results suggest that nonphosphorylatable HSP27 inhibits smooth muscle contraction. Inhibition of the contraction is possibly a consequence of the inhibition of association of HSP27 with signaling proteins and the contractile proteins. Evidence also suggests that there is direct interaction of RhoA with HSP27 and with PKC{alpha}.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acetylcholine was purchased from Sigma (St. Louis, MO). Constructs of phosphomic and nonphosphomic HSP27 were a gift from Dr. Benndorf (Univ. of Michigan). Calfectin was purchased from Quiagen (Valencia, CA). Polyclonal anti-PKC{alpha} antibody was from Panvera (Madison, WI), anti-RhoA monoclonal antibody was from Cytoskeleton (Denver, CO), and anti-HSP27 monoclonal antibody was produced in our laboratory (5). Recombinant GST-RhoA and recombinant hPKC{alpha} were purchased from Cytoskeleton. Recombinant hHSP27 was purchased from Stressgen (San Diego, CA). All other reagents mentioned were of analytical grade unless otherwise mentioned.

Direct association of GST-RhoA with HSP27 and with PKC{alpha}. GST-RhoA (24 µg) was mixed with 200 µl of glutathione-agarose beads and rocked for 1 h at 4°C. The mixture was washed three times with PBS containing 1% Triton X-100. The washes were retained for further analysis. The beads were mixed with either 10 µg of PKC{alpha} or 24 µg of HSP27 and were further rocked for 2 h at 4°C. The mixture was washed for unbound proteins several times with PBS containing 1% Triton X-100. The washes were retained for further analysis. The bound proteins were eluted with 10 mM reduced glutathione buffer (pH 7.4) at least three times. All the fractions of washes and the eluates were spotted on a nitrocellulose membrane. The membranes were blocked with nonfat skimmed milk and were subjected to immunoblotting against anti-GST antibody, anti-PKC{alpha} antibody, anti-RhoA antibody, or anti-HSP27 antibody. The spots were detected by chemiluminescence.

Transfection of smooth muscle cells with HSP27 mutants. We studied the gain of function and loss of function of HSP27 phosphorylation mutants in smooth muscle cells. We transfected smooth muscle cells with phosphorylation mutants of HSP27: 3G or phosphomimic (3D) hHSP27 as described earlier (2). In the 3G HSP27 mutants, all three serine phosphorylation sites (Ser-15, Ser-78, and Ser-82) of the hHSP27 cDNA were replaced with glycine to mimic nonphosphorylatable serine residues. In the 3D mutant, Ser-15, Ser-78, and Ser-82 were mutated into aspartate to mimic constitutively phosphorylated residues. These mutant constructs were expressed in smooth muscle cells. Smooth muscle cells were cultured in DMEM with 10% FBS and 3% penicillin/streptomycin and 0.6% L-glutamine on collagen IV-coated dishes. The cells were passed on the day before transfection and allowed to reach 70% confluence on the day of transfection. The cells were washed with PBS twice. 3D or 3G hHSP27 mutant cDNA was transfected into the cells using Qiagen Effectene transfection kit. After 2 days of transfection, the cells were selected with G-418 (3 mg/ml) for 2 days. The transfections were successful and stable as demonstrated by detection of the mutant hHSP27 protein via immunoblotting of the transfected cell lysates with hHSP27-specific antibody, which has been shown not to have any cross-activity in rabbit and mouse.

Detergent-soluble membrane fractions. Confluent smooth muscle cells in culture were either untreated or treated with acetylcholine (0.1 µM) for up to 4 min. After stimulation, the cells were washed twice with buffer A [in mM: 150 NaCl, 16 Na2HPO4, 4 NaH2PO4, and 1 sodium orthovanadate (pH 7.4)] and sonicated in buffer B [in mM: 150 NaCl, 1 NaF, 2 phenylmethylsulfonyl fluoride, 5 EDTA, 1 Na4MoO4, 1 dithiothreitol, 20 NaH2PO4, 20 Na2HPO4, and 20 Na4P2O7·10H2O, with 50 µl/ml DNase-RNase, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml antipain (pH 7.4)]. The cell sonicates were centrifuged at 100,000 g for 60 min. The supernatant material from the high-speed centrifugation was collected as cytosolic fraction. The pellet material was resuspended by sonication twice for 30 s in the lysis buffer plus 1% Triton X-100 and collected as detergent-soluble membrane fraction. The protein content was determined by using BioRad protein assay reagent.

Immunoprecipitation and immunoblotting. Each sample (400–500 µg protein) obtained as described above was subjected to immunoprecipitation with monoclonal anti-HSP27 antibody, anti-RhoA antibody, or polyclonal anti-PKC{alpha} antibody overnight at a ratio of 1:250. The protein G-Sepharose beads were then added and rocked for 2 h. The beads were washed in Tris-buffered saline twice and boiled in 2x Laemmli sample buffer with 2-mercaptoethanol.

SDS-PAGE and electrophoretic transfer. For one-dimensional SDS-PAGE, the membrane samples or immunoprecipitates were mixed in an equal volume of 2x sample buffer [50 mM Tris, 10% (vol/vol) glycerol, 2% (wt/vol) SDS, and 0.1% (wt/vol) bromophenol blue, pH 6.8]. The proteins were separated by 12.5 or 15% SDS-PAGE and transferred onto nitrocellulose or PVDF membranes. Immunoblotting was performed using a monoclonal anti-RhoA antibody (1:200) or a rabbit polyclonal anti-PKC{alpha} antibody (1:100) as primary antibody. The membrane was reacted with peroxidase-conjugated goat anti-mouse/anti-rabbit IgG antibody (1:2,500 dilution) for 1 h at 24°C. The enzymes on the membrane were detected with luminescent substrates. As a negative control, blots were incubated in the secondary antibody only. Proteins were identified by chemiluminescence.

Measurement of contraction. Smooth muscle cells isolated from rabbit colon were cultured to confluence. Fresh medium was added to the culture flask. Cells were then scraped off with a "policeman" and allowed to float freely for 48 h in a standing flask with occasional shaking to prevent further settling and sticking to the bottom of the flask. Aliquots of cultured cell suspension (2.5 x 104 cells/0.5 ml) were stimulated with acetylcholine (10-7 M) for 30 s or 4 min. The reaction was allowed to proceed for 30 s or 4 min and stopped by the addition of 0.1 ml of acrolein at a final concentration of 1% (vol/vol). Individual cell length was measured by computerized image micrometry. The average length of cells in the control state or after addition of test agents was obtained from 50 cells encountered randomly in successive microscopic fields. The contractile response is defined as the decrease in the average length of the 50 cells and is expressed as the absolute change or the percent change from control length (4).

Data analysis. Bands were quantitated using a densitometer (model GS-700, BioRad Laboratories), and band volumes (absorbance units x mm2) were calculated and expressed as percentages of the total volume. Blotting data are within the linear range of detection for each antibody used.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Direct interaction of GST-RhoA with recombinant hPKC{alpha}. We have previously shown that PKC{alpha} associates with RhoA in the membrane fraction of smooth muscle cells stimulated with acetylcholine (3). To test whether the observed association of RhoA with PKC{alpha} is a direct or due to an interaction with other intermediary protein(s), we tested the interaction of the fusion protein GST-RhoA with recombinant hPKC{alpha}. We have taken the advantage of specific affinity of GST with glutathione agarose beads to immobilize the fusion protein, GST-RhoA. GST-RhoA (24 µg) was incubated with 200 µl of 50% suspension of glutathione-agarose in PBS/0.1% {beta}-mercaptoethanol at 4°C for 1 h. The mixture was washed and further incubated with 24 µg of recombinant hPKC{alpha} for 3 h at 4°C. Incubation of hPKC{alpha} allowed its binding to the immobilized GST-RhoA. The nonspecific binding was washed off using PBS (pH 7.4) containing detergent (1% Triton X-100). Addition of reduced glutathione buffer (pH 8.0) resulted in elution of the GST-putative protein complexes from the glutathione agarose beads. Examination of the dot blots with specific antibodies to RhoA, PKC{alpha}, and GST indicated that there was coelution of PKC{alpha} and GST-RhoA in the glutathione eluates (fractions 11–16), indicating a direct association of recombinant hPKC{alpha} with GST-RhoA (Fig. 1A). GST alone was used with the glutathione beads as control to confirm that the binding of hPKC{alpha} was due to its binding with RhoA and not to GST (Fig. 1B).



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Fig. 1. A: direct interaction of GST-RhoA with recombinant human (h) PKC{alpha}. GST-RhoA (24 µg) was incubated with 200 µl of 50% suspension of glutathione agarose in PBS/0.1% {beta}-mercaptoethanol at 4°C for 30 min. Fractions 1–5 are washings of unbound GST-RhoA. Recombinant hPKC{alpha} (24 µg) was added to the washed beads and incubated at room temperature. Fractions 6–10 are washings of unbound PKC{alpha}. GST-RhoA bound to the glutathione agarose was eluted with 10 mM glutathione in fractions 11–16. a–c: Dot Western blots of the fractions (in duplicates) with antibodies specific for PKC{alpha}, RhoA, and GST, respectively. In fractions 11–16, there was a coelution of PKC{alpha} with GST-RhoA, an indication of direct association of recombinant hPKC{alpha} with GST-RhoA. B: experiments were conducted whereby 24 µg of GST were incubated with 200 µl of 50% suspension of glutathione agarose in PBS/0.1% {beta}-mercaptoethanol at 4°C for 30 min. Fractions 1–3 are washings of unbound GST. Recombinant hPKC{alpha} (24 µg) was added to the washed beads and incubated at room temperature. Fractions 6–10 are washings of unbound PKC{alpha}. GST bound to the glutathione agarose was eluted with 10 mM glutathione in fractions 7–10. a and b: Dot Western blots of the fractions (in duplicates) with antibodies specific for GST and PKC{alpha}, respectively. In fractions 7–10, there was no coelution of PKC{alpha} with GST, indicating that GST did not directly bind with recombinant hPKC{alpha}. HRP, horseradish peroxidase; TX-100, Triton X-100.

 

Direct interaction of GST-RhoA with recombinant HSP27. We have previously shown (3) that RhoA associates with HSP27 in the membrane fraction of smooth muscle cells stimulated with acetylcholine. To test whether the observed association is direct, 24 µg of GST-RhoA were incubated with 200 µl of 50% suspension of glutathione-agarose in PBS/0.1% {beta}-mercaptoethanol at 4°C for 1 h. The mixture was washed and further incubated with 24 µg of recombinant hHSP27 for 3 h at 4°C. After washes, the slurry was eluted with 10 mM glutathione as detailed in MATERIALS AND METHODS. Examination of dot blots indicated that there is coelution of HSP27 and GST-RhoA in the glutathione eluates (fractions 9–11), indicating direct association of recombinant hHSP27 with GST-RhoA (Fig. 2). In control experiments, we confirmed that neither PKC{alpha} nor HSP27 did coelute with GST alone, confirming the direct binding of PKC{alpha} and of HSP27 to RhoA.



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Fig. 2. Direct interaction of GST-RhoA with recombinant hHSP27. GST-RhoA (24 µg) was incubated with 200 µl of 50% suspension of glutathione agarose in PBS/0.1% {beta}-mercaptoethanol at 4°C for 30 min. Fractions 1–4 are washings of unbound GST-RhoA. Recombinant human heat shock protein (HSP) 27 (24 µg) was added to the washed beads and incubated at room temperature. Fractions 5–8 are washings of unbound HSP27. GST-RhoA bound to the glutathione-agarose was eluted with 10 mM glutathione in fractions 9–11. a–c: Dot Western blots in duplicates with antibodies specific for HSP27, RhoA, and GST, respectively. In fractions 9–11, there was a coelution of HSP27 with GST-RhoA, an indication of direct association of recombinant human HSP27 with GST-RhoA.

 

Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on acetylcholine-induced translocation of PKC{alpha} to the membrane fraction. We have previously shown that acetylcholine induces translocation of PKC{alpha} to the membrane fraction. We had also shown that HSP27 is phosphorylated on stimulation with contractile agonists and that the phosphorylation of HSP27 seems to mediate agonist-induced association of actin-myosin in smooth muscle cells of the colon. To examine whether the observed effect on the association of actin with myosin is a direct effect of the interaction of these two proteins or due to an effect on upstream signaling pathways (i.e., the agonist-induced translocation of PKC{alpha}), confluent smooth muscle cells in culture were transfected with either 3D or 3G mutants of HSP27. Membrane fractions of cells were then subjected to SDS-PAGE and Western blotted with anti-PKC{alpha} antibody (1:100). Stimulation with acetylcholine resulted in a significant increase in translocation of PKC{alpha} at 30 s and 4 min in 3D cells compared with normal cells (146.77 ± 11.59 and 123.07 ± 6.96% in 3D-transfected cells compared with 152.53 ± 26.63 and 150.93 ± 21.26% in normal cells at 30 s and 4 min, respectively), indicating a role for phosphorylated HSP27 in modulating agonist-induced translocation of PKC{alpha}. There was no change in the cells transfected with 3G construct (92.93 ± 14.98 and 96.08 ± 14.64 at 30 s and 4 min, respectively; *P <= 0.05). To examine whether inhibition of agonist-induced activation of PKC{alpha} would inhibit its translocation in cells transfected with 3D construct of HSP27, cells were preincubated with calphostinC (1 µM) for 20 min before stimulation with acetylcholine for 30 s or 4 min. The membrane fractions from these cells were separated by SDS-PAGE and were subjected to Western blot against anti-PKC{alpha} antibody (1:100). In nontransfected cells, preincubation with calphostin C resulted in inhibition of acetylcholine-induced translocation of PKC{alpha} to the membrane fraction (159.98 ± 24.69 and 165.28 ± 35.67 vs. 95.89 ± 6.12 and 75.64 ± 7.58% increase in cells preincubated with calphostin C at 30 s and 4 min, respectively). Preincubation of 3D cells with calphostin C resulted in similar inhibition of acetylcholine-induced translocation of PKC{alpha} to the membrane (129.04 ± 12.57 and 135.79 ± 5.82 vs. 83.27 ± 12.68 and 90.09 ± 1.51% increase in cells preincubated with calphostin C at 30 s and 4 min, respectively; Fig. 3C).



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Fig. 3. Effect of transfection of smooth muscle cells with phospomimic (3D) or nonphosphomimic (3G) constructs of HSP27 on ACh-induced translocation of PKC{alpha} to the membrane fraction. Smooth muscle cells from the rabbit colon maintained in culture were transfected or nontransfected with 3D or 3G HSP27 and were stimulated with ACh (0.1 µM) for 30 s or 4 min. Equal amounts (20 µg) of membrane fractions prepared as described in MATERIALS AND METHODS were separated by SDS-PAGE and Western blotted with anti-PKC{alpha} antibody (1:100). A: representative blot indicating an increase in translocation of PKC{alpha} in normal cells (N) and in cells transfected with 3D HSP27 cDNA. No difference was observed in cells transfected with the 3G form of HSP27. B: stimulation with ACh resulted in a significant increase in translocation of PKC{alpha} in normal cells (152.53 ± 26.63 and 150.93 ± 21.26%) and in 3D-transfected cells (146.77 ± 11.59 and 123.07 ± 6.96%) at 30 s and 4 min, respectively. There was no change in the cells transfected with 3G construct (92.93 ± 14.98 and 96.08 ± 14.64% at 30 s and 4 min, respectively; *P <= 0.05). C: effect of calphostin C on ACh-induced increases in the translocation of PKC{alpha} to the membrane fraction normal cells (a) or of smooth muscle cells transfected with 3D (b) constructs of HSP27. Cultured cells were preincubated with calphostin C (1 µM) for 20 min and were further stimulated with ACh (10-7 M) for 30 s or 4 min. Equal amounts (20 µg) of membrane fractions were separated by SDS-PAGE and Western blotted with anti-PKC{alpha} antibody (1:100). Calphostin C (1 µM) inhibited ACh-induced increases in translocation of PKC{alpha} to the membrane fraction both in normal cells and in cells transfected with 3D construct. Normal cells: 159.98 ± 24.69 and 165.28 ± 35.67 vs. 95.89 ± 6.12 and 75.64 ± 7.58% in cells preincubated with calphostin C at 30 s and 4 min, respectively. 3D cells: 129.04 ± 12.57 and 135.79 ± 5.82 vs. 83.27 ± 12.68 and 90.09 ± 1.51% in cells preincubated with calphostin C at 30 s and 4 min, respectively.

 

Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on acetylcholine-induced translocation of RhoA to the membrane fraction. We have previously shown that acetylcholine induces translocation of RhoA to the membrane fraction and induces an increase in the association of HSP27 with RhoA in the membrane fraction (3). To examine whether the phosphorylation of HSP27 affects agonist-induced translocation of RhoA, confluent smooth muscle cells in culture were stimulated with 0.1 µM acetylcholine for 30 s or 4 min. Membrane fractions of cells were subjected to SDS-PAGE and Western blotted with anti-RhoA antibody (1:200; Fig. 4A). Stimulation with acetylcholine resulted in a significant increase in translocation of RhoA compared with normal cells (124.61 ± 14.09 and 107.45 ± 35.46 in 3D-transfected cells and 140.41 ± 20.67 and 115.78 ± 8.99% in normal cells at 30 s and 4 min, respectively). There was no increase in the cells transfected with 3G construct at 30 s (114.56 ± 4.84 and 90.29 ± 3.18% at 30 s and 4 min, respectively; Fig. 4B), indicating a role for phosphorylation of HSP27 in modulating acetylcholine-induced translocation of RhoA.



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Fig. 4. Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on ACh-induced increases in the translocation of RhoA to the membrane fraction. Smooth muscle cells from the rabbit colon maintained in culture were transfected or nontransfected with 3D or 3G HSP27 and were stimulated with ACh (0.1 µM) for 30 s or 4 min. Equal amounts (20 µg) of membrane fractions prepared as described in MATERIALS AND METHODS were separated by SDS-PAGE, and Western blot was prepared with anti-RhoH antibody (1:200). There was no change in the amount of RhoA in 3G transfected cells. A: representative blot indicating an increased translocation of RhoA in the membrane fraction is seen in normal cells and in 3D cells hHSP27 cDNA. B: stimulation with ACh resulted in a significant increase in translocation of RhoA in normal (140.41 ± 20.67 and 115.78 ± 8.99%) and in 3D-transfected cells (124.61 ± 14.09 and 107.45 ± 35.46) at 30 s and 4 min, respectively. There was no increase in the cells transfected with 3G construct after stimulation with ACh (114.56 ± 4.84 and 90.29 ± 3.18% at 30 s and 4 min, respectively) *P <= 0.05.

 

Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on acetylcholine-induced association of RhoA with PKC{alpha} in the fraction. Confluent muscle cells in culture were stimulated with acetylcholine (0.1 µM) for 30 s or 4 min, and immunoprecipitates of RhoA from 500 µg of fractions were subjected to SDS-PAGE and Western blotted with anti-PKC{alpha} antibody (1:100; Fig. 5A). Stimulation with acetylcholine resulted in a significant increase in translocation and association of RhoA with PKC{alpha}. (128.68 ± 13.13 and 228.78 ± 38.94% in 3D-transfected cells compared with 160.01 ± 25.56 and 150.06 ± 22.25% in normal cells at 30 s and 4 min, respectively). There was no change in cells transfected with 3G construct (93.96 ± 10.01 and 110.17 ± 17.34% at 30 s and 4 min, respectively; P <= 0.05; Fig. 5B).



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Fig. 5. Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on ACh-induced association of RhoA with PKC{alpha} in the membrane fraction. Smooth muscle cells from the rabbit colon maintained in culture were transfected or nontransfected with 3D or 3G HSP27 and were stimulated with ACh (0.1 µM) for 30 s or 4 min. Membrane fractions were prepared as described in MATERIALS AND METHODS. Equal amounts (500 µg) of membrane fractions were immunoprecipitated with anti-RhoA antibody and were further separated by SDS-PAGE, and Western blot was prepared with anti-PKC{alpha} antibody (1:100). A: increase in association of RhoA with PKC{alpha} was seen in normal cells and in cells transfected with 3D HSP27 cDNA, whereas minimal association was observed in cells transfected with the 3G form of HSP27. B: stimulation with ACh resulted in a significant increase in translocation and association of RhoA with PKC{alpha} (128.68 ± 13.13 and 228.78 ± 38.94% in 3D-transfected cells compared with 160.01 ± 25.56 and 150.06 ± 22.25% in normal cells at 30 s and 4 min, respectively). There was no change in cells transfected with 3G construct (93.96 ± 10.01 and 110.17 ± 17.34% at 30 s and 4 min, respectively) *P <= 0.05.

 

To test whether immunoprecipitation using antibody reflected a change in the amount of proteins translocated, membrane fractions were immunoprecipitated with anti-PKC{alpha} and immunoblotted with anti-PKC{alpha} antibody (1:100; Fig. 6A). Increased translocation of PKC{alpha} to the membrane fraction is seen in normal cells and in cells transfected with 3D hHsp27 cDNA (136.09 ± 22.92 and 112.94 ± 3.81% in normal cells and 133.48 ± 25.75 and 168.40 ± 36.32% in 3D-transfected cells at 30 s and 4 min, respectively). There was no observed acetylcholine-induced translocation in the 3G-transfected cells (119.55 ± 17.10 and 115.31 ± 23.4% at 30 s and 4 min, respectively; Fig. 6B). Similarly, membrane fractions were immunoprecipitated with anti-RhoA antibody and immunoblotted with anti-RhoA antibody (1:200). There was also an observed decrease in the amount of RhoA translocated to the membrane fractions of cells transfected with 3G cells (Fig. 7). These data confirmed the results obtained from Western blots alone, indicating a reduced amount of the agonist-induced translocated PKC{alpha} or agonist-induced translocated RhoA present in the membrane fractions of muscle cells transfected with the 3G form of HSP27 and an increase in the cells transfected with the 3D form of HSP27. The results also confirm that the antibody used is sufficient for immunoprecipitation of maximum protein present in the 500 µg of the membrane fraction.



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Fig. 6. Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on ACh-induced increases in the translocation of PKC{alpha} to the membrane fraction. A: equal amounts (500 µg) of membrane fractions were immunoprecipitated with anti-RhoA antibody and were further separated by SDS-PAGE, and Western blot was prepared with anti-PKC{alpha} antibody (1:100). Note that the IGG bands indicate that equal amounts of antibody were used in immunoprecipitation experiments. B: ACh (0.1 µM) induced a significant increase in translocation of PKC{alpha} to the membrane fraction in normal cells and in cells transfected with 3D hHSP27 cDNA (136.09 ± 22.92 and 112.94 ± 3.81% in normal cells) and in 3D-transfected cells (133.48 ± 25.75 and 168.40 ± 36.32%) at 30 s and 4 min, respectively. There was no significant change in translocation in the 3G-transfected cells (119.55 ± 17.10 and 115.31 ± 23.4 at 30 s and 4 min, respectively; *P <= 0.05).

 


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Fig. 7. Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on ACh-induced increases in the translocation of RhoA to the membrane fraction. Equal amounts (20 µg) of membrane fractions were separated by SDS-PAGE, and Western blot was prepared with anti-RhoA antibody (1:200). ACh (0.1 µM) induced a significant increase in translocation of RhoA to the membrane fraction in normal cells and in cells transfected with 3D hHSP27 cDNA. No significant differences in amount of RhoA were seen in the 3G-transfected cells.

 

Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on acetylcholine-induced association of PKC{alpha} with HSP27 in the membrane fraction. To test whether the state of phosphorylated HSP27 influences its association with PKC{alpha}, confluent muscle cells in culture were stimulated with 0.1 µM acetylcholine for 30 s or 4 min. Membrane preparations were immunoprecipitated with anti-HSP27 antibody further separated by SDS-PAGE, and Western blot was performed with anti-PKC{alpha} antibody (1:100). Increased association of PKC{alpha} with HSP27 is seen in the cells transfected with 3D hHSP27 cDNA compared with normal cells (323.17 ± 44.93 and 272.99 ± 38.94% in 3D-transfected cells and 178.39 ± 20.66 and 126.73 ± 21.96% in normal cells at 30 s and 4 min, respectively; P <= 0.05; Fig. 8). There was no change in acetylcholine-induced translocation and association of PKC{alpha} with HSP27 in the 3G-transfected cells (73.69 ± 26.02 and 68.13 ± 3.10% at 30 s and 4 min, respectively; P <= 0.05).



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Fig. 8. Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on ACh-induced association of HSP27 with PKC{alpha} in the membrane fraction. Smooth muscle cells from the rabbit colon maintained in culture were transfected or with 3D or 3G HSP27 and were stimulated with ACh (0.1 µM) for 30 s or 4 min. Membrane fractions were prepared as described in MATERIALS AND METHODS. Equal amounts (20 µg) of membrane fractions were immunoprecipitated with anti-HSP antibody and were further separated by SDS-PAGE, and Western blot was prepared with anti-PKC{alpha} antibody (1:100). Graphical representation showing increased association of PKC{alpha} with HSP27 in the membrane fractions of normal cells (178.39 ± 20.66 and 126.73 ± 21.96%) and in cells transfected with 3D hHSP27 cDNA (323.17 ± 44.93 and 272.99 ± 38.94%) at 30 s and 4 min, respectively. There was no change in the cells transfected with 3G construct (73.69 ± 26.02 and 68.13 ± 3.10% at 30 s and 4 min, respectively; *P <= 0.05).

 

Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on acetylcholine-induced translocation and association of RhoA with HSP27 in the membrane fraction. To test whether the state of phosphorylated HSP27 influences its association with RhoA, confluent muscle cells in culture were stimulated with acetylcholine (0.1 µM) for 30 s or 4 min. Membrane fractions (500 µg) were immunoprecipitated with anti-HSP27 antibody and subjected to SDS-PAGE followed by Western blot with anti-RhoA antibody (1:200; Fig. 9A). Stimulation with the contractile agonist acetylcholine (0.1 µM) resulted in a significant increase (P <= 0.05) in the association of RhoA with HSP27 at 30 s and 4 min. Increased association RhoA with HSP27 is seen in the cells transfected with 3D hHSP27 cDNA compared with normal cells (132.12 ± 8.41 and 128.75 ± 12.77% in normal cells vs. 124.08 ± 4.92 and 110.4 ± 4.62% in 3D cells at 30 s and 4 min, respectively; P <= 0.05; Fig. 9B). An inhibition of the association of translocated RhoA with HSP27 was seen in cells transfected with 3G construct (83.79 ± 6.15 and 110.75 ± 24.39%; P <= 0.05).



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Fig. 9. Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on ACh-induced translocation and association of HSP27 with RhoA in the membrane fraction. Smooth muscle cells from the rabbit colon maintained in culture were transfected with 3D or 3G HSP27 and stimulated with ACh (0.1 µM) for 30 s or 4 min. Membrane fractions were immunoprecipitated as described in MATERIALS AND METHODS. Immunoprecipitates of RhoA from 500 µg of membrane fractions were subjected to SDS-PAGE, and Western blot was prepared with anti-HSP antibody (1:1,000). A: representative blot showing increased association of RhoA with HSP27 in the membrane fraction of the cells transfected with 3D HSP27 cDNA compared with normal cells. No significant differences in the association of RhoA with HSP27 were seen in the 3G-transfected cells. B: graphical representation of association of RhoA with HSP27. Increased association of RhoA with HSP27 is seen in the normal cells (160.42 ± 8.10 and 161.71 ± 17.82%) and in cells transfected with 3D HSP27 cDNA (124.08 ± 4.92 and 110.4 ± 4.62%) at 30 s and 4 min, respectively. There was a decrease in cells transfected with 3G construct (83.79 ± 6.15 and 110.75 ± 24.39 in 3G cells at 30 s and 4 min, respectively; *P <= 0.05).

 

Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on acetylcholine-induced contraction of colonic smooth muscle cells. To test whether expression of the mutated HSP27 altered contractile properties, cells grown to confluence were stimulated with acetylcholine (10-7 M) for 30 s or 4 min. Normal cells exhibited a sustained contraction in response to acetylcholine (32.4 ± 2.7% decrease in cell length at 30 s and 34.2 ± 3.3% at 4 min; Fig. 10). Examination of the cells under microscope suggested that cells transfected with the mutant HSP27 (3D) exhibited a contractile pattern of sustained contraction (29.8 ± 1.9% decrease in cell length at 30 s and 31.8 ± 3.4% at 4 min; Fig. 10) similar to normal cells. Sustained contraction was greatly inhibited (75% inhibition) in cells transfected with the 3G constructs: 9.3 ± 0.9% decreases in cell length (n = 4) at 4 min, whereas the initial peak response at 30 s was affected to a much lesser extent [25% inhibition; 24.0 ± 4.5% decrease in cell length (n = 4) at 30 s]. Preincubation of cells with calphostin C had no effect on smooth muscle cell length in normal cells and in cells transfected with 3D construct (length of normal cells 45.28 ± 1.08 and 47.32 ± 0.88 µm in the absence and presence of calphostin C, respectively; length of 3D-transfected cells 43.5 ± 1.12 and 42.8 ± 1.16 µm in the absence and presence of calphostin C, respectively). Preincubation of the cells with calphostin C resulted in inhibition of acetylcholine-induced contraction both in normal cells and in cells transfected with the 3D construct (cell length 47.19 ± 0.87 and 43.62 ± 1.07 µm in normal cells at 30 s and 4 min after stimulation with acetylcholine, respectively; 43.21 ± 1.19 and 43.39 ± 1.08 µm in cells transfected with 3D construct at 30 s and 4 min after stimulation with acetylcholine, respectively).



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Fig. 10. Effect of transfection of smooth muscle cells with 3D or 3G constructs of HSP27 on ACh-induced contraction of colonic smooth muscle cells. Cultured rabbit colon cells transfected or nontransfected with the 3D HSP27 constructs or with the 3G constructs were allowed to reach confluence. After confluence, they were dispersed and maintained in suspension for 48 h before being subjected to contraction with ACh (0.1 µM). Colonic smooth muscle cells exhibited a sustained contraction in response to ACh [32.4 ± 2.7% (n = 4) decrease in cell length at 30 s and 34.2 ± 3.3% (n = 4) at 4 min]. Cells transfected with the 3D constructs exhibited a similar contractile pattern of sustained contraction [29.8 ± 1.9% (n = 4) at 30 s and 31.8 ± 3.4% (n = 4) at 4 min]. Sustained contraction was greatly inhibited (75% inhibition) in cells transfected with the 3G constructs [9.3. ± 0.9% decrease in cell length (n = 4) at 4 min], whereas the initial peak response at 30 s was affected to a much lesser extent [25% inhibition; 24.0 ± 4.5% decrease in cell length (n = 4)].

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There are distinct functional differences between the proximal and the distal colon. All segments of the colon have the capacity to propel fecal material and to store, mix, and absorb fluid. The distal colon serves mainly to expel the fecal bolus, displaying a pattern of intense peristaltic contractions and mass action associated with the propulsion of dehydrated feces. The different patterns of contractions have been attributed to regional differences in the gene transcription and synthesis of nitric oxide synthetase in the colonic myenteric plexus (33). On the basis of their functional behavior, visceral smooth muscles have been classified into sphincteric and nonsphincteric smooth muscles that represent tonic and phasic muscles (11, 16, 31). Phasic muscle shows a very wide range of contractile activity that varies from a fully relaxed basal state to a large-amplitude rapid contraction and rapid relaxation response. Smooth muscle cells isolated from the circular layer of the rabbit colon exhibit a sustained contractile response to acetylcholine (18, 36). Tonic contraction is activated by activation of PKC (24). It was previously shown (1) that during acetylcholine-induced smooth muscle contraction, HSP27 phosphorylation is mediated by activation of PKC. We and others (3, 32) have also shown that in smooth muscle cells, PKC{alpha} redistributes on stimulation with contractile agonists at 30 s and remains sustained at 4 min. Translocation of PKC{alpha} is an early and sustained event in acetylcholine-induced contraction of circular smooth muscle cells from the rabbit colon. Preincubation of smooth muscle cells with calphostin C results in inhibition of PKC{alpha} translocation to the membrane and inhibition of acetylcholine-induced contraction.

In smooth muscle, agonist-induced contraction is also accompanied by activation and phosphorylation of upstream MAPK cascade resulting in phosphorylation and translocation of HSP27 to the membrane (28). In intact smooth muscle, under physiological conditions, HSP27 could regulate actin cytoskeleton structure and may modulate the interaction of actin and myosin. HSP27 has significant effects on the actin cytoskeleton that are regulated by phosphorylation and dephosphorylation (10). Recently, we have shown (2) that agonist-induced phosphorylation of HSP27 modulates actin-myosin interaction through thin-filament regulation of tropomyosin and that HSP27 phosphorylation is independent of myosin light chain (MLC)20 phosphorylation. Inhibition of HSP27 phosphorylation substantially inhibited angiotensin II-induced contraction but had no effect on phenylephrine-induced contraction (23). Anti-HSP27 antibodies inhibited endothelin-1-induced Ca2+ sensitization of chemically permeabilized canine pulmonary artery strips (37) and inhibited PKC-mediated contraction of smooth muscle cells of the colon (3). Thus phosphorylated HSP27 may have an important role in facilitating the agonist-induced increase in translocation and association of RhoA with PKC{alpha} during agonist-induced contraction of smooth muscle. To test the role of phosphorylated HSP27 in translocation and association of RhoA with PKC{alpha}, we used smooth muscle cells transfected with constructs of 3D or 3G forms of hHSP27.

Agonist-induced smooth muscle contraction results in translocation of PKC{alpha} to the membrane and colocalization with contractile proteins (18). Similarly, RhoA translocates to the membrane in response to contractile agonists as seen in confocal microscopy (36). Furthermore, agonist-induced contraction is also accompanied by increased association of RhoA with PKC{alpha} in the membrane fraction (3). Agonist-induced contraction is also accompanied by an increase in association of RhoA with HSP27 and of PKC{alpha} with HSP27 in the membrane fraction. This increase was shown to be due to an increase in translocation of PKC{alpha} and of RhoA (3). This indicates that the translocation and association of RhoA with PKC{alpha} could be mediated by HSP27. However, it was not clear whether the observed association of RhoA with PKC{alpha} and with HSP27 was a direct association or was mediated through a complexing with other protein(s).

In the present studies, we have tested the association of recombinant RhoA with recombinant hPKC{alpha} using GST-RhoA immobilized on glutathione agarose beads. We have taken the advantage of the specific affinity of GST with glutathione to immobilize the fusion protein GST-RhoA on agarose beads cross-linked with glutathione. Incubation of hPKC{alpha} with the beads allowed its binding to the immobilized GST-RhoA. The nonspecific binding was washed off using a buffer containing detergent (pH 7.4; 1% Triton X-100). Coelution of GST-RhoA with hPKC{alpha} with reduced glutathione buffer (pH 8.0) indicated that RhoA interacted directly with hPKC{alpha}. GST alone was used with the glutathione beads as control to confirm that the binding of hPKC{alpha} was specifically due to its binding with RhoA and not GST. These results clarify earlier reports using coimmunoprecipitations that the association of RhoA with PKC{alpha} is due to a direct interaction between these two molecules (3). Similar experiments using recombinant hHSP27 indicate that there is a direct interaction of GST-RhoA with HSP27. This is the first evidence demonstrating a direct interaction of RhoA with hPKC{alpha} and with HSP27. Thus, during agonist-induced smooth muscle contraction, the translocation and association of RhoA with PKC{alpha} and with HSP27 is due to a direct association of RhoA with PKC{alpha} and with HSP27.

Interrelation between RhoA and PKC(s) has been reported in different cell systems (6, 25, 26, 32). A negative relationship between RhoA and PKC has previously been shown in the vascular smooth muscle A7r5 cell line. RhoA was directly responsible for actin reorganization in these cells. Activation of PKC resulted in the disassembly of actin stress fibers concomitant with the appearance of membrane ruffles (6). There is a strong evidence to implicate Rho kinase as a downstream target in the RhoA-linked pathway (35). Rho kinase phosphorylates MYPT1 and inhibits its activity, which, in turn, increases MLC phosphorylation (9, 12, 19). In a parallel mechanism, PKC activates CPI-17. Phosphorylation of MYPT by several kinases, such as PKC, increases the inhibitory potency of CPI-17, which further inhibits myosin phosphatase activity (8, 20). Thus it appears that, either directly or indirectly, both PKC and RhoA pathways target the inhibition of MYPT activity and thus maintain the phosphorylated state of MLC20. However, whether PKC and RhoA work together to increase MLC phosphorylation or decrease MYPT activity is difficult to ascertain. Our current results show that there is a direct interaction of RhoA with PKC{alpha} in vitro and that their association is decreased in 3G cells. In addition, the contractile response of 3G cells to acetylcholine was decreased. These results also suggest that the association of RhoA with PKC{alpha} mediated by phosphorylated HSP27 is important during agonist-induced smooth muscle contraction.

Western blot analysis with anti-PKC{alpha} and with anti-RhoA antibodies of membrane fractions from normal, 3D, and 3G cells in the control unstimulated state indicate that 3D cells exhibited the highest level of basal translocated PKC{alpha} and translocated RhoA. Furthermore, significant increases in the amount of RhoA and PKC{alpha} in the membrane fraction of 3D cells were seen at 30 s and 4 min after stimulation with acetylcholine. Immunoprecipitations followed by Western blot indicated an increased association of PKC{alpha} with RhoA in the membranes of nontransfected control cells, and the increase was further evident after 30 s or 4 min of stimulation of 3D cells with acetylcholine. Thus the presence of the phosphorylated form of HSP27 did accentuate the increase in the translocation of PKC{alpha} and RhoA to the membrane fraction of the cells.

The above suggests that phosphorylated HSP27 increases translocation and association of PKC{alpha} with RhoA in the membrane fraction of smooth muscle cells on stimulation with acetylcholine. The results also confirm that the observed increase at 30 s or 4 min after stimulation with acetylcholine in normal cells must be due to phosphorylation of HSP27. In endothelial and epithelial cells, Rho inhibitors have been shown to block PKC{alpha} translocation and activation, suggesting the activation of RhoA requirement for PKC{alpha} activation and/or translocation (13). Taken together, the results confirm that 1) the association of RhoA with PKC{alpha} and with HSP27 is direct, 2) phosphorylation of HSP27 is crucial for translocation and association of PKC{alpha} and RhoA to the membrane fraction, and 3) HSP27-mediated translocation and association of RhoA with PKC{alpha} is necessary for agonist-induced smooth muscle contraction.


    ACKNOWLEDGMENTS
 
We thank D. Thomas for assistance with technical editing and figure preparation.

GRANTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2-RO1-DK-42876.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. N. Bitar, Division of Pediatric Gastroenterology, Univ. of Michigan Medical School, 1150 West Medical Center Dr. MSRB 1, Rm. A520, Ann Arbor, MI 48109–0656 (E-mail: bitar{at}umich.edu).

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.


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