Role of SM22 in the differential regulation of phasic vs. tonic smooth muscle

Abstract

Preliminary proteomics studies between tonic vs. phasic smooth muscles identified three distinct protein spots identified to be those of transgelin (SM22). The latter was found to be distinctly downregulated in the internal anal sphincter (IAS) vs. rectal smooth muscle (RSM) SMC. The major focus of the present studies was to examine the differential molecular control mechanisms by SM22 in the functionality of truly tonic smooth muscle of the IAS vs. the adjoining phasic smooth muscle of the RSM. We monitored SMC lengths before and after incubation with pFLAG-SM22 (for SM22 overexpression), and SM22 small-interfering RNA. pFLAG-SM22 caused concentration-dependent and significantly greater relaxation in the IAS vs. the RSM SMCs. Conversely, temporary silencing of SM22 caused contraction in both types of the SMCs. Further studies revealed a significant reverse relationship between the levels of SM22 phosphorylation and the amount of SM22-actin binding in the IAS and RSM SMC. Data showed higher phospho-SM22 levels and decreased SM22-actin binding in the IAS, and reverse to be the case in the RSM SMCs. Experiments determining the mechanism for SM22 phosphorylation in these smooth muscles revealed that Y-27632 (Rho kinase inhibitor) but not Gö-6850 (protein kinase C inhibitor) caused concentration-dependent decreased phosphorylation of SM22. We speculate that SM22 plays an important role in the regulation of basal tone via Rho kinase-induced phosphorylation of SM22.

Keywords: actin-myosin binding; proteomics; transgelin-actin binding.

Fig. 1.

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 Cy3 in green) of the rat. Protein spots with significant quantitative expression differences and that have been identified by ProteomeX Electrospray Ion-Trap mass spectrometry (MS) are circled, numbered, and named (see enlarged inset of actual gel). Spots with the predominant red and green color are highly upregulated in IAS and RSM, respectively. Samples were depleted of actin via precipitation with agarose-conjugated actin antibody before run on the gels. The first dimension was run on an immobilized pH gradient (–10), and the second dimension resolved on a 12.5% SDS-PAGE. Molecular weight range is shown on the left (y-axis), and pH range is shown at the bottom (x-axis). Individual protein spots showing difference in the expression in IAS vs. RSM as shown in gel with a circle. Three spots showing lower intensity in IAS vs. RSM SMC are identified as transgelin (SM22).

Fig. 2.

A: transfection of IAS SMC with pFLAG-SM22 significantly (*P < 0.05; n = 4) and concentration dependently increases the IAS SMC length (considered SMC relaxation), but the increase was not significant (P > 0.05; n = 4) for RSM SMC length. Percent increase in SMC length in pFLAG-SM22 transfected SMC is calculated relative to mean SMC length from the control group of SMC transfected with empty vector. B: SM22 small-interfering RNA (siRNA) causes a concentration-dependent decrease of IAS and RSM SMC length (considered SMC contraction), not significantly different from each other (P > 0.05; n = 4). Percent decrease of SMC length in siRNA-transfected SMC is calculated relative to mean SMC length from control groups (45.6 ± 4.5 and 61.4 ± 5.4 μm in the case of the IAS and RSM, respectively) similarly transfected with control siRNA.

Fig. 3.

A: confocal microscopy reveals immunofluorescence intensity (IFI) levels of actin (a) vs. SM22 (b) in the IAS SMC in control experiments. Incubation of the IAS SMC with SM22 siRNA causes contraction of the SMCs (as indicated in α-actin-stained SMC, c) with decrease in the IFI of SM22 in the IAS SMC (d). B: corresponding data in the RSM SMCs show higher IFI levels of SM22 in control experiments (b) and decrease in the levels of SM22 following SM22 siRNA.

Fig. 4.

Effect of Rho kinase (ROCK) inhibitor Y-27632 (10⁻⁸ to 10⁻⁵ M) on p^ser-SM22 in IAS smooth muscle. Y-27632 causes significant (*P < 0.05; n = 4) and concentration-dependent decrease in the phosphorylation of SM22 in the IAS. Phosphorylation of SM22 is almost absent in the RSM. Levels of total SM22 do not show significant change following incubation with Y-27632. Levels of total SM22 are higher in RSM vs. IAS.

Fig. 5.

Effect of ROCK inhibitor Y-27632 (on SM22-actin binding). Y-27632 cause significant (*P < 0.05; n = 4) and concentration-dependent increase in binding of SM22 to actin. Actin-SM22 complexes precipitated by using agarose-conjugated actin antibody and SM22 levels in the precipitates detected via Western blot analysis. Note highest binding of SM22 to actin in the RSM.

Fig. 6.

PKC inhibitor Gö-6850 in contrast with the ROCK inhibitor Y-27632 does not cause significant change in the SM22 phosphorylation in the IAS smooth muscle (P > 0.05; n = 4).

Fig. 7.

PKC inhibitor Gö-6850 does not cause significant effect on the SM22-actin binding in the IAS smooth muscle (P > 0.05; n = 4).

Fig. 8.

A cartoon explaining the role of SM22 in the tonic vs. phasic smooth muscle. In phasic smooth muscles, e.g., RSM (left), SM22 may inhibit actin-myosin interaction via its actin-binding properties. In tonic smooth muscles, e.g., IAS (right), ROCK decreases SM22 binding to actin via phosphorylation of SM22.