Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues

Abstract

Histone deacetylases (HDACs) are a family of enzymes that mediate nucleosomal histone deacetylation and gene expression. Some members of the HDAC family have also been implicated in nonhistone protein deacetylation, which modulates cell-cycle control, differentiation, and cell migration. However, the role of HDACs in smooth muscle contraction is largely unknown. Here, HDAC8 was localized both in the cytoplasm and the nucleus of mouse and human smooth muscle cells. Knockdown of HDAC8 by lentivirus-encoding HDAC8 shRNA inhibited force development in response to acetylcholine. Treatment of smooth muscle tissues with HDAC8 inhibitor XXIV (OSU-HDAC-44) induced relaxation of precontracted smooth muscle tissues. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3T3 cells. In this study, acetylcholine stimulation induced cortactin deacetylation in mouse and human smooth muscle tissues, as evidenced by immunoblot analysis using antibody against acetylated lysine. Knockdown of HDAC8 by RNAi or treatment with the inhibitor attenuated cortactin deacetylation and actin polymerization without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibited contraction and actin dynamics during contractile activation. These results suggest a novel mechanism for the regulation of smooth muscle contraction. In response to contractile stimulation, HDAC8 may mediate cortactin deacetylation, which subsequently promotes actin filament polymerization and smooth muscle contraction.

histone deacetylases (HDACs) are a family of enzymes that are originally identified as key regulators of histone deacetylation, nucleosome stability, and gene transcription (7, 21). There are four classes of mammalian HDACs: class I (HDACs 1, 2, 3, and 8), class II (HDACs 4, 5, 6, 7, 9, and 10), class III (SIRTs 1, 2, 3, 4, 5, 6, and 7) and class IV (HDAC11). HDACs are also known as lysine deacetylases (KDACs) because they primarily catalyze deacetylation of lysine on proteins.

The HDAC family members have long been thought to regulate nucleosomal histone acetylation solely. HDACs typically induce histone deacetylation and repress gene transcription (5). However, recent studies suggest that some HDACs, such as HDAC6, are present in the cytoplasm of nonmuscle cells, which has been implicated in regulating cell migration and microtubule dynamics (10, 37). More importantly, HDAC8 is predominantly expressed in smooth muscle cells/tissues (31). Silencing of HDAC8 by RNA interference (RNAi) impairs the capacity of cultured smooth muscle cells to contract collagen lattices (31). However, the role and mechanisms of HDAC8 in smooth muscle tissues are largely unknown.

Cortactin is an adapter protein that is able to regulate actin filament assembly in in vitro studies as well as adhesion, migration, and endocytosis of nonmuscle cells (1, 3). Cortactin may regulate actin polymerization by affecting the functional state of N-WASP, the Arp2/3 complex, and Nck in nonmuscle cells such as mouse embryonic fibroblasts and mouse 3T3 cells (1, 16). Furthermore, stimulation with growth factors induces cortactin deacetylation in NIH 3T3 cells, which has been implicated in the regulation of cell migration (37).

Structural analysis reveals that cortactin contains an N-terminal acidic domain, a repeat domain, and a Src homology 3 domain at the COOH terminus (1, 3). Among these domains, the repeat region of cortactin is sufficient to bind F-actin, which is essential for the regulation of actin dynamics (1, 3). Moreover, in vitro biochemical studies show that cortactin gets acetylated on nine lysines within the repeat region by PCAF (an acetyltransferase) (37, 38). Acetylation on these nine lysines decreases the association of cortactin with F-actin, which inhibits actin polymerization (37). In contrast, deacetylated cortactin increases its interaction with F-actin by altering a “charge patch” in its repeat region, which promotes actin dynamics (37, 38). It is largely unknown whether cortactin undergoes acetylation/deacetylation in smooth muscle during contractile activation.

In addition to myosin activation (14, 22), actin filament polymerization has recently emerged as a critical cellular mechanism that regulates smooth muscle contraction. Contractile stimulation induces actin polymerization in smooth muscle cells/tissues. Blockage of actin dynamics by inhibitors or molecular approach diminishes smooth muscle contraction without affecting myosin phosphorylation (2, 9, 15, 23, 26, 36). Reorganization of the actin cytoskeleton may facilitate force transmission between contractile units and extracellular matrix and thus promote smooth muscle contraction (9, 26, 30, 36). Therefore, both myosin light chain phosphorylation and actin cytoskeletal remodeling are essential for smooth muscle contraction. Myosin may serve as an “engine” for smooth muscle contraction, whereas the actin cytoskeleton may function as a “transmission system” in smooth muscle. Although myosin activation has been extensively investigated (14, 22), the mechanisms that regulate actin dynamics in smooth muscle in response to contractile stimulation are not fully understood.

The objective of this study was to evaluate the role of HDAC8 in contraction in smooth muscle tissues. Furthermore, we assessed whether HDAC8 affects cortactin acetylation level and actin polymerization in response to contractile activation.

MATERIALS AND METHODS

Measurement of force development.

All experimental protocols were approved by the Institutional Animal Care and Use Committee. C57BL/6 mice (25 ± 5 g) were killed with overdosed pentobarbital sodium. Tracheal rings (5 mm long) were immediately removed and placed at room temperature in physiological saline solution (PSS) containing 110 mM NaCl, 3.4 mM KCl, 2.4 mM CaCl₂, 0.8 mM MgSO₄, 25.8 mM NaHCO₃, 1.2 mM KH₂PO₄, and 5.6 mM glucose. The solution was aerated with 95% O₂-5% CO₂ to maintain a pH of 7.4. Tracheal rings were then placed in PSS at 37°C in a 25-ml organ bath and attached to a Grass force transducer that had been connected to a Gould recorder or a computer with A/D converter (Grass). At the beginning of each experiment, 0.5 g passive tension was applied to tracheal rings. After 60-min equilibrium, they were stimulated with 10⁻⁵ M acetylcholine (ACh) repeatedly until contractile responses and passive tension reached stability. For lentivirus-mediated RNAi in tissues, the thin epithelium layer of tracheal rings was removed by using forceps. They were then transduced with lentivirus encoding HDAC8 shRNA or control shRNA (Santa Cruz Biotechnology) for 4 days. Force development in response to contractile activation was compared before and after lentivirus transduction. For biochemical analysis, tissues were frozen using liquid nitrogen and pulverized as previously described (33, 34, 36).

In addition, human bronchial rings (diameter, 5 mm) were prepared from human lungs that were obtained from the International Institute for Advanced Medicine. Human tissues were nontransplantable and consented for research. This study was approved by the Albany Medical College Committee on Research Involving Human Subjects. Similarly, human bronchial rings were placed in PSS at 37°C in a 25-ml organ bath and attached to a Grass force transducer for the measurement of force development. HDAC8 inhibitor XXIV (OSU-HDAC-44) was purchased from Millipore.

Immunoblot analysis.

Pulverized tissues were lysed in SDS sample buffer composed of 1.5% dithiothreitol, 2% SDS, 80 mM Tris·HCl (pH 6.8), 10% glycerol, 0.01% bromophenol blue, phosphatase inhibitors (2 mM sodium orthovanadate, 2 mM molybdate, and 2 mM sodium pyrophosphate), and protease inhibitors (2 mM benzamidine, 0.5 mM aprotinin and 1 mM phenylmethylsulfonyl fluoride). The lysates were boiled in the buffer for 5 min and separated by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane. The membrane was treated with blockers for 1 h and probed with the use of primary antibody followed by horseradish peroxidase-conjugated secondary antibody (Fisher Scientific). Proteins were visualized by enhanced chemiluminescence (Fisher Scientific) using the LAS-4000 Fuji Image System. Antibodies against HDAC8, cortactin, phospho-myosin light chain (Ser-19), and myosin light chain were purchased from Santa Cruz Biotechnology. Acetyl lysine antibody was purchased from Millipore. GAPDH antibody was purchased from Fitzgerald. Antibody against α-actin was acquired from Sigma-Aldrich. The levels of proteins were quantified by scanning densitometry of immunoblots (Fuji Multigauge Software). The luminescent signals from all immunoblots were within the linear range.

Coimmunoprecipitation analysis.

Coimmunoprecipitation analysis was used to evaluate protein-protein interactions as previously described (2, 20, 32). Briefly, tissue extracts were incubated overnight with corresponding antibodies and then incubated for 2–3 h with 125 μl of a 10% suspension of protein A-Sepharose beads. Immunocomplexes were washed four times in buffer containing 50 mM Tris·HCl (pH 7.6), 150 mM NaCl, and 0.1% Triton X-100. The immunoprecipitates were separated by SDS-PAGE followed by transfer to nitrocellulose membranes. The membranes of immunoprecipitates were probed with corresponding antibodies.

Cell culture.

Human airway smooth muscle (HASM) cells were prepared from HASM tissues using the methods previously described (35). Mouse airway smooth muscle (MASM) cells were prepared from C57BL/6 mice. Briefly, tracheas were incubated for 10–20 min with dissociation solution [130 mM NaCl, 5 mM KCl, 1.0 mM CaCl₂, 1.0 mM MgCl₂, 10 mM Hepes, 0.25 mM EDTA, 10 mM d-glucose, 10 mM taurine, pH 7, 4.5 mg collagenase (type I), 10 mg papain (type IV), 1 mg/ml BSA and 1 mM dithiothreitol]. All enzymes were obtained from Sigma-Aldrich. The tissues were then washed with Hepes-buffered saline solution (composition in mM: 10 Hepes, 130 NaCl, 5 KCl, 10 glucose, 1 CaCl₂, 1 MgCl₂, 0.25 EDTA, 10 taurine, pH 7). The cell suspension was mixed with DMEM medium supplemented with 10% (vol/vol) FBS and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin). Cells were cultured at 37°C in the presence of 5% CO₂ in the same medium. The medium was changed every 3–4 days until the cells reached confluence, and confluent cells were passaged with trypsin/EDTA solution (13, 19, 20, 35).

Immunofluorescent analysis.

Cells cultured on coverslips were fixed and permeabilized using the method previously described (13, 20, 27, 35). These cells were immunofluorescently stained using primary antibody followed by appropriate secondary antibody conjugated to fluorophores (Invitrogen). The cellular localization of fluorescently labeled proteins was viewed under a high-resolution digital fluorescent microscopy (Leica, DMI6000 system). The times of image capturing, intensity gaining, and image contrast levels were optimally adjusted and kept constant for all experiments to standardize the fluorescence intensity measurements among experiments.

Reversible permeabilization.

We used reversible permeabilization (2, 12, 39) to introduce the constructs of wild-type (WT) or mutant 9KQ cortactin (37, 38) into tissues. Briefly, contractile responses of mouse tracheal rings were determined, after which they were placed in 0.5-ml tubes and incubated successively in each of the following solutions: Solution 1 (at 4°C for 120 min) containing 10 mM EGTA, 5 mM Na₂ ATP, 120 mM KCl, 2 mM MgCl₂, and 20 mM N-tris (hydroxymethyl)methyl-2-aminoethane sulfonic acid; Solution 2 (at 4°C overnight) containing 0.1 mM EGTA, 5 mM Na₂ATP, 120 mM KCl, 2 mM MgCl₂, 20 mM TES, and 10 μg/ml plasmids; Solution 3 (at 4°C for 30 min) containing 0.1 mM EGTA, 5 mM Na₂ATP, 120 mM KCl, 10 mM MgCl₂, 20 mM TES; and Solution 4 (at 22°C for 60 min) containing 110 mM NaCl, 3.4 mM KCl, 0.8 mM MgSO₄, 25.8 mM NaHCO₃, 1.2 mM KH₂PO₄, and 5.6 mM dextrose. Solutions 1–3 were maintained at pH 7.1 and aerated with 100% O₂. Solution 4 was maintained at pH 7.4 and was aerated with 95% O₂-5% CO₂. After 30 min in Solution 4, CaCl₂ was added gradually to reach a final concentration of 2.4 mM. The tissues were then incubated in a CO₂ incubator at 37°C for 3 days in DMEM containing 5 mM Na₂ATP, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 μg/ml plasmids.

Analysis of F-actin/G-actin ratios.

The content of F-actin and G-actin in smooth muscle was measured using a method as previously described (2, 36). Briefly, smooth muscle tissues were treated with F-actin stabilization buffer (50 mM PIPES, pH 6.9, 50 mM NaCl, 5 mM MgCl₂, 5 mM EGTA, 5% glycerol, 0.1% Triton X-100, 0.1% Nonidet P40, 0.1% Tween 20, 0.1% β-mercapto-ethanol, 1 mM ATP, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 10 μg/ml benzamidine). The supernatants of protein extracts were collected after centrifugation at 151,000 g for 60 min at 37°C. The pellets were resuspended in ice-cold H₂O plus 1 μM cytochalasin D and then incubated on ice for 1 h to dissociate F-actin. The resuspended pellets were gently mixed every 15 min. The supernatant of the resuspended pellets was collected after centrifugation at 16,100 g for 2 min at 4°C. Equal volume of the first supernatant (G-actin) or second supernatant (F-actin) was subjected to immunoblot analysis using α-smooth muscle actin antibody (Sigma). The amount of F-actin and G-actin was determined by scanning densitometry.

Statistical analysis.

All statistical analysis was performed using Prism 6 software (GraphPad Software). Comparison among multiple groups was performed by one-way analysis of variance followed by Tukey's multiple-comparison test. Differences between pairs of groups were analyzed by Student-Newman-Keuls test or Dunn's method. Values of n refer to the number of experiments used to obtain each value. P < 0.05 was considered to be significant.

RESULTS

HDAC8 regulates smooth muscle contraction.

HDACs have been implicated in gene transcription, cell-cycle control, cell differentiation, and apoptosis (7, 10, 21, 37). It has been shown that HDAC8 is predominantly expressed in smooth muscle cells (31). However, the role of HDAC8 in smooth muscle contraction is not well elucidated. We used lentivirus-mediated RNAi (36) to silence HDAC8 expression and then evaluated the effects of HDAC8 silencing on smooth muscle contraction. Briefly, contraction of mouse tracheal rings was determined, after which they were transduced with lentivirus encoding HDAC8 shRNA or scramble (control) shRNA for 4 days. Force development in tissues was then determined. Immunoblot analysis verified HDAC8 knockdown in the tissues (Fig. 1A). Furthermore, contractile force was lower in tissues transduced with virus for HDAC8 shRNA than in uninfected tissues and tissues infected with virus encoding control shRNA (Fig. 1B). These results suggest that HDAC8 is necessary for smooth muscle contraction. Moreover, knockdown of HDAC8 had minor effects on basal tone.

Fig. 1.

Histone deacetylase 8 (HDAC8) promotes smooth muscle contraction and force maintenance. A: mouse tracheal rings were transduced with lentivirus encoding control (scramble) shRNA or HDAC8 shRNA for 4 days. Immunoblot analysis was used to assess protein expression. Ratios of HDAC8/GAPDH in tissues infected with viruses are normalized to those in uninfected tissues. *P < 0.05 compared with uninfected tissues and tissues infected with virus for control shRNA (n = 7). UI, uninfected; Ctrl, control shRNA; HDAC8, HDAC8 shRNA. B: knockdown of HDAC8 inhibits contractile response in mouse tracheal rings. Contractile responses of tracheal tissues to acetylcholine (ACh) were determined, after which lentivirus-encoding control shRNA or HDAC8 shRNA was introduced into the tissues, and they were incubated for 4 days. Force development in bronchial rings was then determined. (*P < 0.05 compared with uninfected tissues and tissues infected with virus for control shRNA, n = 7). C: mouse tracheal rings were stimulated with 10⁻⁵ M ACh or 5-hydroxytrypdamine (5-HT) for 5 min. Different concentrations of HDAC8 inhibitor XXIV (OSU-HDAC-44) were then applied (5 min for each concentration). Treatment with the inhibitor induces muscle relaxation (*P < 0.05, **P < 0.01 compared with control; n = 4–6). D: HDAC8 inhibitor XXIV (10⁻⁴ M) attenuates human bronchial rings precontracted by ACh (*P < 0.05 compared with untreated tissues). Values represent means ± SE (n = 3).

We also assessed whether HDAC8 is able to affect smooth muscle force maintenance. Mouse tracheal rings were precontracted with 10⁻⁵ M ACh or 5-hydroxytrypdamine (5-HT). ACh and 5-HT have been implicated in the pathogenesis of asthma (4, 17, 20, 33). Different concentrations of HDAC8 inhibitor XXIV (OSU-HDAC-44) were then imposed to precontracted tissues. The addition of HDAC8 inhibitor XXIV induced tracheal ring relaxation, which was dose dependent (Fig. 1C). Moreover, treatment of human bronchial rings with HDAC8 inhibitor XXIV also reduced force induced by the agonist (Fig. 1D).

Cortactin undergoes deacetylation in response to contractile activation.

Cortactin is an actin-binding protein that has been implicated in the regulation of the actin cytoskeleton. In response to stimulation with growth factors, cortactin undergoes deacetylation in nonmuscle cells, which has been implicated in regulating actin dynamics and cell migration (37). Because smooth muscle contraction is also regulated by actin dynamics (2, 9, 15, 23, 26, 36), we evaluated whether contractile activation affects the acetylation level of cortactin. Mouse tracheal rings were treated with ACh or left unstimulated. Cortactin acetylation in these tissues was determined by immunoblot analysis using acetyl lysine antibody. In unstimulated tissues, the level of cortactin acetylation was relatively high. In contrast, the level of cortactin acetylation was reduced in response to ACh stimulation (Fig. 2).

Fig. 2.

Stimulation with ACh induces cortactin deacetylation in smooth muscle. A: representative immunoblots illustrating the effects of contractile stimulation on cortactin deacetylation. Mouse tracheal rings were stimulated with ACh for 5 min. Cortactin acetylation in tissues was determined by immunoblotting using acetyl lysine antibody. Ac-cortactin, acetylated cortactin. B: acetylation level in tissues treated with ACh is normalized to the level in unstimulated tissues. *P < 0.05 compared with unstimulated tissues. All values represent means ± SE (n = 5).

Spatial localization and interaction of HDAC8 and cortactin in smooth muscle cells.

We evaluated the spatial distribution of HDAC8 and cortactin in smooth muscle cells. MASM cells and HASM cells were immunostained for HDAC8 and cortactin. HDAC8 was localized in the nucleus as well as in the cytoplasm of smooth muscle cells. In contrast, cortactin was localized only in the cytoplasm of smooth muscle cells (Fig. 3A). Moreover, stimulation with ACh increased the ratios of HDAC8/cortactin in tissues (Fig. 3B). These results suggest that both HDAC8 and cortactin are localized in the cytoplasm of smooth muscle cells, and contractile stimulation promotes the association of HADC8 with cortactin.

Fig. 3.

Spatial distribution and interaction of HDAC8 and cortactin in smooth muscle cells. A: mouse airway smooth muscle cells (MASMCs) and human airway smooth muscle cells (HASMCs) were immunostained for HDAC8 and cortactin. Cells were also stained with DAPI to visualize the nucleus. HDAC8 is localized both in the cytoplasm and in the nucleus, whereas cortactin is localized only in the nucleus. B: blots of cortactin immunoprecipitates (IP) from mouse tracheal rings treated with or without ACh (10⁻⁴ M, 5 min) were probed with antibodies against cortactin and HDAC8. The ratios of HDAC8/cortactin are significantly higher in stimulated tissues than in unstimulated (US) tissues (*P < 0.05). Data are means ± SE (n = 4).

HDAC8 mediates cortactin deacetylation in smooth muscle.

Because both HDAC8 and cortactin are localized in the cytoplasm of smooth muscle cells, and ACh activation enhances their interaction, this raises a possibility that HDAC8 may affect cortactin acetylation. To test this, we evaluated the effects of knockdown of HDAC8 on cortactin acetylation by immunoblot analysis. In uninfected mouse tracheal rings and tissues expressing control shRNA, ACh stimulation reduced the level of cortactin acetylation. In contrast, the ACh-mediated reduction in cortactin acetylation was inhibited in tissues expressing HDAC8 shRNA (Fig. 4, A and B).

Fig. 4.

HDAC8 mediates cortactin deacetylation in smooth muscle during contraction. A: representative immunoblots illustrating the effects of HDAC8 knockdown on cortactin deacetylation. Uninfected (UI) mouse tracheal rings, rings infected with lentiviruses encoding control (Ctrl) shRNA, or HDAC8 shRNA were stimulated with ACh (10⁻⁴ M, 5 min) or left unstimulated. Cortactin acetylation in these tissues was assessed by immunoblot analysis. B: level of cortactin acetylation in tissues infected with lentiviruses encoding control or HDAC8 shRNA is normalized to that in uninfected tissues. *Significantly lower ACh-stimulated cortactin acetylation compared with corresponding unstimulated tissues (P < 0.05). **Significantly higher ACh-stimulated cortactin acetylation in tissues expressing HDAC8 shRNA compared with uninfected tissues and tissues producing control shRNA (P < 0.05). All values represent means ± SE (n = 4). C: mouse tracheal rings were unstimulated or stimulated with ACh (10⁻⁵ M, 5 min). They were then treated with the HDAC8 inhibitor (10⁻⁴ M, 5 min) to induce relaxation. Cortactin acetylation in tissues was evaluated by immunoblotting (*P < 0.05, n = 5). D: treatment with human bronchial rings with the HDAC8 inhibitor enhances cortactin acetylation induced by ACh (10⁻⁵ M, 5 min) (*P < 0.05, n = 3).

We also assessed the effects of HDAC8 inhibitor XXIV on cortactin acetylation. Mouse tracheal rings or human bronchial rings were treated with ACh in the absence or presence of HDAC8 inhibitor XXIV. Cortactin acetylation in the tissues was evaluated by immunoblot analysis. Treatment with the HDAC8 inhibitor increased cortactin acetylation in mouse or human tissues during ACh stimulation (Fig. 4, C and D).

HDAC8 affects actin polymerization in smooth muscle during contractile activation.

Recent studies suggest that actin polymerization is critical for smooth muscle force development in response to contractile activation (2, 9, 15, 23, 26, 36). Because HDAC8 affects smooth muscle contraction, we evaluated the effects of HDAC8 knockdown on actin polymerization by using the fractionation assay. Knockdown of HDAC8 inhibits actin polymerization in response to contractile activation. The ACh-stimulated increases in F/G α-actin ratios were lower in mouse tissues infected with virus encoding for HDAC8 shRNA than in uninfected tissues and rings infected with virus for control shRNA (Fig. 5A).

Fig. 5.

Knockdown or inhibition of HDAC8 attenuates actin polymerization. A: HDAC8 knockdown by shRNA inhibits ACh-induced actin polymerization in response to contractile activation. Uninfected (UI) mouse tracheal rings, rings infected with lentiviruses encoding control (Ctrl) shRNA, or HDAC8 shRNA were stimulated with ACh or left unstimulated. F/G-actin ratios were evaluated by using the fractionation assay. *Significantly higher F/G-actin ratios in tissues stimulated with ACh compared with corresponding unstimulated tissues (P < 0.05, n = 7). **Significantly lower ACh-induced F/G-actin ratios in HDAC8 shRNA-treated tissues compared with uninfected tissues and tissues expressing control shRNA (P < 0.05, n = 7). B: mouse tracheal rings were unstimulated or stimulated with ACh (10⁻⁵ M, 5 min). They were then treated with HDAC8 inhibitor XXIV (10⁻⁴ M, 5 min). F/G-actin ratios were evaluated using the method described in materials and methods (*P < 0.05, n = 6). C: treatment with human bronchial rings with the HDAC8 inhibitor decreases actin polymerization induced by ACh (*P < 0.05, n = 6). All values represent means ± SE.

We assessed the effects of HDAC8 inhibition on F/G-actin ratios (an indication of actin polymerization) in smooth muscle. Mouse tracheal rings or human bronchial rings were treated with ACh in the absence or presence of HDAC8 inhibitor XXIV. F/G actin ratios were determined by using the fractionation assay. Treatment with HDAC8 inhibitor XXIV attenuated an increase in F/G actin ratios in tissues during ACh stimulation (Fig. 5, B and C).

Myosin light chain phosphorylation is not affected by HDAC8 in smooth muscle.

Because myosin light chain phosphorylation is one of major biochemical changes in smooth muscle in response to contractile activation (14, 24), we also determined the effects of HDAC8 knockdown or inhibition on myosin light chain phosphorylation. Although force development was reduced, knockdown or inhibition of HDAC8 did not affect myosin light chain phosphorylation during ACh stimulation (Fig. 6).

Fig. 6.

Silencing or inhibition of HDAC8 does not affect myosin light chain (MLC) phosphorylation at Ser-19. A: uninfected (UI) mouse tracheal rings, rings infected with lentiviruses encoding control (Ctrl) shRNA, or HDAC8 shRNA were stimulated with ACh or left unstimulated. MLC phosphorylation was evaluated by immunoblot analysis. HDAC8 silencing by shRNA does not affect MLC phosphorylation. All values represent means ± SE (n = 4). B: mouse tracheal rings were stimulated with ACh (10⁻⁵ M, 5 min) followed by the treatment with HDAC8 inhibitor XXIV (10⁻⁴ M, 5 min). MLC phosphorylation at Ser-19 was assessed by immunoblot analysis. NS, not significant (n = 5). C: treatment with human bronchial rings with the HDAC8 inhibitor does not affect MLC phosphorylation in response to ACh stimulation. NS, not significant (n = 3).

Expression of cortactin 9KQ mutant inhibits smooth muscle contraction upon contractile activation.

To determine the role of cortactin deacetylation, we introduced cortactin 9KQ mutant (glutamine substitution at lysine-87, -124, -161, -189, -198, -235, -272, -309, and -319) (37) into tissues by reversible permeabilization (2, 12, 39). Cortactin 9KQ mutant is a charge-neutralizing mutant that acts as a dominant-negative mutant mimicking cortactin acetylation (37, 38). Contractile response of mouse tracheal rings to ACh was evaluated. WT or mutant cortactin was introduced into tissues by reversible permeabilization. These tissues were incubated in the medium for 3 days. Contractile force of these tissues was then evaluated. Immunoblot analysis verified the expression of cortactin in tissues treated with plasmids encoding WT or 9KQ cortactin (Fig. 7A). More importantly, contractile responses were lower in tissues expressing 9KQ mutant of cortactin than in tissues producing WT cortactin (Fig. 7B). Furthermore, expression of the cortactin mutant reduced the ACh-induced F/G-actin ratios (Fig. 7C). However, myosin light chain phosphorylation at Ser-19 was not affected by cortactin 9KQ mutant (Fig. 7D).

Fig. 7.

Expression of mutant 9KQ cortactin attenuates smooth muscle contraction and actin polymerization. A: contractile responses of mouse tracheal rings to ACh (10⁻⁴ M) were determined, after which plasmids encoding wild-type (WT) or mutant 9KQ cortactin were transduced into the tissues by reversible permeabilization, and they were incubated for 3 days. Immunoblot analysis shows higher expression of WT or mutant cortactin in tissues. *Significantly higher protein ratios of cortactin/GAPDH in tissues transfected with plasmids encoding WT or mutant cortactin than in untreated (UT) tissues (P < 0.05, n = 4). B: contraction of mouse tracheal rings to ACh was evaluated, after which they were treated with plasmids as described above. Contractile responses of tissues expressing WT or mutant 9KQ cortactin are normalized to untransfected tissues. *Significantly lower active force in tissues expressing 9KQ mutant compared with untransfected tissues or tissues expressing WT cortactin (P < 0.05, n = 4). C: untransfected tissues or tissues expressing WT or mutant cortactin were stimulated with ACh (10 μM, 5 min), or left unstimulated. F/G-actin ratios in tissues were evaluated using the fractionation assay. *Significantly lower F/G-actin ratios during ACh stimulation in tissues expressing 9KQ mutant compared with untransfected tissues or tissues expressing WT cortactin (P < 0.05). Values represent means ± SE (n = 4). D: MLC phosphorylation in tissues treated with the plasmids was assessed by immunoblot analysis. Myosin phosphorylation was similar in untransfected tissues and in tissues expressing WT or mutant 9KQ cortactin (P > 0.05). Values represent means ± SE (n = 4).

DISCUSSION

HDACs regulate nucleosomal histone deacetylation and nonhistone protein deacetylation, which has a role in gene expression, cell-cycle control, differentiation, and cell migration (7, 10, 21, 37). In this study, HDAC8 was found in the cytoplasm as well as in the nucleus of MASM and HASM cells. The result is supported by a previous study by other investigators (31). These results suggest that HDAC8 in smooth muscle may exert its activity in the cytoplasm in addition to the nucleus. Furthermore, we demonstrate that HDAC8 is necessary for smooth muscle contraction at tissue level. This is because knockdown of HDAC8 inhibited smooth muscle contraction upon agonist stimulation. Moreover, HDAC8 is essential for force maintenance; treatment with the HDAC8 inhibitor induced relaxation of smooth muscle precontracted by the agonists.

Cortactin is an adapter protein that is able to regulate actin dynamics in in vitro studies as well as adhesion, migration, and endocytosis of nonmuscle cells (1, 3). Tyrosine phosphorylation on cortactin has been proposed to modulate its functional state (1, 16). In addition, recent studies show that cortactin undergoes deacetylation during cell migration or activation with growth factors (37). Here, activation with ACh induced cortactin deacetylation in smooth muscle tissues. To the best of our knowledge, this is the first evidence to suggest that contractile stimulation is able to induce changes in cortactin acetylation in smooth muscle tissues.

In this report, both cortactin and HDAC8 were localized in the cytoplasm of smooth muscle cells. Furthermore, HDAC8 mediated cortactin deacetylation in smooth muscle during contractile activation. The results suggest that HDAC8 is critical for the regulation of cortactin deacetylation in smooth muscle. In ovarian cancer cells, cortactin deacetylation is mediated by SIRT1 (a class III histone deacetylase), which has been implicated in cancer cell migration (38). On the other hand, HDAC6 is able to regulate cortactin deacetylation in Hela cells (37). It is likely that SIRT1, HDAC6, or other HDACs may be also expressed in smooth muscle, which may regulate cortactin deacetylation in smooth muscle. Future studies are required to investigate these possibilities.

Cortactin deacetylation has been implicated in promoting actin polymerization (37, 38). Because HDAC8 mediates cortactin deacetylation in smooth muscle, we evaluated the effects of HDAC8 knockdown or inhibition on actin polymerization. Knockdown or inhibition of HDAC8 attenuated the agonist-induced actin polymerization. These findings indicate that HDAC8 may mediate actin filament polymerization during contractile activation, which may facilitate an increase in the numbers of contractile units and the length of actin filaments and provide more efficient contractile elements for force development (11, 25, 26, 36). In addition, actin polymerization may enhance the linkage of actin filaments to integrins strengthening the transduction of mechanical force between contractile units and extracellular matrix (6, 8, 9, 18, 26, 28, 29).

In this report, knockdown or inhibition of HDAC8 did not affect myosin light chain phosphorylation although contractile force was reduced under the condition. Thus HDAC8 is not involved in the regulation of myosin activation. These findings are also consistent with previous studies to suggest that actin polymerization is necessary for smooth muscle contraction (2, 9, 15, 23, 26, 36).

In addition to activating N-WASP, cortactin promotes actin dynamics by binding to F-actin (1, 37, 38). As described above, in vitro biochemical studies show that cortactin gets acetylated on nine lysines within the repeat region (37, 38). Cortactin acetylation on these nine lysines inhibits its association with F-actin, which in turn attenuates actin polymerization (1, 3, 37, 38). In NIH 3T3 cells, expression of the charge-neutralizing 9KQ cortactin mutant (a dominant-negative mutant mimicking acetylated cortactin) inhibits its localization in membrane ruffles and attenuates cell migration (37). Here, we found that the expression of 9KQ cortactin mutant inhibited smooth muscle contraction and actin polymerization. The results suggest that cortactin deacetylation is critical for actin polymerization and smooth muscle contraction. It is likely that the acetylated nine lysines alter a “charge patch” within the repeat domain of cortactin, which subsequently decreases the association of cortactin with F-actin and inhibits actin polymerization (37, 38) and force development in smooth muscle during contractile activation.

Here, we discover a novel mechanism for the regulation of smooth muscle contraction. In addition to myosin light chain phosphorylation, contractile stimulation activates HDAC8, which mediates cortactin deacetylation. Deacetylated cortactin promotes actin polymerization and smooth muscle contraction (Fig. 8).

Fig. 8.

Proposed mechanism. Besides MLC phosphorylation, contractile stimulation activates HDAC8, which mediates cortactin (CTTN) deacetylation. Deacetylated cortactin promotes actin polymerization. Both MLC phosphorylation and actin polymerization facilitate smooth muscle contraction.

GRANTS

This work was supported by NHLBI Grants HL-110951 (to D. Tang) and HL-113208 (to D. Tang) from the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: J.L. and D.D.T. conception and design of research; J.L., S.C., R.A.C., R.W., O.J.G., and E.S. performed experiments; J.L., S.C., R.A.C., R.W., O.J.G., E.S., and D.D.T. analyzed data; J.L., E.S., and D.D.T. interpreted results of experiments; J.L., S.C., and D.D.T. prepared figures; J.L. drafted manuscript; J.L., S.C., R.A.C., R.W., O.J.G., E.S., and D.D.T. approved final version of manuscript; D.D.T. edited and revised manuscript.