Yidi Wu

Actin Depolymerization Factor/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that ∼40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.

Integrin-linked Kinase Regulates N-WASp-mediated Actin Polymerization and Tension Development in Tracheal Smooth Muscle

The contractile stimulation of smooth muscle tissues stimulates the recruitment of proteins to membrane adhesion complexes and the initiation of actin polymerization. We hypothesized that integrin-linked kinase (ILK), a β-integrin-binding scaffolding protein and serine/threonine kinase, and its binding proteins, PINCH, and α-parvin may be recruited to membrane adhesion sites during contractile stimulation of tracheal smooth muscle to mediate cytoskeletal processes required for tension development. Immunoprecipitation analysis indicted that ILK, PINCH, and α-parvin form a stable cytosolic complex and that the ILK·PINCH·α-parvin complex is recruited to integrin adhesion complexes in response to acetylcholine (ACh) stimulation where it associates with paxillin and vinculin. Green fluorescent protein (GFP)-ILK and GFP-PINCH were expressed in tracheal muscle tissues and both endogenous and recombinant ILK and PINCH were recruited to the membrane in response to ACh stimulation. The N-terminal LIM1 domain of PINCH binds to ILK and is required for the targeting of the ILK-PINCH complex to focal adhesion sites in fibroblasts during cell adhesion. We expressed the GFP-PINCH LIM1-2 fragment, consisting only of LIM1-2 domains, in tracheal smooth muscle tissues to competitively inhibit the interaction of ILK with PINCH. The PINCH LIM1-2 fragment inhibited the recruitment of endogenous ILK and PINCH to integrin adhesion sites and prevented their association of ILK with β-integrins, paxillin, and vinculin. The PINCH LIM1-2 fragment also inhibited tension development, actin polymerization, and activation of the actin nucleation initiator, N-WASp. We conclude that the recruitment of the ILK·PINCH·α-parvin complex to membrane adhesion complexes is required to initiate cytoskeletal processes required for tension development in smooth muscle.

Integrin-linked kinase regulates smooth muscle differentiation marker gene expression in airway tissue.

Phenotypic changes in airway smooth muscle occur with airway inflammation and asthma. These changes may be induced by alterations in the extracellular matrix that initiate signaling pathways mediated by integrin receptors. We hypothesized that integrin-linked kinase (ILK), a multidomain protein kinase that binds to the cytoplasmic tail of beta-integrins, may be an important mediator of signaling pathways that regulate the growth and differentiation state of airway smooth muscle. We disrupted signaling pathways mediated by ILK in intact differentiated tracheal muscle tissues by depleting ILK protein using ILK antisense. The depletion of ILK protein increased the expression of the smooth muscle differentiation marker genes myosin heavy chain (SmMHC), SM22alpha, and calponin and increased the expression of SmMHC protein. Conversely, the overexpression of ILK protein reduced the mRNA levels of SmMHC, SM22alpha, and calponin and SmMHC protein. Analysis by chromatin immunoprecipitation showed that the binding of the transcriptional regulator serum response factor (SRF) to the promoters of SmMHC, SM22alpha, and calponin genes was increased in ILK-depleted tissues and decreased in tissues overexpressing ILK. ILK depletion also increased the amount of SRF that localized within the nucleus. ILK depletion and overexpression, respectively, decreased and increased the activation of its downstream substrate protein kinase B (PKB/Akt). The pharmacological inhibition of Akt activity also increased SRF binding to the promoters of smooth muscle-specific genes and increased expression of smooth muscle proteins, suggesting that ILK may exert its effects by regulating the activity of Akt. We conclude that ILK is a critical regulator of airway smooth muscle differentiation. ILK may mediate signals from integrin receptors that control airway smooth muscle differentiation in response to alterations in the extracellular matrix.

A novel role for RhoA GTPase in the regulation of airway smooth muscle contraction.

Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin light chain (MLC) phosphorylation and actin polymerization are required for active tension generation. RhoA inactivation dramatically suppresses agonist-induced tension development and completely inhibits agonist-induced actin polymerization, but only slightly reduces MLC phosphorylation. The inhibition of MLC phosphatase does not reverse the effects of RhoA inactivation on contraction or actin polymerization. Thus, RhoA regulates ASM contraction through its effects on actin polymerization rather than MLC phosphorylation. Contractile stimulation of ASM induces the recruitment and assembly of paxillin, vinculin, and focal adhesion kinase (FAK) into membrane adhesion complexes (adhesomes) that regulate actin polymerization by catalyzing the activation of cdc42 GTPase by the G-protein-coupled receptor kinase-interacting target (GIT) - p21-activated kinase (PAK) - PAK-interacting exchange factor (PIX) complex. Cdc42 is a necessary and specific activator of the actin filament nucleation activator, N-WASp. The recruitment and activation of paxillin, vinculin, and FAK is prevented by RhoA inactivation, thus preventing cdc42 and N-WASp activation. We conclude that RhoA regulates ASM contraction by catalyzing the assembly and activation of membrane adhesome signaling modules that regulate actin polymerization, and that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to a contractile agonist.

Mechanical stimuli and IL-13 interact at integrin adhesion complexes to regulate expression of smooth muscle myosin heavy chain in airway smooth muscle tissue.

Airway smooth muscle phenotype may be modulated in response to external stimuli under physiological and pathophysiological conditions. The effect of mechanical forces on airway smooth muscle phenotype were evaluated in vitro by suspending weights of 0.5 or 1 g from the ends of canine tracheal smooth muscle tissues, incubating the weighted tissues for 6 h, and then measuring the expression of the phenotypic marker protein, smooth muscle myosin heavy chain (SmMHC). Incubation of the tissues at a high load significantly increased expression of SmMHC compared with incubation at low load. Incubation of the tissues at a high load also decreased activation of PKB/Akt, as indicated by its phosphorylation at Ser 473. Inhibition of Akt or phosphatidylinositol-3,4,5 triphosphate-kinase increased SmMHC expression in tissues at low load but did not affect SmMHC expression at high load. IL-13 induced a significant increase in Akt activation and suppressed the expression of SmMHC protein at both low and high loads. The role of integrin signaling in mechanotransduction was evaluated by expressing a PINCH (LIM1-2) fragment in the muscle tissues that prevents the membrane localization of the integrin-binding IPP complex (ILK/PINCH/α-parvin), and also by expressing an inactive integrin-linked kinase mutant (ILK S343A) that inhibits endogenous ILK activity. Both mutants inhibited Akt activation and increased expression of SmMHC protein at low load but had no effect at high load. These results suggest that mechanical stress and IL-13 both act through an integrin-mediated signaling pathway to oppositely regulate the expression of phenotypic marker proteins in intact airway smooth muscle tissues. The stimulatory effects of mechanical stress on contractile protein expression oppose the suppression of contractile protein expression mediated by IL-13; thus the imposition of mechanical strain may inhibit changes in airway smooth muscle phenotype induced by inflammatory mediators.

Vasodilator-stimulated phosphoprotein (VASP) regulates actin polymerization and contraction in airway smooth muscle by a vinculin-dependent mechanism.

Background: The function of vasodilator-stimulated phosphoprotein (VASP) in regulating actin polymerization during airway smooth muscle contraction is unknown. Results: VASP activity requires phosphorylation at Ser157, recruitment to the membrane, and interaction with activated vinculin. Conclusion: VASP regulates actin polymerization and contraction in smooth muscle by a unique mechanism. Significance: The mechanism of VASP function is important for understanding actin dynamics in cells. Vasodilator-stimulated phosphoprotein (VASP) can catalyze actin polymerization by elongating actin filaments. The elongation mechanism involves VASP oligomerization and its binding to profilin, a G-actin chaperone. Actin polymerization is required for tension generation during the contraction of airway smooth muscle (ASM); however, the role of VASP in regulating actin dynamics in ASM is not known. We stimulated ASM cells and tissues with the contractile agonist acetylcholine (ACh) or the adenylyl cyclase activator, forskolin (FSK), a dilatory agent. ACh and FSK stimulated VASP Ser157 phosphorylation by different kinases. Inhibition of VASP Ser157 phosphorylation by expression of the mutant VASP S157A in ASM tissues suppressed VASP phosphorylation and membrane localization in response to ACh, and also inhibited contraction and actin polymerization. ACh but not FSK triggered the formation of VASP-VASP complexes as well as VASP-vinculin and VASP-profilin complexes at membrane sites. VASP-VASP complex formation and the interaction of VASP with vinculin and profilin were inhibited by expression of the inactive vinculin mutant, vinculin Y1065F, but VASP phosphorylation and membrane localization were unaffected. We conclude that VASP phosphorylation at Ser157 mediates its localization at the membrane, but that VASP Ser157 phosphorylation and membrane localization are not sufficient to activate its actin catalytic activity. The interaction of VASP with activated vinculin at membrane adhesion sites is a necessary prerequisite for VASP-mediated molecular processes necessary for actin polymerization. Our results show that VASP is a critical regulator of actin dynamics and tension generation during the contractile activation of ASM.

Focal adhesion kinase (FAK) and mechanical stimulation negatively regulate the transition of airway smooth muscle tissues to a synthetic phenotype

The effects of mechanical forces and focal adhesion kinase (FAK) in regulating the inflammatory responses of airway smooth muscle (ASM) tissues to stimulation with interleukin (IL)-13 were investigated. Canine tracheal tissues were subjected to different mechanical loads in vitro, and the effects of mechanical load on eotaxin secretion and inflammatory signaling pathways in response to IL-13 were determined. Eotaxin secretion by tissues in response to IL-13 was significantly inhibited in muscles maintained at a higher (+) load compared with those at a lower (-) load as assessed by ELISA, and Akt activation was also reduced in the higher (+) loaded tissues. Conversely the (+) mechanical load increased activation of the focal adhesion proteins FAK and paxillin in the tissues. The role of FAK in regulating the mechanosensitive responses was assessed by overexpressing FAK-related nonkinase in the tissues, by expressing the FAK kinase-dead mutant FAK Y397F, or by treating tissues with the FAK inhibitor PF-573228. FAK inactivation potentiated Akt activity and increased eotaxin secretion in response to IL-13. FAK inhibition also suppressed the mechanosensitivity of Akt activation and eotaxin secretion. In addition, FAK inactivation suppressed smooth muscle myosin heavy chain expression induced by the higher (+) mechanical load. The results demonstrate that the imposition of a higher mechanical load on airway smooth muscle stimulates FAK activation, which promotes the expression of the differentiated contractile phenotype and suppresses the synthetic phenotype and the inflammatory responses of the muscle tissue.

Elastase Alters Contractility and Promotes an Inflammatory Synthetic Phenotype in Airway Smooth Muscle Tissues

Neutrophil elastase is secreted by inflammatory cells during airway inflammation and can elicit airway hyperreactivity in vivo. Elastase can degrade multiple components of the extracellular matrix. We hypothesized that elastase might disrupt the connections between airway smooth muscle (ASM) cells and the extracellular matrix and that this might have direct effects on ASM tissue responsiveness and inflammation. The effect of elastase treatment on ASM contractility was assessed in vitro in isolated strips of canine tracheal smooth muscle by stimulation of tissues with cumulatively increasing concentrations of acetylcholine (ACh) and measurement of contractile force. Elastase treatment potentiated contractile responses to ACh at low concentrations but suppressed the maximal contractile force generated by the tissues without affecting the phosphorylation of myosin regulatory light chain (RLC). Elastase also promoted the secretion of eotaxin and the activation of Akt in ASM tissues and decreased expression of smooth muscle myosin heavy chain, consistent with promotion of a synthetic inflammatory phenotype. As the degradation of matrix proteins can alter integrin engagement, we evaluated the effect of elastase on the assembly and activation of integrin-associated adhesion junction complexes in ASM tissues. Elastase led to talin cleavage, reduced talin binding to vinculin, and suppressed activation of the adhesome proteins paxillin, focal adhesion kinase, and vinculin, indicating that elastase causes the disassembly of adhesion junction complexes and the inactivation of adhesome signaling proteins. We conclude that elastase promotes an inflammatory phenotype and increased sensitivity to ACh in ASM tissues by disrupting signaling pathways mediated by integrin-associated adhesion complexes.