Tracheal Muscle Tissues Research Articles

S100A4 is secreted by airway smooth muscle tissues and activates inflammatory signaling pathways via receptors for advanced glycation end products.

S100A4 is a low-molecular-mass (12 kDa) EF-hand Ca2+-binding S100 protein that is expressed in a broad range of normal tissue and cell types. S100A4 can be secreted from some cells to act in an autocrine or paracrine fashion on target cells and tissues. S100A4 has been reported in the extracellular fluids of subjects with several inflammatory diseases, including asthma. Airway smooth muscle plays a critical role in airway inflammation by synthesizing and secreting inflammatory cytokines. We hypothesized that S100A4 may play an immunomodulatory role in airway smooth muscle. Trachealis smooth muscle tissues were stimulated with recombinant His-S100A4, and the effects on inflammatory responses were evaluated. S100A4 induced the activation of Akt and NF-κB and stimulated eotaxin secretion. It also increased the expression of RAGE and endogenous S100A4 in airway tissues. Stimulation of airway smooth muscle tissues with IL-13 or TNF-α induced the secretion of S100A4 from the tissues and promoted the expression of endogenous receptors for advanced glycation end products (RAGE) and S100A4. The role of RAGE in mediating the responses to S100A4A was evaluated by expressing a mutant nonfunctional RAGE (RAGEΔcyto) in tracheal muscle tissues and by treating tissues with a RAGE inhibitor. S100A4 did not activate NF-κB or Akt in tissues that were expressing RAGEΔcyto or treated with a RAGE inhibitor, indicating that S100A4 mediates its effects by acting on RAGE. Our results demonstrate that inflammatory mediators stimulate the synthesis and secretion of S100A4 in airway smooth muscle tissues and that extracellular S100A4 acts via RAGE to mediate airway smooth muscle inflammation.

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 beta-integrin-binding scaffolding protein and serine/threonine kinase, and its binding proteins, PINCH, and alpha-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 alpha-parvin form a stable cytosolic complex and that the ILK.PINCH.alpha-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 beta-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.alpha-parvin complex to membrane adhesion complexes is required to initiate cytoskeletal processes required for tension development in smooth muscle.

Integrin Linked Kinase (ILK) modulates SmMHC expression in tracheal smooth muscle tissues by regulating the activity of Serum Response Factor (SRF)

Integrin-linked kinase (ILK) is a multi-domain protein kinase that binds to the cytoplasmic tail of beta integrins and signals to pathways that regulate cell growth and differentiation. We modulated ILK expression in tracheal muscle tissues and evaluated the effect on smooth muscle myosin heavy chain (SmMHC) expression. ILK antisense was used to depress ILK protein expression, and FLAG-ILK was expressed in smooth muscle tissues to increase ILK protein expression. ILK antisense treatment for 24 hrs caused the depletion of ILK protein by 30–40% and stimulated a two-fold increase in the expression of SmMHC protein. Real time RT-PCR of tissue extracts demonstrated a significant increase in SmMHC mRNA. Transfection with ILK-FLAG plasmids induced a 40–50% increase in ILK protein expression, inhibited SmMHC expression by 30–40% and decreased SmMHC mRNA. Immunofluorescence analysis and cell fractionation were used to evaluate the effect of modifications in ILK expression in the tissues on the cellular localization of the transcriptional regulator, SRF. More SRF fluorescence was observed in the nucleus of cells dissociated from ILK-depleted tissues relative to untreated tissues and more SRF was detected in nuclear extracts from ILK-depleted tissues. A ChIP assay confirmed increased binding of SRF to the SmMHC promoter in extracts from ILK-depleted tissues relative to undepleted tissues. Our results suggest that ILK is a negative modulator of SmMHC expression in airway smooth muscle, and that ILK decreases the amount of SRF that localizes to the nucleus and reduces SRF binding to the SmMHC promoter. Supported by HL 29289, HL074099, and AHA