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Cyclic Strain–Induced HSP27 Phosphorylation Modulates Actin Filaments in Airway Smooth Muscle Cells

¹ Division of Pharmacology and Critical Care, Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio

Correspondence and requests for reprints should be addressed to Subhendu Chaudhuri, M.S., Ph.D., Division of Pharmacology and Critical Care, Department of Pediatrics, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106. E-mail: sxc30{at}case.edu

Mechanical stress (cyclic deformational strain) increases proteins of cytoskeletal and contractile domains in airway smooth muscle (ASM) cells in a manner that increases cell contractility. Here we studied the role of HSP27 in strain-induced microfilament formation and stability. Cultured ASM cells showed rapid phosphorylation of HSP27 upon cyclic strain within a few minutes that continued for 30 to 40 minutes. Such increases in HSP27 phosphorylation were abolished with SB 202190, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by PD 98059 (an inhibitor of extracellular regulated kinase), GF109203X (an inhibitor of protein kinase C), or Y27632 (an inhibitor of Rho kinase). Direct activation of RhoA by GTPS did not alter the level of HSP27 phosphorylation. Confocal microscopy revealed that cells pre-incubated with SB 202190, and/or Y27632 resulted in disorganization of stress fibers upon strain, unlike PD 98059 and GF 1092030X, suggesting that both p38 MAPK and Rho kinase were necessary for strain-induced microfilament formation. To determine the relationship between HSP27 and RhoA in strain-induced microfilament formation, cells were transfected with various isoforms of HSP27 and RhoA before strain. Co-expression of inactive HSP27 (3A-HSP27) with constitutively active EGF-RhoA (RhoV14) caused diminution of microfilaments compared with constitutive active EGFP-RhoA (RhoV14) alone, suggesting that HSP27 is necessary for microfilament stability. Similarly, expression of phosphomimicking HSP27 (3D-HSP27) was sufficient for retaining microfilament formation even when co-expressed with the dominant-negative RhoA (EGFP-RhoN17). Thus, HSP27 activation is necessary for microfilament stability independently of RhoA activation.

Key Words: HSP27 • p38 MAP kinase • Rho • mechanical strain • smooth muscle cells

CLINICAL RELEVANCE Mechanical stress reorganizes cytoskeletal apparatus in airway smooth muscle. Understanding signaling mechanisms that control these processes might lead to therapeutic means to attenuate smooth muscle hyperresponsiveness.