HSP20 phosphorylation and airway smooth muscle relaxation

Mariam Ba¹, Cherie A Singer¹, Manoj Tyagi², Colleen Brophy³, Josh E Baker⁴, Christine Cremo⁴, Andrew Halayko⁵, William T Gerthoffer²

¹Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA; ²Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA; ³Harrington Department of Biochemistry, Arizona State University, Tempe, AZ, USA; ⁴Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA; ⁵Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, Canada

Abstract: HSP20 (HSPB6) is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM) we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ) inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1), and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200–500 kDa) to smaller complexes (<60 kDa). The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway smooth muscle. HSP20 is upregulated by beta agonists and downregulated by proinflammatory cytokines. HSP20 is phosphorylated in vivo in a cAMP-dependent manner and the phosphorylated form promotes airway smooth muscle relaxation, possibly through depolymerization of F-actin as well as inhibition of myosin binding to actin.

Keywords: actin, bronchodilator, forskolin, myosin, salmeterol, trachea