Small heat shock proteins in smooth muscle

Abstract

The small heat shock proteins (HSPs) HSP20, HSP27 and alphaB-crystallin are chaperone proteins that are abundantly expressed in smooth muscles are important modulators of muscle contraction, cell migration and cell survival. This review focuses on factors regulating expression of small HSPs in smooth muscle, signaling pathways that regulate macromolecular structure and the biochemical and cellular functions of small HSPs. Cellular processes regulated by small HSPs include chaperoning denatured proteins, maintaining cellular redox state and modifying filamentous actin polymerization. These processes influence smooth muscle proliferation, cell migration, cell survival, muscle contraction and synthesis of signaling proteins. Understanding functions of small heat shock proteins is relevant to mechanisms of disease in which dysfunctional smooth muscle causes symptoms, or is a target of drug therapy. One example is that secreted HSP27 may be a useful marker of inflammation during atherogenesis. Another is that phosphorylated HSP20 which relaxes smooth muscle may prove to be highly relevant to treatment of hypertension, vasospasm, asthma, premature labor and overactive bladder. Because small HSPs also modulate smooth muscle proliferation and cell migration they may prove to be targets for developing effective, novel treatments of clinical problems arising from remodeling of smooth muscle in vascular, respiratory and urogenital systems.

Figure 1. Cellular processes modified by HSP20 and HSP27 in smooth muscles

Multiple extracellular signals impinge on smooth muscle cells to modify the macromolecular structure of small HSPs. Key protein kinases are shown that catalyze phosphorylation of these proteins which changes their molecular associations thus modifying a variety of downstream processes. HSP27 phosphorylation is catalyzed by MAPKAP kinase 2 (MK2) and HSP20 phosphorylation is catalyzed by cyclic nucleotide-dependent protein kinases (PKA and PKG). HSP27 has been shown to promote muscle contraction, increase cell migration and enhance cell survival under stress conditions. Activation of HSP20 causes smooth muscle relaxation and is cardioprotective.

Figure 2. Domain structure of human HSP27 and HSP20

A. The N terminus of HSP27 contains a WDPF motif (grey box) necessary for multimer formation and may influence actin binding. Multimers are reduced to smaller complexes and ultimately to dimers by phosphorylation of consensus MK2 phosphorylation sites at Serines 15, 78 and 82. HSP22 has been shown in cardiac muscle to bind phosphorylated HSP27. The C terminus includes an α-crystallin motif (black box) that is highly conserved among species. The crystallin domain is necessary for dimer formation. The extreme C-terminus is a flexible region of the sequence which varies with species. B. HSP20 contains an α-crystallin motif (black box) highly homologous to that in HSP27. Serine 16 is an important site for phosphorylation by cyclic nucleotide-dependent protein kinases (PKA and PKG). A motif similar to the minimal inhibitory region of cardiac troponin I is present at residues Gly¹¹¹-Leu¹²³. In rat HSP20 there is also a PKC phosphorylation site at Ser¹⁵⁷ that is not conserved in human HSP20.

Figure 3. Signaling pathways regulating HSP27 structure and function

HSP27 is phosphorylated in response to diverse stimuli in smooth muscles including neurotransmitters, cytokines and growth factors. These agents act via GPCR and tyrosine kinase receptors to activate the p38 MAP kinase cascade culminating in activation of MK2. MK2 and homologs MK3 and MK5 phosphorylate HSP27 thus promoting dissociation from large macromolecular assemblies. Only a 20 mer and dimer is shown in figure, but there is a range of complexes with Mr from 50 to >500 kDa. The complexes are not necessarily homomultimers, but can include several other small HSPs such as HSP20 and αB-crystallin. Phosphorylation of HSP27 favors stabilization of F-actin filaments by undefined molecular mechanisms. One notion is HSP27 acts as a capping protein and phosphorylation dissociates the cap thus allowing polymerization to proceed. HSP27 may also sequester G actin. F-actin formation is necessary to support contraction, cell migration and a variety of biochemical processes that control cell redox state, focal contact formation and cell survival.

Figure 4. Hypothesized mechanisms of smooth muscle relaxation by HSP20

Agents that inhibit contraction by cyclic nucleotides may do so in part by activating adenylate cyclase (AC) which in turn activates protein kinases A and G (PKA, PKG). HSP20 is phosphorylated on Ser16 by these kinases, and phosphorylated HSP20 is sufficient to relax smooth muscle, in some cases independent of changes in myosin light chain phosphorylation. Two proposed mechanisms for relaxation are: 1. Depolymerization of actin via an indirect activation of cofilin involving activation of slingshot phosphatase. Phosphorylated HSP20 is thought to compete with slingshot for binding to 14-3-3 proteins. Unbound slingshot keeps cofilin active and favors actin depolymerization. 2. HSP20 has also been proposed to interact with and inhibit actomyosin by virtue of a troponin I motif.