INTRODUCTION

TOP ABSTRACT INTRODUCTION SIGNAL TRANSDUCTION BY INTEGRIN... ROLE OF FAK AND... SIGNALING PATHWAYS TO... SMALL HEAT SHOCK PROTEINS... SUMMARY AND CONCLUSIONS REFERENCES

RECENT STUDIES SUGGEST THAT smooth muscle tissues possess remarkable capabilities for the rapid adapta   tion of their structural and functional properties to changes in their external physical environment. Smooth muscle cells may acutely adjust their functional properties by reorganizing their cytoskeleton and by altering signaling pathways that regulate contractile protein activation and function. The signaling pathways that mediate these adaptive responses of the cytoskeleton appear to be closely integrated with those that regulate gene expression, protein synthesis, and cell phenotype, suggesting that the immediate adaptive responses of smooth muscle cells may precede or initiate longer term modifications in cell phenotype and tissue structure.

Transmembrane integrins, membrane-spanning proteins that ligate extracellular matrix proteins on the outside of the cells and link to the actin cytoskeleton on the inside, are uniquely situated to mediate the transduction of environmental signals to pathways that regulate the adaptive responses of cells. In nonmuscle cells, integrin-mediated signal transduction pathways regulate processes of actin dynamics and reorganization that enable cells to adjust to external stimuli by changes in their shape, stiffness, contractility, and motility. Recent evidence suggests that analogous signal transduction pathways to the actin cytoskeleton are present in differentiated smooth muscle tissues and that these pathways may be important in adapting the structural organization, phenotype, and contractile properties of the tissues to external forces, ultimately enabling the smooth muscle tissues of hollow organs to respond to a changing environment. The focus of this review will be to describe recent advances in our understanding of the functions of focal adhesion signaling proteins and downstream regulators of actin remodeling in differentiated smooth muscle cells and of the possible role of these proteins as mediators of the acute mechanical adaptation of differentiated smooth muscle tissues to environmental stimuli.

	SIGNAL TRANSDUCTION BY INTEGRIN PROTEINS IN DIFFERENTIATED SMOOTH MUSCLE

TOP ABSTRACT INTRODUCTION SIGNAL TRANSDUCTION BY INTEGRIN... ROLE OF FAK AND... SIGNALING PATHWAYS TO... SMALL HEAT SHOCK PROTEINS... SUMMARY AND CONCLUSIONS REFERENCES

Integrins comprise a family of cell surface receptors that mediate the transmission of tension between the cytoskeletal apparatus and the extracellular matrix. Integrin proteins can transduce both chemical and mechanical signals into intracellular signaling pathways (outside-in signaling). Conversely, cytoskeletal events can regulate integrin activation and their binding to extracellular matrix proteins (inside-out signaling). The mechanical and chemical signals transduced by integrin-mediated signaling pathways are known to modulate many cellular functions, including growth and proliferation, cytoskeletal organization and motility, ion channel function, gene and protein expression, and extracellular matrix production and autocrine secretion.

In smooth muscle cells, integrins localize to membrane-associated dense plaques, which are structurally similar to the focal adhesion sites of cultured cells (9, 10). These membrane sites have long been identified as the sites of tension transmission between the contractile apparatus and the extracellular matrix (19). At these membrane plaque sites, actin filaments anchor to transmembrane integrins via a series of linker proteins that include talin, vinculin, and -actinin (9, 10, 20, 23, 90). Many kinases and signaling proteins also associate with these sites. The functional role of many of the signaling proteins that have been previously identified as components of focal adhesion sites are now being elucidated in differentiated smooth muscle cells and tissues.

There is substantial evidence that integrin proteins themselves act as mechanotransducers, sensing mechanical perturbations and transducing these into cytosolic signaling cascades that lead to the adaptative responses of cells (45, 89). Direct evidence for the involvement of integrin proteins in mediating mechanically induced signaling to the cytoskeleton has been obtained in many cell types. In cultured nonmuscle cells, the application of torsional stress to ferromagnetic beads bound to integrins using integrin-specific ligands elicits stiffening of the actin cytoskeleton (57, 103). The imposition of strain on integrin-bound collagen beads stimulates the tyrosine phosphorylation of cytosolic signaling proteins known to associate closely with integrins, such as paxillin, and stimulates increases in intracellular Ca²⁺ in cultured fibroblasts (26). Conversely, the application of contractile agonists to cultured airway smooth muscle cells results in the increased torsional resistance of integrin-bound ferromagnetic beads, indicating that changes in cytoskeletal tension are transmitted across the membrane via integrin proteins (39).

There is evidence that interactions between extracellular matrix proteins and integrin subunits initiate signaling events that acutely alter the contractility of smooth muscle tissues and that at least some of these effects result from the activation of signal transduction pathways that regulate membrane ion channel conductance. Synthetic Arg-Gly-Asp (RGD)-containing peptides are ligands that bind to many integrin subtypes (82). The binding of RGD peptides to _v3-integrins, vitronectin receptors expressed by vascular smooth muscle, causes the dilation of rat skeletal muscle arterioles and a reduction in intracellular Ca²⁺ (16, 68). In the same tissues, ligands of _v3-integrin inhibit whole cell L-type Ca²⁺ current and increase K⁺ channel activity, which can account for the vasodilation and concurrent decline in intracellular Ca²⁺ elicited by these ligands (74, 108). In contrast, ligands of the fibronectin receptor, ₅₁, enhance L-type Ca²⁺ current in these tissues (108). Integrins may also be involved in regulating pathways that mediate Ca²⁺ release from intracellular store sites in smooth muscle. In renal vascular smooth muscle cells, RGD peptides induce the release of intracellular Ca²⁺ from ryanodine-sensitive Ca²⁺ stores (12).

Current evidence suggests that many signal transduction events mediated by integrins involve activation of two nonreceptor cytoplasmic tyrosine kinases: the 120-kDa focal adhesion kinase (FAK) and the 60-kDa c-Src (27, 33). Although other nonreceptor tyrosine kinases such as Pyk2 have been identified as participants in integrin-mediated signaling, FAK and c-Src are currently the most well-characterized of these kinases and are widely expressed in many cell types. FAK is activated by autophosphorylation on Tyr³⁹⁷ in response to ligand engagement with integrins (85, 86). Both FAK and c-Src undergo phosphorylation in response to adhesion-induced integrin activation, stimulation by growth factors, and the activation of heterotrimeric G-protein-coupled receptors (9, 42, 80, 109). The molecular mechanisms involved in the coupling of the heterotrimeric G-protein-coupled receptors to the activation of tyrosine kinases are not understood; however, a synergistic relationship between receptor occupancy and integrin aggregation has been proposed (67). A large body of evidence has been developed that implicates both FAK and c-Src in signaling pathways that regulate cytoskeletal organization and cell motility (6, 18, 24, 25, 27, 43, 47, 69, 77).

	ROLE OF FAK AND C-SRC IN SIGNALING PATHWAYS REGULATING CYTOSKELETAL PROCESSES IN SMOOTH MUSCLE

TOP ABSTRACT INTRODUCTION SIGNAL TRANSDUCTION BY INTEGRIN... ROLE OF FAK AND... SIGNALING PATHWAYS TO... SMALL HEAT SHOCK PROTEINS... SUMMARY AND CONCLUSIONS REFERENCES

The involvement of both FAK and c-Src in integrin-activated signal transduction pathways that regulate Ca²⁺ channels has been well documented in differentiated smooth muscle. ₅-Integrin antibody or fibronectin elicits an increase in Ca²⁺ current in arteriolar smooth muscle cells that is blocked by the inactivation of c-Src; also, the inhibition of tyrosine phosphatase enhances the effects of integrin activation on intracellular Ca²⁺ (108). Integrin-enhanced Ca²⁺ current is also suppressed in arteriolar smooth muscle cells when they are dialyzed with monoclonal antibody to FAK (108). The injection of pp60 c-Src into smooth muscle cells from rabbit ear artery increases voltage-dependent Ca²⁺ channel currents by a protein kinase C-dependent mechanism, and a peptide that activates Src family kinases increases Ca²⁺ channel currents (106). In differentiated colonic smooth muscle, activated c-Src has been shown to interact with FAK to mediate the regulation of basal Ca²⁺ channel activity and the platelet-derived growth factor (PDGF)-induced enhancement of L-type Ca²⁺ currents (38). In these cells, the ₁-subunit of the voltage-operated Ca²⁺ channel coimmunoprecipitates with c-Src kinase and FAK, and it undergoes tyrosine phosphorylation following the application of PDGF.

The stimulation of smooth muscle cells with contractile agonists stimulates the tyrosine phosphorylation and activation of both FAK and c-Src. In vascular smooth muscle cells, angiotensin stimulates an increase in c-Src kinase activity and an increase in its phosphorylation on Tyr⁴¹⁹, the autophosphorylation site required for its activation (4, 46, 47, 58). In these cells, c-Src activation mediated by angiotensin stimulation leads to the phosphorylation of phospholipase C-, which has been linked to downstream activation of Rho GTPase. c-Src activation also mediates the formation of inositol trisphosphate, which stimulates the release of intracellular Ca²⁺ (58). In vascular smooth muscle cells, stimulation with angiotensin has also been shown to elicit an increase in the tyrosine phosphorylation of FAK (4, 101).

Muscarinic activation of tracheal smooth muscle tissue strips also stimulates the tyrosine phosphorylation of FAK (95). In these tissues, the time course of the increase in FAK tyrosine phosphorylation in response to contractile stimulation closely follows that of tension development. The role of FAK in the contractile activation of tracheal muscle has been evaluated by introducing specific antisense to FAK into the strips to selectively deplete FAK protein (94). The depletion of FAK inhibited the increases in force, intracellular Ca²⁺, and myosin light-chain phosphorylation in response to contractile stimulation with acetylcholine. However, when the FAK-depleted tissues were permeabilized with -toxin and then stimulated by increasing intracellular Ca²⁺, the FAK-depleted tissues developed active tension that was comparable to that in tissues that had not been depleted of FAK. This demonstrates that the primary cause for the suppression of contractile activation in the FAK-depleted tissues is the disruption of Ca²⁺ signaling. Thus these studies are consistent with electrophysiological studies in vascular smooth muscle cells, studies that suggest a critical role for FAK in the regulation of Ca²⁺ channels.

Current evidence suggests that the roles of FAK and c-Src in regulating signal transduction pathways may be closely intertwined. An early downstream event following FAK activation and its autophosphorylation is its binding to c-Src (71, 83, 86). Integrin engagement with fibronectin and the consequent autophosphorylation of FAK at Tyr³⁹⁷ creates a high-affinity binding site on FAK for c-Src that results in the formation of a stable FAK-Src complex (15, 83, 86). This in turn leads to the phosphorylation of the associated "docking" proteins paxillin and p130cas (33). p130cas has been shown to undergo tyrosine phosphorylation and interact with c-Src in cultured vascular smooth muscle cells (78, 81). Both p130cas and paxillin bind to FAK and can be phosphorylated by FAK or c-Src in vitro (2, 36, 84, 85, 102). Thus interventions that interfere with the activation of either FAK or c-Src or with the formation of the FAK-Src complex may interrupt downstream pathways common to both proteins.

The contractile activation of tracheal smooth muscle tissues with acetylcholine stimulates the tyrosine phosphorylation of paxillin concurrently with the phosphorylation of FAK and active tension development (72, 95, 105). The tyrosine phosphorylation of both FAK and paxillin is also elicited by acetylcholine in Ca²⁺-depleted tracheal muscle strips and in permeabilized tracheal muscle strips at very low levels of intracellular Ca²⁺ (pCa 8) (62, 95). Under these conditions of low intracellular Ca²⁺, neither tension development nor myosin light-chain phosphorylation is stimulated by acetylcholine. Thus, in tracheal smooth muscle, the tyrosine phosphorylation of these proteins that is stimulated by acetylcholine does not depend on Ca²⁺ signaling nor is it dependent on myosin light-chain phosphorylation or tension development. This is consistent with a role for these proteins as upstream mediators of Ca²⁺ signaling. Thus FAK and paxillin may be part of a complex of proteins that mediate Ca²⁺-insensitive signaling pathways that regulate contractile protein activation and cytoskeletal dynamics in smooth muscle. These functions may be mediated via the downstream activation of members of the Rac family of GTPases (see below).

When tracheal muscle strips that have been depleted of FAK are stimulated with acetylcholine, paxillin phosphorylation does not increase to normal levels (94). This effect of FAK depletion on paxillin phosphorylation is also present in antisense-treated strips depleted of intracellular Ca²⁺, suggesting that FAK may be a direct catalyst of paxillin phosphorylation in tracheal smooth muscle. The depletion of paxillin in tracheal smooth muscle strips by paxillin antisense inhibits force development in response to acetylcholine in tracheal muscle strips (96). However, paxillin depletion does not result in the inhibition of Ca²⁺ signaling and myosin light-chain phosphorylation, suggesting that the role of paxillin in tension development is different from that of FAK in tracheal smooth muscle (96).

Both FAK and paxillin have been implicated in mechanotransduction pathways in smooth muscle. The application of cyclical mechanical strain to cultured smooth muscle cells and other cell types leads to an increase in the tyrosine phosphorylation of FAK and paxillin as well as to increases in intracellular Ca²⁺ (92, 113). In cultured substratum-adherent cells, cyclical mechanical strain also induces the alignment of stress fibers along the vector of the imposed strain (40, 91). The tyrosine phosphorylation of FAK and paxillin is also acutely sensitive to muscle length in tracheal smooth muscle tissues activated by acetylcholine (95). Isometric contraction of the muscle at longer lengths is associated with higher levels of tyrosine phosphorylation of these proteins than contraction at a shorter length. Myosin light-chain phosphorylation and intracellular Ca²⁺ have been shown to be similarly length sensitive in smooth muscle tissues (1, 32, 63, 76). In tracheal smooth muscle, the length sensitivity of FAK and paxillin phosphorylation is retained even when the tissues are depleted of intracellular Ca²⁺ before stimulation with acetylcholine (95). Under these conditions, tension development and myosin light-chain phosphorylation are inhibited. Thus paxillin and FAK may be components of an integrin-associated protein complex in smooth muscle that senses mechanical strain and initiates downstream signaling to proteins that regulate cytoskeletal structure and contractility (95).

	SIGNALING PATHWAYS TO DOWNSTREAM EFFECTORS OF CYTOSKELETAL REMODELING IN SMOOTH MUSCLE

TOP ABSTRACT INTRODUCTION SIGNAL TRANSDUCTION BY INTEGRIN... ROLE OF FAK AND... SIGNALING PATHWAYS TO... SMALL HEAT SHOCK PROTEINS... SUMMARY AND CONCLUSIONS REFERENCES

There is growing evidence that actin polymerization and the dynamic remodeling of the actin cytoskeleton play important roles in the regulation of smooth muscle contraction. Force development in smooth muscle tissues is dramatically depressed by agents that inhibit actin polymerization (cytochalasin and latrunculin) (13, 70, 99, 114). In tracheal and intestinal smooth muscles, the inhibition of contraction by cytochalasin or latrunculin does not result from the disruption of signaling pathways that mediate myosin light-chain phosphorylation or the interruption of Ca²⁺ signaling or from cytoskeletal disorganization (61, 70). The contractile activation of tracheal smooth muscle tissues decreases the concentration of G actin, providing further evidence that the polymerization of actin is involved in contractile activation in this tissue (61). The process of actin polymerization may play a role in the mechanosensitive regulation of smooth muscle contraction. Actin polymerization is a significant contributor to the development of myogenic tone in cerebral arteries after forced dilation (13), and the inhibition of actin polymerization by latrunculin inhibits the length sensitivity of tension development in airway smooth muscle (61). Actin remodeling has been proposed as a mechanism for the mechanical plasticity of airway smooth muscle (28-30). Airway smooth muscle mechanical plasticity may be a fundamental mechanism underlying the changes in airway tone induced by lung volume changes during breathing (29, 30, 88).

There is currently evidence for the involvement of several major signaling pathways for the regulation of actin dynamics and cytoskeletal function in smooth muscle. Members of the Rac family of GTPases are widely recognized as mediators of the assembly and disassembly of the actin cytoskeleton and of actin remodeling in response to extracellular signals in many cell types (14, 97, 115). Hirshman and colleagues (37, 98) have demonstrated that actin reorganization in primary cultures of airway smooth muscle cells requires the activation of Rho and that Rho-mediated actin reorganization is coupled to the muscarinic receptor activation via heterotrimeric G proteins. RhoA is also well known as a regulator of myosin light-chain phosphorylation in smooth muscle (73, 93). There is also growing evidence that the p38 mitogen-activated protein (MAP) kinase pathways regulate actin dynamics via this downstream effector, the 27-kDa heat shock protein (HSP27) (35, 54, 112).

There is considerable evidence for the involvement of the integrin-associated signaling proteins FAK, c-Src, and paxillin in the regulation of cytoskeletal dynamics and cell motility in other cell types; however, direct evidence supporting their role as a regulator of actin dynamics in smooth muscle tissues is currently lacking. However, there are a number of potential mechanisms by which nonreceptor tyrosine kinases may mediate the activation of pathways involved in cytoskeletal remodeling (Fig. 1). Activation of the extracellular regulated kinase/MAP kinase signaling cascades has been linked to FAK and c-Src through the GRB2 adaptor protein, which binds directly to activated FAK (86) and via phosphatidylinositol-3 kinase, which is activated by the FAK-Src complex (4, 8, 24, 111, 112). Both c-Src and phosphatidylinositol-3 kinase have been shown to contribute to actin-dependent functions, including spreading and migration of cultured vascular smooth muscle cells (44, 47, 110). Several potential pathways have also been described by which the FAK may mediate downstream activation of members of the Ras family of GTPases. FAK binds to a member of the GTPase-activating protein family of GTPase regulators, termed GRAF (GTPase-activating factor for Rho that binds to FAK) (86), which could function as a mediator of cross talk between FAK and Rho family GTPases. Paxillin, a substrate of FAK, has also been implicated as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization (34, 100). Paxillin- is a substrate for p21-activated kinase isoform PAK3 and links PAK3 to integrins (34). Paxillin is also linked indirectly to PAK through a novel protein p95 paxillin-kinase linker (100). More work will be needed to elucidate the role of these proteins in the regulation of actin dynamics in smooth muscle.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Potential pathways for the mediation of actin remodeling in response to agonists and mechanical stimuli in smooth muscle. ECM, extracellular matrix; FAK, focal adhesion kinase; GRAF, GTPase-activating factor for Rho that binds to FAK; HSP27, 27-kDa heat shock protein; MAPK, mitogen-activated protein kinase; PAK, p21-activated kinase; PI-3 kinase, phosphatidylinositol 3-kinase.

	SMALL HEAT SHOCK PROTEINS AS EFFECTORS OF ACTIN REMODELING IN SMOOTH MUSCLE

TOP ABSTRACT INTRODUCTION SIGNAL TRANSDUCTION BY INTEGRIN... ROLE OF FAK AND... SIGNALING PATHWAYS TO... SMALL HEAT SHOCK PROTEINS... SUMMARY AND CONCLUSIONS REFERENCES

There are several families of proteins that are likely to be effectors of actin remodeling, including capping proteins, severing proteins such as gelsolin (22, 31), bundling proteins including -actinin, and cross-linking proteins including filamin. In addition to these rather well-characterized effector proteins, recent work from several groups suggests that small heat shock proteins might also be effectors of actin remodeling or cross-bridge function that contribute to mechanical plasticity of smooth muscle. HSP27 and its homologs HSP25 (mouse) and inhibitor of actin polymerization (chicken) are members of the small heat shock protein family that have a high sequence and functional homology with B-crystallins, major proteins of the lens of the eye. HSP27 exists in vivo as multimers of varying sizes, ranging from dimers to 700-kDa oligimers with large (>500 kDa) multimers predominating. Multimers form from interaction of unphosphorylated dimers. Phosphorylation of Ser⁸² (Ser⁹⁰ in hamster HSP27) promotes dissociation of multimers (3), and further phosphorylation of Ser¹⁵ may regulate interaction of dimers with actin filaments (51).

HSP27 is thought to serve multiple functions in a variety of cell types. HSP27 acts as a chaperone by binding to denatured proteins to promote proper refolding in conjunction with higher molecular weight stress proteins (48). Human HSP27 also maintains intracellular levels of reduced glutathione, possibly by contributing to structures necessary for activity of glucose-6-phosphate dehydrogenase and other enzymes that control glutathione reduction (59, 75). Both mechanisms are thought to confer tolerance to a variety of physical and chemical stresses, including heat, oxidative stress, interleukins, and tumor necrosis factor (52, 60).

In addition to enhancing stress tolerance, HSP27 has significant effects on the actin cytoskeleton that are regulated by phosphorylation and dephosphorylation. Purified unphosphorylated mouse and chicken HSP27 homologs inhibit actin polymerization in vitro (3, 65). Phosphorylation reverses the inhibition, presumably favoring formation of F actin in vivo. This is supported by studies of cultured cells in which phosphorylation of HSP27 was shown to be necessary for growth factor stimulation of F-actin formation (55, 56), stabilization of focal adhesions (87), and promotion of cell migration (79).

Signaling pathways regulating HSP27 phosphorylation are fairly well defined (Fig. 2). Studies of cultured fibroblasts and other nonmuscle cells show that phosphorylation of HSP27 by MAP kinase-activated protein (MAPKAP) kinase 2 (MK2) is necessary for formation of F actin. Phosphorylation of HSP27 by MK2 increases the rate and extent of actin polymerization in vitro (11). This suggests that in vivo the extent of actin polymerization can be increased by phosphorylation of HSP27 by MK2. MK2 activity is stimulated by phosphorylation catalyzed by p38 MAP kinases. p38 MAP kinases are activated by upstream activators MKK3, MKK4, and MKK6. Coupling between the MKKs and surface receptors is less certain, with several nonexclusive pathways having been described. Dechert et al. (17) showed that PAKs 1, 2, and 3 are expressed in airway smooth muscle and that PAK1 appears to be required for coupling cytokine activation to p38 MAP kinase phosphorylation. RhoA is also reported to be an upstream activator of HSP27 in smooth muscle (104), but the coupling of Rho-dependent mechanisms to the p38 MAP kinase pathway is undefined. It also remains to be determined whether other kinases such as TAK1 and the mixed-lineage kinases contribute to activation of the p38 MAP kinase pathway in smooth muscles. cGMP-dependent protein kinase (PKG) also phosphorylates HSP27 at Thr¹⁴³ (11) in platelets, and a T143E mutant reduces the stimulation of actin polymerization by S15D, S78D, S82D mutant HSP27. There is no evidence yet for or against this phosphorylation event in smooth muscles, but this is an obvious point of interest given the fundamental importance of PKG signaling in smooth muscle relaxation, proliferation, and phenotype determination. The full effects of HSP27 phosphorylation on actin structure and function in smooth muscle remain undefined, but they are likely to be diverse based on what is known of the varied roles for actin filaments.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Signal transduction pathway for regulating HSP27 phosphorylation and interaction of HSP27 with actin filaments. Signals to the p38 MAPK pathway are thought to culminate in phosphorylation of HSP27 polymers by MK2, causing dissociation of the polymers to HSP27 dimers or monomers. Phosphorylated HSP27 is a substrate for type 2 phosphatases (PP2A), but the details of this pathway at the level of actin filaments in forming focal contacts are unknown. Actin capping activity of the nonphosphorylated HSP27 dimer/monomer is speculative, but phosphorylation of HSP27 is known to favor formation of F actin at various sites and to promote formation of focal contacts.

In intact smooth muscle under physiological conditions, HSP27 probably regulates actin cytoskeleton structure and may modulate the interaction of actin and myosin. This is not surprising given the biochemical properties of the protein and the high levels of expression in smooth muscle. HSP27 is constitutively expressed in smooth muscles at relatively high concentrations (2-8 µg HSP27/mg total protein) (35, 66). On a mass basis, this is similar to the amount of h1 calponin and tropomyosins that can be extracted from airway smooth muscle (Gerthoffer, unpublished observation). HSP27 has been colocalized to contractile proteins in freshly dispersed intestinal smooth muscle cells stimulated with ceramide, and it coprecipitates with actin, tropomyosin, and caldesmon, suggesting some molecular association with the contractile element (41).

There is functional evidence in vascular and visceral smooth muscle that HSP27 can modify contraction, consistent with the biochemical associations of HSP27 and contractile proteins. Treating permeabilized intestinal smooth muscle cells with anti-HSP27 antibodies inhibits bombesin-induced contraction (5). Anti-HSP27 antibodies also partially inhibit endothelin-1-induced Ca²⁺ sensitization of chemically permeabilized canine pulmonary artery strips (111). Meloche et al. (64) used SB-203580 to block the p38 MAP kinase/MK2 pathway and block HSP27 phosphorylation in rat aorta cultured cells. Blocking HSP27 phosphorylation substantially inhibited angiotensin II-induced contraction but, interestingly, had no effect on phenylephrine-induced contraction. This raises the possibility that the function of HSP27 is differentially regulated by various receptors and their downstream signaling pathways. The molecular mechanisms responsible for agonist-dependent differences are difficult to predict because a variety of contractile stimuli activate p38 MAP kinases in smooth muscle. These include -adrenergic and muscarinic agonists, angiotensin II, and endothelin 1 (35, 54, 111).

In addition to G-protein-coupled receptors, there is evidence that "stress" stimuli also activate signal transduction pathways, leading to HSP27 phosphorylation in smooth muscle. In two studies of vascular smooth muscle relaxation, Brophy and colleagues (21, 50) found that behavioral stress in intact animals and stressing isolated vascular smooth muscle with arsenite or heat stress markedly reduced relaxation induced by forskolin or nitroprusside. These results and the author's earlier biochemical studies (7, 107) suggest that cyclic nucleotides activate protein kinase A to phosphorylate HSP20, which inhibits force by an unknown molecular mechanism. It was suggested that stimulation of the p38/MK2 pathway by arsenite or heat stress results in phosphorylation of HSP27, which then colocalizes with HSP20 and inhibits phosphorylation of HSP20 by protein kinase A, thus antagonizing relaxation.

Contraction and relaxation of smooth muscle clearly depend on both stable F-actin structure as well as G-to-F actin transformation (49, 61), and HSP27 may well be one of the key modulators of this process. Migration of cells is another function that depends critically on G-to-F actin transition. Migration of airway smooth muscle cells also depends on phosphorylation of HSP27 via the p38 MAP kinase pathway (35). Blocking p38 MAP kinase with SB-203580 inhibited activation of MK2, prevented phosphorylation of HSP27, and blocked smooth muscle cell migration (35). Expression of dominant-negative p38 MAP kinase or nonphosphorylatable HSP27 also inhibits migration, and expressing active MKK6 increases p38 MAP phosphorylation, HSP27 phosphorylation, and cell migration. Using an adenoviral vector to express dominant-negative PAK1, Dechert et al. (17) showed dominant-negative PAK1 completely blocked spontaneous and PDGF-stimulated migration. It appears that activation of PAK1 leads to the activation of p38 MAP kinase, possibly through MKK3/6. Activation of p38 MAP kinase and MK2 is coupled to actin cytoskeleton remodeling necessary for cell migration via HSP27 phosphorylation. These results in smooth muscle are similar to previous studies of p38 MAP kinase and HSP27 phosphorylation in vascular endothelial cells (53, 79).

	SUMMARY AND CONCLUSIONS

TOP ABSTRACT INTRODUCTION SIGNAL TRANSDUCTION BY INTEGRIN... ROLE OF FAK AND... SIGNALING PATHWAYS TO... SMALL HEAT SHOCK PROTEINS... SUMMARY AND CONCLUSIONS REFERENCES

Recent biochemical and functional studies of contraction and cell migration suggest that smooth muscle cells possess multiple mechanisms for rapid mechanical adaptation to environmental stimuli. Current evidence suggests that dynamic remodeling of the actin cytoskeleton may provide an important mechanism for mechanical adaptation in differentiated smooth muscle. In differentiated contractile smooth muscle, actin polymerization may contribute to force development and maintenance by increasing the number or stiffness of attachments of actomyosin assemblies to membrane-dense plaques and cytoplasmic-dense bodies, as well by some yet to be defined effects on the organization of actomyosin structure or function.

Integrins and focal adhesion proteins, including FAK, Src family tyrosine kinases, and paxillin, probably function as proximal elements of mechanotransduction pathways that are implicated in coupling receptor activation to actin remodeling, but details of the signaling pathways are not fully defined yet. The Rho family GTPases, Rac and RhoA, are implicated in coupling receptor activation to actin remodeling in contractile smooth muscle; however, details of the signaling pathways that link these proteins to effectors of cytoskeletal remodeling remain to be established for smooth muscle. There is emerging evidence that small heat shock proteins may function as downstream effectors of actin filament remodeling in smooth muscle cells. The p38 MAP kinase pathway, a known downstream target of Rac and Cdc42, may also regulate actin remodeling through phosphorylation of HSP27. Phosphorylation of HSP27 promotes actin remodeling by enhancing actin polymerization.

A potential pathophysiological consequence of actin remodeling may be that, in the hyperreactive airway, actin remodeling and cell stiffening contribute to persistent contractions induced by inflammatory mediators. Actin remodeling is also required for cell migration during development and in the proliferative response to injury. Hypertrophic and hyperplastic responses of airway smooth muscle in asthma may be viewed as responses to injury perpetrated by chronic exposure to mitogenic and chemotactic stimuli or to abnormal mechanical stresses. These chronic chemical or mechanical "stressors" may well affect actin filament dynamics in airway smooth muscle at a variety of sites, including attachment at membrane-dense plaques and cytoplasmic-dense bodies, at filament nucleation sites, and even at the actomyosin cross bridge. Despite significant recent progress, the exact molecular functions of integrins, focal adhesion proteins, and small heat shock proteins in smooth muscles remain poorly defined. Future directions might include defining the cytoskeletal binding partners and regulatory pathways for the various components of focal contacts and small heat shock proteins in smooth muscle and a better understanding of the fundamental features of actin filament regulation, including nucleation, polymerization, cross linking, and interactions with other actin binding proteins.