doi: 10.1161/01.RES.0000093183.00556.D5
© 2003 American Heart Association, Inc.
Editorial |
Rho Signaling
Agonist Stimulation and Depolarization Come Together
From the Departments of Physiology and Biophysics, and Medicine (Cardiology), Case Western Reserve University, Cleveland, Ohio.
Correspondence to Frank V. Brozovich, Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106-4970. E-mail fxb9{at}cwru.edu
Key Words: force enhancement • smooth muscle • Rho-kinase • signaling
Force development in smooth muscle is dependent on Ca2+/calmodulin (Ca2+/CaM) activation of myosin light chain kinase (MLCK) and the subsequent phosphorylation of the regulatory myosin light chain (MLC20).1 However, when smooth muscle is stimulated with agonists2 or by direct activation of the G-proteins,3–6 a higher force is developed for a given [Ca2+] than for depolarization. Although the mechanism for this Ca2+ sensitization of the contractile filaments is still the subject of investigation, evidence suggests that the signaling pathway may involve either the activation of Rho (for reviews see7–9) and/or protein kinase C (PKC),10,11 although a pathway involving Rho and subsequent activation of Rho-kinase is more widely accepted. Agonist stimulation of smooth muscle can be divided into two phases: an initial rapid, transient increase in Ca2+ and MLC20 phosphorylation, and a subsequent phase of a sustained force despite lower levels of Ca2+ and MLC20 phosphorylation.12 There is evidence that the sustained phase of force maintenance is dependent on the activation of Rho.13
Although uncertainty exists regarding the mechanism by which receptor-mediated activation of the G-proteins leads to force enhancement, it is generally accepted that membrane depolarization leads to an increase in intracellular [Ca2+] and a Ca2+-dependent activation of MLCK to increase MLC20 phosphorylation and force. Thus, it has become dogma that depolarization leads to only an increase in Ca2+ and a subsequent activation of MLCK, without the activation of additional signaling pathways. Challenging these generally accepted principles is recent evidence that Rho-kinase inhibition almost completely eliminates the sustained portion of the phasic force response to 60 mmol/L KCl depolarization in rat tail artery without affecting the Ca2+ transient.14 The reduction in force was accompanied by a decrease in MLC20 phosphorylation. These results suggest that Rho-kinase is activated during depolarization of smooth muscle, and its activation leads to an inhibition of MLC phosphatase activity.
In this issue of Circulation Research, Sakurada et al15 extend and further delineate the mechanism by which inhibition of Rho-kinase depresses force during depolarization. During both agonist and KCl depolarization of rabbit aortic smooth muscle, these investigators demonstrated that the GTP-bound active form of Rho increases in a concentration-dependent manner and the inhibition of Rho-kinase with either Y27632 or HA1077 reduced force and MLC20 phosphorylation with similar concentration dependencies. These results suggest not only that Rho is activated during depolarization but that its activation increases force. Additionally, the group demonstrated that activation of Rho during KCl depolarization requires transmembrane Ca2+ flux and results in phosphorylation of the myosin binding subunit (MYPT1) of MLC phosphatase at Thr695. MYPT1 phosphorylation at this site has been previously demonstrated to inhibit MLC phosphatase activity,16 resulting in an increase in MLC20 phosphorylation and force.6,17 Taken together, the data suggest that depolarization leads to an activation of Rho, Rho-kinase, and subsequent MYPT1 phosphorylation resulting in an inhibition of MLC phosphatase activity and Ca2+ sensitization, similar to the pathway suggested for agonist-induced force enhancement.6,17
This Ca2+-dependent activation of Rho is a novel finding and establishes a dual role for Ca2+/CaM: activation of MLCK and inhibition of MLC phosphatase, both of which will lead to an increase in the level of MLC20 phosphorylation (Figure). This signaling pathway probably requires an intact cell membrane and thus would not be present in permeabilized and/or skinned preparations, explaining the absence of an effect of Rho-kinase inhibition on Ca2+ activation of skinned smooth muscle strips.18,19 However, Ca2+ activation of Rho is consistent with the observations that in permeabilized vascular smooth muscle, Ca2+ induces a translocation of Rho to the membrane20 and the inhibition of force produced by depolarization when Rho is inactivated.21
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) and a 20-kDa subunit of unknown function.It has been demonstrated that the magnitude of the change in MLC20 phosphorylation produced by depolarization is less than that produced by agonist stimulation for any given level of intracellular Ca2+.2,4,6,22,23 This suggests that either depolarization only results in a Ca2+-dependent activation of MLCK or the inhibition of MLC phosphatase activity produced by depolarization is significantly less than produced by agonist activation. The results discussed above14,15 would argue for the latter. However if Rho-kinase inhibition produces similar decreases of force during activation by both agonists and depolarization,15 it is unclear how MLC20 phosphorylation can differ for the two forms of activation. One possibility is that there could be an additional factor required in the signaling pathway for the Ca2+-dependent activation of Rho. Sakurada et al15 have data that could implicate Ca2+/CaM-dependent protein kinase II (CAMKII). The CAMKII inhibitor KN93 depressed both force and Rho activation during membrane depolarization but had no effect on the activation of Rho or increase in force produced by ionomycin. These results could argue that for depolarization, transmembrane Ca2+ flux and either transmembrane signaling or signaling at the smooth muscle cell membrane is required for the subsequent activation of the Rho pathway.
Tissue-specific expression of a protein(s) required for this signaling cascade could explain the differences in the magnitude of Rho inhibition observed for different forms of smooth muscle activation. Changes in the level of MYPT1 phosphorylation have been shown to be tissue,13,17,24 agonist,25 and MYPT1 isoform19 specific. Additionally, the expression of both MYPT1 isoforms26 and the MLC phosphatase inhibitor CPI-1727 is tissue specific. These results could suggest that a Ca2+/CaM-dependent inhibition of MLC phosphatase is also tissue specific. It is possible that the Ca2+-induced translocation of Rho to the membrane20 occurs in all smooth muscle, but the subsequent activation of Rho requires the expression of another protein(s). Whether the activation of Rho requires Gq, as suggested by Sakurada et al,15 or some other factor is unclear, but the elucidation of the mechanism for Ca2+-dependent activation of Rho will require further studies.
The Rho/Rho-kinase signaling pathway is not only important for the regulation of smooth muscle contractility but also for other regulatory processes. Activation of the Rho/Rho-kinase pathway has been demonstrated to mediate actin organization and remodeling of the cytoskeleton in nonmuscle cells.28,29 During integrin-mediated signaling in Rat1 cells, evidence suggests that there is a Rho dependence of focal adhesion kinase and paxillin phosphorylation, and that the formation of large focal adhesions from which actin stress fibers radiate during cell spreading and membrane ruffling is dependent on the activation of Rho.29 In addition, in smooth muscle, data suggest that paxillin phosphorylation may participate in force maintenance.30 The results reported by Sakurada et al15 thus implicate Ca2+ both in the mechanism for integrin signaling and reorganization of the smooth muscle cytoskeleton.
The results of this study15 establish a dual role for Ca2+ in the regulation of smooth muscle contraction, activation of MLCK, and a Rho-mediated inhibition of MLC phosphatase. The inhibition of MLC phosphatase would produce a Ca2+ sensitization, a phenomenon that was previously thought to be restricted to only receptor-mediated forms of smooth muscle activation. The inhibition of MLC phosphatase activity has been implicated in the pathogenesis of hypertension31 and in the migration of malignant cells.32 Further, the activation of Rho is important in the regulation of cell morphology.28,29 Therefore, the regulation of Rho and its signaling pathway promises to be an active area of investigation.
Acknowledgments
This work was supported by grants from the NIH (HL44181 and HL64137).
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
References
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