Cellular Redistribution of PKCα, rhoA, and ROKα Following Smooth Muscle Agonist Stimulation

doi:10.1006/excr.1999.4565

Experimental Cell Research

Volume 251, Issue 1, 25 August 1999, Pages 92-101

https://doi.org/10.1006/excr.1999.4565 Get rights and content

Abstract

Efficient receptor-coupled activation of smooth muscle requires discrete coordination of many signal transducing events from the plasma membrane to the myofilaments. Recruitment of key factors to the plasma membrane is thought to be crucial for transduction of extracellular signals leading to contractility. We investigated, therefore, for the first time in intact differentiated smooth muscle cells, the distributions of three molecules important for receptor-coupled excitation: protein kinase Cα (PKCα), rhoA, and rho kinase (ROK). We also directly confirmed, by single cell force measurements, carbachol-induced [Ca²⁺]_i sensitization of contractility. Laser scanning confocal immunofluorescent microscopy of central smooth muscle cell sections determined that, at rest, PKCα, rhoA, and ROKα were distributed predominantly throughout the cytosol. Muscarinic stimulation resulted in significant redistribution of each protein to the cell membrane. By digital image analysis, peripheral:cytosolic distributions of PKCα, rhoA, and ROKα were calculated as, respectively, 1.05 ± 0.03 (8), 1.09 ± 0.03 (5), and 1.26 ± 0.04 (12) at rest, increasing significantly following stimulation to 2.09 ± 0.22 (6), 2.02 ± 0.12 (8), and 1.93 ± 0.05 (10). It is proposed that this receptor-coupled recruitment to the cell periphery of the downstream signaling molecules PKCα, rhoA, and ROKα contributes to the efficacy of agonist-induced contractile activation of smooth muscle.

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A potential explanation for this is the increased expression of Rnd-1, which is a well-described endogenous antagonist of the rhoA-ROK pathway.28 The suppression of uterine contractility by ROK inhibition is in keeping with previous findings.29-33 Further, in mice, lipopolysaccharide-induced preterm delivery was delayed significantly with the ROK inhibitor Y-27632.34
Endogenous uterine agonists can activate numerous signaling pathways to effect increased force. Our objective was to assess expression of key constituents of these pathways, in alliance with contractile function, through late gestation and during term and preterm labor.
Using myography, we measured the response to 3 agonists compared with depolarization alone (K⁺, 124 mEq/L) and calculated agonist/depolarization ratio. We measured gene expression using quantitative reverse transcription–polymerase chain reaction.
Contractile responsiveness to depolarization alone, oxytocin, or endothelin-1 increased during pregnancy compared with nonpregnant animals. The agonist/depolarization ratio did not change during uterine activation or parturition. Inhibition of rhoA-associated kinase decreased responses to oxytocin in all tissues, but significantly more during uterine activation. Expression of rhoA and rhoA-associated kinase was increased significantly in active labor at term or preterm.
The rhoA/rhoA-associated kinase pathway is a key regulator of uterine activation during labor and may be a useful target for the prevention of spontaneous preterm birth.
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In addition, activation of RhoA is strongly dependent on its post-translational modification (isoprenylation) mediated through the activity of geranylgeranyltransferases (Casey, 1995). This process allows translocation to specific plasma membrane sites that can be blocked by caveolin-1 peptide mimetics in smooth muscle cells (Taggart et al., 1999; Taggart et al., 2000), and is required for membrane anchoring of the small GTPase and for subsequent association with its effectors (Fig. 2). GDIs can prevent membrane anchoring and nucleotide exchange by forming cytoplasmic complexes with GDP-bound Rho and keep the protein soluble by shielding the hydrophobic post-translationally modified moiety (Olofsson, 1999)(Fig. 2).
In asthma, inflammatory mediators that are released in the airways by recruited inflammatory cells and by resident structural cells result in airway hyperresponsiveness caused by increased bronchoconstriction. In addition, chronic inflammation appears to drive remodelling of the airways that contributes to the development of fixed airway obstruction and airway hyperresponsiveness in chronic asthma. Airway remodelling includes several key features such as excessive deposition of extracellular matrix proteins in the airway wall (fibrosis) and increased abundance of contractile airway smooth muscle encircling the airways. Current asthma therapy fails to inhibit these features satisfactorily. This review focuses on Rho kinase as a potential drug target in asthma, as compelling evidence from animal models and ex vivo studies suggests a central role for this enzyme and its associated signalling in acute and chronic airway hyperresponsiveness.
Caveolin-1 and Lipid Rafts in Confluent BeWo Trophoblasts: Evidence for Rock-1 Association with Caveolin-1
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Western blotting of the gradient fractions also showed an additional, major pool of Rock-1 in the Triton-soluble cytosolic fractions, supporting the immunofluorescence data indicating abundant cytoplasmic Rock-1. Rock protein has in fact previously been observed to be partially localised to the plasma membrane in uterine smooth muscle cells at rest, with prominent relocation from the cytoplasm to the vicinity of the plasma membrane upon stimulation [65]. Although the latter studies did not extend to the demonstration that Rock and caveolin were associated, it has been proposed that Rock contains two putative CSD-interacting sequences in its catalytic domain through which direct regulatory interaction by caveolin may be possible [58].
Lipid rafts are detergent-insoluble, low-density membrane domains that are rich in cholesterol and sphingolipids; caveolae are a subdomain of the biochemically defined glycolipid raft whose expression is associated with the protein caveolin-1. This protein associates with numerous signalling molecules, regulating their activity by holding them inactive. Human villous cytotrophoblasts contain caveolin-1, but levels reduce greatly during their differentiation into syncytiotrophoblast. Since caveolin-1 is a known regulator of apoptosis and trophoblast syncytialisation involves the apoptotic cascade, we hypothesised that cytotrophoblast caveolin-1 may also play a role in regulating fusion events involved in syncytium formation. The BeWo choriocarcinoma cell line has previously proved valuable for studying trophoblast syncytialisation, hence the present work was carried out to determine whether BeWo cells could be used as a model for the exploration of caveolin-1's role in regulating the syncytialisation process.
Undifferentiated BeWo cells were found to express caveolin-1 in similar amounts to villous cytotrophoblasts isolated from term placenta. Lipid raft fractions prepared from these BeWo cells at confluence contained the raft-associated proteins caveolin-1 and -2, flotillin-1 and -2, stomatin and the heterotrimeric G protein, Gαq. Confocal immunofluorescence studies revealed that caveolin-1 is internalized to the mitochondria, but not to the Golgi or endoplasmic reticulum, in subconfluent BeWo and that the protein relocates to the plasma membrane upon confluence, an observation confirmed by caveolin-1 and cytochrome c Western blotting of lipid raft fractions and mitochondria purified from confluent and subconfluent cells. Western blotting and immunofluorescence experiments comparing undifferentiated cells and those induced to differentiate using the cAMP analogue, dibutyryl cAMP, showed that BeWo syncytialisation was accompanied by a reduction in caveolin-1 levels, similar to the situation in primary villous cytotrophoblasts. Confluent, undifferentiated BeWo cultures were then used to investigate the cellular localisation of Rock-1, a protein which promotes cytoskeletal re-organisation important for syncytialisation and apoptosis. Its association with caveolin-1 was evidenced by the demonstration that the 160 kDa proenzyme form of Rock-1 co-immunoprecipitates with caveolin-1 and vice versa, as well as by the co-localisation of the two proteins at the plasma membrane, as shown in immunofluorescence studies. A proportion of the total cell Rock-1 content was found in BeWo lipid raft fractions, confirming its membrane presence in confluent cells. This close association of plasmalemmal caveolin-1 with Rock-1 protein raises the possibility that caveolin-1 may regulate Rock-1 in these trophoblasts. We conclude that cell–cell contact is required for BeWo trophoblast to exhibit plasmalemmal caveolin-1; BeWo cells at confluence offer a useful model for the study of trophoblast raft behaviour during syncytialisation and for the exploration of the potential Rock-1-regulating role of caveolin-1 in this process.
Rho proteins, pregnancy and labour
2009, Fetal and Maternal Medicine Review

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¹: To whom reprint requests should be addressed at University Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester M13, Great Britain. Fax: +44 161 274 4833. E-mail: [email protected].

²: Present address: Department of Physiology, Yonsei University College of Medicine, Korea.

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Regular ArticleCellular Redistribution of PKCα, rhoA, and ROKα Following Smooth Muscle Agonist Stimulation

Abstract

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Am. J. Obstet. Gynecol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Mechanisms of smooth muscle contraction

Physiol. Rev.

Signal transduction and regulation in smooth muscle

Nature

In situ determination of [Ca2+]i threshold for translocation of the alpha-protein kinase C isoform

Am. J. Physiol.

Role of rho proteins in carbachol-induced contractions in intact and permeabilized guinea-pig intestinal smooth muscle

J. Physiol.

A novel mechanism for the Ca2+ sensitizing effect of protein kinase C on vascular smooth muscle: Inhibition of myosin light chain phosphatase

J. Gen. Physiol.

Myosin phosphatases: Subunits and interactions

Acta Physiol. Scand.

Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit arterial smooth muscle

J. Physiol.

Regular Article
Cellular Redistribution of PKCα, rhoA, and ROKα Following Smooth Muscle Agonist Stimulation

In situ determination of [Ca²⁺]_i threshold for translocation of the alpha-protein kinase C isoform

A novel mechanism for the Ca²⁺ sensitizing effect of protein kinase C on vascular smooth muscle: Inhibition of myosin light chain phosphatase