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Ravichandran VS, Patel HJ, Pagani FD, Westfall MV. Cardiac contractile dysfunction and protein kinase C-mediated myofilament phosphorylation in disease and aging. J Gen Physiol 2019; 151:1070-1080. [PMID: 31366607 PMCID: PMC6719401 DOI: 10.1085/jgp.201912353] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2019] [Revised: 05/25/2019] [Accepted: 06/19/2019] [Indexed: 01/10/2023] Open
Abstract
Increases in protein kinase C (PKC) are associated with diminished cardiac function, but the contribution of downstream myofilament phosphorylation is debated in human and animal models of heart failure. The current experiments evaluated PKC isoform expression, downstream cardiac troponin I (cTnI) S44 phosphorylation (p-S44), and contractile function in failing (F) human myocardium, and in rat models of cardiac dysfunction caused by pressure overload and aging. In F human myocardium, elevated PKCα expression and cTnI p-S44 developed before ventricular assist device implantation. Circulatory support partially reduced PKCα expression and cTnI p-S44 levels and improved cellular contractile function. Gene transfer of dominant negative PKCα (PKCαDN) into F human myocytes also improved contractile function and reduced cTnI p-S44. Heightened cTnI phosphorylation of the analogous residue accompanied reduced myocyte contractile function in a rat model of pressure overload and in aged Fischer 344 × Brown Norway F1 rats (≥26 mo). Together, these results indicate PKC-targeted cTnI p-S44 accompanies cardiac cellular dysfunction in human and animal models. Interfering with PKCα activity reduces downstream cTnI p-S44 levels and partially restores function, suggesting cTnI p-S44 may be a useful target to improve contractile function in the future.
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Affiliation(s)
- Vani S Ravichandran
- Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI
- Department of Cardiac Surgery, University of Michigan, Ann Arbor, MI
| | - Himanshu J Patel
- Department of Cardiac Surgery, University of Michigan, Ann Arbor, MI
| | - Francis D Pagani
- Department of Cardiac Surgery, University of Michigan, Ann Arbor, MI
| | - Margaret V Westfall
- Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI
- Department of Cardiac Surgery, University of Michigan, Ann Arbor, MI
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2
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Eddinger TJ. Smooth muscle-protein translocation and tissue function. Anat Rec (Hoboken) 2015; 297:1734-46. [PMID: 25125185 DOI: 10.1002/ar.22970] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2014] [Revised: 03/18/2014] [Accepted: 03/18/2014] [Indexed: 01/25/2023]
Abstract
Smooth muscle (SM) tissue is a complex organization of multiple cell types and is regulated by numerous signaling molecules (neurotransmitters, hormones, cytokines, etc.). SM contractile function can be regulated via expression and distribution of the contractile and cytoskeletal proteins, and activation of any of the second messenger pathways that regulate them. Spatial-temporal changes in the contractile, cytoskeletal or regulatory components of SM cells (SMCs) have been proposed to alter SM contractile activity. Ca(2+) sensitization/desensitization can occur as a result of changes at any of these levels, and specific pathways have been identified at all of these levels. Understanding when and how proteins can translocate within the cytoplasm, or to-and-from the plasmalemma and the cytoplasm to alter contractile activity is critical. Numerous studies have reported translocation of proteins associated with the adherens junction and G protein-coupled receptor activation pathways in isolated SMC systems. Specific examples of translocation of vinculin to and from the adherens junction and protein kinase C (PKC) and 17 kDa PKC-potentiated inhibitor of myosin light chain phosphatase (CPI-17) to and from the plasmalemma in isolated SMC systems but not in intact SM tissues are discussed. Using both isolated SMC systems and SM tissues in parallel to pursue these studies will advance our understanding of both the role and mechanism of these pathways as well as their possible significance for Ca(2+) sensitization in intact SM tissues and organ systems.
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Affiliation(s)
- Thomas J Eddinger
- Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin
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3
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Zhang Y, Hermanson ME, Eddinger TJ. Tonic and phasic smooth muscle contraction is not regulated by the PKCα - CPI-17 pathway in swine stomach antrum and fundus. PLoS One 2013; 8:e74608. [PMID: 24058600 PMCID: PMC3776813 DOI: 10.1371/journal.pone.0074608] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2013] [Accepted: 08/04/2013] [Indexed: 01/31/2023] Open
Abstract
Regulation of myosin light chain phosphatase (MLCP) via protein kinase C (PKC) and the 17 kDa PKC-potentiated inhibitor of myosin light chain phosphatase (CPI-17) has been reported as a Ca2+ sensitization signaling pathway in smooth muscle (SM), and thus may be involved in tonic vs. phasic contractions. This study examined the protein expression and spatial-temporal distribution of PKCα and CPI-17 in intact SM tissues. KCl or carbachol (CCh) stimulation of tonic stomach fundus SM generates a sustained contraction while the phasic stomach antrum generates a transient contraction. In addition, the tonic fundus generates greater relative force than phasic antrum with 1 µM phorbol 12, 13-dibutyrate (PDBu) stimulation which is reported to activate the PKCα – CPI-17 pathway. Western blot analyses demonstrated that this contractile difference was not caused by a difference in the protein expression of PKCα or CPI-17 between these two tissues. Immunohistochemical results show that the distribution of PKCα in the longitudinal and circular layers of the fundus and antrum do not differ, being predominantly localized near the SM cell plasma membrane. Stimulation of either tissue with 1 µM PDBu or 1 µM CCh does not alter this peripheral PKCα distribution. There are no differences between these two tissues for the CPI-17 distribution, but unlike the PKCα distribution, CPI-17 appears to be diffusely distributed throughout the cytoplasm under relaxed tissue conditions but shifts to a primarily peripheral distribution at the plasma membrane with stimulation of the tissues with 1 µM PDBu or 1 µM CCh. Results from double labeling show that neither PKCα nor CPI-17 co-localize at the adherens junction (vinculin/talin) at the membrane but they do co-localize with each other and with caveoli (caveolin) at the membrane. This lack of difference suggests that the PKCα - CPI-17 pathway is not responsible for the tonic vs. phasic contractions observed in stomach fundus and antrum.
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Affiliation(s)
- Yu Zhang
- Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin, United States of America
| | - Meghan E. Hermanson
- Department of Biology, Bradley University, Peoria, Illinois, United States of America
| | - Thomas J. Eddinger
- Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin, United States of America
- * E-mail:
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4
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Modulation of Ca(2+) release through ryanodine receptors in vascular smooth muscle by protein kinase Calpha. Pflugers Arch 2010; 460:791-802. [PMID: 20571823 DOI: 10.1007/s00424-010-0850-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2009] [Revised: 04/15/2010] [Accepted: 05/11/2010] [Indexed: 01/23/2023]
Abstract
The role of protein kinase C (PKC) in Ca(2+) release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (SMCs) is not well understood. Caffeine was used to activate RyRs and the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in both freshly isolated and cultured mouse aortic SMCs (ASMCs). Pre-activation of PKC with 1,2-dioctanoyl-sn-glycerol (DOG) prevented caffeine-induced [Ca(2+)](i) transients. Application of the PKC inhibitor calphostin C caused [Ca(2+)](i) transients which were not blocked by nifedipine or by removing extracellular Ca(2+) but were abolished after inhibition of the SR Ca(2+)-ATPase with thapsigargin or after inhibition of RyRs with ryanodine. In addition, chelerythrine and GF109203X also elevated resting [Ca(2+)](i) but no further [Ca(2+)](i) increase was seen with subsequent application of caffeine. Selective inhibition of PKCalpha with safingol blocked caffeine-induced [Ca(2+)](i) transients, but the PKCepsilon inhibitory peptide V1-2 did not. In cells expressing a EGFP-tagged PKCalpha, caffeine-induced [Ca(2+)](i) transients were associated with a rapid focal translocation near the cell periphery, while application of ionomycin and DOG caused translocation to the plasma membrane. Western blot showed that caffeine increased the relative amount of PKCalpha in the particulate fraction in a time-dependent manner. Co-immunoprecipitation of RyRs and PKCalpha indicated that they interact. In conclusion, our studies suggest that PKC activation can inhibit the gating activity of RyRs in the SR of ASMCs, and this regulation is most likely mediated by the Ca(2+)-dependent PKCalpha isoform.
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5
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Nelson CP, Willets JM, Davies NW, Challiss RAJ, Standen NB. Visualizing the temporal effects of vasoconstrictors on PKC translocation and Ca2+ signaling in single resistance arterial smooth muscle cells. Am J Physiol Cell Physiol 2008; 295:C1590-601. [PMID: 18829899 DOI: 10.1152/ajpcell.00365.2008] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Arterial smooth muscle (ASM) contraction plays a critical role in regulating blood distribution and blood pressure. Vasoconstrictors activate cell surface receptors to initiate signaling cascades involving increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and recruitment of protein kinase C (PKC), leading to ASM contraction, though the PKC isoenzymes involved vary between different vasoconstrictors and their actions. Here, we have used confocal microscopy of enhanced green fluorescence protein (eGFP)-labeled PKC isoenzymes to visualize PKC translocation in primary rat mesenteric ASM cells in response to physiological vasoconstrictors, with simultaneous imaging of Ca(2+) signaling. Endothelin-1, angiotensin II, and uridine triphosphate all caused translocation of each of the PKC isoenzymes alpha, delta, and epsilon; however, the kinetics of translocation varied between agonists and PKC isoenzymes. Translocation of eGFP-PKCalpha mirrored the rise in [Ca(2+)](i), while that of eGFP-PKCdelta or -epsilon occurred more slowly. Endothelin-induced translocation of eGFP-PKCepsilon was often sustained for several minutes, while responses to angiotensin II were always transient. In addition, preventing [Ca(2+)](i) increases using 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester prevented eGFP-PKCalpha translocation, while eGFP-PKCdelta translocated more rapidly. Our results suggest that PKC isoenzyme specificity of vasoconstrictor actions occurs downstream of PKC recruitment and demonstrate the varied kinetics and complex interplay between Ca(2+) and PKC responses to different vasoconstrictors in ASM.
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Affiliation(s)
- Carl P Nelson
- Department of Cell Physiology & Pharmacology, Univ. of Leicester, LE1 9HN, UK.
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6
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Ho KK, Anderson AA, Rosivatz E, Lam EWF, Woscholski R, Mann DJ. Identification of Cyclin A2 as the Downstream Effector of the Nuclear Phosphatidylinositol 4,5-Bisphosphate Signaling Network. J Biol Chem 2008; 283:5477-85. [DOI: 10.1074/jbc.m706623200] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
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7
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Gasser PJ, Orchinik M. Vasopressin-induced translocation and proteolysis of protein kinase Cα in an amphibian brain: Modulation by corticosterone. Brain Res 2007; 1134:18-26. [PMID: 17196180 DOI: 10.1016/j.brainres.2006.11.071] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2006] [Revised: 11/15/2006] [Accepted: 11/28/2006] [Indexed: 02/07/2023]
Abstract
In urodele amphibians, the hypothalamic neuropeptide arginine vasotocin and the adrenal steroid corticosterone interact to regulate reproductive behavior by actions in the brain. The present study investigated signal transduction pathways underlying acute effects of vasotocin and corticosterone, presumably mediated via "non-genomic" steroid action, in an amphibian brain. We used Western blot to examine the effects of corticosterone and the vasotocin receptor agonist arginine vasopressin, alone and in combination, on the subcellular localization and proteolytic processing of protein kinase C-alpha (PKCalpha) in tiger salamander brain tissue. Treatment of whole brain minces with vasopressin or vasotocin led to increases in PKCalpha in membrane fractions and concurrent decreases in PKCalpha in cytosolic fractions. Vasopressin or vasotocin treatment also induced the appearance in membrane and cytosolic fractions of a PKCalpha-immunoreactive band that corresponds to PKMalpha, the proteolytically generated, free catalytic subunit of PKCalpha. Treatment with corticosterone alone had no consistent effect on either PKCalpha or PKMalpha in either fraction. However, pretreatment with corticosterone reliably blocked vasopressin-induced increases in cytosolic PKMalpha. These data provide new information about the cellular mechanisms of action of vasopressin and corticosterone in the vertebrate brain and suggest a cellular mechanism by which the two hormones interact to regulate neuronal physiology and behavior.
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Affiliation(s)
- Paul J Gasser
- School of Life Sciences, Arizona State University, Tempe, AZ 85287-4601, USA.
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8
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Rybczynski R, Gilbert LI. Protein kinase C modulates ecdysteroidogenesis in the prothoracic gland of the tobacco hornworm, Manduca sexta. Mol Cell Endocrinol 2006; 251:78-87. [PMID: 16621234 DOI: 10.1016/j.mce.2006.02.015] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2005] [Revised: 02/20/2006] [Accepted: 02/27/2006] [Indexed: 11/27/2022]
Abstract
The prothoracic gland is the primary source of ecdysteroid hormones in the immature insect. Ecdysteroids coordinate gene expression necessary for growth, molting and metamorphosis. Prothoracicotropic hormone (PTTH), a brain neuropeptide, regulates ecdysteroid synthesis in the prothoracic gland. PTTH stimulates ecdysteroid synthesis through a signal transduction cascade that involves at least four protein kinases: protein kinase A (PKA), p70 S6 kinase, an unidentified tyrosine kinase, and the extracellular signal-regulated kinase (ERK). In this report, the participation of protein kinase C (PKC) in PTTH signalling is demonstrated and characterized. PTTH stimulates PKC activity through a PLC and Ca(2+)-dependent pathway that is not cAMP regulated. Inhibition of PKC inhibits PTTH-stimulated ecdysteroidogenesis as well as PTTH-stimulated phosphorylation of ERK and its upstream regulator, MAP/ERK kinase (MEK). These observations reveal that the acute regulation of prothoracic gland steroidogenesis is dependent on a web of interacting kinase pathways, which probably converge on factors that regulate translation.
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Affiliation(s)
- Robert Rybczynski
- Department of Biology, University of North Carolina at Chapel Hill, 27599-3280, USA.
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9
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Affiliation(s)
- John F Di Mari
- Department of Internal Medicine, University of Texas Medical Branch, Galveston 77555-1064, USA.
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10
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Maddali KK, Korzick DH, Turk JR, Bowles DK. Isoform-specific modulation of coronary artery PKC by glucocorticoids. Vascul Pharmacol 2005; 42:153-62. [PMID: 15820441 DOI: 10.1016/j.vph.2004.11.008] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2003] [Revised: 11/23/2004] [Accepted: 11/23/2004] [Indexed: 10/25/2022]
Abstract
Glucocorticoids (GC) exert diverse cellular effects in response to both acute and chronic stress, the functional consequences of which have been implicated in the development of cardiovascular pathology such as hypertension and atherosclerosis. However, the mechanisms by which GCs activate divergent signaling pathways are poorly understood. The present study examined the direct effects of natural (cortisol) and synthetic (dexamethasone) GCs on protein kinase C (PKC) isoform expression in coronary arteries. Porcine right coronary arteries were treated in vitro for 18 h in the presence and absence of either dexamethasone (10, 100, or 500 nM) or cortisol (50, 125, 250, or 500 nM). PKC isoform levels and subcellular distribution were determined by immmunoblotting of whole cell homogenates and immunocytofluorescence using PKC-alpha, -betaII, -epsilon, -delta, and -zeta specific antibodies. Dexamethasone caused a approximately 4-fold increase in PKC-alpha, a approximately 2.5-fold increase in PKC-betaII, and a 2-fold increase in PKC-epsilon (p<0.05). In contrast, dexamethasone had no effect on PKC-delta or PKC- zeta levels. Dexamethasone also caused an increase in the activity of PKC-alpha (285%), -betaII (170%), and -epsilon (210%). Cortisol produced similar effects on PKC isoform expression. Confocal microscopy revealed that while dexamethasone altered localization patterns for PKC-alpha, -betaII and -epsilon, no such effect was observed for PKC-delta or PKC-zeta. The stimulatory effects of dexamethasone and cortisol on coronary PKC levels and translocation were prevented by the GC receptor (GR) blocker, RU486. These results demonstrate, for the first time, that GCs modulate coronary PKC expression and subcellular distribution in an isoform-specific manner through a GR-dependent mechanism.
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Affiliation(s)
- K K Maddali
- Department of Biomedical Sciences, University of Missouri, Columbia, MO 65211, USA
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11
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Baluch DP, Koeneman BA, Hatch KR, McGaughey RW, Capco DG. PKC isotypes in post-activated and fertilized mouse eggs: association with the meiotic spindle. Dev Biol 2004; 274:45-55. [PMID: 15355787 DOI: 10.1016/j.ydbio.2004.05.030] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2003] [Revised: 05/14/2004] [Accepted: 05/28/2004] [Indexed: 11/27/2022]
Abstract
Several isotypes of protein kinase C (PKC) have been reported to be expressed in mammalian eggs, but it is unknown whether these isotypes have a common function in the egg during or within the first few hours of fertilization. Here we show that the isotypes of PKC exhibit distinct patterns of enrichment immediately after mouse egg activation. PKCalpha and gamma accumulate in the egg cortex 25 min post-activation, while only PKCalpha accumulates at the contractile ring of the forming second polar body about 1.5 h post-activation. PKCzeta exhibits some unique features that resulted in it being the focus of more extensive analysis. PKCzeta is tightly associated with the meiotic spindle as determined by detergent extraction and is closely associated with alpha-tubulin as determined by FRET analysis in the metaphase II (MII) egg. In addition, after egg activation, PKCzeta remains associated with the spindle as it transits into anaphase II and later telophase II, becoming associated with the midzone microtubules. Antibodies to the active form of PKCzeta are enriched on the spindle poles and later in development on the midzone microtubules. Active PKCzeta also is enriched in both pronuclei in the 6-h post-fertilization and in the 14-h post-fertilization embryo as well as in the nuclei of the two-cell embryo. Inhibition of PKCzeta, but not inhibition of other isotypes of PKC, results in rapid disruption of the meiotic spindle. This study suggests that PKCzeta has a role in spindle stability, while other PKC isotypes have different roles in the conversion of the egg to the zygote.
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Affiliation(s)
- D Page Baluch
- School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA
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12
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Cárdenas C, Müller M, Jaimovich E, Pérez F, Buchuk D, Quest AFG, Carrasco MA. Depolarization of skeletal muscle cells induces phosphorylation of cAMP response element binding protein via calcium and protein kinase Calpha. J Biol Chem 2004; 279:39122-31. [PMID: 15262987 DOI: 10.1074/jbc.m401044200] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Membrane depolarization of skeletal muscle cells induces slow inositol trisphosphate-mediated calcium signals that regulate the activity of transcription factors such as the cAMP-response element-binding protein (CREB), jun, and fos. Here we investigated whether such signals regulate CREB phosphorylation via protein kinase C (PKC)-dependent pathways. Western blot analysis revealed the presence of seven isoforms (PKCalpha, -betaI, -betaII, -delta, -epsilon, -, and -zeta) in rat primary myotubes. The PKC inhibitors bisindolymaleimide I and Gö6976, blocked CREB phosphorylation. Chronic exposure to phorbol ester triggered complete down-regulation of several isoforms, but reduced PKCalpha levels to only 40%, and did not prevent CREB phosphorylation upon myotube depolarization. Immunocytochemical analysis revealed selective and rapid PKCalpha translocation to the nucleus following depolarization, which was blocked by 2-amino-ethoxydiphenyl borate, an inositol trisphosphate receptor inhibitor, and by the phospholipase C inhibitor U73122. In C2C12 cells, which expressed PKCalpha,-epsilon, and -zeta, CREB phosphorylation also depended on PKCalpha. These results strongly implicate nuclear PKCalpha translocation in CREB phosphorylation induced by skeletal muscle membrane depolarization.
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MESH Headings
- Active Transport, Cell Nucleus
- Animals
- Animals, Newborn
- Blotting, Western
- Cell Line
- Cell Nucleus/metabolism
- Cells, Cultured
- Cyclic AMP Response Element-Binding Protein/metabolism
- Down-Regulation
- Estrenes/pharmacology
- Immunohistochemistry
- Inositol 1,4,5-Trisphosphate/chemistry
- Mice
- Microscopy, Confocal
- Microscopy, Fluorescence
- Mitogen-Activated Protein Kinase 1/metabolism
- Mitogen-Activated Protein Kinase 3
- Mitogen-Activated Protein Kinases/metabolism
- Muscle, Skeletal/metabolism
- Phorbol Esters/pharmacology
- Phosphorylation
- Potassium/chemistry
- Precipitin Tests
- Protein Isoforms
- Protein Kinase C/metabolism
- Pyrrolidinones/pharmacology
- Rats
- Rats, Sprague-Dawley
- Reverse Transcriptase Polymerase Chain Reaction
- Time Factors
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Affiliation(s)
- César Cárdenas
- Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago 7, Chile
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13
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Ma T, Qi QH, Xu J, Dong ZL, Yang WX. Signal pathways involved in emodin-induced contraction of smooth muscle cells from rat colon. World J Gastroenterol 2004; 10:1476-9. [PMID: 15133857 PMCID: PMC4656288 DOI: 10.3748/wjg.v10.i10.1476] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the effects induced by emodin on single smooth muscle cells from rat colon in vitro, and to determine the signal pathways involved.
METHODS: Cells were isolated from the muscle layers of Wistar rat colon by enzymatic digestion. Cell length was measured by computerized image micrometry. Intracellular Ca2+ ([Ca2+]i) signals were studied using the fluorescent Ca2+ indicator fluo-3 and confocal microscopy. PKCα distribution at rest state or after stimulation was measured with immunofluorescence confocal microscopy.
RESULTS: (1) Emodin dose-dependently caused colonic smooth muscle cells contraction; (2) emodin induced an increase in intracellular Ca2+ concentration; (3) the contractile responses induced by emodin were respectively inhibited by preincubation of the cells with ML-7 (an inhibitor of MLCK) and calphostin C (an inhibitor of PKC); and (4) Incubation of cells with emodin caused translocation of PKCα from cytosolic area to the membrane.
CONCLUSION: Emodin has a direct contractile effect on colonic smooth muscle cell. This signal cascade induced by emodin is initiated by increased [Ca2+]i and PKCα translocation, which in turn lead to the activation of MLCK and the suppression of MLCP. Both of them contribute to the emodin-induced contraction.
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Affiliation(s)
- Tao Ma
- Department of Surgery, General Hospital of Tianjin Medical University, Tianjin 300052, China.
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14
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Dykes AC, Fultz ME, Norton ML, Wright GL. Microtubule-dependent PKC-alpha localization in A7r5 smooth muscle cells. Am J Physiol Cell Physiol 2003; 285:C76-87. [PMID: 12637266 DOI: 10.1152/ajpcell.00515.2002] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Using laser scanning confocal, fluorescence resonance energy transfer (FRET), and atomic force (AFM) microscopy, we investigated association of protein kinase C (PKC)-alpha with microtubules during stimulus-induced relocalization in A7r5 smooth muscle cells. Confocal microscopy with standard immunostaining techniques confirmed earlier observations that colchicine disruption of microtubules blocked PKC-alpha localization in the perinuclear region of the cell caused by phorbol 12,13-dibutyrate (PDBu; 10-6M). Dual immunostaining suggested colocalization of PKC-alpha and beta-tubulin in both unstimulated and PDBu-treated cells. This finding was verified by FRET microscopy, which indicated that association of PKC-alpha was heterogeneous in distribution and confined primarily to microtubules in the perinuclear region. FRET analysis further showed that association between the molecules was not lost during colchicine-induced dissolution of microtubules, suggesting formation of tubulin-PKC-alpha complexes in the cytosol. Confocal imaging indicated that perinuclear microtubular structure was more highly sensitive to colchicine dissolution than other regions of the cell. Topographic imaging of fixed cells by AFM indicated a well-defined elevated structure surrounding the nucleus that was absent in colchicine-treated cells. It was calculated that the volume of the nuclear sleevelike structure of microtubules increased approximately fivefold in PDBu-treated cells, suggesting a probable increase in microtubular mass. In light of PKC-alpha localization, increased colchicine sensitivity, and their volume change in stimulated cells, the results suggest that perinuclear microtubules form a specialized structure that may be more dynamically robust than in other regions of the cell. PKC-alpha could contribute to this dynamic activity. Alternatively, perinuclear microtubules could act as a scaffold for regulatory molecule interaction at the cell center.
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Affiliation(s)
- A C Dykes
- Joan Edwards School of Medicine, Marshall University, Huntington, WV 25704, USA
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15
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Gimona M, Kaverina I, Resch GP, Vignal E, Burgstaller G. Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells. Mol Biol Cell 2003; 14:2482-91. [PMID: 12808045 PMCID: PMC194896 DOI: 10.1091/mbc.e02-11-0743] [Citation(s) in RCA: 130] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2002] [Revised: 12/11/2002] [Accepted: 01/30/2003] [Indexed: 01/04/2023] Open
Abstract
Phorbol ester induces actin cytoskeleton rearrangements in cultured vascular smooth muscle cells. Calponin and SM22 alpha are major components of differentiated smooth muscle and potential regulators of actin cytoskeleton interactions. Here we show that actin fibers decorated with h1 CaP remain stable, whereas SM22 alpha-decorated actin bundles undergo rapid reorganization into podosomes within 30 min of PDBu exposure. Ectopic expression of GFP alpha-actinin had no effect on the stability of the actin cytoskeleton and alpha-actinin was transported rapidly into PDBu-induced podosomes. Our results demonstrate the involvement of CaP and SM22 alpha in coordinating the balance between stabilization and dynamics of the actin cytoskeleton in mammalian smooth muscle. We provide evidence for the existence of two functionally distinct actin filament populations and introduce a molecular mechanism for the stabilization of the actin cytoskeleton by the unique actin-binding interface formed by calponin family-specific CLIK23 repeats.
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Affiliation(s)
- Mario Gimona
- Institute of Molecular Biology, Department of Cell Biology, Austrian Academy of Sciences, A-5020 Salzburg, Austria.
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16
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Ganitkevich V, Hasse V, Pfitzer G. Ca2+-dependent and Ca2+-independent regulation of smooth muscle contraction. J Muscle Res Cell Motil 2003; 23:47-52. [PMID: 12363284 DOI: 10.1023/a:1019956529549] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
An increase in the cytosolic Ca2+ concentration is a prerequisite in activation of contractile activity of smooth muscle. The shape of the Ca2+-signal is determined by spatial distribution and kinetics of Ca2+-binding sites in the cell. The increase in cytosolic Ca2+ activates myosin light chain kinase (MLCK) which in turn phosphorylates the regulatory light chains of myosin II. This Ca2+-dependent MLC20 phosphorylation is modulated in a Ca2+-independent manner by inhibiting the constitutive active myosin light chain phosphatase mediated by the monomeric GTPase Rho and the Rho-associated kinase as well as protein kinase C or by increasing its activity through cGMP. Furthermore, the activity of MLCK may be decreased due to phosphorylation by CaM kinase II and perhaps p21 activated protein kinase. Hence, smooth muscle tone appears to be regulated by a network of activating and inactivating intracellular signaling cascades which not only show a temporal but also a spatial activation pattern.
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