Roman Ginnan

Interplay Between Calcium and Reactive Oxygen/Nitrogen Species: An Essential Paradigm for Vascular Smooth Muscle Signaling

Signaling cascades initiated or regulated by calcium (Ca(2+)), reactive oxygen (ROS), and nitrogen (RNS) species are essential to diverse physiological and pathological processes in vascular smooth muscle. Stimuli-induced changes in intracellular Ca(2+) regulate the activity of primary ROS and RNS, producing enzymes including NADPH oxidases (Nox) and nitric oxide synthases (NOS). At the same time, alteration in intracellular ROS and RNS production reciprocates through redox-based post-translational modifications altering Ca(2+) signaling networks. These may include Ca(2+) pumps such as sarcoplasmic endoplasmic reticulum Ca(2+)-ATPase (SERCA), voltage-gated channels, transient receptor potential canonical (TRPC), melastatin2 (TRPM2), and ankyrin1 (TRPA1) channels, store operated Ca(2+) channels such as Orai1/stromal interaction molecule 1 (STIM1), and Ca(2+) effectors such as Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In this review, we summarize and highlight current experimental evidence supporting the idea that cross-talk between Ca(2+) and ROS/RNS may represent a well-integrated signaling network in vascular smooth muscle.

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases.

Inflammation plays a critical role in promoting smooth muscle migration and proliferation during vascular diseases such as postangioplasty restenosis and atherosclerosis. Another common feature of many vascular diseases is the contribution of reactive oxygen (ROS) and reactive nitrogen (RNS) species to vascular injury. Primary sources of ROS and RNS in smooth muscle are several isoforms of NADPH oxidase (Nox) and the cytokine-regulated inducible nitric oxide (NO) synthase (iNOS). One important example of the interaction between NO and ROS is the reaction of NO with superoxide to yield peroxynitrite, which may contribute to the pathogenesis of hypertension. In this review, we discuss the literature that supports an alternate possibility: Nox-derived ROS modulate NO bioavailability by altering the expression of iNOS. We highlight data showing coexpression of iNOS and Nox in vascular smooth muscle demonstrating the functional consequences of iNOS and Nox during vascular injury. We describe the relevant literature demonstrating that the mitogen-activated protein kinases are important modulators of proinflammatory cytokine-dependent expression of iNOS. A central hypothesis discussed is that ROS-dependent regulation of the serine/threonine kinase protein kinase Cdelta is essential to understanding how Nox may regulate signaling pathways leading to iNOS expression. Overall, the integration of nonphagocytic NADPH oxidase with cytokine signaling in general and in vascular smooth muscle in particular is poorly understood and merits further investigation.

CaM kinase IIδ2-dependent regulation of vascular smooth muscle cell polarization and migration

Previous studies indicate involvement of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vascular smooth muscle (VSM) cell migration. In the present study, molecular loss-of-function studies were used specifically to assess the role of the predominant CaMKII delta2 isoform on VSM cell migration using a scratch wound healing assay. Targeted CaMKII delta2 knockdown using siRNA or inhibition of activity by overexpressing a kinase-negative mutant resulted in attenuation of VSM cell migration. Temporal and spatial assessments of kinase autophosphorylation indicated rapid and transient activation in response to wounding, in addition to a sustained activation in the leading edge of migrating and spreading cells. Furthermore, siRNA-mediated suppression of CaMKII delta2 resulted in the inhibition of wound-induced Rac activation and Golgi reorganization, and disruption of leading edge morphology, indicating an important function for CaMKII delta2 in regulating VSM cell polarization. Numerous previous reports link activation of CaMKII to ERK1/2 signaling in VSM. Wound-induced ERK1/2 activation was also found to be dependent on CaMKII; however, ERK activity did not account for effects of CaMKII in regulating Golgi polarization, indicating alternative mechanisms by which CaMKII affects the complex events involved in cell migration. Wounding a VSM cell monolayer results in CaMKII delta2 activation, which positively regulates VSM cell polarization and downstream signaling, including Rac and ERK1/2 activation, leading to cell migration.

Calcium/Calmodulin-dependent Protein Kinase II Delta 6 (CaMKIIδ6) and RhoA Involvement in Thrombin-induced Endothelial Barrier Dysfunction

Multiple Ca2+ release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca2+ signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKIIδ6 isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nm thrombin maximally increased CaMKIIδ6 activation assessed by Thr287 autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIδ (siCaMKIIδ) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nm thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIδ inhibited 2.5 nm thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKIIδ6 mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIδ6/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.

MEF2 is regulated by CaMKIIδ2 and a HDAC4-HDAC5 heterodimer in vascular smooth muscle cells.

VSMCs (vascular smooth muscle cells) dedifferentiate from the contractile to the synthetic phenotype in response to acute vascular diseases such as restenosis and chronic vascular diseases such as atherosclerosis, and contribute to growth of the neointima. We demonstrated previously that balloon catheter injury of rat carotid arteries resulted in increased expression of CaMKII (Ca(2+)/calmodulin-dependent protein kinase) IIδ(2) in the medial wall and the expanding neointima [House and Singer (2008) Arterioscler. Thromb. Vasc. Biol. 28, 441-447]. These findings led us to hypothesize that increased expression of CaMKIIδ(2) is a positive mediator of synthetic VSMCs. HDAC (histone deacetylase) 4 and HDAC5 function as transcriptional co-repressors and are regulated in a CaMKII-dependent manner. In the present paper, we report that endogenous HDAC4 and HDAC5 in VSMCs are activated in a Ca(2+)- and CaMKIIδ(2)-dependent manner. We show further that AngII (angiotensin II)- and PDGF (platelet-derived growth factor)-dependent phosphorylation of HDAC4 and HDAC5 is reduced when CaMKIIδ(2) expression is suppressed or CaMKIIδ(2) activity is attenuated. The transcriptional activator MEF2 (myocyte-enhancer factor 2) is an important determinant of VSMC phenotype and is regulated in an HDAC-dependent manner. In the present paper, we report that stimulation of VSMCs with ionomycin or AngII potentiates MEF2s ability to bind DNA and increases the expression of established MEF2 target genes Nur77 (nuclear receptor 77) (NR4A1) and MCP1 (monocyte chemotactic protein 1) (CCL2). Suppression of CaMKIIδ(2) attenuates increased MEF2 DNA-binding activity and up-regulation of Nur77 and MCP1. Finally, we show that HDAC5 is regulated by HDAC4 in VSMCs. Suppression of HDAC4 expression and activity prevents AngII- and PDGF-dependent phosphorylation of HDAC5. Taken together, these results illustrate a mechanism by which CaMKIIδ(2) mediates MEF2-dependent gene transcription in VSMCs through regulation of HDAC4 and HDAC5.

Specificity of G protein alpha-gamma subunit interactions. N-terminal 15 amino acids of gamma subunit specifies interaction with alpha subunit.

The existence of multiple α, β, and subunits raises questions regarding the assembly of particular G proteins. Based on the results of a previous study (Rahmatullah, M., and Robishaw, J. D.(1994) J. Biol. Chem. 269, 3574-3580), we hypothesized that the assembly of G proteins may be determined by the interactions of the more structurally diverse α and subunits. This hypothesis was confirmed in the present study by showing striking differences in the abilities of the 1 and 2 subunits to interact with the the αo subunit. Chimeras of the 1 and 2 subunits were used to delineate which region is responsible. Support for the importance of the N-terminal region of the subunit comes from our observations that 1) the 2 subunit and the chimera bound strongly to the αo-agarose matrix, but the 1 subunit and the chimera bound weakly, if at all; 2) an N-terminal peptide made to the 2 subunit blocked the binding of the chimera to the αo-agarose matrix; 3) both the chimera and the N-terminal peptide were able to partially protect the αo subunit against tryptic cleavage; and 4) the chimera, but not the chimera, supported ADP-ribosylation of the αo subunit.

CaMKIIδ-dependent Inhibition of cAMP-response Element-binding Protein Activity in Vascular Smooth Muscle

Background: CaMKII is implicated in both positive and negative regulation of cAMP-response element-binding protein (CREB) activity. Results: CaMKIIδ increases CREB-Ser142 phosphorylation and inhibits binding of CREB to Sik1 and Rgs2 promoters in vascular smooth muscle (VSM). Conclusion: In VSM CaMKIIδ negatively regulates CREB activity and target gene transcription by phosphorylating CREB-Ser142. Significance: Negative regulation of CREB by CaMKIIδ is a mechanism mediating Ca2+ signal-dependent regulation of gene transcription in VSM. One transcription factor mediator of Ca2+-signals is cAMP response element-binding protein (CREB). CREB expression and/or activity negatively correlates with vascular smooth muscle (VSM) cell proliferation and migration. Multifunctional Ca2+/calmodulin-dependent protein kinases, including CaMKII, have been demonstrated to regulate CREB activity through both positive and negative phosphorylation events in vitro, but the function of CaMKII as a proximal regulator of CREB in intact cell systems, including VSM, is not clear. In this study, we used gain- and loss-of-function approaches to determine the function of CaMKIIδ in regulating CREB phosphorylation, localization, and activity in VSM. Overexpression of constitutively active CaMKIIδ specifically increased CREB phosphorylation on Ser142 and silencing CaMKIIδ expression by siRNA or blocking endogenous CaMKII activity with KN93 abolished thrombin- or ionomycin-induced CREB phosphorylation on Ser142 without affecting Ser133 phosphorylation. CREB-Ser142 phosphorylation correlated with transient nucleocytoplasmic translocation of CREB. Thrombin-induced CREB promoter activity, CREB binding to Sik1 and Rgs2 promoters, and Sik1/Rgs2 transcription were enhanced by a kinase-negative CaMKIIδ2 (K43A) mutant and inhibited by a constitutively active (T287D) mutant. Taken together, these studies establish negative regulation of CREB activity by endogenous CaMKIIδ-dependent CREB-Ser142 phosphorylation and suggest a potential mechanism for CaMKIIδ/CREB signaling in modulating proliferation and migration in VSM cells.

Ca2+/calmodulin-dependent protein kinase II-γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling

Vascular smooth muscle (VSM) expresses calcium/calmodulin‐dependent protein kinase II (CaMKII)‐δ and ‐γ isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIγ function in vascular remodeling after injury. CaMKIIγ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIγC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell‐cycle inhibitor p21 (>2‐fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIγ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIγ in smooth muscle resulted in a 20‐fold increase in neointimal area, with a 3‐fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIγ overexpression induced p53 mRNA (1.7 fold) and protein (1.8‐fold) expression; induced the p53 target gene p21 (3‐fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury‐induced vasculoproliferative response and that CaMKIIγ and ‐δ isoforms have non‐equivalent, opposing functions.—Saddouk, F. Z., Sun, L.‐Y., Liu, Y. F., Jiang, M., Singer, D. V., Backs, J., Van Riper, D., Ginnan, R., Schwarz, J. J., Singer, H. A., Ca2+/calmodulin‐dependent protein kinase II‐γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling. FASEB J. 30, 1051–1064 (2016). www.fasebj.org

MicroRNA-30 inhibits neointimal hyperplasia by targeting Ca(2+)/calmodulin-dependent protein kinase IIδ (CaMKIIδ).

The multifunctional Ca2+/calmodulin-dependent protein kinase II δ-isoform (CaMKIIδ) promotes vascular smooth muscle (VSM) proliferation, migration, and injury-induced vascular wall neointima formation. The objective of this study was to test if microRNA-30 (miR-30) family members are endogenous regulators of CaMKIIδ expression following vascular injury and whether ectopic expression of miR-30 can inhibit CaMKIIδ-dependent VSM cell function and neointimal VSM hyperplasia induced by vascular injury. The CaMKIIδ 3′UTR contains a consensus miR-30 binding sequence that is highly conserved across species. A significant decrease in miR-30 family members and increase in CaMKIIδ2 protein expression, with no change in CaMKIIδ mRNA expression, was observed in medial layers of VSM 7 days post-injury. In vitro, overexpression of miR-30c or miR-30e inhibited CaMKIIδ2 protein expression by ~50% in cultured rat aortic VSM cells, and inhibited VSM cell proliferation and migration. In vivo, lenti-viral delivery of miR-30c into injured rat carotid arteries prevented the injury-induced increase in CaMKIIδ2. Furthermore, neointima formation was dramatically inhibited by lenti-viral delivery of miR-30c in the injured medial smooth muscle. These studies define a novel mechanism for regulating CaMKIIδ expression in VSM and provide a new potential therapeutic strategy to reduce progression of vascular proliferative diseases, including atherosclerosis and restenosis.

Vascular Smooth Muscle Cell Motility Is Mediated by a Physical and Functional Interaction of Ca2+/Calmodulin-dependent Protein Kinase IIδ2 and Fyn

Background: Increased vascular smooth muscle cell motility results in neointimal formation. Results: CaMKIIδ2 and Fyn physically interact, and CaMKIIδ2 activity regulates complex formation, Fyn activity, and motility. Conclusion: CaMKIIδ2 and Fyn regulate the motility of VSM cells due to their physical and functional interaction. Significance: Coupling CaMKIIδ2 and Fyn in VSM cells provides a defined mechanism for increases in intracellular calcium to activate tyrosine kinases required for cell motility. In vascular smooth muscle (VSM) cells, Ca2+/calmodulin-dependent protein kinase IIδ2 (CaMKIIδ2) activates non-receptor tyrosine kinases and EGF receptor, with a Src family kinase as a required intermediate. siRNA-mediated suppression of Fyn, a Src family kinase, inhibited VSM cell motility. Simultaneous suppression of both Fyn and CaMKIIδ2 was non-additive, suggesting coordinated regulation of cell motility. Confocal immunofluorescence microscopy indicated that CaMKIIδ2 and Fyn selectively (compared with Src) co-localized with the Golgi in quiescent cultured VSM cells. Stimulation with PDGF resulted in a rapid (<5 min) partial redistribution and co-localization of both kinases in peripheral membrane regions. Furthermore, CaMKIIδ2 and Fyn selectively (compared with Src) co-immunoprecipitated, suggesting a physical interaction in a signaling complex. Stimulation of VSM cells with ionomycin, a calcium ionophore, resulted in activation of CaMKIIδ2 and Fyn and disruption of the complex. Pretreatment with KN-93, a pharmacological inhibitor of CaMKII, prevented activation-dependent disruption of CaMKIIδ2 and Fyn, implicating CaMKIIδ2 as an upstream mediator of Fyn. Overexpression of constitutively active CaMKII resulted in the dephosphorylation of Fyn at Tyr-527, which is required for Fyn activation. Taken together, these data demonstrate a dynamic interaction between CaMKIIδ2 and Fyn in VSM cells and indicate a mechanism by which CaMKIIδ2 and Fyn may coordinately regulate VSM cell motility.