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Calreticulin Regulation of Lung Endothelial NOS Activity

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Increased synthesis of a multifunctional calcium binding protein calreticulin has been reported under diverse physiologic and pathophysiologic conditions in various tissues in cluding stimulation of vascular endothelium by angiotensin IV (Ang-IV), a metabolic product of Ang-II. Ang-IV-mediated early and sustained activation of lung endothelial cell nitric oxide synthase (eNOS) is mediated through increased mobilization of intracellular calcium and by increased expression of calreticulin. Immunoprecipitation and confocal imaging studies revealed that eNOS and calreticulin are co-localized in Ang-IV-stimulated lung endothelial cells. Catalytic activity of purified eNOS in the absence of calmodulin was increased in a concentration-dependent fashion by calreticulin. The studies monitoring the effect of calreticulin on the rate of electron transfer from the reductase to the oxygenase domain of eNOS revealed that the calreticulin/eNOS interaction promotes electron transfer and mimics eNOS activation in the absence of exogenous calmodulin and enhances electron transfer and the catalytic activity of eNOS in the presence of calmodulin. Thus, calreticulin/eNOS protein:protein interaction enhances the rate of electron transfer, a critical event in the regulation of the catalytic activity of eNOS.

Introduction

Vascular endothelium is a metabolically active tissue that regulates pulmonary and systemic vascular tone through metabolism and/or generation and release of vasoactive mediators such as angiotensin peptides and nitric oxide (NO).1,2 For example, lung endothelial cells metabolize angiotensin I (Ang- I) —> Ang-II —> Ang-III —> Ang-IV by catalytic action of Ang-converting enzyme, aminopeptidase A, and aminopeptidase M, respectively.3 A number of recent studies have identified and characterized the presence of Ang-IV-specific receptors in a variety of tissues including lung.48 Receptor-mediated activation of signal transduction pathways are know to result in direct and/or G protein-coupled activation of phospholipases C and D (PLC/PLD), leading to a rapid increase in intracellular calcium release. Increased intracellular calcium is regulated by multiple mechanisms. One of the most ubiquitous of these mechanisms involves the PLC-phosphatidylinositol 3,4,5-triphosphate (PIP3) pathway, which releases calcium from intracellular stores, namely the endoplasmic reticulum (ER).911 These signaling mechanisms are associated with activation of several calcium-dependent enzymes including eNOS.12,13 Agonist-mediated intracellular calcium mobilization can also play a critical role in modulation of cell function such as the regulation of ER resident calcium binding proteins including calreticulin.1416 Metabolic pathways of Ang and Ang-IV receptor-mediated activation of signaling events leading to activation of eNOS and expression of calreticulin are shown in Figure 19.1.

Figure 1. Angiotensin (Ang) peptide metabolism and Ang-IV receptor-mediated signaling pathway leading to activation of lung endothelial cell eNOS, calreticulin expression, and nitric oxide (NO)/guanosine 3'5'-cyclic monophosphate (cGMP)-linked vasorelaxation.

Figure 1

Angiotensin (Ang) peptide metabolism and Ang-IV receptor-mediated signaling pathway leading to activation of lung endothelial cell eNOS, calreticulin expression, and nitric oxide (NO)/guanosine 3'5'-cyclic monophosphate (cGMP)-linked vasorelaxation. Phospholipase (more...)

The catalytic activity of eNOS is calcium- and calmodulin-dependent and is transiently activated by agonist-mediated signaling pathways that increase intracellular calcium mobilization. 1720 The catalytic activity of eNOS is regulated by multiple post-transcriptional mechanisms including protein:protein interaction. The present review focuses on molecular events involved in Ang-IV-mediated regulation of the catalytic activity of eNOS in context with intracellular calcium mobilization, calreticulin expression, and calreticulin:eNOS protein:protein interaction in lung endothelial cells.

Biochemistry and Physiology of Ang-IV

The vascular endothelium processes a variety of biologically active substances in the circulation, including Ang-IV, a metabolic product of the renin-angiotensin system as shown below in animals and humans.2123

Angiotensinogen NH2-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-COOH

_------------------------------------------------------------------------------_ Renin

Ang-I NH2-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-COOH

_------------------------------------------------------_ ACE

Ang-II NH2-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-COOH

_--------------------_ Aminopeptidase A

Ang-III NH2-Arg-Val-Tyr-Ile-His-Pro-Phe-COOH

_--------------------_ Aminopeptidase M

Ang-IV NH2-Val-Tyr-Ile-His-Pro-Phe-COOH

The presence of Ang-IV-specific receptor sites has been identified in several extra-pulmonary tissues including brain, heart, and kidney2426 as well as in cultured coronary microvascular, aortic, and lung endothelial cells.3,7,8 Although the functional role of Ang-IV is not fully defined, it has been reported that Ang-IV increases blood flow in brain and kidney27,28 and causes endothelium-dependent vasodilation of porcine pulmonary artery and rabbit brain arterioles.3,29 In fact, Ang-IV has been shown to activate the lung endothelial cell isoform of nitric oxide (NO) synthase (eNOS) by a receptor-mediated pathway, leading to increases in NO release, production of guanosine 3',5'-cyclic monophosphate (cGMP), and NO-cGMP-mediated pulmonary artery vasodilation.3,11 Agonist-mediated activation of signaling pathways that increase mobilization of intracellular calcium is known to transiently activate eNOS.1 However, Ang-IV-stimulation of lung endothelial cells results in transient and sustained activation of eNOS.3 Agonist-mediated intracellular calcium mobilization is also known to play a critical role as a second messenger in the regulation of a variety of functions, including cell proliferation, gene expression, protein expression, and protein:protein interaction.1416 Depletion of calcium from the ER under diverse physiologic and pathophysiologic conditions can facilitate a process that results in upregulation of a group of calcium binding proteins, including calreticulin.11,3037 Ang-IV-mediated transient and sustained activation of eNOS appear to be associated with intracellular calcium mobilization as well as with increased expression of the calcium binding protein calreticulin.

Calreticulin Expression and Function: Role of Cell Stimulation/Injury

Calreticulin is a 60-kDa, highly conserved ubiquitous calcium binding protein of the endoplasmic reticulum (ER) membrane with three distinct structural and functional domains (N-/P-/C-domains). The C- and P-domains consist of low and high affinity calcium binding sites, respectively3840 (Fig. 19.2). The N-terminal domain of calreticulin does not bind calcium, but it is believed to be one of the major sites of protein:protein interaction in diverse cell types that results in functional modulation of the binding proteins.4145 C-terminal KDEL (Lys-Asp-Glu-Leu) ER retention signal is responsible for retention of calreticulin in the ER lumen.1,2 Despite the presence of the KDEL ER retention signal in the C-terminal, calreticulin is also localized in various subcellular compartments, including the cytoplasm, the nucleus, and the cell surface membrane,4649 and is also know to be secreted from various mammalian cells including vascular endothelium.5052 Agonist or chemical-induced depletion of calcium from the ER or sarcoplasmic reticulum, known intracellular calcium storage sites, can facilitate a process that results in increased synthesis of a group of ER resident proteins including calreticulin. For example, it is increasingly clear from a number of studies that up-regulation of calreticulin expression occurs in response to a variety of cellular stress/injury or stimuli including amino acid deprivation,30,31 heat shock,32,33 exposure to heavy metals34, irradiation and viral infection,35 cytotoxic T-cell stimulation,36 inhibition of N-linked glucosylation by tunicamycin,37 and depletion of ER calcium stores due to stimulation by agonists such as Ang-IV.11,52 As such, the wide distribution and varying degree of expression of calreticulin under diverse pathophysiologic conditions provide unique features enabiling calreticulin to be associated with a broad array of cellular functions.

Figure 2. Structure of human calreticulin and its N-/P-/C-domains.

Figure 2

Structure of human calreticulin and its N-/P-/C-domains.

In the last 10 years, support for important physiological roles for calreticulin has been documented by numerous studies. For example, calreticulin has been recognized as a multifunctional protein involved in gene expression, receptor regulation, cell adhesion, autoimmunity, apoptosis, vascular remodeling and angiogenesis, endothelial cell NO release, viral RNA binding and replication, cytotoxic T cell as well as neutrophil activation, and modulation of signaling events leading to altered calcium homeostasis.38,39,46,5361 The role of calreticulin in the regulation of vascular endothelial-dependent responses such as NO release, remodeling, and angiogenesis are of particular interest. In this context, we focused on Ang-IV-mediated responses involving intracellular calcium mobilization, calreticulin expression, and NOS activation in lung vascular endothelial cells.

Structure, Function, and Regulation of eNOS Activity

NO is synthesized by mammalian cells by three major isoforms of NOS namely, constitutive endothelial and neuronal isoforms eNOS and nNOS, respectively, and an inducible isoform iNOS.17,18 These isomers are members of a novel family of enzymes containing heme oxygenase and cytochrome P-450 reductase domains that catalyze the oxidative metabolism of cationic amino acid L-arginine to generate L-citrulline, NO, and H2O.19,20 The amino terminal oxygenase domain of eNOS protein contains bindings sites for heme, tetrahydrobiopterin, zinc, and L-arginine and the carboxy-terminal reductase domain consists of sites for NADPH, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and electron transfer control element (ETE) (Fig. 19.3).1720,62,63 These two domains are linked by a calcium/calmodulin binding region. The catalytic activity of eNOS is dependent on the rate of electron transfer from NADPH in the reductase domain to the oxygenase domain, a required process for the oxidative metabolism of L-arginine to generate NO.62,63

Figure 3. Oxygenase and reductase domains of eNOS: binding site or distinct region for calmodulin (CaM), electron transfer control element (ETE), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and NADPH in the reductase domain, and heme, tertahydrobiopterin (BH4), zinc (Zn), and L-arginine (L-Arg) in the oxygenase domain.

Figure 3

Oxygenase and reductase domains of eNOS: binding site or distinct region for calmodulin (CaM), electron transfer control element (ETE), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and NADPH in the reductase domain, and heme, tertahydrobiopterin (more...)

The catalytic activity of eNOS is regulated by transcriptional and post-transcriptional mechanisms. For example, shear stress as well as bioactive agents such as cyclosporine A, vascular endothelial growth factor, insulin, oxidized LDL, lysophosphatidylcholine, and transforming growth factor-_ are known to upregulate eNOS expression through modulation of putative cis-elements including GATA and Sp1 motifs and/or specific regulatory elements such as activator protein-1, nuclear factor-1 element, and sterol regulatory element in the eNOS gene.13,6569

The catalytic activity of eNOS is also regulated by multiple post-transcriptional mechanisms involving a variety of factors including phosphorylation/dephosphorylation state, active site cysteine modulation in oxygenase and reductase domains, myristylation/palmitolation and subcellular localization, and by protein:protein interaction with calmodulin, caveolin, heat shock protein 90, and components of the cytoskeleton.7076,79 All of these known mechanisms involve a limited or transient, but not sustained, activation of eNOS through modulation of phosphorylation of critical amino acid residues or by translocation of eNOS within intracellular domains, as the Golgi, ER, and plasmalemmal caveolae, through calcium mobilization that promotes eNOS caveolin dissociation and calmodulin association.7679 Although calmodulin is an essential regulator of inter- and intra-domain electron transfer, the catalytic activity of eNOS can be transiently facilitated through increased intracellular calcium release (calcium-dependent) or at basal levels of calcium (calcium-independent pathways) by various stimuli including agonists, fluid shear stress, or growth factors.6971 A recent report indicates that mutation of serine 1179 to the negatively charged aspartate increases eNOS acitivity by increasing electron transfer at basal calcium levels in purified bovine

eNOS.80 Thus, it is clear from these reports that the catalytic activity of eNOS is greatly influenced by critical events such as calcium modulation, phosphorylation of target sites, and calmodulin binding characteristics which in turn appear to regulate the rate of electron transfer which is controlled by ETE.

A number of recent reports using molecular biology approaches and purified NOS preparations have characterized the role of ETE present in the reductase domain of eNOS and nNOS but absent in iNOS for its putative autoinhibitory role in electron transfer and its control of catalytic activity of NOS isoforms.62,63 The fact that deletion of ETE from human or bovine eNOS does not alter the structure or active center of eNOS but drastically changes its dependence on calcium or calmodulin binding with enhanced electron transfer and catalytic activity provides experimental evidence that ETE plays a critical role in the regulation of eNOS activity. 62,63 However, nothing is known about the mechanisms by which ETE regulates electron transfer and whether endogenously generated factors such as increased expression of calreticulin and modulation of ETE's function affect eNOS activity.

Ang-IV eNOS Activation: Link to Cellular Calcium and Calreticulin

As stated above the catalytic activity of eNOS is regulated by multiple mechanisms involving transcriptional and post-transcriptional processes. Ang-IV-mediated activation of eNOS is regulated by a post-transcriptional mechanism since the expression of eNOS protein in 4 to 12 hr Ang-IV-stimulated lung endothelial cells was comparable to that in control cells.3 Endothelial cell release of NO is enhanced by receptor-mediated agonists such as acetylcholine, bradykinin, histamine, serotonin, and substance P via signal transduction-mediated transient activation of eNOS.1 However, unlike other agonists described to date, Ang-IV-receptor-mediated activation of eNOS persists for up to 12 hr (Fig. 19.4A). Ang-IV-stimulation in the absence of extracellular calcium causes a rapid increase in intracellular calcium release in a dose-dependent manner which is significantly greater than the basal level of calcium.11 This Ang-IV-mediated activation of eNOS is clearly dependent on intracellular calcium release since pre-incubation of cells with the calcium chelator BAPTA-AM blocked Ang-IV-mediated activation of eNOS (Fig. 19.4B).

Figure 4. Time-dependent activation of eNOS by Ang-IV (panel A) and the effect of the intracellular calcium chelator BAPTA-AM on Ang-IV activated eNOS (panel B) in lung endothelial cells.

Figure 4

Time-dependent activation of eNOS by Ang-IV (panel A) and the effect of the intracellular calcium chelator BAPTA-AM on Ang-IV activated eNOS (panel B) in lung endothelial cells. Cell monolayers were incubated in RPMI 1640 medium alone (control) and with (more...)

Although Ang-IV-stimulated transient activation of eNOS through intracellular mobilization of calcium is consistent with other agonist-mediated responses, Ang-IV-mediated sustained activation of eNOS is unique in the context of known post-transcriptional mechanisms regulating the catalytic activity of eNOS. Agonist and/or chemical-mediated depletion of ER calcium stores have been linked with increased expression of ER resident calcium binding proteins.3037,52 In addition, Ang-IV has been reported to induce protein synthesis of plasminogen activator inhibitor 1 in bovine aortic endothelial cells81 and calreticulin expression in porcine pulmonary artery endothelial cells.52 Ang-IV has also been reported to increase DNA and RNA synthesis in mammalian cells including lung endothelial cells.8284 Ang-IV-mediated expression of calreticulin was significantly increased as early as 2 hr and remained elevated for 6 hr in lung endothelial cells.52 As shown in Figure 19.5, increased levels of calreticulin were observed in the medium of cells stimulated with Ang-IV. Ang-IV-mediated intracellular calcium release is critical to the increased calreticulin expression as the calcium chelator BAPTA-AM blocked Ang-IV-induced expression of calreticulin in lung endothelial cells (Fig. 19.6). Thus, Ang-IV-mediated release of calcium from intracellular stores plays a major role in the regulation of eNOS activity and expression of calreticulin.

Figure 5. Ang-IV-stimulated expression of calreticulin increases its secretion into the extracellular medium.

Figure 5

Ang-IV-stimulated expression of calreticulin increases its secretion into the extracellular medium. Lung endothelial cell monolayers in RPMI 1640 containing 1μM Ang-IV or RPMI 1640 only (Con) were incubated for 2 hr at 37° C. After incubation, (more...)

Figure 6. Effect of the intracellular calcium chelator BAPTA-AM on Ang-IV-mediated calreticulin expression.

Figure 6

Effect of the intracellular calcium chelator BAPTA-AM on Ang-IV-mediated calreticulin expression. Lung endothelial cell monolayers were incubated in Tyrode solution without calcium with or without 50 _M BAPTA-AM for 30 min and were then incubated for (more...)

Some of the distinct characteristics of calreticulin include in vitro and in vivo peptide-binding and/or protein:protein interaction as well as its ability to bind specifically to vascular endothelium with significant implications for the regulation of vascular remodeling and angiogenesis. 4244,52,55,58,59,85,86 We postulated that these responses are likely are mediated, at least in part, through modulation of eNOS function and NO release. To test this, we examined the potential role of calreticulin in the regulation of the catalytic activity of eNOS, focusing on protein:protein interaction and the comparative effects of calmodulin and calreticulin on the rate of electron transfer from the reductase to the oxygenase domain of eNOS. To determine whether a protein:protein interaction exists between calreticulin and eNOS in intact lung endothelial cells, eNOS from control and Ang-IV-stimulated cells was immunoprecipitated using anti-eNOS monoclonal antibody, and the immunoprecipitates were analyzed for the presence of eNOS and calreticulin proteins. As shown in Figure 19.7, the presence of eNOS and calreticulin proteins in the immunoprecipitates indicates that eNOS is present in an immunoprecipitable complex with calreticulin in lung endothelial cells. To support this, a confocal imaging technique was used to determine whether calreticulin co-localizes with eNOS in Ang-IV-stimulated cells. As shown in Figure 19.8, the presence of eNOS (panel A) and calreticulin (panel B) were identified by independent labeling with respective antibodies. Panel C shows co-localized eNOS/calreticulin following double labeling with calreticulin and eNOS antibodies. Thus, interaction of calreticulin with eNOS protein may be responsible for modulation of the catalytic activity of eNOS.

Figure 7. Interaction between eNOS and calreticulin.

Figure 7

Interaction between eNOS and calreticulin. Lung endothelial cell monolayers in RPMI 1640 containing 1 μM Ang-IV or RPMI 1640 alone (control, C) were incubated for 4 hr at 37°C. After incubation, cell lysate protein (50 μg) or buffer (more...)

Figure 8. Confocal image of eNOS (panel A, red labeling), calreticulin (panel B, green labeling), and eNOS/calreticulin (panel C, yellow/orange color represents co-localization of eNOS/calreticulin) in Ang-IV-stimulated lung endothelial cells.

Figure 8

Confocal image of eNOS (panel A, red labeling), calreticulin (panel B, green labeling), and eNOS/calreticulin (panel C, yellow/orange color represents co-localization of eNOS/calreticulin) in Ang-IV-stimulated lung endothelial cells. Blue color represents (more...)

Previous work by several groups has clearly demonstrated that a protein:protein interaction with calmodulin, a calcium-binding protein, is absolutely critical for the regulation of eNOS activity in the presence of elevated levels of intracellular calcium (calcium-dependent) and in the presence of basal levels of calcium (calcium-independent pathway).13,70,71 In addition, calmodulin is also required to shuttle the electrons transferred under the control of the electron transfer element from the reductase to the oxygenase domain. For example, in the absence of calmodulin the electron transfer process is abolished resulting in near total inhibition of the catalytic activity of eNOS.6264 To identify possible competitive effects between calreticulin and calmodulin on eNOS activity, the catalytic activity of purified eNOS was determined in the presence of increasing or decreasing concentrations of calmodulin and calreticulin. As shown in Figure 19.9A, in the absence of calreticulin, the catalytic activity of eNOS was increased by calmodulin in a concentration-dependent fashion with maximal activation observed at 50 nM calmodulin. In the absence of calmodulin, 60 nM calreticulin resulted in only limited activation of eNOS (Fig. 19.9B), whereas calreticulin-to-calmodulin ratios of 60:10, 50:20, 40:30, and 30:40 significantly increased the catalytic activity of eNOS in comparison to eNOS activities at 10, 20, 30, and 40 nM calmodulin alone. This indicates that calreticulin enhances the catalytic activity of eNOS in the presence of calmodulin. However, the more interesting observation is that calreticulin alone at 60 nM increases the catalytic activity of eNOS. This suggests that a protein:protein interaction of calreticulin and eNOS, specifically at the calmodulin binding site on eNOS, may be responsible for modulation of the catalytic activity of eNOS. Although the precise nature of the interaction between calreticulin and eNOS remains to be determined, it is possible that calreticulin can also bind at the site of the electron transfer control element in the reducase domain of eNOS to influence the rate of electron transfer and eNOS activity.

Figure 9. Effect of calreticulin-to-calmodulin ratio on catalytic activity of eNOS.

Figure 9

Effect of calreticulin-to-calmodulin ratio on catalytic activity of eNOS. A: purified eNOS was incubated in the absence of calreticulin but in the presence of increasing concentrations (10−70 nM) of calmodulin. B: purified eNOS was incubated with (more...)

To identify the involvement of the electron transfer control element in the regulation of eNOS activity in context with calreticulin/eNOS interaction and whether such interaction promotes electron transfer and enhances the catalytic activity of eNOS, the catalytic activity of NADPH cytochrome c reductase was monitored as an index of electron transfer from reductase domain to the oxygenase domain with and without the presence of calmodulin, calreticulin, or calmodulin + calreticulin. Figure 19.10 A and B show the effects of calmodulin, calreticulin, and calmodulin + calreticulin on the catalytic activities of eNOS and NADPH cytochrome c reductase, respectively. In the absence of calmodulin or calreticulin, the catalytic activities of eNOS and NADPH cytochrome c reductase are nearly zero. However, in the presence of calmodulin or calreticulin the activities of eNOS and NADPH cytochrome c reductase are significantly enhanced. Moreover, the presence of both calmodulin and calreticulin resulted in further enhancement of these activities. These results indicate that the calreticulin/eNOS interaction promotes electron transfer and mimics eNOS activation in the absence of exogenous calmodulin. The interaction of calreticulin and eNOS also enhances electron transfer and the catalytic activity of eNOS in the presence of calmodulin.

Figure 10. Calmodulin/calreticulin-mediated activities of eNOS (panel A) and NADPH cytochrome c reductase (panel B).

Figure 10

Calmodulin/calreticulin-mediated activities of eNOS (panel A) and NADPH cytochrome c reductase (panel B). For eNOS activity, purified eNOS (5 μg protein) was incubated in Tris-HCl buffer (50 mM, pH 7.4) containing calmodulin or calreticulin (30 (more...)

Concluding Remarks

Vascular endothelial eNOS-catalyzed release of NO plays a critical role mediating diverse physiologic and pathophysiologic events in animals and humans.87,88 Endothelium-derived NO regulates pulmonary and extra-pulmonary vascular function under physiologic conditions. The amount and the rate of NO release under various pathophysiologic conditions are believed to be major factors that determine NO-mediated physiologic responses such as vasorelaxation, angiogenesis, apoptosis, and cell proliferation and migration.8791 Multiple factors are responsible for the regulation of the catalytic activity of eNOS including post-transcriptional mechanisms such as protein:protein interaction and the regulation of electron transfer by the intra-molecular electron transfer control element in the reductase domain. The physiologic significance of Ang-IV-mediated intracellular calcium release, calreticulin expression, and eNOS is important in relation to NO biology and endothelium-dependent vascular function. Similarly, increased expression of calreticulin under diverse pathophysiologic conditions and calreticulin-mediated activation of eNOS may help restore impaired NO production due to diminished or reduced catalytic activity of eNOS associated with a variety of δ pulmonary diseases and/or injuries.

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