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* Department of Internal Medicine and Center for Human Genomics and
Department of Pathology, Wake Forest University Health Science, Winston-Salem, North Carolina; and
Departments of Physiology and Biochemistry, Queen’s University, Kingston, Ontario, Canada
1Correspondence: Center for Human Genomics, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA. E-mail: rpenn{at}wfubmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: G protein-coupled receptor • cysteinyl leukotriene • protein kinase C • desensitization • airway smooth muscle
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
and cardiovascular disease (3)
. Despite its physiological importance and the fact that this receptor was cloned over 7 yr ago (4
, 5)
, only one study to date has identified regulatory features of the CysLT1R. Naik et al. (6)
established that unlike most G protein-coupled receptors (GPCRs), whose agonist-induced responsiveness and endocytosis are regulated by GPCR kinases (GRKs) and arrestins (7)
, the CysLT1R is unique in that both agonist-induced desensitization and internalization are dependent primarily on protein kinase C (PKC) activity.
Because most studies of GPCR regulation involve analyses of overexpressed receptors in artificial expression systems such as COS or HEK293 cells, insight into the physiological significance of receptor regulation is often lacking. In only a handful of instances have paradigms of GPCR regulation been shown to be of physiological relevance through demonstration of their impact on cell function. The most compelling evidence comes from Koch, Rockman, and colleagues. Through a series of elegant studies using a combination of cell and in vivo systems, GRK-mediated desensitization of beta-adrenergic receptors in cardiomyocytes has been shown to not only mediate cardiac tachyphylaxis to infused or endogenous beta-agonist and diminish cardiac contractility but also constitutes a pathogenic mechanism of heart failure (8
9
10
11)
.
Because cellular functions are often the result of the integration of multiple signaling events, the influence of a specific mechanism regulating the GPCR locus on such functions can be constrained. Moreover, multiple, redundant mechanisms of receptor desensitization can exist, and signaling elements downstream of the receptor are also subject to regulation. Thus, the impact of receptor modifications and regulation of proximal signaling events on cellular functions can be offset or overshadowed by numerous other control systems. These factors, in addition to other homeostatic mechanisms that constrain responses of low abundance, endogenously expressed proteins in primary cell types, contribute to the difficulty in demonstrating the relevance of GPCR regulatory mechanisms that target the receptor locus.
In the present study, we demonstrate that PKC-mediated desensitization of the CysLT1R translates into the attenuation of leukotriene D4- (LTD4-) regulated cellular functions in multiple physiological systems. PKC inhibition is shown to enhance contraction of airway smooth muscle (ASM), as well as migration and chemokine production in monocytic cells in response to LTD4, whereas these same functions mediated by other receptors are either unaffected or slightly inhibited with PKC inhibition. Thus, PKC-dependent agonist-specific desensitization of the CysLT1R is a robust phenomenon of physiological relevance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Transgene expression was assessed by PCR analysis of total genomic DNA isolated from tail clips, as described previously (12)
. Ex vivo measurement of tracheal contraction and isolation of ASM cells were performed under protocols approved by the Animal Care and Use Committee at Wake Forest University Health Sciences. Mice were sacrificed by CO2 inhalation and tracheae were excised and cleaned of surrounding connective tissue. For contractile studies, tracheae were sectioned (horizontally) in half, and each section mounted onto a multiwire myograph system consisting of myograph chambers, a block interface for connecting to force transducers, and Powerlab (ADInstruments, Colorado Springs, CO) for data recording. Data collection and analysis were carried out using Chart5 software for Windows (ADInstruments). Tracheal rings were bathed in Krebs-Henseleit solution (pH 7.40–7.45) and maintained at 37°C with 5% CO2 and 95% O2 with frequent changing of the solution. The tracheal segments were allowed to stabilize and subsequently set at a preload tension of 0.5 g. The rings were stimulated with 10 µM methacholine (MCh) for 5 min, then washed and reset to 0.5 g preload, which was maintained through a 30-min equilibration period. Rings were then pretreated with either 10 µM bisindolylmaleimide (Bis) I, 10 µM Bis V, 10 µM RO-31–8220, or vehicle (DMSO) for 10 min. The rings were then stimulated with various different concentrations of LTD4 or MCh, and the responses were recorded for 10 min. In a select set of experiments, the rings were washed thoroughly with K-H buffer and allowed to recover for 10 min. The rings were rechallenged with LTD4 or MCh, and tensions were recorded for 10 min. The net contractile responses were determined by subtracting the baseline tension from that of the peak tension on agonist stimulation, and the change in the tension was normalized to the wet weight of the tracheal ring segment.
Generation of murine and human ASM cultures
Human ASM cell cultures were generated from tissue obtained at autopsy, as described by Panettieri et al. (13)
under a protocol approved by the Wake Forest University Health Sciences Institutional Review Board. For generation of murine ASM cultures, tracheae harvested from 4 or 5 mice were cleaned of surrounding tissues and pooled to isolate ASM cells using the above procedure for human ASM cultures with minor modifications as described previously (14)
. ASM cells were characterized by immunostaining using anti-
-actin and anti- smooth muscle myosin heavy-chain antibodies.
Purification of human monocytes from peripheral blood
Blood was collected by venipuncture from healthy human volunteers and peripheral blood mononuclear cells (PBMCs) were isolated as described previously (15)
. PBMCs were plated onto a 10-cm dish, and monocytes were allowed to attach to the surface for 2 h. The adherent monocytes were washed twice with ice-cold PBS (pH 7.4) and detached by incubation with PBS/5 mM EDTA on ice for 15 min. The recovered cells were centrifuged at 2000 rpm and resuspended in plain RPMI medium. Cell count and viability were determined using an automated cell counter (Vi-CELL; Beckman Coulter, Fullerton, CA).
Intracellular calcium ([Ca2+]i) measurements
1st and 2nd passage human and murine ASM cells and freshly isolated human monocytes were used in experiments assessing regulation of CysLT1R-mediated Ca2+ flux. Cells grown on glass coverslips were washed and loaded with 5 µM Fura-2 AM for 30 min at 37°C. The cells were washed and maintained in Hank’s Balanced Salt Solution containing 10 mM HEPES, 11 mM glucose, 2.5 mM CaCl2, and 1.2 mM MgCl2 (HBSS; pH 7.4). The coverslips were mounted onto an open slide chamber and [Ca2+]i flux was assessed using a dual excitation fluorescence photomultiplier system (IonOptix, Milton, MA) as described previously (6)
. The fluorescence intensities were converted into absolute calcium concentration using a calibration curve derived from maximum (ionophore) and minimum (EGTA) calcium flux in these cells, as per the software. The cells were treated with Veh or 10 µM Bis I for 10 min, and basal [Ca2+]i was determined. The cells were subsequently stimulated with LTD4, histamine, or MCh. The net calcium response was calculated by subtracting the basal from peak [Ca2+]i on agonist stimulation. For analyses of human ASM cells and monocytes, experiments were repeated using cells obtained from different donors.
Measurement of phosphoinositide (PI) generation
First and second passage human and murine ASM cells were grown to confluence on 24-well plates and loaded with 2 µCi/ml myo-[3H]inositol for 18 h. After washing with phosphate-buffered saline the cells were incubated with DMEM containing 5 mM LiCl for 10 min. The cells were then treated with vehicle or 10 µM Bis I for 10 min and stimulated with vehicle (basal), LTD4, histamine or MCh for 30 min. Reactions were quenched with 20 mM formic acid and inositol, and phosphoinositide (PI) fractions were separated by anion exchange chromatography as described previously (6)
. PI production was calculated by dividing PI fraction by total (PI + inositol) inositol fraction and the data were reported as fold basal (agonist-stimulated/vehicle).
Monocyte chemotaxis assay
Chemotaxis of monocytes was determined using Costar Transwell plates fitted with polycarbonate membrane of 5-µM pore size, as described previously (16)
. Human monocytes isolated from peripheral blood were incubated with Veh or 10 µM Bis I for 10 min. RPMI medium containing Veh, 100 nM LTD4, or 100 ng/ml monocyte chemotactic protein-1 (MCP-1) was added to the bottom chamber of the plate. 200,000 cells per well were added to the upper chamber also containing the pretreatment agent and allowed to incubate at 37°C for 1 h, after which cell number in the bottom chamber was determined.
Regulation of MCP-1 production in THP-1 cells
Because analysis of regulation of MCP-1 in monocytes production requires a large number of cells, experiments were performed using the human monocytic cell line THP-1. THP-1 cells grown in RPMI medium supplemented with 10% FBS were centrifuged and resuspended in serum-free RPMI media. The cells were treated with vehicle or 10 µM Bis I for 10 min and subsequently stimulated with vehicle, 100 nM LTD4, or 100 ng/ml LPS. The supernatant was harvested before and 3 h after the addition of the stimulants and frozen at –20°C. MCP-1 levels were subsequently measured by ELISA (R&D Systems, MN) as per manufacturer’s protocol.
Statistical analysis
Data analysis was carried out using GraphPad Prism, and data were expressed as mean ± SE. Group comparisons were performed using one-way ANOVA or Student’s t test where appropriate, with a P value of < 0.05 sufficient to reject the null hypothesis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Wild-type C57BL/6 mice express an extremely low level of endogenous CysLT1R, and tracheal rings exhibit a weak contractile response to LTD4. Transgenic expression of human CysLT1R at physiological levels (20 fmol/mg protein) increases LTD4-mediated ASM tension development to approximately one-half to two-thirds that stimulated by m3 mAChR activation (Fig. 1
and (12)
). As shown in Fig. 1A
, inhibition of conventional PKC isoforms by pretreatment with Bis I significantly increased LTD4-stimulated tension development in tracheal rings. Pretreatment with the PKC inhibitor RO-31–8220 had a similar effect (not shown), whereas pretreatment with Bis V (structurally similar to Bis I but lacking the ability to inhibit PKC) had no effect (Fig. 1B
). Moreover, and in contrast to the effect on LTD4-stimulated contraction, pretreatment with Bis I caused a small but significant decrease in MCh-induced contraction (Fig. 1C
). Interestingly, pretreatment with Bis I also significantly augmented LTD4-stimulated contraction of the endogenous murine CysLT1R (Fig. 1D
), which possesses the same PKC-consensus sites (12)
contained in the human CysLT1R (6)
. LTD4-mediated contraction of rings from transgenic mice was antagonized in a dose-dependent manner by the selective CysLT1R antagonist MK-571 (Fig. 1E
).
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Further evidence of the capacity of PKC inhibition to attenuate the functional consequences of CysLT1R desensitization in ASM is provided when tracheal rings from hCysLT1R mice are repeatedly challenged with LTD4. In preparations pretreated with vehicle, a second challenge with LTD4 resulted in a loss of tension development (Fig. 2
A, E). However, this loss was significantly attenuated in rings pretreated with Bis I (Fig. 2B, E
). Conversely, MCh-stimulated tension development was maintained on rechallenge and was unaffected by PKC inhibition (Fig. 2C, D, E
).
Analysis of agonist-induced PI production and Ca2+ flux in early passage murine ASM cells from hCysLT1R mice demonstrates that the effects of PKC inhibition on contraction-promoting proximal signaling events are consistent with the functional effects observed in tracheae. Bis I pretreatment significantly increased both LTD4-stimulated PI hydrolysis and peak Ca2+ flux in cultures derived from murine trachea from hCysLT1R transgenic mice, whereas a trend toward inhibiting MCh-stimulated PI production (P=0.2) and Ca2+ flux (P=0.09, n=8) was observed (Fig. 3
A, B). With human ASM cells CysLT1R expression wanes rapidly in culture (R. B. Penn, unpublished observation), but LTD4-stimulated signaling can be assessed in early passage cells. As shown in Fig. 3C and D
, PKC inhibition significantly increased LTD4-stimulated PI production and Ca2+ flux in human ASM cells whereas H1 histamine receptor signaling is slightly but not significantly inhibited. Collectively, these experiments demonstrate that despite what appears to be a capacity of PKC inhibition to inhibit downstream signaling that promotes PI generation and Ca2+ flux in ASM, the relief of PKC-mediated desensitization of the CysLT1R is sufficient to promote an increase in LTD4-stimulated Ca2+ flux that translates into an increase in ASM tension development.
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Functional consequences of CysLT1R regulation in human monocytes
Previous studies have shown that human monocytes express CysLT1Rs (16
, 17)
and that LTD4 stimulates monocyte chemotaxis that is inhibited by the CysLT1R antagonist monteleukast (18)
. To characterize regulation of LTD4-mediated chemotaxis, human monocytes were isolated from peripheral blood and PKC-dependent regulation of migration assessed in classical Transwell chamber experiments. As shown in Fig. 4
A, Bis I pretreatment significantly increased LTD4-mediated migration, while slightly inhibiting migration promoted by MCP-1 (P=0.209, n=11). LTD4-stimulated Ca2+ flux in monocytes was also significantly increased in human monocytes by pretreatment with Bis I (Fig. 4B
).
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LTD4 can also stimulate induction of MCP-1 production in monocytic cells, an effect reversed by the CysLT1R selective antagonist pranlukast (19)
. We therefore characterized PKC-dependent regulation of LTD4-stimulated MCP-1 production in the monocytic cell line THP-1. Pretreatment with Bis I significantly increased LTD4-stimulated MCP production by >2 fold (Fig. 4C
). Conversely, MCP-1 production stimulated by LPS was unaffected by Bis I pretreatment. LTD4-stimulated MCP-1 production in THP-1 cells was antagonized in a dose-dependent manner by the selective CysLT1R antagonist MK-571 (Fig. 4D
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously demonstrated the role of PKC in regulating both the responsiveness and internalization of the LTD4-stimulated CysLT1R (6)
. Employing both COS and HEK293 cells as expression systems for recombinant CysLT1R constructs, we found that inhibition of conventional PKC isoforms (but not arrestin inhibition, or inhibition of other protein kinases) significantly enhanced multiple measures of CysLT1R signaling while dramatically inhibiting receptor internalization. Moreover, mutation of putative PKC phosphorylation sites in the C terminus of the CysLT1R had the same effect and were redundant with pharmacological PKC inhibition. The dependence on COS and HEK293 cells for expression of the (difficult to express) CysLT1R limited our studies to analyses of receptor trafficking and signaling events.
Although agonist-specific modification and desensitization is a well-documented phenomenon for the majority of GPCRs, establishing its relevance in physiological systems has been problematic. Several possible reasons exist for the failure to demonstrate a clear impact of GPCR regulatory processes on cell/tissue/organism function. One is that those regulatory features characterized in more artificial systems may be obscured or absent in primary cell types because of differences in regulatory molecule:receptor stoichiometries. Another involves the influence of compartmentalization effects that can be absent or overwhelmed in overexpression systems. In both artificial and physiological systems, the redundancy of GPCR desensitization mechanisms (GRK- and second messenger kinase-dependent mechanisms are often concomitantly evoked) and the molecules they employ (many cells express multiple GRK and arrestin isoforms) is also well established, making it difficult to implicate a specific mechanism/molecule. Perhaps most important is the presence of other, more dominant control systems that serve critical cell or organism needs. Indeed, this latter phenomenon tends to constrain the effects of experimental perturbations in many primary cell types, reflecting a hierarchical system of controls absent in un- or de- differentiated cells that constitute convenient expression systems. Technical issues also contribute, in that endogenous levels of many receptors and regulatory proteins are low, are not easily characterized with existing reagents (antibodies), and provide poor signal-to-noise ratios for receptor-mediated signaling events and cellular functions. The application of transfection or infection strategies in primary cell types and tissues is often problematic, thus limiting the scope of approaches for understanding regulatory mechanisms.
The impact of antithetical control systems invoked by a common upstream effector is evident in our current studies. In both the regulation of calcium flux and calcium-dependent functions, PKC activity has a positive regulatory role through actions on signaling pathway elements downstream of the receptor. This is most clearly evidenced by the ability of PKC inhibition to reduce calcium flux and contraction in airway smooth muscle cells stimulated with either histamine or MCh. Previous studies have demonstrated that the activities of phospholamban, myosin light chain, heat shock protein 27, myosin light chain kinase, caldesmon, CPI-17 of myosin phosphatase, TRPC channels, and nonselective cation channels are modulated by PKC-dependent phosphorylation (20
21
22
23)
. However, relief of PKC-mediated CysLT1R desensitization is sufficient to override these downstream mechanisms and effect an increase in LTD4-stimulated PI generation, Ca2+ flux, and consequently, contraction (Fig. 5
). The inability of PKC inhibition to affect the H1 HR and m3 mAChR loci has been previously established in studies demonstrating that agonist-specific desensitization of these receptors is dependent on GRKs and arrestins (24
25
26)
.
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Despite the paucity of studies directly linking mechanisms of GPCR desensitization to functional consequences in cells or more integrative systems, progress in two specific areas is noteworthy. For both cardiac and neuronal function, considerable evidence suggests that homologous desensitization of GPCRs mediated by GRKs and arrestins plays an important role. Koch, Rockman, and colleagues have demonstrated through use of transgenic mice a clear role for GRK-mediated ßAR desensitization in the diminished contractile response of failing heart to endogenous and exogenous beta-agonist. In murine models of heart failure, elevated levels of circulating catecholamines induce increased expression of GRK2 and promote ßAR desensitization; inhibition of GRK-mediated ßAR desensitization by transgenic expression of the C-terminus of GRK2 (GRK2-CT) reverses ßAR desensitization, improves beta-agonist-stimulated cardiac function, and reverses disease phenotype (8
9
10)
. With respect to neuronal function, several labs have now demonstrated that knockout of various GRK or arrestin isoforms attenuates desensitization or internalization of various GPCRs expressed in neuronal cells and alters behavioral phenotypes. For example, ablation of the arrestin3 (also termed beta-arrestin2) gene impairs agonist-induced mu opioid receptor desensitization, increases the antinociceptive effect of morphine, and deters the onset of opioid tolerance (27
, 28)
. Similarly, opioid-induced analgesic tolerance is significantly less in GRK3-knockout mice (29
, 30)
. Ablation of the GRK5 gene attenuates muscarinic (primarily m2 mAChR) receptor desensitization in mouse brain and is associated with enhanced cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation (31)
, whereas GRK6 knockout enhances striatal D2 dopamine receptor-G protein coupling and is associated with supersensitivity to the locomotor-stimulating effect of cocaine and amphetamine (32)
. Although these studies suggest an important role for GRKs and arrestins in mediating neuronal function, it should be noted that the phenotypes observed cannot be unequivocally linked to altered receptor regulation per se, in light of the (increasingly appreciated) pleiotropic nature of GRKs and arrestins (33
34
35)
.
The obvious benefit of understanding the relative importance of the multiple regulatory mechanisms affecting GPCR-modulated functions is in establishing the feasibility of selectively targeting mechanisms in order to deter disease progression or improve treatment efficacy. Findings from the current study suggest that PKC helps quench CysLT1R activation but otherwise promotes downstream calcium-dependent signaling in ASM, suggesting that any antiasthma therapy targeting PKC in ASM should strive to block PKC-aided calcium-release and signaling while preserving PKC-mediated desensitization of the CysLT1R. Toward this end, advances in gene/drug delivery systems enabling precise regulation of protein-protein interactions or chaperone functions (36
, 37)
could ultimately provide the means for selectively targeting these antithetical actions of PKC.
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ACKNOWLEDGMENTS |
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Received for publication December 29, 2006. Accepted for publication March 1, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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