University of Groningen

(Endo) signaling in human bronchial epithelial and smooth muscle cells Gkoumassi, Effimia

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Download date: 25-09-2021 Chapter 5

Reciprocal regulation of virodhamine induced attenuation of human bronchial epithelial cell survival by CB1 and CB2 cannabinoid receptors

Effimia Gkoumassi, Carolina R.S. Elzinga, Rutger E. Hasenbosch, Hans J. van der Horn, Erwin L.P. van Streun, Bart G.J. Dekkers, Herman Meurs, Johan Zaagsma, Martina Schmidt

Department of Molecular Pharmacology, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands

______Chapter 5

Abstract

Endo() are considered to possess immunomodulatory properties and may be involved in airway pathophysiology. In human bronchial epithelial (16HBE14o-) cells we reported that the endocannabinoid virodhamine known to act as a non-selective on the two cannabinoid (CB) receptors modulates the cellular level of cAMP and Ca2+, both of which might control cell survival. Here we report that virodhamine reduced 16HBE14o- cell survival, apparently largely independent of CB driven cAMP and Ca2+ - alterations. To dissect CB1R and CB2R functions in 16HBE14o cells, we suppressed cellular CB1R or CB2R expression by transient transfection of

specific siRNAs. Protein levels of CB1R and CB2R were substantially reduced in these cells as assessed by using specific antibodies, whereas expression

of β-actin was not altered. Silencing of CB1R enhanced virodhamine-induced

reduction in cell survival, whereas silencing of CB2R had the opposite effect. Similar changes were observed with regard to the phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in CB receptor - silenced 16HBE14o cells. In conclusion, our data indicate that CB1R and - CB2R regulate virodhamine induced attenuation of 16HBE14o cell survival in a reciprocal fashion, a process probably involving ERK1/2.

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Introduction

In the past decade two cannabinoid receptors, CB1R and CB2R, have been identified, cloned, expressed and functionally investigated (Matsuda et al., 1990; Munro et al., 1993). The CB receptors together with their endogenous ligands are referred to as the (Mackie, 2006; Pacher et al., 2006; Pertwee, 2006). Currently, the endocannabinoid system has been implicated both in the central and peripheral nervous system as well as in peripheral organs in many physiological and pathophysiological processes, including metabolic regulation, craving, pain, anxiety, bone growth, and immune function (Mackie, 2006; Pacher et al., 2006; Pertwee, 2006). Such signaling diversity is reflected by CB receptor expression profiles, the

CB1R being expressed not only in central and peripheral nervous structures (Tsou et al., 1998) but also in some peripheral tissues (Liu et al., 2002; Wagner et al., 2001), whereas the CB2R is mainly expressed in immune cells (Berdyshev, 2000; Galiegue et al., 1995; Munro et al., 1993; Schatz et al., 1997).

As members of the G-protein-coupled receptor superfamily both CB1R and

CB2R couple to heterotrimeric G proteins of the pertussis toxin (PTX)-

sensitive Gi/o family, however coupling to Gs proteins has been reported for the CB1R as well (Demuth and Molleman, 2006; Howlett et al., 2002; Matsuda et al., 1990; Munro et al., 1993). In most tissues and cells, inhibition

of cAMP formation by CB receptor coupling to Gi proteins has been shown in most tissues and cells, (Howlett et al., 2002). In addition, inhibition of voltage- operated Ca2+ channels and activation of inwardly-rectifying K+ channels upon stimulation of CB1Rs has been reported (Mackie, 2006); however, opposing effects of the endocannabinoid system on intracellular Ca2+ have also been revealed (Oz, 2006). CB receptor signaling properties comprise not only modulation of adenylyl cyclase and ion channels but also phosphorylation and activation of members of the mitogen-activated protein (MAP) kinase family, such as the extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 MAP kinase and c-Jun N- terminal kinase (Demuth and Molleman, 2006; Howlett et al., 2002).

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Modulation of MAP kinase signaling may contribute to the control of cell survival by the endocannabinoid system (Guzman et al., 2002). In chapter 4 we showed that human bronchial epithelial cells (16HBE14o-)

endogenously express both CB1R and CB2R which were functionally coupled to adenylyl cyclase (Gkoumassi et al., 2007). Using the endocannabinoid

virodhamine we demonstrated that CB2R activation inhibits cAMP formation and interleukin-8 (IL-8) release from 16HBE14o- cells. Pre-treatment with PTX

unmasked stimulation of cAMP formation, a process involving the CB1R. The endocannabinoid virodhamine also profoundly altered cellular Ca2+ handling in 16HBE14o- cells by inducing, in particular at concentrations above 10-30 μM, a clear cellular Ca2+ overload (chapter 4). As it is generally believed that regulation of cellular Ca2+, either acting alone or in concert with cAMP, is of major importance to control cell survival, in particular under conditions of cellular Ca2+ overload (Mayr and Montminy, 2001; Orrenius et al., 2003), the aim of the present study was to analyze whether and, if so, by which mechanisms cell survival of 16HBE14o- cells is modulated by the endocannabinoid virodhamine. Here we report for the first time that virodhamine alters cell survival of 16HBE14o- cells. Surprisingly, this cellular response was largely independent of CB receptor induced alterations in cAMP 2+ and Ca . Following silencing of CB1R and CB2R using siRNAs, we found that

CB1R silencing enhanced virodhamine-induced reduction of cell survival,

whereas CB2R silencing had the opposite effect. Importantly, alterations of cell survival in CBR silenced 16HBE14o- cells were accompanied by changes on cellular ERK1/2 responses.

Materials and Methods

Chemicals and drugs - All compounds were of analytical grade and obtained from Sigma (Zwijndrecht, The Netherlands), unless stated otherwise. Virodhamine [O-(2-Aminoethyl)-5Z,8Z,11Z,14Z-eicosatetraenoate] was obtained from Tocris-Cookson (Avonmouth, UK). Stock solutions of virodhamine and pertussis toxin were prepared in ethanol, shielded from light and stored at -20°C. Trypsin was obtained from Lonza (Belgium). The anti-β- actin antibody was from Sigma (Zwijndrecht, The Netherlands), the anti-

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phospho-ERK1/2 (P-ERK1/2) and the anti-ERK1/2 (ERK1/2) antibodies were from Cell Signaling (via Bioke, Leiden, The Netherlands).

Cell Culture - The immortal human bronchial epithelial cell line 16HBE14o- was kindly donated by Dr. D. C. Gruenert, University of Vermont, Burlington VT, USA) (Cozens et al., 1994). Cells from passages 70-90 were grown at 37°C in minimum essential medium (MEM), supplemented with 10% fetal- bovine serum from PerbioScience (Etten-Leur, The Netherlands), penicillin (50 µg/ml), streptomycin (50 µg/ml) and L-glutamine (2 mM) in fibronectin/collagen type 1 (Cohesion Technologies Palo Alto, CA) coated flasks to near confluency and in an atmosphere of 5% CO2/ 95% O2. All culture media and supplements were obtained from Life Technologies (Breda, The Netherlands). Cell culture dishes were from Costar (Badhoevedorp, The Netherlands) and flasks from Greiner (Alphen a/d Rijn, The Netherlands). To - study the role of Gi proteins, 16HBE14o cells were pretreated with PTX essentially as described before. Briefly, PTX was added at a concentration of 100 ng/ml medium for 20 h, as described also in chapter 4 (Gkoumassi et al., 2007).

Cell survival protocol - 16HBE14o- cells were washed twice with HEPES buffered saline (HBS), trypsinised and subjected to two additional washing steps with HBS. 16HBE14o- cells were resuspended in a buffer containing 145

mM NaCl, 5 mM KCl, 0.5 mM MgSO4, 1 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.4, plus 0.2 % (w/v) bovine serum albumine (BSA), centrifuged for 5 min at room tempurature (1,000 rpm) and washed twice with the same solution. Prior to cell counting the cells were incubated for the indicated periods of time at 37°C with the indicated concentrations of virodhamine or its solvent ethanol. For cell counting, cell suspensions were mixed with equal amounts of trypan blue solution (Fluka) (Berd, 2003). Thereafter, living cells and dead cells, together comprising total cells, were counted using a Zeiss Axiovert 25 microscope. Cell survival is expressed as the ratio of living cells / total cells.

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siRNA transfection - 16HBE14o- cells were passaged 1 day before

transfection with CB1R and CB2R specific siRNAs and allowed to grow to about 50% confluence in MEM with L-glutamine containing serum and antibiotics. For transfection of 16HBE14o- cells with siRNAs followed by cAMP or Ca2+ measurements, the cells were cultured in T80 culture flasks in 7000 μl of the medium mentioned above. In a total volume of 1000 μl EC-R buffer (Qiagen), 2 µg of siRNA (final siRNA concentration in the flask, 20 nM) were mixed by vortexing, then 12 µl of RNAifect transfection reagent (Qiagen, The Netherlands) was added and the final solution was allowed to stand for 15 min at room temperature to allow complex formation with siRNA according to the manufacturer’s instructions (Qiagen). Thereafter, the siRNA containing solution was added to the 16HBE14o- cells and the cells were incubated with 72 h at 37°C. For transfection of 16HBE14o- cells with siRNAs followed by measurements of ERK1/2 phosphorylation, the cells were cultured on fibronectin/collagen coated 35 mm culture dishes. In a total volume of 1750 μl EC-R buffer, 0.5 µg of siRNA (final siRNA concentration on the dish, 20 nM) were mixed by vortexing, then 3 µl of RNAifect transfection reagent (Qiagen, The Netherlands) was added. The probes were further processed as described above. The siRNA for the CB1R was developed against exon 1 of the human gene (sense: GCAUGUUUCCCUCUUGUGATT; antisense:

UCACAAGAGGGAAACAUGCTC) and for the CB2R against exon 2 of the human gene (sense: GGAUUAAGGUGUUGACUUGTT; antisense: CAAGUCAACACCUUAAUCCTC) (Ambion, the Netherlands), respectively. Non-silencing siRNA (Alexa Fluor 488, Qiagen) served as unspecific control siRNA.

2+ 2+ Intracellular Ca measurements - [Ca ]i was measured using fura-2 fluorometry as reported in chapter 2 (Demuth et al., 2005). 16HBE14o- cells were trypsinised, cell suspensions were loaded with 3 μM fura-2 acetoxymethylester at 37°C for 45 min in the dark. Thereafter, fluorescence measurements were performed at 37°C.

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cAMP measurements - 16HBE14o- cells were trypsinised and suspended at 106 cells/ml in the following buffer containing: 118 mM NaCl, 4.7 mM KCl, 3

mM CaCl2, 1.2 mM MgSO4, 0.5 mM EDTA, 10 mM glucose, 20 mM HEPES,

1.2 mM KH2PO4, pH 7.4, including 100 µM 3-Isobutyl-1-methylxanthine 3- Isobutyl-1-methyl-2,6(1H,3H)-purinedione (IBMX) to prevent cAMP degradation, and plated in 24 well plates (2x105 cells per well). The experiment was started by adding the indicated concentration of virodhamine 10 min prior to addition of 0.1 µM forskolin for 10 min. After stopping the reaction with perchloric acid (3.5 %) the amount of cAMP produced was measured as described previously, using a competitive [3H]cAMP radioligand binding assay, as also described in chapter 4 (Gkoumassi et al., 2007).

ERK activation assay - For measurement of ERK activation, 16HBE14o- cells were serum-starved overnight and then incubated for 300 sec at 37°C with the indicated agents, followed by cell lysis in a buffer containing 1% SDS and 10 mM Tris/HCl, pH 7.4, and 5 passages through a 25-gauge needle (Keiper et al., 2004). Thereafter, the lysates were cleared by centrifugation, followed by determination of protein concentration (BCA protein assay kit, Pierce, The Netherlands) and incubation in Laemmli buffer for 10 min at 95°C. After SDS- PAGE and transfer to nitrocellulose membranes, phosphorylated ERK1 and ERK2 were detected with the anti-P-ERK1/2 antibody. Densitometric analysis of the bands (P-ERK1/2) was performed with Totallab tm (Nonlineair Dynamics, Newcastle, U.K.).

Immunoblot Analysis - Cell lysate (corresponding to 10-15 µg protein per lane) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using 10% gel at 100V. Proteins in the gel were transferred onto nitrocellulose membranes which were subsequently blocked with blocking buffer (Tris-HCl 50 mM, NaCl 150 mM, Tween-20 0.1%, dried milk powder 5%) for 90 minutes. Membranes were incubated overnight at 4oC using either polyclonal

rabbit anti-CB1R (1:1000) or polyclonal rabbit anti-CB2R (1:500), respectively, as described in chapter 4 (Gkoumassi et al., 2007). Antibodies were kindly provided by Dr. K. Mackie, University of Washington, Seattle WA 98195, USA. In addition, membranes were incubated overnight at 4oC using either

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polyclonal rabbit anti-β-actin (1:2000) or anti-P-ERK1/2 (1:500), respectively (Keiper et al., 2004). After washes of 10 minutes each, membranes were incubated with horseradish peroxidase-labelled secondary antibodies (1:3000 in blocking buffer) for 90 minutes at room temperature, followed by another 3 washes. Antibodies were visualized by enhanced chemiluminescence. Before incubation with polyclonal rabbit anti-ERK1/2 (1:500), membranes were kept overnight at 4 oC in Tris-HCl 50 mM, NaCl 150 mM, Tween-20 0.1% (w/v) and washed three times. The membranes were then incubated with 10 ml of RestoreTM Western blot stripping buffer (Pierce, the Netherlands) for 60 minutes at 37 oC followed by another 4 washes 15 minutes each. Subsequently followed by visualization from the antibodies through enhanced chemiluminescence.

Data Analysis - Values are expressed as mean values ± s.e. mean. Data were analyzed, curves drawn and fitted using SigmaPlot 10 (SYSTAT Software Inc., Chicago, USA). Comparison of pairs of treatments was made using unpaired Student’s t-test; p<0.05 was considered significant.

Results

In chapter 4, we reported that 16HBE14o- cells endogenously express both

CB1R and CB2R and that activation by the endocannabinoid virodhamine induces cellular cAMP and Ca2+ alterations (Gkoumassi et al., 2007). As shown in Figures 1 and 5, virodhamine modulates 16HBE14o- cell survival in a concentration-dependent fashion reaching its maximum of about 50 % inhibition at 100 μM virodhamine. Reduction of cell survival induced by virodhamine increased over time (Figure 1A versus Figure 1B). After 300 sec the reduction amounted to about 40 %, whereas after 600 sec a reduction of about 70 % was observed (see also Figure 5).

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1,2 B 1,2 A 300 sec 1 600 sec

1,0 11,0

0,8 00,8

0,6 00,6

0,4 00,4 Living cells/ total cells Living cells/ total cells Living cells/ total 0,2 00,2

0,0 00,0 basal 6 5 4 basal 6 5 4 [Virodhamine] (-log M) [Virodhamine] (-logM)

Figure 1. Attenuation of 16HBE14o- cell survival by virodhamine. 16HBE14o- cells were stimulated without (basal) or with the indicated concentrations of virodhamine for 300 sec (A) and for 600 sec (B). Thereafter, 16HBE14o- cell survival was determined as described in “Materials and Methods“. Data are presented as mean values ± s.e.mean from 4 experiments.

A B 1,2 11,2 300 sec 600 sec

1,0 11,0

0,8 00,8

0,6 0,6 0

0,4 0,4 0 Living cells/ total cells

0,2 Living cells/ total cells 0,2 0 PTX PTX 0,0 0,0 0 basal 6 5 4 basal 6 5 4 [Virodhamine] (-log M) [Virodhamine] (-logM)

Figure 2. Effect of PTX on virodhamine induced attenuation of 16HBE14o- cell survival. 16HBE14o- cells were pretreated with 100 ng/ml PTX for 20h. Thereafter, 16HBE14o- cell survival was measured without (basal) or with the indicated concentrations of virodhamine for 300 sec (A) and for 600 sec (B) as described in “Materials and Methods“. Data are presented as mean values ± s.e.mean from 4 experiments.

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To study whether virodhamine-induced attenuation of 16HBE14o-cell survival

involves CB receptor coupling to Gi/o proteins the cells were pretreated for 20 h with 100 ng/ml of PTX. However, cell pretreatment with PTX did not profoundly change virodhamine induced attenuation of cell survival (Figure 1

versus Figure 2). As we demonstrated in chapter 4, Gi/o protein coupling to - CB2Rs inhibits cAMP production in 16HBE14o cells and cell pretreatment

with PTX unmasked Gs-mediated stimulation of cAMP formation by CB1Rs (Gkoumassi et al., 2007), CB receptor driven cAMP alterations seem to be less important for virodhamine induced attenuation of 16HBE14o- cell survival.

A B

CB1R CB2R

β-actin β-actin

Sham siRNA Sham siRNA CB1R CB2R

Figure 3. Silencing of CB1R and CB2R expression by CB receptor specific siRNA in 16HBE14o- cells. 16HBE14o- cells were transfected with unspecific siRNA (sham) or with the CB receptor specific siRNAs, as indicated. After 72 h, 16HBE14o- cell lysates were lysed and

probed as described in “Materials and Methods“. Immunoblot detection of CB1R (A), CB2R (B) and β-actin (A and B) in lysates of control cells (sham) and CB receptor specific siRNA transfected 16HBE14o- cells. Blots shown are representative of 3-5 experiments.

Although CB1R and CB2R selective antagonists, SR141716A and SR144528 respectively, either in the presence or absence of PTX, reversed the modulatory effects of virodhamine on cAMP accumulation in 16HBE14o- cells (Gkoumassi et al., 2007), these compounds did not reduce virodhamine induced Ca2+ alterations (chapter 4). To decipher the involvement of individual CB receptors on the above mentioned cellular response, in particular to study whether CB receptor driven Ca2+ responses alter virodhamine induced

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reduction of 16HBE14o- cell survival, their endogenous expression was suppressed by specific siRNAs. As shown in Figure 3, such maneuver diminished cellular expression of CB1R and CB2R leaving the expression of β-

actin unaffected. Silencing of CB1R and CB2R only slightly reduced (10-20%) virodhamine-induced Ca2+ alterations in 16HBE14o- cells (Figure 4). Silencing

of CB2R (and CB1R) also slightly altered virodhamine induced inhibition of cAMP production (not shown). Most importantly, however, in the presence of virodhamine concentrations above 10 μM, able to reduce of 16HBE14o- cell survival (see above), no difference between sham and silenced CB1R and

CB2R became evident (Figure 4). Taken together virodhamine induced reduction of 16HBE14o- cell survival also seems to be largely independent of CB receptor mediated Ca2+ alterations.

Intriguingly, silencing of CB receptor expression differentially altered virodhamine induced attenuation of 16HBE14o- cell survival. As shown in

Figure 5, silencing of CB1R expression strongly enhanced 100 μM virodhamine induced reduction of cell survival, resulting in almost complete inhibition of cell survival. At lower virodhamine concentrations, however, no differences with control cells were observed independent of the time interval

being studied. In contrast, silencing of CB2R expression diminished the impact of virodhamine on 16HBE14o- cell survival (Figure 5). In the presence of 100 μM virodhamine inhibition of 16HBE14o- cell survival amounted to approximately 20 % and to 30 % after 300 sec and 600 sec, respectively, in

CB2R silenced cells.

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200 A 10 µM Virodhamine 100000 150 80000

(nM)

i 60000 ] 100 2+ (AUC 10 min) (AUC [Ca 40000

2+ siRNA CB1

50 Ca siRNA CB2 20000 Sham

0 0 0 100 200 300 400 500 600 Sham siRNA CB1 siRNA CB2 Time (sec) B 1000 20 µM Virodhamine 400000 800

300000 600 (nM)

i ] 2+ 400 200000 [Ca min) (AUC 10 2+ siRNA CB 1 Ca 200 100000 siRNA CB2 Sham

0 0 0 100 200 300 400 500 600 Sham siRNA CB1 siRNA CB2 C Time (sec) 10000 30 µM Virodhamine 3000000 8000 2500000 6000 2000000 (nM) i ] 4000

2+ 1500000 [Ca 2000 (AUC 10 min) 2+ 1000000

siRNA CB1 Ca siRNA CB 0 2 500000 Sham

0 0 100 200 300 400 500 600 Sham siRNA CB1 siRNA CB2 Time (sec) Figure 4. Effect of CB receptor silencing on virodhamine induced intracellular Ca2+ alterations in 16HBE14o- cells. 16HBE14o- cells were transfected with unspecific siRNA (sham) or with the CB receptor specific siRNAs as indicated. After 72 h, the cells were stimulated with 10 μM virodhamine (A), 20 μM virodhamine (B) or 30 μM virodhamine (C) each added at t=60 sec. Representative traces (left panels) and mean values ± s.e.mean from 4 experiments (right panels) are depicted.

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1,2 A 300 sec

1,0

0,8

0,6

0,4

Living cells/ total cells siRNA CB1 0,2 siRNA CB 2 *

Sham 0,0

654,74,54

[Virodhamine] (-log M) B 11,2 600 sec

11,0

00,8

00,6 #

00,4 Livingcells/ total cells siRNA CB1 00,2 siRNA CB2 Sham * 00,0

654,74,54 [Virodhamine] (-logM)

Figure 5. Effect of CB receptor silencing on virodhamine induced attenuation of 16HBE14o- cell survival. 16HBE14o- cells were transfected with unspecific siRNA (sham) or with the CB receptor specific siRNAs as indicated. After 72 h, the cells were stimulated without (basal) or with the indicated concentrations of virodhamine for 300 sec (A) or 600 sec (B) followed by cell survival measurements as described in “Materials and Methods“. Data are presented as mean values ± s.e.mean from 3-5 experiments. * P<0.05, # P=0.057

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A Sham P-ERK1 Figure 6. Effect of CB receptor silencing P-ERK2 on ERK1/2 responses in 16HBE14o- cells. 16HBE14o- cells were transfected ERK1 with unspecific siRNA (Sham) or with the ERK2 CB receptor specific siRNAs as indicated. B After 72 h, the cells were stimulated siRNA CB1-R P-ERK1 without (Basal) or with 3 μM ATP, 30 μM P-ERK2 virodhamine and the indicated combination (Both). Phosphorylation of ERK1 ERK1/2 (P-ERK1/2) and the total cellular ERK2 content of ERK1/2 were detected in cell lysates with an anti-phospho-ERK1/2 and C siRNA CB2-R an anti-ERK antibody, respectively. Blots P-ERK1 P-ERK2 shown are representative for 3 experiments. ERK1 ERK2

l P e h a t T n s i o a A m B B a h d o r i V

As it is now generally accepted that control of cell survival by the endocannabinoid system involves members of the MAP kinase family (Guzman et al., 2002), we studied ERK1/2 responses in 16HBE14o- cells as well. Previous unpublished studies from our laboratory had revealed that - 2+ 16HBE14o cells respond to purine nucleotides by elevating [Ca ]i (not shown). As purinergic receptors are now believed to control cell survival via ERK1/2 as well (Ahmad et al., 2006), activation of ERK1/2 by ATP was used as control for the present virodhamine experiments. Virodhamine by itself did not phosphorylate ERK1/2 (Figure 6A).

Stimulation of 16HBE14o- cells with ATP strongly increased ERK1/2 phosphorylation, an effect being completely blocked in the presence of virodhamine (Figure 6A). As illustrated in Figures 6B and 6C, silencing of CB

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receptors reciprocally changed ERK1/2 responses in 16HBE14o- cells. In

CB1R silenced cells stimuli induced phosphorylation of ERK1/2 was

completely abolished (Figure 6B). In contrast, CB2R silencing unmasked a stimulatory component of virodhamine on ERK1/2 phosphorylation, which was not dramatically altered in the presence of ATP (Figure 6C). CB receptor silencing did not alter expression of ERK1/2 in 16HBE14o- cells. Effects of

CB1R and CB2R silencing on cellular ERK1/2 phosphorylation matched the effects on virodhamine induced attenuation of 16HBE14o- cell survival.

Discussion

Here we report for the first time that the endocannabinoid virodhamine, known - to act on both CB1R and CB2R being endogenously expressed in 16HBE14o cells, as we discussed in chapter 4 (Gkoumassi et al., 2007), profoundly affects cell survival. Virodhamine reduced 16HBE14o- cell survival in a concentration- and time-dependent manner. Worthwhile to note is that the virodhamine concentration being required to attenuate 16HBE14o- cell survival was about 100 times higher than the concentration being used to inhibit cellular cAMP accumulation and interleukin-8 release (chapter 4) (Gkoumassi et al., 2007). Such difference might explain the ineffectiveness of PTX (Figures 1 and 2), known to efficiently alter cAMP formation in 16HBE14o- cells (Gkoumassi et al., 2007), to alter virodhamine-induced

attenuation of cell survival. Both CB1R and CB2R are differentially coupled to adenylyl cyclase, hence, it is rather unlikely that these signaling properties are of major importance for virodhamine-induced attenuation of 16HBE14o- cell survival. As reported before and also shown in this paper, virodhamine induced at concentrations above 10 μM Ca2+ overload in 16HBE14o- cells (see Figure 4). Remarkably, such virodhamine concentrations (Figures 1 and 5) concentration- and time-dependently reduced 16HBE14o- cell survival implying that virodhamine induced cellular Ca2+ overload might be responsible for its effect on cell survival. Such mechanism would perfectly fit to numerous reports in the literature indicating that Ca2+ ions modulate cell survival (Orrenius et al., 2003). Therefore we aimed to unravel the impact of Ca2+

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overload induced by virodhamine on 16HBE14o- cell survival and in particular

to gain insight into the potential involvement of CB1R and CB2R in such

scenario. Although CB1R and CB2R antagonists, SR141716A and SR144528 respectively, as shown in chapter 4, selectively altered cAMP responses in 16HBE14o- cells (Gkoumassi et al., 2007), these compounds failed to antagonise virodhamine induced Ca2+ alterations. Based on these findings we

decided to interfere on the level of CB1R and CB2R expression using the siRNA technology (Dykhoorn et al., 2003).

To our knowledge this is the first report on silencing of CB1R and CB2R in 16HBE14o- cells and its impact on virodhamine induced cellular responses.

The CB1R and CB2R specific siRNA sequences being used in the present study were rather specific as expression of β-actin was not changed in CB

receptor silenced cells; in addition, expression of CB1R vs. CB2R remained

unchanged in CB2R vs. CB1R silenced cells, respectively (not shown).

Silencing of CB1R and CB2R, however, did not dramatically alter virodhamine induced Ca2+ responses and the same was true with inhibition of cAMP formation (not shown); most strikingly there was no difference between CB1R 2+ and CB2R silencing (Figure 4). The second messengers cAMP and Ca are importantly involved in the control of cell fate (Beavo and Brunton, 2002; Berridge et al., 2004), it would be rather unlikely that virodhamine induced attenuation of 16HBE14o- cell survival is independent of cAMP and Ca2+; however, based on the findings reported here would suggest that CB receptor induced cAMP and Ca2+ alterations are less relevant.

Strikingly, silencing of CB1R and CB2R exerted opposing effects on virodhamine induced attenuation of 16HBE14o- cell survival. These findings further emphasize the CB receptor specificity of the siRNAs being used in the

present study. Whereas silencing of CB1R induced a further reduction of cell

survival, silencing of CB2R had the opposite effect (Figure 5). As silencing of

CB1R and CB2R had similar reciprocal effects on ERK1/2 phosphorylation in 16HBE14o- cells (Figure 6), it is tempting to speculate that virodhamine induced attenuation of cell survival involve ERK1/2. Indeed members of the MAP kinase family like ERK1/2 seem to control cellular fate by

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endocannabinoids (Guzman et al., 2002). Although ERK signaling has initially been reported to positively control cell survival, recent findings indicate that ERK signaling might also reduce cell survival; such distinct signaling properties seem to involve various scaffold proteins and inhibitors (Kolch, 2005; Raman et al., 2007; Zhuang and Schnellmann, 2006).

A C + - cAMP

cellular effects cytokine release CB2

Stimulatory Ca2+ Inhibitory Signal Signal

ERK1

Cell survival

Figure 7. Hypothetical scheme of virodhamine induced attenuation of 16HBE14o- cell survival by CB receptors. Virodhamine modulates 16HBE14o- cell survival largely independent of 2+ cAMP and Ca alterations. Silencing of CB1B R increased virodhamine-induced reduction of

cell survival, whereas silencing of CB2R had the opposite effect. Silencing of CB receptors

altered cellular ERK1/2 responses in similar fashions. Whereas CB1R signaling seems to - enhance cell survival, CB2R signaling diminishes 16HBE14o cell survival; both effects probably involve ERK1/2.

(Endo)cannabinoids acting through the CB1R and/or the CB2R have also been reported to enhance or to reduce cell survival in various cell types, and these processes seemed to involve cAMP and Ca2+ signals. (Davis et al., 2003; Ellert-Miklaszewska et al., 2005; Galve-Roperh et al., 2002; Giuliano et al., 2006; Hsu et al., 2007; Melck et al., 1999; Rueda et al., 2002; Sanchez et al., 2003; Zhao et al., 2005). However, the same reports indicated, using phospho-specific antibodies, that (endo)cannabinoids activate ERK1/2 and/or p38 MAP kinase as well. We report here that virodhamine by itself did not

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induce ERK1/2 phosphorylation in 16HBE14o- cells, but rather dimished cellular ERK1/2 phosphorylation. Importantly, however, modulation of cellular

CB1R and CB2R dramatically altered ERK1/2 responses. In particular, in - CB2R silenced 16HBE14o cells virodhamine substantially phosphorylated

ERK1/2, whereas in CB1R silenced cells phosphorylation of ERK1/2 was completely blunted. In line with the findings described in chapter 4

(Gkoumassi et al., 2007), the CB2R seems to be superior to the CB1R to control ERK1/2 responses in 16HBE14o- cells. That ERK1/2 responses in 16HBE14o- cells are tightly controlled by the cannabinoid system.

As depicted in the scheme (Figure 7), CB2R signaling might convey a signal leading to ERK1/2 inhibition, whereas the opposite scenario seems to account for CB1R signaling. The nature of such signals is presently unknown, however it is tempting to speculate that members of the β-arrestin family might be involved. Although being initially characterized as regulators of G-protein- coupled receptor desensitization and internalization, recent evidence indicate that β-arrestins bear signaling properties independently of G protein activation (De Wire et al., 2007). It is now accepted that β-arrestins, in particular β- arrestin2, function as multiprotein scaffolds for an increasing amount of signaling molecules including ERK1/2 (De Wire et al., 2007; Kolch, 2005). As CB receptors seem to associate with β-arrestin2, lipid rafts and adaptor proteins (Bari et al., 2005; Jin et al., 1999; Sanchez et al., 2001), such

mechanisms might contribute to the impact of the CB1R and CB2R on ERK1/2 responses in 16HBE14o- cells, regulating cell survival by the endocannabinoid virodhamine.

In conclusion, we report here on regulation of 16HBE14o- cell survival by the endocannabinoid virodhamine being apparently largely independent of CB receptor dependent cAMP and Ca2+ alterations. The findings reported here also indicate that the CB1Rs and CB2Rs reciprocally regulate virodhamine- induced attenuation of 16HBE14o- cell survival, and such effects probably converge on the level of ERK1/2.

Acknowledgements - We would like to thank Dr K. Mackie (grant NIH DA11322) for providing the CB receptor-specific antibodies.

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References 1. Ahmad S, Ahmad A, White GC, 2006. Purinergic signaling and kinase activation for survival in pulmonary oxidative stress and disease. Free Rad Biol Med 41: 29-40.

2. Bari M, Battista N, Fezza F, Finazzi-Agro A, Maccarrone M, 2005. Lipid rafts control signaling of type-1 cannabinoid receptors in neuronal cells. Implications for -induced apoptosis. J Biol Chem 280: 12212-12220.

3. Beavo JA, Brunton LL, 2002. Cyclic nucleotide research - still expanding after half a century. Nat Rev Mol Cell Biol 3: 710-718.

4. Berd D, 2003. Contribution of dead cells to the immunogenicity of an autologous, hapten-modified melanoma vaccine. Vaccine 21: 795-797.

5. Berdyshev EV, 2000. Cannabinoid receptors and the regulation of immune response. Chem Phys 108: 169-180.

6. Berridge MJ, Bootman MD, Roderick HL, 2004. Calcium signalling: Dynamics,Homeostasis and Remodelling. Nat Rev Mol Cell Biol 4: 517-529.

7. Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner WE, Widdicombe JH, Gruenert DC, 1994. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 10: 38- 47.

8. Davis MI, Ronesi J, Lovinger DM, 2003. A predominant role for inhibition of the adenylate cyclase/protein kinase A pahtway in ERK activation by 1 in N1E-115 neuroblastoma cells. J Biol Chem 278: 48973-48980.

9. De Wire SM, Ahn S, Lefkowitz RJ, Shenoy SK, 2007. b-arrestins and cell signaling. Annu Rev Physiol 69: 483-510.

10. Demuth DG, Gkoumassi E, Droge MJ, Dekkers BG, Esselink HJ, van Ree RM, Parson ME, Zaagsma J, Molleman A, Nelemans SA, 2005. mediates non-capacitative calcium entry evoked by CB1-cannabinoid receptor activation in DDT1 MF-2 smooth muscle cells. J Cell Physiol 205: 58-67.

11. Demuth DG, Molleman A, 2006. Cannabinoid signalling. Life sciences 78: 549-563.

12. Dykhoorn DM, Novina CD, Sharp PA, 2003. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4: 457-467.

13. Ellert-Miklaszewska A, Kaminska B, Konarska L, 2005. Cannabinoids down-regulate PI3K/Akt and Erk signalling pathways and activate proapoptotic function of Bad proteins. Cell signal 17: 25-37.

14. Galiegue S, Mary S, Marchand J, Dussossoy D, Carriere D, Carayon P, Bouaboula M, Shire D, Casellas P, 1995. Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem 232: 54-61.

15. Galve-Roperh I, Rueda D, Gomez del Pulgar T, Velasco G, Guzman M, 2002. Mechanism of extracellular signal-regulated kinase activation by the CB1 cannabinoid receptor. Mol Pharmacol 62: 1385-1392.

16. Giuliano M, Calvarusco G, Pellerito O, Portanova P, Carlisi D, Vento R, Tesoriere G, 2006. Anandamide-induced apoptosis in Chang liver cells involves ceramide and JNK/AP-1 pathway. Int J Mol Med 17: 811-819.

117 ______Chapter 5

17. Gkoumassi E, Dekkers BGJ, Droge MJ, Elzinga CRS, Schmidt M, Meurs H, Zaagsma J, Nelemans SA, 2007. Virodhamine and CP55,940 modulate cAMP production and IL-8 release in human bronchial epithelial cells. Brit J Pharmacol 151: 1041-1048.

18. Guzman M, Sanchez C, Galve-Roperh I, 2002. Cannabinoids and cell fate. Pharmacology Therapeutics 95: 175-184.

19. Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devanne WA, Felder CC, Herkenham M, Mackie K, Martin BR, Mechoulam R, Pertwee RG, 2002. Classification of cannabinoid receptors. Pharmacological Reviews 54: 161-202.

20. Hsu S-S, Huang C-J, Cheng H-H, Chou C-T, Lee H-Y, Wang J-L, Chen I-S, Liu S-I, Lu Y-C, Chang H-T, Huang J-K, Chen J-S, Jan C-R, 2007. Anandamide-induced Ca2+ elevation leading to p38 MAPK phosphorylation and subsequent cell death via apoptosis in human osteosarcoma cells. Toxicol 231: 21-29.

21. Jin W, Brown S, Roche JP, Hsieh C, Celver JP, Kovoor A, Chavkin C, Mackie K, 1999. Distinct domains of the CB1 cannabinoid receptor mediate desensitization and internalization. J Neurosci 19: 3773-3780.

22. Keiper M, Stope MB, Szatkowski D, Böhm A, Tysack K, vom Dorp F, Saur O, Oude Weernink PA, Evellin S, Jakobs KH, Schmidt M, 2004. Epac- and Ca2+-controlled activation of Ras and extracellular signal-regulated kinases by Gs-coupled receptors. J. Biol. Chem. 279: 46497-46508.

23. Kolch W, 2005. Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nat Rev Mol Cell Biol 6: 827-837.

24. Liu WM, Duan EK, Cao YJ, 2002. Effects of anandamide on embryo implantation in the mouse. Life Sci. 71: 1623-1632.

25. Mackie K, 2006. Cannabinoid receptors as therapeutic targets. Annu Rev Pharmacol Toxicol 46: 101-122.

26. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI, 1990. Structure of a cannabinoid receptor and functional expression of cloned cANA. Nature 346: 561- 564.

27. Mayr B, Montminy M, 2001. Transcriptional regulation by the phosphorylation- dependent factor CREB. Nat. Rev. Mol. Cell. Biol. 2: 599-609.

28. Melck D, Rueda D, Galve-Roperh I, De Petrocellis L, Guzman M, Di Marzo V, 1999. Involvement of the cAMP/protein kinase A pathway and of mitogen-activated protein kinase in the anti-proliferative effects of anandamide in human breast cancer. FEBS Lett 463: 235-240.

29. Munro S, Thomas KL, bu-Shaar M, 1993. Molecular characterization of a peripeheral receptor for cannabinoids. Nature 365: 61-65.

30. Orrenius S, Zhivotovsky B, Nicotera P, 2003. Regulation of cell death: The calcium- apoptosis link. Nat. Rev. Mol. Cell. Biol. 4: 552-565.

31. Oz M, 2006. Receptor-independent actions of cannabinoids on cell membranes: focus on endocannabinoids. Pharmacological Therapeutics 111: 114-144.

32. Pacher P, Batkai S, Kunos G, 2006. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacological Reviews 58: 389-462.

118 ______Chapter 5

33. Pertwee RG, 2006. The pharmacology of cannabinoid receptors and their ligands: an overview. International Journal of Obesity 30: 513-518.

34. Raman M, Chen W, Cobb MH, 2007. Differential regulation and properties of MAPKs. Oncogene 26: 3100-3112.

35. Rueda D, Navarro B, Martinez-Serrano A, Guzman M, Galve-Roperh I, 2002. The endocannabinoid anandamide inhibits neuronal progenitor cell differentiation through attenuation of the Rap1/B-Raf/ERK pathway. J Biol Chem 277: 46645-46650.

36. Sanchez C, Rueda D, Segui B, Galve-Roperh I, Lavade T, Guzman M, 2001. The CB1 cannabinoid receptor of astrocytes is coupled to sphingomyelin hydrolysis through the adaptor protein Fan. Molecular Pharmacology 59: 955-959.

37. Sanchez MG, Ruiz-Llorente L, Sancez AM, Diaz-Laviada I, 2003. Activation of phosphoinositide 3-kinase/PKB pathway by CB1 and CB2 cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction. Cell signal 15: 851-859.

38. Schatz AR, Lee M, Condie RB, Pulaski JT, Kaminski NE, 1997. Cannabinoid receptor CB1 and CB2: a characterization of expression and adenylate cyclase modulation within the immune system. Toxicol Appl Pharmacol 142: 278-287.

39. Tsou K, Brown S, Sanudo-Pena MC, Mackie K, Walker JM, 1998. Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience 83: 393-411.

40. Wagner JA, Jarai Z, Batkai S, Kunos G, 2001. Hemodynamic effects of cannabinoids: coronary and cerebral mediated by cannabinoid CB1 receptors. Eur J Pharmacol 423: 203-210.

41. Zhao Q, He Z, Chen N, Cho Y-Y, Lu C, Ma W, Bode AM, Dong Z, 2005. 2- arachidonoylglycerol stimulates activator protein-1-dependent transcriptional activity and enhances epidermal growth factor-induced cell transformation in JB6P+ cells. J Biol Chem 280: 26735-26742.

42. Zhuang S, Schnellmann RG, 2006. A death-promoting role for extracellular signal- regulated kinase. J Pharmacol Exp Therap 319: 991-997.

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