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Gut 47:242-250 doi:10.1136/gut.47.2.242
  • Epithelial proliferation

Alpha2 adrenoceptors regulate proliferation of human intestinal epithelial cells

  1. S Schaak,
  2. D Cussac,
  3. C Cayla,
  4. J C Devedjian,
  5. R Guyot,
  6. H Paris,
  7. C Denis
  1. Institut National de la Santé et de la Recherche Médicale, Unité INSERM388, Institut Louis Bugnard, CHU Rangueil, 31403 Toulouse Cedex 4, France
  1. Dr C Denis, INSERM U388, Institut Louis Bugnard, CHU Rangueil, Bat. L3, 31403 Toulouse Cedex 4, France. Email:denis{at}rangueil.inserm.fr
  • Accepted 8 February 2000

Abstract

BACKGROUND AND AIMS Previous studies on rodents have suggested that catecholamines stimulate proliferation of the intestinal epithelium through activation of α2 adrenoceptors located on crypt cells. The occurrence of this effect awaits demonstration in humans and the molecular mechanisms involved have not yet been elucidated. Here, we examined the effect of α2 agonists on a clone of Caco2 cells expressing the human α2Aadrenoceptor.

METHODS Cells were transfected with a bicistronic plasmid containing the α2C10 and neomycin phosphotransferase genes. G418 resistant clones were assayed for receptor expression using radioligand binding. Receptor functionality was assessed by testing its ability to couple Gi proteins and to inhibit cAMP production. Mitogen activated protein kinase (MAPK) phosphorylation was followed by western blot, and cell proliferation was estimated by measuring protein and DNA content.

RESULTS Permanent transfection of Caco2 cells allowed us to obtain a clone (Caco2-3B) expressing α2A adrenoceptors at a density similar to that found in normal human intestinal epithelium. Caco2-3B retained morphological features and brush border enzyme expression characteristic of enterocytic differentiation. The receptor was coupled to Gi2/Gi3 proteins and its stimulation caused marked diminution of forskolin induced cAMP production. Treatment of Caco2-3B with UK14304 (α2 agonist) induced a rapid increase in the phosphorylation state of MAPK, extracellular regulated protein kinase 1 (Erk1), and 2 (Erk2). This event was totally abolished in pertussis toxin treated cells and in the presence of kinase inhibitors (genistein or PD98059). It was unaffected by protein kinase C downregulation but correlated with a transient increase in Shc tyrosine phosphorylation. Finally, sustained exposure of Caco2-3B to UK14304 resulted in modest but significant acceleration of cell proliferation. None of these effects was observed in the parental cell line Caco2.

CONCLUSION The results obtained in the present study support a regulatory role for α2 adrenoceptors in intestinal cell proliferation.

The gut mucosa is extensively innervated by noradrenergic neurones, and catecholamines modulate several key functions of the intestinal barrier.1 2 Among them, modification of electrolyte movements is undoubtedly the best documented effect. Noradrenaline promotes Na+, Cl, and water absorption in both the small intestine and colon.3 In addition, the transepithelial short circuit current is decreased and net HCO3 secretion is abolished. As shown by the use of selective adrenergic drugs, this antisecretory action is primarily mediated by postsynaptic α2 adrenoceptors located on epithelial cells.4 5 In agreement with this, α2 adrenoceptors of the α2A subtype were identified on enterocytes and colonocytes isolated from different species, including humans.6-9 The precise mechanisms whereby α2 agonists stimulate net absorption of Na+ and Cl are still debated.10Studies on human colonocytes,9-11 rat enterocytes,12 and the adenocarcinoma cell line HT2913 have shown that α2 adrenoceptors are coupled to Gi2/Gi3 proteins and that stimulation inhibits accumulation of intracellular cAMP induced by vasoactive intestinal peptide or forskolin. Consequently, α2 mediated attenuation of Cl secretion may, at least partially, result from a direct decrease in the activity of the cystic fibrosis transmembrane conductance regulator. Other pathways independent of cAMP were also proposed. They may include interference with Ca++ dependent mechanisms and/or direct coupling of G proteins to ion channels.14 15

Aside from effects on transepithelial ion transport, several in vivo studies suggest that α2 adrenoceptors are also responsible for stimulation of proliferation of intestinal crypt cells by catecholamines.16 So far, the mitogenic effects of α2 agonists have been observed only in the jejunum and colon of rats and mice. Moreover, the molecular mechanisms whereby they occur also remain to be elucidated. Putative involvement of α2 adrenoceptors in the regulation of intestinal proliferative activity in other species however matches several recent findings. Firstly, in rat,7 rabbit,8 and human9 intestinal mucosa, α2 adrenoceptors are preferentially expressed in crypts where cell division occurs and where most noradrenergic nerve endings project. Secondly, α2 adrenergic agonists were found to act as co-mitogens and to activate the mitogen activated protein kinase (MAPK) pathway in cells transfected with the gene encoding the human α2Aadrenoceptor.17-20 This observation, which was originally restricted to cellular models of fibroblastic origin, was recently extended to epithelial cells. In particular, α2adrenoceptor stimulation was reported to increase MAPK activity and DNA synthesis in the OK cell line, derived from the proximal tubule of opossum kidney,21 suggesting that such an effect could also take place in epithelial cells of intestinal origin.

The aim of the present study was to examine the putative growth regulating properties of the α2A adrenoceptor in human intestinal cells. To this end, we developed a clone derived from the enterocyte-like differentiated cell line Caco2 that permanently expresses α2A adrenoceptors at a density similar to that noted in normal human intestinal crypt cells. The α2adrenoceptor was functionally coupled to Gi2 and/or Gi3 in this model. Stimulation caused activation of MAPK and increased cell growth. Further use of this model may also be helpful for investigating the molecular mechanisms responsible for alteration of transepithelial transport by α2 agonists.

Materials and methods

DRUGS AND REAGENTS

[3H]2-(2-methoxy-1,4-benzodioxan-2-yl)-2- imidazoline (RX821002) (59 Ci/mmol) and [3H]clonidine (66 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Courtaboeuf, France), [32P]NAD+ (800 Ci/mmol) from New England Nuclear (Boston, Massachusetts, USA) and [α32P]UTP (800 Ci/mmol) from ICN (Costa Mesa, California, USA). Phentolamine was donated by Ciba Geigy (Basel, Switzerland), and prazosin hydrochloride and 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK14304) tartrate by Pfizer (Sandwich, Kent, UK). Oxymetazoline, idazoxan, chlorpromazine, forskolin, pertussis toxin, GppNHp, phorbol 12-myristate 13-acetate (PMA), and all other chemicals were from Sigma (St Louis, Missouri, USA). Fetal calf serum (FCS) and G418 sulphate were purchased from Gibco BRL (Cergy Pontoise, France). 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) and genistein were obtained from Calbiochem (La Jolla, California, USA). Radioimmunoassay kits for cAMP determination were from Immunotech (Luminy, France). The human α2A adrenoceptor gene (α2C10) was kindly provided by Dr R J Lefkowitz (Duke University, Durham, North Carolina, USA).

EXPRESSION VECTOR

The expression vector used to transfect Caco2 cells (pα2C10Eneo) was a bicistronic plasmid obtained as follows. The HPalpha2GEN construct,22 which contains the BamHI-BamHI fragment (5.5 kb) of the α2C10 gene, was digested with KpnI and HindIII restriction enzymes. The KpnI-HindIII fragment corresponding to nucleotides −1280/+1526 relative to the translation start was subcloned into pKS+ (pBluescriptII KS+, Stratagene, La Jolla, California, USA). This construct was then digested by NheI at position −201 of the α2C10 sequence and NotI in the pKS+ polylinker. The purified insert was cloned into the XbaI and NotI sites of the bicistronic vector, pEN.23 The resulting pα2C10Eneo vector (fig 1) contains an expression cassette comprising the human cytomegalovirus early promoter/enhancer (pCMV), the entire ORF encoding for α2Aadrenoceptor (α2C10), an internal ribosome entry site derived from encephalomyocarditis virus (EMCV), the neomycin phosphotransferase gene, and a rabbit β globin genomic sequence containing an intron and a polyadenylation signal (IVS2-β).

Figure 1

Schematic map of the bicistronic expression vector used for transfection of Caco2 cells. The pα2C10Eneo vector is 7.63 kb in size. It contains an expression cassette comprising the human cytomegalovirus early promoter/enhancer (pCMV), the gene encoding human α2A adrenoceptor subtype (α2C10), the internal ribosomal entry site derived from the encephalomyocarditis virus (EMCV), the neomycin phosphotransferase gene (Neo), a rabbit β globin genomic fragment containing sequences for mRNA stabilisation and polyadenylation (IVS2-β).

CELL CULTURE AND TRANSFECTION

The human colon adenocarcinoma cell line, Caco2, was routinely subcultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and non-essential amino acids. Cells were transfected using the calcium-phosphate method. Two days after transfection, they were subcultured in the presence of G418 sulphate (1 mg/ml), and antibiotic resistant clones were collected individually using cloning cylinders.

ENTEROCYTIC DIFFERENTIATION AND HYDROLASE ACTIVITIES

Immunofluorescence studies were performed on post-confluent cells cultured on Costar Transwell filters (Dutscher, Brumath, France). Cell monolayers were fixed with 3.7% paraformaldehyde-30 mM sucrose in phosphate buffered saline (PBS) for 15 minutes and treated for 10 minutes with 50 mM NH4Cl in PBS to reduce non-specific background. The cells were permeated with 0.05% saponin in PBS containing 1% skimmed milk and all subsequent steps were performed in the same buffer. The filters were cut out and incubated for one hour with phalloidin-FITC conjugate. After extensive washing, samples were mounted with Mowiol and viewed under a confocal laser microscope (Zeiss LSM). Brush border associated enzyme activities were measured on whole cell homogenates. Alkaline phosphatase and dipeptidyl peptidase IV were assayed usingp-nitrophenyl phosphate and glycyl-l-proline-4-nitroanilide, respectively, as substrates.24 25 Values are expressed as milliunits per mg of protein, one unit being the activity that hydrolyses 1 μmol of substrate per minute at 37°C.

RNA EXTRACTION AND RPA

Total cellular RNAs were isolated using the guanidine isothiocyanate/phenol chloroform extraction method.26Synthesis of the α2C10 antisense has been described previously.27 RNase protection assays (RPA) were performed as follows: 100 μg of RNA were added to 30 μl of hybridisation buffer (80% deionised formamide, 0.4 M NaCl, 1 mM EDTA, 40 mM Pipes, pH 6.7) containing an excess of [32P] labelled riboprobe. Samples were heated to 95°C for five minutes and then immediately kept at 55°C for 14 hours. Non-hybridised probe was eliminated by addition of 0.3 ml of RNase A (40 μg/ml) and RNase T1 (2 μg/ml) in 300 mM NaCl, 5 mM EDTA, and 10 mM Tris HCl (pH 7.5). After two hours at 37°C, digestion was stopped by addition of 5 μl of proteinase K (10 mg/ml) and the samples were further incubated for 15 minutes at 37°C. Carrier tRNA (10 μg) and 0.3 ml of solution D (4 M guanidine isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.1 M 2-mercaptoethanol and 0.5% sarkosyl) were added to each tube and protected hybrids were precipitated with isopropyl alcohol. RNA pellets were washed with 70% ethanol, air dried, taken up in sample buffer (97% deionised formamide, 0.1% sodium dodecyl sulphate (SDS), 10 mM Tris HCl, pH 7.0), and run on a 5% acrylamide gel containing 7 M urea. The gels were exposed for 48 hours at −80°C to xray film for autoradiography.

RECEPTOR QUANTIFICATION AND Gi PROTEIN IDENTIFICATION

Receptors were quantified on crude membrane preparations using the selective radioligands [3H]RX821002 (α2antagonist) and [3H]clonidine (α2agonist).28 Specific binding was defined as the difference between total and non-specific binding measured in the presence of 10 μM phentolamine. Saturation isotherms and inhibition curves were analysed using EBDA-LIGAND computer programs.29 ADP ribosylation was carried out essentially as described previously.30 Membranes (50–75 μg of protein) suspended in Tris HCl buffer containing 1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.05% lubrol (v/v) were incubated for 60 minutes at 30°C in the presence of 1 μCi [32P]NAD+ and 100 ng of preactivated pertussis toxin in a final volume of 60 μl. The reaction mixture contained 70 mM Tris HCl (pH 8.0), 0.5 μM NAD+, 1 mM ATP, 10 mM thymidine, 1 mM EDTA, 0.1 mM MgCl2, 1 mg/mll-myristyl phosphatidylcholine, 10 mM nicotinamide, and 25 mM DTT. The reaction was stopped by addition of 2 μg of bovine serum albumin (BSA) in 0.04% SDS and the proteins were precipitated with 70 μl of 10% trichloracetic acid. After centrifugation, pellets were washed twice with diethylether and finally dissolved in Laemmli sample buffer. The labelled proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and revealed by autoradiography.

cAMP MEASUREMENT

Cells were detached in PBS containing 0.6 mM EDTA and collected by gentle centrifugation (400 g, five minutes). The pellet was suspended in DMEM buffered with 25 mM Hepes (pH 7.4). Aliquots of the cell suspension were incubated in a 200 μl final volume of Hepes buffered DMEM containing 0.2 mM IBMX and the indicated concentration of the drug to be tested. After 15 minutes at 37°C, the reaction was stopped by adding 1.8 ml of methanol/formic acid (95/5, v/v). The alcoholic extract was centrifuged (3000g, 10 minutes, 4°C) and an aliquot of supernatant evaporated. The dry samples were taken up in acetate buffer containing 0.1% NaN3, and cAMP content was determined by radioimmunoassay.

DETECTION OF PHOSPHORYLATED Erk1/Erk2 AND Shc

Cells were seeded at low density (105cells/cm2) in 100 mm culture dishes. Three days later, they were rendered quiescent by incubation in FCS free DMEM for 48 hours. They were then treated for the indicated period of time with the compound to be tested, rapidly rinsed with ice cold PBS, and lysed with 1 ml of radio-immunoprecipitation (RIPA) buffer (10 mM Tris HCl, pH 7.4, 1% Triton X100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM phenylmethylsulphonyl fluoride, and 0.5 μM aprotinin). Cell lysates were sonicated, clarified by centrifugation for 15 minutes at 15 000 g, and stored at −80°C until analysis.

The extent of MAPK phosphorylation was measured either on immunoprecipitated tyrosyl phosphorylated proteins using antibodies to extracellular regulated protein kinase 1 (Erk1) and 2 (Erk2) or directly on total cell protein extract using antibody to active MAPK. Tyrosyl phosphorylated proteins were immunoprecipitated (three hours at room temperature) by incubating 2 mg of total cell proteins with protein G-sepharose beads conjugated with an antiphosphotyrosine monoclonal antibody (PY20, Transduction Laboratories, Lexington, Kentucky, USA). The beads were washed four times with RIPA, dried, suspended in 50 μl of Laemmli buffer, boiled for five minutes, and centrifuged for 10 minutes at 15 000 g. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Blots were incubated for two hours at room temperature in TBST (10 mM Tris HCl buffer, pH 8.0, 150 mM NaCl, 0.2% Tween 20) containing 5% skimmed milk and then overnight at 4°C in the same buffer containing rabbit polyclonal antibodies to Erk1 and Erk2 (anti-Erk1, 1:250; anti-Erk2, 1:400; Santa Cruz Biotechnologies, Santa Cruz, California, USA). After extensive washing, blots were exposed for one hour to horseradish peroxidase conjugated donkey antirabbit IgG. Immunoreactive proteins were visualised by chemiluminescence (ECL Western blotting system, Amersham Pharmacia Biotech, Courtaboeuf, France). The intensity of the bands was analysed using ImageQuant software (Molecular Dynamics, Sunnyvale, California, USA). In some experiments, activated Erk1/Erk2 were detected with a rabbit polyclonal antibody recognising the phosphorylated sequence of the active forms of MAPK (Promega, Madison, Wisconsin, USA). In this case, total cell proteins (30 μg) were separated by SDS-PAGE and transferred onto a Polyscreen/polyvinylidene difluoride (PVDF) membrane (NEN Life Science, Belgium). Blots were treated for one hour in TBST containing 0.1% BSA and then incubated overnight at 4°C in the same buffer containing antiactive MAPK antibody (1:3000). After extensive washing, bound antibody was detected with the horseradish peroxidase conjugated secondary antibody and revealed by chemiluminescence. The membrane was then stripped of Ig and reprobed using anti-Erk2 antibody to assess equal loading. For determination of Shc phosphorylation, 500 μg of cell proteins were immunoprecipitated using 5 μg of rabbit polyclonal Shc antibody (Upstate Biotechnology, Lake Placid, New York, USA). After overnight incubation at 4°C, the immunocomplex was precipitated by addition of 50 μl of protein A-agarose beads (Transduction Laboratories, Lexington, Kentucky, USA). After several washes in RIPA, beads were dried, suspended in Laemmli sample buffer, boiled for five minutes, and centrifuged for 10 minutes at 15 000 g. The proteins were subjected to SDS-PAGE, electrotransferred to a PVDF membrane, and probed with an antiphosphotyrosine-horseradish peroxidase conjugated monoclonal antibody (ECL phosphorylation detection system, Amersham Pharmacia Biotech, Courtaboeuf, France). The membrane was stripped of Ig and reprobed using anti-Shc antibody to assess equal loading.

CELL PROLIFERATION ASSAY

Cells were seeded at a density of 105cells/cm2 in 24 well plates. Three days after seeding they were rendered quiescent by incubation in FCS free culture medium for 48 hours. Experiments were started when replacing the cells in DMEM supplemented with 0.5% FCS with or without 1 μM UK14304. The medium was changed every day. At the indicated time following reinitiation of cell proliferation, the effect of the α2 agonist was estimated by measuring total cellular protein and DNA content. Protein concentration was determined using the Coomassie blue method.31 DNA was measured by a fluorometric method using DAPI.32

STATISTICAL ANALYSIS

Results are expressed as mean (SEM) for the number of observations indicated (n). Data were analysed using the Student'st test, and a value of p<0.05 was considered statistically significant.

Results

EXPRESSION AND FUNCTIONAL ACTIVITY OF HUMAN α2A ADRENOCEPTOR IN Caco2 CELL TRANSFECTANTS

The Caco2 human colonic adenocarcinoma cell line undergoes enterocytic differentiation under standard culture conditions. This process is growth related. In the latter stages of the culture, cells are organised as a polarised monolayer with tight junctions and an apical brush border,33 and form domes that are indicative of transepithelial transport properties.34 RPA and RT-PCR experiments have previously demonstrated that Caco2 cells do not spontaneously contain α2adrenoceptors.27 35 In order for this model to express the same receptor subtype as normal human mucosa, the Caco2 cells were transfected with the bicistronic construct pα2C10Eneo (fig 1). Of the 20, G418 resistant clones which were isolated and subcultured, five were submitted to a first round of screening using RPA with a specific riboprobe derived from the α2C10-AR gene. As shown in the autoradiogram in fig 2, one of the five clones (clone 1A) was negative whereas the others contained substantial amounts of transcript. Binding experiments using [3H]RX821002 (α2antagonist) were thus performed to check the level of receptor expression. As expected, no specific radioligand binding was detected in the parental cell line or in clone 1A. All other clones displayed radioligand binding sites but at densities ranging from 28 to 300 fmol/mg of membrane protein. The clone 3B (Caco2-3B), which expresses the receptor at a level similar to that in epithelial cells from normal human intestinal mucosa,9 was selected for further studies. Data from a typical saturation binding experiment on Caco2-3B membranes is presented in fig 3. The number of [3H]RX821002 binding sites was not dependent on the stage of culture (230 (15) and 195 (23) fmol/mg of protein in pre-confluent and post-confluent cells, respectively) and remained stable over at least 30 successive cell passages. From phase contrast and confocal microscopy, Caco2-3B was seen to exhibit the same pattern of enterocytic differentiation as the parental cell line. In addition, measurement of enzymatic activities in cell extracts showed that brush border associated hydrolases were expressed (alkaline phosphatase 85 (12) mU/mg protein; dipeptidyl peptidase IV, 59 (7) mU/mg protein, n=3). Inhibition of [3H]RX821002 binding by various adrenergic compounds indicated that the Ki values for yohimbine (2.9 (1.3) nM), idazoxan (3.7 (1.2) nM), phentolamine (9.2 (3.1) nM), chlorpromazine (2630 (350) nM), and prazosin (2200 (420) nM) matched the pharmacological properties expected for an α2Aadrenoceptor subtype.

Figure 2

Screening of positive clones by RPA. RNA from HT29 (used as a positive control), Caco2, and G418 resistant clones were hybridised with [32P]labelled α2C10 probe. Samples were digested with a mixture of RNases A and T1. The resistant hybrids were analysed by electrophoresis. A representative autoradiogram is shown. Lanes P and (−) correspond to the undigested probe and negative control, respectively, carried out on 100 μg of tRNA.

Figure 3

Saturation isotherm of [3H]RX821002 binding to Caco2 and Caco2-3B cell membranes. Membranes prepared from Caco2 and Caco2-3B were incubated in the presence of various concentrations of radioligand. The amount of specifically bound [3H]RX821002 was determined using 10 μM phentolamine to estimate non-specific binding. The data presented are from a typical experiment. The inset shows the corresponding Scatchard plot. Computer assisted analysis of the results from this specific experiment indicated that the Bmax and Kd values of [3H]RX821002 were, respectively, 198 (12) fmol/mg of protein and 1.05 (0.11) nM in membranes from Caco2-3B. No specific binding was detected on membranes from Caco2 cells.

The degree of Gi protein coupling was analysed by examining the ability of the receptor to exist under a high affinity state for agonists (fig4). In contrast with the situation with antagonists, the UK14304 competition curve was shallow and was better fitted to a two site inhibition model. According to computer assisted analysis of the data from three independent experiments, the Ki values for UK14304 for the high and low affinity state receptor were, respectively, 1.05 (0.23) and 108 (30) nM. The proportion with the high affinity conformation for agonist represented 62 (6)% of the whole receptor population. This fraction was abolished in the presence of Gpp(NH)p/Na+ or in membranes prepared from cells treated for 16 hours with pertussis toxin (250 ng/ml). Furthermore, [32P]ADP ribosylation demonstrated that Caco2-3B expressed the same pertussis toxin sensitive G proteins as the parental cell line (fig 4, inset). Two peptides with apparent molecular masses of 40 and 41 kDa were labelled. Immunoblotting with specific antibodies indicated that they corresponded to αi2 and αi3 subunits (not shown). Further evidence for receptor coupling to Gi proteins was provided by the direct study of [3H]clonidine binding. Indeed, this [3H]agonist labelled 122 (18) fmol of sites/mg of protein with high affinity (Kd 2.5 (0.9) nM). As expected, no significant [3H]clonidine binding was detected in membranes from Caco2-3B cells treated with pertussis toxin. In cells isolated from human and rat colonic crypts,11 12 or in the human adenocarcinoma cell line HT29,13 α2 adrenoceptors were negatively coupled to adenylyl cyclase. In the next series of experiments we asked whether this pathway of signal transduction is also efficient in Caco2-3B. Measurement of intracellular cAMP levels indicated that Caco2 and Caco2-3B cells were similarly responsive to forskolin exposure (table 1). UK14304, clonidine, or (−)adrenaline were ineffective in the parental cell line but significantly inhibited forskolin induced cAMP accumulation in Caco2-3B. It is worth mentioning that neither UK14304 nor any of the other tested agonists lowered basal cAMP levels. Further experiments demonstrated that the inhibitory effect of UK14304 on forskolin induced cAMP production was dose dependent (EC50 12 (5) nM) and abolished by addition of α2 antagonist (not shown).

Figure 4

Analysis of receptor coupling and identification of Gi proteins. Caco2-3B cells were or were not treated with 250 ng/ml of pertussis toxin for 16 hours. Cell membrane preparations were incubated in the presence of 3 nM [3H]RX821002 and increasing concentrations of UK14304. The experiments were carried out in the absence or presence of 10 μM GppNHp with 100 mM NaCl. The curves presented are from a specific experiment. Inset: membranes from rat brain, Caco2, and Caco2-3B were ADP ribosylated with pertussis toxin in the presence of [32P]NAD+. The labelled proteins were resolved on SDS-PAGE and autoradiographed as described in materials and methods.

Table 1

Effect of α2 agonists on basal and forskolin induced cAMP accumulation. Intact cell suspensions prepared from Caco2 and Caco2-3B culture dishes were incubated in serum free Dulbecco's modified Eagle's medium buffered with 25 mM Hepes (pH 7.4) containing vehicle (basal), 1 μM UK14304 (UK), 10 μM forskolin (FK), 10 μM forskolin plus 1 μM UK14304 (FK+UK), 10 μM forskolin plus 10 μM clonidine (FK+clo) or 10 μM forskolin plus 10 μM adrenaline (FK+adre). cAMP was extracted and measured by radioimmunoassay as described in materials and methods. Concentrations of cAMP are expressed as nanomoles of cAMP/mg of cellular protein and are mean (SEM) (n=8)

EFFECT OF α2ADRENOCEPTOR STIMULATION ON MAPK ACTIVATION AND CELL PROLIFERATION

The effect of receptor stimulation on MAPK was investigated by immunoprecipitation of tyrosine phosphorylated proteins followed by immunodetection with anti-Erk1/Erk2 antibodies (fig 5). In Caco2 and Caco2-3B, these antibodies recognised two proteins with apparent molecular masses of 44 and 42 kDa. Treatment of Caco2-3B with UK14304 resulted in a rapid increase in the extent of phosphorylation of the two forms of MAPK. Phosphorylation was observed as early as two minutes after treatment, was maximal at five minutes, and persisted for at least 15 minutes. On the basis of the analysis of seven independent experiments, the maximal effect observed after five minutes of exposure represents a twofold increase in phosphorylation. No significant change in phosphorylation of MAPK was detected in Caco2 cells, demonstrating that the effect of UK14304 was primarily due to stimulation of α2 adrenoceptors. The mechanisms whereby stimulation of α2A adrenoceptor increases MAPK phosphorylation were then investigated directly using antiactive MAPK antibody on total cellular extracts. Unlike anti-Erk antibodies on immunoprecipitated phosphoproteins (as in fig 5), the antiactive MAPK antibody revealed a major band corresponding to phosphorylated Erk2 (see figs 6, 7). The western blots presented in fig 6 clearly show that the increase in Erk2 phosphorylation following exposure to UK14304 was totally abolished by pretreatment of the cells with pertussis toxin (250 ng/ml, 16 hours). Thus the integrity of Gi proteins is necessary for the effect of the α2 agonist to occur. In the same manner, addition of the inhibitor of protein tyrosine kinases, genistein (25 μM), or addition of the inhibitor of the MEK1 form of MAPK kinases, PD98059 (50 μM), prevented the effect of UK14304. As shown in fig 7, acute stimulation of protein kinase C (PKC) by addition of 2.5 μM PMA for five minutes also caused marked activation of MAPK. Long term treatment of cells with PMA totally abolished any further response to the phorbol ester but did not affect the response to UK14304, suggesting that α2 adrenoceptors trigger their effects through a PKC independent pathway. As activation of MAPK by several G protein coupled receptors was shown to correlate with Shc phosphorylation in Rat-1 fibroblasts,36 we examined if such a phosphorylation also occurs on α2A adrenoceptor stimulation in Caco2-3B cells. The use of anti-Shc antibody on extracts prepared from A431 and Caco2-3B cells (fig 8) indicated that our model predominantly expressed the 46 and 52 kDa isoforms of Shc. Furthermore, treatment of the cells with 1 μM UK14304 caused increased phosphorylation of both isoforms of Shc. Activation was notable after two minutes of treatment with the α2 agonist and was transient, a decrease in phosphorylation being observed after 10 minutes. The time course of Shc phosphorylation was thus compatible with activation of Shc preceding that of MAPK.

Figure 5

Effect of UK14304 on mitogen activated protein kinase (MAPK) phosphorylation. Caco2 and Caco2-3B cells were seeded at low density (105 cells/cm2) and grown for three days in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. Pre-confluent cell layers were then maintained for 48 hours in serum free medium and treated for the indicated time with 1 μM UK14304. Left: tyrosine phosphorylated proteins were immunoprecipitated, separated by SDS-PAGE, and MAPK revealed by immunoblotting with anti-Erk1 and anti-Erk2 antibodies as described in materials and methods. Right: the extent of MAPK phosphorylation is expressed as a per cent of that in control cells (untreated). Results are mean (SEM) (n=3 for Caco2, n=7 for Caco2-3B). Significantly different from untreated cells: *p<0.05, **p<0.02.

Figure 6

Effect of pertussis toxin (PTX), genistein, and PD98059 on mitogen activated protein kinase (MAPK) phosphorylation. Caco2-3B cells were grown and rendered quiescent as indicated in the legend of fig 5. The cells were treated for 16 hours with pertussis toxin (250 ng/ml) or for 30 minutes with either genistein (25 μM) or PD98059 (50 μM). They were then exposed (+) or not (−) to 1 μM UK14304 for five minutes. Top: MAPK phosphorylation was studied using the anti-active MAPK antibody. Bottom: the membrane was stripped of Ig and reprobed with anti-Erk2 antibody to assess equal protein loading.

Figure 7

Effect of phorbol 12-myristate 13-acetate (PMA) on UK14304 induced mitogen activated protein kinase (MAPK) phosphorylation. The Caco2-3B cells were grown and rendered quiescent, as indicated in the legend of fig 5, and treated for 16 hours with 2.5 μM PMA (treated) or vehicle (control). They were then exposed for five minutes to vehicle (−), 2.5 μM PMA (PMA), or 1 μM UK14304 (+). Top: MAPK phosphorylation was studied using the anti-active MAPK antibody. Bottom: the membrane was stripped of Ig and reprobed with anti-Erk2 antibody to assess equal protein loading.

Figure 8

Identification of Shc isoforms and effect of UK14304 on phosphorylation. Left: identification of the Shc isoforms expressed in Caco2-3B cells. Protein extracts from Caco2-3B and A431 cells (used here as positive control) were subjected to SDS-PAGE and Shc isoforms were revealed by western blot using anti-Shc antibody. Right: UK14304 induced phosphorylation of Shc. The Caco2-3B cells were grown, rendered quiescent as indicated in the legend of fig 5 and then treated for the indicated time with 1 μM UK14304. Shc proteins were immunoprecipitated with anti-Shc antibody and the extent of their phosphorylation estimated by western blot with an antiphosphotyrosine antibody (right upper panel). The membrane was stripped of Ig and reprobed using anti-Shc antibody to assess equal protein loading (right lower panel).

Activation of MAPK being generally associated with cellular proliferation, we finally examined the effect of α2adrenoceptor stimulation on the growth rate of Caco2-3B. No effect was detected when UK14304 was tested in serum free or in 10% FCS medium. However, an accelerated proliferation was repeatedly observed when assays were carried out in culture medium containing 0.5% FCS. The proliferative effect of the α2 agonist was modest but significant. As estimated by measurement of total cellular protein and DNA content, it represented a 20% increase in cell proliferation after 16 days of culture (fig 9). Furthermore, this effect was solely due to α2 adrenoceptor activation because it was not observed in the parental cell line Caco2 (not shown).

Figure 9

Effect of UK14304 on cell growth. Caco2-3B cells were seeded at low density (105 cells/cm2). Three days after seeding, they were rendered quiescent in serum free medium for 48 hours. Cellular growth was then reinitiated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.5% fetal calf serum with or without 1 μM UK14304. Protein content (left) and DNA content (right) per well were determined as described in materials and methods. Data presented are from a typical experiment and the results are mean (SEM) (n=3). Significantly different from untreated cells: *p<0.05, **p<0.01.

Discussion

The α2 adrenoceptors regulate a number of physiological functions including neurotransmitter release, insulin secretion, vasoconstriction, platelet aggregation, renal Na+ reabsorption, and intestinal Clsecretion.37 In addition to these actions, recent findings have demonstrated that these G protein coupled receptors can also activate the MAPK pathway and behave as mitogens or co-mitogens on transfected cells.17-21 This latter effect is reminiscent of former observations which indicated that administration of noradrenaline to rodents activated proliferation of intestinal crypt cells via stimulation of α2 adrenoceptors.16As yet the occurrence of this mitogenic effect awaits demonstration in other species and the molecular mechanisms involved remain to be elucidated. As it is impossible to carry out in vivo studies in humans or to maintain crypt cells isolated from normal mucosa in culture, the putative action of α2 adrenoceptors on proliferation can only be investigated in transformed cells. The human colon adenocarcinoma HT29, which spontaneously expresses α2adrenoceptors, may have represented an alternative model to approach this question. However, receptor expression is growth related in this cell line.38 More precisely, receptor density is very low during the exponential phase of proliferation and increases at confluence, making HT29 inadequate. To circumvent this problem, we developed a model in which receptor expression was independent of culture conditions. The host cells we retained for this purpose were Caco2 cells. The reason for this choice lies in the fact that in addition to proliferation studies, this spontaneously differentiated model represents a valuable in vitro system to investigate the regulatory effects of α2 adrenoceptor on intestinal ion transport mechanisms.

Transfection of Caco2 cells gave a permanent clone (Caco2-3B) expressing α2A adrenoceptors at a density similar to that of human intestinal epithelium.9 As in normal enterocytes or in HT29,9 39 the receptor is coupled to the pertussis toxin sensitive G proteins Gi2 and/or Gi3, and stimulation caused a marked reduction in forskolin stimulated adenylyl cyclase. Exposure to UK14304 induced an increase in the phosphorylation state of Erk1 and Erk2. As expected, this effect was blocked by addition of RX821002 (α2 antagonist). This was also observed with the endogenous catecholamine (−)adrenaline (not shown). MAPK phosphorylation was rapid (peak at five minutes) and similar to that found in other cell systems in which α2 adrenoceptors had mitogenic properties.19 21 Previous studies have established that the mechanisms whereby G protein coupled receptors activate MAPK vary according to the receptor or cell type examined. Indeed, Erk1/2 phosphorylation may be mediated by either Gq or Gi/o and be dependent on PKC or Ras activation.20 40 As α2 adrenoceptors stimulate PKC via a Gi dependent pathway in OK cells or in human platelets,41 42 and as they increase phospholipase C activity and intracellular Ca++ in HEL or transfected CHO cells,43 we investigated if the effect of UK14304 involved PKC activation. Short term exposure of Caco2-3B cells to the PKC activator PMA resulted in an increase in Erk phosphorylation. However, the effect of UK14304 was not significantly influenced after desensitisation of PKC activity. In contrast, it was totally abolished by pertussis toxin treatment, indicating that it is fully dependent on activation of Gi proteins. Nevertheless, it is clear that inhibition of adenylyl cyclase is not responsible for the action of the α2 agonist because under conditions where effects on Erk are observed (that is, in the presence of UK14304 alone) intracellular levels of cAMP are unchanged. In contrast, the change in MAPK phosphorylation was abolished by genistein and PD98059, indicating that tyrosine kinases and MEK 1 are necessary for UK14304 to act. To further elucidate the signalling pathway leading to MAPK activation, phosphorylation of Shc was measured. The two major isoforms of Shc (46 kDa and 52 kDa), which are expressed in Caco2-3B, are transiently phosphorylated following agonist exposure. As in transfected COS7 cells,44 it is therefore likely that α2A adrenoceptors in Caco2-3B stimulate a cascade of events comprising recruitment of Gi proteins, Gβγ subunit mediated formation of Shc-Grb2-SOS complex, and subsequent activation of MEK 1 and MAPK.

A recent study on a subclone of HT29 cells (HT29-N2) showed that a decrease in MAPK activity plays a critical role in the biochemical and morphological differentiation of intestinal cells.45 It was thus conceivable that the reverse may hold true and that activation of MAPK by UK14304 enhances proliferation of Caco2-3B. In fact, no stimulatory effect of UK14304 was observed when proliferation tests were conducted in either the presence or absence of high concentrations of FCS. However, a significant increase in growth was found when cells were placed in medium containing trace amounts of growth factors (0.5% FCS). Thus if the α2 agonist is inefficient in quiescent cells and cannot be considered per se as a mitogen, it acts on slowly growing cells and therefore behaves as a co-mitogen on Caco2-3B cells. In this respect, our results differ from those found in transfected 3T3F442A cells or in OK cells in which α2 agonists accelerate the growth rate, even in the absence of FCS.19 21 They are, however, consistent with earlier observations on CCL39 cells transfected with the human α2A adrenoceptor.17 Indeed, α2agonists cannot stimulate the proliferation of these fibroblastic cells when tested alone, but they do stimulate [3H]thymidine incorporation when assayed in combination with fibroblast growth factor. The reason why UK14304 does not increase Caco2-3B proliferation in serum free medium is unclear. It is likely that MAPK activation is not persistent enough and/or not sufficient to result in exit from the Go phase and entry into the cell cycle.46 It is also noteworthy that in rat aortic smooth muscle cells,47activation of MAPK by an α2 agonist is not associated with an increase in cellular proliferation but rather results in cell migration due to F-actin depolymerisation. Conversely, α2agonists were demonstrated to promote actin polymerisation and cell spreading in mouse preadipocytes.48 In this latter case, the effect of α2 agonists on cell adhesion correlated with activation of the RhoA/FAK pathway.49 Interestingly, a previous study on Caco2 cells showed that Rho proteins play a crucial role in the mechanisms whereby growth factors stimulate migration of intestinal cells and thus contribute to the maintenance of epithelium integrity.50 The question of whether α2agonists also stimulate migration or attachment of Caco2-3B deserves further attention. Finally, the clone Caco2-3B retained the morphological features of a polarised epithelium and in the future may represent a suitable model to study the effects of α2adrenoceptor on transepithelial transport. Preliminary results indicate that α2 agonists increase the activity of the peptide transporter PepT1 in these cells,51 suggesting that at least part of the effects of clonidine observed in vivo52are due to direct stimulation of α2 adrenoceptors located on intestinal epithelial cells.

In conclusion, the present work shows that α2adrenoceptors activate the MAPK pathway and act as co-mitogens on a cell line derived from human intestinal epithelium. Further studies are necessary to clarify the precise molecular mechanisms responsible for this action and to assess the action of these receptors on other epithelial functions. However, together with previous findings, our results support the participation of the α2 adrenoceptor in the regulation of intestinal cell proliferation.

Acknowledgments

This work was supported by the BIOMED 2 Programme PL963373 (European Commission, Brussels, Belgium) and by a grant from the Fondation pour la Recherche Médicale (Paris, France). During the time of his participation in this study, JCD was the recipient of a fellowship from the Fondation IPSEN (Paris, France). We thank F Quinchon and E Fonta for helpful technical assistance.

Footnotes

  • Abbreviations used in this paper:
    DMEM
    Dulbecco's modified Eagle's medium
    PBS
    phosphate buffered saline
    Erk
    extracellular regulated protein kinase
    FCS
    fetal calf serum
    MAPK
    mitogen activated protein kinase
    pCMV
    cytomegalovirus early promoter/enhancer
    EMCV
    encephalomyocarditis virus
    PMA
    phorbol 12-myristate 13-acetate
    PD98059
    2-(2-amino-3-methoxyphenyl)-4H-1- benzopyran-4-one
    RX821002
    2-(2-methoxy-1,4-benzodioxan-2-yl)-2 -imidazoline
    UK14304
    5-bromo-6-(2-imidazoline-2-ylamino)- quinoxaline
    SDS-PAGE
    sodium dodecyl sulphate-polyacrylamide gel electrophoresis
    PKC
    protein kinase C
    BSA
    bovine serum albumin
    RPA
    RNase protection assays
    DTT
    dithiothreitol
    RIPA
    radio immunoprecipitation
    PVDF
    polyvinylidene difluoride

References