Protein Kinase Cγ Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B*

Activation of the 5-hydroxytryptamine receptor 2B (5-HT2B), a Gq/11 protein-coupled receptor, results in proliferation of various cell types. The 5-HT2B receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT2B receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT2B receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by LY294002 decreased base-line proliferation but had no effect on 5-HT2B receptor-mediated proliferation. Proliferation of ICC through the 5-HT2B receptor was inhibited by the phospholipase C inhibitor U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the α, β, γ, and μ protein kinase C (PKC) inhibitor Gö6976, and the α, β, γ, δ, and ζ PKC inhibitor Gö6983 inhibited 5-HT2B receptor-mediated proliferation, indicating the involvement of PKC α, β, or γ. Of all the PKC isoforms blocked by Gö6976, PKCγ and μ mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT2B receptor activation in primary cell cultures obtained from PKCγ−/− mice did not result in a proliferative response, further indicating the requirement for PKCγ in the proliferative response to 5-HT2B receptor activation. The data demonstrate that the 5-HT2B receptor-induced proliferative response of ICC is through phospholipase C, [Ca2+]i, and PKCγ, implicating this PKC isoform in the regulation of cellular proliferation.

Tight control of cell proliferation is essential to maintain organ size and function. Proliferation needs to be tightly regulated to maintain a critical mass of a particular cell type while preventing dysplasia or malignancy. Cell proliferation is regulated by a complex interaction between extrinsic and intrinsic factors. Extrinsic factors usually signal through cell surface receptors such as various growth factor receptors. 5-Hy-droxytryptamine (5-HT, 2 serotonin) is well established as a neurotransmitter and a paracrine factor with over 90% of 5-HT produced by the gastrointestinal tract (1,2). There is now substantial evidence that, together with these established functions, 5-HT is involved in the control of cell proliferation through various 5-HT receptors, in particular the 5-hydroxytryptamine receptor 2B (5-HT 2B (3)(4)(5)(6)(7)(8)(9)). The 5-HT 2B receptor is G q/11 protein-coupled. Activation of the 5-HT 2B receptor regulates cardiac function, smooth muscle contractility, vascular physiology, and mood control. Recently it was demonstrated that activation of the 5-HT 2B receptor also induces proliferation of neurons, retinal cells (3,4), hepatocytes (5), osteoblasts (8), and interstitial cells of Cajal (ICC) (9). ICC express the 5-HT 2B receptor, and activation by 5-HT induces proliferation of ICC (9). ICC are specialized, mesoderm-derived mesenchymal cells in the gastrointestinal tract. Their best known function is the generation of slow waves (10), but they also conduct and amplify neuronal signals (11,12), release carbon monoxide to set the intestinal smooth muscle membrane potential gradient (13), and act as mechanosensors (14,15). Loss of ICC has been associated with pathological conditions such as gastroparesis (16 -18), infantile pyloric stenosis (19,20), pseudo-obstruction (21,22), and slow transit constipation (23), whereas increased proliferation of ICC or their precursors is associated with gastrointestinal stromal tumors (24).
The mechanisms by which activation of the 5-HT 2B receptor results in increased proliferation are not well understood. In cultured cardiomyocytes, stimulation of the 5-HT 2B receptor activated both phosphatidylinositol 3-kinase (PI3Ј-K)/Akt and ERK1/2/mitogen-activated protein kinase (MAPK) signaling pathways to protect cardiomyocytes from apoptosis (25). On the other hand, the 5-HT2 subfamily of receptors are also known to couple to phospholipase C (PLC) (26 -28).
The objective of this study was to utilize the known expression of the 5-HT 2B receptor on ICC to determine whether proliferation through the 5-HT 2B receptor required PI3Ј-K or PLC. This study demonstrates that proliferation mediated by the 5-HT 2B receptor requires PLC, intracellular calcium release, and the ERK/MAPK signaling pathway and identifies the PKC isoform activated by the 5-HT 2B receptor in ICC as PKC␥.

Animals
The mice were maintained and the experiments were performed with approval from the Institutional Animal Care and Use Committee of the Mayo Clinic. BALB/c mice were obtained from Harlan Sprague-Dawley (Indianapolis, IN). PKC␥ knock-out (PKC␥ Ϫ/Ϫ ) mice were obtained from Jackson Laboratory (Bar Harbor, ME). 2-4-day-old mice were killed by CO 2 inhalation and cervical dislocation. The jejunum was quickly dissected out, flushed with ice-cold calcium-free Hanks' balanced salt solution (Invitrogen), and pinned onto a Sylgard-lined Petri dish, and the mucosa and mesentery were removed.

Reverse Transcription-PCR
To determine the expression of different PKC isoforms, PCR was performed on adult mouse jejunal muscle strip RNA. Adult mouse brain served as a positive control. Whole mouse brain was quickly dissected out on ice. Total RNA from jejunal muscle strips and brain was isolated using RNAbee (Tel-Test, Friendswood, TX) according to the manufacturer's instructions. Reverse transcription was performed using Gene Amp Gold RNA PCR reagent kit (Applied Biosystems, Foster City, CA) as described by the manufacturer. Polymerase chain reaction for PKC␣, ␤, ␥, and message was performed on 50 ng of sample cDNA using 300 nmol/liter of gene-specific primers (supplemental Table S1) and AmpiTaq Gold (Applied Biosystems) according to the manufacturer's instructions.

Enzymatic Dissociation of ICC
To obtain freshly dispersed jejunal cells, muscle strips from three mice were pooled in a collagenase-based dissociation mixture. The mixture contained 2500 units of collagenase type II (Worthington Biochemical Company), 20 mg of bovine serum albumin (Sigma), 20 mg of trypsin inhibitor (Sigma), and 5 mg of adenosine triphosphate (Roche Applied Science) in 10 ml of calcium-free Hank's balanced salt solution. The pH was adjusted to 7.0 with 0.1 M NaOH. After 15 min of incubation at 32°C in a gently shaken water bath, the tissue was triturated and spun down at 800 ϫ g for 6 min. The cells were resuspended in 2 ml of Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum (Hyclone, Logan, UT), 1% sodium pyruvate (Invitrogen), and 1% antibiotic-antimycotic (Invitrogen).

Mouse Interstitial Cells of Cajal and Fibroblast Co-cultures
Freshly dispersed cells obtained from the mouse small intestine were cultured on 22-mm glass coverslips covered with mouse fibroblasts as described before (29). These fibroblasts support ICC survival because of being genetically engineered to produce murine stem cell factor, the ligand for the Kit receptor tyrosine kinase protein expressed on ICC. Briefly, Sl/Sl 4 mSCF248, murine stem cell factor secreting fibroblasts (provided by Dr. David Williams, Indianapolis), were plated on 22-mm human fibronectin-coated glass coverslips at 4.5 ϫ 10 4 cells/coverslip in high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1% sodium pyruvate, and 1% antibiotic-antimycotic (all from Invitrogen). After 30 h, fibroblast cell division was arrested by irradiation at 16,000 grays. After a 24-h recovery period, 250 l of suspensions containing cells freshly dissociated from mouse jejunum were plated onto the Sl/Sl 4 mSCF248 fibroblasts at a cell density of ϳ3 ϫ 10 5 cells/coverslip. The cells were allowed to sit for 30 min at 37°C/5% CO 2 before adding 2 ml of the culture medium to the well. These culture conditions result in cell cultures highly enriched in ICC.

Isolation of Single ICC
Single ICC were selected on the basis of detection of the ICC marker kit. This procedure was carried out on ice. Freshly dissociated cells were stained for Kit expression with the phycoerythrin-labeled anti-mouse CD117 (ACK2; eBioscience, San Diego, CA) diluted at 1:300 in flow cytometry staining buffer (eBioscience). The cells were incubated for 20 min in the dark on a shaker and washed three times with ice-cold staining buffer. Single ICC were selected based on the phycoerythrin-ACK2 immunolabeling and collected by gentle aspiration into a 30-m-wide patch clamp pipette tip. Two to three cells were lifted out of the chamber and immediately expelled into a 200-l tube on dry ice as described previously (30). For controls, two to three single cells not positive for phycoerythrin-ACK2 labeling were collected per tube.

Single-cell PCR
0.5 g of yeast tRNA (Ambion, Austin, TX) and 10 g of proteinase K (Roche Applied Science) were added per isolated cell. The sample was centrifuged at 3300 ϫ g at 4°C for 30 s to release RNA. Nucleases and proteinases were destroyed by incubation at 90°C for 10 min, 55°C for 30 min, and 90°C for 10 min. Two-step reverse transcription and PCR were performed using the TaqMan Gold reverse transcription-PCR kit (Applied Biosystems) as described by the manufacturer. 25-l PCRs were set up by dividing all solutions by 2. 5 l of the first PCR (94°C for 5 min, 35 times 94°C for 20 s, melting temp (T m ) for 20 s, 72°C for extension time, and 72°C for 10 min) was used for the nested PCR (94°C for 5 min, 35 times 94°C for 20 s, T m for 20 s, 72°C for extension time, and 72°C for 10 min). The optimized PCR programs are reported in supplemental Table S1.

Data Analysis
All of the data analysis was carried out on the raw non-normalized data. For cell culture experiments, analysis of variance with a Tukey-Kramer multiple comparisons post test was done to compare groups of data. For experiments with two groups, Student's t test was used. All of the data are expressed as the means Ϯ S.E., where n represents the number of experiments on individual dissociations.

Immunohistochemistry
Immunohistochemical Staining of Primary Cell Cultures-Interstitial cells of Cajal were immunolabeled using the rat monoclonal anti-c-Kit antibody ACK2 (eBioscience, San Diego, CA) as described previously (29). Briefly, acetone-fixed coverslips (4°C, 10 min) were washed with PBS and incubated with 10% normal donkey serum (NDS; Jackson Immunoresearch Laboratories, West Grove, PA) and 0.2% Triton X-100 (Sigma) in PBS for 1 h to minimize nonspecific antibody binding followed by primary antibody (1/150, 120 g/ml, in 5% NDS) incubation overnight at 4°C. Next, the sections were rinsed in PBS.
Immunolabeling using a monoclonal antibody to Ki67 (Novus Biologicals, Littleton, CO) was used to identify proliferating cells. After the ACK2 staining procedure, the coverslips were post-fixed with 4% paraformaldehyde for 10 min, washed with PBS, and incubated with anti-Ki67 (2 g/ml, in 5% NDS) for 4 h at room temperature. Next, the sections were rinsed in PBS and incubated in the dark for 1 h at room temperature with donkey anti-rat IgG conjugated to Cy3 (Jackson Immunoresearch Laboratories; 1.8 g/ml in 2.5% NDS) and donkey antirabbit conjugated to fluorescein isothiocyanate (Jackson Immunoresearch Laboratories; 7.5 g/ml in 2.5% NDS).
Mounting and Analysis-Sections and immunostained cultures were mounted with Slow Fade Gold containing 4Ј,6-diamidino-2-phenylindole from Invitrogen and examined using a fluorescence microscope. Immunostained cultures were examined for Kit-positive cells with the use of a fluorescent microscope (Olympus BX51WI; Olympus America Inc., Center Valley, PA). A 20ϫ (numeral aperture, 0.5) objective was used to count the number of Kit-positive cells/high power field. One field covered 0.94 mm 2 . At least 35 fields were counted per culture. Sampling from 35 fields was done because, based on previous studies, this sample size gives an accurate measure of the mean density of the cells and variation in the cell density.
Base-line proliferation of ICC in culture was not affected by the PLC inhibitor U73122 (Fig. 2B). In contrast, the proliferative response of ICC to 5-HT 2B receptor activation was completely blocked by the U73122 (Fig. 2B), indicating that proliferation mediated by activation of the 5-HT 2B receptor requires PLC. U73343, the inactive analogue of U73122, did not block the proliferative response to BW723C86 (control, 16 AUGUST 7, 2009 • VOLUME 284 • NUMBER 32

5-HT 2B -mediated Proliferation Requires PKC␥
Proliferation of ICC through the 5-HT 2B Receptor Is Dependent upon Intracellular Calcium Release-Activation of phospholipase C mediates generation of diacylglycerol and inositol 1,4,5-trisphosphate, resulting in intracellular calcium release through opening of inositol 1,4,5-trisphosphate receptor channels. To test whether the proliferative effect of 5-HT 2B receptor activation requires intracellular calcium release, the effect of the inositol 1,4,5-trisphosphate receptor antagonist Xestospongin C (31) on proliferation was assayed.
Xestospongin C had no effect on base-line proliferation of ICC (Fig. 3A). Proliferation of ICC through the 5-HT 2B receptor was completely inhibited by 333 nM and 1 M Xestospongin C (Fig. 3A). 5-HT 2B receptor activation in ICC increased intracellular Ca 2ϩ (supplemental Fig. S1), and 5-HT 2B receptor-mediated responses were abolished in the presence of Xestospongin C (supplemental Fig. S2).
A Calcium-dependent Protein Kinase C Is Required for Proliferation of ICC through the 5-HT 2B Receptor-To examine whether the proliferative effect of the 5-HT 2B receptor is mediated by protein kinase C, cells were treated with calphostin C, a nonspecific PKC inhibitor that inhibits all PKC isoforms except for PKC. In the presence of calphostin C, base-line proliferation of ICC was unchanged (Fig. 3B). In contrast, proliferation of ICC through the 5-HT 2B receptor was dose-dependently inhibited by 50 and 150 nM calphostin C (Fig. 3B). These data show that activation of a PKC is required for the proliferative effects of the 5-HT 2B receptor and that the isoform involved is not PKC.
Next we studied the effect of the more specific PKC inhibitors Gö6976 and Gö6983. In the presence of the ␣, ␤, ␥, and PKC inhibitor Gö6976 (100 nM), base-line proliferation of ICC was reduced (Fig. 4A). The proliferative response of ICC to BW723C86 was inhibited by Gö6976 (Fig. 4A). In contrast to Gö6976, the ␣, ␤, ␥, ␦, and PKC inhibitor Gö6983 (30 and 100  nM), which does not inhibit PKC (IC 50 ϭ 20,000 nM), did not change base-line proliferation of ICC (Fig. 4B). Proliferation of ICC through the 5-HT 2B receptor was inhibited by Gö6983 (Fig. 4B). Taken together, the PKC inhibitor data indicate that 5-HT 2B receptor-mediated proliferation requires a calcium-dependent conventional PKC. The data also demonstrate that PKC is involved in non-5-HT 2B -mediated proliferation of ICC.
Proliferation through the 5-HT 2B Receptor Requires ERK/ MAPK Activation-To test whether proliferation through the 5-HT 2B receptor requires ERK/MAPK activation, primary cultures were treated with 25 M of ERK inhibitor, which inhibits ERK binding. The proliferative response of ICC to 5-HT 2B receptor activation was inhibited in the presence of the drug (Fig. 5).
Single ICC Expressed Gö6976-sensitive PKC Isoforms ␥ and -Experiments were directed toward determining which calcium-dependent PKCs are expressed in the intestinal smooth muscle wall and whether PKC is also expressed. Mouse brain cDNA was used as a positive control. Using PCR, PKC␣, ␤, ␥, and mRNAs of the expected size, 414, 557, 528, and 426 nt, respectively, were amplified from total RNA of mouse brain and jejunal muscle strips (Fig. 6).
Next, nested PCR for PKC␣, ␤, ␥, and was performed on two or three single Kit-positive neonatal ICC and on two or three Kit-negative cells to identify the specific PKC isoforms expressed on ICC. mRNA for PKC␣ was amplified (223-nt product) from Kit-negative cells but not from ICC (Fig. 7A). mRNA for PKC␤ could not be amplified from ICC nor from Kit-negative cells (data not shown). On the other hand, prod-    AUGUST 7, 2009 • VOLUME 284 • NUMBER 32 ucts of the expected length for PKC␥ (334 and 528 nt; Fig. 7B) and PKC (248 nt; Fig. 7C) were amplified from mRNA extracted from ICC but not from Kit-negative cells. ␤-Actin mRNA was amplified (453 nt) from ICC and Kit-negative cells (Fig. 7D). As a control, a product of the expected length for Kit (285 nt; Fig. 7E) was amplified from mRNA isolated from ICC but not from Kit-negative cells. The identities of all bands were confirmed by sequencing. These data, taken with the PKC inhibitor data, suggest that 5-HT 2B receptor signals through PKC␥.

5-HT 2B -mediated Proliferation Requires PKC␥
The Proliferative Response of ICC by Activation of the 5-HT 2B Receptor Is Absent in ICC Lacking PKC␥-To confirm the PKC inhibitor data, ICC cultures were obtained from PKC␥ Ϫ/Ϫ mice. Activation of the 5-HT 2B receptor by its agonist BW723C86 had no proliferative effect on ICC (Fig. 8). Functionality of the 5-HT 2B receptor in the PKC␥ Ϫ/Ϫ mice was confirmed by measuring contractility of jejunal circular muscle strips in response to the 5-HT 2B receptor agonist BW723C86.

DISCUSSION
This study provides evidence that activation of the 5-HT 2B receptor increases proliferation through activation of PLC, inositol 1,4,5-trisphosphate receptor-mediated intracellular calcium release, activation of the calcium-dependent PKC␥, and ERK/MAPK activation.
The role of 5-HT in regulating gastrointestinal motility has been extensively studied. However, accumulating evidence, mostly from outside the gastrointestinal tract, shows that 5-HT can also regulate cell survival and proliferation. Four serotonin receptors have been implicated in transduction of the 5-HT signal to regulate cell survival and proliferation: 5-HT 1A , 5-HT 1D , 5-HT 2B , and 5-HT 2C receptors. Activation of 5-HT 1A and 5-HT 2C receptors results in increased numbers of newly formed neurons in rat brain (32)(33)(34), whereas 5-HT 1A and 5-HT 1D receptors regulate mitogenesis in human small cell carcinoma of the lung (35). 5-HT 1A receptors are also involved in proliferation of T and B cells (36) and rat blood lymphocytes (37). A role for 5-HT 2B receptors in cell differentiation and proliferation has been shown in various cell types and species including mouse enteric neurons, hepatocytes, mouse cardiomyocytes, and retinal cells in Xenopus (3)(4)(5)(6)(7)38). We demonstrated previously that activation of the 5-HT 2B receptor on murine ICC induces proliferation of ICC (9).
The 5-HT 2B receptor is a G q/11 protein-coupled receptor that can activate at least two mitogenic pathways including PI3Ј-K/ Akt (25) and PLC (26 -28). The effect of the PI3Ј-K inhibitor LY294002 and the PLC inhibitor U73122 on proliferation induced by activation of the 5-HT 2B receptor was studied on primary ICC cultures. Although LY294002 dose-dependently decreased the percentage of base-line proliferating ICC, activation of the 5-HT 2B receptor in the presence of LY294002 still increased ICC proliferation. These results demonstrate that proliferation of ICC through the 5-HT 2B receptor is not dependent upon PI3Ј-K and that another signaling cascade, downstream of a so far unidentified receptor, induces proliferation of ICC through PI3Ј-K. Proliferation of ICC activated by The faint band slightly above the expected size seen in the ICC lane was sequenced and was found to be nonspecific amplification. B and C, PKC␥ (B, 334 and 528 nt) and (C, 248 nt) mRNAs were amplified from ICC but not from nonlabeled cells. D, ␤-actin mRNA was amplified from ICC and nonlabeled cells (453 nt). E, as a control, c-Kit mRNA was amplified from ICC but not from nonlabeled cells (285 nt). The identity of all amplification products was confirmed by sequencing. Ladders, DNA molecular weight marker XIV (Roche Applied Science). BW723C86 was completely inhibited by U73122 and Xestospongin C, the inositol 1,4,5-trisphosphate receptor inhibitor, and was therefore dependent upon PLC and intracellular Ca 2ϩ release. In support of this observation, U73343, the inactive analogue of U73122, did not affect the proliferative response to BW723C86, and although both of these compounds do have some nonspecific effects, these observations are most consistent with their differential effects on PLC.
A role for PKC␥ in mediating proliferation has not previously been established. Although neuronal PKC␥ was implicated in the proliferation of astrocytes following repeated morphine administration in vivo, the mechanism of action was not studied (46). In contrast, overexpression of PKC␥ reduced proliferation and resulted in differentiation of lens epithelial cells (47) and glioma cells (48), whereas mutations in PKC␥ reduced its kinase activity, resulting in lower ERK phosphorylation and nuclear ERK translocation, reduced MAPK signaling, and reduced survival of Purkinje cells leading to spinocerebellar ataxia-14 (49). This is the first study demonstrating that 5-HT 2B receptor-mediated proliferation is regulated by PKC␥.
Based on literature, ICC express various PKC isoforms including PKC␥ (50,51), PKC (50 -52), and PKC⑀ (53). In our hands, PKC␣ and PKC␤ mRNAs were lacking in ICC, in line with Furness et al. (50). We confirmed the expression of PKC␥ mRNA in ICC, as described in guinea pig (51) and human (50), and we demonstrated for the first time the expression of PKC mRNA in ICC.
The use of PKC inhibitors to determine specific PKC isoforms is fraught with difficulties because of the conflicting literature and the need to use correct drug concentrations based on the IC 50 values of the different isoforms. We therefore used three different PKC inhibitors and confirmed our results in PKC␥ Ϫ/Ϫ mice before concluding that the effects of 5-HT 2B on proliferation are mediated through PKC␥. Calphostin C was used as a nonspecific PKC antagonist (inhibits all isoforms except for PKC). Gö6976 is used as a potent inhibitor of PKC ␣, ␤, and . However, 158 nM Gö6976 has been shown to inhibit all PKC activity in the brain (IC 50 ϭ 12.5 nM). PKC isoforms expressed in the brain include PKC␣, ␤I, ␤II, and ␥. Therefore PKC␥ is also inhibited by Gö6976 in this concentration range, making Gö6976 an inhibitor of all the conventional PKCs and also of PKC (54 -56). Gö6983 inhibits PKC␣, ␤, ␥, ␦, and at nanomolar range and PKC at 20 M and can therefore be used to discriminate the role of PKC in the presence of other PKC isoforms (56). We found that calphostin C dose-dependently inhibited the proliferative effect of BW723C86 and had no effect on base-line proliferation. 100 nM Gö6976 and 30 and 100 nM Gö6983 fully inhibited proliferation of ICC induced by the 5-HT 2B receptor agonist BW723C86, indicating that PKC␣, ␤, and/or ␥ are required. Gö6976, which also inhibits PKC, decreased non-5-HT 2B receptor-mediated proliferation as well. Based on these PKC inhibitor data and the fact that of the three possible isoforms only PKC␥ mRNA was expressed in ICC, we concluded that 5-HT 2B receptor-mediated proliferation was regulated by PKC␥. This result was confirmed in ICC obtained from PKC␥ Ϫ/Ϫ mice, where activation of the 5-HT 2B receptor did not result in a proliferative response despite the 5-HT 2B receptor appearing to be functionally active. We found that base-line proliferation of ICC required PKC. It is known that overexpression of PKC activates MAPK (57) and inhibits the c-Jun N-terminal kinase signaling pathway (58,59), thereby inducing cellular proliferation. PKC is also known to be activated by PI3Ј-K (60). Based on the experiments with LY294002 and the PKC inhibitor results, we can state that base-line proliferation of ICC likely requires PI3Ј-K and PKC, but the growth receptor(s) underlying this proliferative pathway remain unknown.
ICC are important regulators of gastrointestinal motility because they generate slow waves and determine the frequency of contractions, they act as amplifiers of neuronal signals and as mechanosensors, and they set the smooth muscle membrane potential gradient. A decreased number of ICC or a disrupted ICC network is associated with pathologic conditions such as slow transit constipation, diabetic gastroparesis, and pseudoobstruction. Understanding the factors that underlie ICC survival and proliferation is important in determining how ICC networks are maintained and how to limit ICC loss or induce recovery in disease states associated with the loss of ICC. Recent observations indicate that turnover of ICC occurs in immature and adult ICC. We now know that there are several substances that help modulate ICC numbers in the gastrointestinal tract including a functional c-Kit-stem cell factor pathway (29), the insulin/insulin growth factor I signaling pathway (61), and cryoprotective molecules including nitric oxide (62) and heme-oxygenase 1 (16).
In summary, we showed that activation of the 5-HT 2B receptor on ICC results in proliferation of ICC. This proliferative effect of the 5-HT 2B receptor is mediated by activation of PLC, intracellular calcium release, and PKC␥.