The Glucagon-like Peptide-2 Receptor Mediates Direct Inhibition of Cellular Apoptosis via a cAMP-dependent Protein Kinase-independent Pathway*

Glucagon and the glucagon-like peptides regulate metabolic functions via signaling through a glucagon receptor subfamily of G protein-coupled receptors. Activation of glucagon-like peptide-2 receptor (GLP-2R) signaling maintains the integrity of the intestinal epithelial mucosa via regulation of crypt cell proliferation. Because GLP-2 decreases mortality and reduces intestinal apoptosis in rodents after experimental injury, we examined whether GLP-2R signaling directly modifies the cellular response to external injury. We show here that activation of GLP-2R signaling inhibits cycloheximide-induced apoptosis in baby hamster kidney fibroblasts expressing a transfected GLP-2 receptor. GLP-2 reduced DNA fragmentation and improved cell survival, in association with reduced activation of caspase-3 and decreased poly(ADP-ribose) polymerase cleavage and reduced caspase-8 and caspase-9-like activities. Both GLP-2 and forskolin reduced mitochondrial cytochrome crelease and decreased the cycloheximide-induced cleavage of caspase-3 in the presence or absence of the PKA inhibitor H-89. Similarly, GLP-2 increased cell survival following cycloheximide in the presence of the kinase inhibitors PD98054 and LY294002. These findings provide evidence that signaling through G protein-coupled receptors of the glucagon superfamily is directly linked to regulation of apoptosis and suggest the existence of a cAMP-dependent protein kinase-, phosphatidylinositol 3-kinase-, and mitogen-activated protein kinase-independent pathway coupling GLP-2R signaling to caspase inhibition and cell survival.

Glucagon and the glucagon-like peptides are co-encoded within a common precursor, proglucagon, that is expressed in a tissue-specific manner, giving rise to glucagon in the pancreatic A cells and glucagon-like peptides-1 and 2 in the endocrine cells of the gastrointestinal tract (1,2). Proglucagon-derived peptides play important roles in regulation of metabolic function following nutrient assimilation (2). Glucagon regulates hepatic glucose production and maintains plasma glucose in a narrowly defined physiological range by opposing the actions of insulin at the hepatocyte. Glucagon-like peptide-1 (GLP-1) 1 is secreted from the gut following nutrient ingestion and controls glycemia via stimulatory and inhibitory effects on insulin and glucagon, respectively (2,3). In contrast, glucagon-like peptide-2 (GLP-2) exerts its principal actions on nutrient homeostasis proximal to nutrient absorption via regulation of the integrity of the mucosal epithelium (4,5).
In addition to metabolic effects regulating fuel homeostasis, glucagon-like peptides also exert specific actions on cell proliferation and tissue regeneration. Glucagon potentiates proliferation of rat hepatocytes (6) and stimulates hepatic DNA synthesis following partial hepatectomy in vivo (7). GLP-1 increases islet proliferation in mice (8) and enhances islet regeneration and lowers blood glucose in rats following partial pancreatectomy (9). GLP-1 also appears to stimulate islet neogenesis via induction of pdx-1 expression in normal and diabetic rodents (10).
GLP-2 stimulates intestinal crypt cell proliferation in normal rats and mice leading to villus hyperplasia and expansion of the mucosal epithelium (4,11,12). The beneficial effects of GLP-2 in experimental models of intestinal injury have largely been attributed to enhancement of mucosal regeneration via GLP-2-dependent stimulation of crypt proliferation (13)(14)(15)(16). Although GLP-2 inhibits apoptosis in the crypt compartment following administration of the nonsteroidal anti-inflammatory agent indomethacin (14), the mechanisms coupling GLP-2 signaling to anti-apoptotic effects in a direct or indirect manner remain unknown. Because intestinal cell lines expressing the endogenous GLP-2 receptor have not yet been identified, we have now examined the effects of GLP-2 receptor signaling on cell death in heterologous cells expressing the transfected rat GLP-2 receptor.

EXPERIMENTAL PROCEDURES
Materials-Tissue culture medium, serum, and other supplements, including G418, were from Life Technologies, Inc. Cycloheximide, forskolin, protease inhibitor mixture (P-2714), and 4Ј,6-diamidino-2-phenylindole were purchased from Sigma. Recombinant human [Gly2]-GLP-2 was a kind gift from NPS Allelix Inc. (Mississauga, Canada). The caspase inhibitors Z-VAD-fmk and Z-YVAD-fmk and the kinase inhibitors H89 and LY294002 were obtained from Calbiochem (San Diego, CA). Ac-IETD-pNA and Ac-LEHD-pNA were from BIOSOURCE International (Camarillo, CA). PD98059 was obtained from New England * This work was supported in part by grants from the Medical Research Council of Canada and the Ontario Research and Development Challenge Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  Biolabs (Beverly, MA). X-gal was purchased from BioShop Canada Inc. (Burlington, Canada). All electrophoresis and immunoblotting reagents were purchased from Bio-Rad. The pCRE/␤-galactosidase reporter plasmid (17) and the expression vector MtR(AB) (18) were gifts from R. D. Cone (Portland, OR) and G. S. McKnight (Seattle, WA), respectively.
Cell Culture, Apoptosis Induction, and Drug Treatments-Baby hamster kidney (BHK) fibroblasts containing the stably integrated pcDNA3.1 plasmid (BHK-pcDNA3) (Invitrogen, Carlsbad, CA) or the identical plasmid directing expression of the rat GLP-2 receptor (BHK-GLP-2R) were propagated as described previously (19). Stock cultures were trypsinized, and the cells were replated in culture medium lacking G418. Upon reaching 80 -90% confluency, the cultures were starved in Dulbecco's modified Eagle's medium supplemented with 0.2% calf serum for 15-17 h prior to apoptosis induction by cycloheximide in the same medium, in the presence or absence of the indicated peptides or drugs for the indicated period of time. Adherent cells were then scraped off the culture dishes in PBS, combined with detached cells floating in the medium and spun at 12,000 rpm for 30 s. Depending on the type of assay to be performed, cell pellets were either stored at Ϫ70°C or used immediately. Cycloheximide was dissolved in anhydrous ethyl alcohol, h[Gly2]-GLP-2 in PBS, pH 7.4, and forskolin, H89, LY294002, PD98059, Z-VAD-fmk, and Z-YVAD-fmk in dimethyl sulfoxide. Drug stock solutions were stored at Ϫ70°C and diluted as required in their corresponding solvents, immediately before being added either alone or in combination to the cultures at the desired final concentrations. Control cultures were subjected to the same manipulations as treated cells but in the absence of the drugs. Ethanol and dimethyl sulfoxide final concentrations were identical in every culture irrespective of the particular treatment group.
Microscopy-Morphological assessment of cell death was performed by examination of the cultures under phase contrast microscopy for the presence of cell detachment and the appearance of plasma membrane blebbing. Phase contrast as well as bright field microscopy were performed on a PhotoZoom Leica inverted microscope. Images of representative areas of the cultures were acquired with a JVC TK-1280U color video camera and the Leica Q500MC Image Analysis System. To visualize apoptotic nuclear morphology and chromatin condensation, cultures were fixed with 2% paraformaldehyde and then stained with 1 g/ml 4Ј,6-diamidino-2-phenylindole in PBS. The cells were washed once with PBS before mounting with 50% glycerol and viewed under a Olympus BX60 System fluorescence microscope. Images were recorded using a CoolSNAP CCD video camera (Photometrics GmbH, Munich) and CoolSNAP software.
Analysis of Genomic DNA Fragmentation by Agarose Gel Electrophoresis-For the detection of DNA fragmentation associated with apoptosis, genomic DNA was extracted from lysed cells and treated with proteinase K and RNase A, according to standard protocols. DNA samples were size fractionated by electrophoresis in 1.5% agarose gel in 1ϫ TAE buffer, and visualized by UV light after ethidium bromide staining. Densitometry was performed on Polaroid pictures of the ethidium bromide stained gels using a Hewlett Packard ScanJet 3p scanner and the NIH Image software.
Cell Viability Assay-Cells were exposed to either vehicle alone or cycloheximide in the presence or absence of the indicated drugs and inhibitors. At the indicated periods of time following apoptosis induction, the number of viable cells in each condition was assessed by measuring the bioreduction of a MTS tetrazolium salt at 490 nm using the CellTiter 96 aqueous assay (Promega, Madison, WI).

SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analysis-Cell pellets obtained as indicated above were lysed at 4°C in PBS containing 1% Triton X-100 and protease inhibitor mixture (1:100 dilution). Lysates were cleared at 12,000 rpm for 15 min at 4°C, treated for 60 min at 35°C with sample buffer containing ␤-mercaptoethanol and stored at Ϫ70°C until use. Protein concentration was determined using a Coomassie dye assay (Bio-Rad) and bovine serum albumin as a standard. 40 g of cell lysate were separated by discontinuous SDSpolyacrylamide gel electrophoresis under reducing conditions and electrotransferred onto Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech) using standard procedures. The resulting blot was blocked with 5% skim milk in PBS containing 0.2% Tween 20 and incubated with a designed primary antibody overnight at room temperature. Proteins were detected with a secondary antibody conjugated to horseradish peroxidase and an enhanced chemiluminescence commercial kit (Amersham Pharmacia Biotech). Primary antibodies reactive to procaspase-3/active caspase-3 p17 subunit (1:5,000 dilution, gift of RP Sekaly, Montreal, PQ), poly(ADP-ribose) polymerase (PARP) (1:4,000 dilution; Pharmingen Canada Inc., Mississauga, Canada), cytochrome c (1 g/ml; BIOSOURCE International), porin/VDAC 31HL (2 g/ml; Calbiochem), phospho-Erk1/Erk2, Erk1/Erk-2, phospho-Akt, Akt (1:1,000; New England Biolabs), and anti-actin (1:5,000 dilution; Sigma) were used in this study. The anti-actin polyclonal antibody was utilized to monitor loading and transfer conditions.
Caspase Activity Assay-Caspase enzymatic activity was assessed by the cleavage of site-selected tetrapeptide chromogenic reporter substrates with the specificity of IETD (caspase-8) and LEHD (caspase-9like). Cells were lysed at 4°C in 50 mM Hepes-KOH, pH 7.4, 1 mM EDTA buffer containing 75 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml pepstatin A, and 100 KIU/ml aprotinin (Trasylol, Bayer) and spun at 12,000 rpm for 20 min at 4°C, and the supernatant was recovered. Enzymatic reactions (final volume, 0.25 ml) were performed at 37°C and contained 50 -75 g of cell lysate and 100 M of chromogenic reporter substrate in 50 mM Hepes-KOH, pH 7.4, buffer containing 75 mM NaCl, 2 mM dithiothreitol, and 0.1% CHAPS. Caspase catalyzed release of the chromophore pnitroanilide was monitored spectrophotometrically at 405 nm. Optical density readings were corrected for background and standardized to equivalent protein.
Apoptosis Assessment in Transfected Cells-Transient transfection of BHK-GLP-2R cells was done by calcium phosphate coprecipitation as described previously (19). Cell cultures were transfected with 6.5 g of a RSV-␤-galactosidase expression plasmid, as a marker for transfection, plus 13.5 g of pBluescript II (Stratagene, La Jolla, CA) carrier DNA or with the ␤-galactosidase marker plasmid plus 10 g of MtR(AB) expression vector and carrier DNA for a total of 20 g. After glycerol shock, cells were cultured in serum-depleted medium for 15-17 h (Dulbecco's modified Eagle's medium with 0.2% calf serum), induced to undergo apoptosis by cycloheximide in the presence or absence of the indicated drugs, and then fixed in 2% paraformaldehyde. Transfected, ␤-galactosidase-expressing cells were identified by histochemistry with X-gal (2 mg/ml in PBS containing 5 mM potassium ferrycianide, 5 mM potassium ferrocyanide, and 100 mM MgCl 2 ). The percentage of ␤-galactosidase-positive cells remaining in the plate that showed evidence of apoptosis was determined by counting Ͼ200 cells from at least 15-20 different fields. Transfected cells were scored as apoptotic when appearing rounded-up with a shrunken morphology, as opposed to the flat extended appearance of viable, healthy cells (see Fig. 5C, upper right panel).
cAMP-dependent Protein Kinase Activity Measurement-The extent of PKA inhibition achieved by the transfected MtR(AB) dominant negative expression plasmid was assessed indirectly by determining its ability to inhibit both h[Gly2]-GLP-2 and forskolin-induced transcriptional activation of a CRE/␤-galactosidase reporter plasmid, as described previously (19). The inhibitory effect of H89 on both basal and h[Gly2]-GLP-2 or forskolin-induced cAMP-dependent protein kinase activity was measured in vitro using a fluorescent-labeled kemptide (PepTag nonradioactive cAMP-dependent protein kinase assay; Promega). Phosphorylation of the kemptide alters the net charge of the peptide, allowing the phosphorylated form to be separated from the nonphosphorylated form on an agarose gel at pH 8. PKA activity was defined as kemptide phosphorylation that could be inhibited by 10 M protein kinase inhibitor-(5-22)-amide peptide (Upstate Biotechnology, Lake Placid, NY).
Mitochondria and Cytosol Isolation-Cells were resuspended in icecold mitochondrial isolation buffer (10 mM Hepes-KOH, pH 7.4, 200 mM mannitol, 70 mM sucrose, 1 mM EGTA, and protease inhibitor mixture (1:100 dilution)) and lysed at 4°C with a glass Dounce homogenizer. After pelleting nuclei and unbroken cells at 770 ϫ g for 10 min, the supernatant was centrifuged for 15 min at 10,000 ϫ g to collect the heavy membrane fraction enriched for mitochondria. The newly obtained supernatant containing the cytosol was further centrifuged for 15 min at 20,800 ϫ g to remove the light membrane fraction and then stored at Ϫ70°C. The mitochondria-enriched pellet was washed once in mitochondrial isolation buffer and then resuspended in the same buffer and stored at Ϫ70°C until use. Protein concentration in both cytosolic and mitochondrial fractions was determined before preparing the samples for immunoblot analysis as described above.
Statistical Analysis-For assessment of statistical significance, data were analyzed using analysis of variance, and group comparisons were done using the Bonferroni multiple comparison post-test.

RESULTS
The glucagon-like peptide-2 receptor is expressed in a highly tissue-specific pattern primarily in the intestine and brain (20). Despite ongoing attempts by our laboratory and others, intestinal cell lines that express the endogenous GLP-2 receptor have not yet been identified. Accordingly we have studied GLP-2R signaling and its putative coupling to anti-apoptotic pathways in BHK fibroblasts stably transfected with the rat GLP-2 receptor (19). Incubation of wild type BHK cells or BHK-GLP-2R cells with cycloheximide alone at concentrations (5-80 M) that inhibit protein synthesis by greater than 95% produced a visibly detectable decrease in cell viability. In contrast, incubation of cells with cycloheximide and h[Gly2]-GLP-2 or forskolin (or 8-bromo-cyclic AMP) resulted in significantly improved cell survival (Fig. 1, A and B). Furthermore, 1,9dideoxyforskolin, a forskolin analog that does not stimulate adenylate cyclase, had no effect on the cycloheximide-induced decrease in cell viability (data not shown). Cycloheximide-induced cell death was reduced by the pancaspase inhibitor Z-VAD-fmk but not by Z-YVAD-fmk, an inhibitor of caspase-1like activity (Fig. 1C). The extent of cycloheximide-induced cell death was markedly attenuated in the presence of ZnCl 2 , a known inhibitor of caspase-3-mediated apoptosis (21). Morphologically, BHK-GLP-2R cells exhibited characteristic nuclear features consistent with apoptosis including chromatin condensation and margination as well as nuclear fragmentation following treatment with cycloheximide (Fig. 1D). DNA fragmentation was induced by cycloheximide and reduced by treatment of cells with h[Gly2]-GLP-2 (Fig. 1E). Taken together, these findings are consistent with the cycloheximide-dependent activation of apoptosis in BHK-GLP-2R cells in vitro.
Consistent with the importance of caspase activation for cycloheximide-induced cellular injury, the levels of the p17 active subunit of caspase-3 were increased in a time-dependent manner following treatment with cycloheximide and reduced following treatment of cells with either h[Gly2]-GLP-2 or forskolin ( Fig. 2A). Similarly, the cycloheximide-induced cleavage of PARP was clearly attenuated following treatment with either h[Gly2]-GLP-2 or forskolin ( Fig. 2A). In contrast, the protective effects of h[Gly2]-GLP-2 but not forskolin on both caspase-3 and PARP cleavage were absent in control BHK-pcDNA3 cells (Fig. 2B), consistent with the importance of the GLP-2R for transduction of the GLP-2-mediated signal to downstream apoptosis pathways.
Because caspase-3 processing and PARP cleavage are distal events that follow activation of upstream signaling molecules, we assessed the effects of GLP-2 on the activation of more proximal caspase enzymes following cycloheximide treatment of BHK cells. Analysis of initiator caspase activity using synthetic tetrapeptide substrates that are preferentially cleaved by caspase-8 (IETD-pNA) or caspase-9-like (LEHD-pNA) proteases showed that both h[Gly2]-GLP-2 and forskolin significantly inhibited the induction of IETD-pNA and LEHD-pNA cleaving activity following cycloheximide treatment of BHK-GLP-2R cells (Fig. 3). In contrast, only forskolin, but not h[Gly2]-GLP-2 inhibited activation of caspase-8-and caspase-9-like activity in control BHK-pcDNA3 cells (Fig. 3), consistent with the importance of the GLP-2R for transduction of the anti-apoptotic effects of h[Gly2]-GLP-2 in vitro. The specificity of caspase induction was further illustrated by findings that caspase-1-like activity, as assessed using YVAD-pNA as a substrate, was not induced following treatment of BHK cells with similar concentrations of cycloheximide (data not shown).
The finding that GLP-2R signaling was coupled to inhibition of cycloheximide-induced caspase-9-like activity prompted us to assess mitochondrial cytochrome c release, a known upstream effector of caspase-9 activation (22). Cycloheximide treatment of BHK-GLP-2R cells was associated with a marked time-dependent induction in the levels of cytosolic cytochrome c (Fig. 4). Both h[Gly2]-GLP-2 and forskolin markedly attenuated the cycloheximide induction of cytosolic cytochrome c re-lease from BHK-GLP-2R cells (Fig. 4). Because both GLP-2 and forskolin are known to exert their effects via activation of adenylate cyclase and downstream PKA-dependent signaling pathways (19,20), we assessed the importance of PKA signaling for cellular apoptosis in the presence or absence of H-89, a pharmacological inhibitor of protein kinase A. H-89 alone reduced cell viability in BHK-GLP-2R cells, consistent with the importance of basal PKA signaling for cell survival in vitro (Fig. 5A). However, both h[Gly2]-GLP-2 and forskolin significantly increased cell survival after cycloheximide in the presence of H-89 (Fig. 5A), demonstrating that stimulation of PKAdependent signaling pathways is not required for the antiapoptotic actions of these agents (Fig. 5A). To verify that H-89 inhibited PKA activity in BHK-GLP-2R cells, we assessed basal and stimulated PKA activity following incubation of cells with h[Gly2]-GLP-2 or forskolin. PKA activity was induced following treatment of BHK-GLP-2R cells with h[Gly2]-GLP-2 or forskolin, and PKA activation by these agents was effectively blocked by H-89 in the same experiments (Fig. 5A).
Because activation of both the glucagon and GLP-1 receptors has been associated with induction of mitogen-activated protein kinase activity (23,24), we assessed the effects of PD98059, a known inhibitor of Erk1/2 activity, on cycloheximide-induced apoptosis. Incubation of BHK-GLP-2R cells with PD98059 alone had no effect on the number of viable cells in the presence or absence of cycloheximide, demonstrating that basal Erk 1/2 activity is not required for cell survival in BHK-GLP-2R cells (Fig. 6A). Furthermore, both h[Gly2]-GLP-2 and forskolin significantly increased cell viability following cycloheximide in the presence of PD98059. In contrast, the activation of phosphorylated Erk1/2 by lysophosphatidic acid or fetal calf serum was markedly attenuated in the presence of PD98059 (Fig. 6A). These findings demonstrate that the effects of GLP-2R signaling on cell survival following cycloheximide treatment are independent of Erk1/2 activation in BHK-GLP-2R cells.
Because GLP-1 activates phosphatidylinositol 3-kinase (PI 3-kinase) in INS-1 cells (25) and PI 3-kinase is known to exert anti-apoptotic effects via induction of Akt (26), we examined whether the anti-apoptotic effects of GLP-2 might be mediated via a PI 3-kinase-dependent pathway. Treatment of BHK cells with LY294002, an inhibitor of phosphatidylinositol 3-kinase signaling, significantly reduced BHK cell survival, and co-incubation of cells with both LY294002 and cycloheximide produced a further diminution in survival beyond that seen with either agent alone (Fig. 6B). In contrast, cell survival was significantly increased in cycloheximide-treated cells incubated with either h[Gly2]-GLP-2 or forskolin in the presence of LY294002 at concentrations that completely block Akt activation by either lysophosphatidic acid or fetal calf serum (Fig.  6B). These results imply that although basal PI 3-kinase activity is required for BHK-GLP-2R cell survival, the effects of h[Gly2]-GLP-2 on cycloheximide-induced cell death are mediated through a phosphatidylinositol 3-kinase-independent pathway.

DISCUSSION
Although signaling through G protein-coupled receptors (GPCRs) of the glucagon/GLP-1/GLP-2 receptor superfamily has not previously been reported to directly modify apoptotic pathways, recent experiments provide increasing evidence linking GPCR activation and signal transduction pathways modulating cell death. Activation of the somatostatin receptor modulates pH-dependent apoptosis in heterologous cell types (27,28) and signaling through the PTH/PTHrP (parathyroid hormone/parathyroid hormone-related protein) receptor activates apoptotic pathways in cells of the chondrocyte and osteo-blast lineages (29). Activation of the pituitary adenylate cyclase-activating peptide receptor prevented apoptosis in cerebellar neurons, and the effects of pituitary adenylate cyclase-activating peptide signaling on apoptosis were reversed following transfection of cells with a dominant negative inhibitor of protein kinase A (30), consistent with the importance of the PKA pathway for antiapoptotic action in this cell type. In contrast, our data clearly show that the antiapoptotic effects of GLP-2 are PKA-independent. Similarly, stimulation of thymocyte apoptosis following activation of ␤-adrenergic receptor signaling is mediated by a PKA-independent, G s ␣-dependent pathway (31). These findings illustrate the diversity of signal- ing pathways downstream of GPCR activation and emphasize that GPCRdependent effects on apoptotic pathways are highly cell-and receptor-specific.
GPCR-dependent modulation of cell death has been extensively studied in the heart, and stimulation of cardiac myocytes with ␤-adrenergic agonists such as isoproterenol leads to induction of apoptosis that is prevented by inhibitors of protein kinase A (32). Similarly, stimulation of ␣-adrenergic receptor signaling with agents such as norepinephrine also leads to cardiomyocyte apoptosis, and these effects were abolished by co-incubation with the protein kinase A inhibitor H-89 (33). Although adrenergic stimulation leads to activation of Erk1/2 in rat ventricular myocytes, inhibition of the Erk1/2 pathway had no effect on adrenergic receptor-mediated apoptosis (34), in agreement with our findings that Erk1/2 activation was not essential for GLP-2 inhibition of apoptosis in BHK-GLP-2R cells.
Although receptors for glucagon, GLP-1, and GLP-2 classically transduce their effects via activation of adenylate cyclase and protein kinase A (20,35,36), studies of both glucagon and GLP-1 action have provided evidence for PKA-independent signaling following activation of their cognate receptors (4,12,23,25,37). Similarly, our data clearly demonstrate that although GLP-2 activates a PKA-dependent pathway in transfected fibroblasts expressing the human or rat GLP-2 receptor (19,20), the effects of GLP-2R signaling on cell survival and downstream caspase activation are mediated via PKA-independent pathways. The observations that GPCR ligands may also activate metalloproteinase activity leading to transactivation of growth factor receptor activity (38) further expands the complexity of intracellular signaling pathways residing downstream of GPCR activation.
In previous studies of GLP-2 action in BHK-GLP-2R cells in the absence of cycloheximide-induced cellular injury, we did not detect GLP-2-dependent activation of intracellular calcium influx, and GLP-2 stimulation failed to activate Erk1/2, p70 S6 kinase or Akt1 kinase activity (19). The findings on the lack of Erk induction in BHK-GLP-2R cells differ from studies of NGF action in PC 12 cells, where activation of Erk activity prevents apoptosis induced by NGF withdrawal (39). Similarly, the PI 3-kinase pathway is required for the antiapoptotic effects of NGF in PC 12 cells because PI 3-kinase inhibitors such as LY294002 also inhibit the effects of NGF on cell survival (26). In contrast, activation of GLP-2R signaling reduced cell death and decreased caspase activation in the presence of H-89 or MtR(AB), LY294002, or PD98059. Hence, although elevated levels of cAMP and/or PKA may negatively regulate apoptosis in some cell types (40), the available evidence from our studies points to the existence of a GLP-2-dependent, PKA-independent pathway coupled to inhibition of caspase-3 cleavage and prevention of cell death. Similarly, the lack of effect of the inhibitors LY294002 and PD98059 on GLP-2-mediated enhancement of cell survival strongly suggest that the anti-apoptotic effects of GLP-2R are mediated via a PI 3-kinase-and mitogen-activated protein kinase-independent pathway.
The cellular targets utilized by cycloheximide for induction of cell death appear cell type-specific, remain incompletely understood, and may involve both mitochondrial and death receptorassociated pathways. Although activation of Fas leading to caspase-8 cleavage and caspase-3 activation may occur independent of the mitochondrial pathway, recent evidence suggests that caspase-8 activation may also lead to mitochondrial cytochrome c release via a cycloheximide-sensitive pathway in are shown. Anti-Erk1/2 and anti-Akt polyclonal antibodies were used to monitor loading and transfer conditions. specific cell types (41). Although Fas ligand was not expressed in cycloheximide-sensitive Jurkat cells prior to or following induction of apoptosis, a dominant negative Fas-associated death domain protein completely inhibited cycloheximide-induced apoptosis, strongly implicating the importance of the Fas-associated death domain adapter protein in cycloheximideinduced cell death (42). The observation that GLP-2 inhibits the cycloheximide induction of both cytochrome c release and IETD-pNA and LEHD-pNA cleaving activity is consistent with previous studies of cycloheximide action (41) and suggests that GLP-2R signaling may interact with multiple signaling pathways upstream of caspase-3 cleavage.
GLP-2 ameliorates experimental intestinal injury in rats following nutritional deprivation, intestinal resection, and vascular ischemia (15,16,43) and in mice following chemically induced injury to the large and small bowel (13,14). Although the beneficial effects of GLP-2 on intestinal mucosa were originally attributed to stimulation of crypt cell proliferation (4,12,23), we detected reduced apoptosis in the crypt compartment following GLP-2 treatment of mice with intestinal injury (14). Because the cellular localization of intestinal GLP-2 receptor expression has not yet been identified, our data generated in a fibroblast cell line may not necessarily be directly applicable to GLP-2 action in the intestine in vivo. Nevertheless, the observations that the transfected GLP-2 receptor confers resistance to caspase activation and apoptosis in heterologous cells in the presence of GLP-2 clearly suggest that intestinal cells expressing the endogenous GLP-2R may also be protected from cell death associated with exposure to genotoxic stress in vivo. The finding that GLP-2 improves cell survival and reduces caspase activation provides new evidence linking direct activation of GLP-2 receptor signaling to signaling pathways regulating apoptosis in vitro. Identification of the signaling mechanisms underlying the coupling of the GLP-2 receptor to inhibition of caspase activity and enhanced cell survival may facilitate identification of targets for prevention of cell death in the mucosal epithelium of the gastrointestinal tract.