Coordinate Down-regulation of Adenylyl Cyclase Isoforms and the Stimulatory G Protein (Gs) in Intestinal Epithelial Cell Differentiation*

The intestinal epithelium is dynamic, with proliferation of undifferentiated crypt cells balanced by terminal differentiation and cell death at the colon surface or small intestinal villus tips. Cyclic AMP, induced by agonists such as prostaglandin E2 and vasoactive intestinal polypeptide, promotes proliferation and ion secretion and suppresses apoptosis in intestinal epithelial cells. Here, we show that cell differentiation in a model intestinal epithelium leads to attenuation of cAMP production in response to G protein-coupled receptor and receptor-independent agonists. Concomitantly, key components of the cAMP cascade, the α subunit of the stimulatory G protein, Gs, and adenylyl cyclase (AC) isoforms 3, 4, 6, and 7 are down-regulated. By contrast, AC1, AC2, AC8, and AC9, and the receptors for prostaglandin E2 and vasoactive intestinal polypeptide, are not expressed or not affected by differentiation. We confirmed key findings in normal murine colon epithelium, in which the major AC isoforms and Gsα are markedly down-regulated in differentiated surface cells. Suppression of AC isoforms and Gsα is functionally important, because their constitutive expression completely reverses differentiation-induced cAMP attenuation. Thus, down-regulation of AC isoforms and Gsα is an integral part of the intestinal epithelial differentiation program, perhaps serving to release cells from cAMP-promoted anti-apoptosis as a prerequisite for cell death upon terminal differentiation.

The intestinal epithelium, a single-cell layer that separates and protects the body from microbes and toxins in the intestinal lumen, is critical for digestion and uptake of nutrients and is characterized by rapid turnover and spatial specialization. Epithelial cells at the bottom of crypts proliferate and migrate upwards toward the tips of the villi in the small intestine or the surface in the colon, where they die and slough off or are taken up by underlying phagocytic cells. The migration of cells is accompanied by cell differentiation that is characterized by loss of proliferative capacity and gain of digestive and absorptive functions (1,2). The differentiated cells express high levels of digestive enzymes, such as alkaline phosphatase and sucrase/ isomaltase (3,4). In the colon, differentiated surface epithelial cells, but not crypt cells, express the ion transporters, sodium/ hydrogen exchanger-2, and SLC26A3/DRA (5,6), which are needed for electroneutral absorption of NaCl, the driving force behind the critical function of the colon, i.e. uptake of water.
Beyond changes in digestive and absorptive activities, differentiated epithelial cells exhibit alterations in signaling pathways that impact on many functions in health and disease. Freshly isolated differentiated villus epithelial cells show diminished agonist-stimulated synthesis of the second messenger cAMP, compared with undifferentiated crypt cells (7), and in vitro induction of epithelial cell differentiation attenuates cAMP production in response to PGE 2 (8). Cyclic AMP, a key second messenger in intestinal epithelial cells, regulates ion transport (9), cell growth (10), and cell death (11). Activation of cAMP synthesis by adenylyl cyclase (AC) 4 -mediated catalysis of ATP is physiologically initiated by ligand binding to G proteincoupled receptor (GPCR) family members. In intestinal epithelial cells, receptors for PGE 2 , and vasoactive intestinal polypeptide are linked to cAMP synthesis (12,13) through activation of the ␣ subunit of the stimulatory G protein (G s ) and stimulation of AC activity. In addition to levels of agonists, the concentration of cAMP in a given cell can be influenced by several factors, including levels of expression of receptors, G s , or AC, and the activity of cAMP-degrading phosphodiesterases (14). Analysis of the stoichiometry of the three key signaling components, receptor, G s , and AC, in cardiac myocytes has revealed that AC is the least abundant and thereby is the rate-limiting component for agonist-stimulated cAMP synthesis (15)(16)(17). Whether this concept applies to other cells, in particular intestinal epithelial cells, is not known.
Ten AC isoforms exist in humans, of which nine are membrane-bound and linked to transmembrane receptors (18). The AC isoforms differ in their expression pattern and regulation by various signaling pathways (19 -21). For example, AC1, AC3, and AC8 are the predominant isoforms in neuronal cells (22). Furthermore, treatment of P19 teratocarcinoma stem cells with retinoic acid, which induces neuronal cell differentiation, is accompanied by up-regulation of AC2, AC5, and AC8 and down-regulation of AC3 (23). The AC isoforms have been categorized into four major groups based on sequence homology and regulation by other second messenger pathways (24). Group I isoforms (AC1, AC3, and AC8) are stimulated by calcium/calmodulin; group II isoforms (AC2, AC4 and AC7) are stimulated by G␤␥ subunits; group III isoforms (AC5 and AC6) are inhibited by calcium and protein kinase A, and the group IV isoform, AC9, is the only isoform that is inhibited by the calciumdependent phosphatase calcineurin (25). Differences in AC isoform expression and regulation likely help to determine the ability of target cells to integrate signals from different pathways in the production of cAMP (25).
Intestinal epithelial cells express AC isoforms that can be regulated by cytokines and nitric oxide (26,27). For example, interferon-␥ inhibits the expression of AC5 and AC7 and decreases cAMP synthesis (26). However, the impact of epithelial cell differentiation on AC isoform expression is not known. We thus sought to define this impact and the functional consequences of changes in AC isoform expression in intestinal epithelial cells.

EXPERIMENTAL PROCEDURES
Cell Culture-T 84 human colon epithelial cells (ATCC CCL-248) were grown in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with 5% newborn calf serum and penicillin/streptomycin, in 5% CO 2 , 95% air at 37°C. Cell differentiation was induced by treatment with 10 mM butyrate for varying periods.
Cyclic AMP Assays-Confluent T 84 cells, undifferentiated or after butyrate-induced differentiation, were stimulated with various cAMP-elevating agonists as follows: PGE 2 (10 M, 20 min), cholera toxin (CTX; 250 ng/ml, 2 h), forskolin (30 M, 30 min), or vasoactive intestinal polypeptide (10 nM, 30 min). At the end of the stimulation, 250 l of ice-cold 7.5% trichloroacetic acid were added, and cAMP was measured by radioimmunoassay. Samples were acetylated using triethylamine and acetic anhydride. Fifty microliters were incubated with ϳ5000 cpm of 125 I-cAMP (PerkinElmer Life Sciences) and 25 l of rabbit anti-cAMP antibody (Chemicon International) overnight at 4°C. Fifty microliters of secondary antibody (goat anti-rabbit IgG, BioMag) were added, and the mixture was incubated with constant shaking for 1 h at 4°C. Samples were washed with 100 l of 12% polyethylene glycol solution using a Millipore filter apparatus. Levels of cAMP were determined by counting bound radioactivity with a ␥-counter and comparing values to those of a standard curve. Cyclic AMP levels were normalized to the amount of total protein in each sample.
PCR Analysis-Total RNA was extracted using TRIzol reagent (Invitrogen) and was analyzed by qualitative or real time RT-PCR. For qualitative RT-PCR, the number of amplification cycles for each gene was optimized to maximize discrimination of differences in mRNA expression in control and butyratetreated cells. As a positive control, RNA from cells or tissues known to express the respective mRNAs was used. As a negative control, RNA was omitted from reverse transcription and PCR amplification. Real time RT-PCR analysis was conducted with SYBR Green reagents using 40 cycles of amplification. Relative changes in expression were determined by the 2 ⌬⌬Ct method (28). Absolute mRNA levels (per mass of total RNA) were determined by employing known amounts of cDNA standards in parallel reactions (29). The PCR primers and amplification conditions are listed in Table 1. Generation of Transgene Expression Vectors-Human AC6 cDNA was obtained from the Mammalian Gene Collection (ATCC). Using restriction enzymes, EcoRI and NotI, the complete AC6 cDNA was inserted into the expression vector, pIRESneo3 (Clontech), containing a constitutively active human cytomegalovirus promoter/enhancer and a neomycin resistance marker for selection of stable transfectants. The human G s ␣ short variant cDNA was obtained by PCR from T 84 cells and inserted, using AgeI and NheI restriction sites, into the pIRESpuro3 (Clontech) expression vector, which contains a puromycin resistance gene for selection. The same expression vectors without a cDNA insert or containing an irrelevant insert, Escherichia coli ␤-galactosidase, were used as controls. All expression vectors were confirmed by restriction digests and sequencing.
Generation of Stable Transfectants-T 84 cells were transfected with the AC6 expression vector or the control vector, and AC6 transgenic cells were subsequently transfected with the Gs␣ expression vector or the control plasmid vector, using FuGENE transfection reagent (Roche Applied Sciences). Geneticin (0.25 mg/ml) and/or puromycin (10 g/ml) was used to select for stable transfectants. Clones were evaluated for transgene expression by real time RT-PCR and immunoblot analysis. Data were combined and analyzed from at least two separate clones of each of the transgenic cells.
Preparation of Murine Colon Epithelial Cells-The colon from normal adult C57BL/6 mice was removed, opened longitudinally, washed with ice-cold phosphate-buffered saline, cut into 5-mm pieces, placed into 2 mM EDTA in phosphate-buffered saline, and vortexed at 37°C for 6 min. Detached cells were removed, and incubation and vortexing were repeated several times to obtain progressively less differentiated epithelial cell populations (30). Detached cells were pelleted and snap-frozen in liquid nitrogen. All animal studies were reviewed and approved by the University of California, San Diego Institutional Animal Care and Use Committee.

Suppression of Agonist-stimulated cAMP Production upon
Differentiation of Colon Epithelial Cells-Treatment of cultured colon epithelial cells with the short-chain fatty acid, butyrate, is a commonly used strategy to induce cell differentiation (31)(32)(33). Consistent with this, we found that butyrate treatment of human T 84 colon epithelial cells increases expres-sion of the sodium/hydrogen exchanger-2 and down-regulates expression of the Na-K-2Cl cotransporter SLC12A2/NKCC1 (Fig. 1A). In addition, intestinal alkaline phosphatase activity was induced 5-fold after 72 h of butyrate stimulation (0.64 Ϯ 0.02 mIU/mg protein in controls versus 3.18 Ϯ 0.13 in 72-h butyrate-treated cells; mean Ϯ S.E., n ϭ 3). These gene expression changes are characteristic of differentiated human colon epithelial cells in vivo (33)(34)(35). Our previous work had shown that butyrate treatment of T 84 cells markedly suppresses cAMP production in response to the agonist PGE 2 (8), which is consistent with the findings in freshly isolated intestinal epithelial cells (7). Furthermore, cAMP suppression was not related to increased cAMP degradation, because a broadly active phosphodiesterase inhibitor did not reverse the attenuation (8). In this study, we found that attenuation of cAMP in differentiated epithelial cells was not limited to PGE 2 stimulation, as butyratetreated T 84 cells also showed a Ͼ80% decrease in cAMP generation after stimulation with three other cAMP agonists, vasoactive intestinal polypeptide, CTX, and forskolin (Fig. 1). Maximal attenuation of cAMP formation required at least 48 h of butyrate treatment, which paralleled the characteristic changes in the expression of differentiation-associated genes (Fig. 1A). These results demonstrate that cell differentiation in intestinal epithelial cells is accompanied by down-regulation of cAMP responses to multiple stimulatory agonists.
Differential Down-regulation of AC Isoforms in Differentiated Human Colon Epithelial Cells-Attenuation of cAMP production is accompanied by a down-regulation of total AC levels in differentiated intestinal epithelial cells (8). However, the mechanism underlying the decrease in AC expression and its functional consequences in these cells are unknown. We first determined which AC isoforms are predominantly expressed in colon epithelial cells and how cell differentiation affects their expression. We thus assayed mRNA expression of each of the membrane-bound AC isoforms, AC1-9, by qualitative and quantitative RT-PCR in control and butyrate-treated T 84 cells. AC6 and AC7, as well as AC3, AC4, and AC9, were the most abundant AC isoforms in undifferentiated cells, whereas AC1 and AC5 were expressed at 5-30-fold lower levels; AC2 and AC8 were not significantly expressed (Fig. 2, A and B). Induction of cell differentiation by butyrate down-regulated mRNA expression of AC3, AC4, AC6, and AC7 by 70 -90%, whereas AC9 mRNA levels were essentially unchanged (Fig.  2C). AC1 and AC5 mRNA levels increased slightly but remained a minor fraction of the total AC mRNA pool (Fig.  2, C and D). Total AC mRNA levels, derived by summing the mRNA levels of individual AC isoforms, was decreased Ͼ70% in differentiated cells (Fig. 2D), consistent with previously observed decreases in total AC protein and activity levels (8,36). These data demonstrate that colonic epithelial cells express a distinct profile of AC isoforms under resting conditions and that specific isoforms are differentially down-regulated upon cell differentiation.
Differential AC Isoform Expression in Normal Murine Colon Epithelium-To validate and extend the data obtained in human T 84 colon epithelial cells, we isolated epithelial cells from normal murine colon in four different fractions that represent different degrees of cell differentiation (30). Crypt epithelial cells (fraction F4) showed a high level of expression of the epithelial stem cell marker, Lgr5, and low expression of the solute transporter, SLC26A3/DRA, whereas surface epithelial cells (fraction F1) exhibited a reverse pattern of expression (Fig.  3). These findings are consistent with the expression patterns of Lgr5 (37) and SLC26A3/DRA (38), thus demonstrating the utility of our epithelial cell isolation procedure. Crypt epithelial cells showed a distinct profile of AC isoform expression, with AC6 being the most abundant, followed by AC5, AC2, AC3, and AC9 (Fig. 3A). AC1 and AC7 were expressed at intermediate levels, and AC4 and AC8 were the least expressed isoforms. Many of the AC isoforms were expressed in murine colon crypt cells with a pattern similar to that in human T 84 cells (e.g. AC6, AC5, and AC8), although we found several differences, most notably for AC2, AC4, and AC7 (compare Fig. 2B and Fig. 3A). Furthermore, the dynamic range of AC expression levels was markedly greater in the murine cells (ϳ10 5 -fold) relative to T 84 cells (ϳ500-fold), which may be related to the limited differentiation capabilities of the latter cells in culture.
Comparison of the AC profile in murine crypt cells with other organs also revealed interesting differences. Total brain tissue showed higher expression of almost all AC isoforms (with the exception of AC6) compared with spleen and colon crypt epithelium, which is consistent with the importance of cAMP in neuronal cell signaling and brain activity (19). By comparison, splenic AC isoform expression was more limited and selective, with only low levels of expression of AC2, AC7, AC8, and AC9 (Fig. 3A). These results show that AC isoforms are expressed in different organs over a wide dynamic range and  (B-D). A, qualitative PCR analysis. PCR cycle numbers were optimized for each gene to maximize discrimination of mRNA expression differences. As PCR controls, RNA was used from cells and tissues known to express the respective mRNA. GADD45 was employed as a differentiation marker and GAPDH as a control for equal RNA amounts in the reactions. B, AC isoform expression levels in control T 84 cells not treated with butyrate. Data are mean Ϯ S.E. of three more experiments. C, time course of AC isoform expression in differentiating cells. AC1, AC2, and AC8 were omitted because they were not expressed or were expressed at levels too low for biological significance. Data are means Ϯ S.E. of three more experiments. D, AC isoform distribution. The stacked bar graph illustrates the distribution of AC isoform mRNAs at different times after butyrate addition. AC1, AC2, and AC8 together comprised Ͻ1% of total AC expression and were omitted from the graph for clarity.
with distinct profiles, with AC6 being the most abundant in colonic crypt epithelial cells.
Analysis of the AC isoform levels in the different colon epithelial cell fractions revealed consistent down-regulation of all major AC isoforms in the most differentiated surface cells compared with the least differentiated crypt cells. AC6 expression was decreased by ϳ10-fold, AC5 and AC2 by 50-fold, and AC9 by Ͼ500-fold (Fig. 3B). The minor epithelial AC isoforms were also decreased by Ͼ40-fold (AC1, AC7, and AC8) or were not consistently altered (AC3 and AC4) with epithelial cell differentiation. Taken together, the overall expression pattern in murine surface colon epithelial cells resembled that in butyrate-treated T 84 cells, as most AC isoforms were markedly down-regulated, and none of the AC isoforms were increased in differentiated cells in either system.
Functional Significance of AC Down-regulation in Attenuating cAMP Production-Prior stoichiometric analysis of the components of the cAMP signaling cascade suggested that AC is the least abundant and hence the critical rate-limiting component in cAMP production (15,16). Based on such results, we hypothesized that down-regulation of AC isoform expression in differentiated colon epithelial cells was responsible for the attenuation of cAMP responses in these cells. To test this hypothesis, we generated AC-expressing transgenic cells, in which AC expression was not decreased after induction of cell differentiation. Because AC6 was one of the most abundant AC isoforms in T 84 cells (Fig. 2B) and the most abundant isoform in murine crypt epithelial cells (Fig. 3A), and AC6 mRNA expression was markedly down-regulated by butyrate treatment in T 84 cells (Fig. 2C) and in differentiated murine surface colon epithelial cells (Fig. 3B), we selected AC6 for transgenic expression. Stable AC6 transfectants and the respective vector controls were established in T 84 cells, and clones were evaluated for transgene expression. In the AC6 transgenic cells, AC6 expression was no longer down-regulated by butyrate-induced differentiation, whereas the control transfectants showed the expected suppression of AC6 expression (Fig. 4A). AC3, examined as a control, was decreased in both AC6 transgenic and control cells in response to differentiation (Fig. 4A), whereas the expression of the differentiation marker, intestinal alkaline phosphatase, was increased in both groups of cells (data not shown), indicating that differentiation occurred normally in the transfectants.
The AC6 transgenic cells exhibited a complete reversal in the differentiation-induced attenuation of CTX-and forskolinstimulated cAMP production (Fig. 4B), indicating that AC down-regulation (most importantly that of AC6) accounted for the limitation in maximal cAMP response to direct stimulation of G s or AC under these conditions. In contrast, differentiationinduced attenuation of PGE 2 -stimulated cAMP production was only partly restored in AC6 transgenic cells (Fig. 4B). These results suggest that AC6 down-regulation is functionally significant, but not solely responsible, for attenuation of GPCR-pro-  moted cAMP production in differentiated intestinal epithelial cells.
Down-regulation of G s ␣ in Differentiated Intestinal Epithelial Cells-Because uncoupling of AC6 expression from cell differentiation only partially reversed differentiation-related cAMP attenuation after receptor stimulation, we next asked whether other components of the cAMP synthesis cascade limit maximal cAMP synthesis in differentiated cells. Using RT-PCR analysis we assessed changes in mRNA expression of different GPCRs linked to cAMP synthesis, including the PGE 2 receptors, EP2 and EP4, and the vasoactive intestinal polypeptide receptors, VPAC1R and VPAC2R, in control and butyratetreated T 84 cells. We observed constitutive expression of EP2, EP4, and VPAC1R in resting cells (Fig. 5A). Expression of VPAC2R was detected (data not shown). Butyrate-induced differentiation was associated with an increase in EP2 and EP4 mRNA levels, which is consistent with histological observations in the murine small intestine (39), whereas VPAC1R expression was not affected (Fig. 5A). These results suggest that changes in receptor expression do not explain the attenuation of cAMP synthesis in differentiated cells. In contrast, mRNA levels of G s ␣, which together with ubiquitous G␤␥ subunits forms the central link between membrane receptors and AC isoforms, were down-regulated by 3-8-fold after butyrate-in-duced differentiation (Fig. 5, A and B). In parallel, G s ␣ protein levels decreased after butyrate treatment (Fig. 5C). The attenuation of G s ␣ expression was selective for this particular G protein, because the inhibitory G proteins, G i ␣ 1 , G i ␣ 2 , and G i ␣ 3 , were up-regulated after butyrate-induced differentiation; the ratios of mRNA levels in butyrate-treated (72 h) relative to untreated cells were 3.5, 24.7, and 2.8 (means, n ϭ 2) for G i ␣ 1 , G i ␣ 2 , and G i ␣ 3 , respectively.
To confirm that differentiation down-regulated G s ␣ under physiological conditions, we isolated epithelial cells from murine colon (30). The least differentiated crypt epithelial cells had the highest levels of G s ␣, whereas expression decreased progressively in the more differentiated cell fractions (Fig. 5D). Thus, G s ␣ is physiologically down-regulated upon intestinal epithelial differentiation.
AC6 and G s ␣ Act Synergistically in Determining Maximal cAMP Production in Intestinal Epithelial Cells-Because several AC isoforms and G s ␣ were markedly down-regulated upon butyrate-induced differentiation, we hypothesized that AC and G s ␣ act synergistically in determining maximal cAMP production in differentiated cells. To test this idea, we established stable T 84 double transfectants for AC6 and G s ␣. The latter exists in short and long variants (40), of which the shorter variant is the predominant isoform in T 84 cells (data not shown). We therefore used this G s ␣ isoform for the construction of AC6/ G s ␣ double transgenic cells. To confirm stable expression of AC6 and G s ␣, we assayed AC6 and G s ␣ expression in the double transgenic and vector control cells before and after butyrate treatment. Both AC6 and G s ␣ expressions escaped the inhibitory effect of differentiation in the double transgenic cells (Fig.  6A). AC3 expression, which is normally down-regulated with  cell differentiation, was similar in control and double transgenic cells (Fig. 6A).
We then assayed cAMP accumulation in AC6/G s ␣ double transgenic cells in response to PGE 2 or CTX. Compared with control T 84 cells and with single AC6 or G s ␣ transgenic cells, which showed prominent attenuation of PGE 2 -stimulated cAMP production with butyrate-induced differentiation, the AC6/G s ␣ double transgenic cells exhibited complete reversal of the differentiation-related inhibition of PGE 2 -stimulated cAMP production (Fig. 6B). Moreover, cAMP production was increased above controls in the double transgenic cells after butyrate treatment in CTX-stimulated cells (Fig. 6B). These results lead us to conclude that the expression levels of both AC6 and G s ␣ critically determine maximal receptor-stimulated cAMP response in differentiated intestinal epithelial cells.

DISCUSSION
This study demonstrates that undifferentiated colon epithelial cells constitutively express an AC isoform profile that differs from that of other cell types and tissues. Expression of the AC isoforms could be divided into three major groups as follows: AC3, AC4, AC6, AC7, and AC9 were most highly expressed; AC1 and AC5 were expressed at medium levels; and AC2 and AC8 were not expressed in the T 84 intestinal epithelial cell model. These quantitative data are consistent with results obtained by qualitative RT-PCR in normal human colon biopsies and two epithelial cell line models, although modest discrepancies exist for AC1 and AC9 (27). By comparison, large duct cholangiocytes express AC5, AC6, AC8, and AC9 (41); fetal rat skin keratinocytes express AC1-4, AC6, and AC8 but not AC5, AC7, or AC9 (42); bronchial epithelial cells produce AC1, AC3, AC4, AC7, and AC8 (43,44); and AC1, AC3, and AC8 are the predominant isoforms in neuronal cells (22). Thus, each cell type appears to have a unique AC isoform pattern. The functional implications of these distinct patterns are not clear, but it seems likely that different types of regulation result from expression of the different AC isoforms. Interestingly, the five major AC isoforms in T 84 colon epithelial cells represent all four AC groups, namely AC3 of group I, AC4 and AC7 of group II, AC6 of group III, and AC9 of group IV (25). Similarly, murine crypt epithelial cells expressed high levels of members of all four AC groups, although with some differences in the specific isoforms compared with human T 84 cells. Thus, in the murine cells, groups I, III, and IV were represented by AC3, AC5 and AC6, and AC9, respectively, whereas group II was represented by AC2 (rather than AC4 and AC7 in T 84 cells). This comprehensive representation of the AC isoform groups suggests that despite the selective AC isoform expression pattern, a very diverse pattern of AC regulation by various signaling pathways is present in intestinal epithelial cells. In contrast, bronchial epithelial cells only appear to express group I (AC1 and AC8) and group II (AC4 and AC7) AC isoforms (43,44), implying that AC coregulation has more restrictive requirements in those cells.
We found that differentiation of intestinal epithelial cells is accompanied by differential down-regulation of AC isoforms, because expression of all but one of the major AC isoforms decreased in differentiated T 84 cells, and all but two isoforms were markedly reduced in differentiated murine surface colon epithelial cells compared with undifferentiated crypt epithelial cells. Consistent with these data, immunohistological studies have found decreased AC5/6 expression in surface, compared with crypt, epithelial cells in the murine colon (27), and biochemical studies have detected diminished total AC activity in differentiated villus cells compared with undifferentiated crypt epithelial cells (36). These results, along with those observed in interferon-␥-stimulated colon epithelial cells (26), indicate that AC isoform expression is dynamic and responsive to environmental cues. Importantly, the nature of the environmental stimulus determines the specificity of the AC isoform expression. Thus, interferon-␥ stimulation selectively decreases expression of AC5 and AC7, but not AC6 or AC9 (26), while we found that cell differentiation down-regulates AC3, AC4, AC6, and AC7 but not AC9 in T 84 cells. The underlying mechanisms of differential AC isoform expression are not understood, partly because little is currently known about the transcriptional control elements in the promoter regions of these genes. However, the observations regarding AC isoform expression may have functional implications, because differentiated epithelial cells not only have less total AC expression (8) but lose virtually entire groups of AC isoforms. For example, the group II AC isoforms, AC2, AC4, and AC7, and the group III AC isoforms, AC5 and AC6, are either weakly or not at all expressed or are strongly down-regulated upon cell differentiation in T 84 cells. Similarly, both group III isoforms, AC5 and AC6, are markedly reduced in murine colon surface epithelial cells. These results suggest that the stimulatory regulation of these two AC groups by dissociated G␤␥ subunits is selectively lost in differentiated epithelial cells (25).
The regulated synthesis of cAMP requires all components of the cAMP signaling cascade, including GPCR, G protein, and AC, but the abundance of these components determines their relative importance for maximal cAMP production upon agonist stimulation (17). AC levels are the primary determinant of cAMP production in cardiac myocytes, whereas the levels of receptors or G s protein has relatively little impact on cAMP in these cells, presumably because they are in stoichiometric excess over AC (16). Consistent with this, we found that AC levels are limiting for maximal cAMP production in differentiated epithelial cells. However, reduced AC levels alone cannot fully account for the cAMP attenuation upon differentiation of these cells. The current studies revealed that down-regulation of G s occurs in differentiated cells and is likely to be as least partially responsible for limiting cAMP production. Thus, intestinal epithelial cell differentiation is associated with coordinate suppression of the two common components involved in cAMP generation, G s and AC, thereby contributing synergistically to attenuation of cAMP formation. In addition, our results showing increased expression of several G i ␣ isoforms suggest that such changes may further contribute, along with those in G s and AC, to the differentiation-associated loss of cAMP responsiveness to multiple agonists and receptor ligands in intestinal epithelial cells.
Given that cAMP production is profoundly altered at several loci in the cAMP signaling cascade, the question arises as to why differentiated epithelial cells limit maximal cAMP produc-tion. Multiple functions are mediated by cAMP in epithelial cells that may need to be down-regulated for normal intestinal physiology. For instance, cAMP stimulates proliferation and activates anti-apoptotic pathways in intestinal epithelial cells (11,45). Down-regulation of cAMP responses may be necessary for proliferation to cease and for apoptosis to occur in cells at the small intestinal villus tips or on the colonic surface. Furthermore, under conditions of mucosal inflammation and epithelial loss and regeneration, which are associated with a relatively greater number of undifferentiated epithelial cells, it may be beneficial that cAMP synthesis is less attenuated, as maintenance of cAMP formation would allow for greater control of cAMP-dependent ion transport that might be disturbed by epithelial barrier loss (9). However, the ultimate physiological and pathophysiological significance of differentiation-related attenuation of cAMP synthesis remains to be defined both for normal conditions and settings in which cAMP pathways are dysregulated, such as in the development of colon adenomas and cancers (46,47).