Coordinate down-regulation of adenylyl cyclase isoforms and the stimulatory G protein (G(s)) in intestinal epithelial cell differentiation

Abstract

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 E(2) 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 alpha subunit of the stimulatory G protein, G(s), 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 E(2) 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 G(s)alpha are markedly down-regulated in differentiated surface cells. Suppression of AC isoforms and G(s)alpha is functionally important, because their constitutive expression completely reverses differentiation-induced cAMP attenuation. Thus, down-regulation of AC isoforms and G(s)alpha 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.

FIGURE 1.

Attenuation of cAMP responses in differentiated intestinal epithelial cells. A, T₈₄ human colon epithelial cells were treated with butyrate for the indicated times, and gene expression was examined by real time PCR analysis. Expression levels are shown relative to those in cells not treated with butyrate. Data are mean ± S.E. (n = 4). B, T₈₄ cells, undifferentiated or after butyrate-induced differentiation, were stimulated with the indicated agonists that increase cellular cAMP levels, which were determined by radioimmunoassay. Data are mean ± S.E. of three or more experiments; *, p < 0.05 (by t test) compared with cells not treated with butyrate (0 h). FSK, forskolin; VIP, vasoactive intestinal polypeptide.

FIGURE 2.

Differential AC isoform expression in human T₈₄ colon epithelial cells upon cell differentiation. T₈₄ cells were treated with butyrate for 48 h (A, T84 + butyrate) or the indicated times (C and D), or they were left untreated (T84, 0 h), and mRNA levels of the indicated AC isoforms were determined by qualitative PCR (A) or quantitative real time PCR with cDNA standards (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 T₈₄ cells not treated with butyrate. Data are mean ± 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 ± 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 <1% of total AC expression and were omitted from the graph for clarity.

FIGURE 3.

AC isoform expression in murine colon epithelium. Fractions of murine colon epithelial cells were obtained by differential EDTA-mediated detachment, with the first fraction (F1) representing the most differentiated (surface) cells and the last fraction (F4) the least differentiated (deep crypt) cells. RNA was extracted from each fraction, as well as from total brain and spleen, and AC isoform expression was determined by quantitative real time PCR. A, AC isoform expression in crypt epithelial cells (fraction F4), spleen, and brain. Data are means ± S.E. (epithelium, n = 4) or means (spleen and brain, n = 2). B, expression levels of the major epithelial AC isoforms, the solute transporter, SLC26A3, and the epithelial stem cell marker, Lgr5, were determined for all four colon epithelial cell fractions. GAPDH was used as an expression control for all fractions. Data are means ± S.E. (n = 4).

FIGURE 4.

AC expression levels limit maximal cAMP production in differentiated intestinal epithelial cells. T₈₄ cells expressing AC6 as a transgene (+AC6 tg) or carrying an expression vector without cDNA insert (−AC6 tg) were generated and were treated with butyrate for 48 h (differentiated (diff)) or were left untreated (undifferentiated (undiff)). A, expression levels of the sum of endogenous and transgenic AC6 and, as a control, of AC3 were determined by real time PCR and are expressed as the ratio of the levels in differentiated versus undifferentiated cells. Data are mean ± S.E. of three more experiments. B, levels of cAMP after stimulation of differentiated and undifferentiated cells with the indicated agonists were determined by radioimmunoassay and are expressed as the percentage of the levels in differentiated cells versus those in undifferentiated cells. Data are mean ± S.E. of three more experiments using two different clones each of transgenic and control cells. *, p < 0.05 relative to undifferentiated cells treated with the same agonist; #, p < 0.05 relative to differentiated cells without the AC6 transgene and treated with the same agonist. FSK, forskolin.

FIGURE 5.

Differentiation-associated down-regulation of G_sα in colon epithelial cells. T₈₄ cells were treated with butyrate for 72 h (A, differentiated (diff); C, T84 + Bu) or the indicated times (B), or were left untreated (A, undifferentiated (undiff); B, 0 h; C, T₈₄). A, levels of the indicated mRNAs were determined by real time PCR and are expressed as the ratio of the levels in differentiated versus undifferentiated cells. Data are means ± S.E., n = 3. B, levels of G_sα and, as a control, GAPDH were assayed by quantitative real time PCR with cDNA standards. Data are mean ± S.E., n = 3. C, immunoblot analysis of G_sα in T₈₄ cells. D, immunoblot analysis of G_sα in freshly isolated mouse colon epithelial cells. Fractions of epithelial cells were obtained by differential EDTA-mediated detachment, with the first fraction (F1) representing the most differentiated (surface) cells and the last fraction (F4) the least differentiated (deep crypt) cells. The solute transporter, SLC26A3, was used as a control for differentiated cells; actin was used as a loading control for all fractions.

FIGURE 6.

Cooperative control of maximal cAMP responses by G_sα and AC in intestinal epithelial cells. T₈₄ cells expressing AC6 as a single transgene (+AC6 tg), G_sα as a single transgene (+G_sα), AC6 and G_sα as double transgenes (dtg, or +AC6 tg + G_sα tg), or carrying the respective expression vectors without cDNA inserts (−AC6 tg −G_sαtg) were generated. Cells were treated with butyrate for 72 h (differentiated (diff)) or left untreated (undifferentiated (undiff)). A, expression levels of the sums of endogenous and transgenic AC6, endogenous and transgenic G_sα, and, as a control, of AC3 were determined by real time PCR and are expressed as ratio of the levels in differentiated versus undifferentiated cells. Data are means ± S.E., n ≥3. B, levels of cAMP after stimulation with the indicated agonists were determined by radioimmunoassay and are expressed as the percentage of the levels in differentiated cells versus those in undifferentiated cells. Data are mean ± S.E. of three more experiments using three different clones each of transgenic and control cells. *, p < 0.05 relative to undifferentiated cells treated with the same agonist; #, p < 0.05 relative to differentiated cells not expressing the transgenes and treated with the same agonist.