EGF receptor transactivation mediates ANG II-stimulated mitogenesis in intestinal epithelial cells through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway

Terence Chiu, Chintda Santiskulvong, Enrique Rozengurt

Abstract

The role of epidermal growth factor receptor (EGFR) tyrosine kinase and its downstream targets in the regulation of the transition from the G0/G1 phase into DNA synthesis in response to ANG II has not been previously investigated in intestinal epithelial IEC-18 cells. ANG II induced a rapid and striking EGFR tyrosine phosphorylation, which was prevented by selective inhibitors of EGFR tyrosine kinase activity (e.g., AG-1478) or by broad-spectrum matrix metalloproteinase (MMP) inhibitor GM-6001. Pretreatment of these cells with either AG-1478 or GM-6001 reduced ANG II-stimulated DNA synthesis by ∼50%. To elucidate the downstream targets of EGFR, we demonstrated that ANG II stimulated phosphorylation of Akt at Ser473, mTOR at Ser2448, p70S6K1 at Thr389, and S6 ribosomal protein at Ser235/236. Pretreatment with AG-1478 inhibited Akt, p70S6K1, and S6 ribosomal protein phosphorylation. Inhibition of phosphatidylinositol (PI)3-kinase with LY-294002 or mTOR/p70S6K1 with rapamycin reduced [3H]thymidine incorporation by 50%, i.e., to levels comparable to those achieved by addition of either AG-1478 or GM-6001. Utilizing Akt small-interfering RNA targeted to Akt1 and Akt2, Akt protein knockdown dramatically inhibited p70S6K1 and S6 ribosomal protein phosphorylation. In contrast, AG-1478 or Akt gene silencing exerted no detectable inhibitory effect on ANG II-induced extracellular signal-regulated kinase 1/2 phosphorylation in IEC-18 cells. Taken together, our results demonstrate that EGFR transactivation mediates ANG II-stimulated mitogenesis through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway in IEC-18 cells.

  • IEC-18 cells
  • mitogen-activated protein kinase
  • mitogen/extracellular signal-regulated kinase
  • protein kinase B
  • epidermal growth factor receptor
  • phosphatidylinositol 3-kinase

the sequential proliferation, lineage-specific differentiation, migration, and cell death of epithelial cells of the intestinal mucosa is a tightly regulated process that is modulated by a broad spectrum of regulatory peptides (5, 26, 50), but the signal transduction pathways involved remain incompletely understood. The nontransformed IEC-18 cells, derived from rat small intestinal crypt (40), have provided an in vitro model to examine intestinal epithelial cell migration, differentiation, and proliferation (16, 17, 28). These cells are undifferentiated, as judged by morphological and functional criteria; they resemble stem cells and thus may serve as a model to study the crypt stem cell (51). The importance of examining this compartment is highlighted by recent reports suggesting that the initial mutant cell in the pathogenesis of colorectal adenoma may arise from the crypt stem cell compartment (37, 55).

Neuropeptides and vasoactive peptides that signal through G protein-coupled receptors (GPCRs), characterized by seven-transmembrane helixes, act as potent cellular growth factors for a variety of cell types (43, 44, 45, 47). In particular, GPCRs and their agonists play a crucial role in the regulation of multiple functions in the gastrointestinal tract, including cell proliferation, motility, and transport. We have recently demonstrated that the vasoactive peptide ANG II and the neuropeptide arginine vasopressin induce DNA synthesis and cell proliferation in intestinal epithelial IEC-18 cells (8, 9). Some of the key molecules that mediate the growth-promoting signal of these GPCR agonists in IEC-18 cells have been identified. ANG II induces a dramatic increase in protein kinase C (PKC)-dependent protein kinase D (PKD) activation in IEC-18 cells via the angiotensin type 1 (AT1) receptor coupled to Gq (7). ANG II also stimulates PKC-dependent tyrosine phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), a nonreceptor tyrosine kinase that has been implicated as an upstream element in extracellular signal-regulated kinase (ERK) 1/2 activation, in IEC-18 cells (56). Accordingly, this GPCR agonist stimulates PKC-dependent ERK activation in these cells. Interestingly, inhibition of the PKC or ERK pathway in these cells reduces ANG II-induced DNA synthesis by 50–60% (8, 9). These findings not only indicated that ANG II-induced DNA synthesis is partially dependent on ERK and PKC but also prompted us to hypothesize that another pathway, in addition to ERK and PKC, plays a substantial role in AT1-mediated mitogenesis of IEC-18 cells.

Recently, it has been shown that a variety of GPCR agonists also induce a rapid increase in epidermal growth factor receptor (EGFR) tyrosine autophosphorylation in several cell types (1012, 29, 52), termed transactivation (6, 36). This receptor cross talk can occur via extracellular release of EGFR ligands at the cell surface (36) by matrix metalloproteases that belong to the ADAM (a disintegrin and metalloprotease) family of zinc-dependent proteases. GPCR-induced EGFR transactivation leads to the phosphorylation of specific tyrosine residues within the EGFR cytoplasmic domain that act as docking sites for effector molecules, resulting in activation of downstream signaling pathways. However, it is increasingly recognized that the contribution of EGFR transactivation to agonist-induced mitogenic signaling is strongly dependent on cell context. The experiments presented in this study were designed to examine the role of EGFR tyrosine kinase and its downstream targets in mediating the transition from the G0/G1 phase into S phase in response to the potent mitogen ANG II in the normal intestinal epithelial IEC-18 cells.

In contrast to many cell types in which EGFR contributes to mitogenesis via the Ras/Raf/MEK/ERK pathway, the results presented in this study demonstrate that EGFR transactivation mediates ANG II-induced DNA synthesis in IEC-18 cells via activation of the phosphatidylinositol (PI)3-kinase/Akt/mTOR/p70S6K1 pathway.

MATERIALS AND METHODS

Cell culture.

IEC-18 cells were purchased from American Type Culture Collection. Stock cultures were maintained in DMEM supplemented with 5% FBS in a humidified atmosphere containing 10% CO2-90% air at 37°C. For experiments, cells were plated in 100-mm dishes at 3 × 105 cells/dish in DMEM containing 5% FBS, allowed to grow to confluency (5–7 days), and then changed to serum-free DMEM for 18–24 h before the experiment.

Western blot analysis for protein kinase phosphorylation.

Serum-starved cultures of IEC-18 cells grown on 100-mm dishes were washed two times with DMEM and then treated as described in the individual experiments. The cells were lysed in 2× SDS-PAGE sample buffer. After SDS-PAGE, proteins were transferred to Immobilon-P membranes (Millipore) and blocked by 3- to 6-h incubation with 5% nonfat milk in PBS, pH 7.2. Membranes were then incubated overnight with the respective phosphospecific antibodies (see Materials). The same membranes were stripped and probed in a similar fashion with the respective polyclonal antibody for total protein.

Bound primary antibodies to immunoreactive bands were visualized by enhanced chemiluminescence detection with horseradish peroxidase-conjugated anti-mouse, anti-rabbit, or anti-goat antibodies. Autoradiograms were scanned using a GS-710 scanner (Bio-Rad), and the labeled bands were quantified using the Quantity One software program (Bio-Rad).

Assays of EGFR tyrosine phosphorylation.

Serum-starved cultures of IEC-18 cells were washed two times with serum-free DMEM and then incubated for 1 h at 37°C with or without inhibitors, as indicated. Cells were then treated for 2 min with agonists. Cell stimulation was terminated by transferring the cultures to ice, aspirating the medium, and lysing the cells in 1 ml ice-cold modified radioimmunoprecipitation (RIPA) buffer (50 mM Tris·HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM Pefabloc SC, 1 mg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM Na3VO4, and 1 mM NaF). The cell suspension was then mixed for 20 min at 4°C to lyse the cells, and cell lysates were clarified by centrifugation at 15,000 rpm for 10 min. To measure EGFR tyrosine phosphorylation, the supernatants were transferred to new microtubes and mixed with 10 μg/ml polyclonal α-EGFR at 4°C overnight. Immunocomplexes were captured by adding 100 μl protein A-agarose beads for 1 h, and then agarose beads were collected by pulse centrifugation (0.5 min at 14,000 rpm). The beads were washed three times with ice-cold RIPA buffer and subsequently solubilized in 100 μl 2× SDS-PAGE sample buffer. Tyrosine-phosphorylated EGFR was detected by immunoblotting using 4G10 monoclonal antibody (MAb).

Assay of DNA synthesis.

Confluent and serum-starved cultures of IEC-18 cells were washed two times with DMEM and incubated with DMEM/Waymouth’s medium (1:1, vol/vol) and various additions, as described in the legends for Figs. 19. After 15 h of incubation at 37°C, [3H]thymidine (0.2 μCi/ml, 1 μM) was added, and the cultures were incubated for a further 4 h at 37°C. Cultures were then washed two times with PBS and incubated in 5% TCA at 4°C for 20 min to remove acid-soluble radioactivity, washed with ethanol, and solubilized in 1 ml of 2% Na2CO3 and 0.1 M NaOH. The acid-insoluble radioactivity was determined by scintillation counting in 6 ml Beckman Readysafe.

Fig. 1.

ANG II-stimulated epidermal growth factor receptor (EGFR) transactivation is dependent on matrix metalloprotease activity in IEC-18 cells. A: time course of ANG II-stimulated EGFR tyrosine phosphorylation. Confluent IEC-18 cells were washed and incubated at 37°C with 50 nM ANG II for various times as indicated. The EGFR was immunoprecipitated (IP) using a polyclonal EGFR antibody and resolved by SDS-PAGE. Proteins were transferred to Immobilon-P membranes and then Western blotted (WB) using 4G10 anti-Tyr(P) monoclonal antibody (MAb; PY). Membranes were then stripped and reprobed with anti-EGFR. B: treatment with AG-1478, compound 56, and GM-6001 prevents ANG II-induced EGFR tyrosine phosphorylation. Confluent and serum-starved cultures of IEC-18 cells were stimulated for 2 min with 50 nM ANG II after 1 h preincubation with 250 nM AG-1478 (AG), 250 nM compound 56 (56), or 10 μM GM-6001 (GM), as indicated. C: treatment with heparin differentially inhibits EGFR tyrosine phosphorylation induced by either ANG II or heparin-binding (HB)-epidermal growth factor (EGF) but not EGF. Serum-starved cultures of IEC-18 cells were incubated for 30 min with or without 50 μg/ml heparin. The cultures were subsequently left unstimulated (−) or stimulated with 50 nM ANG II, 1 ng/ml HB-EGF, or 10 ng/ml EGF. EGFR immunoprecipitates were analyzed for tyrosine phosphorylation as described above. The results presented are representative of 3 independent experiments.

Flow cytometric/cell cycle analysis.

The proportion of cells in the G0/G1, S, G2, and M phases of the cell cycle was determined by flow cytometric analysis. Confluent and serum-starved cultures of IEC-18 cells were washed two times with DMEM and incubated with DMEM/Waymouth’s medium (1:1, vol/vol) containing various additions, as described in the legends for Figs. 19. After 19 h of incubation at 37°C, cultures were washed two times with PBS. Cells were then detached by treatment with trypsin (0.025%), suspended in DMEM containing 10% FBS, and centrifuged at 1,000 g for 5 min. Cells (106) were then resuspended and stained by adding 1 ml of a hypotonic DNA staining buffer containing propidium iodide (0.1 mg/ml), sodium citrate (1 mg/ml), RNase A (20 μg/ml), and Triton X-100 (0.3%). Samples were kept at 4°C, protected from light for 30 min, and analyzed on a FACScan (Becton-Dickinson, Franklin Lakes, NJ), using the software CELLQuest version 3.3 and Modfit 3.1 (Verity Software House, Topsham, ME).

Akt small-interfering RNA.

To inhibit Akt protein expression, subconfluent cultures of IEC-18 cells were transfected with small-interfering RNA (siRNA). The synthetic 21-mer oligonucleotide RNA duplex corresponding to the consensus sequence of Akt1 and Akt2 that is identical in both human and rat was synthesized by Cell Signaling (Beverly, MA; see Ref. 27). A fluorescein-labeled nontargeted negative control duplex purchased from Cell Signaling was used as a control. Transfection of duplex siRNAs at 100 nM was performed using TransIT-TKO Reagent (Mirus, Madison, WI) according to the manufacturer’s protocol. After transfection (48 h), cells were used for experiments and subsequent Western blot analysis.

Materials.

[3H]thymidine was from Amersham Pharmacia Biotech (Piscataway, NJ). Tyrphostin AG-1478, compound 56, PD-98059, U-0126, LY-294002, wortmannin, and heparin were from Calbiochem. Protein A-agarose was from Boehringer Mannheim (Indianapolis, IN). ANG II, epidermal growth factor (EGF), heparin-binding (HB)-EGF, and trypsin-EDTA solution (1×) were obtained from Sigma (St. Louis, MO). Anti-ERK2 polyclonal antibody was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). 4G10 anti-Tyr(P) MAb and anti-EGFR antibody were from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-ERK-1/ERK-2 MAb, phospho-Ser473 Akt MAb, polyclonal Akt antibody, phospho-Ser2448 mTOR MAb, phospho-Thr389 p70S6 kinase1 MAb, polyclonal p70S6 kinase1 antibody, phospho-S6 ribosomal protein (Ser235/236) antibody, and SignalSilence Akt siRNA Kit were obtained from Cell Signaling. Other items were from standard suppliers or as indicated in the text.

RESULTS

ANG II-stimulated EGFR transactivation is dependent on matrix metalloprotease activity in IEC-18 cells.

To determine whether ANG II stimulates EGFR tyrosine phosphorylation in IEC-18 cells, serum-starved cultures of these cells were stimulated with 50 nM ANG II for various times and then lysed. The extracts were immunoprecipitated with anti-EGFR polyclonal antibody (Fig. 1A, top), separated by SDS-PAGE, and further analyzed by Western blotting using anti-Tyr(P) MAb. As shown in Fig. 1A, ANG II stimulated a rapid EGFR tyrosine phosphorylation in IEC-18 cells, reaching a maximum within 30 s and remaining elevated for at least 60 min.

Pretreatment of IEC-18 cells with the selective EGFR tyrosine kinase inhibitors tyrphostin AG-1478 or the structurally unrelated compound 56 (20), both at 250 nM, completely prevented ANG II-induced increase in tyrosine phosphorylation of EGFR induced by ANG II. Stripping the same blots and reprobing with anti-EGFR antibody confirmed that similar amounts of EGFR protein were recovered after ANG II stimulation (Fig. 1, A and B, bottom). The results presented in Fig. 1, A and B, demonstrate that ANG II markedly stimulates EGFR tyrosine kinase activity in IEC-18 cells.

To determine whether the cross talk between EGFR and GPCRs is mediated by extracellular release of EGFR ligands at the cell surface in IEC-18 cells, cultures of these cells were preincubated with GM-6001 (also known as Ilomastat or galardin), a broad-spectrum matrix metalloproteinase (MMP) inhibitor (1, 21, 49). Serum-starved cultures of IEC-18 cells were preincubated with either control medium or medium containing 10 μM GM-6001 for 1 h and then stimulated with ANG II. The concentration of GM-6001 used in these experiments (10 μM) was shown to inhibit MMP-1, -2, and -3 (21). Pretreatment of cells with GM-6001 suppressed the increase in EGFR tyrosine phosphorylation induced by ANG II stimulation (Fig. 1B). This result indicates that ANG II-stimulated EGFR tyrosine phosphorylation depends on the activity of GM-6001-sensitive metalloprotease(s) to mediate extracellular release of EGFR ligands.

Exogenously added heparin has been shown to inhibit EGFR transactivation by competing with cell surface-associated heparan sulfate proteoglycans for HB-EGF-binding (25, 41). Here we used heparin to determine whether ANG II stimulates EGFR tyrosine phosphorylation through HB-EGF release in IEC-18 cells. Serum-starved cultures of these cells were pretreated with 50 μg/ml heparin before stimulation with ANG II, HB-EGF, or EGF. We verified that heparin inhibited HB- and EGF-induced EGFR tyrosine phosphorylation, whereas EGF-induced EGFR tyrosine phosphorylation was not affected in IEC-18 cells (Fig. 1C). Interestingly, pretreatment with heparin significantly reduced ANG II-induced EGFR tyrosine phosphorylation. This result combined with our results with GM-6001 suggests that HB-EGF is required for ANG II-stimulated EGFR transactivation in IEC-18 cells.

ANG II-stimulated DNA synthesis is dependent on EGFR tyrosine kinase activity.

We recently demonstrated that ANG II acts as a potent growth factor for IEC-18 cells, promoting exit from G0/G1 and entry into the S phase in IEC-18 cells (8), but the precise mechanism(s) that mediate ANG II-induced mitogenesis remains incompletely understood. To examine whether EGFR transactivation contributes to ANG II-induced DNA synthesis, serum-deprived cultures of IEC-18 cells were preincubated for 1 h with the selective EGFR tyrosine kinase inhibitor tyrphostin AG-1478 at various concentrations (30–500 nM) before stimulation with ANG II. As shown in Fig. 2A, treatment with tyrphostin AG-1478 markedly attenuated [3H]thymidine incorporation in response to ANG II stimulation in a concentration-dependent fashion. A maximal inhibitory effect (∼50%) was achieved at 100 nM tyrphostin AG-1478.

Fig. 2.

ANG II-stimulated DNA synthesis is dependent on EGFR transactivation and matrix metalloprotease activity. A: tyrphostin AG-1478 inhibits ANG II-induced DNA synthesis in a concentration-dependent manner. Confluent and serum-starved cultures of IEC-18 cells were washed and incubated at 37°C in 2 ml DMEM/Waymouth’s medium containing 50 nM ANG II either without or with AG-1478 at various concentrations, as indicated. [3H]thymidine incorporation into acid-precipitable material was measured as described. Results are expressed as a percentage of the maximum increase ± SE (n = 3) obtained with 50 nM ANG II after subtraction of the basal [3H]thymidine incorporation from the respective controls. B: treatment with the selective EGFR tyrosine kinase inhibitor attenuates the stimulation of cell cycle progression induced by ANG II. Confluent and serum-starved cultures of IEC-18 cells were washed and incubated at 37°C in 2 ml DMEM/Waymouth’s medium containing 50 nM ANG II either in the absence or in the presence of 250 nM AG-1478. After 19 h of incubation at 37°C, cultures were washed two times with PBS, and cells were detached by treatment with trypsin (0.025%). Cells (106) were then resuspended and stained by 1 ml of a hypotonic DNA staining buffer containing propidium iodide (0.1 mg/ml), sodium citrate (1 mg/ml), RNase A (20 μg/ml), and Triton X-100 (0.3%). Samples were kept at 4°C, protected from light for 30 min, and analyzed on a FACScan (Becton-Dickinson). The profiles represent the number of cells as a function of relative DNA content. In each case, the percentages of cells in G0/G1 (first peak), S (hatched area well-defined in ANG II-treated cells), and G2/M (second peak) are indicated. Results shown are representative of 3 independent experiments. C: effect of AG-1478 and GM-6001 on DNA synthesis in response to ANG II. Confluent and serum-starved cultures of IEC-18 cells were washed and incubated at 37°C in 2 ml DMEM/Waymouth’s medium containing 50 nM ANG II either without or with 250 nM AG-1478 and/or 10 μM GM-6001, as indicated. [3H]thymidine incorporation into acid-precipitable material was measured as described. Results are expressed as a percentage of the maximum increase ± SE (n = 3) obtained with 50 nM ANG II.

To substantiate that the effects on [3H]thymidine incorporation reflect an increase in DNA replication through the S phase of the cell cycle rather than alterations in transport and/or phosphorylation of [3H]thymidine, we used flow cytometric analysis to determine the proportion of cells in the various phases (G0/G1, S, and G2/M) of the cell cycle. Confluent and serum-starved cultures of IEC-18 cells were subjected to identical conditions as those for the [3H]thymidine incorporation experiments and were preincubated for 1 h with AG-1478 before stimulation with ANG II. As shown in Fig. 2B, serum starvation of IEC-18 cells resulted in 85.4 ± 2% of cells in G0/G1, comparable with previous published results, confirming that serum starvation induced the accumulation of IEC-18 cells into quiescence. Addition of 50 nM ANG II induced a marked increase (from 9 to 20%) of cells into S phase of the cell cycle. Consistent with [3H]thymidine incorporation data, treatment with tyrphostin AG-1478 reduced the shift toward S phase in response to ANG II stimulation by ∼50% (Fig. 2B).

Because our results showed that the broad-spectrum metalloprotease inhibitor GM-6001 also prevented EGFR transactivation in response to ANG II, we tested whether ANG II-stimulated DNA synthesis would be similarly inhibited by cell exposure to this antagonist. As shown in Fig. 2C, pretreatment with GM-6001 reduced [3H]thymidine incorporation in response to ANG II stimulation by ∼50%, an inhibitory effect identical to that induced by the EGFR kinase inhibitor tyrphostin AG-1478. The results presented in Fig. 2 indicate that a substantial component (∼50%) of the mitogenic stimulus induced by ANG II in intestinal epithelial IEC-18 cells is mediated by EGFR transactivation.

ANG II stimulates EGFR-dependent Akt activation.

Next, we attempted to identify the mechanism(s) by which EGFR contributes to ANG II-induced DNA synthesis in IEC-18 cells. In a variety of cell types, EGFR transactivation mediates mitogenic stimulation by GPCRs through ERK1/2 activation via a well-defined mechanism involving SOS-Grb2/Ras/Raf and mitogen/extracellular signal-regulated kinase (MEK; see Refs. 46 and 54). However, our recent results with IEC-18 cells suggested that a different mechanism is operational downstream of EGFR in these intestinal epithelial cells. Specifically, ANG II-induced ERK activation in IEC-18 cells was mediated primarily by PKCs rather than EGFR, and inhibition of the ERK pathway attenuated (but did not eliminate) ANG II-induced DNA synthesis (also see below). These findings suggested that a pathway other than ERK plays a substantial role in mitogenesis in response to ANG II in these epithelial cells.

In addition to the well-recognized role of the ERKs in mitogenic signaling, the PI3-kinase pathway has also been implicated in signal transduction linked to cell division, differentiation, and tumorigenesis (4). Signaling by PI3-kinases is mediated by 3-phosphoinositide binding to the PH domains of both Akt and 3′-phosphoinositide-dependent kinase (PDK) 1, thereby inducing their translocation to the plasma membrane where PDK1 phosphorylates and activates Akt. These findings prompted us to determine whether ANG II-induced EGFR tyrosine kinase acts via the PI3-kinase/Akt pathway in IEC-18 cells.

To determine whether ANG II stimulates the PI3-kinase pathway in IEC-18 cells, we first examined whether ANG II induces the activation of the key PI3-kinase target Akt in these cells. Akt, translocated to the plasma membrane in response to products of PI3-kinase, is activated by phosphorylation at Thr308 by PDK1 (2) and by phosphorylation within the COOH terminus at Ser473 by a less well-defined protein kinase referred to as PDK2 (33). Recently, DNA-dependent protein kinase has been implicated as PDK2 (15). Serum-starved cultures of IEC-18 cells were stimulated with 50 nM ANG II for various times (0–60 min) and lysed, and Western blot analyses were performed using an MAb that recognizes Akt phosphorylated at Ser473, a major regulatory phosphorylation site commonly used as a marker for increased signaling via this pathway. As shown in Fig. 3A, ANG II stimulation induced phosphorylation at Ser473 rapidly, reaching a maximum within 2.5 min.

Fig. 3.

ANG II stimulates EGFR-dependent Akt activation in IEC-18 cells. A: ANG II induces Akt activation in a time-dependent manner. Confluent and quiescent cultures of IEC-18 cells were treated for various times with 50 nM ANG II at 37°C as indicated. Western blot analysis using a specific anti-phospho-Akt MAb that recognizes only the activated form phosphorylated on Ser473 was performed after lysis of the cells with 2× sample buffer as described in materials and methods. B: tyrphostin AG-1478 and compound 56 inhibit ANG II-induced Akt activation. Top, confluent and quiescent IEC-18 cells were incubated for 1 h with 250 nM AG-1478 and 500 nM compound 56 (Cpd 56). The cultures were subsequently left unstimulated (−) or stimulated (+) for 2.5 min with 50 nM ANG II at 37°C. Middle, Western blot was also probed for total Akt, showing equal loading. C: phosphatidylinositol (PI)3-kinase inhibitors LY-294002 (LY) and wortmannin (Wort) inhibit ANG II-induced Akt activation. Top, confluent and quiescent IEC-18 cells were incubated for 1 h with either 10 μM LY-294002 or 100 nM wortmannin. The cultures were subsequently left unstimulated (−) or stimulated (+) for 2.5 min with 50 nM ANG II at 37°C. Middle, the Western blot was also probed for total Akt, showing equal loading. B and C, bottom: values (means ± SE, n = 3) of the level of Akt phosphorylation obtained from scanning densitometry expressed as a percentage of the maximum increase in phosphorylation obtained with 50 nM ANG II. The Western blots shown above of the bar graphs are representative of 3 independent experiments.

Next, we investigated whether a functional EGFR is required for the ANG II-induced activation of Akt in IEC-18 cells. Serum-starved cultures of these cells were pretreated with the EGFR tyrosine kinase inhibitors tyrphostin AG-1478 (250 nM) or compound 56 (500 nM) before stimulation with 50 nM ANG II. As shown in Fig. 3B, ANG II-induced Akt phosphorylation at Ser473 was dramatically reduced by treatment with each of the selective EGFR tyrosine kinase inhibitors. Similarly, treatment with two structurally unrelated PI3-kinase inhibitors, LY-294002 and wortmannin, completely blocked ANG II-induced Akt phosphorylation at Ser473 (Fig. 3C). These results indicate that ANG II-stimulated activation of Akt is mediated via EGFR transactivation and PI3-kinase in IEC-18 cells.

ANG II stimulates mTOR phosphorylation and EGFR-dependent p70S6K1 activation.

Akt has been shown to mediate mTOR phosphorylation at Ser2448 (34), a site of importance in the regulation of mTOR function (30, 42), via the tuberous sclerosis complex/Ras homolog enriched in brain pathway (32). To determine whether ANG II can stimulate mTOR activation in IEC-18 cells, lysates of these cells stimulated with 50 nM ANG II for various times (0–60 min) were subjected to Western blot analyses using an antibody that detects mTOR phosphorylated at Ser2448. As shown in Fig. 4A, ANG II stimulation rapidly induced mTOR Ser2448 phosphorylation in IEC-18 cells. A half-maximal effect was achieved at 2.5 min, and a maximum was reached within 5 min.

Fig. 4.

ANG II stimulates mTOR phosphorylation and EGFR-dependent p70S6K1 activation in IEC-18 cells. A: ANG II induces mTOR phosphorylation in a time-dependent manner. Confluent and quiescent cultures of IEC-18 cells were treated for various times with 50 nM ANG II at 37°C as indicated. Western blot analysis using a specific anti-phospho-mTOR polyclonal antibody that recognizes only the activated form phosphorylated (p) on Ser2448 was performed after lysis of the cells with 2× sample buffer, as described in materials and methods. B: ANG II induces p70S6K1 activation in a time-dependent manner. Confluent and quiescent cultures of IEC-18 cells were treated for various times with 50 nM ANG II at 37°C as indicated. Western blot analysis using a specific anti-phospho-p70S6K1 MAb that recognizes only the activated form phosphorylated on Thr389 was performed after lysis of the cells with 2× sample buffer, as described in materials and methods. The Western blot was stripped and also probed for total p70S6K1, showing similar loading. C: effects of PI3-kinase inhibitors (LY-294002 and wortmannin) and mitogen/extracellular signal-regulated kinase (MEK) inhibitors [U-0126 and PD-98059 (PD)] on ANG II-induced p70S6K1 activation. Top, confluent and quiescent IEC-18 cells were incubated for 1 h with 10 μM LY-294002, 100 nM wortmannin, 2.5 μM U-0126, or 10 μM PD-98059. The cultures were subsequently left unstimulated (−) or stimulated (+) for 10 min with 50 nM ANG II at 37°C. Western blot analysis using a specific anti-phospho-p70S6K1 MAb that recognizes only the activated form phosphorylated on Thr389 was performed after lysis of the cells with 2× sample buffer as described. The Western blot was also probed for total p70S6K1 after stripping. Note that p70S6K1 protein was present in all lanes, including those that did not show any phosphorylation. Bottom, values (means ± SE, n = 3) of the level of p70S6K1 phosphorylation obtained from scanning densitometry expressed as a percentage of the maximum increase in phosphorylation obtained with 50 nM ANG II. Western blots are representative of 3 independent experiments.

p70S6K1 is a major downstream target of the PI3-kinase/Akt/mTOR pathway that is implicated in cell growth and G1 cell cycle progression (39). Rapamycin, a macrolide immunosuppressant that complexes with FK506-binding protein (FKBP12), is a potent and selective inhibitor of TOR proteins and signaling to p70S6K1. The activity of p70S6K1 is controlled by multiple phosphorylation events located within the catalytic, linker, and pseudosubstrate domains (39). Rapamycin-sensitive phosphorylation of Thr389 by mTOR in the linker region is thought to be required for subsequent PDK1-mediated phosphorylation of Thr229 in the catalytic domain (35, 38).

To determine whether ANG II activates p70S6K1 in IEC-18 cells, serum-starved cultures of these cells were stimulated with 50 nM ANG II for various times (0–60 min) and lysed, and the extracts were subjected to Western blot analyses using an antibody that recognizes p70S6K1 phosphorylated at Thr389, a major phosphorylation site that correlates closely with kinase activity (53). As shown in Fig. 4B, ANG II stimulation rapidly induced p70S6K1 phosphorylation at Thr389. A half-maximal effect was achieved at 2.5 min, and maximal phosphorylation was reached within 10 min. Thus the kinetics of p70S6K1 phosphorylation at Thr389 parallels that of mTOR phosphorylated at Ser2448. To determine whether a functional mTOR was required for the ANG II-induced phosphorylation of p70S6K1 at Thr389, serum-starved cultures of IEC-18 cells were pretreated with rapamycin before stimulation with ANG II. As shown in Fig. 5A, rapamycin completely blocked ANG II-induced p70S6K1 phosphorylation at Thr389.

Fig. 5.

Effects of inhibition of EGFR tyrosine kinase and HB-EGF binding on p70S6K1 phosphorylation. A: EGFR tyrosine kinase inhibitors (AG-1478 and compound 56) and rapamycin (Rap) inhibit ANG II-induced p70S6K1 activation. Top, confluent and quiescent IEC-18 cells were incubated for 1 h with 250 nM AG-1478, 500 nM compound 56, or 1 nM rapamycin. The cultures were subsequently left unstimulated (−) or stimulated (+) for 10 min with 50 nM ANG II at 37°C. Western blot analysis for phospho-Thr389 p70S6K1 was performed as described. After stripping the same blot, it was also reprobed for total p70S6K1. Bottom, values (means ± SE, n = 3) of the level of p70S6K1 phosphorylation obtained from scanning densitometry expressed as a percentage of the maximum increase in phosphorylation obtained with 50 nM ANG II. B: heparin inhibits ANG II-induced p70S6K1 phosphorylation. Serum-starved cultures of IEC-18 cells were preincubated for 30 min with or without 50 μg/ml heparin. The cultures were subsequently left unstimulated (−) or stimulated (+) with 50 nM ANG II, 1 ng/ml HB-EGF, or 10 ng/ml EGF, as indicated. Western blot analysis for phospho-Thr389 p70S6K1 and phospho-Thr202/Tyr204 ERK1/2 were performed on the same blot. The Western blot was also probed for total ERK, showing equal loading. The results presented are representative of 3 independent experiments.

To substantiate that p70S6K1 activation occurs downstream of PI3-kinase in ANG II-stimulated IEC-18 cells, serum-starved cultures of IEC-18 cells were pretreated with either LY-294002 or wortmannin before stimulation with 50 nM ANG II. As shown in Fig. 4C, ANG II-induced p70S6K1 phosphorylation at Thr389 was nearly abolished by cell exposure to the PI3-kinase inhibitors. In contrast, treatment of parallel cultures with the MEK inhibitors U-0126 or PD-98059 exerted no discernible inhibitory effect on the phosphorylation of this site in response to ANG II. These results indicate that ANG II-induced p70S6K1 phosphorylation at Thr389 is downstream of PI3-kinase and is not dependent on MEK-mediated ERK activation.

Because our results demonstrated that the activation of the PI3-kinase/Akt pathway by ANG II is mediated by EGFR transactivation, we next tested whether ANG II-induced p70S6K1 activation also requires a functional EGFR pathway. As shown in Fig. 5A, ANG II-induced p70S6K1 phosphorylation at Thr389 was dramatically reduced by prior treatment of the cells with either tyrphostin AG-1478 (250 nM) or compound 56 (500 nM). The Western blot was also probed for total p70S6K1 after stripping. Note that p70S6K1 protein was present in all lanes, including those that did not show any phosphorylation.

Because our results demonstrated that the activation of the PI3-kinase/Akt/p70S6K1 pathway by ANG II is mediated by EGFR transactivation, we next tested whether ANG II-induced p70S6K1 activation also requires HB-EGF activity. Serum-starved cultures of IEC-18 cells were pretreated with heparin before ANG II, HB-EGF, or EGF stimulation. As shown in Fig. 5B, heparin inhibited ANG II- and HB-EGF-induced p70S6K1 phosphorylation at Thr389, whereas EGF-induced p70S6K1 phosphorylation was not affected. This result, combined with the experiments shown in Fig. 1, suggests that HB-EGF is required for ANG II-stimulated HB- and EGF-mediated EGFR transactivation in IEC-18 cells.

ANG II induces S6 ribosomal protein phosphorylation through EGFR, PI3-kinase, and p70S6K1 in IEC-18 cells.

To substantiate that phosphorylation of p70S6K1 at Thr389 faithfully reflects p70S6K1 catalytic activity in IEC-18 cells stimulated with ANG II, we next examined the phosphorylation of the 40S ribosomal protein subunit S6, which is a well-established downstream target of p70S6K1 in intact cells. Phosphorylation of S6 by p70S6K1 correlates with an increase in translation, particularly of mRNAs with an oligopyrimidine tract in their 5′-untranslated regions (3). The main in vivo S6 ribosomal protein phosphorylation sites, including Ser235, Ser236, Ser240, and Ser244, have been identified and are located within a small 19-amino-acid region (3).

To determine the kinetics of ANG II-stimulated S6 ribosomal protein phosphorylation, serum-starved cultures of IEC-18 cells were stimulated with 50 nM ANG II for various times (0–30 min) and then lysed. The extracts were subjected to Western blot analysis using a polyclonal antibody that recognizes S6 ribosomal protein phosphorylated at Ser235 and Ser236. As shown in Fig. 6A, ANG II stimulation rapidly induced S6 ribosomal protein phosphorylation at Ser235 and Ser236. A half-maximal effect was achieved at 5 min and reached a maximum within 15 min. This kinetic profile of S6 ribosomal protein phosphorylation parallels that of p70S6K1 activation, as judged by phosphorylation at Thr389. As shown in Fig. 6B, treatment with the EGFR tyrosine kinase inhibitors AG-1478 or compound 56 completely abrogated ANG II-induced S6 ribosomal protein Ser235 and Ser236 phosphorylation. Furthermore, congruent with the notion that EGFR transactivation leads to the activation of the PI3-kinase/Akt/mTOR/p70S6K1 pathway, treatment with either LY-294002 or rapamycin completely blocked ANG II-induced S6 ribosomal protein phosphorylation at Ser235 and Ser236 (Fig. 6B).

Fig. 6.

Effects of inhibition of EGFR tyrosine kinase, Akt, and mTOR on p70S6K1 and S6 phosphorylation. A: ANG II induces S6 activation in a time-dependent manner. Confluent and quiescent cultures of IEC-18 cells were treated for various times with 50 nM ANG II at 37°C as indicated. Western blot analysis using a specific anti-phospho-S6 polyclonal antibody that recognizes only the activated form phosphorylated on Ser235 and Ser236 was performed after lysis of the cells with 2× sample buffer, as described. B: AG-1478, compound 56, LY-294002, and rapamycin inhibit ANG II-induced S6 phosphorylation. Top, confluent and quiescent IEC-18 cells were incubated for 1 h with 250 nM AG-1478, 500 nM compound 56, 1 nM rapamycin, or 10 μM LY-294002. The cultures were subsequently left unstimulated (−) or stimulated (+) for 10 min with 50 nM ANG II at 37°C. Western blot analysis using a specific anti-phospho-S6 polyclonal antibody that recognizes only the activated form phosphorylated on Ser235 and Ser236 was performed after lysis of the cells with 2× sample buffer, as described. Western blots are representative of 3 independent experiments. C: effects of Akt knockdown on ANG II-induced signaling in IEC-18 cells. IEC-18 cells were transfected with Akt small-interfering RNA (siRNA) or nontargeted negative control duplex, as described in materials and methods. After 48 h, cells were incubated in the absence (−) or presence (+) of 50 M ANG II for 5 min. Next, the cells were lysed and subjected to Western blot analysis, using polyclonal anti-Akt antibody, anti-phospho-p70S6K1 MAb, anti-phospho-S6 polyclonal antibody, or anti-phospho-ERK-1/ERK-2 MAb. The Western blot was also probed for total ERK, showing equal loading. The blots are representative of 3 independent experiments.

Akt gene silencing inhibits ANG II-stimulated p70S6K1 and S6 ribosomal protein phosphorylation in IEC-18 cells.

To substantiate that Akt mediates p70S6K1 activation and S6 phosphorylation, we utilized gene silencing by RNA interference. Subconfluent cultures of IEC-18 cells were transiently transfected with Akt siRNA that targets Akt1 and Akt2 (27). To test the specificity of the siRNA, subconfluent cultures of IEC-18 cells were transfected with a nontargeted negative control duplex. As shown in Fig. 6C, Akt protein level in IEC-18 cells transfected with Akt siRNA was dramatically reduced compared with cells transfected with nontargeted negative control duplex. Akt gene silencing abrogated ANG II-induced p70S6K1 Thr389 phosphorylation (Fig. 6C) and dramatically reduced ANG II-induced S6 ribosomal protein phosphorylation at Ser235 and Ser236 compared with nontargeted negative control duplex transfected cells (Fig. 6C). In contrast, ANG II-induced ERK1/2 phosphorylation was not affected in IEC-18 cells transfected either with Akt siRNA or nontargeted negative control duplex (Fig. 6C). These results further demonstrate the selectivity of Akt gene silencing and indicate that ANG II induces ERK1/2 phosphorylation via a distinct signaling pathway, separate from the PI3-kinase/Akt/mTOR/p70S6K1 pathway.

ANG II induces EGFR transactivation and ERK activation through parallel pathways.

If PI3-kinase and p70S6K1 lie downstream of EGFR in ANG II-stimulated IEC-18 cells, inhibitors of these kinases should not interfere with EGFR transactivation. As shown in Fig. 7A, stimulation of IEC-18 cell cultures with ANG II induced a marked increase in the tyrosine phosphorylation of the EGFR, in agreement with the results presented in Fig. 1. Pretreatment of these cells with either LY-294002 or rapamycin did not produce any detectable effect on the increase in EGFR tyrosine phosphorylation induced by ANG II. These results substantiate that PI3-kinase and p70S6K1 lie downstream of EGFR in ANG II-stimulated cells.

Fig. 7.

Effects of inhibition of EGFR tyrosine kinase, PI3-kinase, mTOR, and MEK on EGFR tyrosine phosphorylation and ERK activation in IEC-18 cells. A: ANG II-induced EGFR tyrosine phosphorylation is not affected by inhibition of PI3-kinase or mTOR. Confluent and serum-starved cultures of IEC-18 cells were stimulated for 2 min with 50 nM ANG II after 1 h preincubation with 10 μM LY-294002 or 10 nM rapamycin, as indicated. The EGFR was immunoprecipitated using a polyclonal EGFR antibody and resolved by SDS-PAGE. Proteins were transferred to Immobilon-P membranes, and then tyrosine phosphorylated EGFR was detected using 4G10 anti-Tyr(P) MAb. Membranes were then stripped and reprobed with anti-EGFR. The results are representative of 3 independent experiments. B: ANG II induces p42mapk (ERK2) and p44mapk (ERK1) phosphorylation in a time-dependent manner. Confluent and serum-starved cultures of IEC-18 cells were incubated for 1 h with 250 nM AG-1478, 1 nM rapamycin, 10 μM LY-294002, or 2.5 μM U-0126. Control cells received equivalent amount of solvent. The cultures were subsequently left unstimulated (−) or stimulated (+) for 2.5 min with 50 nM ANG II at 37°C. Western blot analysis using a specific anti-phospho-ERK1/2 MAb that recognizes only the activated forms phosphorylated on Thr202 and Tyr204 was performed after lysis of the cells with 2× sample buffer, as described in materials and methods. The Western blot was also probed for total ERK, showing equal loading. C: ANG II-induced EGFR tyrosine phosphorylation is not affected by MEK inhibitors. Confluent and serum-starved cultures of IEC-18 cells were stimulated for 2 min with 50 nM ANG II after 1 h preincubation with 2.5 μM U-0126 or 10 μM PD-98059, as indicated. The EGFR was immunoprecipitated using a polyclonal EGFR antibody and resolved by SDS-PAGE. Proteins were transferred to Immobilon-P membranes, and then tyrosine-phosphorylated EGFR was detected using 4G10 anti-Tyr(P) MAb. Membranes were then stripped and reprobed with anti-EGFR. The results are representative of 3 independent experiments.

In many cellular systems, EGFR transactivation mediates activation of the ERK pathway in response to GPCR agonists. It is conceivable that ANG II-induced EGFR transactivation not only leads to the activation of the PI3-kinase/Akt/TOR/p70S6K1 pathway but also mediates ERK activation. To directly test this possibility, we examined whether EGFR transactivation mediates ANG II-induced ERK1/2 phosphorylation. Serum-deprived cultures of IEC-18 cells were pretreated with either tyrphostin AG-1478 or compound 56 before addition of ANG II. The activated forms of ERK1/2 were monitored by using a specific anti-phospho-p44/p42mapk MAb that recognizes only the activated forms phosphorylated on Thr202 and Tyr204. As shown in Fig. 7B, ANG II-induced ERK1/2 activation was unaffected by either of the EGFR tyrosine kinase inhibitors, demonstrating that ERK is not rapidly activated by ANG II via EGFR transactivation. Furthermore, inhibition of PI3-kinase or p70S6K1 had no effect on ANG II-induced ERK1/2 activation, supporting the notion that these pathways act in parallel (Fig. 7B). In addition, ANG II-induced EGFR transactivation was not prevented by cell treatment with the MEK inhibitors U-0126 or PD-98059, demonstrating that the ERKs are not upstream of EGFR transactivation (Fig. 7C). The results shown in Fig. 7 provide further support for the notion that the EGFR/PI3-kinase/Akt/mTOR/p70S6K1 pathway and the MEK/ERK pathway act in parallel in ANG II-stimulated IEC-18 cells.

Rapamycin attenuates ANG II-induced DNA synthesis in IEC-18 cells.

Given our results demonstrating that ANG II-induced p70S6K1 activation is mediated by EGFR transactivation, we next tested whether suppression of mTOR signaling by the potent and specific inhibitor rapamycin attenuates ANG II-induced DNA synthesis. Cultures of IEC-18 cells were treated with increasing concentrations of rapamycin, stimulated with ANG II, and labeled with [3H]thymidine to determine the level of DNA synthesis. As shown in Fig. 8A, rapamycin markedly reduced ANG II-induced [3H]thymidine incorporation in IEC-18 cells in a concentration-dependent fashion. A maximal inhibitory effect (∼50%) of rapamycin was achieved at a concentration of 1 nM.

Fig. 8.

Effects of inhibition of EGFR tyrosine kinase, PI3-kinase, p70S6K1, and MEK on DNA synthesis in IEC-18 cells. A: rapamycin inhibits ANG II-induced DNA synthesis in a concentration-dependent manner. Confluent and serum-starved cultures of IEC-18 cells were washed and incubated at 37°C in 2 ml DMEM/Waymouth’s medium containing 50 nM ANG II either without or with rapamycin at various concentrations, as indicated. [3H]thymidine incorporation into acid-precipitable material was measured as described. B: inhibition of EGFR tyrosine kinase and PI3-kinase did not have an additive inhibitory effect on ANG II-induced DNA synthesis. Confluent and serum-starved cultures of IEC-18 cells were washed and incubated at 37°C in 2 ml DMEM/Waymouth’s medium containing 50 nM ANG II with 250 nM AG-1478, 10 μM LY-294002, both inhibitors, or vehicle (control). C: U-0126 inhibits ANG II-induced DNA synthesis in a concentration-dependent manner. Confluent and serum-starved cultures of IEC-18 cells were washed and incubated at 37°C in 2 ml DMEM/Waymouth’s medium containing 50 nM ANG II either without or with U-0126 at various concentrations, as indicated. [3H]thymidine incorporation into acid-precipitable material was measured as described. Results shown are expressed as a percentage of the maximum increase ± SE (n = 3) obtained with 50 nM ANG II after subtraction of the basal [3H]thymidine incorporation from the respective controls. D: pretreatment with the combination of AG-1478 and U-0126 gave an additive inhibitory effect that nearly abolished ANG II-induced DNA synthesis. Confluent and serum-starved cultures of IEC-18 cells were washed and incubated at 37°C in 2 ml DMEM/Waymouth’s medium containing 50 nM ANG II with 250 nM AG-1478, 2.5 μM U-0126, both inhibitors, or vehicle (control). [3H]thymidine incorporation was determined as described. Results shown are in counts/min ± SE (n = 3). *P < 0.01.

The results presented above suggest that PI3-kinase plays a critical role in mediating the downstream effects of EGFR transactivation induced by ANG II in IEC-18 cells. If EGFR and PI3-kinase lie in the same pathway, concomitant inhibition of EGFR tyrosine kinase and PI3-kinase should not produce any additional inhibitory effect on [3H]thymidine incorporation in response to ANG II beyond that achieved by inhibition of EGFR alone. In agreement with this prediction, treatment with both the EGFR tyrosine kinase inhibitor tyrphostin AG-1478 and the PI3-kinase inhibitor LY-294002 did not inhibit ANG II-induced [3H]thymidine incorporation to a greater extent than that induced by tyrphostin AG-1478 alone (Fig. 8B). The results presented in Fig. 8 substantiate the notion that EGFR transactivation induced by ANG II in IEC-18 cells contributes to the mitogenic stimulation promoted by this GPCR agonist through the PI3-kinase/Akt/TOR/p70S6K1 pathway.

ANG II induces DNA synthesis through EGFR and ERK pathways in IEC-18 cells.

Our results in Fig. 1B demonstrate that selective EGFR tyrosine kinase inhibitor tyrphostin AG-1478 could reduce [3H]thymidine incorporation in response to ANG II stimulation in a concentration-dependent fashion, with a maximal inhibitory effect at ∼50%. The results presented in Fig. 8C show that inhibition of the ERK pathway by treatment of IEC-18 cells with the MEK inhibitor U-0126 also attenuated [3H]thymidine incorporation in response to ANG II stimulation by ∼50%. Given these results, we hypothesized that inhibition of both EGFR tyrosine kinase and ERK would lead to an additive inhibitory effect, suggesting that these distinct pathways contribute to ANG II-induced mitogenesis in IEC-18 cells.

As shown in Fig. 8D, pretreatment with the combination of selective EGFR tyrosine kinase inhibitor tyrphostin AG-1478 and selective MEK inhibitor U-0126 gave an additive inhibitory effect that nearly abolished ANG II-induced DNA synthesis. Taken together, these results strongly suggest that ANG II-induced DNA synthesis requires two distinct pathways involving the MEK/ERK and the EGFR/PI3-kinase/Akt/mTOR/ p70S6K1, as shown schematically in Fig. 9.

Fig. 9.

Signal transduction pathways involved in ANG II-induced mitogenesis in IEC-18 cells. This scheme summarizes our results demonstrating that ANG II-induced mitogenesis is dependent on 2 independent, distinct pathways, involving the protein kinase C (PKC)/MEK/p44/p42 mitogen-activated protein kinase [MAPK; extracellular signal-regulated kinase (ERK)] and the EGFR/PI3-kinase/Akt/mTOR/p70S6K1 pathways. ANG II also stimulates calcium- and PKC-dependent tyrosine phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), a nonreceptor tyrosine kinase that has been implicated as an upstream element in p44/p42 MAPK activation, in IEC-18 cells (56). The inhibitors used in this study to block specific pathways are shown as marked by boxes. The dotted lines imply the existence of intermediary steps leading to the subsequent event; solid lines represent direct consequence of the previous effector. GF-1 or GF-109203X (also known as bisindolylmaleimide I) is a PKC inhibitor. PLC, phospholipase C; PI3K, PI3-kinase; DAG, diacylglycerol; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; [Ca2+]i, intracellular Ca2+ concentration.

DISCUSSION

The proliferation of epithelial cells of the intestinal mucosa is regulated by a broad spectrum of regulatory peptides acting through both tyrosine kinase receptors and heptahelical GPCRs (5, 26, 50). The understanding of the mechanisms involved requires the identification of the signal transduction pathways that mediate intestinal epithelial cell proliferation.

We have recently demonstrated that the addition of ANG II, at nanomolar concentrations, induces DNA synthesis and cell proliferation in intestinal epithelial IEC-18 cells via the AT1 receptor in the absence of any other exogenously added growth factor. ANG II induces a dramatic increase in PKC-dependent PKD activation in these cells (7) and stimulates PKC-dependent tyrosine phosphorylation of Pyk2, a nonreceptor tyrosine kinase that has been implicated as an upstream element in ERK1/2 activation (56). Accordingly, ANG II stimulates PKC-dependent ERK activation, a major pathway leading to cell cycle activation and cell proliferation. Interestingly, inhibition of the ERK pathway in these cells reduces ANG II-induced DNA synthesis by 50–60% (8). These findings not only indicated that ANG II-induced DNA synthesis is partially dependent on ERK and PKC but also raise the intriguing possibility that another pathway, in addition to ERK and PKC, plays a substantial role in AT1-mediated mitogenesis of IEC-18 cells. The experiments presented in this study were designed to identify the pathway that, together with ERK, drives cell cycle progression in ANG II-stimulated IEC-18 cells.

Activation of a variety of GPCRs has been shown to stimulate the tyrosine kinase activity of the EGFR in several cell types (1012, 29, 52), a process termed transactivation (6, 36). As a first step to evaluate the role of EGFR transactivation in mediating ANG II-induced mitogenesis in IEC-18 cells, we demonstrated that stimulation with this agonist induced a striking increase in EGFR tyrosine phosphorylation. This response was prevented by cell exposure to the selective EGFR tyrosine kinase inhibitor AG-1478 or by the broad-spectrum metalloprotease inhibitor GM-6001, suggesting that proteolytic release of EGFR ligand(s) is involved. We have further shown that heparin pretreatment significantly reduced ANG II-induced EGFR tyrosine phosphorylation (Fig. 1C) and ANG II-induced EGFR signaling (p70S6K1 phosphorylation), whereas ANG II-induced ERK activation was not affected (Fig. 5B). These results combined with those obtained by using the broad-spectrum metalloproteinase inhibitor GM-6001 suggest that HB-EGF is required for ANG II-stimulated EGFR transactivation and EGFR-dependent downstream signaling in IEC-18 cells.

Furthermore, treatment with EGFR tyrosine kinase inhibitors or GM-6001 also attenuated (by ∼50%) DNA synthesis induced by ANG II in IEC-18 cells, indicating that EGFR transactivation plays a role in mediating the proliferative response induced by ANG II in IEC-18 cells. These initial findings prompted us to identify the pathway(s) downstream of EGFR that promotes mitogenesis in IEC-18 cells.

In many cell types, the EGFR has been proposed to mediate Ras/ERK1/2 activation in response to GPCR activation, and the ERK pathway has been implicated in mitogenic signal transduction by many stimuli (13, 14, 18, 19). For example, in vascular smooth muscle cells (13, 14), pancreatic stellate cells (19), and breast cancer cells (18), ANG II has been shown to stimulate cell cycle progression through EGFR-dependent ERK activation.

In contrast, our results with IEC-18 cells indicate that EGFR transactivation contributes to ANG II-induced mitogenesis primarily through activation of the PI3-kinase/Akt/p70S6K1 pathway, whereas PKC (not EGFR tyrosine kinase) mediates the ANG II-stimulated ERK pathway. Specifically, several lines of evidence support this conclusion as follows: 1) suppression of EGFR tyrosine kinase had no effect on ANG II-stimulated ERK1/2 activation in IEC-18 cells, in agreement with our recent results indicating that ANG II-induced ERK activation is mediated primarily by PKCs in these cells; 2) activation of PI3-kinase results in production of lipid-derived second messengers that promote PDK1-mediated activation of Akt. We demonstrated here that stimulation of IEC-18 cells with ANG II induced Akt phosphorylation at Ser473, a major regulatory phosphorylation site required for kinase activity. Akt activation in response to ANG II was completely inhibited by prior exposure of the cells to selective inhibitors of PI3-kinase (wortmannin, LY-294002) or EGFR tyrosine kinase (tyrphostin AG-1478); 3) another downstream effector of PI3-kinase implicated in cell cycle progression is p70S6K1. We showed that ANG II markedly stimulated p70S6K1 phosphorylation at Thr389, a major phosphorylation site that correlates closely with kinase activity, through an EGFR-, PI3-kinase- and mTOR-dependent pathway; and 4) siRNAs targeting Akt1 and Akt2 dramatically inhibited ANG II-induced p70S6K1 phosphorylation at Thr389 and the phosphorylation at Ser235/236 of its downstream target, the S6 ribosomal protein. In contrast, ANG II-induced ERK1/2 activation was not prevented by treatment with LY-294002, rapamycin, or Akt siRNA. Reciprocally, blocking MEK-mediated ERK activation prevented neither EGFR transactivation nor p70S6K1. Taken together, these results demonstrate that PI3-kinase, Akt, and p70S6K1 are downstream effectors of EGFR tyrosine kinase and lie in a pathway distinct from the ERKs in ANG II-stimulated IEC-18 cells.

We also produced several lines of evidence indicating that ANG II-induced activation of the PI3-kinase pathway contributes to cell cycle progression into DNA synthesis in IEC-18 cells. Specifically, we demonstrated that inhibition of PI3-kinase or mTOR/p70S6K1 by the specific immunosuppressant rapamycin reduced [3H]thymidine incorporation by 50%, i.e., to levels comparable to those achieved by addition of either selective EGFR tyrosine kinase inhibitors or the broad-spectrum metalloprotease GM-6001. If PKC-dependent ERK activation and EGFR-dependent PI3-kinase signaling operate as independent pathways that contribute to ANG II-induced DNA synthesis in IEC-18 cells, we expect that simultaneous blockade of these pathways should produce additive inhibition of DNA synthesis. In line with this prediction, we show that treatment of IEC-18 cells with a combination of the EGFR tyrosine kinase inhibitor tyrphostin AG-1478 and the selective MEK inhibitor U-0126 produced an additive inhibitory effect that nearly abolished ANG II-induced DNA synthesis. Inhibition of either pathway alone reduced ANG II-stimulated DNA synthesis by ∼50%. As depicted in Fig. 9, we conclude that ANG II-stimulated mitogenesis in intestinal epithelial IEC-18 cells is mediated by the following two distinct signaling pathways: PKC-dependent MEK/ERK and an EGFR-dependent PI3-kinase/Akt/mTOR/p70S6K1, dissected in the present study.

Early studies demonstrated that AT1 is the predominant ANG II receptor in the gastrointestinal tract, as revealed by in situ autoradiography (22). ANG II has been shown to mediate epithelial sodium and water absorption in the jejunum, ileum, and distal colon (23, 24), but the role of ANG II-induced AT1 receptor signaling in intestinal epithelial cell proliferation and differentiation is much less defined. Collectively, our previous and present results demonstrate that ANG II is a potent growth factor for intestinal epithelial cells and identify some of the critical molecular events that mediate the mitogenic effect of ANG II in these cells. These results assume an added significance in view of recent reports demonstrating that 1) drugs blocking the biological activity of ANG II enhanced the antitumor effect of cyclooxygenase-2 inhibitors in colon carcinoma models, 2) the high frequency of activating mutations of PI3-kinase gene in human colorectal cancers, and 3) the evidence suggesting that inhibitors of ANG II generation (i.e., angiotensin I-converting enzyme) protect against risk of multiple cancers (31, 48, 57).

GRANTS

This work was supported by an American Gastroenterological Association/AstraZeneca Fellowship/Faculty Transition Award, a Howard Hughes Medical Institute Research Resources Faculty Development Award, and National Institutes of Health (NIH) Grant K08-DK-063983 (to T. Chiu). This work was also supported by NIH Grants DK-56930 and DK-55003 to E. Rozengurt. Flow cytometry was performed in the University of California Los Angeles (UCLA) Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility, which was supported by NIH Grant CA-16042 and AI-28697, by the Jonsson Cancer Center, the UCLA AIDS Institute, and David Geffen School of Medicine at UCLA.

Acknowledgments

We thank Drs. Sushovan Guha, Cliff Hurd, J. Adrian Lunn, and Richard Waldron for thoughtful comments.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

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