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J. Biol. Chem., Vol. 279, Issue 10, 9233-9247, March 5, 2004

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Involvement of the ERK Signaling Cascade in Protein Kinase C-mediated Cell Cycle Arrest in Intestinal Epithelial Cells^*

Jennifer A. Clark, Adrian R. Black, Olga V. Leontieva, Mark R. Frey, Marybeth A. Pysz, Laura Kunneva, Anna Woloszynska-Read, Durga Roy, and Jennifer D. Black

From the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263

Received for publication, November 10, 2003 , and in revised form, December 10, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have reported previously that protein kinase C (PKC) signaling can mediate a program of cell cycle withdrawal in IEC-18 nontransformed intestinal crypt cells, involving rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of pocket proteins (Frey, M. R., Clark, J. A., Leontieva, O., Uronis, J. M., Black, A. R., and Black, J. D. (2000) J. Cell Biol. 151, 763–777). In the current study, we present evidence to support a requisite role for PKC in mediating these effects. Furthermore, analysis of the signaling events linking PKC/PKC activation to changes in the cell cycle regulatory machinery implicate the Ras/Raf/MEK/ERK cascade. PKC/PKC activity promoted GTP loading of Ras, activation of Raf-1, and phosphorylation/activation of ERK. ERK activation was found to be required for critical downstream effects of PKC/PKC activation, including cyclin D1 down-regulation, p21^Waf1/Cip1 induction, and cell cycle arrest. PKC-induced ERK activation was strong and sustained relative to that produced by proliferative signals, and the growth inhibitory effects of PKC agonists were dominant over proliferative events when these opposing stimuli were administered simultaneously. PKC signaling promoted cytoplasmic and nuclear accumulation of ERK activity, whereas growth factor-induced phospho-ERK was localized only in the cytoplasm. Comparison of the effects of PKC agonists that differ in their ability to sustain PKC activation and growth arrest in IEC-18 cells, together with the use of selective kinase inhibitors, indicated that the length of PKC-mediated cell cycle exit is dictated by the magnitude/duration of input signal (i.e. PKC activity) and of activation of the ERK cascade. The extent/duration of phospho-ERK nuclear localization may also be important determinants of the duration of PKC agonist-induced growth arrest in this system. Taken together, the data point to PKC and the Ras/Raf/MEK/ERK cascade as key regulators of cell cycle withdrawal in intestinal epithelial cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Members of the protein kinase C (PKC)¹ family of signal transduction molecules have been implicated in the regulation of a wide variety of cellular processes, including cell growth and cell cycle progression, differentiation, survival/apoptosis, and transformation (1–4). The PKC family consists of at least 10 distinct isozymes (, I, II, , , , , , , and ) that share the same basic structure but differ with respect to activator and cofactor requirements, substrate specificity, tissue expression, and subcellular distribution (3). Studies in several systems, including self-renewing epithelial tissues (i.e. intestinal mucosa and epidermis), several leukemic cell lines, and melanoma cells increasingly point to a role for sustained PKC signaling in mediating cell cycle exit and cell differentiation (see Ref. 1 for review). Mechanistic studies in intestinal (5) and epidermal (6) epithelial systems have shown that activation of PKC , in particular, is sufficient to trigger a program of cell cycle withdrawal, involving inhibition of G₁/S cyclin-dependent kinase activity and coordinated alterations in the expression/activity of members of the pocket protein family (i.e. p107, pRb, and p130). Studies in erythroleukemia cells (7), myeloid cells (8), and melanoma cells (9) have further demonstrated the importance of PKC in cell differentiation. Whereas the cell cycle-specific and growth regulatory effects of PKC signaling have been well characterized in these systems, the events linking PKC activation to changes in the cell cycle regulatory machinery remain largely unknown.

The current study investigated the downstream events of negative growth regulatory PKC signaling using intestinal epithelial cells as a model system. Based on (a) evidence for the ability of PKC agonists (10) and individual members of the PKC family (11, 12) to activate the extracellular signal-regulated kinase (ERK) pathway, and (b) recent reports demonstrating a role for ERK activity in growth arrest/differentiation of intestinal epithelial cells (13–16), we focused on the ERK signaling cascade. This three-kinase cascade, consisting of Raf, MAP kinase/ERK kinase (MEK), and ERKs 1 and 2, is ubiquitously expressed in mammalian cells and, like PKC, has been widely implicated in control of cell proliferation, differentiation, survival, and transformation (10, 17, 18). ERK1/2 and their upstream regulators are acutely stimulated by the interaction of growth or differentiation factors with cell surface receptor tyrosine kinases, heterotrimeric G protein-coupled receptors, or cytokine receptors (10, 17, 18). Stimulation of the pathway is often dependent on the activity of the monomeric G protein Ras, which can play an important role in the activation of Raf (19). A large body of evidence indicates that the strength and duration of the ERK signal plays a critical role in determining cellular response to activation of this pathway (20–22). Transient or cyclical ERK activation has been linked to cell cycle progression, whereas sustained levels of ERK activity can lead to cell growth arrest and differentiation. ERK activation can influence both nuclear and cytosolic events. On stimulation, ERKs can translocate to the nucleus where they phosphorylate transcription factors and thus regulate gene expression (23). Other ERK targets include membrane and cytoplasmic proteins, such as downstream kinases and cytoskeletal proteins (10, 17).

Previous studies from our laboratory have demonstrated that activation of PKC/PKC in IEC-18 nontransformed intestinal crypt cells results in cell cycle withdrawal. By using pharmacological inhibitors of PKC and ERK signaling, we now show that PKC plays a key role in PKC agonist-induced cell cycle arrest in IEC-18 cells and that ERK signaling is required for critical downstream cell cycle-specific effects of PKC/PKC activation, including down-regulation of cyclin D1, induction of p21^Waf1/Cip1, and cell cycle exit. We further demonstrate that PKC-mediated IEC-18 cell cycle arrest involves strong and sustained ERK signaling and that the duration of growth arrest is determined by the extent/duration of input signal, i.e. PKC activity, and of activation of the ERK pathway. The magnitude and duration of nuclear phospho-ERK activity also appear to be important determinants of the length of the effect. Notably, PKC/PKC stimulation is shown to promote both Ras and Raf activity in IEC-18 cells, and maintenance of ERK signaling in this system correlates with the duration of activation of these molecules.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Mouse monoclonal antibody specific for PKC was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal rabbit anti-PKC (C-17) and anti-PKC (C-15) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies used in this study have been characterized previously for the absence of cross-reactivity with other PKC isozymes (5, 24). Anti-phospho-p44/p42 ERK (anti-phospho-ERK1/2) monoclonal antibody (E10) was purchased from Cell Signaling Technology (Beverly, MA) and rabbit polyclonal anti-total ERK1/2 was from Santa Cruz Biotechnology. Rabbit polyclonal anti-cyclin D1 (H-295) and anti-MAP kinase phosphatase 1 (MKP-1) (M-18) antibodies were purchased from Santa Cruz Biotechnology, and mouse monoclonal anti-p21^Waf1/Cip1 (G3–245) was obtained from BD Pharmingen (San Diego, CA). Polyclonal anti-Raf-1 (residues 253–269) was from Upstate Biotechnology, Inc. Horseradish peroxidase-conjugated rat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA) and Chemicon International (Temecula, CA), respectively. TRITC-labeled goat anti-mouse IgG was purchased from Jackson ImmunoResearch. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PDBu), and 1,2-dioctanoyl-sn-glycerol (DiC₈) were obtained from Sigma, and bryostatin-1 (Bryo) was from Biomol (Plymouth Meeting, PA) or LC Laboratories (Woodburn, MA). The PKC inhibitors bisindolylmaleimide 1 (BIM I) and Gö6976 were obtained from LC Laboratories (Woburn, MA) and Calbiochem, respectively. The MEK inhibitors U0126 and PD098059 were purchased from Alexis Biochemicals (San Diego, CA).

Cell Culture, Cell Synchronization, and PKC Activation/Depletion Protocols—The IEC-18 cell line (ATCC CRL-1589) is an immature, nontransformed cell line derived from rat ileal epithelium that maintains many characteristics of proliferating intestinal crypt cells (25). These cells express the phorbol ester/diacylglycerol-responsive PKC isozymes , , and , and the atypical PKC isozymes and (26). IEC-18 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 4 mM glutamine, 10 µg/ml insulin, and 5% fetal bovine serum (FBS). Cells were synchronized in G₀/G₁ by incubation in 0.5% serum for 72 h as we have described previously (26). More than 90% of cells were arrested in G₀/G₁ by this method, as determined by flow cytometric analysis. Cells were released from G₀/G₁ arrest by addition of complete growth medium containing 5% serum and 10 µg/ml insulin.

PKC , , and were activated in IEC-18 cells by treatment with either 100 nM PMA, 20 µg/ml DiC₈, or 100 nM Bryo for various times. PMA and Bryo were dissolved in ethanol, with a final vehicle concentration in the medium of <0.1%; DiC₈ was dissolved in acetone, with a final vehicle concentration of <0.2%. Control cells were treated with the appropriate vehicle alone. Depletion of PKC , , and from IEC-18 cells was accomplished by treatment with 1 µM PDBu or 100 nM PMA for 24 h, as we have described previously (26). Selective down-regulation of PKC and was carried out by pulse treatment of IEC-18 cells with 10 or 100 nM PMA for 15 min, followed by two washes in warm PBS, and return to complete medium for 24 h. We have demonstrated previously (5, 24) that this procedure produces a population of cells expressing PKC as the only phorbol ester-responsive PKC isozyme (see Figs. 2, 8, and 9).

View larger version (27K): [in this window] [in a new window]   FIG. 2. PKC signaling activates the ERK signaling cascade in IEC-18 cells. A, PMA and DiC8 induce ERK1/2 phosphorylation/activation in intestinal epithelial cells. IEC-18 cells were treated with 100 nM PMA for 5 min (upper panel) or 15 min (lower panel) in the presence or absence of 10 µM U0126 or 50 µM PD098059 (added 30 min prior to PMA). Alternatively, cells were treated with 20 µg/ml DiC8 for 5 min as indicated. ERK1/2 activation was assessed by immunoblot analysis using anti-phospho-ERK1/2 (anti-active ERK1/2). Total ERK1/2 levels were determined using anti-total ERK1/2 antibody. B, PMA-induced ERK1/2 activation is PKC-dependent and can be mediated by PKC alone in IEC-18 cells. i, the phorbol ester-responsive PKC isozymes, PKC , , and , were depleted (lane D) from IEC-18 cells by treatment with 1 µM PDBu for 24 h; cells expressing PKC as the only phorbol ester-responsive PKC isozyme (lane ) were generated by treatment with 100 nM PMA for 15 min, followed by two washes in warm PBS, and return to complete medium for 24 h (26). ii, PKC-depleted cells were treated with 100 nM PMA for 30 min, and ERK1/2 activation and expression were assessed by immunoblot analysis as in A. iii, PKC /-depleted cells were treated with 100 nM PMA for 30 min, and ERK1/2 activation was assessed by immunoblot analysis as in ii. Note that the extent of PMA-induced ERK1/2 activation in PKC /-depleted cells varied slightly between experiments and correlated with the levels of PKC remaining in the cells. Data are representative of at least three independent experiments.

View larger version (27K): [in this window] [in a new window]   FIG. 8. PKC/PKC stimulation by PMA or Bryo activates Ras in IEC-18 cells; Ras activation by Bryo is more transient than that induced by PMA. A, PMA and Bryo activate Ras in IEC-18 cells. Asynchronously growing or serum-starved (0.5% serum for 24 h, no insulin) IEC-18 cells were treated with 100 nM PMA (P) or 100 nM Bryo (B) for 5 min. Cell lysates were prepared according to the instructions provided in the Pierce EZ-Detect Ras Activation kit, and affinity precipitation of GTP-bound Ras was performed using GST-tagged Raf-RBD. Levels of Ras in cell lysates (total Ras) or pulled down Ras (Ras-GTP) were determined by anti-Ras immunoblotting. Positive (+) and negative (-) controls for the pull-down procedure were generated by in vitro GTPS or GDP lysate treatments, respectively. Lane C, vehicle-treated cells. Data in asynchronously growing and serum-starved cells are representative of six and two independent experiments, respectively. B, activation of Ras by PMA or Bryo is PKC-dependent. i, the phorbol ester-responsive PKC isozymes, PKC , , and , were depleted from IEC-18 cells by treatment with 1 µM PDBu for 24 h (26), as shown by Western blot analysis. ii, PKC-depleted cells were subsequently treated with 100 nM PMA or 100 nM Bryo for 5 min, and the activation state of Ras was determined as in A. Lane C, vehicle-treated cells. C, PKC is sufficient to mediate Ras activation by PMA or Bryo. Cells expressing PKC as the only phorbol ester/Bryo-responsive PKC isozyme were generated by pulse treatment with PMA for15 min, followed by two washes in warm PBS, and return to complete medium for 24 h (26). PKC /-depleted cells were subsequently treated with 100 nM PMA or 100 nM Bryo for 5 min, and the activation state of Ras and expression of PKC isozymes were determined as in B. D, Bryo-induced Ras activity is down-regulated more rapidly than that induced by PMA. Asynchronously growing IEC-18 cells were treated with 100 nM PMA (P) or 100 nM Bryo (B) for 5 or 75 min, and Ras activity and expression were determined as in A. E, PMA-induced Ras activity is maintained for at least 6 h in IEC-18 cells. Asynchronously growing cells were treated with 100 nM PMA for 1, 2, 4, or 6 h and Ras activity was determined as in A. Data in B–E are representative of at least two independent experiments.

View larger version (37K): [in this window] [in a new window]   FIG. 9. PKC/PKC stimulation by PMA or Bryo activates Raf-1 in IEC-18 cells; Raf-1 activation by Bryo is more transient than that induced by PMA. A, PMA and Bryo activate Raf-1 in IEC-18 cells. Asynchronously growing IEC-18 cells were treated with vehicle (Control), 100 nM PMA, or 100 nM Bryo for 5 or 75 min. Cell lysates were prepared according to the Raf-1 Immunoprecipitation Kinase Cascade Assay kit protocol from Upstate Biotechnology, Inc., and Raf-1-induced phosphorylation of myelin basic protein was determined by scintillation counting. 5- and 75-min data are the average of four and two independent experiments, respectively. B, PKC is sufficient to mediate PMA-induced activation of Raf-1. PKC /-depleted cells (, Pulse), generated by pulse treatment with PMA as described under "Experimental Procedures" (see immunoblot), were treated with 100 nM PMA for 5 min, and cell lysates were examined for Raf-1 activity as described in A. C, PMA-induced activation of Raf-1 is PKC/PKC -dependent. Asynchronously growing IEC-18 cells (i.e. control cells), PKC , , and -depleted cells, and PKC /-depleted (PKC -expressing) cells were incubated with 100 nM PMA for 2 h. Raf-1 was detected in cell lysates by anti-Raf-1 immunoblotting. PMA treatment of cells expressing the full profile of PKC isozymes resulted in a change in the mobility of Raf-1 from a faster migrating form to a slower migrating form (arrow). This mobility shift was abrogated by PKC depletion (D) and could be induced by PKC alone () in these cells. Lane C, control cells; P, control cells treated with PMA; D, PKC-depleted cells; D/P, PKC-depleted cells treated with PMA; , PKC /-depleted cells; /P, PKC /-depleted cells treated with PMA. Data in B and C are representative of two independent experiments.

Inhibition of PKC or ERK Activity—PKC activity was inhibited in IEC-18 cells using either 5 µM BIM I (an inhibitor of all members of the PKC family (27)) or 0.25–1.0 µM Gö6976 (an inhibitor of the Ca²⁺-dependent PKC isozymes , I, II, and (28), i.e. only PKC in IEC-18 cells). Inhibition of ERK signaling was achieved using either 10 µM U0126 or 50 µM PD098059 (inhibitors that block the ability of MEK1 and MEK2 to phosphorylate/activate downstream targets (29–31)). Cells were treated with inhibitors or vehicle at various times either prior to (30 min), at the same time, or after (30, 45, 60, 75, 90, 105, 120, 135, and/or 150 min) the addition of PKC agonists and were maintained in the medium for the duration of PKC agonist treatment.

Silencing of PKC and PKC Using RNA Interference (RNA_i) Technology—The target sequences for rat PKC and PKC were 5'-AAGATTATCGGCCGCTGCACT-3' and 5'-AAGTGCGCTGGGCTAAAGAAA-3', respectively (32). High pressure liquid chromatography-purified and annealed double-stranded short interfering RNA sequences with d(TT) overhangs at the 3' end (siRNAs) were obtained from Qiagen, Inc. siRNAs were transfected into IEC-18 cells at 30% confluence using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. siRNA resuspension buffer was used in place of siRNA for control transfections. Forty two hours after transfection, cells were treated with 100 nM PMA or vehicle (EtOH) for 6 h and harvested for flow cytometric analysis. Selective silencing of the appropriate PKC isozyme was confirmed by subjecting parallel whole cell lysates to anti-PKC , anti-PKC , and anti-PKC immunoblot analysis.

Preparation of Whole Cell Lysates and Western Blot Analysis—For preparation of whole cell lysates, cells were solubilized in boiling SDS lysis buffer (10 mM Tris, pH 7.4, 1% SDS). Cellular DNA was sheared by passing the lysate through a 27-gauge needle, and extracts were cleared by centrifugation (10 min, 12,000 x g) and boiled in Laemmli sample buffer (33). SDS-PAGE and Western blot analysis were performed as described previously (5), using either 10% (PKC isozymes, MKP-1), 15% (Raf-1), or 20% (phospho-ERK1/2, total ERK1/2, Ras, cyclin D1, and p21^Waf1/Cip1) SDS-polyacrylamide minigels. Blots were routinely stained with 0.1% Fast Green (Sigma) immediately after transfer to ensure equal loading and even transfer. Primary antibody dilutions were as follows: 1:500 for MKP-1, 1:1000 for PKC , 1:1500 for Raf-1, 1:2000 for PKC , PKC , phospho-ERK1/2, cyclin D1, and p21^Waf1/Cip1, and 1:10,000 for total ERK1/2. Secondary antibodies were used at 1:2000.

Subcellular Fractionation—IEC-18 cells were partitioned into soluble (cytosolic), membrane, and cytoskeletal fractions as described previously (24, 26). Briefly, digitonin (0.5 mg/ml)-soluble (cytosolic) and digitonin-insoluble (particulate) fractions were separated by ultracentrifugation at 100,000 x g for 40 min at 4 °C. Cytosolic protein in the supernatant was precipitated with 10% trichloroacetic acid for 10 min on ice, pelleted, washed in acetone, solubilized in 100 mM NaOH, and neutralized by the addition of 100 mM HCl. Cellular membranes were extracted from the particulate pellet with 1% Triton X-100. The membrane sample was cleared by centrifugation at 10,000 x g for 30 min at 4 °C. To obtain the cytoskeletal fraction, the Triton X-100-insoluble pellet was resuspended in digitonin lysis buffer containing 0.5% SDS and protease/phosphatase inhibitors, briefly probe-sonicated, and centrifuged at 10,000 x g for 30 min at 4 °C. Soluble, membrane, and cytoskeletal fractions were boiled in Laemmli sample buffer (33) for 5 min before being subjected to SDS-PAGE and immunoblot analysis.

Ras Activation Assays—The activation state of Ras was determined using the EZ-Detect Ras Activation kit from Pierce, and data were confirmed using the Ras Activation Assay kit from Upstate Biotechnology, Inc. These kits use a GST fusion protein containing the Ras-binding domain (RBD) of Raf (GST-Raf-RBD) to pull down active GTP-bound Ras. The pulled down active Ras is detected by anti-Ras immunoblotting. In vitro GTPS or GDP treatments of lysates were performed to generate positive and negative controls for the pull-down procedures, according to the manufacturer's instructions.

Raf-1 Activity Assay—Raf-1 kinase activity was measured using a Raf-1 Immunoprecipitation-Kinase Cascade Assay kit from Upstate Biotechnology, Inc. This kit determines Raf-1 activity through a cascade reaction that uses MEK activation and subsequent ERK phosphorylation as an end point. The assay was performed according to the manufacturer's instructions. Briefly, Raf-1 was immunoprecipitated from IEC-18 cell lysates and incubated with MEK1 and ERK2 in the presence of ATP. Soluble products of this reaction were used to phosphorylate myelin basic protein substrate in the presence of [-³²P]ATP. Samples were spotted onto phosphocellulose squares, and the incorporated radioactivity was measured using a Beckman LS6500 multipurpose scintillation counter.

Immunofluorescence Staining of Phospho-ERK in IEC-18 Cells— Cells were grown on glass coverslips and treated with PMA, Bryo, or 10% serum (double the normal content in growth medium) for various times. Coverslips were then washed in PBS, fixed in methanol/acetone (3:1) at -20 °C for 20 min, and air-dried. The cells were then incubated with anti-phospho-ERK1/2 mouse monoclonal antibody (1:30) in PBS containing 0.2% Triton X-100 (PBS/Triton) for1hat room temperature. Following washes in PBS/Triton, cells were incubated with TRITC-conjugated goat anti-mouse IgG (1:100) for 30 min. The coverslips were then washed in PBS, mounted with Aquamount (Polysciences, Inc., Warrington, PA), and viewed with a Zeiss epifluorescence microscope (OberKochen, Germany).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Previous studies in this laboratory have demonstrated that PKC activation in IEC-18 nontransformed intestinal crypt cells triggers hallmark events of cell cycle withdrawal into G₀, including rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and coordinated alterations in the expression and phosphorylation state of pocket proteins (see Refs. 5 and 26). Treatment of IEC-18 cells with 100 nM PMA activates PKC , , and (the only phorbol ester-responsive PKC isozymes expressed in these cells (see Figs. 2 and 5) and results in transient cell cycle blockade (26) (Fig. 1A). Reversal of growth arrest at 12 h after addition of PMA correlates with depletion of these phorbol ester-responsive PKC isozymes (cf. Figs. 1A and 5D and see Refs. 5 and 26). As confirmed in Fig. 1B, PMA-induced inhibition of IEC-18 cell cycle progression is PKC-dependent; consistent with published data using PKC-depleted cells (26), inhibition of PKC activity using the general PKC inhibitor BIM I (5 µM) blocked PMA-induced cell cycle arrest. Treatment with BIM I also inhibited PMA-induced down-regulation of cyclin D1 and induction of p21^Waf1/Cip1, verifying the PKC dependence of these key consequences of PMA treatment in IEC-18 cells (Fig. 1C). Notably, selective inhibition of PKC (the only Ca²⁺-dependent PKC isozyme in IEC-18 cells) by Gö6976 (28) also abrogated PMA-induced cell cycle blockade, providing the first evidence of a requisite role for PKC in phorbol ester-induced IEC-18 intestinal epithelial cell cycle withdrawal (Fig. 1D). Consistent with this finding, knockdown of PKC or PKC using RNA_i technology failed to prevent PMA-induced cell cycle blockade in this system (Fig. 1E).

View larger version (19K): [in this window] [in a new window]   FIG. 5. Bryo is capable of producing PKC-dependent growth arrest in IEC-18 cells, although the effect is more transient than that produced by PMA. A, Bryo produces PKC/PKC -dependent inhibition of cell cycle progression in IEC-18 cells. Asynchronously growing IEC-18 cells were treated with 100 nM Bryo for6hinthe presence or absence of 5 µM BIMIor1 µM Gö6976 (Gö) as indicated. DNA content was determined by flow cytometric analysis. C indicates control cells. B, both PMA and Bryo transiently inhibit S phase progression in IEC-18 cells; Bryo-induced growth arrest is shorter-lived than that produced by PMA. i, asynchronously growing IEC-18 cells were treated with 100 nM Bryo or 100 nM PMA for the indicated times and subjected to flow cytometric analysis. ii, IEC-18 cells synchronized in G0/G1 by serum starvation for 72 h were released from growth arrest by addition of complete growth medium (time 0) and treated with 100 nM PMA or 100 nM Bryo 8 h later (in mid-G1); cell cycle distribution was determined at 16, 18, and 20 h after serum stimulation (i.e. 8, 10, and 12 h of PKC agonist treatment). C, Bryo induces membrane/cytoskeletal translocation/activation of PKC , , and in IEC-18 cells, paralleling the effects of PMA. IEC-18 cells were treated with 100 nM PMA (P) or 100 nM Bryo (B) for 15 min. Cytosolic, membrane, and cytoskeletal (CSK) fractions were prepared and subjected to immunoblot analysis using isozyme-specific antibodies for PKC , , and . Note the slower membrane translocation of PKC in Bryo-treated cells. Lane C, untreated cells. D, Bryo induces more rapid down-regulation/desensitization of PKC than PMA. i, asynchronously growing IEC-18 cells were treated with 100 nM PMA (P) or 100 nM Bryo (B) for the indicated times, and cell lysates were subjected to immunoblot analysis for PKC , , and expression. Although the slower migrating, activated form of PKC (top arrow) remains detectable following9hofPMA treatment, it is no longer evident by 4–6 h in Bryo-treated cells. Instead, Bryo treatment results in marked accumulation of unphosphorylated, inactive PKC (bottom arrow); this inactive PKC is evident by 30 min (see ii). PKC is protected from down-regulation in Bryo-treated cells for longer than 12 h (see iii) but is markedly down-regulated by6hof treatment with PMA. PKC appears to be down-regulated slightly more rapidly in PMA-treated cells. Data are representative of at least three independent experiments.

View larger version (18K): [in this window] [in a new window]   FIG. 1. PKC activation inhibits cell cycle progression in IEC-18 nontransformed intestinal crypt cells. A, the PKC agonist PMA induces cell cycle arrest in IEC-18 cells. Subconfluent cultures of asynchronously growing IEC-18 cells were treated with 100 nM PMA for the indicated times, and cell cycle distribution was determined by flow cytometric analysis of DNA content in propidium iodide-stained cells. PMA-induced G0/G1 arrest was evident by 6 h and maintained for 12 h of treatment. C indicates control (IEC-18 cells treated with vehicle alone). B, PMA-induced IEC-18 cell cycle arrest is PKC-dependent. IEC-18 cells were pretreated with the general PKC inhibitor BIM I (5 µM) for 30 min before addition of 100 nM PMA for 6 h as indicated. DNA content/cell cycle distribution was assessed by flow cytometric analysis. C, PMA-induced cyclin D1 down-regulation and p21Waf1/Cip1 induction are PKC-dependent. i, IEC-18 cells were treated with 100 nM PMA for 2 h, and cyclin D1 and p21Waf1/Cip1 expression levels were determined by immunoblot analysis. PMA triggers down-regulation of cyclin D1 and induction of p21Waf1/Cip1 in these cells. Lane C indicates cells treated with vehicle alone. ii, IEC-18 cells were pretreated with 5 µM BIM I (or vehicle) for 30 min followed by addition of 100 nM PMA for 2 h as indicated. Cyclin D1 and p21Waf1/Cip1 expression were determined by Western blot analysis. D, PMA-induced IEC-18 cell cycle arrest is abrogated by inhibition of PKC . IEC-18 cells were pretreated with the PKC -selective inhibitor Gö6976 (0.25 or 1.0 µM) for 30 min followed by addition of 100 nM PMA for 6 h as indicated. DNA content/cell cycle distribution was assessed by flow cytometric analysis. Note that Gö6976 alone did not significantly affect G1 S phase progression of IEC-18 cells over 6 h of treatment (see Fig. 5A). E, silencing of PKC (i) or PKC (ii) expression using siRNA does not inhibit PMA-induced growth arrest in IEC-18 cells. PKC isozyme expression was specifically silenced in IEC-18 cells using siRNA as described under "Experimental Procedures." Control and siRNA-treated cells were exposed to vehicle (-) or 100 nM PMA (+) for 6 h and subjected to flow cytometric analysis. Selective knockdown of PKC or PKC (>85%) in siRNA-treated cells was confirmed by immunoblot analysis of the expression of the phorbol ester-responsive isozymes expressed in IEC-18 cells (i.e. PKC , , and ). Data in A–D and in E are representative of three and two independent experiments, respectively.

In order to determine whether the ERK signaling cascade is required for PKC-mediated inhibition of IEC-18 cell cycle progression, cells were treated with 100 nM PMA in the presence of the MEK inhibitor U0126 (10 µM), and effects on cell cycle distribution were evaluated by flow cytometric analysis. Given the known role of ERK signaling in cellular proliferation, the modest inhibition of cell cycle progression produced by 10 µM U0126 in control cells was not unexpected; similar findings have been reported in other cell types (e.g. Ref. 22). It is noteworthy, however, that inhibition of MEK activity consistently abrogated the complete G₁ S phase blockade produced by PMA in IEC-18 cells (Fig. 3A). MEK inhibition by U0126 (Fig. 3B) or PD098059 (data not shown) also inhibited PMA-induced down-regulation of cyclin D1 and induction of p21^Waf1/Cip1, strongly pointing to the requirement for ERK signaling in PKC-dependent negative growth regulation in IEC-18 cells.

View larger version (23K): [in this window] [in a new window]   FIG. 3. The ERK pathway is required for PKC-mediated IEC-18 cell cycle arrest, cyclin D1 down-regulation, and p21Waf1/Cip1 induction in IEC-18 cells. A, inhibition of MEK activity blocks PMA-induced cell cycle arrest. IEC-18 cells were treated with 100 nM PMA for 6 h in the presence or absence of 10 µM U0126. DNA content/cell cycle distribution was determined by flow cytometric analysis. B, MEK inhibition blocks PMA-induced cyclin D1 down-regulation and p21Waf1/Cip1 induction in IEC-18 cells. IEC-18 cells were treated with 100 nM PMA for 2 h in the presence or absence of 10 µM U0126, and cyclin D1 and p21Waf1/Cip1 expression levels were assessed by immunoblot analysis. Data are representative of at least three independent experiments.

View larger version (27K): [in this window] [in a new window]   FIG. 4. PMA-induced ERK activation is stronger and more sustained than that induced by serum in IEC-18 cells, and PMA and serum differentially regulate cyclin D1 and p21Waf1/Cip1 expression in these cells. IEC-18 cells were treated with 100 nM PMA or with a serum boost (10% FBS, double the normal content in growth medium) for the indicated times. Lane C, control cells; P, PMA; S, serum boost, 10% FBS. A, ERK1/2 phosphorylation/activation and total ERK1/2 expression were assessed by immunoblot analysis using anti-phospho-ERK1/2 antibody or anti-total ERK1/2 antibody, respectively. Lane C, vehicle-treated cells collected at 5 min and 6 h as indicated. B, cyclin D1 and p21Waf1/Cip1 expression levels were determined by immunoblot analysis. C, PMA-induced effects on ERK signaling and cell cycle regulatory molecules are dominant over the effects of proliferative signals in IEC-18 cells. IEC-18 cells were simultaneously treated with 100 nM PMA and a serum boost (10% FBS) for the indicated times. Levels of phospho-ERK1/2, total ERK1/2, cyclin D1, and p21Waf1/Cip1 were determined by immunoblot analysis. Data in A and B and in C are representative of three and two independent experiments, respectively.

View larger version (21K): [in this window] [in a new window]   FIG. 6. Bryo-induced IEC-18 cell cycle arrest is ERK-dependent but is characterized by more transient activation of ERK1/2 compared with that induced by PMA. A, inhibition of MEK activity blocks Bryo-induced cell cycle arrest. IEC-18 cells were treated with 100 nM Bryo for6hinthe presence or absence of 10 µM U0126. DNA content/cell cycle distribution was determined by flow cytometric analysis. B, i and ii, Bryo-induced ERK1/2 activation is more transient than that produced by PMA. IEC-18 cells were treated with 100 nM PMA (P) or 100 nM Bryo (B) for the indicated times, and cell lysates were subjected to immunoblot analysis using antibodies specific for phospho-ERK1/2 or total ERK1/2. Lane C, vehicle-treated cells. Control samples shown on the left and right ends of the panel in B, ii, were collected at 5 min and 16 h, respectively. C, the effects of Bryo on expression of cyclin D1 and p21Waf1/Cip1 in IEC-18 cells are weaker than those produced by PMA. Asynchronously growing IEC-18 cells were treated with 100 nM PMA or 100 nM Bryo for the indicated times, and expression of cyclin D1 and p21Waf1/Cip1 was determined by immunoblot analysis. The values below the blots represent the relative levels of cyclin D1 and p21Waf1/Cip1 determined by densitometry and normalized to levels of the loading control (a nonspecific band consistently seen with the p21Waf1/Cip1 antibody that was not affected by PKC agonist treatments; similar values were obtained using total ERK as a loading control, not shown). Data are representative of at least four independent experiments.

To determine the effects of simultaneous treatment with PMA and increased growth factors, IEC-18 cells were incubated with 100 nM PMA and an additional 5% serum (serum boost) for various times. As shown in Fig. 4C, the effects of PMA were dominant over proliferative signals by serum/growth factors when both stimuli were administered simultaneously. The combined treatment resulted in sustained ERK activation for longer than 6 h, down-regulation of cyclin D1, and significant induction of p21^Waf1/Cip1 (although these effects may have been slightly attenuated by the presence of proliferative signals).

In summary, PMA-induced PKC activation in IEC-18 cells resulted in strong and sustained activation of the ERK pathway, down-regulation of cyclin D1, and induction of p21^Waf1/Cip1, whereas serum stimulation produced transient and more modest ERK1/2 activation, transient cyclin D1 induction, and no change in p21^Waf1/Cip1 expression. In addition, PKC-mediated negative growth regulatory signals involving the ERK cascade appear to be dominant over serum-induced proliferative ERK1/2 signaling pathways in this system.

The Duration of PKC-mediated Cell Cycle Arrest Is Linked to the Intensity/Duration of ERK1/2 Signaling in IEC-18 Cells—To characterize further the involvement of ERK signaling in PKC-mediated intestinal epithelial growth arrest, the cell cycle-specific effects of the macrocyclic lactone Bryo were examined and compared with those of PMA. Although a potent activator of PKC, studies in a variety of systems have shown that Bryo often produces only a subset of the typical phorbol ester responses and antagonizes those phorbol ester-mediated effects it cannot itself induce (35). Flow cytometric analysis revealed that, like PMA, Bryo is able to negatively regulate IEC-18 cell cycle progression (Fig. 5). Treatment of asynchronously growing IEC-18 cells with 100 nM Bryo inhibited G₁ S phase progression by 6 h (Fig. 5A); this effect was PKC-dependent, as confirmed by the ability of the general PKC inhibitor BIM I to block Bryo-induced cell cycle arrest. The PKC -selective inhibitor Gö6976 also inhibited the cell cycle effects of Bryo in IEC-18 cells, indicating that PKC plays a key role in both PMA- and Bryo-induced negative growth regulatory effects in this system. Notably, comparison of the extent and duration of the effects of these agents revealed that the cell cycle arrest produced by Bryo is less complete and reverses more rapidly than that induced by PMA. In asynchronously growing cells, cell cycle arrest was evident by 5 h in both Bryo- and PMA-treated cells; however, whereas PMA-induced cell cycle arrest was maintained at 9 h (not reversing until 12 h, see Fig. 1A), the effect produced by Bryo had begun to reverse by 8 h of treatment (Fig. 5B, i). For analysis of the effects of these agents in synchronized cells, IEC-18 cells were arrested in G₀/G₁ by serum deprivation and serum-stimulated for 8 h before addition of 100 nM PMA or 100 nM Bryo for the indicated times. Flow cytometric analysis of untreated cells revealed that cells progress into S phase 15 h after serum stimulation (data not shown). As shown in Fig. 5B, ii, treatment of IEC-18 cells with PMA in mid-G₁ (8 h after serum stimulation) delayed S phase progression by 5–6 h; the release kinetics were similar to those seen in asynchronously growing cells with growth arrest maintained until 12 h after addition of phorbol ester (see Fig. 1). Bryo, on the other hand, produced a more short-lived delay in S phase entry lasting 3 h (9 h from addition of the agent).

To gain insight into the basis for the differential cell cycle effects of Bryo and PMA in IEC-18 cells, the ability of these agents to induce and sustain activation of PKC , , and in these cells was compared. PKC isozyme translocation (i.e. association with the particulate subcellular fraction) and down-regulation were used as measures of agonist-induced isozyme-specific effects (36, 37). As shown in Fig. 5C, Bryo promoted translocation of all three PKC isozymes to the membrane/cytoskeletal fractions by 15 min, paralleling the effects of PMA (26). Notably, however, membrane translocation of PKC was slower in Bryo-treated cells. Comparison of PKC isozyme expression and phosphorylation/activation state at later times after treatment revealed marked differences in the effects of these agents (Fig. 5D). Bryo produced significantly more rapid down-regulation/desensitization of PKC than PMA (Fig. 5D, i and ii), as indicated by more rapid disappearance of the fully phosphorylated slower migrating form of the enzyme (activated PKC ; top arrow) and accumulation of the higher mobility nonphosphorylated protein (bottom arrow) that is inactive (38, 39). This inactive form, which appeared following extensive caveolar internalization of the enzyme (39), was evident as early as 30 min after addition of Bryo (Fig. 5D, ii). Furthermore, whereas the slower migrating, activated form of PKC remained detectable following 9 h of PMA treatment, it was no longer evident by 4–6 h in Bryo-treated cells. In contrast, activated PKC was protected from down-regulation by Bryo (as observed in other systems, where the protected enzyme was confirmed to retain kinase activity (40)). As shown in Fig. 5D, iii, levels of PKC had changed little following 12 h of Bryo treatment, a time when Bryo-induced growth arrest had reversed and cells had progressed into S phase (Fig. 5B). Immunoblot analysis further demonstrated little difference in the effects of PMA and Bryo on PKC expression over 9 h of treatment (Fig. 5D, i). Thus, the duration of PKC activation, but not that of PKC or , correlated with the duration of cell cycle arrest produced by these agents, providing further support for a major role of PKC signaling in PKC agonist-induced IEC-18 cell cycle arrest. Taken together, the data presented above demonstrated that both PMA and Bryo can induce PKC -dependent cell cycle blockade in IEC-18 cells. However, differences are evident in the duration of PKC activation and cell cycle arrest produced by these agents, with PMA inducing more sustained PKC activity and more prolonged growth inhibition (3 h longer) than Bryo in these cells.

To examine the role of ERK signaling in Bryo-induced cell cycle arrest, asynchronously growing IEC-18 cells were treated with 100 nM Bryo in the presence or absence of the MEK inhibitor U0126. As seen in PMA-treated cells, 10 µM U0126 abrogated the inhibition of S phase progression produced by Bryo in these cells (Fig. 6A). Thus, the ERK signaling cascade appears to be required for Bryo-induced cell cycle inhibition in IEC-18 cells. Based on these findings, we next compared the extent and duration of ERK activation induced by PMA and Bryo by using Western blot analysis. As shown in Fig. 6B, i, PMA and Bryo strongly activated ERK1/2 by 5 min of treatment, although the levels of ERK1/2 activation were consistently slightly lower in Bryo-treated cells. While a noticeable decline in phospho-ERK was evident by 45 min in cells treated with Bryo (data not shown and Fig. 6B), cells treated with PMA showed a sustained activation of ERK lasting longer than 6 h (Fig. 6B, ii; also see Fig. 4). Bryo and PMA also differed in their effects on the expression of cyclin D1 and p21^Waf1/Cip1 in IEC-18 cells. Bryo induced a less marked down-regulation of cyclin D1, and levels of p21^Waf1/Cip1, although elevated, did not reach those produced by PMA in these cells (Fig. 6C). The duration of IEC-18 cell cycle arrest induced by PMA and Bryo treatment, therefore, correlated with the extent and duration of ERK1/2 activity produced by these agents which, in turn, was reflected in the extent of cyclin D1 down-regulation and p21^Waf1/Cip1 induction in this system.

The Duration of PKC and ERK1/2 Activation Determines the Duration of PMA-induced Growth Arrest in IEC-18 Cells— Comparison of the effects of PMA and Bryo indicated that the duration of cell cycle arrest produced by these agents was determined by their ability to sustain PKC and ERK1/2 activity in IEC-18 cells. To investigate this idea further, selective inhibitors were used to examine the effect of manipulating the duration of PKC and ERK1/2 stimulation on PMA-induced growth arrest in this system. IEC-18 cells were treated with 100 nM PMA (or vehicle) for 6 or 9 h, and Gö6976 (0.5 µM) or U0126 (10 µM) was added at various times to inhibit PKC and ERK1/2 signaling, respectively. The inhibitors were added either 30 min prior, at the same time, or at different times after the addition of PMA (as indicated) and were left in the medium for the remainder of the 6 or 9 h PMA incubation. Cells were then subjected to flow cytometric analysis, and the degree of PMA-induced growth arrest was evaluated by changes in the percent of cells in S phase. As shown in Fig. 7, to obtain PMA-induced growth arrest at 6 h, less than 30 min of PKC and ERK1/2 activation appears to be required. Addition of the inhibitors at 30 or 45 min provided partial protection from the growth inhibitory effects of PMA, whereas no protection was observed when the inhibitors were added at 60–75 min. In contrast, maintenance of PMA-induced cell cycle blockade at 9 h required continued PKC and ERK1/2 signaling for 60–75 min. Addition of Gö6976 or U0126 up to 60 min after treatment with PMA blocked the ability of the PKC agonist to reduce the percentage of cells in S phase at 9 h; thus, activation of PKC for up to 1 h provided insufficient signal for maintenance of growth arrest at this time point. However, when the inhibitors were added at times later than 60 min, a progressive loss of cells in S phase was noted, with addition at times after 120 min offering no protection from the cell cycle effects of PMA. Thus, maintenance of growth arrest for 9 h following addition of PMA required more prolonged activation of PKC and ERK1/2 than required to sustain growth arrest for 6 h. These findings are consistent with the inability of Bryo, which promotes rapid desensitization of PKC and a marked decline in ERK1/2 signaling by 45 min to 1 h of treatment (see Figs. 5D and 6B), to sustain growth arrest beyond 8–9 h in IEC-18 cells. Taken together, the data clearly demonstrate that the duration of both PKC and ERK1/2 signaling plays an important role in determining the duration of PKC agonist-induced growth arrest in IEC-18 cells.

View larger version (25K): [in this window] [in a new window]   FIG. 7. The duration of PKC and ERK1/2 activation determines the duration of PMA-induced growth arrest in IEC-18 cells. IEC-18 cells were treated with 100 nM PMA or vehicle for 6 or 9 h and Gö6976 (0.5 µM) (A) or U0126 (10 µM) (B) were added either 30 min prior, at the same time, or at various times after (30–150 min) the addition of PMA as indicated. Inhibitors were left in the medium for the remainder of the 6- or 9-h PMA incubation. The arrows indicate time of addition of PMA. Cells were harvested for flow cytometric analysis, and the extent of cell cycle arrest was assessed by determining the percentage of cells in S phase. The broken line in A indicates the % S phase cells in the presence of Gö6976 (0.5 µM) (30%); at the concentration used, Gö6976 had little effect on % S phase cells. In contrast, treatment with U0126 (10 µM) alone reduced the proportion of S phase cells to 22% (B). A 6-h treatment with PMA alone usually reduced the proportion of S phase cells to 10% (not shown). See text for details. Data are representative of more than three independent experiments.

We next examined the ability of PMA and Bryo to activate Raf-1 in IEC-18 cells. Raf-1 is a serine-threonine kinase responsible for activation of MEK1/2. In many systems, Raf-1 activation appears to involve Ras-mediated recruitment of Raf-1 to the plasma membrane, as well as interaction with additional membrane-associated kinases and phospholipids (50). The activity of Raf-1 was determined by measuring its ability to activate MEK and ERK1/2 in a Raf-1 immunoprecipitation kinase cascade assay (Upstate Biotechnology, Inc.). Both PMA and Bryo stimulated Raf-1 kinase activity by 5 min of treatment (Fig. 9A). Notably, although PMA-induced Raf-1 activity was sustained at 75 min, levels in Bryo-treated cells had significantly decreased by this time. Bryo-induced activation of Ras, Raf-1, and ERK1/2 was, therefore, more transient than that induced by PMA in IEC-18 cells, consistent with the more short-lived cell growth arrest produced by this agent.

Treatment of PKC /-depleted cells with PMA also resulted in increased Raf-1 activity (Fig. 9B), demonstrating that PKC is sufficient to mediate PKC agonist-induced Raf-1 activation in IEC-18 cells. The PKC dependence of PKC agonist-induced effects on Raf-1 was further investigated by taking advantage of a mobility shift in Raf-1 protein associated with activation of the kinase (51). Raf-1 activation has been reported to be accompanied by increased Raf-1 phosphorylation, which can be detected by decreased mobility of the protein on SDS-polyacrylamide gels (51). Anti-Raf-1 immunoblot analysis revealed that both PMA and Bryo induce a slower migrating form of Raf-1 in IEC-18 cells (Fig. 9C; data not shown for Bryo-treated cells). This form of Raf-1 was also produced in response to DiC₈ treatment (data not shown). Depletion of PKC , , and from IEC-18 cells by prolonged treatment with 1 µM PDBu abolished the PMA-induced mobility shift in Raf-1, demonstrating that PKC signaling is required for the effect (Fig. 9C). Consistent with data from Raf-1 kinase assays, depletion of PKC and from these cells failed to affect PMA-induced post-translational modification of Raf-1, further indicating that PKC is sufficient to induce phosphorylation/activation of Raf-1 in response to PMA (Fig. 9C).

PMA- and Bryo-induced ERK Activity Accumulates in the Cytoplasm and Nucleus, whereas Serum-induced Phospho-ERK Is Detected Only in the Cytoplasm—To compare the subcellular localization of phospho-ERK1/2 induced by growth factors, PMA, or Bryo, IEC-18 cells grown on glass coverslips were given a serum boost or exposed to PKC agonists for various times. Cells were then fixed and processed for immunofluorescence analysis of phospho-ERK1/2 distribution as described under "Experimental Procedures." As shown in Fig. 10A, untreated cells exhibited low levels of phospho-ERK staining, predominantly in the cytoplasm. Following addition of serum, there was a marked increase in cytoplasmic phospho-ERK fluorescence; this increase was detectable by 20 s of treatment (data not shown) and maintained for 30–45 min (Fig. 10B). Nuclear localization of phospho-ERK1/2 was not detected at any time following addition of growth factors to the cells. PMA and Bryo, on the other hand, produced a more complex pattern of phospho-ERK1/2 compartmentalization (Fig. 10C and Table I). Whereas both agents first promoted cytoplasmic accumulation of activated ERK, by 2–5 min (Fig. 10C, a–d) nuclear phospho-ERK1/2 staining became evident in many of the cells; importantly, the effects of Bryo were significantly slower than those of PMA, consistent with the slower membrane translocation of PKC induced by this agent (see Fig. 5C and Ref. 39). The overall intensity of phospho-ERK staining was also slightly lower in Bryo-treated cells. By 15–30 min of treatment with either PKC agonist (Fig. 10C, e and f), the majority of cells exhibited nuclear accumulation of activated ERK1/2, although the number of cells showing predominantly nuclear staining was considerably higher in response to PMA treatment (Table I). By 45 min to 1 h, many cells exposed to either PMA or Bryo showed uniform staining in both the cytoplasm and nucleus, whereas others began to exhibit predominantly cytoplasmic fluorescence (Fig. 10C, g and h; Table I). As shown in Fig. 10C, h, levels of ERK fluorescence had begun to decline significantly in Bryo-treated cells by this time, and little phospho-ERK1/2 staining could be detected in these cells by 2 h (Fig. 10C, j). In contrast, PMA-treated cells still exhibited nuclear and cytoplasmic staining at 2 h (Fig. 10C, i), and high levels of cytoplasmic, but no nuclear, phospho-ERK were still detectable at 6 h (k). Thus, cytoplasmic ERK activation by PMA or Bryo is followed by transient nuclear accumulation of the active kinase; however, the extent and duration of phospho-ERK nuclear accumulation are significantly lower in response to Bryo treatment. PMA-treated cells subsequently exhibit sustained ERK activity in the cytoplasm, lasting longer than 6 h.

View larger version (44K): [in this window] [in a new window]   FIG. 10. Immunofluorescence localization of phospho-ERK in IEC-18 cells. IEC-18 cells were given a serum boost (10% FBS, double the normal content in growth medium) or treated with 100 nM PMA or 100 nM Bryo for the indicated times. Cells were fixed in methanol/acetone and immunostained for phospho-ERK as described under "Experimental Procedures." A, untreated cells exhibit low levels of cytoplasmic phospho-ERK. IEC-18 cells were treated with vehicle for 5 min (a) or 6 h (b). B, growth factors transiently induce ERK activity in the cytoplasm of IEC-18 cells. Cells exposed to a serum boost for 5 min (a) or 1 h (b). C, PMA and Bryo promote transient nuclear accumulation of phospho-ERK in IEC-18 cells; prolonged cytoplasmic phospho-ERK activity is seen only in PMA-treated cells. IEC-18 cells were treated with PMA (a, c, e, g, i, and k) or Bryo (b, d, f, h, j, and l) for 2 min (a and b), 5 min (c and d), 15 min (e and f), 1 h (g and h), 2 h (i and j), or 6 h (k and l). Note that by 2 min many PMA- and some Bryo-treated cells are beginning to accumulate phospho-ERK in the nucleus (for example, see arrowhead in a). Although the majority of PMA- or Bryo-treated cells shows nuclear accumulation of phospho-ERK by 15 min, the proportion of cells with predominant nuclear staining was significantly higher in cells exposed to PMA (see Table I). By 1 h of PKC agonist treatment, some cells show uniform phospho-ERK staining throughout the nucleus and cytoplasm (closed arrows in g and h), whereas others show predominantly cytoplasmic staining (open arrow in g). A marked decline in Bryo-induced phospho-ERK staining is seen by this time (also see Table I). See text for details. Magnification bars, 10 µm. D, PMA and Bryo induce MKP-1 expression with identical kinetics in IEC-18 cells. Cells were treated with 100 nM Bryo (B) or 100 nM PMA (P) for various times as indicated, and cell lysates were analyzed for MKP-1 expression by Western blotting. Lane C, vehicle-treated control cells. Data in A–D are representative of three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The current report demonstrates that the ERK signaling cascade plays a requisite role in linking PKC/PKC activity to regulation of the cell cycle machinery in IEC-18 nontransformed intestinal epithelial cells. We have reported previously (5, 26) that sustained activation of PKC signaling is sufficient to induce and maintain a program of cell cycle exit in these cells, although a role for PKC and/or in this effect was not excluded in those studies. By using the inhibitor Gö6976 (28) to selectively block PKC activity in IEC-18 cells, we now provide the first evidence for a requisite role of PKC signaling in mediating PKC agonist (PMA or Bryo)-induced cell cycle blockade in intestinal epithelial