September 2003
Volume 44, Issue 9
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Cornea  |   September 2003
Regulatory Role of cAMP on Expression of Cdk4 and p27Kip1 by Inhibiting Phosphatidylinositol 3-kinase in Corneal Endothelial Cells
Author Affiliations
  • Hyung Taek Lee
    Doheny Eye Institute, Los Angeles, California.
  • EunDuck P. Kay
    From the Department of Ophthalmology, University of Southern California, Los Angeles, California; and the
    Doheny Eye Institute, Los Angeles, California.
Investigative Ophthalmology & Visual Science September 2003, Vol.44, 3816-3825. doi:https://doi.org/10.1167/iovs.03-0147
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      Hyung Taek Lee, EunDuck P. Kay; Regulatory Role of cAMP on Expression of Cdk4 and p27Kip1 by Inhibiting Phosphatidylinositol 3-kinase in Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(9):3816-3825. https://doi.org/10.1167/iovs.03-0147.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Fibroblast growth factor (FGF)-2 is a potent mitogen of corneal endothelial cells (CECs). Results in an earlier study showed that FGF-2 activates phosphatidylinositol (PI) 3-kinase to stimulate the cell cycle machinery. The current study was designed to determine whether adenosine 3′,5′-monophosphate (cAMP) antagonizes FGF-2 by inhibiting PI 3-kinase/Akt pathways, thus leading to regulation of cyclin-dependent kinase 4 (Cdk4) and p27Kip1 (p27) expression.

methods. Cell proliferation was assayed by counting cells. Subcellular localization of proteins was determined by immunofluorescent staining and expression of Cdk4, p27, PI 3-kinase, Akt, and β-actin was analyzed by immunoblot analysis. PI 3-kinase activity was determined by measuring production of phosphatidylinositol-3-phosphate.

results. 8-Bromoadenosine cAMP (8-Br-cAMP), a diffusible cAMP analogue, inhibited the PI 3-kinase/Akt signaling pathways. The 8-Br-cAMP and PI 3-kinase inhibitor (LY294002) produced equivalent stimulation and inhibition, respectively, of p27 and Cdk4 protein levels. They also equally inhibited cell proliferation, nuclear translocation of Cdk4, and phosphorylation of p27. Negative regulation of PI 3-kinase by 8-Br-cAMP was mediated by a direct inhibition of PI 3-kinase activity, which subsequently blocked phosphorylation of Akt at both the Ser473 and Thr308 sites. In addition, 8-Br-cAMP promoted a rapid turnover of Akt protein, and 8-Br-cAMP markedly reduced the half-life of Cdk4 protein. This inhibitory activity of cAMP was not mediated by PKA, but 8-Br-cAMP inhibited membrane localization of the p85 regulatory subunit of PI 3-kinase.

conclusions. These data support the hypothesis that cAMP inhibits the proliferation of CECs, preventing them from entering the S phase by negatively regulating PI 3-kinase.

Corneal endothelium is a monolayer of differentiated cells located in the posterior portion of the cornea. The corneal endothelium is essential for maintaining corneal transparency, but its capacity for regeneration after injury is severely limited in humans and primates. 1 In response to certain pathologic conditions, corneal endothelial cells (CECs) in vivo respond by converting to fibroblast-like cells. These morphologically modulated cells simultaneously resume their proliferation ability and deposit a fibrillar extracellular matrix in the basement membrane environment. One clinical example of this process is the development of a retrocorneal fibrous membrane 2 3 that causes blindness by physically blocking vision. In our studies, 4 5 we have reported that fibroblast growth factor (FGF)-2 is the direct mediator for such endothelial mesenchymal transformation. In normal cornea, the 18-kDa FGF-2 isoform is a component of Descemet’s membrane that may be necessary for wound repair. 5 6 7 In additional studies, we have shown that, in response to FGF-2 stimulation, the mitogenic signaling pathway uses cytoskeleton-associated phosphoinositide-specific phospholipase C (PLC)-γ1 as a minor pathway 8 9 and phosphatidylinositol (PI) 3-kinase as a major pathway. 10 We have further demonstrated that PI 3-kinase stimulates cell proliferation by regulating cyclin-dependent kinase 4 (Cdk4) and p27Kip1 (p27) expression and by regulating the events that occur after synthesis, such as translocation of Cdk4 or phosphorylation of p27. 9 10  
Although FGF-2 is known to cause mitogenic activity under some pathologic conditions, human corneal endothelium in vivo remains arrested in the G1 phase of the cell cycle, 11 12 13 14 but the mechanism of this arrest at G1 is not known. Nevertheless, some researchers have proposed that TGF-β2 and cAMP, the two resident components in the aqueous humor 15 16 17 in which CECs constantly bathe, play key roles in maintaining CECs in G1 arrest in vivo and in vitro. 16 18 We have reported that 8-bromoadenosine cAMP (8-Br-cAMP), a nonhydrolysable but diffusible cAMP analogue, inhibits serum-mediated cell proliferation in CECs by upregulating p27; in addition, 8-Br-cAMP blocks phosphorylation of p27, which is a prerequisite for nuclear export of p27 for degradation. 18 Furthermore, numerous studies have reported that cAMP inhibits G1/S transition by downregulating Cdk2, Cdk4, cyclin D1, or cyclin D3 and/or upregulating p21 or p27. 19 20 21 22 23 24 Thus, cAMP directly inhibits cell cycle progression. 
Recent studies demonstrate that cAMP and its effector, cAMP-dependent protein kinase A (PKA), are implicated in a variety of cross talks between intracellular signaling pathways. 25 26 27 28 29 Although the antiproliferative action of cAMP has been well studied, largely in conjunction with hormone receptors linked to guanine nucleotide binding protein (G protein), adenylyl cyclase, and the activation of PKA, the growth-inhibitory effects of cAMP are believed to be at least partially mediated by cAMP-dependent inactivation of mitogen-activated protein kinase (MAPK) pathways, 30 31 32 33 PI 3-kinase/Akt pathways, 26 27 34 or Ras. 35 36 In the current study we used a unique cell system, in which a positive effector (FGF-2) and a negative effector (cAMP) are readily available in the immediate environment. It is crucial to understand how corneal endothelium maintains G1 arrest of the cell cycle under physiological conditions and when such a suppressed phenotype is challenged. In the present study, we sought to determine whether cAMP negatively regulates the PI 3-kinase signaling pathways in response to FGF-2 stimulation. We further investigated the mechanism by which cAMP antagonizes PI 3-kinase. 
Materials and Methods
FGF-2 was purchased from Intergen (Purchase, NY); radiochemicals from ICN (Irvine, CA); anti-p85 subunit of PI 3-kinase antibody from BD Biosciences (San Diego, CA); anti-Akt, anti-phosphorylated Akt (Ser473), and anti-phosphorylated Akt (Thr308) antibodies from Cell Signaling Technology (Beverly, MA); monoclonal antibodies against Cdk4, p27, Myc, and β-actin, LY294002, 8-Br-cAMP, cycloheximide, PD98059, and H89 from Sigma-Aldrich (St. Louis, MO); anti-phosphorylated p27 (Thr187) from Zymed Laboratories Inc. (South San Francisco, CA); fluorescein isothiocyanate (FITC)- and rhodamine-conjugated secondary antibodies from Chemicon (Temecula, CA); and biotinylated secondary antibodies from Vector Laboratories (Burlingame, CA). 
Cell Cultures
Rabbit eyes were purchased from Pel Freeze (Rogers, AR). Rabbit CECs were isolated and established as previously described. 37 Briefly, the Descemet’s membrane-corneal endothelium complex was treated with 0.2% collagenase and 0.05% hyaluronidase (Worthington Biochemical, Lakewood, NJ) for 90 minutes at 37°C. Cultured cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 50 μg/mL gentamicin (DMEM-10) in a 5% CO2 incubator. First-passage CECs were used for all experiments. For subculture, confluent cultures were treated with 0.05% trypsin and 5 mM EDTA in phosphate-buffered saline (PBS) for 5 minutes. We added heparin (10 μg/mL, Sigma-Aldrich) to cultures when cells were treated with FGF-2, because our previous study showed that CECs require supplemental heparin for FGF-2 activity to occur. 5 The following conditions were used in all experiments: When cells reached 60% to 70% confluence, they were placed in serum-free medium (DMEM-0) for 24 hours before treatment with the growth factor with or without inhibitors. To determine posttranslational regulation of Akt or Cdk4 expression, CECs were treated with FGF-2 (10 ng/mL) for 24 hours. Cells were then treated with cycloheximide (20 μM) in the absence of FGF-2 in serum-free medium (DMEM-0), with or without 8-Br-cAMP (1 mM), for 2, 4, or 8 hours. 
Transfection
The cDNA encoding human p85α was cloned into the epitope-tagged mammalian expression vector between the BglII and BamHI sites of the pCMV6-myc vector (BD Biosciences-Clontech, Palo Alto, CA). The pCMV6 p85-myc and the empty vector (pCMV6) were kind gifts from Lewis Cantley (Harvard Medical School, Boston, MA). For transfection, CECs (4 × 104/chamber) were plated on four-well chamber slides. When cells reached approximately 60% to 70% confluence, they were transiently transfected using 1 μg of the expression vector or empty vector, with a commercial system (Effectene; Qiagen, Valencia, CA) with slight modification of the protocols recommended by the manufacturer. Eight microliters of enhancer and 5 μL of transfection reagent were used to promote an optimum transfection efficiency. Eight hours after transfection, cells were further maintained in one of the following experimental conditions for 24 hours: DMEM-0, FGF-2 (10 ng/mL) in DMEM-0, or FGF-2 (10 ng/mL) with 8-Br-cAMP (1 mM) in DMEM-0. Cells transfected with either pCMV6 p85-myc or the empty vector were then analyzed for subcellular localization of the expressed fusion protein using immunocytochemical analysis. 
Cell Proliferation Assay
The serum-starved cells plated in 35-mm dishes were treated with FGF-2 in the presence or absence of inhibitors (8-Br-cAMP, LY294002, PD98059, or H89) under the conditions used for individual experiments. At the end of the incubation period, cells were treated with trypsin-EDTA. Cells were then stained with 0.03% trypan blue to mark the dead cells, and viable cells were counted using a hematocytometer. 
Protein Preparation and Protein Determination
Cells were washed with ice-cold PBS and then lysed with cell lysis buffer I (20 mM HEPES [pH 7.2], 10% glycerol, 10 mM Na3VO4, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.1 mM dithiothreitol, 1 μg/mL leupeptin, 1 μg/mL pepstatin, and 1% Triton X-100) on ice for 30 minutes. The lysate was sonicated, and the cell homogenates were centrifuged at 15,000g for 10 minutes. Protein concentration of the resultant supernatant was assessed with a Bradford reagent. 
Measurement of PI 3-kinase Enzyme Activity
The conditions for measuring PI 3-kinase enzyme activity have been described previously. 10 38 The serum-starved CECs were treated with FGF-2 for a designated period or dose. To study the inhibitory effect of LY294002 or 8-Br-cAMP on PI 3-kinase enzyme activity, the two inhibitors were added alone or together in the presence of FGF-2. After stimulation, cells were washed twice with ice-cold PBS and harvested by scraping into 300 μL of cold lysis buffer II (20 mM Tris-HCl [pH 7.4], 10 mM NaCl, 100 mM iodoacetamide, 10 mM NaF, 1 mM Na3VO4, 1 mM MgCl2, 10% [vol/vol] glycerol, 1% [vol/vol] Nonidet P-40, 1 mM PMSF, 1 μg/mL leupeptin, and 1 mM aprotinin). Enzyme assays were performed as previously described, 38 with a slight modification. The lysates were sonicated briefly, and the insoluble material was pelleted by centrifugation at 14,000g at 4°C for 10 minutes. The p85 subunit of PI 3-kinase (p85) was immunoprecipitated from lysates containing 500 μg of protein by incubation with monoclonal anti-p85 antibody at 4°C for 2 hours, followed by incubation with protein-G agarose (Sigma-Aldrich) at 4°C for 1 hour. The p85 immune complexes were pelleted by centrifugation and washed three times with lysis buffer and once with PI 3-kinase assay buffer (20 mM HEPES [pH 7.4], 100 mM NaCl, 2 mM EGTA, and 12.5 mM MgCl2). The immune complex was resuspended in 20 μL of assay buffer and mixed with 20 μL of lipid/ATP mix containing 500 μg/mL phosphatidylinositol, 80 μM ATP, 200 μM adenosine (PI 4-kinase inhibitor), 10 μCi [γ-32P] ATP (3000 Ci/mmol), 20 mM HEPES [pH 7.4], 100 mM NaCl, 2 mM EGTA, and 12.5 mM MgCl2. Samples were incubated for 30 minutes at 37°C. The reaction was stopped by the addition of 80 μL of 1 N HCl. The phospholipids were extracted with 160 μL of chloroform/methanol (1:1, vol/vol). Phosphatidylinositol monophosphate in organic phase was separated by borate thin-layer chromatography (TLC) on aluminum-backed plates coated with silicone gel (Silica Gel 60; Fisher Scientific, Pittsburgh, PA), as previously described. 38 Phosphatidylinositol-3-phosphate (PI-3-P) was detected using autoradiography. Phosphatidylinositol-4-phosphate was used as a standard for TLC resolution of the lipid and visualized by iodine vapor. The relative density of the PI-3-P spots was estimated using a commercial documentation system (Gel Doc; Bio-Rad Laboratories, Hercules, CA). 
SDS-PAGE and Immunoblot Analysis
The conditions of electrophoresis were as described by Laemmli. 39 Thirty micrograms of protein was electrophoresed on an 8% (Akt, PI 3-kinase, and β-actin), or 12% (Cdk4, p27, and β-actin) SDS-polyacrylamide gels under the reduced condition. The proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane (Bio-Rad Laboratories) and immunoblot analysis was performed using a commercial avidin-biotin complex staining kit (ABC Vectastain; Vector Laboratories), as described previously. 9 10 Nonspecific binding sites of nitrocellulose membrane were blocked by 5% nonfat milk. The incubations were performed with primary antibodies (1:1000 dilution) for 1 hour, with biotinylated secondary antibody (1:5000 dilution) for 1 hour, and with ABC reagent for 30 minutes. The membrane was treated with the enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK), and the ECL-treated membrane was exposed to autoradiograph film. The relative density of protein bands was estimated using a gel documentation system (Bio-Rad Laboratories). 
Immunofluorescent Staining
The conditions of immunofluorescent staining have been described. 9 10 Cells were fixed and permeabilized and then blocked with 2% bovine serum albumin. Cells were incubated with the primary antibodies (1: 200 dilution) for 1 hour at 37°C and then incubated with FITC-conjugated secondary antibody (1:200 dilution) for 1 hour at 37°C in the dark. After extensive washing, the slides were mounted in a drop of antifade mounting medium (Vectashield; Vector Laboratories, Inc.). Control experiments, performed in parallel with omission of the primary antibodies, showed negative staining in all experiments. For double staining, cells were simultaneously incubated with both primary antibodies at 37°C for 1 hour and then rinsed. Cells were then simultaneously incubated with FITC-conjugated secondary antibody (1:100 dilution) and rhodamine-conjugated secondary antibody (1:200 dilution) for 1 hour at 37°C in the dark. 
Confocal Microscopy and Image Analysis
Antibody labeling was examined using a laser scanning confocal microscope (LSM-510; Carl Zeiss Meditec, Thornwood, NY). The 1.8-μm optical slices were taken perpendicular to the cell monolayer (apical to basal orientation). A 488-nm argon laser was used in combination with a 499/505 to 530-excitation/emission-filter set for fluorescein examination. For rhodamine, the 543-nm helium neon laser was used with a 543-nm excitation filter and 560-nm emission filter. Simultaneous images of FITC and rhodamine were captured from the same optical section. The captured images were then pseudocolored: red for rhodamine and green for FITC. Image analysis was performed using the standard system operating software provided with the confocal microscope. All illustrations were assembled and processed digitally (Photoshop, ver. 5.5; Adobe Systems, Mountain View, CA). 
Results
Effect of cAMP on Cell Proliferation Stimulated by FGF-2
We first examined the effect of 8-Br-cAMP on FGF-2-stimulated cell proliferation. When serum-starved CECs were treated with FGF-2 at 10 ng/mL for 24 hours, there was a marked increase in the number of cells (Fig. 1A) . When cells were simultaneously treated with FGF-2 and 8-Br-cAMP, 8-Br-cAMP inhibited cell proliferation in a dose-dependent manner. 8-Br-cAMP at 1 mM and PI 3-kinase inhibitor LY294002 at 20 μM equally inhibited cell proliferation stimulated by FGF-2: both induced approximately a 40% decrease in cell proliferation (Fig. 1A) . Furthermore, treating cells with both 8-Br-cAMP (1 mM) and LY294002 (20 μM) did not synergistically inhibit FGF-2-stimulated cell proliferation, suggesting that cAMP and PI 3-kinase may affect the same signaling pathways in FGF-2–mediated cell proliferation and that one of these molecules is upstream of the other. Figure 1A also shows that 8-Br-cAMP had no effect on the basal activity of CEC proliferation in the absence of FGF-2. Our previous study demonstrated that cell proliferation of CECs requires prolonged and continuous FGF-2 exposure and that activation of PI 3-kinase also requires similar kinetics. 10 Therefore, we determined whether cAMP also required prolonged and continuous exposure to the cells to inhibit FGF-2–stimulated cell proliferation. As shown in Figure 1B , CECs treated with FGF-2 for up to 16 hours showed no proliferation, whereas cells treated for 24 hours demonstrated a marked increase in cell numbers, confirming our previous data. 10 This FGF-2 action was markedly inhibited by simultaneous treatment of 8-Br-cAMP at 1 mM, leading to the basal cell–proliferating level. 
In many cells, extracellular signal-regulated kinase (ERK) activation through Ras and Raf plays a part in cell proliferation by regulating transit through the G1 phase of the cell cycle. 40 41 Therefore, we examined the contribution of this pathway in the FGF-2–mediated mitogenic signaling pathway in CECs. PD98059, a specific inhibitor for ERK pathway, did not inhibit FGF-2–mediated cell proliferation, even at the highest concentration (Fig. 1C) , suggesting that MAPK is not activated in the mitogenic pathways in response to FGF-2 stimulation in CECs. 
Effect of cAMP on PI 3-kinase Pathways
In our previous study, we showed that PI 3-kinase was involved in the major signaling pathway in response to FGF-2 stimulation in CECs. 10 We further investigated whether the antiproliferative effect of cAMP is mediated through inhibiting PI 3-kinase/Akt pathways. We first determined the inhibitory action of 8-Br-cAMP on PI 3-kinase activity (Fig. 2A) . The serum-starved CECs were treated with 8-Br-cAMP for 24 hours in concentrations ranging from 0.1 to 1 mM in the presence of FGF-2. Cell extracts were immune-precipitated with anti-PI 3-kinase (p85 subunit) antibody. They were then assayed for PI 3-kinase activity by measuring the phospholipid product PI-3-P. Cell extracts obtained from FGF-2 treatment contained high levels of PI 3-kinase activity. 8-Br-cAMP inhibited PI 3-kinase enzyme activity in a dose-dependent manner (Fig. 2A) . Both 8-Br-cAMP at 1 mM and LY294002 at 20 μM again equally inhibited PI 3-kinase enzyme activity. Cells treated with both LY294002 (20 μM) and 8-Br-cAMP (1 mM) showed no synergistic effect on FGF-2–stimulated PI 3-kinase activity. Figure 2A also demonstrates that 8-Br-cAMP had no effect on the basal PI 3-kinase activity in the absence of FGF-2 stimulation. These data suggest that cAMP is the upstream molecule to PI 3-kinase in the mitogenic pathway of FGF-2. The activation of PI 3-kinase in response to FGF-2 was further confirmed with PI 3-kinase–mediated Akt phosphorylation, using specific antibodies for phosphorylated Akt (Ser473 or Thr308; Fig. 2B ). Akt phosphorylation at both Thr308 and Ser473 was mediated in response to FGF-2 stimulation, and 8-Br-cAMP markedly reduced Akt phosphorylation at both the Thr308 and Ser 473 sites (Fig. 2B) . This inhibitory effect of cAMP on Akt activation further confirmed that cAMP is the upstream molecule to PI 3-kinase/Akt pathways. This observation is in agreement with previous reports on the inhibitory action of cAMP on Akt phosphorylation. 26 27 We further investigated whether cAMP regulated Akt expression at the protein level (Fig. 2C) . When CECs were treated with FGF-2 alone for 1 hour to 24 hours, Akt level was slightly increased as a function of the duration of FGF-2 stimulation. When cells were simultaneously treated with FGF-2 and 8-Br-cAMP, CECs demonstrated a marked time-dependent reduction of Akt expression: an 8-hour treatment of cells with 8-Br-cAMP reduced the protein level by approximately 40%, and longer treatment of cells with 8-Br-cAMP facilitated further reduction of Akt protein level. These findings are different from the previous report 27 in which total Akt protein levels were not altered by 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) treatment, regardless of the treatment duration. We do not know what causes the discrepancy observed in the two studies, except that two different cAMP analogues were used. However, unlike the unstable Akt level, the p85 protein level of PI 3-kinase was not altered by treating cells with 8-Br-cAMP (Fig. 2C) , suggesting that the reduced levels of Akt after 8-Br-cAMP treatment are not artifacts of the culture system. The reduced levels of Akt mediated by 8-Br-cAMP may be attributable to rapid turnover of the protein. To investigate this possibility, CECs were first stimulated with FGF-2 for 24 hours. Cells were then treated with cycloheximide alone or with 8-Br-cAMP. Figure 2D shows that, in the cells treated with cycloheximide alone, the Akt level was not altered. However, when cells were simultaneously treated with cycloheximide and 8-Br-cAMP, there was a marked reduction of Akt level within 2 hours of treatment. These data indicate that cAMP regulates the stability of Akt protein, thus leading to a rapid turnover. 
cAMP-mediated effects in eukaryotes have traditionally been considered the result of cAMP binding to PKA. 42 We therefore determined whether the inhibition of PI 3-kinase by cAMP is mediated by PKA (Fig. 3) . CECs treated with FGF-2 for 24 hours showed a marked increase of PI 3-kinase activity, whereas 8-Br-cAMP inhibited the FGF-2–stimulated PI 3-kinase activity, confirming Figure 2A . Cells treated with FGF-2 and H89, a PKA inhibitor, demonstrated a marked reduction of PI 3-kinase activity, regardless of the presence or absence of 8-Br-cAMP. Should PKA inhibit PI 3-kinase, H89 could reverse the action of PKA and yield to activation of PI 3-kinase; however, our data suggest that cAMP-mediated inhibition of PI 3-kinase activity is independent of PKA activity. 
After mitogenic stimulation, PI 3-kinase increases the levels of phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol-3, 4-bisphosphate, which recruit both phosphoinositide-dependent kinase (PDK) and Akt to the membrane. 43 It has been reported that membrane localization of PDK1 and subsequent coupling of PDK1 and Akt are downregulated by cAMP. 26 We investigated whether cAMP also affects recruitment of PI 3-kinase to the membrane site as the enzyme complex was targeted to the receptors. To test this hypothesis, CECs were transiently transfected with pCMV6 p85-myc, and the transfected cells were stimulated with FGF-2, with or without 8-Br-cAMP. Cells transfected with empty vector showed negative staining for myc (Fig. 4A) . Cells transfected with pCMV6 p85-myc showed cytoplasmic staining of the expressed fusion protein in the absence of FGF-2 stimulation (Fig. 4B) . FGF-2 induced membrane localization of the p85 subunit of PI 3-kinase (Fig. 4C) , and 8-Br-cAMP abolished the FGF-2-induced membrane localization of the expressed protein (Fig. 4D) . These data suggest that cAMP is able to block membrane targeting of PI 3-kinase mediated by mitogenic stimulation and that membrane localization of PI 3-kinase is prerequisite to the enzyme activation. 
Inhibitory Activity of cAMP on Cell Cycle Regulatory Proteins
Subcellular compartmentalization of cell cycle regulatory proteins plays a key role in regulating cell cycle progression. 44 In a previous study, 10 we have shown that FGF-2 facilitates nuclear translocation of Cdk4 through the action of PI 3-kinase. Therefore, we asked whether translocation of Cdk4 mediated by PI 3-kinase was also blocked by cAMP. Cells maintained under serum-free conditions for 24 hours showed faint cytoplasmic staining of Cdk4 (Fig. 5B) , whereas cells treated with FGF-2 for 24 hours showed strong positive nuclear staining and much less prominent cytoplasmic staining of Cdk4 (Fig. 5C) . Simultaneous treatment of cells with FGF-2 and 8-Br-cAMP at 0.3 mM for 24 hours demonstrated strong nuclear Cdk4 staining in 60% of the cell population (Fig. 5D) , whereas such nuclear staining of Cdk4 was completely absent in the cells treated with 1 mM 8-Br-cAMP, and the diffuse cytoplasmic staining profile of Cdk4 (Fig. 5E) was similar to that observed in serum-starved cells (Fig. 5B) . Likewise, cells treated with LY294002 showed faint cytoplasmic Cdk4 staining in the absence of nuclear Cdk4 staining (Fig. 5F) . These data indicate that cAMP blocks the nuclear translocation of Cdk4 by blocking the action of PI 3-kinase. 
In the same study, we also demonstrated that PI 3-kinase facilitates phosphorylation of p27. 10 Nuclear p27 is phosphorylated at the residue of threonine 187 (Thr187) before nuclear export into the cytoplasm. There, the phosphorylated p27 is subjected to degradation, either by the ubiquitin-proteasome pathway 45 46 or by ubiquitin-independent proteolytic cleavage. 47 We further explored whether cAMP blocks PI 3-kinase–mediated phosphorylation of p27 in response to FGF-2 stimulation. For this purpose, cells simultaneously treated with FGF-2 and inhibitors were double stained with anti-p27 and anti-phosphorylated p27 antibodies. The anti-phosphorylated p27 antibody is specific to the Thr187 phosphorylated form of p27 and does not react with unphosphorylated p27. Cells maintained in DMEM-0 showed strongly positive staining of nuclear p27, whereas anti-phosphorylated p27 antibody did not stain the p27-positive cells (Fig. 6) . Most of the cells stimulated with FGF-2 for 24 hours demonstrated strongly positive staining of phosphorylated p27, whereas most of these cells showed faint staining of nuclear p27. 8-Br-cAMP inhibited phosphorylation of p27 mediated by FGF-2 in a dose-dependent manner: At 0.1 mM 8-Br-cAMP, 30% of cells contained phosphorylated p27, whereas at 1 mM 8-Br-cAMP, phosphorylation of p27 was completely blocked. Likewise, LY294002 completely blocked the phosphorylation of p27 in response to stimulation with FGF-2. 
We have also reported that FGF-2 regulates the expression of Cdk4 and p27 through the action of PI 3-kinase. 10 PI 3-kinase upregulates Cdk4 expression, whereas PI 3-kinase downregulates p27 expression. We therefore explored whether cAMP inhibits the action of PI 3-kinase on the expression of Cdk4 and p27. We tested this hypothesis by treating cells with FGF-2 and 8-Br-cAMP in different concentrations for 24 hours. Upregulation of Cdk4 mediated by FGF-2 stimulation was inhibited by 8-Br-cAMP in a concentration-dependent manner, whereas downregulation of p27 mediated by FGF-2 was blocked by 8-Br-cAMP in a concentration-dependent manner (Fig. 7A) . LY294002 at 20 μM also blocked the upregulation of Cdk4 and downregulation of p27 mediated by FGF-2. Simultaneous treatment of cells with both 8-Br-cAMP and LY294002 did not further inhibit the action of PI 3-kinase. The turnover rate of Cdk4 was determined to elucidate the mechanism underlying the downregulation of Cdk4 expression. CECs were first stimulated with FGF-2 for 24 hours, and cells were then treated with cycloheximide alone or with 8-Br-cAMP in the absence of FGF-2 stimulation for 2, 4, or 8 hours. Figure 7B shows that the Cdk4 level was slightly modulated in cells treated with cycloheximide alone, but cells treated simultaneously with cycloheximide and 8-Br-cAMP demonstrated a marked time-dependent reduction of Cdk4 level. The half-life of Cdk4 in cells treated with both cycloheximide and 8-Br-cAMP is approximately 2 hours, whereas the half-life of Cdk 4 in cells treated with cycloheximide alone is longer than 8 hours. These data further suggest that cAMP also regulates the turnover rate of Cdk4 protein. In an attempt to further confirm that PKA is not involved in the regulation of Cdk4 and p27 expression through PI 3-kinase pathways, we determined Cdk4 and p27 expression in the cells treated with either 8-Br-cAMP, H89 or a combination of the two in the presence of FGF-2 stimulation (Fig. 7C) . Downregulation of Cdk4 and upregulation of p27 by 8-Br-cAMP were again observed. Both cells treated with H89 alone and cells simultaneously treated with 8-Br-cAMP and H89 further downregulated Cdk4 and upregulated p27. Should PKA be activated by cAMP and block PI 3-kinase, H89 could reverse the action of PKA and activate PI 3-kinase, thus leading to upregulation of Cdk4 and simultaneous downregulation of p27. However, the results do not support this hypothesis. Thus, Figure 7C further confirms that PKA may not be involved in the PI 3-kinase pathways. Recently, it has been reported that H89, previously considered to be a selective inhibitor of PKA, inhibits ROCK-II and p70 ribosomal protein S6 kinase (S6K). 48 Our unpublished data showed that rapamycin, an inhibitor to S6K, did not inhibit FGF-2–mediated downregulation of p27, suggesting that S6K may not be activated in response to FGF-2 stimulation. Furthermore, ROCKII, a downstream molecule to the Rho pathway, is not involved in mitogenic pathways. Therefore, although H89 is less specific for PKA, it does not antagonize PI 3-kinase pathways in CECs. 
Discussion
In our previous studies, 9 10 we have shown that FGF-2 uses both PLC-γ1 and PI 3-kinase for its mitogenic signaling pathways in CECs. In those studies, we showed that the level of cell proliferation mediated by PLC-γ1 is approximately 20% of that stimulated by FGF-2, as determined by several experimental approaches. 9 In contrast, PI 3-kinase plays a major role in the mitogenic signaling pathway of FGF-2 in CECs 10 by upregulating Cdk4 expression, facilitating the nuclear import of Cdk4, and sequestering Cdk4 in the nuclei, as it simultaneously downregulates p27 expression and triggers p27 degradation by facilitating phosphorylation of the molecule. 
Corneal endothelium is located posterior to the underlying basement membrane in which 18 kDa FGF-2 is stored. In contrast, corneal endothelium is constantly bathed in aqueous humor containing a high level of cAMP. 17 Thus, corneal endothelium is equally exposed to the opposite effectors that ultimately regulate the behavior of the cells. Therefore, we addressed whether cAMP directly affects FGF-2 activities. In the present study, cAMP inhibited FGF-2 by inhibiting PI 3-kinase activity. 8-Br-cAMP and LY294002 equally inhibited cell proliferation and PI 3-kinase enzyme activity. cAMP further regulated Akt at two distinctive levels. It inhibited phosphorylation of Akt at both the Thr308 and Ser473 sites, and it caused rapid turnover of Akt at the protein level. Taken together, these data suggest that cAMP acts as an upstream molecule to PI 3-kinase/Akt in response to FGF-2 in CECs. 
Recent observations from several independent studies suggest that PI 3-kinase regulates mitogen-induced G1 transit by linking to the cell cycle regulatory machinery. 40 49 50 In our previous study, we showed that PI 3-kinase is directly involved in the regulation of Cdk4 and p27 expression at the protein level in CECs. In the present study, we further explored whether cAMP inhibits the action of PI 3-kinase on cell cycle regulatory proteins. In the present study, upregulation of Cdk4 and downregulation of p27 mediated by FGF-2 was blocked by 8-Br-cAMP in a concentration-dependent manner. An important finding is that cAMP appeared to facilitate a rapid turnover of Cdk4 at the protein level. The elevated Cdk4 by FGF-2 was rapidly decreased by 8-Br-cAMP, reducing the half-life of Cdk4 to 2 hours in contrast to the longer half-life of the much stabilized Cdk4 in the absence of 8-Br-cAMP. Nevertheless, the underlying mechanism by which cAMP regulates the half-life of Cdk4 remains to be elucidated. 
A recent study demonstrated that LY294002 treatment of Chinese hamster embryonic fibroblasts inhibits α-thrombin–mediated nuclear translocation of Cdk2. 44 Those findings suggest that PI 3-kinase is involved in nuclear import of Cdks. Our previous study also demonstrated that PI 3-kinase is directly involved in nuclear translocation of Cdk4. 10 In the present study, the nuclear translocation of Cdk4 mediated by PI 3-kinase was completely blocked by 8-Br-cAMP. Although translocation of Cdk4 was prerequisite for activation of the Cdk4-cyclin D complex, it is unknown how Cdk4 is imported into the nuclei in CECs. It has been reported that ERK acts as a nuclear transport factor for the Cdk2-cyclin E. 51 Likewise, we can assume that Cdk4 is carried into the nucleus while associated with PI 3-kinase. However, this scenario may not occur in CECs, because immunofluorescent staining of PI 3-kinase demonstrates the absence of nuclear staining of the enzyme, even after mitogen-activation (Kay EP, unpublished data). 
In contrast to these findings about the subcellular compartmentalization of Cdk4, FGF-2 did not alter the subcellular localization of p27. Instead, it largely altered the staining potential of nuclear p27. Our previous study 10 demonstrated that PI 3-kinase is directly involved in phosphorylation of p27 in response to FGF-2 stimulation, suggesting that PI 3-kinase triggers the degradation pathway of p27. The initial step for p27 degradation is phosphorylation at the Thr187 residue of p27. To determine whether cAMP inhibits the PI 3-kinase–mediated phosphorylation of p27, we examined double-stained cells for p27 and phosphorylated p27. 8-Br-cAMP blocked phosphorylation of p27 in a dose-dependent manner, suggesting that cAMP may inhibit the degradation pathway of p27 as it antagonizes the action of PI 3-kinase. Together, these data suggest that cells regulated by cAMP favor G1 arrest as they accumulate active G1 inhibitor (p27) and simultaneously block the several activation pathways for S-phase entry, such as induction of cyclin A (Kay EP, unpublished data) and maintaining optimal protein levels of Cdk4. 
In an attempt to elucidate the mechanism by which cAMP downregulates PI 3-kinase activity, we investigated whether PI 3-kinase is targeted to the membrane after FGF-2 stimulation and whether cAMP blocks the membrane localization of PI 3-kinase. Recent studies suggest that cAMP downregulates Akt activity by interfering in the membrane targeting of PDK1 in part. 26 In CECs transiently transfected with pCMV6 p85-myc, FGF-2 induced membrane localization of the p85 subunit of PI 3-kinase and 8-Br-cAMP completely abolished this effect of FGF-2. cAMP-mediated inhibition of PI 3-kinase activity was independent of PKA activity. 
Taken together, these data suggest that PI 3-kinase regulates cell cycle progression by modulating Cdk4 and p27 levels and that cAMP is able to antagonize these actions of PI 3-kinase in the presence of mitogenic stimulation (Fig. 8) . The balancing activity between the positive effectors (FGF-2 via PI 3-kinase) and the negative effectors (cAMP) is especially critical for corneal endothelial cells in vivo because of its unique environment. Thus, a balancing act between these opposite effectors is required for corneal endothelium, not only for homeostasis, but also during the wound repair process. This present study demonstrates that PI 3-kinase and cAMP are the major effector molecules that tightly regulate cell proliferation of corneal endothelial cells in response to mitogenic stimulation. Negative regulation of PI 3-kinase by cAMP may play an important role in the G1 arrest phenotypes of corneal endothelium in vivo. Nevertheless, the findings obtained from the present study apply only to rabbit CECs. At present, no such study has been performed in CECs from other species, including humans. It should also be noted that the egress of cAMP from cells is not known. Furthermore, there are no known cAMP receptors on the cell surface in mammalian systems. However, the extracellular cAMP-adenosine pathway is defined in kidney, in which extracellular cAMP is converted to adenosine by the serial actions of ectophosphodiesterase and ecto-5′-nucleotidase. 52 Adenosine then enters the cells through putative adenosine transporters. Alternatively, adenosine thus formed may interact with A2B receptors expressed by CECs. Such adenosine receptors have recently been identified in bovine CECs (Srinivas S, et al. IOVS 2003;44:ARVO E-Abstract 2085). Therefore, it is likely that an extracellular cAMP-adenosine pathway also operates in rabbit CECs, serving as the entry mechanism of cAMP from the aqueous humor. 
 
Figure 1.
 
Effect of 8-Br-cAMP, LY294002, and PD98059 on cell proliferation stimulated by FGF-2 (10 ng/mL). The serum-starved CECs were treated with (A) FGF-2 and 8-Br-cAMP simultaneously in concentrations ranging from 0.1 to 1.0 mM, 20 μM LY294002, or a combination of the two for 24 hours; (B) FGF-2 for 1, 8, 16, or 24 hours and then the stimulated cells were maintained in DMEM-0 for up to 24 hours; of (C) FGF-2 in the presence or absence of PD98059, in concentrations ranging from 0.1 to 40 μM, for 24 hours. At the end of the treatment, cells were counted. Experiments were performed in triplicate and repeated three times. Data are results of one experiment and are expressed as the mean ± SE.
Figure 1.
 
Effect of 8-Br-cAMP, LY294002, and PD98059 on cell proliferation stimulated by FGF-2 (10 ng/mL). The serum-starved CECs were treated with (A) FGF-2 and 8-Br-cAMP simultaneously in concentrations ranging from 0.1 to 1.0 mM, 20 μM LY294002, or a combination of the two for 24 hours; (B) FGF-2 for 1, 8, 16, or 24 hours and then the stimulated cells were maintained in DMEM-0 for up to 24 hours; of (C) FGF-2 in the presence or absence of PD98059, in concentrations ranging from 0.1 to 40 μM, for 24 hours. At the end of the treatment, cells were counted. Experiments were performed in triplicate and repeated three times. Data are results of one experiment and are expressed as the mean ± SE.
Figure 2.
 
Inhibitory effect of 8-Br-cAMP on PI 3-kinase pathways. The serum-starved CECs were treated as follows: (A) FGF-2 (10 ng/mL) in the presence or absence of various concentration of 8-Br-cAMP, LY294002 (20 μM), or a combination of the two for 24 hours and the PI 3-kinase enzyme activity was then measured; (B) FGF-2 (10 ng/mL) in the presence or absence of 1 mM 8-Br-cAMP for 24 hours, with the phosphorylated forms of Akt at Thr308 or Ser473 determined by immunoblot analysis; (C) FGF-2 (10 ng/mL) in the presence or absence of 8-Br-cAMP (1 mM) for up to 24 hours, with total Akt protein level, the p85 subunit of PI 3-kinase, and β-actin by immunoblot analysis; or (D) FGF-2 (10 ng/mL) for 24 hours and FGF-2 was removed from the cultures. Cells were then treated with cycloheximide (20 μM) in the presence or absence of 1 mM 8-Br-cAMP for 2, 4, or 8 hours. Total Akt protein level and β-actin were determined by immunoblot analysis. β-actin was used for control of protein concentration on Western blot analysis. Data are representative of three or four experiments. Relative density of PI-3-P spots (A) and Akt (C) was determined with a gel documentation system.
Figure 2.
 
Inhibitory effect of 8-Br-cAMP on PI 3-kinase pathways. The serum-starved CECs were treated as follows: (A) FGF-2 (10 ng/mL) in the presence or absence of various concentration of 8-Br-cAMP, LY294002 (20 μM), or a combination of the two for 24 hours and the PI 3-kinase enzyme activity was then measured; (B) FGF-2 (10 ng/mL) in the presence or absence of 1 mM 8-Br-cAMP for 24 hours, with the phosphorylated forms of Akt at Thr308 or Ser473 determined by immunoblot analysis; (C) FGF-2 (10 ng/mL) in the presence or absence of 8-Br-cAMP (1 mM) for up to 24 hours, with total Akt protein level, the p85 subunit of PI 3-kinase, and β-actin by immunoblot analysis; or (D) FGF-2 (10 ng/mL) for 24 hours and FGF-2 was removed from the cultures. Cells were then treated with cycloheximide (20 μM) in the presence or absence of 1 mM 8-Br-cAMP for 2, 4, or 8 hours. Total Akt protein level and β-actin were determined by immunoblot analysis. β-actin was used for control of protein concentration on Western blot analysis. Data are representative of three or four experiments. Relative density of PI-3-P spots (A) and Akt (C) was determined with a gel documentation system.
Figure 3.
 
Effect of H89 on PI 3-kinase. The serum-starved CECs were simultaneously treated with FGF-2 (10 ng/mL) and 8-Br-cAMP (1 mM), H89 (10 μM), or a combination of the two for 24 hours. PI 3-kinase enzyme activity was measured. Data are representative of three experiments. Relative density of PI-3-P spots was determined using a gel documentation system.
Figure 3.
 
Effect of H89 on PI 3-kinase. The serum-starved CECs were simultaneously treated with FGF-2 (10 ng/mL) and 8-Br-cAMP (1 mM), H89 (10 μM), or a combination of the two for 24 hours. PI 3-kinase enzyme activity was measured. Data are representative of three experiments. Relative density of PI-3-P spots was determined using a gel documentation system.
Figure 4.
 
Effect of 8-Br-cAMP on membrane localization of p85 subunit of PI 3-kinase. CECs were transiently transfected with pCMV-p85-myc or empty vector for 8 hours, followed by treatment with FGF-2 (10 ng/mL), with or without 8-Br-cAMP (1 mM) for an additional 24 hours. Cells were then stained with anti-myc antibody. CECs were transfected with (A) empty vector, (B) pCMV-p85-myc, (C) pCMV-p85-myc and treated with FGF-2, or (D) pCMV-p85-myc and treated with FGF-2 and 1 mM 8-Br-cAMP. Arrows: membrane targeted p85-myc. Data are representative of three experiments.Bar, 10 μm.
Figure 4.
 
Effect of 8-Br-cAMP on membrane localization of p85 subunit of PI 3-kinase. CECs were transiently transfected with pCMV-p85-myc or empty vector for 8 hours, followed by treatment with FGF-2 (10 ng/mL), with or without 8-Br-cAMP (1 mM) for an additional 24 hours. Cells were then stained with anti-myc antibody. CECs were transfected with (A) empty vector, (B) pCMV-p85-myc, (C) pCMV-p85-myc and treated with FGF-2, or (D) pCMV-p85-myc and treated with FGF-2 and 1 mM 8-Br-cAMP. Arrows: membrane targeted p85-myc. Data are representative of three experiments.Bar, 10 μm.
Figure 5.
 
Effect of 8-Br-cAMP on subcellular localization of Cdk4 in response to FGF-2 stimulation. The serum-starved CECs were treated with FGF-2 (10 ng/mL) in the presence or absence of 8-Br-cAMP or LY294002 (20 μM) for 24 hours and stained for Cdk4. CECs were (A) stained in the absence of anti-Cdk4 antibody; (B) maintained in DMEM-0 or were treated with (C) FGF-2, (D) FGF-2 and 0.3 mM 8-Br-cAMP, (E) FGF-2 and 1 mM 8-Br-cAMP, or (F) FGF-2 and 20 μM LY294002. The control experiment (A) showed the absence of Cdk4 staining. Data are representative of three experiments. Bar, 20 μm.
Figure 5.
 
Effect of 8-Br-cAMP on subcellular localization of Cdk4 in response to FGF-2 stimulation. The serum-starved CECs were treated with FGF-2 (10 ng/mL) in the presence or absence of 8-Br-cAMP or LY294002 (20 μM) for 24 hours and stained for Cdk4. CECs were (A) stained in the absence of anti-Cdk4 antibody; (B) maintained in DMEM-0 or were treated with (C) FGF-2, (D) FGF-2 and 0.3 mM 8-Br-cAMP, (E) FGF-2 and 1 mM 8-Br-cAMP, or (F) FGF-2 and 20 μM LY294002. The control experiment (A) showed the absence of Cdk4 staining. Data are representative of three experiments. Bar, 20 μm.
Figure 6.
 
Effect of 8-Br-cAMP on phosphorylation of p27 mediated by FGF-2. The serum-starved CECs were treated with FGF-2 (10 ng/mL) for 24 hours. Cells were simultaneously treated with 8-Br-cAMP in various concentrations or 20 μM LY294002. Cells were then fixed and double stained for p27 (green) and phosphorylated p27 (red). Yellow represents the merged images. Data are representative of four experiments. Bar, 20 μm.
Figure 6.
 
Effect of 8-Br-cAMP on phosphorylation of p27 mediated by FGF-2. The serum-starved CECs were treated with FGF-2 (10 ng/mL) for 24 hours. Cells were simultaneously treated with 8-Br-cAMP in various concentrations or 20 μM LY294002. Cells were then fixed and double stained for p27 (green) and phosphorylated p27 (red). Yellow represents the merged images. Data are representative of four experiments. Bar, 20 μm.
Figure 7.
 
Effect of 8-Br-cAMP on expression of Cdk4 and p27 in response to stimulation with FGF-2. The serum-starved CECs were treated with (A) FGF-2 (10 ng/mL) for 24 hours in the presence or absence of various concentrations of 8-Br-cAMP or 20 μM LY294002; (B) FGF-2 (10 ng/mL) for 16 hours and then with 20 μM cycloheximide with or without 1 mM 8-Br-cAMP for 2, 4, or 8 hours in the absence of FGF-2, with relative density of Cdk4 was determined by gel documentation assay; or (C) T FGF-2 (10 ng/mL) for 24 hours in the presence or absence of 1 mM 8-Br-cAMP, 10 μM H89, or a combination of the two, and the extracts were examined by immunoblot analysis. Data are representative of three or four experiments.
Figure 7.
 
Effect of 8-Br-cAMP on expression of Cdk4 and p27 in response to stimulation with FGF-2. The serum-starved CECs were treated with (A) FGF-2 (10 ng/mL) for 24 hours in the presence or absence of various concentrations of 8-Br-cAMP or 20 μM LY294002; (B) FGF-2 (10 ng/mL) for 16 hours and then with 20 μM cycloheximide with or without 1 mM 8-Br-cAMP for 2, 4, or 8 hours in the absence of FGF-2, with relative density of Cdk4 was determined by gel documentation assay; or (C) T FGF-2 (10 ng/mL) for 24 hours in the presence or absence of 1 mM 8-Br-cAMP, 10 μM H89, or a combination of the two, and the extracts were examined by immunoblot analysis. Data are representative of three or four experiments.
Figure 8.
 
Diagram of cAMP’s inhibition of PI 3-kinase and cell proliferation. The PI 3-kinase/Akt pathway is activated on binding of FGF-2 to the FGF receptor, causing upregulation of Cdk4 and downregulation of p27 by increasing the phosphorylation of p27. These simultaneous activities of cell cycle regulatory proteins stimulate G1/S progression, leading to cell proliferation. In contrast, cAMP inhibits the PI 3-kinase pathway by inhibiting membrane targeting of the enzyme and subsequent enzyme activity.
Figure 8.
 
Diagram of cAMP’s inhibition of PI 3-kinase and cell proliferation. The PI 3-kinase/Akt pathway is activated on binding of FGF-2 to the FGF receptor, causing upregulation of Cdk4 and downregulation of p27 by increasing the phosphorylation of p27. These simultaneous activities of cell cycle regulatory proteins stimulate G1/S progression, leading to cell proliferation. In contrast, cAMP inhibits the PI 3-kinase pathway by inhibiting membrane targeting of the enzyme and subsequent enzyme activity.
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Figure 1.
 
Effect of 8-Br-cAMP, LY294002, and PD98059 on cell proliferation stimulated by FGF-2 (10 ng/mL). The serum-starved CECs were treated with (A) FGF-2 and 8-Br-cAMP simultaneously in concentrations ranging from 0.1 to 1.0 mM, 20 μM LY294002, or a combination of the two for 24 hours; (B) FGF-2 for 1, 8, 16, or 24 hours and then the stimulated cells were maintained in DMEM-0 for up to 24 hours; of (C) FGF-2 in the presence or absence of PD98059, in concentrations ranging from 0.1 to 40 μM, for 24 hours. At the end of the treatment, cells were counted. Experiments were performed in triplicate and repeated three times. Data are results of one experiment and are expressed as the mean ± SE.
Figure 1.
 
Effect of 8-Br-cAMP, LY294002, and PD98059 on cell proliferation stimulated by FGF-2 (10 ng/mL). The serum-starved CECs were treated with (A) FGF-2 and 8-Br-cAMP simultaneously in concentrations ranging from 0.1 to 1.0 mM, 20 μM LY294002, or a combination of the two for 24 hours; (B) FGF-2 for 1, 8, 16, or 24 hours and then the stimulated cells were maintained in DMEM-0 for up to 24 hours; of (C) FGF-2 in the presence or absence of PD98059, in concentrations ranging from 0.1 to 40 μM, for 24 hours. At the end of the treatment, cells were counted. Experiments were performed in triplicate and repeated three times. Data are results of one experiment and are expressed as the mean ± SE.
Figure 2.
 
Inhibitory effect of 8-Br-cAMP on PI 3-kinase pathways. The serum-starved CECs were treated as follows: (A) FGF-2 (10 ng/mL) in the presence or absence of various concentration of 8-Br-cAMP, LY294002 (20 μM), or a combination of the two for 24 hours and the PI 3-kinase enzyme activity was then measured; (B) FGF-2 (10 ng/mL) in the presence or absence of 1 mM 8-Br-cAMP for 24 hours, with the phosphorylated forms of Akt at Thr308 or Ser473 determined by immunoblot analysis; (C) FGF-2 (10 ng/mL) in the presence or absence of 8-Br-cAMP (1 mM) for up to 24 hours, with total Akt protein level, the p85 subunit of PI 3-kinase, and β-actin by immunoblot analysis; or (D) FGF-2 (10 ng/mL) for 24 hours and FGF-2 was removed from the cultures. Cells were then treated with cycloheximide (20 μM) in the presence or absence of 1 mM 8-Br-cAMP for 2, 4, or 8 hours. Total Akt protein level and β-actin were determined by immunoblot analysis. β-actin was used for control of protein concentration on Western blot analysis. Data are representative of three or four experiments. Relative density of PI-3-P spots (A) and Akt (C) was determined with a gel documentation system.
Figure 2.
 
Inhibitory effect of 8-Br-cAMP on PI 3-kinase pathways. The serum-starved CECs were treated as follows: (A) FGF-2 (10 ng/mL) in the presence or absence of various concentration of 8-Br-cAMP, LY294002 (20 μM), or a combination of the two for 24 hours and the PI 3-kinase enzyme activity was then measured; (B) FGF-2 (10 ng/mL) in the presence or absence of 1 mM 8-Br-cAMP for 24 hours, with the phosphorylated forms of Akt at Thr308 or Ser473 determined by immunoblot analysis; (C) FGF-2 (10 ng/mL) in the presence or absence of 8-Br-cAMP (1 mM) for up to 24 hours, with total Akt protein level, the p85 subunit of PI 3-kinase, and β-actin by immunoblot analysis; or (D) FGF-2 (10 ng/mL) for 24 hours and FGF-2 was removed from the cultures. Cells were then treated with cycloheximide (20 μM) in the presence or absence of 1 mM 8-Br-cAMP for 2, 4, or 8 hours. Total Akt protein level and β-actin were determined by immunoblot analysis. β-actin was used for control of protein concentration on Western blot analysis. Data are representative of three or four experiments. Relative density of PI-3-P spots (A) and Akt (C) was determined with a gel documentation system.
Figure 3.
 
Effect of H89 on PI 3-kinase. The serum-starved CECs were simultaneously treated with FGF-2 (10 ng/mL) and 8-Br-cAMP (1 mM), H89 (10 μM), or a combination of the two for 24 hours. PI 3-kinase enzyme activity was measured. Data are representative of three experiments. Relative density of PI-3-P spots was determined using a gel documentation system.
Figure 3.
 
Effect of H89 on PI 3-kinase. The serum-starved CECs were simultaneously treated with FGF-2 (10 ng/mL) and 8-Br-cAMP (1 mM), H89 (10 μM), or a combination of the two for 24 hours. PI 3-kinase enzyme activity was measured. Data are representative of three experiments. Relative density of PI-3-P spots was determined using a gel documentation system.
Figure 4.
 
Effect of 8-Br-cAMP on membrane localization of p85 subunit of PI 3-kinase. CECs were transiently transfected with pCMV-p85-myc or empty vector for 8 hours, followed by treatment with FGF-2 (10 ng/mL), with or without 8-Br-cAMP (1 mM) for an additional 24 hours. Cells were then stained with anti-myc antibody. CECs were transfected with (A) empty vector, (B) pCMV-p85-myc, (C) pCMV-p85-myc and treated with FGF-2, or (D) pCMV-p85-myc and treated with FGF-2 and 1 mM 8-Br-cAMP. Arrows: membrane targeted p85-myc. Data are representative of three experiments.Bar, 10 μm.
Figure 4.
 
Effect of 8-Br-cAMP on membrane localization of p85 subunit of PI 3-kinase. CECs were transiently transfected with pCMV-p85-myc or empty vector for 8 hours, followed by treatment with FGF-2 (10 ng/mL), with or without 8-Br-cAMP (1 mM) for an additional 24 hours. Cells were then stained with anti-myc antibody. CECs were transfected with (A) empty vector, (B) pCMV-p85-myc, (C) pCMV-p85-myc and treated with FGF-2, or (D) pCMV-p85-myc and treated with FGF-2 and 1 mM 8-Br-cAMP. Arrows: membrane targeted p85-myc. Data are representative of three experiments.Bar, 10 μm.
Figure 5.
 
Effect of 8-Br-cAMP on subcellular localization of Cdk4 in response to FGF-2 stimulation. The serum-starved CECs were treated with FGF-2 (10 ng/mL) in the presence or absence of 8-Br-cAMP or LY294002 (20 μM) for 24 hours and stained for Cdk4. CECs were (A) stained in the absence of anti-Cdk4 antibody; (B) maintained in DMEM-0 or were treated with (C) FGF-2, (D) FGF-2 and 0.3 mM 8-Br-cAMP, (E) FGF-2 and 1 mM 8-Br-cAMP, or (F) FGF-2 and 20 μM LY294002. The control experiment (A) showed the absence of Cdk4 staining. Data are representative of three experiments. Bar, 20 μm.
Figure 5.
 
Effect of 8-Br-cAMP on subcellular localization of Cdk4 in response to FGF-2 stimulation. The serum-starved CECs were treated with FGF-2 (10 ng/mL) in the presence or absence of 8-Br-cAMP or LY294002 (20 μM) for 24 hours and stained for Cdk4. CECs were (A) stained in the absence of anti-Cdk4 antibody; (B) maintained in DMEM-0 or were treated with (C) FGF-2, (D) FGF-2 and 0.3 mM 8-Br-cAMP, (E) FGF-2 and 1 mM 8-Br-cAMP, or (F) FGF-2 and 20 μM LY294002. The control experiment (A) showed the absence of Cdk4 staining. Data are representative of three experiments. Bar, 20 μm.
Figure 6.
 
Effect of 8-Br-cAMP on phosphorylation of p27 mediated by FGF-2. The serum-starved CECs were treated with FGF-2 (10 ng/mL) for 24 hours. Cells were simultaneously treated with 8-Br-cAMP in various concentrations or 20 μM LY294002. Cells were then fixed and double stained for p27 (green) and phosphorylated p27 (red). Yellow represents the merged images. Data are representative of four experiments. Bar, 20 μm.
Figure 6.
 
Effect of 8-Br-cAMP on phosphorylation of p27 mediated by FGF-2. The serum-starved CECs were treated with FGF-2 (10 ng/mL) for 24 hours. Cells were simultaneously treated with 8-Br-cAMP in various concentrations or 20 μM LY294002. Cells were then fixed and double stained for p27 (green) and phosphorylated p27 (red). Yellow represents the merged images. Data are representative of four experiments. Bar, 20 μm.
Figure 7.
 
Effect of 8-Br-cAMP on expression of Cdk4 and p27 in response to stimulation with FGF-2. The serum-starved CECs were treated with (A) FGF-2 (10 ng/mL) for 24 hours in the presence or absence of various concentrations of 8-Br-cAMP or 20 μM LY294002; (B) FGF-2 (10 ng/mL) for 16 hours and then with 20 μM cycloheximide with or without 1 mM 8-Br-cAMP for 2, 4, or 8 hours in the absence of FGF-2, with relative density of Cdk4 was determined by gel documentation assay; or (C) T FGF-2 (10 ng/mL) for 24 hours in the presence or absence of 1 mM 8-Br-cAMP, 10 μM H89, or a combination of the two, and the extracts were examined by immunoblot analysis. Data are representative of three or four experiments.
Figure 7.
 
Effect of 8-Br-cAMP on expression of Cdk4 and p27 in response to stimulation with FGF-2. The serum-starved CECs were treated with (A) FGF-2 (10 ng/mL) for 24 hours in the presence or absence of various concentrations of 8-Br-cAMP or 20 μM LY294002; (B) FGF-2 (10 ng/mL) for 16 hours and then with 20 μM cycloheximide with or without 1 mM 8-Br-cAMP for 2, 4, or 8 hours in the absence of FGF-2, with relative density of Cdk4 was determined by gel documentation assay; or (C) T FGF-2 (10 ng/mL) for 24 hours in the presence or absence of 1 mM 8-Br-cAMP, 10 μM H89, or a combination of the two, and the extracts were examined by immunoblot analysis. Data are representative of three or four experiments.
Figure 8.
 
Diagram of cAMP’s inhibition of PI 3-kinase and cell proliferation. The PI 3-kinase/Akt pathway is activated on binding of FGF-2 to the FGF receptor, causing upregulation of Cdk4 and downregulation of p27 by increasing the phosphorylation of p27. These simultaneous activities of cell cycle regulatory proteins stimulate G1/S progression, leading to cell proliferation. In contrast, cAMP inhibits the PI 3-kinase pathway by inhibiting membrane targeting of the enzyme and subsequent enzyme activity.
Figure 8.
 
Diagram of cAMP’s inhibition of PI 3-kinase and cell proliferation. The PI 3-kinase/Akt pathway is activated on binding of FGF-2 to the FGF receptor, causing upregulation of Cdk4 and downregulation of p27 by increasing the phosphorylation of p27. These simultaneous activities of cell cycle regulatory proteins stimulate G1/S progression, leading to cell proliferation. In contrast, cAMP inhibits the PI 3-kinase pathway by inhibiting membrane targeting of the enzyme and subsequent enzyme activity.
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