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Vol. 19, Issue 12, 5082-5092, December 2008
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Department of Biochemistry, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, N-0317 Oslo, Norway
Submitted January 29, 2008;
Revised August 19, 2008;
Accepted September 10, 2008
Monitoring Editor: Carl-Henrik Heldin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-irradiation–induced apoptosis, an effect abolished by knockdown of cyclin E. Moreover, cAMP induces early activation of ERK, leading to reduced degradation of cyclin E. The cAMP-mediated up-regulation of cyclin E was blocked by knockdown of ERK or by an inhibitor of the ERK kinase MEK. We conclude that cAMP inhibits cdk2 activity and pRB phosphorylation, leading to reduced ASC proliferation. Concomitant with this growth inhibition, however, cyclin E levels are increased in a MEK/ERK-dependent manner. Our results suggest that cyclin E plays an important, cdk2-independent role in genotoxic stress–induced apoptosis in mesenchymal stem cells. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Baksh et al., 2004; Boquest et al., 2006a; Schaffler and Buchler, 2007). The pluripotent nature of MSCs makes them potentially ideal candidates for tissue engineering, and they have already demonstrated some efficacy in cell therapy and regenerative medicine (Kassem et al., 2004; Kassem, 2004; Leo and Grande, 2006; Mimeault and Batra, 2006). Adipose tissue contains a supportive stroma that can be easily isolated and provides a rich source of MSCs (Zuk et al., 2002; Boquest et al., 2006a). Thus, adipose tissue represents a valuable source of multilineage stem cells. It is therefore important to understand the biology of adipose stem cells (ASCs).
Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates a number of different cellular processes, including metabolism, growth, differentiation, and gene regulation (Krebs and Beavo, 1979; McKnight, 1991; Vossler et al., 1997; Daniel et al., 1998; Kim et al., 2005). Most effects of cAMP are mediated through activation of protein kinase A (PKA) or the Epac (exchange protein directly activated by cAMP) family of exchange proteins (Walsh et al., 1968; Bos, 2003). Elevation of intracellular cAMP has both proliferative and antiproliferative effects depending on the cell type. For instance, cAMP stimulates the proliferation of thyroid cells, neurons, and Swiss 3T3 cells, while inhibiting the proliferation of lymphocytes, fibroblasts, and adipocytes (Rozengurt et al., 1981; Blomhoff et al., 1987; Burgering et al., 1993; Sevetson et al., 1993; Dugan et al., 1999; Iacovelli et al., 2001). One target of cAMP that regulates cell proliferation is the extracellular signal–regulated kinase (ERK; also called mitogen-activated protein kinase, or MAPK) cascade. Several studies have demonstrated a cross-talk between the cAMP signaling pathway and the Ras-Raf-MEK-ERK pathway (Stork and Schmitt, 2002; Dumaz and Marais, 2005). In a cell-specific manner, cAMP can either inhibit (e.g., astrocytes and adipocytes) or activate (e.g., neurons and bone marrow–derived MSCs) ERK (Sevetson et al., 1993; Jaiswal et al., 1994; Stork and Schmitt, 2002; Kim et al., 2005).
Control of the G1/S phase transition at the restriction point plays a crucial role in the regulation of cell proliferation (Zetterberg et al., 1995; Bartek et al., 1996). At the restriction point, cyclin/cdkb complexes inactivate retinoblastoma protein (pRB) by hyperphosphorylation, resulting in E2F release and transcription of S-phase genes (Weinberg, 1995). D-type cyclins are the first group of cyclins to be expressed in response to growth or mitotic signals (Sherr, 1993). They assemble with either cdk4 or cdk6 to promote G1 progression by partial inactivation of pRB, resulting in E2F-mediated transcription of cyclin E, and by titrating the cdk inhibitors p21Cip1 and p27Kip1 to prevent them from inactivating cyclin E/cdk2 complexes (Sherr, 1996; Sherr and Roberts, 1999; Cheng et al., 1999). Cyclin E/cdk2 complexes then complete pRB inactivation leading to S-phase entry (Zarkowska and Mittnacht, 1997; Lundberg and Weinberg, 1998). pRB remains in a hyperphosphorylated state in S phase by the combined actions of cyclin E/cdk2 and cyclin A/cdk2 complexes (Mittnacht, 1998).
Each phase of the cell cycle contains checkpoints that allow cell cycle arrest and activation of repair mechanisms when a defect is detected. If repairs cannot be made, for instance due to a large amount of DNA damage, the apoptotic cascade is activated, leading to programmed cell death (Rowinsky, 2005; Siegel, 2006). Thus, apoptosis is an element of cell cycle checkpoints, allowing for the removal of damaged and abnormal cells. Besides the key regulatory function of cyclin E in the G1/S transition and the initiation of DNA replication, cyclin E also plays an important role in apoptosis of tumor cells of hematopoietic origin. Mazumder et al. (2000) demonstrated that genotoxic stress, such as -irradiation, increased the levels of cyclin E, leading to amplification of apoptosis through activation of the caspase cascade.
The differentiation capacity of MSCs, including ASCs (Boquest et al., 2005), has been widely studied, yet little is known about the cell cycle–related events controlling proliferation and differentiation of these cells. As cAMP is one of the established differentiation factors of MSCs, we unraveled the cell cycle–related events downstream of cAMP signaling in ASCs. In the present study, we demonstrate that elevation of intracellular cAMP strongly inhibits proliferation of ASCs and that this antiproliferative effect is associated with a reduction in both cdk2 activity and pRB phosphorylation. Surprisingly however, the level of cyclin E is increased under these conditions, indicating that cAMP leads to uncoupling of cyclin E from cdk2 activity and cell cycle progression. This suggests that cyclin E may have other functions in ASCs besides its role as a cdk2 activator. Indeed, we show that cAMP promotes a cdk2-independent effect of cyclin E in DNA damage–induced apoptosis of MSCs and that cyclin E is induced in an ERK-dependent manner.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Treatment
Adipose tissue was obtained by liposuction from abdominal, hip, and thigh regions of healthy female donors after formal consent, and the stromal vascular portion was used for subsequent isolation of ASCs essentially as previously described (Boquest et al., 2006b). Briefly, lipoaspirate (300–400 ml) was washed and digested with 0.2% collagenase (Sigma) for 2 h at 37°C with shaking. Floating adipocytes were separated from the stromal vascular fraction by centrifugation. After lysis of erythrocytes and sedimentation, the cellular pellet was resuspended and strained through 100- and 40-µm sieves. Magnetic beads were used to remove CD45+ and CD31+ cells, and as previously reported, the remaining stem cells expressed a CD45–CD31–CD34+CD105+ phenotype (Boquest et al., 2005). Immediately after separation, cells were washed and resuspended in DMEM/F12 supplemented with 20% fetal bovine serum (FBS), antibiotics, and 2.5 µg/ml amphotericin B. After 7 d in culture, attached cells were passaged by trypsinization and cultured further in DMEM/F12 supplemented with 20% FBS and antibiotics. ASCs (polyclonal) were cultured at a density of 2000–6000 cells/cm2 and passaged at 70–80% confluency. To induce proliferation, treatment was carried out in high-glucose DMEM (4.5 g/l; Invitrogen-BRL, Carlsbad, CA) containing 10% FBS, 10 ng/ml epidermal growth factor (EGF; Sigma, St. Louis, MO), 20 ng/ml basic fibroblast growth factor (bFGF; Sigma), B27 (1:50; Invitrogen), and antibiotics. Cells at passages 5–12, i.e., in the log expansion phase, were used in all experiments.
Cell Proliferation and BrdU Incorporation
Cell proliferation was determined by measuring the incorporation of [3H]thymidine (Amersham Biosciences, Piscataway, NJ) into DNA and by counting the number of viable cells. For the [3H]thymidine assay, ASCs were seeded in 25-cm2 flasks at a density of 4 x 104 cells/flask. Cells were pulsed with 0.2 mCi of [3H]thymidine for the last 20 h of a 72-h incubation. Cells were harvested by trypsinization and collected by centrifugation (300 x g for 10 min). After transferring to a microtiter plate, cells were harvested on a cell harvester and counted in a liquid scintillation counter (Topcount; Packard Instrument, Meriden, CT), according to the manual of the instruments. For direct cell counting, ASCs were seeded in 75-cm2 flasks at 1.5 x 105 cells/flask. After 4 d, cells were collected before counting using a Bürker chamber. For the BrdU incorporation study, ASCs were seeded in 150-cm2 flasks at 3 x 105 cells/flask. Cells were pulse-labeled with BrdU (10 µM) for the last 90 min of a 72-h incubation, harvested by trypsinization, and analyzed for BrdU incorporation by fluorescence-activated cell sorting (FACS) as described by Naderi et al. (2005).
TUNEL Assay
The in situ cell death detection kit fluorescein (Roche Diagnostics, Alameda, CA) was used to detect DNA strand breaks generated during apoptosis. TUNEL was performed as recommended by the manufacturer and the proportion of TUNEL-positive cells was determined by fluorescence microscopy.
Immunoblot Analysis
ASCs (400,000 cells/175-cm2 flask) were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 10 mM β-glycerophosphate, 0.2 mM PMSF, 10 µg/ml leupeptin, and 0.5% aprotinin) before protein concentration was determined using the Bradford method (Bio-Rad, Richmond, CA). Equal amounts of protein (50 µg) were subjected to SDS-PAGE and transferred to nitrocellulose (Amersham Biosciences) using a semidry transfer cell (Bio-Rad). Proteins were detected using appropriate primary antibodies and the enhanced chemiluminescence detection system (ECL Plus, Amersham Biosciences).
Subcellular Fractionation
Nuclear and cytoplasmic extracts from 1.0 x 106 ASCs were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA; Cat. No. 40010) according to the manufacturer's instruction. Equal amounts of protein (50 µg) were then subjected to SDS-PAGE and examined by Western blotting.
Immunoprecipitation and Kinase Assay
ASCs (400,000 cells/175 cm2 flask) were lysed in Triton X-100 lysis buffer (20 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.1% Triton X-100, 10 mM NaF, 5 mM β-glycerophosphate, 0.1 mM Na3VO4, 0.2 mM PMSF, 10 µg/ml leupeptin, and 0.5% aprotinin) for 1 h on ice with occasional vortexing, followed by sonication. After centrifugation, total cell lysate (300 µg) was incubated with anti-cdk2 or anti-cyclin E antibodies (2 µg) overnight at 4°C with rotation. A 1:1 slurry of protein G Sepharose beads (30 µl; Upstate Biotechnology, Lake Placid, NY) was added to the lysate, and the incubation was continued for another hour at the same conditions. For immunoprecipitation, the resulting immune complexes were washed three times in lysis buffer before they were resuspended in 1x SDS sample buffer, boiled for 10 min, and subjected to Western blot analysis. For detection of cdk2 activity, the immune complexes were washed twice in lysis buffer and once in kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, 2 mM EGTA, 1 mM NaF, 5 mM β-glycerophosphate, and 0.1 mM Na3VO4) before they were resuspended in 20 µl kinase buffer containing 30 µM ATP, 5 µg histone H1 (Upstate Biotechnology) and 10 µCi of [-32P]ATP (Amersham Biosciences). After 15 min at 30°C, the reactions were terminated by adding 10 µl of 3x SDS sample buffer, followed by boiling for 5 min. Samples were subjected to SDS-PAGE, and the gels were dried before phosphorylated histone H1 was detected by autoradiography.
Immunocytochemistry
For immunocytochemistry, ASCs were plated onto coverslips at 1–1.5 x 104 cells/coverslip into six-well plates. After 2 d (for detection of cyclin E) or 5 d (for detection of NF200) of culture, cells were fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, washed three times with PBS followed by permeabilization with PBS containing 0.1% Triton X-100 for 15 min. After washing, the cells were incubated with blocking solution (PBS containing 0.01% Tween-20 and 2% BSA) for 15 min, before incubation with cyclin E antibodies (1:50 dilution in blocking solution overnight at 4°C) or NF200 antibodies (1:200 dilution in blocking solution for 30 min). The cells were washed four times with blocking solution and then further incubated with goat anti-rabbit or goat anti-mouse antibodies conjugated to Cy3 (1:300 in blocking solution) for 30 min. Cells were washed twice in PBS followed by DNA-staining with 0.25 µg/ml DAPI for 10 min. After washing twice with PBS and once with dH2O, coverslips were mounted and observed with an Olympus BX51 microscope (Melville, NY) using AnalySIS (Soft Imaging Systems, Münster, Germany), or an Olympus Fluoview 1000 laser scanning confocal microscope (60x objective).
siRNA and Transient Transfection
Stealth RNA interference (RNAi) duplex oligoribonucleotides (siRNA) for human cyclin E1 (HSS101458 and HSS101460) and ERK/MAPK (12935-025), and control small interfering RNAs (siRNAs; 12935-200 and 12935-300, respectively) were purchased from Invitrogen. Stealth RNAi oligos are 25-mer double-strand RNA (dsRNA) molecules with chemical modifications that increase stability and reduce off-target effects by limiting sense strand activity. Two nonoverlapping Stealth RNAi duplexes of each siRNA were used in our experiments. The plasmids encoding cdk2 and cdk2-D146N (dominant negative cdk2) were constructed by Dr. Sander van den Heuvel (Utrecht University, The Netherlands; van den Heuvel and Harlow, 1993) and obtained from Addgene (Addgene plasmids 1884 and 1885, respectively). The siRNAs/plasmids were transiently transfected into ASCs using the Human MSC Nucleofector Kit (Amaxa Biosystems, Gaithersburg, MD). For each nucleofection sample, 5 x 105 cells were subjected to nucleofection with 5 µg of siRNA/plasmid according to the manufacturer's protocol. The nucleofection was carried out under the program U-23 of the Nucleofector device, and after nucleofection the cells were incubated in DMEM/F12 supplemented with 20% FBS and antibiotics for 24–40 h before treatment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine whether other inducers of intracellular cAMP had the same effect as 8-CPT on proliferation of ASCs, we tested the effects of PGE2, forskolin, and the PKA-specific cAMP analog 6-Bnz-cAMP (6-Bnz). The PGE2 receptors EP2 and EP4 activate G-proteins, which in turn stimulate adenylate cyclase activity (Regan, 2003), whereas the diterpene forskolin directly activates the catalytic subunit of adenylate cyclase (Seamon and Daly, 1981). As shown in Figure 1E, all cAMP-elevating agents were able to inhibit DNA synthesis. Similar observations were made in a polyclonal culture of ASCs from three different donors (data not shown).
Effect of cAMP on Protein Expression of Cell Cycle–regulating Genes
To study the mechanisms underlying growth inhibition of ASCs by cAMP, we analyzed the expression of various cell cycle–regulating proteins after 48 h of treatment with cAMP inducers. As shown in Figure 2A, pRB was highly phosphorylated in stimulated ASCs. Addition of different cAMP-increasing agents resulted in a prominent inhibition of pRB phosphorylation, consistent with the observed cAMP-induced growth arrest. The changes in pRB phosphorylation appeared after 12 h of treatment with 8-CPT (Figure 2B). Reduced phosphorylation of pRB was associated with reduced levels of cdk2, especially of the lower band, and reduced expression of cyclin A was also observed (Figure 2A). No or minor effects were seen on the levels of D-type cyclins and of the two cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. Interestingly however, the level of cyclin E markedly increased upon cAMP induction despite reduced phosphorylation of pRB. The time-dependent effect of 8-CPT on cdk2 expression correlated temporally with the kinetics of pRB phosphorylation, whereas the effect on cyclin E was noted at 3 h of treatment (Figure 2B).
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). Because we have shown that cAMP reduced the expression of cdk2 and cyclin A, concomitant with an increase in cyclin E (see Figure 2A), we determined the effect of cAMP on cdk2 activity. This was addressed by analyzing anti-cdk2- and anti-cyclin E immunoprecipitates from stimulated ASCs for cdk2 activity using histone H1 as substrate (Figure 3). Cdk2 activity was significantly reduced by 8-CPT and PGE2, and this could be explained by the reduced cdk2 level observed in IPs of cdk2 (Figure 3, top panels). Reduced kinase activity was also observed when cyclin E–associated kinase activity was measured, and in IPs of cyclin E the cdk2 level in 8-CPT–treated cells was reduced relative to cyclin E (Figure 3, middle panels). Western blot analysis of the same lysates confirmed the cAMP-induced down-regulation of cdk2, inhibition of pRB phosphorylation, and up-regulation of cyclin E (Figure 3, bottom panels). The same effects of cAMP on the cell cycle machinery were observed in a polyclonal culture of ASCs from three different donors (data not shown). We conclude therefore that in ASCs, cAMP leads to reduced proliferation, reduced cdk2 activity, and reduced pRB phosphorylation, despite markedly induced levels of cyclin E.
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). However, a recent report showed that cyclin E shuttles between the nucleus and cytoplasm and that its apparent nuclear localization is a reflection of slow nuclear export compared with a more rapid import (Jackman et al., 2002). Therefore, we explored the possibility that cAMP might affect this shuttling, leading to translocation of cyclin E from the nucleus. Immunofluorescence staining of cyclin E in stimulated ASCs treated with or without 8-CPT showed that in control cells, staining of cyclin E was predominantly nuclear (Figure 4AI). In contrast, no nuclear staining was observed in cells treated with 8-CPT (Figure 4AII), suggesting that cyclin E was shuttled to the cytoplasm. In line with this assumption, Western blot analysis of the cytosolic fraction confirmed up-regulation of cyclin E in the cytoplasm after 8-CPT-treatment (Figure 4B). The expected reduced nuclear expression of cyclin E upon cAMP treatment was probably masked by contamination of cytosolic cyclin E in the nuclear fraction, because the cytoplasmic marker was expressed in both fractions. When cells were analyzed by immunofluorescence staining, we did not detect increased staining of cyclin E in the cytoplasm upon cAMP induction. The weak cytoplasmic staining in Figures 4A, I and II, seemed to be background staining, because similar staining was detected in cells transfected with cyclin E siRNA (see Figure 6). No translocation of cdk2 from the nucleus was observed upon 8-CPT treatment (Figures 4A, III and IV). Therefore, the results indicate that cAMP induces a shift in the subcellular localization of cyclin E. This might explain why elevation of cyclin E seems to be uncoupled from cdk2 activation in ASCs.
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), cAMP induced differentiation of ASCs into neuron-like cells with distinct changes in cell morphology (Figure 5A; compact cell bodies and long processes) and with increased levels of the neuron-specific protein NF200 (Figure 5B). After 4 d of treatment, 
30% of the cells treated with 8-CPT expressed NF200, compared with 8% in control cells. Having shown that cyclin E was induced by cAMP in ASCs and apparently was uncoupled from cdk2 activation, we postulated that cyclin E might have cdk2-independent function in these cells. Cyclin E has been shown to promote development of the CNS in Drosophila melanogaster (Berger et al., 2005); thus we investigated whether cyclin E could play a role in the observed cAMP-induced differentiation of ASCs. Expression of cyclin E was silenced by transient transfection with siRNA targeted to cyclin E, and as shown in Figure 6, A and B, cyclin E levels were markedly reduced. In accordance with its role as a regulator of the G1/S transition, knocking down cyclin E in the control cells reduced [3H]thymidine incorporation to 34% compared with the mock-transfected cells (Figure 6C). However, no effect of cyclin E siRNA was noted on the cAMP-mediated growth-inhibition and on cAMP-mediated differentiation, as demonstrated by NF200 staining (Figure 6D) and by morphology (data not shown).
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). Further, recent investigations have revealed that genotoxic stress increases the levels of cyclin E in hematopoietic cells, leading to amplification of apoptosis (Mazumder et al., 2000, 2002, 2004). To study whether the cAMP-mediated high levels of cyclin E sensitized ASCs to genotoxic stress, cells were first treated with 8-CPT for 24 h to induce cyclin E expression and then were exposed to 25 Gy -irradiation for 48 h. Apoptotic cells were assessed by TUNEL assay. Treatment with 8-CPT alone did not induce apoptosis (Figure 7A), consistent with the other viability tests that we performed (see above). However, 8-CPT–treated ASCs were sensitized to -irradiation–induced apoptosis, revealing a sevenfold induction in the number of TUNEL-positive cells upon irradiation compared with untreated cells (Figure 7A). No effect of -irradiation was noted on the 8-CPT–induced up-regulation of cyclin E (Figure 7A, bottom panels); however, when cyclin E was knocked down by siRNA, the proportion of TUNEL-positive cells after -irradiation was markedly reduced compared with mock-transfected cells (Figure 7, B and C) as well as cells transfected with control siRNA (data not shown). Furthermore, reducing the activity of cdk2 by transfecting the cells with a construct of dominant negative cdk2 did not affect cAMP-mediated induction of apoptosis (Figure 7D). Taken together, these results indicate that cyclin E plays an important cdk2-independent role in -irradiation–induced apoptosis in ASCs and that this effect is promoted by cAMP, through elevation of cyclin E levels.
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; Grewal et al., 1999; Iacovelli et al., 2001). Because many targets of the MEK-ERK pathway are cell cycle regulatory proteins (Meloche and Pouyssegur, 2007), we assessed the possible involvement of this pathway in cAMP-mediated induction of cyclin E in stimulated ASCs. Pretreatment with the MEK inhibitor U0126 markedly reduced the 8-CPT–induced elevation of cyclin E expression (Figure 8A). The inhibitor did not reverse the effect of 8-CPT on any of the other cell cycle parameters and rather inhibited the levels of cdk2 alone. To confirm the involvement of the MEK-ERK pathway in cAMP-mediated signaling in ASCs, we directly analyzed the activity of ERK. The level of phosphorylated (activated) ERK was analyzed by Western blotting using phospho-specific antibodies (Thr202/Tyr204-phosphorylated p44/p42 MAPK). As shown in Figure 8B, cAMP markedly induced phosphorylation (activation) of ERK, and the effect was noticeable by 3 h of treatment (Figure 8C). As a control of equal loading, the lysates were also analyzed for expression of total ERK (Figure 8, B and C, bottom panels). The time-dependent effect of cAMP on cyclin E expression (see Figure 2B) closely resembled that of ERK phosphorylation, supporting a causative relationship between these events. To further demonstrate the requirement of ERK for the cAMP-mediated up-regulation of cyclin E, ERK was knocked down by siRNA. Indeed, reducing the levels of ERK prevented the induction of cyclin E upon 8-CPT treatment and totally prevented the induction of apoptosis (Figure 8D).
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; Kim et al., 2005; Jori et al., 2005), we also assessed the role of MEK-ERK pathway in cAMP-mediated differentiation of ASCs into NF200-positive cells. However, because pretreatment of ASCs with U0126 did not reduce the number of NF200-positive cells induced by elevation of cAMP (data not shown), we concluded that the MEK-ERK pathway does not play a role in cAMP-mediated neuronal differentiation of ASCs. This conclusion was supported by the inability of siRNA against the ERK-induced cyclin E to prevent differentiation of ASCs by cAMP (Figure 6).
cAMP Inhibits the Degradation of Cyclin E
To further investigate the mechanisms whereby cAMP up-regulates cyclin E expression, we analyzed whether cAMP affects the levels of cyclin E mRNA. RT-PCR analysis showed that 8-CPT did not affect the steady state level of cyclin E mRNA (data not shown), indicating that cAMP regulates expression of cyclin E posttranscriptionally. Cyclin E is an unstable protein, and its expression is also regulated by ubiquitin-dependent proteolysis (Hwang and Clurman, 2005). To address whether cAMP affected the rate of cyclin E degradation, ASCs pretreated with or without 8-CPT for 48 h were treated with the protein synthesis inhibitor cycloheximide in a time-course experiment. Western blot analysis revealed that cyclin E was rapidly degraded in control cells, whereas 8-CPT completely stabilized cyclin E protein levels (Figure 9), indicating that cAMP prevents degradation of cyclin E.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Zhao et al., 2006; Wang et al., 2006), and activation of the cAMP/PKA pathway has been shown to drive these cells in neurogenic direction (Rydel and Greene, 1988; Deng et al., 2001; Kim et al., 2005; Shi et al., 2006; Wang et al., 2007). We have identified a hitherto unknown role and regulation of cyclin E downstream of cAMP signaling in ASCs. Thus, although the cells were markedly growth inhibited by cAMP, cyclin E levels were potently induced and rendered the cells susceptible to DNA damage–induced apoptosis.
The antiproliferative effect of cAMP was associated with a reduction in both cdk2 activity and pRB phosphorylation. Induced expression of cyclin E was therefore unexpected, given the critical role of cyclin E in cell cycle progression. However, induction of cyclin E concomitant with reduced DNA synthesis has previously been demonstrated in HT29 human colon carcinoma cells (Leonce et al., 2001), as well as in growth-arrested hepatocytes and bone marrow–derived MSCs (Oliva et al., 2003; Mullany et al., 2007). In the latter studies, the growth arrest was explained by increased levels of cdk inhibitors, whereas in our experiments, treatment with cAMP-increasing agents did not change the expression of cdk inhibitors. Instead, the reduced activity of cdk2 and thereby reduced proliferation could be the result of the reduced protein levels of cdk2 and cyclin A. The discrepancy between cyclin E induction and reduced cdk2 activity could further be explained by the decreased nuclear localization of cyclin E in response to elevated levels of cAMP, leading to different subcellular localization of cyclin E and cdk2 and thereby making cyclin E unable to activate cdk2.
The apparent uncoupling of cyclin E from cdk2 activity and cell cycle function in cAMP-treated ASCs lead us to postulate that cyclin E might have cdk2-independent functions in these cells. Knockout experiments have shown that cdk2–/– mice are viable, whereas cyclin E1–/–, E2–/– mice die in utero, suggesting that cyclin E may have cdk2-independent roles (Berthet et al., 2003; Geng et al., 2003; Ortega et al., 2003; Parisi et al., 2003). In fact, recent reports have demonstrated kinase-independent functions of cyclin E in cell cycle progression, as well as in other cellular processes (Mazumder et al., 2002; Matsumoto and Maller, 2004; Geng et al., 2007). Thus, cyclin E can act as a cell fate determinant in the developing CNS of D. melanogaster (Berger et al., 2005), and cyclin E2 is required for embryogenesis in Xenopus laevis (Gotoh et al., 2007). Given the role of cyclin E in differentiation and because cAMP induced differentiation of ASCs into neuron-like cells, the possibility existed that the role of cAMP-induced elevation of cyclin E expression was to drive differentiation of ASCs into neurogenic direction. However, knocking down cyclin E by siRNA did not inhibit the cAMP-mediated neurogenic differentiation of the cells, ruling out this possibility.
Cyclin E has also been reported to play a role in apoptosis (Leonce et al., 2001; Mazumder et al., 2004). We did not observe any induction of apoptosis in response to 8-CPT alone, a result supported by the fact that the growth-inhibitory effect of cAMP on ASCs was reversible. It was still possible, however, that the increased level of cyclin E rendered the cells susceptible to apoptosis-inducing agents, because this would be in accordance with reports showing that cyclin E plays a critical role in amplification of genotoxic stress–induced apoptosis of hematopoietic cells (Mazumder et al., 2000, 2002). To address a possible role of cyclin E in DNA damage–induced apoptosis, ASCs pretreated with 8-CPT to induce cyclin E expression were exposed to 25 Gy -irradiation for 48 h. Indeed, elevated levels of cAMP enhanced the number of TUNEL-positive cells upon irradiation, thus sensitizing these cells to DNA damage–induced apoptosis. This sensitization by cAMP was dependent on cyclin E expression, thus we concluded that cyclin E induction seems to play an important role in -irradiation–induced apoptosis of ASCs. The proapoptotic effect of cyclin E was apparently independent of cdk2 activity, which is in accordance with other reports (Leonce et al., 2001; Mazumder et al., 2007). Cyclin E expression has been shown to decline late in the apoptotic process, due to caspase-dependent proteolysis of cyclin E (Mazumder et al., 2002). Thus, cyclin E induction is likely to play an important role in the initiation phase of cell death. The mechanism behind cyclin E–mediated amplification of DNA-damage–induced apoptosis in adipose stem cells is the subject of further studies.
Although expression of cyclin E usually depends on the pRB/E2F pathway (Ohtani et al., 1995; Le et al., 1999; Moroy and Geisen, 2004), cyclin E may also be a direct target of extracellular signals, such as the MEK/ERK pathway (Nourse et al., 1994; Mohapatra et al., 2001). Several studies have demonstrated a cross-talk between the cAMP signaling pathway and the Ras-Raf-MEK-ERK pathway (Stork and Schmitt, 2002; Dumaz and Marais, 2005). In a cell-specific manner, cAMP can either inhibit or activate ERK, depending on the relative amounts of Rap1 (a GTPase of the Ras superfamily) and the Raf isoforms B-Raf and Raf-1 expressed by the cell (Stork and Schmitt, 2002). In B-Raf–positive cells, such as neurons, Rap1 associates with B-Raf, leading to activation of ERK. Activation of ERK by cAMP has also been reported for bone marrow–derived MSCs (Kim et al., 2005). Thus in these cells, cAMP induced neuronal differentiation due to PKA/B-Raf–mediated activation of ERK. To examine the possibility of ERK being involved in cAMP-mediated up-regulation of cyclin E, ERK was knocked down by siRNA or cells were pretreated with the MEK inhibitor U0126 before addition of 8-CPT. Both the siRNA and the inhibitor markedly reduced the induction of cyclin E upon 8-CPT treatment, suggesting the involvement of the Ras-Raf-MEK-ERK pathway in this process. This was further supported by the enhanced activity of ERK in response to increased levels of intracellular cAMP. Moreover, the time-dependent effect of cAMP on ERK closely resembled that on cyclin E, supporting a causative relationship between these events. Our results are consistent with the recent finding that treatment of rat mesangial cells with the MEK inhibitor U0126 completely inhibited the stimulation-induced cyclin E expression in these cells (Bokemeyer et al., 2007).
Expression of cyclin E is primarily regulated at the level of transcription or by ubiquitin-dependent proteolysis (Ohtani et al., 1995; Le et al., 1999; Moroy and Geisen, 2004). As expected, because cdk2 activation and pRB phosphorylation were reduced in 8-CPT–treated ASCs, we did not observe transcriptional induction of cyclin E. Instead, we demonstrated that cAMP induced a pronounced stabilization of cyclin E at the protein level. Cyclin E is an unstable protein that is degraded by two distinct pathways: 1) ubiquitination of monomeric cyclin E (by the Cul-3 ubiquitin-ligase) and 2) ubiquitination of phosphorylated cyclin E in complex with cdk2 (by the SCF-Fbw7 ubiquitin-ligase; Clurman et al., 1996; Singer et al., 1999; Welcker et al., 2003; Hwang and Clurman, 2005). We are currently investigating which of these pathways are disrupted by cAMP induction in ASCs.
In conclusion, we have demonstrated that cAMP induction in ASCs leads to uncoupling of cyclin E from its established role as an obligate driving force in cell cycle progression and that cAMP-signaling in stead directs cyclin E to render these cells sensitive to DNA damage–induced apoptosis. It is well established that stem cells in general are resistant to such apoptosis (Guo et al., 2006; Hong et al., 2006), and this might be important for preserving a pool of self-renewing stem cells. However, by promoting differentiation into neuron-like cells, it is possible that cAMP also must ensure that resistance to DNA damage–induced apoptosis is relieved. Thus, it might be important for neuron-like cells to more readily undergo cell death when exposed to threatening DNA-damaging agents.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work. 
Address correspondence to: Heidi Kiil Blomhoff (h.k.blomhoff{at}medisin.uio.no)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bartek, J., Bartkova, J., and Lukas, J. (1996). The retinoblastoma protein pathway and the restriction point. Curr. Opin. Cell Biol 8, 805–814.[CrossRef][Medline]
Berger, C., Pallavi, S. K., Prasad, M., Shashidhara, L. S., and Technau, G. M. (2005). A critical role for cyclin E in cell fate determination in the central nervous system of Drosophila melanogaster. Nat. Cell Biol 7, 56–62.[CrossRef][Medline]
Berthet, C., Aleem, E., Coppola, V., Tessarollo, L., and Kaldis, P. (2003). Cdk2 knockout mice are viable. Curr. Biol 13, 1775–1785.[CrossRef][Medline]
Blomhoff, H. K. et al. (1987). Cyclic AMP-mediated suppression of normal and neoplastic B cell proliferation is associated with regulation of myc and Ha-ras protooncogenes. J. Cell. Physiol 131, 426–433.[CrossRef][Medline]
Bokemeyer, D. et al. (2007). The map kinase ERK regulates renal activity of cyclin-dependent kinase 2 in experimental glomerulonephritis. Nephrol. Dial. Transplant 22, 3431–3441.
Boquest, A. C., Noer, A., and Collas, P. (2006a). Epigenetic programming of mesenchymal stem cells from human adipose tissue. Stem Cell Rev 2, 319–329.[CrossRef][Medline]
Boquest, A. C., Shahdadfar, A., Brinchmann, J. E., and Collas, P. (2006b). Isolation of stromal stem cells from human adipose tissue. Methods Mol. Biol 325, 35–46.[Medline]
Boquest, A. C., Shahdadfar, A., Fronsdal, K., Sigurjonsson, O., Tunheim, S. H., Collas, P., and Brinchmann, J. E. (2005). Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture. Mol. Biol. Cell 16, 1131–1141.
Bos, J. L. (2003). Epac: a new cAMP target and new avenues in cAMP research. Nat. Rev. Mol. Cell Biol 4, 733–738.[CrossRef][Medline]
Burgering, B. M., Pronk, G. J., van Weeren, P. C., Chardin, P., and Bos, J. L. (1993). cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor. EMBO J 12, 4211–4220.[Medline]
Cheng, M., Olivier, P., Diehl, J. A., Fero, M., Roussel, M. F., Roberts, J. M., and Sherr, C. J. (1999). The p21(Cip1) and p27(Kip1) CDK ‘inhibitors’ are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J 18, 1571–1583.[CrossRef][Medline]
Clurman, B. E., Sheaff, R. J., Thress, K., Groudine, M., and Roberts, J. M. (1996). Turnover of cyclin E by the ubiquitin-proteasome pathway is regulated by cdk2 binding and cyclin phosphorylation. Genes Dev 10, 1979–1990.
Daniel, P. B., Walker, W. H., and Habener, J. F. (1998). Cyclic AMP signaling and gene regulation. Annu. Rev. Nutr 18, 353–383.[CrossRef][Medline]
Deng, W., Obrocka, M., Fischer, I., and Prockop, D. J. (2001). In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem. Biophys. Res. Commun 282, 148–152.[CrossRef][Medline]
Dugan, L. L., Kim, J. S., Zhang, Y., Bart, R. D., Sun, Y., Holtzman, D. M., and Gutmann, D. H. (1999). Differential effects of cAMP in neurons and astrocytes. Role of B-raf. J. Biol. Chem 274, 25842–25848.
Dumaz, N., and Marais, R. (2005). Integrating signals between cAMP and the RAS/RAF/MEK/ERK signalling pathways. FEBS J 272, 3491–3504. Based on the anniversary prize of the Gesellschaft fur Biochemie und Molekularbiologie Lecture delivered on 5 July 2003 at the Special FEBS Meeting in Brussels.[CrossRef][Medline]
Geng, Y. et al. (2007). Kinase-independent function of cyclin E. Mol. Cell 25, 127–139.[CrossRef][Medline]
Geng, Y. et al. (2003). Cyclin E ablation in the mouse. Cell 114, 431–443.[CrossRef][Medline]
Gotoh, T., Shigemoto, N., and Kishimoto, T. (2007). Cyclin E2 is required for embryogenesis in Xenopus laevis. Dev. Biol 310, 341–347.[CrossRef][Medline]
Grewal, S. S., York, R. D., and Stork, P. J. (1999). Extracellular-signal-regulated kinase signalling in neurons. Curr. Opin. Neurobiol 9, 544–553.[CrossRef][Medline]
Guo, Y., Graham-Evans, B., and Broxmeyer, H. E. (2006). Murine embryonic stem cells secrete cytokines/growth modulators that enhance cell survival/anti-apoptosis and stimulate colony formation of murine hematopoietic progenitor cells. Stem Cells 24, 850–856.
Hong, Y., Cervantes, R. B., and Stambrook, P. J. (2006). DNA damage response and mutagenesis in mouse embryonic stem cells. Methods Mol. Biol 329, 313–326.[Medline]
Hwang, H. C., and Clurman, B. E. (2005). Cyclin E in normal and neoplastic cell cycles. Oncogene 24, 2776–2786.[CrossRef][Medline]
Iacovelli, L., Capobianco, L., Salvatore, L., Sallese, M., D'Ancona, G. M., and De, B. A. (2001). Thyrotropin activates mitogen-activated protein kinase pathway in FRTL-5 by a cAMP-dependent protein kinase A-independent mechanism. Mol. Pharmacol 60, 924–933.
Jackman, M., Kubota, Y., den, E. N., Hagting, A., and Pines, J. (2002). Cyclin A- and cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm. Mol. Biol. Cell 13, 1030–1045.
Jaiswal, R. K., Moodie, S. A., Wolfman, A., and Landreth, G. E. (1994). The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras. Mol. Cell Biol 14, 6944–6953.
Jori, F. P., Napolitano, M. A., Melone, M. A., Cipollaro, M., Cascino, A., Altucci, L., Peluso, G., Giordano, A., and Galderisi, U. (2005). Molecular pathways involved in neural in vitro differentiation of marrow stromal stem cells. J. Cell. Biochem 94, 645–655.[CrossRef][Medline]
Kassem, M. (2004). Mesenchymal stem cells: biological characteristics and potential clinical applications. Cloning Stem Cells 6, 369–374.[CrossRef][Medline]
Kassem, M., Kristiansen, M., and Abdallah, B. M. (2004). Mesenchymal stem cells: cell biology and potential use in therapy. Basic Clin. Pharmacol. Toxicol 95, 209–214.[CrossRef][Medline]
Kim, S. S. et al. (2005). cAMP induces neuronal differentiation of mesenchymal stem cells via activation of extracellular signal-regulated kinase/MAPK. Neuroreport 16, 1357–1361.[CrossRef][Medline]
Krebs, E. G., and Beavo, J. A. (1979). Phosphorylation-dephosphorylation of enzymes. Annu. Rev. Biochem 48, 923–959.[CrossRef][Medline]
Le, C. L., Polanowska, J., Fabbrizio, E., Olivier, M., Philips, A., Ng, E. E., Classon, M., Geng, Y., and Sardet, C. (1999). Timing of cyclin E gene expression depends on the regulated association of a bipartite repressor element with a novel E2F complex. EMBO J 18, 1878–1890.[CrossRef][Medline]
Leo, A. J., and Grande, D. A. (2006). Mesenchymal stem cells in tissue engineering. Cells Tissues. Organs 183, 112–122.[CrossRef][Medline]
Leonce, S. et al. (2001). Induction of cyclin E and inhibition of DNA synthesis by the novel acronycine derivative S23906–1 precede the irreversible arrest of tumor cells in S phase leading to apoptosis. Mol. Pharmacol 60, 1383–1391.
Lundberg, A. S., and Weinberg, R. A. (1998). Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol. Cell Biol 18, 753–761.
Matsumoto, Y., and Maller, J. L. (2004). A centrosomal localization signal in cyclin E required for Cdk2-independent S phase entry. Science 306, 885–888.
Mazumder, S., DuPree, E. L., and Almasan, A. (2004). A dual role of cyclin E in cell proliferation and apoptosis may provide a target for cancer therapy. Curr. Cancer Drug Targets 4, 65–75.[CrossRef][Medline]
Mazumder, S., Gong, B., and Almasan, A. (2000). Cyclin E induction by genotoxic stress leads to apoptosis of hematopoietic cells. Oncogene 19, 2828–2835.[CrossRef][Medline]
Mazumder, S., Gong, B., Chen, Q., Drazba, J. A., Buchsbaum, J. C., and Almasan, A. (2002). Proteolytic cleavage of cyclin E leads to inactivation of associated kinase activity and amplification of apoptosis in hematopoietic cells. Mol. Cell Biol 22, 2398–2409.
Mazumder, S., Plesca, D., and Almasan, A. (2007). A Jekyll and hyde role of cyclin E in the genotoxic stress response: switching from cell cycle control to apoptosis regulation. Cell Cycle 6, 1437–1442.[Medline]
McKnight, G. S. (1991). Cyclic AMP second messenger systems. Curr. Opin. Cell Biol 3, 213–217.[CrossRef][Medline]
Meloche, S., and Pouyssegur, J. (2007). The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition. Oncogene 26, 3227–3239.[CrossRef][Medline]
Mimeault, M., and Batra, S. K. (2006). Concise review: recent advances on the significance of stem cells in tissue regeneration and cancer therapies. Stem Cells 24, 2319–2345.
Mittnacht, S. (1998). Control of pRB phosphorylation. Curr. Opin. Genet. Dev 8, 21–27.[CrossRef][Medline]
Mohapatra, S., Agrawal, D., and Pledger, W. J. (2001). p27Kip1 regulates T cell proliferation. J. Biol. Chem 276, 21976–21983.
Morgan, D. O. (1997). Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu. Rev. Cell Dev. Biol 13, 261–291.[CrossRef][Medline]
Moroy, T., and Geisen, C. (2004). Cyclin E. Int. J. Biochem. Cell Biol 36, 1424–1439.[CrossRef][Medline]
Mullany, L. K. et al. (2007). Akt-mediated liver growth promotes induction of cyclin E through a novel translational mechanism and a p21-mediated cell cycle arrest. J. Biol. Chem 282, 21244–21252.
Naderi, S., Wang, J. Y., Chen, T. T., Gutzkow, K. B., and Blomhoff, H. K. (2005). cAMP-mediated inhibition of DNA replication and S phase progression: involvement of Rb, p21C:P1, and PCNA. Mol. Biol. Cell 16, 1527–1542.
Nourse, J., Firpo, E., Flanagan, W. M., Coats, S., Polyak, K., Lee, M. H., Massague, J., Crabtree, G. R., and Roberts, J. M. (1994). Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 372, 570–573.[CrossRef][Medline]
Ohtani, K., DeGregori, J., and Nevins, J. R. (1995). Regulation of the cyclin E gene by transcription factor E2F1. Proc. Natl. Acad. Sci. USA 92, 12146–12150.
Ohtsubo, M., Theodoras, A. M., Schumacher, J., Roberts, J. M., and Pagano, M. (1995). Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol. Cell Biol 15, 2612–2624.[Abstract]
Oliva, A., Borriello, A., Zeppetelli, S., Di, F. A., Cortellazzi, P., Ventriglia, V., Criscuolo, M., Zappia, V., and Della, R. F. (2003). Retinoic acid inhibits the growth of bone marrow mesenchymal stem cells and induces p27Kip1 and p16INK4A up-regulation. Mol. Cell Biochem 247, 55–60.[CrossRef][Medline]
Ortega, S., Prieto, I., Odajima, J., Martin, A., Dubus, P., Sotillo, R., Barbero, J. L., Malumbres, M., and Barbacid, M. (2003). Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice. Nat. Genet 35, 25–31.[CrossRef][Medline]
Parisi, T., Beck, A. R., Rougier, N., McNeil, T., Lucian, L., Werb, Z., and Amati, B. (2003). Cyclins E1 and E2 are required for endoreplication in placental trophoblast giant cells. EMBO J 22, 4794–4803.[CrossRef][Medline]
Regan, J. W. (2003). EP2 and EP4 prostanoid receptor signaling. Life Sci 74, 143–153.[CrossRef][Medline]
Rowinsky, E. K. (2005). Targeted induction of apoptosis in cancer management: the emerging role of tumor necrosis factor-related apoptosis-inducing ligand receptor activating agents. J. Clin. Oncol 23, 9394–9407.
Rozengurt, E., Legg, A., Strang, G., and Courtenay-Luck, N. (1981). Cyclic AMP: a mitogenic signal for Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA 78, 4392–4396.
Rydel, R. E., and Greene, L. A. (1988). cAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor. Proc. Natl. Acad. Sci. USA 85, 1257–1261.
Schaffler, A., and Buchler, C. (2007). Concise review: adipose tissue-derived stromal cells–basic and clinical implications for novel cell-based therapies. Stem Cells 25, 818–827.
Seamon, K. B., and Daly, J. W. (1981). Forskolin: a unique diterpene activator of cyclic AMP-generating systems. J. Cyclic. Nucleotide. Res 7, 201–224.[Medline]
Sevetson, B. R., Kong, X., and Lawrence, J. C., Jr. (1993). Increasing cAMP attenuates activation of mitogen-activated protein kinase. Proc. Natl. Acad. Sci. USA 90, 10305–10309.
Sherr, C. J. (1993). Mammalian G1 cyclins. Cell 73, 1059–1065.[Medline]
Sherr, C. J. (1996). Cancer cell cycles. Science 274, 1672–1677.
Sherr, C. J., and Roberts, J. M. (1999). CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13, 1501–1512.
Shi, G. X., Rehmann, H., and Andres, D. A. (2006). A novel cyclic AMP-dependent Epac-Rit signaling pathway contributes to PACAP38-mediated neuronal differentiation. Mol. Cell. Biol 26, 9136–9147.
Siegel, R. M. (2006). Caspases at the crossroads of immune-cell life and death. Nat. Rev. Immunol 6, 308–317.[CrossRef][Medline]
Singer, J. D., Gurian-West, M., Clurman, B., and Roberts, J. M. (1999). Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells. Genes Dev 13, 2375–2387.
Stork, P. J., and Schmitt, J. M. (2002). Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol 12, 258–266.[CrossRef][Medline]
Strickland, S., Smith, K. K., and Marotti, K. R. (1980). Hormonal induction of differentiation in teratocarcinoma stem cells: generation of parietal endoderm by retinoic acid and dibutyryl cAMP. Cell 21, 347–355.[CrossRef][Medline]
van den Heuvel, S., and Harlow, E. (1993). Distinct roles for cyclin-dependent kinases in cell cycle control. Science 262, 2050–2054.
Vossler, M. R., Yao, H., York, R. D., Pan, M. G., Rim, C. S., and Stork, P. J. (1997). cAMP activates MAP kinase and Elk-1 through a B-Raf- and Rap1-dependent pathway. Cell 89, 73–82.[CrossRef][Medline]
Walsh, D. A., Perkins, J. P., and Krebs, E. G. (1968). An adenosine 3',5'-monophosphate-dependant protein kinase from rabbit skeletal muscle. J. Biol. Chem 243, 3763–3765.
Wang, T. T., Tio, M., Lee, W., Beerheide, W., and Udolph, G. (2007). Neural differentiation of mesenchymal-like stem cells from cord blood is mediated by PKA. Biochem. Biophys. Res. Commun 357, 1021–1027.[CrossRef][Medline]
Wang, Y., Maciejewski, B. S., Lee, N., Silbert, O., McKnight, N. L., Frangos, J. A., and Sanchez-Esteban, J. (2006). Strain-induced fetal type II epithelial cell differentiation is mediated via cAMP-PKA-dependent signaling pathway. Am. J. Physiol. Lung Cell Mol. Physiol 291, L820–L827.
Weinberg, R. A. (1995). The retinoblastoma protein and cell cycle control. Cell 81, 323–330.[CrossRef][Medline]
Welcker, M., Singer, J., Loeb, K. R., Grim, J., Bloecher, A., Gurien-West, M., Clurman, B. E., and Roberts, J. M. (2003). Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation. Mol. Cell 12, 381–392.[CrossRef][Medline]
Zarkowska, T., and Mittnacht, S. (1997). Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J. Biol. Chem 272, 12738–12746.
Zetterberg, A., Larsson, O., and Wiman, K. G. (1995). What is the restriction point? Curr. Opin. Cell Biol 7, 835–842.[CrossRef][Medline]
Zhao, L., Yang, S., Zhou, G. Q., Yang, J., Ji, D., Sabatakos, G., and Zhu, T. (2006). Downregulation of cAMP-dependent protein kinase inhibitor gamma is required for BMP-2-induced osteoblastic differentiation. Int. J. Biochem. Cell Biol 38, 2064–2073.[CrossRef][Medline]
Zuk, P. A. et al. (2002). Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell 13, 4279–4295.
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0.001. (B) ASCs were grown for 4 d in the presence or absence of 8-CPT (250 µM). The cells were collected, and the number of cells was determined using a Bürker chamber. The cell counts are presented as mean fold induction ± SD (n = 4). * p 
