cAMP signaling regulates histone H3 phosphorylation and mitotic entry through a disruption of G2 progression

cAMP signaling is known to have significant effects on cell growth, either inhibitory or stimulatory depending on the cell type. Study of cAMP-induced growth inhibition in mammalian somatic cells has focused mainly on the combined role of protein kinase A (PKA) and mitogen-activated protein (MAP) kinases in regulation of progression through the G1 phase of the cell cycle. Here we show that cAMP signaling regulates histone H3 phosphorylation in a cell cycle-dependent fashion, increasing it in quiescent cells but dramatically reducing it in cycling cells. The latter is due to a rapid and dramatic loss of mitotic histone H3 phosphorylation caused by a disruption in G2 progression, as evidenced by the inhibition of mitotic entry and decreased activity of the CyclinB/Cdk1 kinase. The inhibition of G2 progression induced through cAMP signaling is dependent on expression of the catalytic subunit of PKA and is highly sensitive to intracellular cAMP concentration. The mechanism by which G2 progression is inhibited is independent of both DNA damage and MAP kinase signaling. Our results suggest that cAMP signaling activates a G2 checkpoint by a unique mechanism and provide new insight into normal cellular regulation of G2 progression.     

Genistein affects histone modifications on Dickkopf-related protein 1 (DKK1) gene in SW480 human colon cancer cell line

Genistein (GEN) is a plant-derived isoflavone and can block uncontrolled cell growth in colon cancer by inhibiting the WNT signaling pathway. This study aimed to test the hypothesis that the enhanced gene expression of the WNT signaling pathway antagonist, DKK1 by genistein treatment is associated with epigenetic modifications of the gene in colon cancer cells. Genistein treatment induced a concentration-dependent G2 phase arrest in the human colon cancer cell line SW480 and reduced cell proliferation. Results from several other human colon cancer cell lines confirmed the growth inhibitory effects of genistein. Overexpression of DKK1 confirmed its involvement in growth inhibition. Knockdown of DKK1 expression by siRNA slightly induced cell growth. DKK1 gene expression was increased by genistein in SW480 and HCT15 cells. DNA methylation at the DKK1 promoter was not affected by genistein treatment in all the cell lines tested. On the other hand, genistein induced histone H3 acetylation of the DKK1 promoter region in SW480 and HCT15 cells. This indicates that increased histone acetylation is associated with the genistein-induced DKK1 expression. The association between histone acetylation and DKK1 gene expression is confirmed by the histone deacetylase inhibitor trichostatin A (TSA) treatment. In conclusion, genistein treatment decreases cell growth and proliferation in colon cancer cell lines. The effect is associated with the increased DKK1 expression through the induction of histone acetylation at the DKK1 promoter region.     

8-Chloro-cyclic AMP and protein kinase A I-selective cyclic AMP analogs inhibit cancer cell growth through different mechanisms

Cyclic AMP (cAMP) inhibits the proliferation of several tumor cells. We previously reported an antiproliferative effect of PKA I-selective cAMP analogs (8-PIP-cAMP and 8-HA-cAMP) on two human cancer cell lines of different origin. 8-Cl-cAMP, another cAMP analog with known antiproliferative properties, has been investigated as a potential anticancer drug. Here, we compared the antiproliferative effect of 8-Cl-cAMP and the PKA I-selective cAMP analogs in three human cancer cell lines (ARO, NPA and WRO). 8-Cl-cAMP and the PKA I-selective cAMP analogs had similarly potent antiproliferative effects on the BRAF-positive ARO and NPA cells, but not on the BRAF-negative WRO cells, in which only 8-Cl-cAMP consistently inhibited cell growth. While treatment with the PKA I-selective cAMP analogs was associated with growth arrest, 8-Cl-cAMP induced apoptosis. To further investigate the actions of 8-Cl-cAMP and the PKA I-selective cAMP analogs, we analyzed their effects on signaling pathways involved in cell proliferation and apoptosis. Interestingly, the PKA I-selective cAMP analogs, but not 8-Cl-cAMP, inhibited ERK phosphorylation, whereas 8-Cl-cAMP alone induced a progressive phosphorylation of the p38 mitogen-activated protein kinase (MAPK), via activation of AMPK by its metabolite 8-Cl-adenosine. Importantly, the pro-apoptotic effect of 8-Cl-cAMP could be largely prevented by pharmacological inhibition of the p38 MAPK. Altogether, these data suggest that 8-Cl-cAMP and the PKA I-selective cAMP analogs, though of comparable antiproliferative potency, act through different mechanisms. PKA I-selective cAMP analogs induce growth arrest in cells carrying the BRAF oncogene, whereas 8-Cl-cAMP induce apoptosis, apparently through activation of the p38 MAPK pathway.     

Influence of irradiation and pentoxifylline on histone H3 phosphorylation in human tumour cell lines

Phosphorylation of histone H3 at Ser-10 correlates with chromatin condensation and this amino terminal modification is now recognized as a specific marker of mitosis. We have monitored the appearance of cells showing histone H3 phosphorylation in four human tumour cell lines to identify cell cycle progression after irradiation. In the human melanoma cell lines Be11 and MeWo and in the squamous cell carcinoma lines 4197 and 4451 a dose of 7 Gy of Co-gamma irradiation increases the number of cells binding anti-histone H3-P antibody 1-8-fold in a p53-independent manner. In the p53 mutant cell lines MeWo and 4451 H3-P phosphorylated cells can be detected as early as 30 min and show a maximum 1 h post-irradiation. In the cell lines Be11, 4197 and 4451 the early wave of H3 phosphorylated cells is followed by a second wave, which reaches a maximum 4.5-7 h post-irradiation and then declines. These events are attributed to damage-induced cell cycle blocks in the G1 and G2 phase of the cell cycle. Addition of the dose modifying drug pentoxifylline before irradiation increases the appearance of cells showing early and the late H3 phosphorylation. When pentoxifylline is added 12-24 h post-irradiation when the cell cycle blocks have reached their maximum the appearance of cells with phosphorylated H3 increases 3-5-fold in the p53 mutant cell lines MeWo and 4451. These observations are consistent with the function of the drug as a G2 block abrogator. The large H3 phosphorylation signal in p53 mutant cells is consistent with early entry of a cohort of G2 cells into mitosis. The smaller H3-P signal in p53 wild type cells correlates with the lower proportion of stable G2 populations in G1 blocked cells. These results indicate that pentoxifylline influences the appearance of histone H3 phosphorylated cells in a manner strongly dependent on the number of cells in G2 phase. This suggests that addition of pentoxifylline indeed abrogates the G2 block and thereby facilitates early entry into mitosis.     

Mitotic Phosphorylation of Histone H3: Spatio-Temporal Regulation by Mammalian Aurora Kinases

Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G(2) phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Mutation of Ser-10 is essential in Tetrahymena, since it results in abnormal chromosome segregation and extensive chromosome loss during mitosis and meiosis, establishing a strong link between signaling and chromosome dynamics. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. Here we show that the expression of Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G(2)/M transition. During the G(2) phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, our findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system.     

Cooperative regulation of GSK-3 by muscarinic and PDGF receptors is associated with airway myocyte proliferation

Muscarinic receptors and platelet-derived growth factor (PDGF) receptors synergistically induce proliferation of airway smooth muscle (ASM), but the pathways that regulate these effects are not yet completely identified. We hypothesized that glycogen synthase kinase-3 (GSK-3), a kinase that represses several promitogenic signaling pathways in its unphosphorylated form, is cooperatively inhibited by PDGF and muscarinic receptors in immortalized human ASM cell lines. PDGF or methacholine alone induced rapid GSK-3 phosphorylation. This phosphorylation was sustained only for PDGF; however, methacholine potentiated PDGF-induced sustained GSK-3 phosphorylation. Synergistic effects of methacholine also were observed on PDGF-induced retinoblastoma protein (Rb) phosphorylation and cell proliferation. Suppression of GSK-3 inhibitory function using SB 216763 also augmented PDGF-induced Rb phosphorylation and cell cycle progression; this synergy was similar in magnitude to that seen for methacholine with PDGF. GSK-3 phosphorylation induced by methacholine required PKC, since it was abolished by GF 109203X and G? 6976; however, inhibition of PKC had no effect on cell responses to PDGF. PKC inhibition also specifically abolished the synergistic effect of methacholine on PDGF-induced GSK-3 phosphorylation and cell proliferation. Collectively, these results show that GSK-3 plays a key repressive role in ASM cell proliferation. Moreover, muscarinic receptors mediate PKC-dependent GSK-3 inhibition, and this appears to be a primary mechanism underpinning augmentation of PDGF-induced cell growth.     

Intracellular events mediating insulin-like growth factor I-induced oligodendrocyte development: modulation by cyclic AMP

Insulin-like growth factor I (IGF-I) is a potent inducer of oligodendrocyte development and myelination. Although IGF-I intracellular signaling has been well described in several cell types, intracellular mechanisms for IGF-I-induced oligodendrocyte development have not been defined. By using specific inhibitors of intracellular signaling pathways, we report here that the MAPK and phosphatidylinositol 3-kinase signaling pathways are required for the full effect of IGF-I on oligodendrocyte development in primary mixed rat cerebrocortical cell cultures. The MAPK activation, but not the phosphatidylinositol 3-kinase activation, leads to phosphorylation of the cAMP response element-binding protein, which is necessary for IGF-I to induce oligodendrocyte development. cAMP, although it does not show any effect on oligodendrocyte development, has an inhibitory effect on IGF-I-induced oligodendrocyte development that is mediated by the cAMP-dependent protein kinase. Furthermore, cAMP also has an inhibitory effect on IGF-I-dependent MAPK activation. This is a cAMP-dependent protein kinase-independent effect and probably contributes to the cAMP action on IGF-I-induced oligodendrocyte development.     

Phosphorylation of ICBP90 by protein kinase A enhances topoisomerase IIalpha expression

Inverted CCAAT box binding protein of 90kDa (ICBP90) is a nuclear protein involved in the topoisomerase IIalpha (TopoIIalpha) gene expression. It belongs to a family of E3 ligases of the RING finger type and its expression is deregulated in cancer cells. Previous studies have shown that high expression of ICBP90 may impair the control of G1/S transition of the cell cycle in various cancer cell lines. Since PKA signaling pathway is involved in G1/S transition of the cell cycle, the aim of the present study was to investigate whether cAMP signaling pathways involve phosphorylation of ICBP90. Here, we show that phosphorylation of ICBP90 through the cAMP signaling pathway accelerates exit of forskolin-treated cells from the G1 phase and increases binding of ICBP90 to the ICB2 element of the TopoIIalpha gene promoter with a subsequent increase of TopoIIalpha expression. We identify S298 of ICBP90 as target for PKA. We propose that cAMP signaling pathway enhances TopoIIalpha expression through ICBP90 phosphorylation, which may be one of the major events involved in the G1/S transition.     

Site-specific loss of acetylation upon phosphorylation of histone H3

Post-translational modification of histones is a central aspect of gene regulation. Emerging data indicate that modification at one site can influence modification of a second site. As one example, histone H3 phosphorylation at serine 10 (Ser(10)) facilitates acetylation of lysine 14 (Lys(14)) by Gcn5 in vitro (, ). In vivo, phosphorylation of H3 precedes acetylation at certain promoters. Whether H3 phosphorylation globally affects acetylation, or whether it affects all acetylation sites in H3 equally, is not known. We have taken a genetic approach to this question by mutating Ser(10) in H3 to fix either a negative or a neutral charge at this position, followed by analysis of the acetylation states of the mutant histones using site-specific antibodies. Surprisingly, we find that conversion of Ser(10) to glutamate (S10E) or aspartate (S10D) causes almost complete loss of H3 acetylation at lysine 9 (Lys(9)) in vivo. Acetylation of Lys(9) is also significantly reduced in cells bearing mutations in the Glc7 phosphatase that increase H3 phosphorylation levels. Mutation of Ser(10) in H3 and the concomitant loss of Lys(9) acetylation has minimal effects on expression of a Gcn5-dependent reporter gene. However, synergistic growth defects are observed upon loss of GCN5 in cells bearing H3 Ser(10) mutations that are reminiscent of delays in G(2)/M progression caused by combined loss of GCN5 and acetylation site mutations. Together these results demonstrate that H3 phosphorylation directly causes site-specific and opposite changes in acetylation levels of two residues within this histone, Lys(9) and Lys(14), and they highlight the importance of these histone modifications to normal cell functions.     

The isoflavonoids genistein and quercetin activate different stress signaling pathways as shown by analysis of site-specific phosphorylation of ATM, p53 and histone H2AX

The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53. Here, we show that genistein also induces phosphorylation of p53 at serines 6, 9, 20, 46, and 392, and that genistein-induced accumulation and phosphorylation of p53 is reduced in two ATM-deficient human cell lines. Also, we show that genistein induces phosphorylation of ATM on serine 1981 and phosphorylation of histone H2AX on serine 139. The related bioflavonoids, daidzein and biochanin A, did not induce either phosphorylation of p53 or ATM at these sites. Like genistein, quercetin induced phosphorylation of ATM on serine 1981, and ATM-dependent phosphorylation of histone H2AX on serine 139; however, p53 accumulation and phosphorylation on serines 6, 9, 15, 20, 46, and 392 occurred in ATM-deficient cells, indicating that ATM is not required for quercetin-induced phosphorylation of p53. Our data suggest that genistein and quercetin induce different DNA-damage induced signaling pathways that, in the case of genistein, are highly ATM-dependent but, in the case of quercetin, may be ATM-dependent only for some downstream targets.     

RGS19 enhances cell proliferation through its C-terminal PDZ motif

Regulator of G protein signaling 19 (RGS19), also known as Gα-interacting protein (GAIP), is a GTPase activating protein (GAP) for Gα_i subunits. Apart from its GAP function, RGS19 has been implicated in growth factor signaling through binding to GAIP-interacting protein C-terminus (GIPC) via its C-terminal PDZ-binding motif. To gain additional insight on its function, we have stably expressed RGS19 in a number of mammalian cell lines and examined its effect on cell proliferation. Interestingly, overexpression of RGS19 stimulated the growth of HEK293, PC12, Caco2, and NIH3T3 cells. This growth promoting effect was not shared by other RGS proteins including RGS4, RGS10 and RGS20. Despite its ability to stimulate cell proliferation, RGS19 failed to induce neoplastic transformation in NIH3T3 cells as determined by focus formation and soft-agar assays, and it did not induce tumor growth in athymic nude mice. Deletion mutants of RGS19 lacking the PDZ-binding motif failed to complex with GIPC and did not exhibit any growth promoting effect. Overexpression of GIPC alone in HEK293 cells stimulated cell proliferation whereas its knockdown in H1299 non-small cell lung carcinomas suppressed cell proliferation. This study demonstrates that RGS19, in addition to acting as a GAP, is able to stimulate cell proliferation in a GIPC-dependent manner.     

Parietal cell hyperstimulation and autoimmune gastritis in cholera toxin transgenic mice

The stimulation of gastric acid secretion from parietal cells involves both intracellular calcium and cAMP signaling. To understand the effect of increased cAMP on parietal cell function, we engineered transgenic mice expressing cholera toxin (Ctox), an irreversible stimulator of adenylate cyclase. The parietal cell-specific H(+),K(+)-ATPase beta-subunit promoter was used to drive expression of the cholera toxin A1 subunit (CtoxA1). Transgenic lines were established and tested for Ctox expression, acid content, plasma gastrin, tissue morphology, and cellular composition of the gastric mucosa. Four lines were generated, with Ctox-7 expressing approximately 50-fold higher Ctox than the other lines. Enhanced cAMP signaling in parietal cells was confirmed by observation of hyperphosphorylation of the protein kinase A-regulated proteins LASP-1 and CREB. Basal acid content was elevated and circulating gastrin was reduced in Ctox transgenic lines. Analysis of gastric morphology revealed a progressive cellular transformation in Ctox-7. Expanded patches of mucous neck cells were observed as early as 3 mo of age, and by 15 mo, extensive mucous cell metaplasia was observed in parallel with almost complete loss of parietal and chief cells. Detection of anti-parietal cell antibodies, inflammatory cell infiltrates, and increased expression of the Th1 cytokine IFN-gamma in Ctox-7 mice suggested that autoimmune destruction of the tissue caused atrophic gastritis. Thus constitutively high parietal cell cAMP results in high acid secretion and a compensatory reduction in circulating gastrin. High Ctox in parietal cells can also induce progressive changes in the cellular architecture of the gastric glands, corresponding to the development of anti-parietal cell antibodies and autoimmune gastritis.     

Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3′-5′-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity.     

Inorganic phosphate inhibits growth of human osteosarcoma U2OS cells via adenylate cyclase/cAMP pathway

In order to elucidate how phosphate regulates cellular functions, we investigated the effects of inorganic phosphate (Pi) on adenylate cyclase (AC)/cyclic AMP (cAMP) axis. Here we describe that Pi treatment of human osteosarcoma U2OS cells results in a decrease of both intracellular cAMP levels and AC activity, and in a cell growth inhibition. The phosphate-triggered effects observed in U2OS cells are not a widespread phenomenon regarding all cell lines, since other cell lines screened respond differently to parallel Pi treatments. In U2OS cell line, the AC activity/cAMP downregulation is accompanied by significant variations in the levels of some membrane proteins belonging to the AC system. Remarkably, the above effects are blunted by pharmacological inhibition of sodium-dependent phosphate transport. Moreover, 8-Br-cAMP and other cAMP-elevating agents, such as IBMX and forskolin, interestingly, prevent the cell growth inhibition in response to phosphate. Our results enforce the increasing evidences of phosphate as a signaling molecule, identifying in U2OS cell line the AC/cAMP axis, as a novel-signaling pathway modulated by phosphate to ultimately affect cell growth.