腹膜淋巴孔与淋巴转归NO-cGMP-Ca2+信号转导途径研究

为研究一氧化氮(nitric oxide,NO)调节大鼠腹膜淋巴孔和淋巴吸收的细胞内信号转导机制,在体外培养的间皮细胞上,利用cGMP检测试剂盒和激光共聚焦扫描显微镜,研究NO对间皮细胞内cGMP和游离钙离子浓度(Ca^2 )的影响;并利用动物实验研究NO-cGMP-Ca^2 通路对大鼠腹膜淋巴孔和淋巴吸收的影响。结果发现：(1)与对照组相比,NO供体Sper NO （spermine／nitric oxide complex)可以剂量依赖性地升高间皮细胞内cGMP的浓度(P＜0．01)。此作用可被可溶性鸟苷酸环化酶(soluble guanylyl cyclase,sGC)特异性抑制剂1H-[1,2,4]噁二唑[4．3-a]喹喔啉-1-one酮(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one,ODQ)阻断(P＜0．05,P＜0．01)。Sper／NO可使间皮细胞内[Ca^2 ],相对水平降低(P＜0．05),但此效应可被ODQ阻断;L-型电压依赖性钙通道阻滞剂nifedipine,可使细胞内的[Ca^2 ];在短时间内迅速明显下降(P＜0．05),达平衡后再加入Sper／NO并不能引起[Ca^2 ];的进一步改变(P＞0．05);(2)NO可以剂量依赖性地提高大鼠膈组织淋巴孔最大分布面积(P＜0．01)和对示踪剂的吸收量(P＜0．05),ODQ可明显抑制NO引起的淋巴孔和淋巴吸收的改变(P＜0．01)。该结果首次发现nifedipine能显著增加淋巴孔最大分布面积(P＜0．01)及膈组织对台盼蓝的吸收量(P＜O．05),而且nifedipine预处理后Sper／NO并不能使淋巴孔的淋巴吸收进一步升高(P＞0．05)。结果提示,NO可以通过降低cGMP水平降低大鼠腹膜间皮[Ca^2 ],且此过程和L-型电压依赖性钙通道有关;NO可通过NO-cGMP-[Ca^2 ],通路,促进淋巴孔的开放和吸收。     

Flow cytometric analysis of X-ray sensitivity in ataxia telangiectasia

Flow cytometric analysis of 5-bromodeoxyuridine (BrdU) incorporation during DNA synthesis was used to characterize the effects of X-rays on cell-cycle kinetics in the DNA-repair deficiency disease ataxia telangiectasia (AT). Cultured fibroblasts from homozygotes (at/at), heterozygotes (at/+) and normal controls (+/+) were either: (1) irradiated, cultured, then pulsed with BrdU and harvested, or (2) pulsed with BrdU, irradiated, cultured and then harvested. Cells were then fixed and stained with both a fluoresceinated monoclonal antibody against BrdU to identify S-phase cells and with propidium diiodide to measure total DNA content. Irradiation of +/+ and at/+ cells induced a similar, transient G2/M arrest detectable within 8 h, which subsequently delayed by 6-8 h the passage of cells into G1 and depleted early S phase. In contrast, at/at cells failed to arrest in G2/M phase and entered the next cell cycle without pausing to repair radiation-induced damage. X-Rays also blocked entry of +/+ G1 cells into S phase, subsequently reducing the total S-phase population. This effect was not observed in at/at cells. These cell-cycle responses to radiation may be of diagnostic use and ultimately may help explain the basic defect in AT.     

cAMP-mediated growth inhibition of a B-lymphoid precursor cell line Reh is associated with an early transient delay in G2/M, followed by an accumulation of cells in G1

This study was undertaken to gain more insight into the effects of cyclic adenosine monophosphate (cAMP) on cell-cycle progression in the B-lymphoid precursor cell line Reh. The adenylate cyclase activator forskolin reduced the proliferation of asynchronously growing Reh cells by 50% after 72 hr culture. Growth inhibition was associated with an accumulation of cells in G1. Furthermore, we demonstrated that forskolin provoked a delay of cells for approximately 10 hr in G2/M prior to the G1 arrest. Two different methods were applied to elucidate how cells in different phases of the cell cycle were affected by an elevated cAMP level. One method was based on centrifugal elutriation, whereby synchronous cell populations from the different phases of the cell cycle were isolated. By the other method, S-phase cells were selectively stained by pulsing asynchronously growing cells with bromo-deoxyuridine (BrdU). The data demonstrate that the position of a cell in the cell cycle is critical in determining how the cell will respond to an elevated cAMP level. Thus cells in G1 at the time forskolin is added are not delayed in G2/M, but they will subsequently accumulate in G1 after 48 hr. Cells given forskolin in G2/m, however, are delayed for 10 hr in G2/M, but they do not accumulate in G1. Cells given forskolin in the S phase are delayed in G2/M as well as arrested in G1. The results suggest that cAMP inhibits growth of the Reh cells by preventing the cells from passing important restriction points located in the G1 and G2 phases of the cell cycle.     

Calcium ion-dependent proliferation of L1210 cells in culture

Maximum growth of L1210 cells in culture required the presence of free extracellular calcium ions. Reducing the free extracellular calcium ion concentration with EGTA served to decrease the growth rate of the cells. The decrease in cell growth was not due to cell death but rather due to the "pile-up" of the L1210 cells in the GO/Gl phase of the cell cycle. With the readdition of excess calcium ions, there was a lag period of 3 to 6 hours before the L1210 cells initiated DNA synthesis or transited from the G0/G1 phase to S-phase. Cells enriched for S and G2/M phase by elutriation and which were incubated in EGTA-containing culture medium, continued through the cell cycle and were blocked in GO/Gl. These data indicate that the proliferation of L1210 cells in culture requires a calcium ion-dependent process to allow movement from the G0/G1 to S-phase of the cell cycle.     

Modulatory effect of linoleic and oleic acid on cell proliferation and lipid metabolism gene expressions in primary bovine satellite cells

This study was performed to elucidate the effects of linoleic acid (LA), oleic acid (OA) and their combination (LA?+?OA) on cell proliferation, apoptosis, necrosis, and the lipid metabolism related gene expression in bovine satellite cells (BSCs), isolated from bovine muscles. Cell viability was significantly increased with the OA and LA treatment. Furthermore, LA?+?OA enhanced cell proliferation in a dose-dependent manner (10 to 100?µM), whereas it lowered at 250?µM. In addition, a cell-cycle analysis showed that 100?µM of LA and OA markedly decreased the G0/G1 phase proportion (62.58% and 61.33%, respectively), compared to controls (68.02%), whereas the S-phase cells’ proportion was increased. The ratio of G2/M phase cells was not significantly different among the groups. Moreover, analyses with AO/EtBr staining showed that no apoptosis occurred. Necrosis were determined by flow cytometry using Annexin V-FITC/PI staining which revealed no early apoptosis in the cells pretreated with LA or OA, but occurred in the LA?+?OA group. We also analyzed the mRNA expression of lipid metabolizing genes such as peroxisome proliferator receptor alfa (PPARα), peroxisome proliferator receptor gamma (PPARγ), acyl-CoA oxidase (ACOX), lipoprotein lipase (LPL), carnitine palmitoyl transferase (CPT-1), and fatty-acid binding protein4 (FABP4), which were upregulated in LA or OA treated cells compared to the control group. In essence, LA and OA alone promote the cell proliferation without any apoptosis and necrosis, which might upregulate the lipid metabolism related gene expressions, and increase fatty-acid oxidation in the BSCs’ lipid metabolism.     

Differential effect of D-penicillamine on the cell kinetic parameters of various normal and transformed cellular types

The cell-growth-inhibitory and phase-specific effects of D-penicillamine on cell-cycle progression were investigated using cell-proliferation patterns, quantitative cell-cycle analysis by flow cytometry, and determination of the mitotic index and binucleate cell fraction of normal (rabbit articular chondrocytes, L 809, rabbit fibroblasts) and transformed (HeLa, L 929) cells. D-penicillamine treatment resulted in an inhibition of growth within a dose range of 5 × 10^?4 M to 7.5 × 10^?3 M. Examination of DNA by flow cytometric analysis revealed that rabbit articular chondrocytes were preferentially arrested in the G0/1 phase of the cell cycle, whereas the other cell lines were blocked in the G2 + M phase; the increase in the proportion of cells with G2 + M DNA content was partially due to an enhancement of binucleate cells, resulting in a cytokinesis perturbation for HeLa and L 929 cells. These results showed that D-penicillamine affects cell proliferation through different events according to cell type.     

Nitric oxide stimulates PC12 cell proliferation via cGMP and inhibits at higher concentrations mainly via energy depletion.

We investigated the mechanisms by which nitric oxide (NO) from an NO donor (DETA/NO) regulates proliferation of pheochromocytoma PC12 cells. The NO donor stimulated proliferation at low concentrations, but reversibly and completely inhibited proliferation at higher concentrations. The stimulation (but not the inhibition) of proliferation was apparently due to NO stimulation of soluble guanylate cyclase to produce cGMP, as it was prevented by a specific cyclase inhibitor (ODQ), and replicated by a cell-permeable form of cGMP. The NO-induced cytostasis was not reversed by inhibitors of MEK kinase or poly(ADP-ribose)polymerase, or by treatments that bypass inhibition of ribonucleotide reductase or ornithine decarboxylase. Cytostatic concentrations of DETA/NO strongly inhibited respiration of PC12 cells, and specific respiratory inhibitors (rotenone, myxothiazol, or azide) caused complete cytostasis. Uridine and pyruvate reversed the cytostasis induced by the specific respiratory inhibitors, but not that induced by DETA/NO. However, the combination of uridine, pyruvate, and N-acetyl-cysteine did reverse DETA/NO-induced cytostasis. DETA/NO strongly and progressively inhibited glycolysis measured by glucose consumption, lactate production, and ATP level, and a specific glycolytic inhibitor (5 mM 2-deoxy-d-glucose) caused complete cytostasis. Our results indicate that NO at low concentrations increases cell proliferation via cGMP, while high concentrations of NO block proliferation via inhibition of both glycolysis and respiration, causing energy depletion.     

Insulin delays the progression of Drosophila cells through G2/M by activating the dTOR/dRaptor complex

In Drosophila and mammals, insulin signalling can increase growth, progression through G1/S, cell size and tissue size. Here, we analyse the way insulin affects cell size and cell-cycle progression in two haemocyte-derived Drosophila cell lines. Surprisingly, we find that although insulin increases cell size, it slows the rate at which these cells increase in number. By using BrdU pulse-chase to label S-phase cells and follow their progression through the cell cycle, we show that insulin delays progression through G2/M, thereby slowing cell division. The ability of insulin to slow progression through G2/M is independent of its ability to stimulate progression through G1/S, so is not a consequence of feedback by the cell-cycle machinery to maintain cell-cycle length. Insulin's effects on progression through G2/M are mediated by dTOR/dRaptor signalling. Partially inhibiting dTOR/dRaptor signalling by dsRNAi or mild rapamycin treatment can increase cell number in cultured haemocytes and the Drosophila wing, respectively. Thus, insulin signalling can influence cell number depending on a balance between its ability to accelerate progression through G1/S and delay progression through G2/M.     

Timed,sequential administration of paclitaxel improves its cytotoxic effectiveness in a cell culture model

Paclitaxel (taxol) is a chemotherapeutic agent frequently used in combination with other anti-neoplastic drugs. It is most effective during the M phase of the cell-cycle and tends to cause synchronization in malignant cells lines. In this study, we investigated whether timed, sequential treatment based on the cell-cycle characteristics could be exploited to enhance the cytotoxic effect of paclitaxel. We characterized the cell-cycle properties of a rapidly multiplying cell line (Sp2, mouse myeloma cells) by propidium-iodide DNA staining such as the lengths of various cell cycle phases and population duplication time. Based on this we designed a paclitaxel treatment protocol that comprised a primary and a secondary, timed treatment. We found that the first paclitaxel treatment synchronized the cells at the G2/M phase but releasing the block by stopping the treatment allowed a large number of cells to enter the next cell-cycle by a synchronized manner. The second treatment was most effective during the time when these cells approached the next G2/M phase and was least effective when it occurred after the peak time of this next G2/M phase. Moreover, we found that after mixing Sp2 cells with another, significantly slower multiplying cell type (Jurkat human T-cell leukemia) at an initial ratio of 1:1, the ratio of the two different cell types could be influenced by timed sequential paclitaxel treatment at will. Our results demonstrate that knowledge of the cell-cycle parameters of a specific malignant cell type could improve the effectivity of the chemotherapy. Implementing timed chemotherapeutic treatments could increase the cytotoxicity on the malignant cells but also decrease the side-effects since other, non-malignant cell types will have different cell-cycle characteristic and be out of synch during the treatment.     

Acetylcholine inhibits cell cycle progression in rat Schwann cells by activation of the M2 receptor subtype

Cultures of Schwann cells from neonatal rat sciatic nerves were treated with acetylcholine agonists and the effects on cell proliferation evaluated. (3)[H]-thymidine incorporation shows that acetylcholine (ACh) receptor agonists inhibit cell proliferation, and FACS analysis demonstrates cell-cycle arrest and accumulation of cells in the G1 phase. The use of arecaidine, a selective agonist of muscarinic M2 receptors reveals that this effect depends mainly on M2 receptor activation. The arecaidine dependent-block in G1 is reversible because removal of arecaidine from the culture medium induces progression to the S phase. The block of the G1-S transition is also characterized by modulation of the expression of several cell-cycle markers. Moreover, treatment with ACh receptor agonist causes both a decrease in the PCNA protein levels in Schwann cell nuclei and an increase in p27 and p53 proteins. Finally, immuno-electron microscopy demonstrates that M2 receptors are expressed by Schwann cells in vivo. These results indicate that ACh, by modulating Schwann cell proliferation through M2 receptor activation, might contribute to their progression to a more differentiated phenotype.     

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.     

Fulfilling the metabolic requirements for cell proliferation

The activity of key metabolic enzymes is regulated by the ubiquitin ligases that control the function of the cyclins; therefore the activity of these ubiquitin ligases explains the coordination of cell-cycle progression with the supply of substrates necessary for cell duplication. APC/C (anaphase-promoting complex/cyclosome)-Cdh1, the ubiquitin ligase that controls G1- to S-phase transition by targeting specific degradation motifs in cell-cycle proteins, also regulates the glycolysis-promoting enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3) and GLS1 (glutaminase 1), a critical enzyme in glutaminolysis. A decrease in the activity of APC/C-Cdh1?in mid-to-late G1 releases both proteins, thus explaining the simultaneous increase in the utilization of glucose and glutamine during cell proliferation. This occurs at a time consistent with the point in G1 that has been described as the nutrient-sensitive restriction point and is responsible for the transition from G1 to S. PFKFB3 is also a substrate at the onset of S-phase for the ubiquitin ligase SCF (Skp1/cullin/F-box)-β-TrCP (β-transducin repeat-containing protein), so that the activity of PFKFB3 is short-lasting, coinciding with a peak in glycolysis in mid-to-late G1, whereas the activity of GLS1 remains high throughout S-phase. The differential regulation of the activity of these proteins indicates that a finely-tuned set of mechanisms is activated to fulfil specific metabolic demands at different stages of the cell cycle. These findings have implications for the understanding of cell proliferation in general and, in particular, of cancer, its prevention and treatment.     

High levels of Cdc7 and Dbf4 proteins can arrest cell-cycle progression

Cdc7-Dbf4 serine/threonine kinase is essential for initiation of DNA replication. It was previously found that overexpression of certain replication proteins such as Cdc6 and Cdt1 in fission yeast resulted in multiple rounds of DNA replication in the absence of mitosis. Since this phenomenon is dependent upon the presence of wild-type Cdc7/Hsk1, we hypothesized that high levels of Cdc7 and/or Dbf4 could also cause multiple rounds of DNA replication, or could facilitate entry into S phase. To test this hypothesis, we transiently overexpressed hamster Cdc7, Dbf4 or both in CHO cells. Direct observations of individual cells by fluorescence microscopy and flow cytometric analysis on cell populations suggest that overexpression of Cdc7 and/or Dbf4 does not result in multiple rounds of DNA replication or facilitating entry into S phase. In contrast, moderately increased levels of Dbf4, but not Cdc7, cause cell-cycle arrest in G2/M. This G2/M arrest coincides with hyperphosphorylation of Cdc2/Cdk1 at Tyr-15, raising the possibility that high levels of Dbf4 may activate a G2/M cell-cycle checkpoint. Further increase in Cdc7 and/or Dbf4 by 2–4 fold can arrest cells in G1 and significantly slow down S-phase progression for the cells already in S phase.     

Essential roles of the nitric oxide (no)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway and the atrial natriuretic peptide (ANP)/cGMP/PKG-Iα autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells

Inappropriate signaling conditions within bone marrow stromal cells (BMSCs) can lead to loss of BMSC survival, contributing to the loss of a proper micro-environmental niche for hematopoietic stem cells (HSCs), ultimately causing bone marrow failure. In the present study, we investigated the novel role of endogenous atrial natriuretic peptide (ANP) and the nitric oxide (NO)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway in regulating BMSC survival and proliferation, using the OP9 BMSC cell line commonly used for facilitating the differentiation of HSCs. Using an ANP-receptor blocker, endogenously produced ANP was found to promote cell proliferation and prevent apoptosis. NO donor SNAP (S-nitroso-N-acetylpenicillamine) at low concentrations (10 and 50 μM), which would moderately stimulate PKG activity, protected these BMSCs against spontaneous apoptosis. YC-1, a soluble guanylyl cyclase (sGC) activator, decreased the levels of apoptosis, similar to the cytoprotective effects of low-level NO. ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one), which blocks endogenous NO-induced activation of sGC and thus lowers endogenous cGMP/PKG activity, significantly elevated apoptotic levels by 2.5- and three-fold. Pre-incubation with 8-Bromo-cGMP or ANP, which bypass the ODQ block, almost completely prevented the ODQ-induced apoptosis. A highly-specific PKG inhibitor, DT-3, at 20, and 30 μM, caused 1.5- and two-fold increases in apoptosis, respectively. ODQ and DT-3 also decreased BMSCs proliferation and colony formation. Small Interfering RNA gene knockdown of PKG-Iα increased apoptosis and decreased proliferation in BMSCs. The data suggest that basal NO/cGMP/PKG-Iα activity and autocrine ANP/cGMP/PKG-Iα are necessary for preserving OP9 cell survival and promoting cell proliferation and migration.     

Cyclin mRNA and protein expression in recombinant interleukin 2-stimulated cloned murine T lymphocytes

Expression of cyclin, a non-histone nuclear protein, during recombinant interleukin 2 (rIL2)-driven cell-cycle progression of cloned T lymphocytes has been assessed. We found that expression of cyclin protein, as detected by immunofluorescence, is tightly associated with proliferation, and not merely S-phase, of L2 cells stimulated with rIL2. Cyclin immunofluorescence was detected in all cell-cycle phases (G1/S/G2/M, as detected by flow cytometry) of proliferating L2 cells. Accumulation of cyclin mRNA levels was induced as early as 1 h after stimulation, was maximal at 25-49 h, and remained elevated throughout stimulation, as detected by Northern blot analysis. A cDNA-encoding murine cyclin was cloned from a cDNA library prepared from IL2-stimulated cloned T cells. The sequence of the 5' end of the murine cyclin cDNA was determined and found to be 88% and 82% similar to the sequences of cDNA clones encoding rat and human cyclin, respectively. The present studies demonstrate that cyclin protein and mRNA accumulation are highly regulated during IL2-induced proliferation of a cloned T cell. These data provide a framework for addressing the molecular mechanisms regulating cyclin gene expression during cellular proliferation.     

Cell cycle function of a Medicago sativa A2-type cyclin interacting with a PSTAIRE-type cyclin-dependent kinase and a retinoblastoma protein

In plants multiple A-type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1-A3), while in animal somatic cells a single type of cyclin A is required for cell-cycle regulation from the S to M phases. We studied the function of an A2-type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A-type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti-Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2-M phase, the protein level after a rapid increase in S-phase reached a plateau during the G2 phase. In the yeast two-hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE-motif-containing cyclin-dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2-associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.     

Modulation of mitochondrial Ca2+ by nitric oxide in cultured bovine vascular endothelial cells

In the present study, we used laser scanning confocal microscopy in combination with fluorescent indicator dyes to investigate the effects of nitric oxide (NO) produced endogenously by stimulation of the mitochondria-specific NO synthase (mtNOS) or applied exogenously through a NO donor, on mitochondrial Ca²⁺ uptake, membrane potential, and gating of mitochondrial permeability transition pore (PTP) in permeabilized cultured calf pulmonary artery endothelial (CPAE) cells. Higher concentrations (100–500 µM) of the NO donor spermine NONOate (Sper/NO) significantly reduced mitochondrial Ca²⁺ uptake and Ca²⁺ extrusion rates, whereas low concentrations of Sper/NO (<100 µM) had no effect on mitochondrial Ca²⁺ levels ([Ca²⁺]_mt). Stimulation of mitochondrial NO production by incubating cells with 1 mM L-arginine also decreased mitochondrial Ca²⁺ uptake, whereas inhibition of mtNOS with 10 µM L-N⁵-(1-iminoethyl)ornithine resulted in a significant increase of [Ca²⁺]_mt. Sper/NO application caused a dose-dependent sustained mitochondrial depolarization as revealed with the voltage-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Blocking mtNOS hyperpolarized basal mitochondrial membrane potential and partially prevented Ca²⁺-induced decrease in TMRE fluorescence. Higher concentrations of Sper/NO (100–500 µM) induced PTP opening, whereas lower concentrations (<100 µM) had no effect. The data demonstrate that in calf pulmonary artery endothelial cells, stimulation of mitochondrial Ca²⁺ uptake can activate NO production in mitochondria that in turn can modulate mitochondrial Ca²⁺ uptake and efflux, demonstrating a negative feedback regulation. This mechanism may be particularly important to protect against mitochondrial Ca²⁺ overload under pathological conditions where cellular [NO] can reach very high levels. nitric oxide synthase; permeability transition pore; endothelium     

MKK7 couples stress signalling to G2/M cell-cycle progression and cellular senescence

During the development of multicellular organisms, concerted actions of molecular signalling networks determine whether cells undergo proliferation, differentiation, death or ageing. Here we show that genetic inactivation of the stress signalling kinase, MKK7, a direct activator of JNKs in mice, results in embryonic lethality and impaired proliferation of hepatocytes. Beginning at passage 4-5, mkk7(-/-) mouse embryonic fibroblasts (MEFs) display impaired proliferation, premature senescence and G2/M cell cycle arrest. Similarly, loss of c-Jun or expression of a c-JunAA mutant in which the JNK phosphorylation sites were replaced with alanine results in a G2/M cell-cycle block. The G2/M cell-cycle kinase CDC2 was identified as a target for the MKK7-JNK-c-Jun pathway. These data show that the MKK7-JNK-c-Jun signalling pathway couples developmental and environmental cues to CDC2 expression, G2/M cell cycle progression and cellular senescence in fibroblasts.