Ginsenosides promote proliferation of chicken primordial germ cells via PKC-involved activation of NF-kappaB

The effect of ginsenosides on proliferation of chicken primordial germ cells (PGCs) was evaluated and involvement of nuclear factor (NF)-kappaB in the signaling pathway was investigated. PGCs were isolated from the genital ridge of 3.5-4 day embryos and cultured in Medium 199 supplemented with 5% FCS and 10 ng/ml LIF. PGCs subcultured on chicken embryonic fibroblast feeder were challenged with ginsenosides alone or in combination with PKC inhibitor H(7) or activator phorbol 12-myristate 13-acetate (PMA) for 24h. Moreover, the translocation of NF-kappaB and degradation level of IkappaBalpha were investigated by Western blot analysis. Results show that PGCs were identified by periodic acid-Schiff, alkaline phosphatase histochemistry as well as c-kit, SSEA-1 and Oct-4 immunocytochemistry. Treatment with ginsenosides at 1-100 microg/ml significantly increased the number and area of PGC colonies in a dose-dependent manner. However, this proliferating effect was obviously attenuated by combined treatment of H(7) (10(-7)-10(-5)M). Similarly, PKC staining of PGC colonies was more intensive after ginsenosides treatment compared with the control group. In addition, treatment with ginsenosides at 1-10 microg/ml stimulated the translocation of NF-kappaB (p65). However, the NF-kappaB translocation and the degradation of IkappaBalpha were significantly blocked by combined treatment with 10(-6)M H(7). These results indicated that ginsenosides promote proliferation of chicken PGCs through activation of PKC-involved NF-kappaB signaling pathway.     

Interactive actions of prostaglandin and epidermal growth factor to enhance proliferation of granulosa cells from chicken prehierarchical follicles

The interactive actions of prostaglandin (PG) and epidermal growth factor (EGF) on proliferation of granulosa cells was investigated in prehierarchical small yellow follicles (SYF) of laying hens. The granulosa layers were dispersed into single cells by 12.5 microg/ml collagenase. After 16 h pre-incubation in 0.5% fetal calf serum-supplemented medium, the medium was replaced with serum-free medium. Immunocytochemical staining showed that granulosa cells expressed EGF and its receptor, and their expression was increased by PGE(1) (1-100 ng/ml) or forskolin (10(-7) to 10(-5)M) treatments. EGF receptor was also induced by its ligand EGF. The specific prostaglandin synthase inhibitors SC560 (for COX-1) and NS398 (for COX-2) suppressed EGF-stimulated increase of the granulosa cell number. Furthermore, the effect of EGF was confirmed by the immunocytochemical staining of the proliferating cell nuclear antigen in granulosa cells. Though EGF promoted the expression of both COX-1 and COX-2, the rescue experiment indicated that combined treatment of PGE(1) showed better rescuing effect on NS398 inhibition than SC560 at 10(-6)M, which implies COX-2 plays the predominant role in mediating EGF action. The above results indicate that reciprocal stimulation of intracellular PG and EGF production may enhance proliferation of granulosa cells, hence to facilitate development of chicken prehierarchical follicles.     

Phosphorylation of Mcl‐1 by CDK1–cyclin B1 initiates its Cdc20‐dependent destruction during mitotic arrest

The balance between cell cycle progression and apoptosis is important for both surveillance against genomic defects and responses to drugs that arrest the cell cycle. In this report, we show that the level of the human anti‐apoptotic protein Mcl‐1 is regulated during the cell cycle and peaks at mitosis. Mcl‐1 is phosphorylated at two sites in mitosis, Ser64 and Thr92. Phosphorylation of Thr92 by cyclin‐dependent kinase 1 (CDK1)–cyclin B1 initiates degradation of Mcl‐1 in cells arrested in mitosis by microtubule poisons. Mcl‐1 destruction during mitotic arrest requires proteasome activity and is dependent on Cdc20/Fizzy, which mediates recognition of mitotic substrates by the anaphase‐promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Stabilisation of Mcl‐1 during mitotic arrest by mutation of either Thr92 or a D‐box destruction motif inhibits the induction of apoptosis by microtubule poisons. Thus, phosphorylation of Mcl‐1 by CDK1–cyclin B1 and its APC/C^Cdc20‐mediated destruction initiates apoptosis if a cell fails to resolve mitosis. Regulation of apoptosis, therefore, is linked intrinsically to progression through mitosis and is governed by a temporal mechanism that distinguishes between normal mitosis and prolonged mitotic arrest.     

The expression of MMP‐1 and MMP‐9 is up‐regulated by smooth muscle cells after their cross‐talk with macrophages in high glucose conditions

Patients with diabetes mellitus have an increased risk of myocardial infarction and coronary artery disease‐related death, exhibiting highly vulnerable plaques. Many studies have highlighted the major role of macrophages (MAC) and smooth muscle cells (SMC) and the essential part of metalloproteases (MMPs) in atherosclerotic plaque vulnerability. We hypothesize that in diabetes, the interplay between MAC and SMC in high glucose conditions may modify the expression of MMPs involved in plaque vulnerability. The SMC‐MAC cross‐talk was achieved using trans‐well chambers, where human SMC were grown at the bottom and human MAC in the upper chamber in normal (NG) or high (HG) glucose concentration. After cross‐talk, the conditioned media and cells were isolated and investigated for the expression of MMPs, MCP‐1 and signalling molecules. We found that upon cross‐talk with MAC in HG, SMC exhibit: (i) augmented expression of MMP‐1 and MMP‐9; (ii) significant increase in the enzymatic activity of MMP‐9; (iii) higher levels of soluble MCP‐1 chemokine which is functionally active and involved in MMPs up‐regulation; (iv) activated PKCα signalling pathway which, together with NF‐kB are responsible for MMP‐1 and MMP‐9 up‐regulation, and (v) impaired function of collagen assembly. Taken together, our data indicate that MCP‐1 released by cell cross‐talk in diabetic conditions binds to CCR2 and triggers MMP‐1 and MMP‐9 over‐expression and activity, features that could explain the high vulnerability of atherosclerotic plaque found at diabetic patients.     

Genistein protects ovarian granulosa cells from oxidative stress via cAMP‐PKA signaling

Genistein is an isoflavone that has estrogen (E₂)‐like activity and is beneficial for follicular development, but little is known regarding its function in oxidative stress (OS)‐mediated granulosa cell (GC) injury. Here, we found that after exposure to H₂O₂, Genistein weakened the elevated levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA), which were regarded as the biomarkers for OS, and rescued glutathione (GSH) content and GSH/GSSG ratio accompanying with a simultaneous increase in cyclic adenosine monophosphate (cAMP) level, whereas addition of protein kinase A (PKA) inhibitor H89 impeded the effects of Genistein on the levels of ROS and MDA. Further analysis evidenced that Genistein enhanced the activities of antioxidant enzymes superoxide dismutase (SOD), GSH‐peroxidase (GSH‐Px), and catalase (CAT) in H₂O₂‐treated GCs, but this enhancement was attenuated by H89. Under OS, Genistein improved cell viability and lessened the apoptotic rate of GCs along with a reduction in the activity of Casp3 and levels of Bax and Bad messenger RNA (mRNA), while H89 reversed the above effects. Moreover, Genistein treatment caused an obvious elevation in mitochondrial membrane potential (MMP) followed by a decline in the levels of intracellular mitochondrial superoxide, but H89 inhibited the regulation of Genistein on MMP and mitochondrial superoxide. Supplementation of Genistein promoted the secretion of E₂ and increased the expression of Star and Cyp19a1 mRNA, whereas suppressed the level of progesterone (P₄) accompanied with a decline in the level of Hsd3b1 mRNA expression. H89 blocked the regulation of Genistein on the secretion of E₂ and P₄, and alleviated the ascending of Star and Cyp19a1 elicited by Genistein. Collectively, Genistein protects GCs from OS via cAMP‐PKA signaling.     

Uncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1-arrested CHO cells

Chinese hamster ovary cells have been engineered to inducibly over-express the p21(CIP1) cyclin-dependent kinase inhibitor, to achieve cell cycle arrest and increase cell productivity. In p21(CIP1)-arrested cells production of antibody from a stably integrated lgG4 gene, was enhanced approximately fourfold. The underlying physiological basis for enhanced productivity was investigated by measuring a range of cellular and metabolic parameters. Interestingly, the average cell volume of arrested cells was approximately fourfold greater than that of proliferating cells. This was accompanied by significant increases in mitochondrial mass, mitochondrial activity, and ribosomal protein S6 levels. Our results suggest that p21(CIP1)-induced cell cycle arrest uncouples cell growth from cell-cycle progression, and provides new insight into how improved productivity can be achieved in a cell line commonly used for large-scale production of pharmaceutical proteins.     

Effects of 2-methoxyestradiol on proliferation, apoptosis and gene expression of cyclin B1 and c-Myc in esophageal carcinoma EC9706 cells

2-Methoxyestradiol (2-ME) is an endogenous metabolite of 17β-estradiol. In this study, we determined the antitumour activities of 2-ME on the well-differentiated EC9706 esophageal carcinoma cells in vitro. 2-ME had a strong antiproliferative effect on EC9706 cells and caused an increase in the population of apoptotic cells, detected by flow cytometry. A significant number of cells were blocked in the G(2)/M phase of the cell cycle. 2-ME-treated cells demonstrated an increase in cyclin B1 and c-Myc protein levels, as well as an increase in the percentage of G(2)/M phase. Their up-regulation may be involved in 2-ME-induced apoptosis and G(2)/M cell cycle arrest of the EC9706 cells, and it precedes the onset of apoptosis.     

The effect of Cyclin‐dependent kinase 5 on voltage‐dependent calcium channels in PC12 cells varies according to channel type and cell differentiation state

Cyclin‐dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre‐synaptic terminals triggered by Ca²⁺ influx into the pre‐synaptic cytoplasm through voltage‐dependent Ca²⁺ channels (VDCCs). It is reported that Cdk5 regulates L‐, P/Q‐, or N‐type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca²⁺‐channel property of VDCCs, using PC12 cells expressing endogenous, functional L‐, P/Q‐, and N‐type VDCCs. The Ca²⁺ influx, induced by membrane depolarization with high K⁺, was monitored with a fluorescent Ca²⁺ indicator protein in both undifferentiated and nerve growth factor (NGF)‐differentiated PC12 cells. Overall, Ca²⁺ influx was increased by expression of Cdk5‐p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5‐p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5‐p35 regulates L‐, P/Q‐, or N‐type VDCCs in a cellular context‐dependent manner.

     

DICER1 regulated let‐7 expression levels in p53‐induced cancer repression requires cyclin D1

Let‐7 miRNAs act as tumour suppressors by directly binding to the 3′UTRs of downstream gene products. The regulatory role of let‐7 in downstream gene expression has gained much interest in the cancer research community, as it controls multiple biological functions and determines cell fates. For example, one target of the let‐7 family is cyclin D1, which promotes G0/S cell cycle progression and oncogenesis, was correlated with endoribonuclease DICER1, another target of let‐7. Down‐regulated let‐7 has been identified in many types of tumours, suggesting a feedback loop may exist between let‐7 and cyclin D1. A potential player in the proposed feedback relationship is Dicer, a central regulator of miRNA expression through sequence‐specific silencing. We first identified that DICER1 is the key downstream gene for cyclin D1‐induced let‐7 expression. In addition, we found that let‐7 miRNAs expression decreased because of the p53‐induced cell death response, with deregulated cyclin D1. Our results also showed that cyclin D1 is required for Nutlin‐3 and TAX‐induced let‐7 expression in cancer repression and the cell death response. For the first time, we provide evidence that let‐7 and cyclin D1 form a feedback loop in regulating therapy response of cancer cells and cancer stem cells, and importantly, that alteration of let‐7 expression, mainly caused by cyclin D1, is a sensitive indicator for better chemotherapies response.     

Secalonic acid D blocks embryonic palatal mesenchymal cell-cycle by altering the activity of CDK2 and the expression of p21 and cyclin E

BACKGROUND: The mycotoxin, secalonic acid D (SAD), a known animal and potential human cleft palate (CP)-inducing agent, is produced by Pencillium oxalicum in corn. SAD selectively inhibits proliferation of murine embryonic palatal mesenchymal (MEPM) cells leading to a reduction in cell numbers. These effects can explain the reduction in shelf size and the resulting CP seen in the offspring of SAD-exposed mice. Ability of SAD to inhibit proliferation as well as to block the progression of cells from G1- to S-phase of the cell-cycle were also shown in the human embryonic palatal mesenchymal (HEPM) cells suggesting the potential CP-inducing effect of SAD in human beings METHODS: Gestation day (GD) 12 mouse embryos and HEPM cells were used to test the hypothesis that the cell-cycle block induced by SAD results from a disruption of stage-specific regulatory components both in vivo and in vitro. The effects of SAD on the activity of various cyclin dependent kinases (CDK) and on the levels of various positive (cyclins and CDK) and negative (CDK inhibitors p15, 16, 18, 19, 21, 27, 57) cell-cycle regulators were assessed by performing kinase assays and immunoblots, respectively. RESULTS: In the murine embryonic palates, SAD specifically inhibited G1/S-phase-specific CDK2 activity, reduced the level of cyclin E and tended to increase the level of the CIP/kip CDK inhibitor, p21. In the HEPM cell cultures, exposure to IC50 of SAD significantly affected all of the above targets. In addition, a reduction in the levels/activity of CDK 4/6, a reduction in the levels of cyclins D1, D2, D3, E, A, and all INK4 family proteins, and an increase in the level of the CIP/kip CDK inhibitor, p57, were also seen. CONCLUSIONS: These results suggest that the S-phase-specific cell-cycle proteins CDK2, cyclin E and possibly p21 are the common targets of SAD in murine palatal shelves in vivo and in human embryonic palatal mesenchymal cells in vitro and may be relevant to the pathogenesis of SAD-induced CP.     

Cyclin‐dependent kinase‐activating kinases CDKD;1 and CDKD;3 are essential for preserving mitotic activity in Arabidopsis thaliana

For the full activation of cyclin‐dependent kinases (CDKs), not only cyclin binding but also CDK phosphorylation is required. This activating phosphorylation is mediated by CDK‐activating kinases (CAKs). Arabidopsis has four genes showing similarity to vertebrate‐type CAKs, three CDKDs (CDKD;1‐CDKD;3) and one CDKF (CDKF;1). We previously found that the cdkf;1 mutant is defective in post‐embryonic development, even though the kinase activities of core CDKs remain unchanged relative to the wild type. This raised a question about the involvement of CDKDs in CDK activation in planta. Here we report that the cdkd;1 cdkd;3 double mutant showed gametophytic lethality. Most cdkd;1‐1 cdkd;3‐1 pollen grains were defective in pollen mitosis I and II, producing one‐cell or two‐cell pollen grains that lacked fertilization ability. We also found that the double knock‐out of CDKD;1 and CDKD;3 caused arrest and/or delay in the progression of female gametogenesis at multiple steps. Our genetic analyses revealed that the functions of CDKF;1 and CDKD;1 or CDKD;3 do not overlap, either during gametophyte and embryo development or in post‐embryonic development. Consistent with these analyses, CDKF;1 expression in the cdkd;1‐1 cdkd;3‐1 mutant could not rescue the gametophytic lethality. These results suggest that, in Arabidopsis, CDKD;1 and CDKD;3 function as CAKs controlling mitosis, whereas CDKF;1 plays a distinct role, mainly in post‐embryonic development. We propose that CDKD;1 and CDKD;3 phosphorylate and activate all core CDKs, CDKA, CDKB1 and CDKB2, thereby governing cell cycle progression throughout plant development.     

Coordinated activation of AMP‐activated protein kinase,extracellular signal‐regulated kinase,and autophagy regulates phorbol myristate acetate‐induced differentiation of SH‐SY5Y neuroblastoma cells

We explored the interplay between the intracellular energy sensor AMP‐activated protein kinase (AMPK), extracellular signal‐regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)‐induced neuronal differentiation of SH‐SY5Y human neuroblastoma cells. PMA‐triggered expression of neuronal markers (dopamine transporter, microtubule‐associated protein 2, β‐tubulin) was associated with an autophagic response, measured by the conversion of microtubule‐associated protein light chain 3 (LC3)‐I to autophagosome‐bound LC3‐II, increase in autophagic flux, and expression of autophagy‐related (Atg) proteins Atg7 and beclin‐1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference‐mediated silencing of AMPK suppressed PMA‐induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA‐induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA‐induced differentiation of SH‐SY5Y cells. Therefore, PMA‐induced neuronal differentiation of SH‐SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response.

     

Overlapped and differential expression of cAMP‐dependent kinase‐inhibitor isoforms during avian organogenesis period

Although cAMP‐dependent kinase (PKA) has been known to regulate many biological systems, including patterning, cell differentiation and proliferation, it is not well understood how the spatial‐temporal specificity of the PKA signaling is generated. While the PKA signal activation is regulated in many ways, a direct visualization of PKA activity in situ is not possible. Thus, examinations of PKA regulators will provide indirect, but nonetheless important information to elucidate the distribution of PKA activity. In the present study, three isoforms of PKA‐inhibitor (PKI) genes were identified from avian genome, and their expression patterns were examined during the organogenesis period. PKI genes were strongly expressed in muscle, liver, and nervous system primordia, suggesting their inhibitory roles on the PKA signaling in the development of these tissues.