Culture of zygotes increases TRP53 [corrected] expression in B6 mouse embryos, which reduces embryo viability

The expression of TRP53 in blastocysts that had been cultured from the zygote stage in vitro for 90 h was compared with that in blastocysts collected from the uterus in C57BL6 (B6) and in F1 hybrid (B6CBF1) strain mice. In both strains, there was little TRP53 detected in blastocysts collected from the uterus. There was some increased expression in cultured embryos from B6CBF1 mice and marked increased expression in cultured B6 blastocysts. In cultured B6 embryos, there was obvious accumulation of TRP53 within the nuclear region of embryonic cells. Cultured B6 zygotes had significantly poorer rates of blastocyst formation and of capacity to undergo implantation or form viable fetuses than cultured zygotes from B6CBF1 mice or B6 blastocysts collected from the uterus. Trp53-/- zygotes (B6 background) were significantly more likely to form blastocysts than sibling wild-type embryos, with Trp53+/- embryos having an intermediate level of viability (P<0.01). On transfer of blastocysts to recipient females, Trp53-/- blastocysts were more likely to form viable fetuses than wild-type or heterozygous sibling blastocysts when the embryos resulted from culture of zygotes (P<0.001). This shift in viability did not occur when embryos were only subjected to 24 h of culture from the compacted embryo stage. Culture in vitro in the B6 strain caused a marked increase in the expression and nuclear accumulation of TRP53. This expression was a significant cause of the loss of viability that occurs on culture of zygotes from this strain in vitro.     

PP2Cdelta (Ppm1d, WIP1), an endogenous inhibitor of p38 MAPK, is regulated along with Trp53 and Cdkn2a following p38 MAPK inhibition during mouse preimplantation development

Preimplantation embryos utilize mitogen-activated protein kinase signaling (MAPK) pathways to relay signals from the external environment to prepare appropriate responses and adaptations to a changing milieu. It is therefore important to investigate how MAPK pathways are regulated during preimplantation development. This study was conducted to investigate whether PP2Cdelta (Ppm1d, WIP1) is expressed during mouse preimplantation development and to determine the influences of p38 MAPK inhibition on expression of Trp53 (p53), Ppm1d, (WIP1), and Cdkn2a (p16) during mouse preimplantation development. Our results indicate that Trp53, Ppm1d, and Cdkn2a mRNAs and TRP53 and PP2Cdelta proteins are expressed throughout mouse preimplantation development. Treatment of 2-cell embryos with SB220025 (potent inhibitor of p38 MAPK alpha/beta/MAPK 14/11) significantly increased Trp53, Ppm1d and Cdkn2a and Mapk14 mRNA levels at 12 and 24 hr. Treatment of 8-cell embryos with SB220025 for 12 hr increased Trp53, Ppm1d, and Cdkn2a mRNA levels, but not Mapk14 mRNA levels. Treatment of 8-cell embryos for 24 hr increased Trp53, and Ppm1d mRNA levels, but decreased Cdkn2a and Mapk14 mRNA levels. Therefore, blockade of p38 MAPK activity is associated with embryo stage specific influences on Trp53, Ppm1d, Cdkn2a, and Mapk14 expression during mouse preimplantation development. These results define downstream targets of p38 MAPK during preimplantation development and indicate that the p38 MAPK pathway regulates Trp53, Ppm1d, and Cdkn2a expression. This study increases our understanding of the mechanisms controlling preimplantation development and of the interactions between preimplantation embryos and their culture environments.     

AKT signaling promotes derivation of embryonic germ cells from primordial germ cells

Primordial germ cells (PGCs) are embryonic germ cell precursors. Although the developmental potency of PGCs is restricted to the germ lineage, PGCs can acquire pluripotency, as verified by the in vitro establishment of embryonic germ (EG) cells and the in vivo production of testicular teratomas. PGC-specific inactivation of PTEN, which is a lipid phosphatase antagonizing phosphoinositide-3 kinase (PI3K), enhances both EG cell production and testicular teratoma formation. Here, we analyzed the effect of the serine/threonine kinase AKT, one of the major downstream effectors of PI3K, on the developmental potency of PGCs. We used transgenic mice that expressed an AKT-MER fusion protein, the kinase activity of which could be regulated by the ligand of modified estrogen receptor (MER), 4-hydroxytamoxifen. We found that hyperactivation of AKT signaling in PGCs at the proliferative phase dramatically augmented the efficiency of EG cell establishment. Furthermore, AKT signaling activation substituted to some extent for the effects of bFGF, an essential growth factor for EG cell establishment. By contrast, AKT activation had no effect on germ cells that were in mitotic arrest or that began meiosis at a later embryonic stage. In the transgenic PGCs, AKT activation induced phosphorylation of GSK3, which inhibits its kinase activity; enhanced the stability and nuclear localization of MDM2; and suppressed p53 phosphorylation, which is required for its activation. The p53 deficiency, but not GSK3 inhibition, recapitulated the effects of AKT hyperactivation on EG cell derivation, suggesting that p53 is one of the crucial downstream targets of the PI3K/AKT signal and that GSK3 is not.     

Direct evidence for the action of phosphatidylinositol (3,4,5)-trisphosphate-mediated signal transduction in the 2-cell mouse embryo

Paf (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine) is a putative autocrine survival factor for the preimplantation embryo. It acts to induce receptor-mediated calcium transients in the early embryo. Inhibitors of 1-o-phosphatidylinositol-3-kinase (PI3kinase), such as wortmannin and LY 294002, blocked these calcium transients, implicating the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in autocrine signal transduction in the early embryo. Perfusion of the embryo cytoplasm with a blocking antibody to PIP3 inhibited paf-induced calcium transients and hyperpolarization of the membrane potential. Furthermore, direct infusion of PIP3 into the embryo induced a nifedipine (10 micromol/L)- and diltiazem (10 micromol/L)-sensitive calcium current in the 2-cell embryo. PIP3 acts as a docking site on membranes for proteins that contain pleckstrin homology domains, such as the thymoma viral proto-oncogene protein (AKT) and phospholipase C gamma. The 2-cell embryo expressed three genes for AKT (Akt 1-3) and two genes for phospholipase C gamma (Plcg1 and Plcg2), and we confirmed the expression of both AKT and phospholipase C gamma 1 by immunolocalization. Paf induced increased accumulation of serine 473-phosphorylated AKT in the region of the plasma membrane, consistent with its recruitment to membrane PIP3. Inhibitors of PI3kinase, such as LY294002, and of AKT, e.g., deguelin and AKT-inhibitor, reduced zygote development in a dose-dependent manner, and this inhibition was partially reversed by the addition of paf to the culture medium. These results provide the first direct evidence that PIP3 and its responsive signaling pathways act in the 2-cell embryo. Since signal transduction via PI3kinase has important roles in governing the cell survival pathways, these results support the hypothesis that autocrine embryotropins, such as paf, act as survival factors.     

A novel site of AKT-mediated phosphorylation in the human MDM2 onco-protein

MDM2 is an E3 ubiquitin ligase which mediates ubiquitylation and proteasome-dependent degradation of the p53 tumor suppressor protein. Phosphorylation of MDM2 by the protein kinase AKT is thought to regulate MDM2 function in response to survival signals, but there has been uncertainty concerning the identity of the sites phosphorylated by AKT. In the present study, we identify Ser-166, a site previously reported as an AKT target, and Ser-188, a novel site which is the major site of phosphorylation of MDM2 by AKT in vitro. Analysis of MDM2 in cultured cells confirms that Ser-166 and Ser-188 are phosphorylated by AKT in a physiological context.     

Variable expressivity of the tumour suppressor protein TRP53 in cryopreserved human blastocysts

In a mouse model, in vitro fertilization or extended embryo culture leads to the increased expression of TRP53 in susceptible embryos. Ablation of the TRP53 gene improved embryo viability indicating that increased expression of TRP53 is a cause of the reduction of embryo viability resulting from in vitro fertilization or embryo culture. This study investigates the status of TRP53 expression in human embryos produced by intracytoplasmic sperm injection. Following fertilization, embryos were cultured for 96 h and then cryopreserved. Immediately upon thawing they were fixed in formaldehyde and subjected to immunostaining for TRP53. Staining was visualized by confocal microscopy. Negative controls were incubated with isotype control immunoglobulin and showed negligible staining. All embryos showed TRP53 staining above negative controls. TRP53 staining was heterogenous within and between embryos. An embryo that showed retarded development showed high levels of TRP53 expression. A blastocyst that had a collapsed blastocoel also showed high levels of TRP53 compared to morphologically normal blastocysts. Most TRP53 staining was in the region of the nucleus. Morphologically normal blastocysts tended to show little nuclear accumulation of stain. However, some cells within these embryos had high levels of nuclear TRP53 expression. The results show that embryos have varying sensitivity to the stresses of production and culture in vitro, and this resulted in variable expressivity of TRP53.     

The effect of Trp53 gene-dosage and parent-of-origin of inheritance on mouse gamete and embryo function in vitro

The transformation-related protein 53 (TRP53) has a canonical role as the "guardian of the genome," serving to protect against the propagation of cells with genomic damage. Autocrine trophic signals act to block the accumulation of TRP53 in the normal preimplantation embryo. Culture of the early embryo at limiting dilutions in simple defined media limits autocrine signaling, resulting in the accumulation of TRP53. This TRP53 reduces the rate of development of embryos. In this study we show that deletion of the Trp53 gene improved development in vitro in a dose-dependent manner. Development to morphological blastocysts increased as the dose of Trp53 was reduced, and this was accompanied by a Trp53-dependent increase in the allocation of cells to the inner cell mass. The intermediate developmental response of heterozygous mice provides evidence for haploinsufficiency of this trait. This haploinsufficiency was evident irrespective of the parent-of-origin of the null allele; however, zygotes with paternal inheritance of the Trp53-null allele had better development in vitro than those with maternal inheritance. There was a beneficial effect of the Trp53-null allele on the number of oocytes released by Trp53(+/-) females, and heterozygous males produced higher fertilization rates than controls, although this was independent of the genotype of the fertilizing sperm. The study shows that ovulation induction or culture of embryos in limiting conditions creates conditions that favor the early development of embryos inheriting loss of Trp53 function. This occurs even in the heterozygous state, showing that the conditions provide a potential basis for accelerated accumulation of deleterious mutations within a population.     

Knockdown of sphingosine kinase 1 inhibits the migration and invasion of human rheumatoid arthritis fibroblast-like synoviocytes by down-regulating the PI3K/AKT activation and MMP-2/9 production in vitro

To investigate the potential regulation of sphingosine kinase 1 (SPHK1) on the migration, invasion, and matrix metalloproteinase (MMP) expression in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). RA-FLS were transfected control siRNA or SPHK1 siRNA. The migration and invasion of unmanipulated control, control siRNA or SPHK1 siRNA- transfected RA-FLS in vitro were measured by the transwell system. The relative levels of SPHK1, PI3K, and AKT as well as AKT phosphorylation in RA-FLS were determined by Western blot. The levels of MMP-2/9 secreted by RA-FLS were detected by ELISA. Knockdown of SPHK1 significantly inhibited the spontaneous migration and invasion of RA-FLS, accompanied by significantly reduced levels of PI3K expression and AKT phosphorylation. Similarly, treatment with LY294002, an inhibitor of the PI3K/AKT pathway, inhibited the migration and invasion of RA-FLS. Knockdown of SPHK1 and treatment with the inhibitor synergistically inhibited the migration and invasion of RA-FLS, by further reducing the levels of PI3K expression and AKT phosphorylation. In addition, knockdown of SPHK1 or treatment with LY294002 inhibited the secretion of MMP-2 and MMP-9, and both synergistically reduced the production of MMP-2 and MMP-9 in RA-FLS in vitro. Knockdown of SPHK1 expression inhibits the PI3K/AKT activation, MMP-2 and MMP-9 expression, and human RA-FLS migration and invasion in vitro. Potentially, SPHK1 may be a novel therapeutic target for RA.     

Endogenously activated mGlu5 metabotropic glutamate receptors sustain the increase in c-Myc expression induced by leukaemia inhibitory factor in cultured mouse embryonic stem cells

We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-3-K, both effects resulting from the activation of PKC.     

Uteroplacental insufficiency increases p53 phosphorylation without triggering the p53-MDM2 functional circuit response in the IUGR rat kidney

Uteroplacental insufficiency (UPI) leads to intrauterine growth restriction (IUGR), which predisposes infants toward renal insufficiency early in life and increases the risk of kidney-related adult morbidities, such as hypertension. This compromised in utero environment has been demonstrated to impair nephrogenesis, as evidenced by a reduced nephron endowment in humans and in rats rendered IUGR by UPI. Concordantly, we have observed that IUGR rats have increased kidney p53 protein levels associated with increased apoptosis. Several factors can regulate p53 gene expression and activity, including posttranslational modifications and protein-protein interactions in the cell. Among these, two important mechanisms are 1) phosphorylation of the amino terminal serine 15 [phospho-p53 (Ser15)], which increases p53 stability and apoptotic activity, and 2) the murine double-minute (MDM2) functional circuit that limits further p53-induced apoptosis by promoting proteosomal degradation of p53. We hypothesize that UPI induces an increase in phospho-p53 (Ser15) in association with an absent MDM2 response, predisposing the kidney to increased apoptosis. To test our hypothesis, we induced IUGR through bilateral uterine artery ligation of the pregnant rat. UPI significantly increased phospho-p53 (Ser15), as well as ataxia teleangiectasia-mutated kinase/A-T-related kinase and dsDNA-activated protein kinase kinase levels, which induce phosphorylation of p53. In contrast, UPI induced no increase in kidney MDM2 mRNA and protein levels in IUGR pups. We conclude that among multiple mechanisms that affect nephrogenesis, UPI induces an increase in p53 phosphorylation without a corresponding increase in MDM2 expression, and we speculate that this response may contribute to the increased apoptosis previously described in the IUGR kidney.     

Involvement of protein kinase B/AKT in early development of mouse fertilized eggs

The activation of AKT (also called protein kinase B) is thought to be a critical step in the phosphoinositide 3-kinase pathway that regulates cell growth and differentiation. In this report, we investigated the role of AKT in the regulation of mouse early embryo development. Injection of mRNA coding for a constitutively active myristoylated AKT (myr-Akt1) into one-cell stage fertilized eggs induced cell division more effectively than injection of wild-type AKT (Akt1-WT) mRNA, whereas microinjection of mRNA of kinase-deficient AKT (Akt1-KD) delayed the first mitotic division. Meanwhile, microinjection of different kinds of mRNA of AKT affected the phosphorylation status of CDC2A-Tyr15 and the activation of M-phase promoting factor (MPF). To investigate the intermediate factor between AKT and MPF, we then injected one-cell stage eggs first with Akt1-WT mRNA or myr-Akt1 mRNA and then with mRNA encoding either wild-type CDC25B (Cdc25b-WT) or a AKT-nonphosphorylatable Ser351 to Ala CDC25B mutant (Cdc25b-S351A). Cdc25b-S351A strongly inhibited the effect of AKT. Therefore, AKT causes the activation of MPF and strongly promotes the development of one-cell stage mouse fertilized eggs by inducing AKT-dependent phosphorylation of CDC25B, a member of the CDC25 phosphatase family. Our finding that CDC25B acts as a potential target of AKT provides new insight into the effect of AKT in the regulation of early development of mouse embryos.     

Rapamycin Inhibits IGF-1-Mediated Up-Regulation of MDM2 and Sensitizes Cancer Cells to Chemotherapy

The Murine Double Minute 2 (MDM2) protein is a key regulator of cell proliferation and apoptosis that acts primarily by inhibiting the p53 tumor suppressor. Similarly, the PI3-Kinase (PI3K)/AKT pathway is critical for growth factor-mediated cell survival. Additionally, it has been reported that AKT can directly phosphorylate and activate MDM2. In this study, we show that IGF-1 up-regulates MDM2 protein levels in a PI3K/AKT-dependent manner. Inhibition of mTOR by rapamycin or expression of a dominant negative eukaryotic initiation factor 4E binding protein 1 (4EBP1) mutant protein, as well as ablation of eukaryotic initiation factor 4E (eIF4E), efficiently abolishes IGF-1-mediated up-regulation of MDM2. In addition, we show that rapamycin effectively inhibits MDM2 expression and sensitizes cancer cells to chemotherapy. Taken together, this study reveals a novel mechanism by which IGF-1 activates MDM2 via the mTOR pathway, and that pharmacologic inhibition of mTOR combined with chemotherapy may be more effective in treatment of a subset of cancers harboring increased MDM2 activation.