The dual roles of geminin during trophoblast proliferation and differentiation

Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles.     

Protein phosphatase 2A in stretch-induced endothelial cell proliferation

We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459–466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic strain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C. © 1996 Wiley-Liss, Inc.     

Immunomodulation of dual specificity phosphatase 4 during visceral leishmaniasis

DUSP4, an inducible protein has a substrate specificity toward ERK1/2, a component of MAP kinase which is enhanced during Leishmania infection. The DUSP4^?/? mice show increased susceptibility towards the infection caused by Toxoplasma gondii and Leishmania mexicana. These observations emphatically established the fact that unlike DUSP1, DUSP4 has host protective role. In our study, it has been Leishmania donovani, the causative agent of visceral leishmaniasis (VL) significantly reduced the expression of DUSP4 during infection. In order to find out the host protective role of DUSP4 in macrophages during VL, we silenced DUSP4 prior to infection and the parasite number within macrophage was counted. Under DUSP4 knock-down condition, phosphorylation of p38 MAPK and generation of pro-inflammatory response like IL-12, TNF-α, and iNOS was decreased significantly. Silencing DUSP4 promoted the phosphorylation of ERK1/2 and the generation of anti-inflammatory response like- IL-10, TGF-β with increased Arginase-1 and Cox-2 activity. Glycyrrhizic Acid (GA), an immunomodulator, already known to suppress L. donovani infection, found to up-regulate DUSP4 expression during L. donovani infection. On the other hand, GA failed to increase Th1 cytokine production and decrease Th2 response during DUSP4 knock-down condition suggesting the key role of DUSP4 while providing protection during L. donovani infection.     

DDAH1 plays dual roles in PM2.5 induced cell death in A549 cells

BackgroundDimethylarginine dimethylaminohydrolase 1 (DDAH1) is an enzyme that can degrade asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor. Emerging evidence suggests that alterations in the ADMA–DDAH1 pathway are involved in environmental pollution induced airway inflammation. However, the role of DDAH1 in protection against cytotoxicity of ambient airborne particulate matter is unclear.MethodsWe examined the influence of DDAH1 expression on oxidative stress and cell apoptosis in human type II alveolar epithelial A549 cells exposed to PM_2.5 (particulate matter with an aerodynamic diameter less than 2.5 μM).ResultsWe found that PM_2.5 exposure for 48 h significantly decreased DDAH1 expression. However, knockdown of DDAH1 prior to PM_2.5 exposure actually attenuated the cytotoxicity of PM_2.5. Cytoprotection in DDAH1 deficient cells was due to increased reactive oxygen species, activation of PI3K–AKT and mitogen-activated protein kinase (MAPK) pathways, subsequent activation of nuclear factor erythroid-2-related factor 2 (Nrf2) and this caused a subsequent reduction in PM_2.5 induced oxidative stress relative to control. DDAH1 depletion also repressed the induction of inducible NOS (iNOS) in PM_2.5-exposed cells and knockdown of iNOS protected cells against PM_2.5 induced cell death. Interestingly, overexpression of DDAH1 also exerted a protective effect against the cytotoxicity of PM_2.5 and this was associated with a reduction in oxidative stress and upregulation of the anti-apoptotic protein Bcl-2.ConclusionsOur data indicate that DDAH1 plays dual roles in protection against cytotoxicity of PM_2.5 exposure, apparently by limiting PM_2.5 induced oxidative stress.General significanceOur findings reveal new insights into the role(s) of the DDAH1/ADMA in pulmonary protection against airborne pollutants. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.     

Hedgehog signaling plays roles in epithelial cell proliferation in neonatal mouse uterus and vagina

Both the uterus and vagina develop from the Müllerian duct but are quite distinct in morphology and function. To investigate factors controlling epithelial differentiation and cell proliferation in neonatal uterus and vagina, we focused on Hedgehog (HH) signaling. In neonatal mice, Sonic hh (Shh) was localized in the vaginal epithelium and Indian hh (Ihh) was slightly expressed in the uterus and vagina, whereas all Glioma-associated oncogene homolog (Gli) genes were mainly expressed in the stroma. The expression of target genes of HH signaling was high in the neonatal vagina and in the uterus, it increased with growth. Thus, in neonatal mice, Shh in the vaginal epithelium and Ihh in the uterus and vagina activated HH signaling in the stroma. Tissue recombinants showed that vaginal Shh expression was inhibited by the vaginal stroma and uterine Ihh expression was stimulated by the uterine stroma. Addition of a HH signaling inhibitor decreased epithelial cell proliferation in organ-cultured uterus and vagina and increased stromal cell proliferation in organ-cultured uterus. However, it did not affect epithelial differentiation or the expression of growth factors in organ-cultured uterus and vagina. Thus, activated HH signaling stimulates epithelial cell proliferation in neonatal uterus and vagina but inhibits stromal cell proliferation in neonatal uterus.     

SHB1 plays dual roles in photoperiodic and autonomous flowering

Flowering was initiated by the integration of environmental signals such as day-length with the internal development status in Arabidopsis, a facultative long-day plant. The photoperiodic flowering involves two key components, CONSTANS and FT, whereas the autonomous flowering is operated through a central quantitative floral repressor, FLC, and several other genes that act upstream of FLC. SOC1 acts downstream to integrate the flowering signals from the two pathways. Here, we report that SHB1 plays dual roles in both photoperiodic and autonomous flowering. shb1-D, a gain-of-function mutant, flowered early and shb1, a loss-of-function allele, flowered late under both long days and short days. The shb1-D mutation activated the expression of CO, FT, and SOC1 under both long and short days, and however, the co-2 mutation attenuated the shb1-D activated expression of FT and SOC1 only under long days but not short days. The shb1-D or shb1 mutations also reduced and increased, respectively, the expression of FLC under both long and short days. Transgenic remedy of FLC to wide-type level in shb1-D background also reverted shb1-D flowering and FT or SOC1 expression to wild type mostly under short days. Furthermore, the shb1-D suppression on FLC expression is likely operated through LD as ld-3 blocked this suppression and SHB1 appears to act upstream of LD. In summary, SHB1 represents signaling steps that regulate CO expression in leaves and LD or FLC expression in either leaves or shoot apical meristem, contributing to a threshold expression of SOC1 in shoot apical meristem for floral initiation.     

Protein kinase C plays an inhibitory role in interleukin 3- and interleukin 4-mediated mast cell proliferation

Interleukin 3 (IL-3) is required for the survival and proliferation of mouse bone marrow derived mast cells (BMMC). Although interleukin 4 (IL-4) has no direct effect on growth activity, it synergizes with IL-3 in promoting the growth of these cells. The intracellular mechanism by which these ligand-receptor interactions promote mast cell growth are not well documented in the literature. Here we present evidence that both IL-3 and IL-4 have been found to activate protein kinase C (PKC) and phosphatidylinositol turnover in BMMC, in a similar time- and dose-dependent manner, indicating that activation of PKC is not sufficient to induce proliferation in these cells. In this work we addressed the question as to whether the activation of PKC is necessary for mast cell proliferation. Activation of PKC by phorbol myristate acetate causes inhibition of IL-3-mediated growth for the first 72 h of incubation. The inhibition in IL-3-mediated proliferation gradually lessens with the stages of PKC depletion, which is complete after 72 h. The enhancement in phorbol myristate acetate-treated cells grows as PKC is depleted. The inactive phorbol ester, 4-alpha-phorbol, had no effect on proliferation of BMMC. Cells, PKC-depleted by chronical phorbol ester treatment, responded to IL-3 or IL-4 with a significant increase in [3H] thymidine uptake over PKC containing cells stimulated with the same lymphokine. Use of antibodies to these lymphokines showed that the enhanced response of the PKC-depleted BMMC was not due to the additional autocrine production of IL-3 or IL-4 by these cells. The PKC-depleted cells retain the capacity to return to almost normal levels of PKC activity and sensitivity to IL-3 and IL-4, after 72 and 120 h, respectively. These results indicate that PKC plays an important inhibitory role in IL-3- and IL-4-mediated proliferation of BMMC.     

The formin DAD domain plays dual roles in autoinhibition and actin nucleation

Formins are a large family of actin assembly-promoting proteins with many important biological roles. However, it has remained unclear how formins nucleate actin polymerization. All other nucleators are known to recruit actin monomers as a central part of their mechanisms. However, the actin-nucleating FH2 domain of formins lacks appreciable affinity for monomeric actin. Here, we found that yeast and mammalian formins bind actin monomers but that this activity requires their C-terminal DAD domains. Furthermore, we observed that the DAD works in concert with the FH2 to enhance nucleation without affecting the rate of filament elongation. We dissected this mechanism in mDia1, mapped nucleation activity to conserved residues in the DAD, and demonstrated that DAD roles in nucleation and autoinhibition are separable. Furthermore, DAD enhancement of nucleation was independent of contributions from the FH1 domain to nucleation. Together, our data show that (1) the DAD has dual functions in autoinhibition and nucleation; (2) the FH1, FH2, and DAD form a tripartite nucleation machine; and (3) formins nucleate by recruiting actin monomers and therefore are more similar to other nucleators than previously thought.     

Protein phosphatase 4 regulates apoptosis, proliferation and mutation rate of human cells

The proteins which regulate apoptosis are of great importance both in normal cell biological processes and in the development of pathology in the diverse diseases which involve apoptosis dysfunction. The activity of many of these proteins is controlled by reversible phosphorylation, so that the relevant kinases and phosphatases play crucial roles in apoptosis control. Here we report the analysis of the role of the serine/threonine protein phosphatase, protein phosphatase 4, in controlling the apoptosis of HEK 293 T cells, using the complementary techniques of gene over-expression and down regulation through RNA interference. This analysis has demonstrated that PP4 regulates both apoptosis and proliferation in human cells and has also shown that the level of PP4 has a strong influence on gene mutation rate, which is crucial to oncogenesis. A parallel proteomic analysis has shown that the phosphorylation status of many relevant protein targets is affected by changes in PP4 and has focused attention particularly on the critical apoptosis regulators Bad and PEA-15. The phosphorylation of both of these proteins is increased when PP4 levels are suppressed, and is reduced when PP4 levels are increased, with striking consequences for the fate of the cell.     

Live cell imaging reveals plant aurora kinase has dual roles during mitosis

The proper segregation of chromosomes during mitosis is required for accurate distribution of genetic information by two daughter cells. Here, we used live cell imaging of microtubules and kinetochores after treatment with an Aurora kinase inhibitor, hesperadin, in tobacco BY-2 cells to analyze the function of plant Aurora kinase during mitosis. Hesperadin treatment induced the delay of CenH3 alignment on the spindle equator. Furthermore, two types of dynamics of lagging CenH3s were observed during chromosome segregation. The findings indicate that the plant Aurora kinase has dual roles; correction of aberrant kinetochore-microtubule attachment and dissociation of cohesin during chromosome alignment and segregation.     

Hepatocyte growth factor plays a dual role in tendon-derived stem cell proliferation,migration, and differentiation

Heterotopic ossification is common in tendon healing after trauma, but the detailed mechanisms remain unknown. Tendon-derived stem cells (TDSCs) are a type of progenitor cell found in the tendon niche, and their incorrect differentiation after trauma may lead to tendon calcification. The expression of hepatocyte growth factor (HGF) presents drastic fluctuations in serum/tissue after trauma and was found to activate quiescent stellate cells and contribute to wound healing; however, its potential role in TDSCs remains elusive. In this study, TDSCs isolated from rats were cultured in media containing HGF with or without a signaling inhibitor, and the proliferation, migration, and differentiation ability of TDSCs were measured to determine the role and mechanism of HGF in TDSCs. We showed that HGF promotes TDSC proliferation and migration but inhibits TDSC osteogenic differentiation ability. HGF activated-HGF/c-Met, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling, which was positively correlated with TDSCs proliferation and migration but negatively related to TDSC osteogenic differentiation ability. The phosphorylation of Smad1/5/8 was also negatively related to HGF/c-Met, MAPK/ERK1/2, and PI3K/AKT signaling, which demonstrated that the inhibition of osteogenic differentiation was dependent on BMP/Smad1/5/8 signaling. Overall, we showed that HGF could promote TDSCs proliferation and migration and inhibit osteogenic differentiation in vitro, suggesting a potential role for HGF as a cytokine treatment of tendon trauma.     

Temporal variations in protein tyrosine phosphatase activity during cell proliferation and differentiation

Cellular oscillations in total extractable protein content and protein tyrosine phosphatase activity were analysed in proliferating and differentiating human acute promyelocytic leukaemic (HL-60) cell extracts. Differentiation was induced along the granulocytic pathway using all-trans retinoic acid (ATRA). High frequency rhythms with distinct, well defined waveforms of varying amplitude were observed for both the total protein content and for the enzyme activity of protein tyrosine phosphatase (PTP). Linear correlation analysis showed that there was no obvious relationship between the protein content and the corresponding PTP activity, suggesting that the periodic variations between these two components are relatively independent of each other. ATRA significantly altered the characteristics of the rhythms with respect to the period and amplitude and had a dampening effect on the oscillatory pattern of PTP activity. Modulation of such characteristics may be of significance with respect to the regulation of the differentiation processes and the possible reversal of transformation.     

Protein tyrosine phosphorylation during phytohormone-stimulated cell proliferation in Arabidopsis Hypocotyls

Very little is known about the molecular events triggering differentiated cells to re-enter the cell cycle. We have investigated the possible role of tyrosine phosphorylation in this process with hypocotyl explants of Arabidopsis thaliana. Phytohormone-stimulated cell cycle reactivation in hypocotyls was accompanied by tyrosine phosphorylation of several proteins. Such regulation of the tyrosine phosphorylation in these proteins was not observed in a callus-formation-deficient mutant, srd2, a result which suggests that the induction of tyrosine phosphorylation occurs as a specific event in callus cell proliferation. The promoter activity of cyclin-dependent kinase, CDKA;1, was also examined in phytohormone-stimulated hypocotyls. This study highlighted that protein tyrosine phosphorylation may play an important regulatory role in phytohormone-stimulated cell proliferation.     

Mitogen-activated protein kinase phosphatase 1/dual specificity phosphatase 1 mediates glucocorticoid inhibition of osteoblast proliferation

Steroid-induced osteoporosis is a common side effect of long-term treatment with glucocorticoid (GC) drugs. GCs have multiple systemic effects that may influence bone metabolism but also directly affect osteoblasts by decreasing proliferation. This may be beneficial at low concentrations, enhancing differentiation. However, high-dose treatment produces a severe deficit in the proliferative osteoblastic compartment. We provide causal evidence that this effect of GC is mediated by induction of the dual-specificity MAPK phosphatase, MKP-1/DUSP1. Excessive MKP-1 production is both necessary and sufficient to account for the impaired osteoblastic response to mitogens. Overexpression of MKP-1 after either GC treatment or transfection ablates the mitogenic response in osteoblasts. Knockdown of MKP-1 using either immunodepletion of MKP-1 before in vitro dephosphorylation assay or short interference RNA transfection prevents inactivation of ERK by GCs. Neither c-jun N-terminal kinase nor p38 MAPK is activated by the mitogenic cocktail in 20% fetal calf serum, but their activation by a DNA-damaging agent (UV irradiation) was inhibited by either GC treatment or overexpression of MKP-1, indicating regulation of all three MAPKs by MKP-1 in osteoblasts. However, an inhibitor of the MAPK/ERK kinase-ERK pathway inhibited osteoblast proliferation whereas inhibitors of c-jun N-terminal kinase or p38 MAPK had no effect, suggesting that ERK is the MAPK that controls osteoblast proliferation. Regulation of ERK by MKP-1 provides a novel mechanism for control of osteoblast proliferation by GCs.     

Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excision

The binding of Tn916 Xis protein to its specific sites at the left and right ends of the transposon was compared using gel mobility shift assays. Xis formed two complexes with different electrophoretic mobilities with both right and left transposon ends. Complex II, with a reduced mobility, formed at higher concentrations of Xis and appeared at an eightfold lower Xis concentration with a DNA fragment from the left end of the transposon rather than with a DNA fragment from the right end of the transposon, indicating that Xis has a higher affinity for the left end of the transposon. Methylation interference was used to identify two G residues that were essential for binding of Xis to the right end of Tn916. Mutations in these residues reduced binding of Xis. In an in vivo assay, these mutations increased the frequency of excision of a minitransposon from a plasmid, indicating that binding of Xis at the right end of Tn916 inhibits transposon excision. A similar mutation in the specific binding site for Xis at the left end of the transposon did not reduce the affinity of Xis for the site but did perturb binding sufficiently to alter the pattern of protection by Xis from nuclease cleavage. This mutation reduced the level of transposon excision, indicating that binding of Xis to the left end of Tn916 is required for transposon excision. Thus, Xis is required for transposon excision and, at elevated concentrations, can also regulate this process.