Involvement of the Interaction of Afadin with ZO-1 in the Formation of Tight Junctions in Madin-Darby Canine Kidney Cells

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with β- and α-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-ΔPR1–2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.     

Double PHD Fingers Protein DPF2 Recognizes Acetylated Histones and Suppresses the Function of Estrogen-related Receptor α through Histone Deacetylase 1

Estrogen-related receptor α (ERRα) is a member of the nuclear receptor superfamily and regulates many physiological functions, including mitochondrial biogenesis and lipid metabolism. ERRα enhances the transactivation function without endogenous ligand by associating with coactivators such as peroxisome proliferator-activated receptor γ coactivator 1 α and β (PGC-1α and -β) and members of the steroid receptor coactivator family. However, the molecular mechanism by which the transactivation function of ERRα is converted from a repressive state to an active state is poorly understood. Here we used biochemical purification techniques to identify ERRα-associated proteins in HeLa cells stably expressing ERRα. Interestingly, we found that double PHD fingers protein DPF2/BAF45d suppressed PGC-1α-dependent transactivation of ERRα by recognizing acetylated histone H3 and associating with HDAC1. DPF2 directly bound to ERRα and suppressed the transactivation function of nuclear receptors such as androgen receptor. DPF2 was recruited to ERR target gene promoters in myoblast cells, and knockdown of DPF2 derepressed the level of mRNA expressed by target genes of ERRα. These results show that DPF2 acts as a nuclear receptor-selective co-repressor for ERRα by associating with both acetylated histone H3 and HDAC1.     

Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex through LL5α/β

LL5β has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)–bound microtubule plus ends to the cell cortex. In this study, we show that LL5β and its homologue LL5α (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins α3β1 and α6β4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin α3 at the basal cell cortex. Activation of integrin α3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin–integrin associations attaches microtubule plus ends to the epithelial basal cell cortex.     

Antiproliferative activity of root extract from gentian plant (Gentiana triflora) on cultured and implanted tumor cells

We describe a novel pharmacological activity of the gentian root, an ingredient of Chinese medicines. Root extract from Gentiana triflora triggered cell death of human Daudi cells in culture. In addition, daily administration of the extract to mice inhibited growth of implanted solid tumors. Extract treatment of cultured cells resulted in the appearance of shranken, fragmented, or condensed cell and nuclear morphologies, and in chromosomal DNA degradation. But, the extract-treated cells did not show DNA fragmentation, which exhibits a nucleosome ladder, suggesting that extract-triggered cell death is not mediated through a typical apoptotic pathway.     

Effect of clutch size on male egg-fanning behavior and hatching success in the goby, Eviota prasina (Klunzinger)

In fish that exhibit paternal care, the females often choose their mates on the basis of male traits that are indicative of the parental ability of the males. In a marine goby, Eviota prasina, males tend their eggs within their nests until hatching, and females prefer males that have longer dorsal fins and exhibit courtship behavior with a higher frequency as their mates. In order to clarify the relationship between these sexually selected traits and the parental ability of males of E. prasina, the factors affecting the hatching success of eggs within male nests and the male parental care behavior were examined in an aquarium experiment. Females spawned their eggs in male nests and the clutch size of females showed a high individual variation (range = 88-833 eggs). The hatching success of eggs within male nests showed a positive correlation with the time spent by males in fanning eggs and the clutch size. In contrast to the prediction, however, the hatching success did not show a significant correlation with the sexually selected traits, i.e., the male dorsal fin length and the frequency of courtship displays. Moreover, multiple regression analysis indicated that the time spent by the males in fanning was the most important factor affecting the survival rate of the eggs. The time spent by males in fanning behavior was influenced by the clutch size within their nests; the fanning behavior of males occurred with a higher frequency when they tended larger clutches. Males are required to invest a greater effort in egg-tending behavior to achieve a higher hatching success when they receive larger clutches, probably due to the greater reward for their parental behavior. Based on their mate choice, females may obtain other benefits such as high quality offspring.     

p51/p63, a novel p53 homologue, potentiates p53 activity and is a human cancer gene therapy candidate

BACKGROUND: p51 (p73L/p63/p40/KET), a recently isolated novel p53 homologue, binds to p53-responsive elements to upregulate some p53 target genes and has been suggested to share partially overlapping functions with p53. p51 may be a promising candidate target molecule for anti-cancer therapy. METHODS: In this study, we adenovirally transduced p51A cDNA into human lung, gastric and pancreatic cancer cells and analyzed the intracellular function of p51 in anti-oncogenesis in vitro and in vivo. RESULTS: Overexpression of p51A revealed an anti-proliferative effect in vitro in all the cancer cells examined in this study. The anchorage-dependent and -independent cell growth of EBC1 cells carrying mutations in both p51 and p53 was suppressed and significant apoptosis following adenoviral transduction with p51 and/or p53 was seen. This growth suppression was cooperatively enhanced by the combined infection with adenoviral vectors encoding both p51 and p53. Furthermore, p51 activated several, but not all, p53-inducible genes, indicating that the mechanisms controlling p51- and p53-mediated tumor suppression differed. CONCLUSIONS: Our observations indicate that, although p51 exhibited reduced anti-oncogenetic effects compared with p53, it cooperatively enhanced the anti-tumor effects of p53. Our results suggest that p51 functions as a tumor suppressor in human cancer cells in vitro and in vivo and may be useful as a potential tool for cancer gene therapy.