Jun Iwashita

mRNA of MUC2 is stimulated by IL-4, IL-13 or TNF-α through a mitogen-activated protein kinase pathway in human colon cancer cells

MUC2 mucin is a secretory glycoprotein which is produced from the intestinal goblet cells and is a major component of the intestinal epithelial mucus. The biological function of MUC2 mucin is considered to be the protection of intestinal epithelial surface, whereas the regulatory mechanism of MUC2 mucin production in immune response is not completely understood. We have studied the effects of cytokines, IL‐4, IL‐13 and TNF‐α, on the regulation of MUC2 mRNA in the human colonic cancer cell lines, LS174T and HT29. The quantitative reverse transcription‐polymerase chain reaction showed that single addition of IL‐4, IL‐13 and TNF‐α to cell culture induced about two‐fold increase of MUC2 mRNA level in LS174T cells. Interleukin‐4 and IL‐13 activated phosphorylation of mitogen‐activated protein kinase in LS174T cells. A specific inhibitor of mitogen‐activated protein kinase pathway, U0126, totally inhibited the increase of MUC2 mRNA by IL‐4 or IL‐13 in those cells. Therefore, mitogen‐activated protein activation of kinase is required for the increase of MUC2 mRNA by IL‐4 or IL‐13 in LS174T cells. In contrast to LS174T cells, only TNF‐α increased MUC2 mRNA through a mitogen‐activated protein kinase pathway in HT29 cells that express low levels of MUC2 mRNA. These findings sustain a novel phenomenon that MUC2 mRNA expression is differently controlled by IL‐4, IL‐13, or TNF‐α in LS174T and HT29 cells, whereas the mitogen‐activated protein kinase pathway plays a role in the MUC2 mRNA expression induced by those cytokines in both cell lines.

MUC5AC production is downregulated in NCI-H292 lung cancer cells cultured on type-IV collagen.

Mucus overproduction is an important feature of bronchial asthma. MUC5AC mucin is a major component of mucus and is overproduced in patients with asthma. Although regulation of MUC5AC production has been well investigated, its regulation through the signals from extracellular matrix (ECM) is less clear. In this study, we investigated whether the signals from ECM regulate MUC5AC production in the human lung epithelial cell line NCI-H292. We found that MUC5AC production is downregulated in NCI-H292 cells cultured on type-IV collagen, a major component of ECM, but shows no obvious changes when cultured on type-I collagen or fibronectin. In contrast, MUC5AC production was upregulated on laminin and on reconstituted basement membrane (Matrigel), a complex of ECM components. Antibody-mediated inhibition of integrin β1-subunit, a major receptor involved in the adherence of cells to type-IV collagen, upregulated the MUC5AC production in NCI-H292 cells, and also in the cells cultured on type-IV collagen. Although the major signaling pathway from integrins is via Src kinase activation, treatment of cells with PP2, a Src kinase inhibitor, did not recover the downregulation of MUC5AC on type-IV collagen. In contrast, on Matrigel, the inhibition of integrin β1-subunit did not abolish the upregulation of MUC5AC production, but PP2 reduced the upregulation. These results suggest that ECM and an integrin/Src pathway play an important role in the regulation of MUC5AC production in the cell line NCI-H292. The production of MUC5AC is downregulated on type-IV collagen through a Src-independent pathway. In contrast, MUC5AC is upregulated on Matrigel through a Src-dependent pathway in NCI-H292 cells.

Inhibition of E-Cadherin Dependent Cell-Cell Contact Promotes MUC5AC Mucin Production through the Activation of Epidermal Growth Factor Receptors

Mucin production by epithelial cells is modulated by many soluble factors, including epidermal growth factor (EGF). E-Cadherin promotes EGF receptor (EGFR)-mediated MUC5AC mucin production in airway epithelial cells in dense cultures, suggesting the involvement of E-cadherin in activating EGFRs and mucin production. However, the role of E-cadherin in modulating mucin production is not completely understood. We examined its role in MUC5AC production in a human lung epithelial cell line, NCI-H292. Treatment of low density NCI-H292 cells with an anti-E-cadherin monoclonal antibody (SHE78-7) inhibited cell-cell contact in the dispersed colonies, but promoted MUC5AC production. Furthermore, treatment of the NCI-H292 cells with anti-E-cadherin antibody stimulated phosphorylation of extracellular signal-regulated kinase (ERK). The enhanced production of MUC5AC was inhibited with an EGFR inhibitor and with a MEK inhibitor, but not with a Src family kinase inhibitor. These results suggest that inhibition of E-cadherin activates EGFRs independently of Src and promotes MUC5AC production through the ERK signaling pathway in sparsely cultured NCI-H292 cells.

Intact structure of EGAM1 homeoproteins and basic amino acid residues in the common homeodomain of EGAM1 and EGAM1C contribute to their nuclear localization in mouse embryonic stem cells

Recently, we identified the structurally related homeoproteins EGAM1, EGAM1N, and EGAM1C in both preimplantation mouse embryos and mouse embryonic stem (ES) cells. These EGAM1 homeoproteins act as positive or negative regulators of differentiation and cell growth in mouse ES cells, such that these proteins are considered transcriptional regulators. In this study, we investigated their nuclear localization and identified the amino acid residues crucial for the nuclear translocation of EGAM1 and EGAM1C. When expressed exogenously in pluripotent ES cells and somatic NIH3T3 cells, all EGAM1 homeoproteins localized to the nucleus. Analysis using the web-based tool PSORTII predicted a potential nuclear localization signal (NLS) motif, RKDLIRSWFITQRHR, in the homeodomain shared by EGAM1 and EGAM1C. The introduction of mutations, such as mutations from K or R, both basic amino acid residues, to A, in this potential NLS resulted in significant impairment of the nuclear localization of both EGAM1 and EGAM1C. In contrast, GFP fusion proteins of all the full-length EGAM1 homeoproteins failed to localize to the nucleus. These results, when taken together, suggest that basic amino acid residues in the common homeodomain of EGAM1 and EGAM1C and the intact structures of the EGAM1 homeoproteins contribute, at least in part, to the nuclear localization of these proteins in mouse ES cells.

Partial inhibition of differentiation associated with elevated protein levels of pluripotency factors in mouse embryonic stem cells expressing exogenous EGAM1N homeoprotein.

We previously reported that transcripts encoding the homeoprotein EGAM1N are expressed in preimplantation mouse embryos and embryonic stem (ES) cells, and the exogenous expression of EGAM1N inhibits the differentiation of ES cells. In order to clarify the relationship between the inhibition of differentiation and EGAM1N, we generated mouse MG1.19 ES cells stably expressing EGAM1N. Control transfectants with an empty vector formed relatively flattened cell colonies similar to those observed in parental MG1.19 cells. In contrast, Egam1n transfectants formed tightly aggregated cell colonies with increased localization of CDH1 at cell-to-cell interfaces. The protein levels of pluripotency factors, including TBX3 and SOX2, were also increased. The expression of Tbx3 transcripts was induced, although the level of Sox2 transcripts was almost unchanged. The expression of EGAM1N resulted in no obvious changes in the expression of genes encoding receptors, protein kinases, transcription factors, and their encoded proteins involved in the LIF-STAT3 signaling pathway. Alkaline phosphatase activity, a marker for the undifferentiated state, in Egam1n transfectants was exhibited in a clonal proliferation assay. When differentiation of Egam1n transfectants was induced, progression was prevented with increases in transcript levels of Pou5f1, Sox2, Nanog, Klf4, Tbx3, and their encoded proteins. However, Egam1n transfectants formed relatively flattened-cell layers as observed in the control, indicating that the expression of EGAM1N could not maintain LIF-independent self-renewal of ES cells. Overall, we suggest that expression of EGAM1N could inhibit differentiation, at least in part, by elevating the protein levels of pluripotency factors in MG1.19 ES cells.

MUC5B mucin production is upregulated by fibronectin and laminin in human lung epithelial cells via the integrin and ERK dependent pathway

MUC5B mucin is a principal component of airway mucus and plays a key role in biodefense. We investigated the regulation of MUC5B production using the signals from extracellular matrix (ECM) components in NCI-H292 human lung epithelial cells. We found that MUC5B production in NCI-H292 cells cultured on fibronectin or laminin increased by 4–5-fold, with the increase occurring in a dose- and time-dependent manner. In contrast, MUC5B production was unchanged on type-IV collagen. Inhibition of integrin β1 induced upregulation of MUC5B and MUC5AC; however, inhibition of p38 MAPK did not show any remarkable change in overproduced MUC5B. Inhibition of extracellular signal-regulated kinase (ERK) pathway or the transcription factor NF-κB induced the recovery of overproduced MUC5B on fibronectin and laminin. These results suggest that MUC5B production can be regulated by ECM components and that MUC5B is upregulated by fibronectin and laminin via the integrin, ERK, and NF-κB dependent pathway. MUC5B mucin plays a key role in biodefense. Our results suggest that a signal from fibronectin and laminin induce upregulation of MUC5B, mediated by integrin pathway and ERK pathway.

Generative cell–specific activation of the histone gH2A gene promoter of Lilium longiflorum in tobacco

The Lilium longiflorum gH2A promoter is active exclusively in the generative cells of mature pollen in transgenic tobacco expressing the gH2A promoter::GUS (β-glucuronidase) construct as a reporter gene. Temporal and spatial aspects of gH2A promoter activity examined during pollen development in transgenic tobacco reveal that GUS reporter activity was not detected until developing pollen entered the early bicellular developmental stage. Activity was first detected in generative cells at early-mid stages and gradually increased to maximum levels at mid-bicellular stages. The patterns of appearance and longevity of GUS activity in tobacco were very similar to those of gH2A mRNA during pollen development in Lilium. Exogenous treatment with colchicine, a well-known microtubule depolymerize, blocked microspore mitosis and inhibited generative cell differentiation. No GUS signal was detected in the resulting anomalous pollen, which lacked generative cell differentiation. These data strongly suggest that normal generative cell development is essential for activation of the gH2A promoter. Furthermore, these results indicate that common transcriptional activator(s) of the gH2A promoter may be present in both Lilium and Nicotiana, and that such putative factor(s) activates the gH2A promoter only when generative cells undergo normal development.

Histone deacetylase induces accelerated maturation in Xenopus laevis oocytes

In oocyte maturation in Xenopus laevis, nuclear material induces rapid maturation and is required for entry into meiosis II. Nuclear material contains a large number of RNAs and proteins, including histone deacetylase (HDAC); however, it is not known which materials induce accelerated maturation. The HDAC activity modifies transcription rate and is required for normal meiosis; however, its function in oocyte maturation is still unclear. We investigated the function of HDAC activity, which is localized in the nuclear material, in the regulation of the speed of oocyte maturation. Inhibition of HDAC activity with trichostatin A (TSA) induced hyperacetylation of histone H3 and prolonged oocyte maturation. In contrast, increase in HDAC activity with an injection of FLAG‐tagged maternal histone deacetylase (HDACm‐FLAG) mRNA induced deacetylation of histone H3 and reduced the duration of oocyte maturation. Cdc2 kinase, Cdc25C or mitogen‐activated protein kinase (MAPK), which are key regulators of the meiosis, were activated coincidently with maturation progression. In oocytes, the mRNA level of Cdc25C, an activator of Cdc2, was increased by HDACm‐FLAG mRNA‐injection; in contrast, the mRNA level of Cdc2 inhibitor Wee1 was increased by TSA treatment. These results suggest that HDAC activity is involved in the control of maturation speed through the regulation of mRNA levels of cell cycle regulators. Thus, HDACm is a candidate for the nuclear material component that induces rapid maturation in Xenopus oocytes.