Type IV collagen reduces MUC5AC secretion in the lungs of ovalbumin-sensitized mice.

Mucin 5AC (MUC5AC) is excessively secreted in the respiratory tract of patients with asthma. Suppressing this secretion is important for improving the air passages, which facilitates easy breathing. We have previously reported that the addition of type IV collagen, a typical extracellular matrix (ECM) protein, to the culture medium for human cell lines and primary cells reduced MUC5AC secretion. In this report, we further investigated the effect of type IV collagen on MUC5AC secretion in vivo. We employed ovalbumin (OVA)-sensitized mice to model of asthma and exposed them to type IV collagen to verify the reducing effect of MUC5AC in vivo. The amount of MUC5AC in bronchoalveolar lavage fluid was examined after nebulization of type IV collagen. Hypersecretion of MUC5AC of the OVA-sensitized mice was suppressed by type IV collagen exposure in a time- and dose-dependent manner. Furthermore, type IV collagen exposure to OVA-sensitized mice decreased integrin α2 and ß1 expression in the lungs and increased the levels of Akt and extracellular signal-regulated kinase (ERK) phosphorylation in the trachea. These results suggest that type IV collagen suppresses MUC5AC hypersecretion via modulating integrin expression and Akt/ERK phosphorylation in the respiratory tract of the OVA-sensitized mice.

Integrin ß1 subunit regulates cellular and secreted MUC5AC and MUC5B production in NCI-H292 human lung epithelial cells.

The surface of the human respiratory tract is covered with a mucus layer containing mucin 5AC (MUC5AC) and mucin 5B (MUC5B) as the main components. This layer contributes to biological defense by eliminating irritants, but excessive MUC5AC secretion by the airway epithelial cells exacerbates asthma. Therefore, regulating mucin production is important for asthma treatment. In this study, the effects of integrin ß1 subunit on MUC5AC and MUC5B production were examined in NCI-H292 human lung cancer epithelial cells. When integrin ß1 was overexpressed, cellular and secreted MUC5AC levels were decreased, whereas cellular MUC5B production was increased. Conversely, integrin ß1 depletion using siRNA increased cellular and secreted MUC5AC production, but decreased cellular MUC5B production. Further, the activity of extracellular signal-regulated kinase (ERK), which promotes MUC5AC production, was decreased by integrin ß1 overexpression and increased by its depletion. These results suggest that integrin ß1 suppresses MUC5AC production and promotes MUC5B production by downregulating ERK.

Type IV collagen reduces mucin 5AC secretion in three-dimensional cultured human primary airway epithelial cells.

Mucin 5AC (MUC5AC) hypersecretion induces airway narrowing in patients with asthma, which leads to breathing problems. We investigated the regulation of MUC5AC secretion by extracellular matrix (ECM) proteins in human primary airway epithelial cells from patients with asthma. The addition of type IV collagen to three-dimensional cultured human primary airway epithelial cells, which mimics the airway surface, reduced MUC5AC secretion in the medium, while the addition of laminin increased MUC5AC secretion. Furthermore, the addition of fibronectin did not affect MUC5AC secretion. In particular, the repeated addition of a low concentration of type IV collagen demonstrated a cumulative effect on the reduction in MUC5AC secretion. Human primary cells incubated with type IV collagen showed downregulated extracellular signal-regulated kinase (ERK) activity, which induced MUC5AC hypersecretion but did not affect Akt activity. These results suggest that the addition of type IV collagen to the apical surface of primary cells downregulates MUC5AC secretion and has a cumulative effect on MUC5AC secretion which might be effected via the ERK signaling pathway.

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 ß1 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.

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.

Akt induces down regulation of MUC5AC production in NCI-H292 human airway epithelial cells cultured on extracellular matrix.

MUC5AC mucin overproduction is a key feature of asthma as contributes to airway obstruction. The production of MUC5AC is regulated in part by signals from extracellular matrix via integrin pathways, but it remains largely unclear. We investigated the role of Akt, a typical signal transducer in the integrin pathway, in the regulation of MUC5AC production. When NCI-H292 human airway epithelial cells were cultured on laminin or Matrigel, we found that the activity of Akt was suppressed, as compared to control cells with upregulated MUC5AC production. In contrast, Akt was activated in cells cultured on type IV collagen with downregulated MUC5AC production. The Akt inhibitor induced upregulation of MUC5AC. In contrast, overexpression of active Akt induced downregulation of MUC5AC production. These results suggest that a signal from laminin or Matrigel induces upregulation of MUC5AC by suppressing Akt activity, whereas a signal from type IV collagen induces downregulation of MUC5AC, mediated by Akt activation.

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.

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.

Differentiation- and apoptosis-inducing activities of rice bran extracts in a human colon cancer cell line.

Rice bran water extract (RBWE) and ethanol extract (RBEE) at 1.0 mg/ml markedly inhibited the proliferation of LS174T human colon cancer cells. RBEE but not RBWE induced apoptosis. RBWE promoted the production of intestinal mucin (MUC2). Real-time RT-PCR demonstrated that RBWE up-regulated the expression of MUC2 and sucrase-isomaltase complex (a differentiation marker of colon cancer cells), and attenuated that of proliferating cell nuclear antigen at the mRNA level in a dose-dependent manner. These findings suggested that RBWE suppress the proliferation of colon cancer cells by inducting differentiation not apoptosis.

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 (ß-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.

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.

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 beta1-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 beta1-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.

The short chain fatty acid, butyrate, stimulates MUC2 mucin production in the human colon cancer cell line, LS174T.

The short fatty acid, butyrate, which is produced by intestinal anaerobic bacteria in the colon, has inhibitory activity on histone deacetylases (HDACs). Treatment of the human colon cancer cell line, LS174T, with 1-2 mM sodium butyrate stimulated MUC2 mucin production, as determined by histological PAS staining of carbohydrate chains of mucin, and confirmed at the protein and mRNA levels by immunoblotting with anti-MUC2 antibody and real-time RT-PCR, respectively. Increases in acetylated histone H3 in the LS174T cells treated with butyrate suggest inhibition of HDACs in these cells. Butyrate-stimulated MUC2 production in the LS174T cells was inhibited by the MEK inhibitor, U0126, implicating the involvement of extracellular signal-regulated kinase (ERK) cascades in this process. Proliferation of the LS174T cells was inhibited by butyrate treatment. Although apoptotic nuclear DNA fragmentation could not be detected, cell-cycle arrest at the G0/G1 phase in the butyrate-treated cells was demonstrated by flow cytometry. Thus butyrate, an HDAC inhibitor, inhibits proliferation of LS174T cells but stimulates MUC2 production in individual cells.

The histone deacetylase inhibitor butyrate inhibits melanoma cell invasion of Matrigel.

BACKGROUND: Histone deacetylase (HDAC) inhibitors have anticancer effects. Their effects on expression of cell adhesion molecules might be related to their effects on tumor cell invasion. MATERIALS AND METHODS: Murine B16-BL6 cells were treated with the HDAC inhibitors, butyrate or trichostatin A. Melanoma cell invasion of the artificial basement membrane, Matrigel, was examined by Transwell chamber assay. RESULTS: Butyrate as well as trichostatin A inhibited the cell growth mainly by arresting the cell cycle. The cell invasion of Matrigel was inhibited by butyrate and trichostatin A. The butyrate treatment increased the cell-cell aggregation, although neither E-cadherin nor N-cadherin mRNA were up-regulated. Both mRNA expression and protein levels of the immunoglobulin superfamily cell adhesion molecules, Mel-CAM and L1-CAM, were increased in the butyrate-treated cells. CONCLUSION: The HDAC inhibitor butyrate blocked the B16-BL6 melanoma cell invasion of Matrigel, although it increased the expression of Mel-CAM and L1-CAM which are important to the metastatic potential.

The tyrosine kinase inhibitor AG490 inhibits growth of cancer cells and activates ERK in LS174T and HT-29 cells.

BACKGROUND: The activity of tyrosine kinases, although strictly regulated in normal cells, is often disturbed in cancer cells. The inhibition of a tyrosine kinase could be a target for treating cancer. MATERIALS AND METHODS: The colon cancer cell lines LS174T and HT-29 and the lung cancer cell line NCI-H292 were used. The cells were incubated with 100 microM of the tyrosine kinase inhibitor AG490 for 1-3 days and were examined for growth. Extracellular signal-regulated kinase (ERK) activation was detected by anti-phospho ERK antibodies. The cell cycle was analyzed by flow cytometry. RESULTS: AG490 inhibited the growth of LS174T, HT-29 and NCI-H292 cells without inducing apoptosis. Short-term treatment with AG490 activated ERK and p38 MAPK in the LS174T and HT-29 cells, but not in NCI-H292 cells. ERK activation, however, was unrelated to the growth inhibition in LS174T cells, because the inhibition persisted even after the prevention of ERK activation. CONCLUSION: AG490 inhibits the growth of some cancer cells and activates ERK in LS174T and HT-29 cells. ERK activation is unrelated to growth inhibition.

Isolation and functional analysis of a chk2 homologue from Entamoeba histolytica.

Mammalian Chk2 is a Ser/Thr kinase required for cell-division arrest induced by DNA damage. We found six new kinase genes of Entamoeba histolytica by analysis in silico. One of the kinase genes was a homologue of human chk2 gene. The chk2 homologue gene (Eh chk2) was expected to encode 398 amino acids and showed nearly 50% homology to human Chk2 in amino acid sequence. Eh chk2 had a catalytic domain of Ser/Thr kinase and a fork head-associated (FHA) domain that is highly conserved among Chk2 homologues in vertebrates. To examine the biological functions of Eh chk2, we synthesized Eh chk2 mRNA in vitro and injected it into immature frog eggs (Xenopus laevis oocytes) as a model system of cell division. Eh chk2 markedly delayed the cell division of frog eggs by disrupting transition of G2 phase to M phase. Eh chk2 also inhibited the activation of p42 MAPK and Cdc2 kinase which are representative events induced by cell division. These results suggest that Eh chk2 gene should be a cell-division regulator in E. histolytica.

mRNA of MUC2 is stimulated by IL-4, IL-13 or TNF-alpha 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-alpha, 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-alpha 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-alpha 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-alpha 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.