Transcriptional networks in the human epididymis.

BACKGROUND: The epithelial lining of the human epididymis is critical for sperm maturation. This process requires distinct specialized functions in the head, body, and tail of the duct. These region-specific properties are maintained by distinct gene expression profiles which are governed by transcription factor networks, non-coding RNAs, and other factors. MATERIALS AND METHODS: We used genome-wide protocols including DNase-seq, RNA-seq and ChIP-seq to characterize open (active) chromatin, the transcriptome and occupancy of specific transcription factors (TFs) respectively, in caput, corpus, and cauda segments of adult human epididymis tissue and primary human epididymis epithelial (HEE) cell cultures derived from them. RNA-seq following TF depletion or activation, combined with gene ontology analysis also determined TF targets. RESULTS: Among regional differentially expressed transcripts were epithelial-selective transcription factors (TFs), microRNAs, and antiviral response genes. Caput-enriched TFs included hepatocyte nuclear factor 1 (HNF1) and the androgen receptor (AR), both of which were also predicted to occupy cis-regulatory elements identified as open chromatin in HEE cells. HNF1 targets were identified genome-wide using ChIP-seq, in HEE cells. Next, siRNA-mediated depletion of HNF1 revealed a pivotal role for this TF in coordinating epithelial water and solute transport in caput epithelium. The importance of AR in HEE cells was shown by AR ChIP-seq, and by RNA-seq after synthetic androgen (R1881) treatment. AR has a distinct transcriptional program in the HEE cells and likely recruits different co-factors (RUNX1 and CEBPß) in comparison to those used in prostate epithelium. DISCUSSION AND CONCLUSION: Our data identify many transcription factors that regulate the development and differentiation of HEE cells. Moreover, a comparison between immature and adult HEE cells showed key TFs in the transition to fully differentiated function of this epithelium. These data may help identify new targets to treat male infertility and have the potential to open new avenues for male contraception.

Matrix metalloproteinase-2 from bronchial epithelial cells induces the proliferation of subepithelial fibroblasts.

BACKGROUND: In bronchial asthma, subepithelial fibrosis in the conducting airways is associated with increased numbers of subepithelial fibroblasts. OBJECTIVE: This study examined the hypothesis that MMP-2 from airway epithelial cells induces the proliferation of subepithelial fibroblasts. METHODS: Using primary bronchial epithelial cells MMP-2, MT1-MMP and TIMP-2 mRNA expression were assessed by Northern blotting and RT-PCR. Primary bronchial epithelial cells transfected with constructs encoding pro-MMP-2 and MT1-MMP (MMP-14). RESULTS: Transfected cells showed enhanced expression of the appropriate mRNA species by RT-PCR and enhanced MMP-2 or MT1-MMP activity by zymography. Active MMP-2 levels in epithelial supernatants were increased most by cotransfection with pro-MMP-2 and MT1-MMP encoding constructs. By measuring tritiated thymidine incorporation, supernatants from transfected cells were found to enhance DNA synthesis of primary airway fibroblast cultures compared with controls. There was a strong correlation (r = 0.9, P < 0.01) between MMP-2 levels in epithelial cell conditioned media and fibroblast proliferation as indicated by DNA synthesis. The MMP inhibitor 1,10-phenanthroline attenuated the increased proliferation, while the addition of exogenous purified MMP-2 alone also increased fibroblast proliferation. CONCLUSIONS: Our results support a role for MMP-2 in mediating cross-talk between epithelial cells and myofibroblasts.

Rhinoviruses infect the lower airways.

Rhinoviruses are the major cause of the common cold and a trigger of acute asthma exacerbations. Whether these exacerbations result from direct infection of the lower airway or from indirect mechanisms consequent on infection of the upper airway alone is currently unknown. Lower respiratory infection was investigated in vitro by exposing primary human bronchial epithelial cells to rhinoviruses and in vivo after experimental upper respiratory infection of human volunteers. Bronchial infection was confirmed by both approaches. Furthermore, rhinoviruses induced production of interleukin-6, -8, and -16 and RANTES and were cytotoxic to cultured respiratory epithelium. This evidence strongly supports a direct lower respiratory epithelial reaction as the initial event in the induction of rhinovirus-mediated asthma exacerbations. The frequency of infection and the nature of the inflammatory response observed are similar to those of the upper respiratory tract, suggesting that rhinovirus infections may be one of the most important causes of lower in addition to upper respiratory disease.

Increased CD44 expression in human bronchial epithelial repair after damage or plating at low cell densities.

We have investigated the effect of mechanical damage, cell density, and cell-derived soluble mediators on CD44 expression in a model of bronchial epithelial repair. CD44 (all isoforms) and variant-containing isoforms (CD44v3, CD44v6, and CD44v9) were identified with flow cytometry and immunocytochemistry with image analysis. After mechanical damage, CD44 expression increased up to 500 microm from the wound edge and for up to 48 h in two human bronchial epithelium-derived cell lines, 16HBE14o- and NCI-H292. CD44 expression was unchanged by interferon-gamma and increased by <50% by tumor necrosis factor-alpha. To exclude other soluble factors, a Vaseline spacer was used to temporarily divide petri dishes, with cells at high density on one side and those at low density on the other. After the spacer was removed, the cells at low cell density growing in the shared medium expressed up to fourfold higher CD44, although cell proliferation was unchanged. Thus increased CD44 expression at low cell density was not mediated by soluble factors and may reflect functional involvement in cell motility, dedifferentiation, or altered cell-substrate adhesion in epithelial repair.

Expression of c-erbB receptors and ligands in human bronchial mucosa.

The epidermal growth factor receptor (EGFR, c-erbB1) plays a pivotal role in maintenance and repair of epithelial tissues; however, little is known about coexpression of c-erbB receptors and their ligands in human bronchial epithelium. We therefore analyzed the expression of these molecules in cultured bronchial epithelial cells and normal bronchial mucosa, using reverse transcription-polymerase chain reaction (RT- PCR), flow cytometry, and immunohistochemistry. Messenger RNA (mRNA) encoding EGFR, c-erbB2, and c-erbB3, but not c-erbB4, was detected in primary cultures of human bronchial epithelial cells, as well as in the human bronchial epithelial-derived cell lines H292 and 16HBE 14o-. Transcripts encoding epidermal growth factor (EGF), heparin binding epidermal growth factor (HB-EGF), transforming growth factor-alpha (TGF-alpha), and amphiregulin (AR) were also detected, and expression of the three receptors and four ligands was confirmed by immunocytochemical staining of the cultured cells. Immunohistochemical analysis of resin- or paraffin-embedded sections from surgical specimens of bronchial mucosa revealed strong membrane staining for EGFR within the bronchial epithelium; this was particularly evident between basal cells and the basal aspect of columnar cells. The patterns of staining for c-erbB2 and c-erbB3 in the bronchial epithelium were similar to those for EGFR. Immunostaining for EGF, TGF-alpha, AR, HB- EGF, and betacellulin (BTC) was intense in the submucosal glands; with the exception of BTC, EGFR ligand immunoreactivity was also observed in the bronchial epithelium, where it paralleled EGFR staining. Colocalization of c-erbB receptors and ligands demonstrates the potential for productive c-erbB receptor interactions in bronchial epithelium. Further study of these interactions may help to define their role in maintenance and repair of the bronchial epithelium.

Antigen-induced anaphylactic death in mice.

Intravenous injection of bovine serum albumin (BSA) into the BSA/CFA-primed ICR mice specifically induced anaphylactic death within 1 h. The anaphylactic death could not be induced until day 8 after sensitization, and sensitization subsisted for more than 3 months. The response was dose dependent; mice challenged with BSA doses higher or equivalent to 25 microgram developed anaphylactic death. The intravenous route was more effective than the intraperitoneal one, while subcutaneous injection was ineffective. Antigen in any of complete Freund's adjuvant, incomplete Freund's adjuvant or aluminum hydroxide could sensitize the mice to develop anaphylactic death. The combination of antigen and the mouse strain or the gender of the mouse determined the susceptibility of the anaphylactic death. AKR, B10.BR, as well as ICR, strains were susceptible. Antigen of HoGG induced a higher mortality rate than that of GAT or lysozyme. Male mice were more susceptible than female ones. The BSA-induced anaphylactic death could be prevented by pretreating ICR mice with cyproheptadine (histamine and serotonin antagonist) or diphenhydramine (histamine antagonist) and ketanserin (serotonin antagonist). Intravenous injection of saline during anaphylaxis also protected the mice from death. Furthermore, immune serum could transfer the anaphylactic death, and heat (56 degrees C, 4 h) did not destroy its activity. The primary IgG subclass induced by GAT, HoGG or lysozyme was IgG1. There was no qualitative difference in the IgG subclass induced in different strains by different antigens. The IgE class of antibodies was not detectable. These results suggest that there is a non-IgE-mediated anaphylactic death which involves the release of histamine and serotonin that cause the increase of vasopermeability and fatal blood volume loss.

Involvement of histamine or tumor necrosis factor in early-type hypersensitivity.

A novel early-type hypersensitivity (ETH) reaction, manifested as capillary congestion, increase of vasopermeability, and plasma protein leakage, can be induced within 1 h after challenge of antigen-sensitized mice. The mediators involved in ETH varied among different strains of mice. The poly(Glu60Ala30Tyr10) (GAT)-induced ETH in BALB/c mice was blocked by diphenhydramine (histamine H1 antagonist) and ketanserine (serotonin antagonist), but not by cimetidine (histamine H2 antagonist). These results indicate that both histamine and serotonin are involved, and that the histamine effect is mediated through a H1 receptor. Meanwhile, GAT-induced ETH in B6 mice was inhibited by anti-TNF alpha antibody suggesting that TNF alpha is involved. The mice can be classified into either histamine or TNF alpha type based on the pattern of mediator involved in ETH. A/J and CBA strains as well as BALB/c mice were classified as histamine type while A. TL, B10.BR, and C3H/He in addition to B6 mice were TNF alpha type. The observation that GAT-induced ETH in (BALB/c x B6)F1 mice was inhibited by both diphenhydramine and anti-TNF alpha suggests that the mediation of the actions of histamine or TNF alpha by GAT was genetically controlled and inherited as the dominant trait in (BALB/c x B6)F1 mice. ETH could be passively transferred by heat (56 degrees C, 4 h)-treated anti-GAT sera. Sera derived from the histamine type transferred ETH across the type barrier and histamine was the mediator, irrespective of the type of the recipient. However, sera derived from TNF alpha type only transferred ETH to the mice of the same TNF alpha type and TNF alpha was the mediator.

Horse gammaglobulin-induced thrombocytopenia in anaphylaxis involving sequestration and activation of platelets.

Thrombocytopenia as well as hemoconcentration and leukopenia followed by leukocytosis were induced after HoGG challenge on HoGG-sensitized mice. Thrombocytopenia was induced within 2 min and sustained for 1 day. HoGG-induced thrombocytopenia was not observed until day 10 after sensitization; mice challenged with HoGG dose > or = 10 micrograms developed thrombocytopenia. Two types of thrombocytopenia were observed in appropriately sensitized mice. HoGG induced thrombocytopenia at 2 min and 60 min, whereas, alpha-macroglobulin induced thrombocytopenia at 2 min, the platelet count of which returned to normal within 60 min. Poly (Glu60Ala30Tyr10) did not induce thrombocytopenia at 2 min or 60 min. The tracing study by 3H-serotonin labelled platelets demonstrated the 2 min-sequestration of platelets in lungs or livers. The HoGG-induced sequestration of platelets at 2 min was blocked by high dose heparin or Cobra Venom factor. Platelet activation at 60 min was partially inhibited by dexamethasone, rhodostomin synthetic peptide 45-59, or platelet activation factor antagonist (WEB 2086). Furthermore, the thrombocytopenia could be transferred by heat (56 degrees C, 4h) treated immune sera. This suggests that HoGG-induced, non-IgE-mediated thrombocytopenia in anaphylaxis involves sequestration and activation of platelets. The sequestion in lungs occurs within 2 min and can be inhibited by high dose heparin or Cobra Venom factor. The activation of platelets involves platelet activation factor, and fibrinogen receptor.