Transcription In Epithelial Cells Research Articles

The Scribble-SGEF-Dlg1 complex regulates E-cadherin and ZO-1 stability, turnover and transcription in epithelial cells.

SGEF (also known as ARHGEF26), a RhoG specific GEF, can form a ternary complex with the Scribble polarity complex proteins Scribble and Dlg1, which regulates the formation and maintenance of adherens junctions and barrier function of epithelial cells. Notably, silencing SGEF results in a dramatic downregulation of both E-cadherin and ZO-1 (also known as TJP1) protein levels. However, the molecular mechanisms involved in the regulation of this pathway are not known. Here, we describe a novel signaling pathway governed by the Scribble-SGEF-Dlg1 complex. Our results show that the three members of the ternary complex are required to maintain the stability of the apical junctions, ZO-1 protein levels and tight junction (TJ) permeability. In contrast, only SGEF is necessary to regulate E-cadherin levels. The absence of SGEF destabilizes the E-cadherin-catenin complex at the membrane, triggering a positive feedback loop that exacerbates the phenotype through the repression of E-cadherin transcription in a process that involves the internalization of E-cadherin by endocytosis, β-catenin signaling and the transcriptional repressor Slug (also known as SNAI2).

Effect of heat exposure on prostaglandin production and expression of COX-2, PGES, PGFS, ITGAV and LGALS15 mRNAs in endometrial epithelial cells of buffalo (Bubalus bubalis).

Early embryonic mortality is one of the major intriguing factors of reproductive failure that causes considerable challenge to the mammalian cell biologists. Heat stress is the major factor responsible for reduced fertility in farm animals. The present study aimed to investigate the influence of heat stress on prostaglandin production and the expression of key genes, including COX-2, PGES, PGFS, ITGAV and LGALS15, in buffalo endometrial epithelial cells. Buffalo genitalia containing ovaries with corpus luteum (CL) were collected immediately post-slaughter. The stages of the estrous cycle were determined based on macroscopic observations of the ovaries. Uterine lumens of the mid-luteal phase (days 6-10 of the estrous cycle) were washed and treated with trypsin to isolate epithelial cells, which were then cultured at control temperature (38.5°C for 24h) or exposed to elevated temperatures [38.5°C for 6h, 40.5°C for 18h; Heat Stressed (HS)]. The supernatant and endometrial epithelial cells were collected at various time points (0, 3, 6, 12, and 24h) from both the control and treatment groups. Although heat stress (40.5°C) significantly (P < 0.05) increased COX-2, PGES, and PGFS transcripts in epithelial cells but it did not affect the in vitro production of PGF2α and PGE2. The expression of ITGAV and LGALS15 mRNAs in endometrial epithelial cells remained unaltered under elevated temperature conditions. It can be concluded that elevated temperature did not directly modulate prostaglandin production but, it promoted the expression of COX-2, PGES and PGFS mRNA in buffalo endometrial epithelial cells.

OTUD6A in tubular epithelial cells mediates angiotensin II-induced kidney injury by targeting STAT3.

Kidney fibrosis is a prominent pathological feature of hypertensive kidney diseases (HKD). Recent studies have highlighted the role of ubiquitinating/deubiquitinating protein modification in kidney pathophysiology. Ovarian tumor domain-containing protein 6 A (OTUD6A) is a deubiquitinating enzyme involved in tumor progression. However, its role in kidney pathophysiology remains elusive. We aimed to investigate the role and underlying mechanism of OTUD6A during kidney fibrosis in HKD. The results revealed higher OTUD6A expression in kidney tissues of nephropathy patients and mice with chronic angiotensin II (Ang II) administration than that from the control ones. OTUD6A was mainly located in tubular epithelial cells. Moreover, OTUD6A deficiency significantly protected mice against Ang II-induced kidney dysfunction and fibrosis. Also, knocking OTUD6A down suppressed Ang II-induced fibrosis in cultured tubular epithelial cells, whereas overexpression of OTUD6A enhanced fibrogenic responses. Mechanistically, OTUD6A bounded to signal transducer and activator of transcription 3 (STAT3) and removed K63-linked-ubiquitin chains to promote STAT3 phosphorylation at tyrosine 705 position and nuclear translocation, which then induced profibrotic gene transcription in epithelial cells. These studies identified STAT3 as a direct substrate of OTUD6A and highlighted the pivotal role of OTUD6A in Ang II-induced kidney injury, indicating OTUD6A as a potential therapeutic target for HKD.NEW & NOTEWORTHY Ovarian tumor domain-containing protein 6 A (OTUD6A) knockout mice are protected against angiotensin II-induced kidney dysfunction and fibrosis. OTUD6A promotes pathological kidney remodeling and dysfunction by deubiquitinating signal transducer and activator of transcription 3 (STAT3). OTUD6A binds to and removes K63-linked-ubiquitin chains of STAT3 to promote its phosphorylation and activation, and subsequently enhances kidney fibrosis.

Localization of phosphatidylinositol phosphate 5 kinase γ, phospholipase β3 and diacylglycerol kinase ζ in corneal epithelium in comparison with conjunctival epithelium of mice

Based on the theory that the phosphoinositide (PI) signal is involved in the physiology of cornea and conjunctiva, we examined the localization in the mouse anterior ocular epithelia of immunoreactivities for phosphatidylinositol 4-phosphate 5-kinase (PIP5K), phospholipase C (PLC) and diacylglycerol kinase (DGK), enzymes that work sequentially in PI cycle. Immunoreactivity for PIP5Kγ in the corneal epithelium, including the limbus, was distinct in adults in contrast to faint or negligible immunoreactivity in the conjunctival epithelium in neonatal mice. This adult localization pattern was first recognized at the postnatal time of eyelid opening. Immunoreactivity for PLCβ3 was rather equally distinct throughout the entire corneal and conjunctival epithelia in adults. DGKζ-immunoreactive nuclei were mainly localized in the basal half domain of the corneal epithelium but in both basal and apical domains of the conjunctival epithelium in adults. This nuclear immunoreactivity was at weak or negligible levels in the peripheral and limbus cornea and in a considerable portion of the bulbar conjunctival epithelium continuous with the limbus. The adult patterns for PLCβ3 and DGKζ were already present at birth. The present findings suggest the following possibilities on the functional significance of the three enzyme molecules. PIP5Kγ is involved in cornea-specific functions such as bright-field vision, including corneal transparency, and in the stability of epithelial junctions, for which there seems to be a much higher requirement in the corneal epithelium than in the conjunctival epithelium. PLCβ3 is involved from birth in as-yet undefined functions exerted ubiquitously from birth in both corneal and conjunctival epithelia. DGKζ is involved in regulation from birth of the transcription in epithelial cells, including apoptosis as well as regulation of mitosis of epithelial cells in both cornea and conjunctiva, with the transcription involvement more apparent in the conjunctiva, although it does not work in stem cells of the corneal limbus.

PIWI-piRNA pathway-mediated transposable element repression in Hydra somatic stem cells.

Transposable elements (TEs) can damage genomes, thus organisms use a variety of mechanisms to repress TE expression. The PIWI–piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the somatic stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations, all of which express PIWI proteins; endodermal and ectodermal epithelial stem cells (ESCs) are somatic, whereas the interstitial stem cells have germline competence. To study somatic function of the pathway, we isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI–piRNA pathway represses TEs in Hydra ESCs. First, epithelial knockdown of the Hydra piwi gene hywi resulted in up-regulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Altogether, our data reveal that the PIWI–piRNA pathway represses TE expression in the somatic cell lineages of Hydra, which we propose contributes to the extreme longevity of the organism. Furthermore, our results, in combination with others, suggest that somatic TE repression is an ancestral function of the PIWI–piRNA pathway.

RORα binds to E2F1 to inhibit cell proliferation and regulate mammary gland branching morphogenesis.

Retinoic acid receptor-related orphan nuclear receptor alpha (RORα) is a potent tumor suppressor that reduces cell proliferation and inhibits tumor growth. However, the molecular mechanism by which it inhibits cell proliferation remains unknown. We demonstrate a noncanonical nuclear receptor pathway in which RORα binds to E2F1 to inhibit cell cycle progression. We showed that RORα bound to the heptad repeat and marked box region of E2F1 and suppressed E2F1-regulated transcription in epithelial cells. Binding of RORα inhibited E2F1 acetylation and its DNA-binding activity by recruiting histone deacetylase 1 (HDAC1) to the protein complexes. Knockdown of HDAC1 or inhibition of HDAC activity at least partially rescued transcription factor activity of E2F1 that was repressed by RORα. Importantly, RORα levels were increased in mammary ducts compared to terminal end buds and inversely correlated with expression of E2F1 target genes and cell proliferation. Silencing RORα in mammary epithelial cells significantly enhanced cell proliferation in the ductal epithelial cells and promoted side branching of the mammary ducts. These results reveal a novel link between RORα and E2F1 in regulating cell cycle progression and mammary tissue morphogenesis.

Regulation and activity of secretory leukoprotease inhibitor (SLPI) is altered in smokers

A hallmark of cigarette smoking is a shift in the protease/antiprotease balance, in favor of protease activity. However, it has recently been shown that smokers have increased expression of a key antiprotease, secretory leukoprotease inhibitor (SLPI), yet the mechanisms involved in SLPI transcriptional regulation and functional activity of SLPI remain unclear. We examined SLPI mRNA and protein secretion in differentiated nasal epithelial cells (NECs) and nasal lavage fluid (NLF) from nonsmokers and smokers and demonstrated that SLPI expression is increased in NECs and NLF from smokers. Transcriptional regulation of SLPI expression was confirmed using SLPI promoter reporter assays followed by chromatin immunoprecipitation. The role of STAT1 in regulating SLPI expression was further elucidated using WT and stat1(-/-) mice. Our data demonstrate that STAT1 regulates SLPI transcription in epithelial cells and slpi protein in the lungs of mice. Additionally, we reveal that NECs from smokers have increased STAT1 mRNA/protein expression. Finally, we demonstrate that SLPI contained in the nasal mucosa of smokers is proteolytically cleaved but retains functional activity against neutrophil elastase. These results demonstrate that smoking enhances expression of SLPI in NECs in vitro and in vivo, and that this response is regulated by STAT1. In addition, despite posttranslational cleavage of SLPI, antiprotease activity against neutrophil elastase is enhanced in smokers. Together, our findings show that SLPI regulation and activity is altered in the nasal mucosa of smokers, which could have broad implications in the context of respiratory inflammation and infection.

Abstract B018: Evaluation of the expression of coding and noncoding transcripts in epithelial cells of invasive ductal breast cancer of patients with and without hormone receptor

Abstract Introduction: Breast cancer is the most common tumor among women and is responsible for a high mortality rate worldwide. Gene expression profiling is vastly used in cancer research, and in breast cancer in particular it is used for diagnostic and prognostic characterization. The most frequent subtype, invasive ductal carcinoma (IDC), is considered a heterogeneous group of tumors with different alterations at the molecular level. Perou et al. in 2000 initially described five groups (Luminal A, Luminal B, HER-enriched, normal and basal-like) which have distinct gene expression patterns with different clinical implications. The use of genomics tools has contributed greatly to the understanding of the biology of breast tumors and the identification of new biomarkers. In recent years, long transcripts named long non-coding RNAs (lncRNAs) have been identified that do not encode proteins and are derived from intronic and intergenic sequences. Analysis of lncRNAs has shown differences in transcript expression in specific tissues and some tumors, showing that this area is promising for a better comprehending of cancer. In this context, the present study aims to evaluate the expression of coding and non-coding transcripts, in epithelial tumor cells of invasive ductal carcinoma. Experimental procedure: Determination of gene expression was performed by hybridization to a customized microarray slide with 244,000 probes, of which 44,000 are directed against protein-coding transcripts and 200,000 against lncRNAs. RNA samples derived from laser microdissected epithelial cells (103 IDCs of all subtypes, characterized by immunohistochemical markers) was twice amplified, labeled and hydribized to the microarray slide. The microarrays were analyzed using GeneSpring GX 12.1 software. Probes close to the background, saturated, or with uncertain reading were eliminated, resulting in 23.215 probes for further analysis. Clustering of the samples was performed according to Perou′s intrinsic groups following the PAM50 UNC microarray database guidelines. Coding and non-coding probes were analyzed separately, searching for differentially expressed transcripts in hormone receptor positive and negative subgroups, using t-test (unpaired, with FDR correction) considering fold change ≥|2| and p≥0.05. We compared the lists searching for genes and non-coding anti-sense transcripts within the same locus and performed Pearson correlation considering sense and anti-sense (SAS) pairs with coefficient ≥|0.5| and p ≥ 0.05. Partial Results: Robustness of microarray data was shown by our ability to replicate clustering of molecular profiles using only PAM50 genes. We identified SAS pairs with inverted expression pattern within the same locus, associated with hormone receptor status in breast tumors. We found 2386 coding probes and 358 non-coding probes differentially expressed, of which 56 were from the same locus and 15 had inverted expression (Pearson correlation ≥|0.5|). We are performing qRT-PCR validation of two pairs of candidate genes (LCOR and RFWD2) and their non-coding transcripts on the opposite strand of the same locus, which were inversely expressed with greater correlation coefficient. Perspective: The influence of non-coding antisense transcripts on the sense transcript′s coding is not yet fully understood, but it is suggestive that there is a role in regulating the expression of the gene encoding protein. Citation Format: Mabel Fernandez Pinilla, Alex de calvalho Fiorini, Dirce Maria Carraro. Evaluation of the expression of coding and noncoding transcripts in epithelial cells of invasive ductal breast cancer of patients with and without hormone receptor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B018.

SIRT1 Positively Regulates Breast Cancer Associated Human Aromatase (CYP19A1) Expression

Breast cancer remains one of the leading causes of death in women diagnosed with cancer. In breast cancer, aberrant expression of the CYP19A1 gene, which encodes the aromatase enzyme, contributes to increased intratumoral levels of estradiol. Regardless of whether this estrogen is produced by peripheral tissues or within specific subpopulations of cells within the breast tumor, it is clear that the aromatase enzymatic activity is critical for the growth of estrogen-dependent tumors. Currently, aromatase inhibitors have proven to be highly effective in blocking the growth of estrogen-dependent forms of breast cancer. CYP19A1 transcription is tightly controlled by 10 tissue-specific promoters. In breast cancer, however, aromatase transcription is driven by multiple promoters that somehow override the tissue-specific regulation of normal tissue. Here, we explore the role that the deacetylase, sirtuin-1 (SIRT1), plays in positively regulating aromatase in breast cancer. We demonstrate that the use of cambinol and the SIRT1/2 inhibitor VII, 2 small molecule inhibitors of SIRT1 and SIRT2, as well as small molecule inhibitors and small interfering RNA specific to SIRT1, all reduce the levels of aromatase mRNA. We further demonstrate that pharmacologic inhibition causes a marked reduction in aromatase protein levels. Additionally, by chromatin immunoprecipitation, we demonstrate that SIRT1 occupies the promoter regions PI.3/PII and PI.4, and its inhibition leads to increased acetylation of estrogen-related receptorα, a transcription factor that positively regulates CYP19A1 transcription in epithelial cells. Finally, we demonstrate by immunohistochemistry that SIRT1 is significantly up-regulated in invasive ductal carcinoma relative to normal tissue adjacent to tumor, further suggesting a role of SIRT1 in breast cancer. This work uncovers a new mechanism for the regulation of aromatase and provides rationale for further investigation of how the inhibition of specific sirtuins may provide a unique strategy for inhibiting aromatase that may complement or synergize with existing therapies.

2010 Winner: Leyla Kocgozlu

![Figure][1] We are pleased to announce that the winner of the 2010 JCS Prize is Leyla Kocgozlu, for her paper entitled ‘Selective and uncoupled role of substrate elasticity in the regulation of replication and transcription in epithelial cells’ ([Kocgozlu et al., 2010][2]). The $1000

E-cadherin regulates MAL-SRF-mediated transcription in epithelial cells

Epithelial junctions are dynamically and functionally linked to the actin cytoskeleton, and their disassembly is a key event during physiological and pathological processes. We recently showed that epithelial disintegration facilitates transcriptional activation via Rac, G-actin, MAL (also known as MRTF) and serum response factor (SRF). Here, we investigate which specific component of the epithelial junction is essential for this MAL-SRF-mediated transcription. The Ca(2+)-dependent dissociation of polarised epithelial cells depleted of ZO proteins - which form adherens junctions (AJs) but completely lack tight junctions (TJs) - fully activated SRF. By contrast, AGS gastric adenocarcinoma epithelial cells, which form TJs but are deficient in E-cadherin, and therefore also in AJs, failed to activate SRF. The introduction of wild-type E-cadherin in AGS cells restored AJ formation and MAL-SRF inducibility. To gain further insight into the membrane-proximal signalling, AGS cells were stably transfected with E-cadherin-alpha-catenin fusions. Despite restored formation of cell-cell contacts containing the nectin-afadin complex and p120-catenin, these cells did not activate SRF upon junction dissociation, suggesting that signal transmission depends on the C-terminal tail of E-cadherin. We conclude that the dissociation of intercellular E-cadherin interactions from AJs, and signals originating from the C-terminal region covering the beta-catenin-binding site of E-cadherin, are essential for transcriptional activation via Rac, MAL and SRF, whereas TJs are not involved.

Selective and uncoupled role of substrate elasticity in the regulation of replication and transcription in epithelial cells

Actin cytoskeleton forms a physical connection between the extracellular matrix, adhesion complexes and nuclear architecture. Because tissue stiffness plays key roles in adhesion and cytoskeletal organization, an important open question concerns the influence of substrate elasticity on replication and transcription. To answer this major question, polyelectrolyte multilayer films were used as substrate models with apparent elastic moduli ranging from 0 to 500 kPa. The sequential relationship between Rac1, vinculin adhesion assembly, and replication becomes efficient at above 200 kPa because activation of Rac1 leads to vinculin assembly, actin fiber formation and, subsequently, to initiation of replication. An optimal window of elasticity (200 kPa) is required for activation of focal adhesion kinase through auto-phosphorylation of tyrosine 397. Transcription, including nuclear recruitment of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), occurred above 50 kPa. Actin fiber and focal adhesion signaling are not required for transcription. Above 50 kPa, transcription was correlated with alphav-integrin engagement together with histone H3 hyperacetylation and chromatin decondensation, allowing little cell spreading. By contrast, soft substrate (below 50 kPa) promoted morphological changes characteristic of apoptosis, including cell rounding, nucleus condensation, loss of focal adhesions and exposure of phosphatidylserine at the outer cell surface. On the basis of our data, we propose a selective and uncoupled contribution from the substrate elasticity to the regulation of replication and transcription activities for an epithelial cell model.

Opposing Effects of KLF5 on the Transcription of MYC in Epithelial Proliferation in the Context of Transforming Growth Factor β

The proto-oncogene MYC plays a critical role in cell proliferation and tumorigenesis, and its down-regulation by transforming growth factor beta (TGFbeta) signaling is necessary for TGFbeta to inhibit cell proliferation. KLF5, on the other hand, is a pro-proliferative basic transcription factor that reverses function to become an anti-proliferative TGFbeta cofactor upon TGFbeta stimulation in epithelial homeostasis. In this study we investigated whether KLF5 directly regulates MYC transcription in epithelial cells in the context of TGFbeta. Knockdown of KLF5 significantly reduced MYC expression in the HaCaT epidermal epithelial cells. When TGFbeta was applied, however, whereas MYC expression was significantly inhibited, knockdown of KLF5 increased MYC expression. Furthermore, re-expression of KLF5 restored the inhibitory effect of TGFbeta on MYC expression in two cancer cell lines. Chromatin immunoprecipitation and oligo pulldown experiments demonstrated that whereas binding of KLF5 to both KLF5 binding element (KBE) and TGFbeta inhibitory element (TIE) DNA elements was necessary for MYC transcription, binding to KBE was decreased by TGFbeta, and binding to TIE was increased by TGFbeta. These results suggest that KLF5 is not only essential for MYC transcription in proliferating epithelial cells but also mediates the inhibitory effect of TGFbeta on MYC transcription. Furthermore, different binding sites mediate different effects of KLF5 in the context of TGFbeta.

Msx and Dlx Homeogene Expression in Epithelial Odontogenic Tumors

Epithelial odontogenic tumors are rare jaw pathologies that raise clinical diagnosis and prognosis dilemmas notably between ameloblastomas and clear cell odontogenic carcinomas (CCOCs). In line with previous studies, the molecular determinants of tooth development-amelogenin, Msx1, Msx2, Dlx2, Dlx3, Bmp2, and Bmp4-were analyzed by RT-PCR, ISH, and immunolabeling in 12 recurrent ameloblastomas and in one case of CCOC. Although Msx1 expression imitates normal cell differentiation in these tumors, other genes showed a distinct pattern depending on the type of tumor and the tissue involved. In benign ameloblastomas, ISH localized Dlx3 transcripts and inconstantly detected Msx2 transcripts in epithelial cells. In the CCOC, ISH established a lack of both Dlx3 and Msx2 transcripts but allowed identification of the antisense transcript of Msx1, which imitates the same scheme of distribution between mesenchyme and epithelium as in the cup stage of tooth development. Furthermore, while exploring the expression pattern of signal molecules by RT-PCR, Bmp2 was shown to be completely inactivated in the CCOC and irregularly noticeable in ameloblastomas. Bmp4 was always expressed in all the tumors. Based on the established roles of Msx and Dlx transcription factors in dental cell fates, these data suggest that their altered expression is a proposed trail to explain the genesis and/or the progression of odontogenic tumors.

NIPA1(SPG6), the Basis for Autosomal Dominant Form of Hereditary Spastic Paraplegia, Encodes a Functional Mg2+ Transporter

Mutations in the NIPA1(SPG6) gene, named for "nonimprinted in Prader-Willi/Angelman" has been implicated in one form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and weakness. However, the function of NIPA1 is unknown. Here, we show that reduced magnesium concentration enhances expression of NIPA1 suggesting a role in cellular magnesium metabolism. Indeed NIPA1 mediates Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with a Michaelis constant of 0.69+/-0.21 mM when expressed in Xenopus oocytes. Subcellular localization with immunofluorescence showed that endogenous NIPA1 protein associates with early endosomes and the cell surface in a variety of neuronal and epithelial cells. As expected of a magnesium-responsive gene, we find that altered magnesium concentration leads to a redistribution between the endosomal compartment and the plasma membrane; high magnesium results in diminished cell surface NIPA1 whereas low magnesium leads to accumulation in early endosomes and recruitment to the plasma membrane. The mouse NIPA1 mutants, T39R and G100R, corresponding to the respective human mutants showed a loss-of-function when expressed in oocytes and altered trafficking in transfected COS7 cells. We conclude that NIPA1 normally encodes a Mg2+ transporter and the loss-of function of NIPA1(SPG6) due to abnormal trafficking of the mutated protein provides the basis of the HSP phenotype.

The C-terminal 26-residue peptide of serpin A1 is an inhibitor of HIV-1

Serpin A1 (α1-proteinase inhibitor) inhibits human immunodeficiency virus 1 (HIV-1) production by mechanisms which remain to be elucidated. The complex formation of serpin A1 with proteinases eliminates the proteolytic activity and generates a fragment corresponding to the serpin C-terminal 36-residue peptide. Here, we show that the C-terminal 26-residue peptide of serpin A1 (A1-C26) inhibits HIV-long terminal repeat (LTR)-driven transcription in epithelial cells transfected with HIV-1 LTR promoter-driven genes. A1-C26 increased STAT1 phosphorylation and strongly reduced viral expression in a monocytic cell line infected with HIV-1 NL4-3. This reduction of expression was also observed in HIV-1 infected, PHA-activated peripheral blood mononuclear cells. In HIV-1 infected cells, the inhibitory activity of HIV-1 caused by B9-C23 and C1-C26, the A1-C26 homologues corresponding to the C-terminal sections of serpin B9 and serpin C1, was much lower than that obtained with A1-C26. These serpin peptides represent a novel class of antiviral agents.

Myeloid Elf-1-like Factor, an ETS Transcription Factor, Up-regulates Lysozyme Transcription in Epithelial Cells through Interaction with Promyelocytic Leukemia Protein

Myeloid elf-1-like factor (MEF) or Elf4, which is a member of the ETS transcription factor family, up-regulates the basal expression of lysozyme gene in epithelial cells and is constitutively localized in the nucleus. The mammalian cell nucleus is organized into distinct nuclear domains or compartments that are essential for diverse physiological processes. Promyelocytic leukemia (PML) nuclear body or nuclear domain 10 is one of the nuclear domains and is involved in tumor suppression and regulation of transcription. Here, we investigate the role of PML nuclear body in MEF transactivation. We show that PML, but not Sp100, induced the accumulation of MEF in PML nuclear bodies and that MEF and PML physically interacted. This interaction stimulated MEF transcriptional activity, resulting in the up-regulation of endogenous lysozyme expression. Amino acids 348-517 of MEF were required for the accumulation of MEF in PML nuclear bodies and up-regulation of lysozyme transcription, which is enhanced by PML. Moreover, the C-terminal region of MEF spanning amino acids 477-517 was the putative region required for interaction between MEF and PML as determined with the use of the mammalian two-hybrid system. In addition, heat-shock treatment induced the accumulation of MEF in endogenous PML nuclear bodies and enhanced MEF transactivation of lysozyme gene. Thus, the recruitment of MEF to PML nuclear bodies may partly regulate lysozyme transcription in epithelial cells.

Induction of Short Interspersed Nuclear Repeat-containing Transcripts in Epithelial Cells upon Infection with a Chicken Adenovirus

Chicken embryo lethal orphan adenovirus (CELO) is used as a vector for expression of exogenous genes in mammalian cells. Here, we analyzed transcriptional alterations in mouse epithelial host cells following infection with CELO using cDNA microarray analysis. Sequence data characterization revealed that a major portion of CELO-induced genes contained short interspersed nuclear elements of the B2 subclass (B2 SINEs). In fact, we could identify SINEs and other repetitive sequences as contributing significantly to the cDNAs used for microarray construction. Moreover, we found that the CELO protein Gam1 was able to mediate transcriptional activation of these B2 SINE-containing RNAs. We hypothesize that upregulation of B2-SINE-containing RNAs could be a novel contribution of Gam1 to CELO host cell infection.

The retinoblastoma protein binds the promoter of the survival gene bcl-2 and regulates its transcription in epithelial cells through transcription factor AP-2.

The retinoblastoma (RB) gene product has been shown to restrict cell proliferation, promote cell differentiation, and inhibit apoptosis. Loss of RB function can induce both p53-dependent apoptosis and p53-independent apoptosis; little is known about the mechanisms of RB-regulated p53-independent apoptosis. Here we show that RB specifically activates transcription of the survival gene bcl-2 in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated by the transcription factor AP-2. By monitoring protein-DNA interactions in living cells using formaldehyde cross-linking and chromatin immunoprecipitation, we show that endogenous RB and AP-2 both bind to the same bcl-2 promoter sequence. In addition, we demonstrate that RB and AP-2 also bind to the E-cadherin gene promoter in vivo, consistent with regulation of this promoter by both AP-2 and RB in epithelial cells. This study provides evidence that RB activates bcl-2 and E-cadherin by binding directly to the respective promoter sequences and not indirectly by repressing an inhibitor. This recruitment is mediated by a transcription factor, in this case AP-2. For the first time, our results suggest a direct molecular mechanism by which RB might inhibit apoptosis independently of p53. The results are discussed in a context where RB and Bcl-2 contribute under nonpathological conditions to the maintenance of cell viability in association with a differentiated phenotype, contributing to the tumor suppressor function of RB and playing important roles in normal development.

Bacterial stimulation of epithelial G-CSF and GM-CSF expression promotes PMN survival in CF airways.

Airway epithelial cells provide an immediate response to bacterial pathogens by producing chemokines and cytokines that recruit polymorphonuclear leukocytes (PMNs) to the site of infection. This response is excessive in patients with cystic fibrosis (CF) who have bacterial contamination of their airways. We postulated that CF airway pathogens, in activating nuclear factor-kappaB-dependent gene transcription in epithelial cells, would promote expression of cytokines that inhibit constitutive apoptosis of recruited PMNs. Epithelial cell culture supernatants from CF (IB-3) and corrected (C-38) epithelial cells stimulated by Staphylococcus aureus or Pseudomonas aeruginosa, increased survival of PMNs by 2- to 5-fold. Enhanced PMN survival was attributed to effects of epithelial granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor expression, which inhibit PMN apoptosis, and was negated by neutralizing antibody to either cytokine. Both CF and normal cells responded to bacteria with increased cytokine production. Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor expression were activated by ligation of asialoGM1, a receptor for P. aeruginosa and S. aureus, and by S. aureus lipoteichoic acid. Lipopolysaccharide was not a potent stimulus of cytokine expression, and P. aeruginosa algC (lipopolysaccharide) and lasR (quorum sensing) mutants were fully capable of activating epithelial cells. Induced expression of cytokines by airway cells repeatedly exposed to bacteria, as occurs in CF, serves not only to recruit and activate PMNs, but also to enhance their survival.