Warren Thomas

How bacteria could cause cancer: one step at a time

Helicobacter pylori highlighted the potential for bacteria to cause cancer. It is becoming clear that chronic infection with other bacteria, notably Salmonella typhi, can also facilitate tumour development. Infections caused by several bacteria (e.g. Bartonella spp., Lawsonia intracellularis and Citrobacter rodentium) can induce cellular proliferation that can be reversed by antibiotic treatment. Other chronic bacterial infections have the effect of blocking apoptosis. However, the underlying cellular mechanisms are far from clear. Conversely, several bacterial toxins interfere with cellular signalling mechanisms in a way that is characteristic of tumour promoters. These include Pasteurella multocida toxin, which uniquely acts as a mitogen, and Escherichia coli cytotoxic necrotizing factor, which activates Rho family signalling. This leads to activation of COX2, which is involved in several stages of tumour development, including inhibition of apoptosis. Such toxins could provide valuable models for bacterial involvement in cancer, but more significantly they could play a direct role in cancer causation and progression.

17β-Estradiol Inhibits IL-8 in Cystic Fibrosis by Up-Regulating Secretory Leucoprotease Inhibitor

RATIONALE An unexplained gender gap is observed in cystic fibrosis (CF). Females have poorer lung function, decreased survival, and earlier Pseudomonas colonization. OBJECTIVES To evaluate the effect of 17beta-estradiol (E(2)) on CF bronchial epithelial cells in vitro and in vivo. METHODS On exposure of CFBE41o- cultures to physiological concentrations of E(2), there was a significant dose-dependent inhibition of IL-8 release induced by toll-like receptor agonists, CF bronchoalveolar lavage fluid, or Pseudomonas-conditioned media. Estrogen receptor (ER)-alpha and -beta expression was quantified in cell lines and bronchial brushings from CF and non-CF patients. MEASUREMENTS AND MAIN RESULTS Both receptors were expressed in vitro and in vivo, although ERbeta expression was significantly higher in CF. Using ER isoform-specific agonists and antagonists, we established that ERbeta mediates the inhibition of CF bronchoalveolar lavage fluid-induced IL-8 release. We also showed that secretory leucoprotease inhibitor gene expression and protein localization to the nucleus increased in response to E(2). Secretory leucoprotease inhibitor knockdown abrogated the inhibitory effects of E(2). CONCLUSIONS E(2) inhibits IL-8 release by ERbeta in CF bronchial epithelial cells through up-regulation of secretory leucoprotease inhibitor, inhibition of nuclear factor (NF)-kappaB, and IL-8 gene expression. These data implicate a novel anti-inflammatory mechanism for E(2) in females with CF, which predisposes to infection and colonization. This could, in part, account for the observed gender dichotomy in CF.

Absence of SHIP-1 Results in Constitutive Phosphorylation of Tank-Binding Kinase 1 and Enhanced TLR3-Dependent IFN-β Production

Autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, result from a loss of tolerance to self-antigens and immune-mediated injury precipitated by the overproduction of type I IFN and inflammatory cytokines. We have identified the inositol 5′ phosphatase SHIP-1 as a negative regulator of TLR3-induced type I IFN production. SHIP-1–deficient macrophages display enhanced TLR-induced IFN-β production, and overexpression of SHIP-1 negatively regulates the ability of TLR3 and its adaptor, Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β, to induce IFN-β promoter activity, indicating that SHIP-1 negatively regulates TLR-induced IFN-β production. Further dissection of the IFN-β pathway implicates TANK-binding kinase 1 (TBK1) as the target for SHIP-1. Critically, in the absence of SHIP-1, TBK1 appears to be hyperphosphorylated both in unstimulated cells and following TLR3 stimulation. In addition, TBK1 appears to be constitutively associated with Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β and TNFR-associated factor 3 in SHIP-1 deficient cells, whereas in wild-type cells this association is inducible following TLR3 stimulation. In support of a role for SHIP-1 in regulating complex formation, confocal microscopy demonstrates that TBK1 distribution in the cell is significantly altered in SHIP-1–deficient cells, with more prominent endosomal staining observed, compared with wild-type controls. Taken together, our results point to SHIP-1 as a critical negative regulator of IFN-β production downstream of TLR3 through the regulation of TBK1 localization and activity.

Aldosterone Regulates Rapid Trafficking of Epithelial Sodium Channel Subunits in Renal Cortical Collecting Duct Cells via Protein Kinase D Activation

Aldosterone elicits rapid physiological responses in target tissues such as the distal nephron through the stimulation of cell signaling cascades. We identified protein kinase D (PKD1) as an early signaling response to aldosterone treatment in the M1-cortical collecting duct (M1-CCD) cell line. PKD1 activation was blocked by the PKC inhibitor chelerythrine chloride and by rottlerin, a specific inhibitor of PKCdelta. The activation of PKCdelta and PKCepsilon coincided with PKD1 activation and while a complex was formed between PKD1 and PKCepsilon after aldosterone treatment, there was a concurrent reduction in PKD1 association with PKCdelta. A stable PKD1 knockdown M1-CCD-derrived clone was developed in which PKD1 expression was 90% suppressed by gene silencing with a PKD1-specific siRNA. The effect of aldosterone treatment on the subcellular distribution of enhanced cyan fluorescent protein (eCFP)-tagged epithelial sodium channel (ENaC) subunits in wild type (WT) and PKD1 suppressed cells was examined using confocal microscopy. In an untreated confluent monolayer of M1-CCD cells, alpha, beta, and gamma ENaC subunits were evenly distributed throughout the cytoplasm of WT and PKD1-suppressed cells. After 2 min treatment, aldosterone stimulated the localization of each of the ENaC subunits to discrete regions within the cytoplasm of WT cells. The translocation of eCFP-ENaC subunits in WT cells was inhibited by rottlerin and the mineralocorticoid receptor (MR) antagonist spironolactone. No subcellular translocation of eCFP-ENaC subunits was observed in PKD1-suppressed cells treated with aldosterone. These data demonstrate the involvement of a novel MR/PKCdelta /PKD1 signaling cascade in the earliest ENaC subunit intracellular trafficking events that follow aldosterone treatment.

Aldosterone rapidly activates protein kinase D via a mineralocorticoid receptor/EGFR trans-activation pathway in the M1 kidney CCD cell line

Aldosterone elicits physiological responses through the modulation of gene expression and by stimulating signaling processes. Here we investigated the activation pathway of protein kinase D1 (PKD1) by aldosterone in the murine M1 renal cortical collecting duct cell line. Aldosterone stimulated a rapid increase in PKD1 activity peaking at 2-5 min and at 30 min after treatment that was insensitive to inhibitors of transcription or translation. PKD1 was not activated by aldosterone in MR null NIH-3T3 fibroblasts or M1-CCD cells propagated without dexamethasone, which did not express MR. PKD1 activation was sensitive to the MR antagonists spironolactone and RU28318 but not to the glucocorticoid receptor antagonist RU486. Aldosterone activation of PKD1 was inhibited by the epidermal growth factor (EGFR) antagonist tyrphostin AG1478 and by the c-Src inhibitor PP2. Western blotting revealed EGFR phosphorylation following aldosterone treatment at the c-Src tyrosine kinase-specific residue Tyr845. The activation of c-Src was dependent on its interaction with HSP84, since HSP84 antagonist 17-AAG inhibited both the phosphorylation of EGFR in response to aldosterone by c-Src and also the subsequent activation of PKD1.

Estrogen induces phospholipase A2 activation through ERK1/2 to mobilize intracellular calcium in MCF-7 cells.

The principal secreted estrogen, 17beta-estradiol rapidly activates signaling cascades that regulate important physiological processes including ion transport across membranes, cytosolic pH and cell proliferation. These effects have been extensively studied in the MCF-7 estrogen-responsive human breast carcinoma cell line. Here, we demonstrate that a physiological concentration of 17beta-estradiol caused a rapid, synchronous and transient increase in intracellular calcium concentration in a confluent monolayer of MCF-7 cells 2-3 min after treatment. This response was abolished when cells were pre-incubated with the phospholipase A(2) (PLA(2)) inhibitor quinacrine or with the cyclooxygenase inhibitor indomethacin. The translocation of GFP-cPLA(2)alpha to perinuclear membranes occurred 1-2 min after 17beta-estradiol treatment; this translocation was concurrent with the transient phosphorylation of cPLA(2)alpha at serine residue 505. The phosphorylation and translocation of cPLA(2) were sensitive to inhibition of the extracellular signal regulated kinase (ERK) signaling cascade and occurred simultaneously with a transient activation of ERK. The phosphorylation of cPLA(2) could be stimulated by membrane impermeable 17beta-estradiol conjugated to bovine serum albumen and was blocked by an antagonist of the classical estrogen receptor. Here we show, for the first time, that PLA(2) and the eicosanoid biosynthetic pathway are involved in the 17beta-estradiol induced rapid calcium responses of breast cancer cells.

Mechanisms Underlying Rapid Aldosterone Effects in the Kidney

The steroid hormone aldosterone is a key regulator of electrolyte transport in the kidney and contributes to both homeostatic whole-body electrolyte balance and the development of renal and cardiovascular pathologies. Aldosterone exerts its action principally through the mineralocorticoid receptor (MR), which acts as a ligand-dependent transcription factor in target tissues. Aldosterone also stimulates the activation of protein kinases and secondary messenger signaling cascades that act independently on specific molecular targets in the cell membrane and also modulate the transcriptional action of aldosterone through MR. This review describes current knowledge regarding the mechanisms and targets of rapid aldosterone action in the nephron and how aldosterone integrates these responses into the regulation of renal physiology.

Rapid responses to aldosterone in the kidney and colon

Aldosterone is a crucial modulator of ion transport across high resistance epithelia and regulates whole body electrolyte balance through its effects on the kidney and colon. The net consequence of aldosterone release is to promote salt conservation. The genomic mechanism of aldosterone action is relatively well characterized and the role of the classical mineralocorticoid receptor as a ligand-dependent transcription factor is well established. The rapid effects of aldosterone on target tissues are less well understood and there is still controversy over the identity of the aldosterone non-genomic receptor. Greater understanding of the physiological consequences of aldosterones rapid responses in the kidney and colon has been achieved through the identification of definite and putative membrane targets and their signaling regulators.

Estrogen Receptor β Exerts Tumor Repressive Functions in Human Malignant Pleural Mesothelioma via EGFR Inactivation and Affects Response to Gefitinib

Background The role of estrogen and estrogen receptors in oncogenesis has been investigated in various malignancies. Recently our group identified estrogen receptor beta (ERβ) expression as an independent prognostic factor in the progression of human Malignant Pleural Mesothelioma (MMe), but the underlying mechanism by which ERβ expression in tumors determines clinical outcome remains largely unknown. This study is aimed at investigating the molecular mechanisms of ERβ action in MMe cells and disclosing the potential translational implications of these results. Methods We modulated ERβ expression in REN and MSTO-211H MMe cell lines and evaluated cell proliferation and EGF receptor (EGFR) activation. Results Our data indicate that ERβ knockdown in ER positive cells confers a more invasive phenotype, increases anchorage independent proliferation and elevates the constitutive activation of EGFR-coupled signal transduction pathways. Conversely, re-expression of ERβ in ER negative cells confers a more epithelioid phenotype, decreases their capacity for anchorage independent growth and down-modulates proliferative signal transduction pathways. We identify a physical interaction between ERβ, EGFR and caveolin 1 that results in an altered internalization and in a selective reduced activation of EGFR-coupled signaling, when ERβ is over-expressed. We also demonstrate that differential expression of ERβ influences MMe tumor cell responsiveness to the therapeutic agent: Gefitinib. Conclusions This study describes a role for ERβ in the modulation of cell proliferation and EGFR activation and provides a rationale to facilitate the targeting of a subgroup of MMe patients who would benefit most from therapy with Gefitinib alone or in combination with Akt inhibitors.

Protein kinase D stabilizes aldosterone-induced ERK1/2 MAP kinase activation in M1 renal cortical collecting duct cells to promote cell proliferation.

Aldosterone elicits transcriptional responses in target tissues and also rapidly stimulates the activation of protein kinase signalling cascades independently of de novo protein synthesis. Here we investigated aldosterone-induced cell proliferation and extra-cellular regulated kinase 1 and 2 (ERK1/2) mitogen activated protein (MAP) kinase signalling in the M1 cortical collecting duct cell line (M1-CCD). Aldosterone promoted the proliferative growth of M1-CCD cells, an effect that was protein kinase D1 (PKD1), PKCdelta and ERK1/2-dependent. Aldosterone induced the rapid activation of ERK1/2 with peaks of activation at 2 and 10 to 30 min after hormone treatment followed by sustained activation lasting beyond 120 min. M1-CCD cells suppressed in PKD1 expression exhibited only the early, transient peaks in ERK1/2 activation without the sustained phase. Aldosterone stimulated the physical association of PKD1 with ERK1/2 within 2 min of treatment. The mineralocorticoid receptor (MR) antagonist RU28318 inhibited the early and late phases of aldosterone-induced ERK1/2 activation, and also aldosterone-induced proliferative cell growth. Aldosterone induced the sub-cellular redistribution of ERK1/2 to the nuclei at 2 min and to cytoplasmic sites, proximal to the nuclei after 30 min. This sub-cellular distribution of ERK1/2 was inhibited in cells suppressed in the expression of PKD1.