Functional expression of TRPV4 in human periodontal ligament cells

Background: Periodontal ligament (PDL) cells are exposed to physical forces in vivo in response to mastication, parafunction, speech and orthodontic tooth movement. Although it has been shown that PDL cells perceive and respond directly to mechanical stimulation, the nature of the ion channels that mediate this mechanotransduction remain to be fully elucidated. The transient receptor potential (TRP) superfamily of ion channels is believed to play a critical role in sensory physiology, where they act as transducers for thermal, chemical and mechanical stimuli. Recent studies have shown that members of the vanilloid (TRPV) and ankyrin (TRPA) subfamilies encode mechanosensitive TRPs. The vanilloid family member TRPV4 is one such non selective calcium permeable cationic channel which has been shown to be activated by chemical ligands, hypotonicity, and mechanical stimuli. Objectives: The objective of the current study was to investigate functional expression of TRPV4 in cultured human PDL cells. Methods: Human PDL cells were grown in Dulbecco's Modified Eagle Medium with L-glutamine supplemented with 10% fetal bovine serum (FBS), 100UI/ml penicillin and 100μg/ml streptomycin. Cells in passage 4-6 were used in all experiments. TRPV4 functional expression was determined using ratiometric calcium imaging. Cultured cells were loaded with intracellular Ca2+ probe fura-2 and cells were then stimulated with the TRPV4 agonists, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), GSK1016790A or hypotonic solution. The TRPV4 antagonist RN 1734 was used to block the corresponding agonist responses. Results: PDL fibroblasts responded to application of TRPV4 agonists and hypotonic stimuli by an increase in intracellular calcium which was attenuated in the presence of the TRPV4 antagonist. Conclusions: We have shown for the first time the functional expression of the mechanosensitive TRPV4 channel in human PDL cells. The molecular identity and mechanisms of activation of mechanosensitive TRP channels in PDL cells merit further investigation.

MicroRNA-155 Promotes Resolution of Hypoxia-Inducible Factor 1{alpha} Activity

The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1a (but not HIF-2a) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxia-inducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1a mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1a in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1a mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1a stabilization and activity. Based on these data and a mathematical model of HIF-1a suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1a activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1a-dependent transcription.

Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2

The Jeryl Lynn (JL) vaccine against mumps virus (MuV) contains two components, MuV(JL5) and MuV(JL2), which differ by over 400 nt. Due to the occurrence of bias in the direction of mutation, these differences and those found in nucleotide sequences of different isolates of the minor component in the vaccine (MuV(JL2)) might be due to the effect of ADAR-like deaminases on MuV grown in tissue-cultured cells. A molecular clone Of MuV(JL2) (pMuV(JL2)) and MuV(JL2) -specific helper plasmids were constructed in order to investigate molecular interactions between MuV(JL5) and MuV(JL2), to augment the existing molecular clone Of MuV(JL)5 (pMuV(JL5)) and MuV(JL5) -specific helper plasmids. Genome and mRNA termini Of MuV(JL2) were characterized, and an unusual oligo-G insertion transcriptional editing event was detected near the F mRNA polyadenylation site of MuV(JL2), but not Of MuV(JL5). Genes encoding glycoproteins of rMuV(JL2) and rMuV(JL5) have been exchanged to characterize the oligo-G insertion, which associated with the specific sequence of the IF gene of MuV(JL2) and not with any other genes or the RNA-dependent RNA polymerase of strain MuV(JL2). The results indicate that a single G-to-A sequence change obliterates the co-transcriptional editing of the F mRNA and that this oligo-G insertion does not affect the growth of the virus.

Expression of the SEPT9_i4 isoform confers resistance to microtubule-interacting drugs

BACKGROUND: The evolutionarily conserved septin family of genes encode GTP binding proteins involved in a variety of cellular functions including cytokinesis, apoptosis, membrane dynamics and vesicle trafficking. Septin proteins can form hetero-oligomeric complexes and interact with other proteins including actin and tubulin. The human SEPT9 gene on chromosome 17q25.3 has a complex genomic architecture with 18 different transcripts that can encode 15 distinct polypeptides. Two distinct transcripts with unique 5' ends (SEPT9_v4 and SEPT9_v4*) encode the same protein. In tumours the ratio of these transcripts changes with elevated levels of SEPT9_v4* mRNA, a transcript that is translated with enhanced efficiency leading to increased SEPT9_i4 protein.

METHODS: We have examined the effect of over-expression of SEPT9_i4 on the dynamics of microtubule polymer mass in cultured cells.

RESULTS: We show that the microtubule network in SEPT9_i4 over-expressing cells resists disruption by paclitaxel or cold incubation but also repolymerises tubulin more slowly after microtubule depolymerisation. Finally we show that SEPT9_i4 over-expressing cells have enhanced survival in the presence of clinically relevant microtubule acting drugs but not after treatment with DNAinteracting agents.

CONCLUSIONS: Given that SEPT9 over-expression is seen in diverse tumours and in particular ovarian and breast cancer, such data indicate that SEPT9_v4 expression may be clinically relevant and contribute to some forms of drug resistance.

Glycosylation of low-density lipoprotein enhances cholesteryl ester synthesis in human monocyte-derived macrophages

Glucose can react with the lysine residues of low-density lipoproteins (LDLs) and convert the lipoprotein to a form with a receptor-mediated uptake by cultured cells that is impaired. However, in contrast to other modified lipoproteins taken up by both murine and human macrophages via the scavenger-receptor pathway that may induce the formation of foam cells, glycosylated LDL is not recognized by murine macrophages, and thus far, it has not been shown to lead to marked intracellular accumulation of cholesterol in human macrophages. This study illustrates that glycosylated LDL incubated with human monocyte-derived macrophages, at a concentration of 100 micrograms LDL/ml medium, stimulates significantly more cholesteryl ester (CE) synthesis than does control LDL (10.65 +/- 1.5 vs. 4.8 +/- 0.13 nmol.mg-1 cell protein.20 h-1; P less than .05). At LDL concentrations similar to those of plasma, the rate of CE synthesis in macrophages incubated with glycosylated LDL is more markedly enhanced than that observed in cells incubated with control LDL (3-fold increase). The marked stimulation of CE synthesis in human macrophages exposed to glycosylated LDL is paralleled by a significant increase in CE accumulation in these cells (P less than .001). The increase in CE synthesis and accumulation seem to be mediated by an increase in the degradation of glycosylated LDL by human macrophages. Glycosylated LDL enters the macrophages and is degraded by the classic LDL-receptor pathway in slightly smaller amounts than control LDL, but its degradation by pathways other than the classic LDL receptor or scavenger receptor is markedly enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

Culture and Characterization of Microglia from the Adult Murine Retina

Purpose. To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells. Method. Retinal single-cell suspensions were prepared from adult MF1 mice. Culture conditions including culture medium, growth factors, seeding cell density, and purification of microglia from the mixed cultures were optimised. Cultured retinal microglial cells were phenotyped using the surface markers CD45, CD11b, and F4/80. Their ability to secrete proinflammatory cytokines in response to lipopolysaccharide (LPS) stimulation was examined using cytometric bead array (CBA) assay. Results. Higher yield was obtained when retinal single-cell suspension was cultured at the density of cells per cm2 in Dulbecco’s modified Eagle medium (DMEM)/F12 + Glutamax supplement with 20% fetal calf serum (FCS) and 20% L929 supernatant. We identified day 10 to be the optimum day of microglial isolation. Over 98% of the cells isolated were positive for CD45, CD11b, and F4/80. After stimulating with LPS they were able to secrete proinflammatory cytokines such as IL-6 and TNF-α and express CD86, CD40, and MHC-II. Conclusion. We have developed a simple method for isolating and culturing retinal microglia from adult mice.

Resistance to chemotherapy in ovarian cancer is miRrored by the microRNA miR-433-dependent dysregulation of the cell cycle.

An understanding of the mechanisms underlying the development of resistance to chemotherapy treatment is a gateway to the introduction of novel therapies and improved outcomes for women presenting with ovarian cancer (OC). The desired apoptotic death post-chemotherapy depends on an intact and fully functioning cell cycle machinery.

In this study we demonstrate that stable expression of miR-433 renders OC cells more resistant to paclitaxel treatment. Interestingly, only cells with the highest miR-433 survived paclitaxel suggesting the possible role of miR-433 in cancer recurrence. Importantly, for the first time we demonstrate that miR 433 induces cellular senescence, exemplified by a flattened morphology, the downregulation of phosphorylated Retinoblastoma (p Rb) and increased β galactosidase activity. Surprisingly, miR 433 induced senescence was independent of two well recognised senescent drivers: p21 and p16. Further in silico analysis followed by in vitro experiments identified CKD6 as a novel miR-433 target gene possibly explaining the observed p21 and p16-independent induction of cellular senescence. Another in silico identified miR-433 target gene was CDC27, a protein involved in the regulation of the cell cycle during mitosis. We demonstrate that the overexpression of pre-miR-433 leads to the downregulation of CDC27 in vitro revealing a novel interaction between miR-433 and CDC27, an integral cell cycle regulating protein.

Interestingly, miR-433 expressing cells also demonstrated an ability to impact their tumour microenvironment. We show that miR-433 is present in exosomes released from miR-433 overexpressing and high miR-433 naïve cells. Moreover, growth condition media (GCM) harvested from cells with high miR-433 have higher levels of IL-6 and IL-8, two key cytokines involved in the senescence associated secretory phenotype (SASP). Importantly, GCM from miR-433-enriched cells repressed the growth of co-cultured cells with initial studies showing a GCM-dependent induction of chemoresistance.

In conclusion, data in this study highlights how the aberrant expression miR-433 contributes to chemoresistance in OC cells. We postulate that standard chemotherapy, particularly paclitaxel, used to treat women with OC may have an attenuated ability to kill cells harbouring increased levels of miR-433, allowing for a subsequent chemoresistant phenotype post-therapy.

Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70

The Wilms tumor suppressor WT1 encodes a zinc finger transcription factor that is expressed in glomerular podocytes during a narrow window in kidney development. By immunoprecipitation and protein microsequencing analysis, we have identified a major cellular protein associated with endogenous WT1 to be the inducible chaperone Hsp70. WT1 and Hsp70 are physically associated in embryonic rat kidney cells, in primary Wilms tumor specimens and in cultured cells with inducible expression of WT1. Colocalization of WT1 and Hsp70 is evident within podocytes of the developing kidney, and Hsp70 is recruited to the characteristic subnuclear clusters that contain WT1. The amino-terminal transactivation domain of WT1 is required for binding to Hsp70, and expression of that domain itself is sufficient to induce expression of Hsp70 through the heat shock element (HSE). Substitution of a heterologous Hsp70-binding domain derived from human DNAJ is sufficient to restore the functional properties of a WT1 protein with an amino-terminal deletion, an effect that is abrogated by a point mutation in DNAJ that reduces binding to Hsp70. These observations indicate that Hsp70 is an important cofactor for the function of WT1, and suggest a potential role for this chaperone during kidney differentiation.

Neuropeptide Receptors in the Dental Pulp

Objectives: The inflammatory response to pulpal injury or infection has major clinical significance. The aim of the study is to investigate the presence and regulation of expression of neuropeptide receptors on human pulp fibroblasts and whole pulp tissue. This study will investigate the expression of Substance P (NK-1) and Neuropeptide Y (NPY-Y1) receptors on pulp fibroblasts, determine the effects of Transforming Growth Factor Beta-1 (TGF-b1) and Interleukin 1-Beta (IL-1b) on the expression of NK-1 and NPY-Y1 receptors on pulp fibroblasts and examine the levels of receptor expression in whole pulp samples. Methods: Primary pulp fibroblast cell lines were obtained from patients undergoing extractions for orthodontic reasons. The cells were grown to confluence and stimulated for 5 days with IL-1b or TGF-b1. Pulp tissue fragments were obtained from freshly extracted sound and carious teeth, snap frozen in liquid nitrogen and cracked open using a vice. The monolayer was removed with cell scrapers and pelleted. The cell membranes of the cultured cells and the whole tissue were isolated using a Mem-PER® Eukaryotic Membrane Protein Extraction Reagent Kit (Pierce, UK). The membrane proteins were separated by SDS-PAGE and Western blotting was used to detect the presence of NK-1 and NPY-Y1. Results: Initial results demonstrated the presence of NK-1 and NPY-Y1 in cultured pulp fibroblasts. Following the 5 day incubation with TGF-b1, the cells appeared not to express NK-1. IL-1b had a slight stimulatory effect on NK-1 expression. The NPY-Y1 expression was not affected by either TGF-b1 or IL-1b. In whole pulp samples, levels of NK-1 were increased in carious teeth compared to caries-free teeth. The NPY-Y1 levels were similar in carious and non-carious teeth. Conclusion: These findings give an insight into how pulp cells react to inflammatory stimuli with regards to neuropeptide receptor expression and their roles in health and disease

Dissecting the role of the Tir:Nck and Tir:IRTKS/IRSp53 signalling pathways in vivo

Attaching and effacing (A/E) lesions and actin polymerization, the hallmark of enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) infections, are dependent on the effector Tir. Phosphorylation of Tir(EPEC/CR) Y474/1 leads to recruitment of Nck and neural Wiskott-Aldrich syndrome protein (N-WASP) and strong actin polymerization in cultured cells. Tir(EPEC/CR) also contains an Asn-Pro-Tyr (NPY(454/1)) motif, which triggers weak actin polymerization. In EHEC the NPY(458) actin polymerization pathway is amplified by TccP/EspF(U), which is recruited to Tir via IRSp53 and/or insulin receptor tyrosine kinase substrate (IRTKS). Here we used C. rodentium to investigate the different Tir signalling pathways in vivo. Following infection with wild-type C. rodentium IRTKS, but not IRSp53, was recruited to the bacterial attachment sites. Similar results were seen after infection of human ileal explants with EHEC. Mutating Y471 or Y451 in Tir(CR) abolished recruitment of Nck and IRTKS respectively, but did not affect recruitment of N-WASP or A/E lesion formation. This suggests that despite their crucial role in actin polymerization in cultured cells the Tir:Nck and Tir:IRTKS pathways are not essential for N-WASP recruitment or A/E lesion formation in vivo. Importantly, wild-type C. rodentium out-competed the tir tyrosine mutants during mixed infections. These results uncouple the Tir:Nck and Tir:IRTKS pathways from A/E lesion formation in vivo but assign them an important in vivo role.

Cortactin recruitment by enterohemorrhagic Escherichia coli O157:H7 during infection in vitro and ex vivo

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen that colonizes the gut mucosa via attaching and effacing (A/E) lesions; A/E lesion formation in vivo and ex vivo is dependent on the type III secretion system (T3SS) effector Tir. Infection of cultured cells by EHEC leads to induction of localized actin polymerization, which is dependent on Tir and a second T3SS effector protein, TccP, also known as EspF(U). Recently, cortactin was shown to bind both the N terminus of Tir and TccP via its SH3 domain and to play a role in EHEC-triggered actin polymerization in vitro. In this study, we investigated the recruitment of cortactin to the site of EHEC adhesion during infection of in vitro-cultured cells and mucosal surfaces ex vivo (using human terminal ileal in vitro organ cultures [IVOC]). We have shown that cortactin is recruited to the site of EHEC adhesion in vitro downstream of TccP and N-WASP. Deletion of the entire N terminus of Tir or replacing the N-terminal polyproline region with alanines did not abrogate actin polymerization or cortactin recruitment. In contrast, recruitment of cortactin to the site of EHEC adhesion in IVOC is TccP independent. These results imply that cortactin is recruited to the site of EHEC adhesion in vitro and ex vivo by different mechanisms and suggest that cortactin might have a role during EHEC infection of mucosal surfaces.

Enteropathogenic Escherichia coli O125:H6 triggers attaching and effacing lesions on human intestinal biopsy specimens independently of Nck and TccP/TccP2

Typical enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) employ either Nck, TccP/TccP2, or Nck and TccP/TccP2 pathways to activate the neuronal Wiskott-Aldrich syndrome protein (N-WASP) and to trigger actin polymerization in cultured cells. This phenotype is used as a marker for the pathogenic potential of EPEC and EHEC strains. In this paper we report that EPEC O125:H6, which represents a large category of strains, lacks the ability to utilize either Nck or TccP/TccP2 and hence triggers actin polymerization in vitro only inefficiently. However, we show that infection of human intestinal biopsies with EPEC O125:H6 results in formation of typical attaching and effacing lesions. Expression of TccP in EPEC O125:H6, which harbors an EHEC O157-like Tir, resulted in efficient actin polymerization in vitro and enhanced colonization of human intestinal in vitro organ cultures with detectable N-WASP and electron-dense material at the site of bacterial adhesion. These results show the existence of a natural category of EPEC that colonizes the gut mucosa using Nck- and TccP-independent mechanisms. Importantly, the results highlight yet again the fact that conclusions made on the basis of in vitro cell culture models cannot be extrapolated wholesale to infection of mucosal surfaces and that the ability to induce actin polymerization on cultured cells should not be used as a definitive marker for EPEC and EHEC virulence.