Heat shock protein A12B (HSPA12B) is a newly discovered member of the HSP70 protein family. This study investigated the effects of HSPA12B on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) and the possible mechanisms involved. A HUVECs inflammatory model was induced by LPS. Overexpression of HSPA12B in HUVECs was achieved by infection with recombinant adenoviruses encoding green fluorescence protein-HSPA12B. Knockdown of HSPA12B was achieved by siRNA technique. Twenty four hours after virus infection or siRNA transfection, HUVECs were stimulated with 1 μg/ml LPS for 4 hrs. Endothelial cell permeability ability was determined by transwell permeability assay. The binding rate of human neutrophilic polymorphonuclear leucocytes (PMN) with HUVECs was examined using myeloperoxidase assay. Cell migrating ability was determined by the wound-healing assay. The mRNA and protein expression levels of interested genes were analyzed by RT-qPCR and Western blot, respectively. The release of cytokines interleukin-6 and tumour necrosis factor-α was measured by ELISA. HSPA12B suppressed LPS-induced HUVEC permeability and reduced PMN adhesion to HUVECs. HSPA12B also inhibited LPS-induced up-regulation of adhesion molecules and inflammatory cytokine expression. By contrast, knockdown of HSPA12B enhanced LPS-induced increases in the expression of adhesion molecules and inflammatory cytokines. Moreover, HSPA12B activated PI3K/Akt signalling pathway and pharmacological inhibition of this pathway by Wortmannin completely abrogated the protection of HSPA12B against inflammatory response in HUVECs. Our results suggest that HSPA12B attenuates LPS-induced inflammatory responses in HUVECs via activation of PI3K/Akt signalling pathway.

Aims Toll-like receptor (TLR)-mediated signalling pathways have been implicated in myocardial ischaemia/reperfusion (I/R) injury. Activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway protects the myocardium from ischaemic injury. We hypothesized that the modulation of TLR2 would induce cardioprotection against I/R injury via activation of the PI3K/Akt signalling. Methods and results Mice were treated with TLR2 ligands, peptidoglycan (PGN) or Pam3CSK4, respectively, 1 h before the hearts were subjected to ischaemia (1 h), followed by reperfusion (4 h). Infarct size was determined by triphenyltetrazolium chloride staining. Cardiac function and haemodynamic performance were evaluated. Infarct size was significantly reduced in PGN-or Pam3CSK4-treated mice compared with untreated I/R mice. Administration of TLR2 ligands improved cardiac function following I/R. PGN treatment increased the levels of phospho-Akt and phospho-GSK-3β (glycogen synthase kinase-3β), compared with untreated I/R hearts. PGN stimulation increased TLR2 tyrosine phosphorylation and association of the p85 subunit of PI3K with TLR2. To investigate the role of PI3K/Akt signalling in PGN-induced cardioprotection, we administered the PI3K inhibitor, Wortmannin, to the mice 15 min before PGN treatment. We also administered PGN to kinase-deficient Akt (kdAkt) transgenic mice 1 h before myocardial I/R. Both PI3K inhibition and kdAkt mice abolished the cardioprotection induced by PGN. To examine the role of TLR2 in PGN-induced cardioprotection, we administrated PGN to TLR2 knockout mice 1 h before the hearts were subjected to I/R. PGN-induced cardioprotection was lost in TLR2-deficient mice. Conclusion These results demonstrate that TLR2 ligands induced cardioprotection, which is mediated through a TLR2/PI3K/Akt-dependent mechanism.

<p>There is increasing evidence that prostate tumours are organized as a hierarchy with rare cancer stem cells (CSCs) implicated in initiating and maintaining the tumour. However, prospective prostate cancer stem cells (PCSCs) have not been thoroughly characterized from primary tissue specimens. Using the DU145 cell line, PCSCs have been propagated as non-adherent spheres <em>in vitro</em>. Approximately 1.25% of monolayer DU145 cells formed primary spheres while 26% of sphere cells formed subsequent spheres; a measure of PCSC self-renewal capacity. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins and CSC markers (CD44, CD24, integrin alpha2beta1). PCSCs initiate xenograft tumours with enhanced capacity compared to monolayer cells. While epidermal growth factor (EGF) promoted PCSC propagation, basic fibroblast growth factor (bFGF) inhibited these events. Activation of EGF receptor (EGFR) signalling, following EGF treatment or expression of constitutively-active EGFR (EGFRvIII), increased sphere formation. Conversely, attenuation of EGFR signalling inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signalling, the MEK-ERK pathway contributes to EGFR-facilitated PCSC propagation. Inhibition of ERK activation following MEK inhibitor treatment, expression of dominant-negative MEK1(K97M), or knockdown of ERK1 or ERK2 reduced PCSC propagation. Therefore, EGFR signalling promotes PCSC self-renewal by activating the MEK-ERK pathway.</p> <p>SOX2 is an essential transcription factor for stem cells, however, its role in PCSCs remains unclear. SOX2 protein is upregulated in PCSCs propagated as spheres, and its expression is regulated by EGFR signalling. EGFR activation, following EGF treatment or expression of constitutively-active EGFRvIII, increased SOX2 expression and PCSC self-renewal, while being attenuated by EGFR inhibitor treatment. Ectopic SOX2 expression enhanced EGF-induced PCSC self-renewal, while SOX2 knockdown renders PCSCs non-responsive to EGF-induced self-renewal and reduced their anchorage-independent growth. Furthermore, SOX2 expression is associated with the ability of PCSCs to form aggressive xenograft tumours. Collectively, SOX2 regulates EGFR-mediated PCSC self-renewal.</p> / Doctor of Philosophy (PhD)

Class I PI3Ks are a family (α, β, δ and γ) of ubiquitous lipid kinases that can be activated by cell surface receptors to 3-phosphorylate PI(4,5)P2 (phosphatidylinositol(4,5)-bisphosphate) and generate the signalling lipid PI(3,4,5)P3. The PI(3,4,5)P3 signal then activates a diverse collection of effector proteins involved in regulation of cell migration, metabolism and growth. The importance of this network is evidenced by the relatively high frequency with which cancers acquire gain-of-function mutations in this pathway and huge efforts to make PI3K inhibitors to treat cancer. The canonical model describing these events suggests class I PI3Ks are activated at the plasma membrane and generate PI(3,4,5)P3 in the inner leaflet of the plasma membrane where its effectors are activated. The PI(3,4,5)P3 signal can be terminated directly, by the tumour-suppressor and PI(3,4,5)P3-3-phosphatase PTEN, or modified to a distinct PI(3,4)P2 signal, by SHIP-family 5-phosphatases. The PI(3,4)P2 is removed by INPP4-family 4-phosphatases. Published work has shown that PI(3,4,5)P3 signalling can also occur in endosomes and nuclei, however, there is very little data defining the intracellular distribution of endogenous class I PI3Ks that supports these ideas; this is as a result of technical problems such as; their very low abundance, poor antibody-based tools and artefacts generated by overexpression of PI3Ks. Past work has indicated that, in PTEN-null mouse models of prostate tumour progression, either PI3Kβ or PI3Ks α and β, have important roles. Furthermore, the cell types and mechanism involved remained unclear. Recent published work in the host laboratory had indicated that there is an unexpectedly large accumulation of PI(3,4)P2 in PTEN-null cells that might be an important part of its status as a major tumour suppressor. The explanation and prevalence of this observation was unclear but potentially a result of PTEN also acting as a PI(3,4)P2 3-phosphatase in vivo. MEFs were derived from genetically-modified mice expressing endogenous, AviTagged class I PI3K subunits and used in experiments to define the subcellular localisation of class I PI3Ks. We found that following stimulation with PDGF, class IA PI3K subunits were unexpectedly depleted from the adherent basal membrane, in contrast, p85α and p110α, but not p85β and p110β, accumulated transiently in the nucleus. Interestingly, p110β, but none of the other subunits, was constitutively localised in the nucleus. These results support the idea that class I PI3K and PI(3,4,5)P3 signalling occurs in the nucleus. In organoids derived from WT, PI3Kγ-null or PTEN-null mouse prostate, application of PI3K-selective inhibitors revealed that PI3Kα had a dominant role in generating PI(3,4,5)P3 in prostate epithelial cells. The levels of PI(3,4)P2 were also elevated substantially in PTEN-null, compared to WT prostate organoids, use of PI3K-selective inhibitors suggested that it was also generated by PI3Kα. These data were consistent with the idea that PTEN can act as a PI(3,4)P2 3-phosphatase. Surprisingly, raising the pH of the organoids medium dramatically increased accumulation of PI(3,4,5)P3 and PI(3,4)P2, although the cause of this effect was unclear, we hypothesised the pH of the local environment may influence signalling via class I PI3Ks.