A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominantnegative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 'gain of function' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53 R175H mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53 R175H , was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial-mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53 R175H mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53 R175H mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.
The molecular mechanisms underlying
Aeromonas hydrophila
-pathogenesis are not well understood. Using head kidney macrophages (HKM) of
Clarias gariepinus
, we previously reported the role of ER-stress in
A. hydrophila
-induced pathogenesis. Here, we report that PI3K/PLC-induced cytosolic-Ca
2+
imbalance induces the expression of pro-apoptotic ER-stress marker, CHOP in
A. hydrophila-
infected HKM. CHOP promotes HKM apoptosis by inhibiting AKT activation and enhancing JNK signaling. Elevated mitochondrial ROS (mtROS) was recorded which declined significantly by ameliorating ER-stress and in the presence of ER-Ca
2+
release modulators (2-APB and dantrolene) and mitochondrial-Ca
2+
uptake inhibitor, Ru360, together suggesting the role of ER-mitochondrial Ca
2+
dynamics in mtROS generation. Inhibiting mtROS production reduced HKM death implicating the pro-apoptotic role of mtROS in
A. hydrophila
-pathogenesis. The expression of autophagic proteins (LC3B, beclin-1, and atg 5) was suppressed in the infected HKM. Our results with autophagy-inducer rapamycin demonstrated that impaired autophagy favored the cytosolic accumulation of mitochondrial DNA (mtDNA) and the process depended on mtROS levels. Enhanced caspase-1 activity and IL-1β production was detected and transfection studies coupled with pharmacological inhibitors implicated mtROS/mtDNA axis to be crucial for activating the caspase-1/IL-1β cascade in infected HKM. RNAi studies further suggested the involvement of IL-1β in generating pro-apoptotic NO in
A. hydrophila
-infected HKM. Our study suggests a novel role of ER-mitochondria cross-talk in regulating
A. hydrophila
pathogenesis. Based on our observations, we conclude that
A. hydrophila
induces ER-stress and inhibits mitophagy resulting in mitochondrial dysfunction which leads to mtROS production and translocation of mtDNA into cytosol triggering the activation of caspase-1/IL-1β-mediated NO production, culminating in HKM apoptosis.
Toll-like receptor 4 (TLR4) plays a critical role in host immunity against Gram-negative bacteria. It transduces signals through two distinct TIR-domain-containing adaptors, MyD88 and TRIF, which function at the plasma membrane and endosomes, respectively. Using zebrafish Aeromonas hydrophila infection model, we demonstrate that synchronization of MyD88 and TRIF dependent pathways is critical for determining the fate of infection. Zebrafish were infected with A. hydrophila, and bacterial recovery studies suggested its effective persistence inside the host. Histopathological assessment elucidates that A. hydrophila did not provoke inflammatory responses in the spleen. Immunofluorescence revealed the presence of TLR4-bound A. hydrophila on the plasma membrane at 3 h post-infection (p.i.), and inside endosomes 1 day p.i. Quantitative PCR studies suggest that TLR4 activates the downstream pathway of MyD88-IRAK4 axis at early stages followed by a shift to TRIF-TRAF6 axis at late stages of infection coupled with fold increase in NFκB. Our results implicated the involvement of p110δ isoform of PI(3)Kinase in this transition. Coupled to this, we noted that the TLR4-TRIF-NFκB axis prompted burgeoned secretion of anti-inflammatory cytokines. We observed that A. hydrophila inhibits endosome maturation and escapes to cytoplasm. Significant downregulation of cytosolic-NLR receptors further suggested that A. hydrophila represses pro-inflammatory responses in cytosol aiding its persistence. Our findings suggest a novel role of 'TLR4 topology' in A. hydrophila-induced pathogenesis. We propose that A. hydrophila manipulates translocation of TLR4 and migrates to endosome, where it triggers TRIF-dependent anti-inflammatory responses, interferes with endosomal maturation and escapes to cytosol. Inside the cytosol, A. hydrophila avoids detection by suppressing NLRs, facilitating its survival and ensuing pathogenesis.
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