Addressing the effects of Salmonella internalization in host cell signaling on a reverse‐phase protein array

Through acute enteric infection, Salmonella invades host enterocytes and reproduces intracellularly into specialized vacuolae. This involves changes in host cell signaling elicited by bacterial proteins delivered via type III secretion systems (TTSS). One of the two TTSSs of Salmonella enterica serovar Typhimurium encoded by the Salmonella pathogenicity island‐1, triggers bacterial internalization. Among the effector proteins translocated by this TTSS, the GTPase modulator SopE/E2 and the phosphoinositide phosphatase SigD are known to play key roles in these processes. To better understand their contribution to re‐programming host cell pathways, we used ZeptoMARK reverse‐phase protein array technology, which allows printing 32‐sample lysate arrays that can be analyzed with phospho‐specific antibodies to evaluate the phosphorylation of signaling proteins. Lysates were obtained at different times after infection of HeLa cells with WT, TTSS‐deficient, sopE/E2 and sigD single and double deletants, as well as different sigD Salmonella mutants. Our analysis detected activation of p38, JNK and ERK mitogen‐activated protein kinases, mainly dependent on SopE/E2, as well as SigD‐dependent phosphorylation of PKB/Akt and its targets GSK‐3β and FKHR/FoxO. This is the first time that reverse‐phase protein array technology is used in the cellular microbiology field, demonstrating its value to screen for host signaling events through bacterial infection.     

LUCAT1 contributes to MYRF‐dependent smooth muscle cell apoptosis and may facilitate aneurysm formation via the sequestration of miR‐199a‐5p

The overwhelming number of interrogations reveals the implication of long noncoding RNAs (lncRNAs) in diverse malignancies, little is unveiled about lncRNAs participation in the abdominal aortic aneurysm (AAA). The study aimed to monitor the role and responsible mechanism of LUCAT1 in AAA. The cellular function of LUCAT1 on smooth muscle cells (SMCs) proliferation and apoptosis were examined through the conduction of CCK‐8, EdU, TUNEL, and caspase‐3 activity assays. LUCAT1 depletion was observed to boost SMCs proliferation or suppress SMCs apoptosis. The opposite results on SMCs proliferation and apoptosis were achieved in response to LUCAT1 promotion. The abundance of LUCAT1 in the cytoplasm was ascertained by subcellular fractionation and FISH analyses on the basis of LncLocator prediction. The binding of LUCAT1 to miR‐199a‐5p predicted by DIANA and starbase was certified by luciferase reporter assay and RIP analysis. Besides, multiple prediction tools unveiled the interaction between miR‐199a‐5p and myelin regulatory factor (MYRF). Quantitative real‐time polymerase chain reaction uncovered the suppressive effect of miR‐199a‐5p and the positive regulation of LUCAT1 on MYRF expression. Rescue experiments revealed that LUCAT1 depletion pose suppression on SMCs apoptosis and MYRF elevation abrogated this suppression induced by LUCAT1 inhibition. These findings unmasked that the pro‐apoptosis impact of LUCAT1 in SMCs via directly targeting miR‐199a‐5p to elevate MYRF expression, which may provide valuable information on AAA prevention.     

Interleukin‐8 induction due to diffusely adherent Escherichia coli possessing Afa/Dr genes depends on flagella and epithelial Toll‐like receptor 5

DAEC is considered potentially diarrheagenic. For diffuse adhesion, the role of the Afa, which was originally identified as a uropathogenic factor, is now understood. However, the role of DAEC in diarrheal disease remains controversial because DAEC is often isolated not only from patients but also from healthy individuals. Previously, we suggested that Afa/Dr DAEC, which can induce high levels of IL‐8 secretion in cultures of human carcinoma epithelial cells (HEp‐2, Caco‐2), is enterovirulent. In the present study, we examined whether IL‐8 secretion induced by certain Afa/Dr DAEC strains was primarily due to flagella via TLR5. All IL‐8 high‐inducing strains were highly motile in swarming tests. Partially purified flagella induced IL‐8 in a dose‐dependent manner. However, IL‐8 induction was inhibited by small‐interfering RNA against TLR5 or by treating flagella with disialoganglioside‐GD1a, a TLR5 blocker. TLR5 is reportedly located on the basolateral side of intestinal epithelia; flagella should not have reached TLR5 from the apical side beyond tight junctions. Reduction in the number of intracellular organisms by wortmannin, a PI3K inhibitor, did not reduce IL‐8 secretion. Afa/Dr DAEC seemed to loosen the tight junctions because it quickly reduced transepithelial electrical resistance after infection. Decreased resistance led to increased IL‐8 production. In conclusion, diffuse adhesion itself is insufficient to induce high levels of IL‐8, and simultaneous stimulation by flagella via TLR5 is likely required for additional induction. Clinically, high motility may be a candidate criterion for predicting the ability of Afa/Dr DAEC strains to induce higher levels of IL‐8 secretion.     

The effect of Cyclin‐dependent kinase 5 on voltage‐dependent calcium channels in PC12 cells varies according to channel type and cell differentiation state

Cyclin‐dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre‐synaptic terminals triggered by Ca²⁺ influx into the pre‐synaptic cytoplasm through voltage‐dependent Ca²⁺ channels (VDCCs). It is reported that Cdk5 regulates L‐, P/Q‐, or N‐type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca²⁺‐channel property of VDCCs, using PC12 cells expressing endogenous, functional L‐, P/Q‐, and N‐type VDCCs. The Ca²⁺ influx, induced by membrane depolarization with high K⁺, was monitored with a fluorescent Ca²⁺ indicator protein in both undifferentiated and nerve growth factor (NGF)‐differentiated PC12 cells. Overall, Ca²⁺ influx was increased by expression of Cdk5‐p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5‐p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5‐p35 regulates L‐, P/Q‐, or N‐type VDCCs in a cellular context‐dependent manner.

     

Intestinal epithelial cancer cell anoikis resistance: EGFR‐mediated sustained activation of Src overrides Fak‐dependent signaling to MEK/Erk and/or PI3‐K/Akt‐1

Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3‐K/Akt‐1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down‐activation, cancer cells nevertheless exhibit sustained Fak–Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3‐K/Akt‐1 down‐activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3‐K/Akt‐1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak‐mediated signaling to MEK/Erk and PI3‐K/Akt‐1 in T84 cells, as in undifferentiated IECs, whereas PI3‐K/Akt‐1 is Src‐independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak–Src interactions and down‐activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3‐K/Akt‐1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR‐mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak–Src interactions and MEK/Erk activation, thus not only overriding Fak‐mediated signaling to MEK/Erk and/or PI3‐K/Akt‐1, but also the requirement of Fak and/or PI3‐K/Akt‐1 for survival. J. Cell. Biochem. 107: 639–654, 2009. © 2009 Wiley‐Liss, Inc.