A Novel TLR4-Mediated Signaling Pathway Leading to IL-6 Responses in Human Bladder Epithelial Cells

The vigorous cytokine response of immune cells to Gram-negative bacteria is primarily mediated by a recognition molecule, Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide (LPS) and initiates a series of intracellular NF-κB–associated signaling events. Recently, bladder epithelial cells (BECs) were reported to express TLR4 and to evoke a vigorous cytokine response upon exposure to LPS. We examined intracellular signaling events in human BECs leading to the production of IL-6, a major urinary cytokine, following activation by Escherichia coli and isolated LPS. We observed that in addition to the classical NF-κB–associated pathway, TLR4 triggers a distinct and more rapid signaling response involving, sequentially, Ca2+, adenylyl cyclase 3–generated cAMP, and a transcriptional factor, cAMP response element–binding protein. This capacity of BECs to mobilize secondary messengers and evoke a more rapid IL-6 response might be critical in their role as first responders to microbial challenge in the urinary tract.

and underlined sequences are forward and reverse sequences, respectively, which correspond to nucleotides 2324-2342 of the human AC-3 gene (AC3a and AC3b, GenBank TM accession number AF033861), nucleotides 2096-2114 of the human AC-4 gene (AC4a and AC4b, GenBank TM accession number AF497516), nucleotides 2085-2103 of the human AC-6 gene (AC6a and AC6b, GenBank TM accession number AF250226), and nucleotides 747-765 of the human AC-7 gene (AC7a and AC7b, GenBank TM accession number NM_001114). The oligos were annealed to form double-stranded DNA and cloned into the BamHI and XbaI sites of pQCXIN-U6 to generate pSi-AC3, pSi-AC4, pSi-AC6, and pSi-AC7. The Amphopack-293 Cell Line (BD Biosciences) was used to produce the viral particles. Production of viral particles, infection of target cell line (5637), and selection of viral infected cells were performed as recommended by the vendor of the pQCXIN vector (BD Biosciences). The geneticin-resistant stabletransfected cell lines were named AC-3 KD, AC-4 KD, AC-6 KD, and AC-7 KD. Knockdowns were verified by RT-PCR using the AC isoform-specific primers listed above.
The boldface and underlined sequences are forward and reverse sequences, respectively, which correspond to nucleotides 1026-1044 of the human TLR4 gene (GenBank TM accession number U88880).

Western blot analysis
BECs were seeded onto 60-mm culture dishes and grown overnight. The cells were uninfected or infected with E. coli (MOI=100) for 1 h, or treated with 100 μg/ml LPS for 6 h. Cells were lysed in a RIPA buffer (Bio-Rad) containing 1 mM PMSF and a 1:100 dilution of mammalian protease inhibitor cocktail (Sigma).
The cell suspension was passed 20 times through a 21-gauge needle and then sonicated. The cell lysate was centrifuged at 10,000 rpm for 10 min with the precipitates then being discarded. Protein concentrations were determined using the Bradford reagent (Bio-Rad) with bovine serum albumin as the standard. Cellular proteins (100 μg per lane) were electrophoresed in 4-15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a PVDF (polyvinylidene fluoride) membrane, which was blocked with 5% non-fat dried milk in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20, pH 7.5) for 1h, and then incubated overnight with 1:250-1:500 diluted anti-AC3 antibody (FabGennix, Inc.) in blocking solution at 4°C. After washing with TBST, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody for 1h at room temperature. The blots were then immunodetected with the enhanced chemiluminescence detection system (Pierce). As a loading control, 20 μg cellular proteins were loaded in 4-15% SDS-PAGE and blotted with anti-β-actin antibody (1:5000 dilution; Sigma). Image J software (National Institutes of Health) was used for densitometry to quantify protein expression for statistical analysis.
Anti-CREB and anti-phospho-CREB antibody were purchased from Cell Signaling technology and CREB western blotting was performed according to the manufacturer's instruction. BECs were cultured onto 6well plates overnight and left uninfected or infected with 100 MOI Ε. coli for 1 hr, or treated with 50 μM forskolin or 10 μM calcium ionophore A23187 for 1 hr. When specified, BECs were treated for 6 hrs with either 2 μg/ml of Lipoteichoic acid (TLR2 ligand) or 25 μg/ml of polyinosine-polycytidylic acid (TLR3 ligand).