A novel TLR4-mediated signaling pathway leading to IL-6 responses in human bladder epithelial cells

Abstract

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-kappaB-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-kappaB-associated pathway, TLR4 triggers a distinct and more rapid signaling response involving, sequentially, Ca(2+), 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.

Figure 1. IL-6 Response of BECs to Type 1 Fimbriated E. coli Is Largely Elicited by LPS and Involves TLR4

(A) IL-6 secretion by BECs in response to E. coli (100 multiplicity of infection) or purified LPS (100 μg/ml). When specified, E. coli and purified LPS were pretreated with 1 μg/ml PMB for 30 min. **, p < 0.001 relative to values of untreated (UT) BECs; *, p < 0.03 relative to E. coli or LPS-treated BECs. (B) RT-PCR of control-transfected BECs (Ctrl) and TLR4 KD BECs. Glyseraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as a loading control. (C) IL-6 secretion of control-transfected BECs (Ctrl) and TLR4 KD BECs after E. coli and LPS stimulation. **, p < 0.05 relative to UT control; *, p < 0.05 relative to E. coli-treated control or LPS control.

Figure 2. IL-6 Response of BECs to E. coli Is Preceded by an Increase in [Ca²⁺]_i

(A) [Ca²⁺]_i tracing of BECs before and after E. coli exposure. E. coli was added at time 0. (B) BEC IL-6 responses after E. coli exposure in the absence or presence of NiCl₂ (2 mM) or BAPTA-AM (5 μM), or after calcium ionophore A23187 (1 μM) treatment without bacterial exposure. *, p < 0.001 relative to untreated (UT) BECs; **, p < 0.001 relative to E. coli (EC)–treated BECs. (C–E) BEC [Ca²⁺]_i tracing before and after purified LPS treatment (C) or PMB pretreated LPS treatment (E). (D) BEC IL-6 responses following exposure to LPS in the absence or presence of NiCl₂ or BAPTA-AM. *, p < 0.001 relative to UT BECs; **, p < 0.01 relative to LPS-treated BECs.

Figure 3. IL-6 Response of BECs to E. coli Is Associated with a Significant Increase in Intracellular cAMP Levels

(A and B) BEC cAMP production before and after E. coli exposure. When specified, BECs were pretreated with MDL-12,330A (MDL) (0.4 mM) or NiCl₂ for 30 min, or E. coli was pretreated with PMB for 30 min. *p < 0.03 relative to untreated (UT) BECs; **p < 0.03 relative to E. coli (EC)–treated BECs. (C) IL-6 secretion by BECs was measured 6 h after exposure to E. coli, in the absence (EC) or presence of MDL-12,330A (EC, MDL), or after 6 h of treatment with 1 mM dibutyryl cAMP (dbcAMP), or 1 mM 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′-5′-cyclic monophosphate (8-CPT-cAMP) without bacterial exposure. *, p < 0.01 relative to UT; **, p < 0.02 relative to EC. (D) IL-6 secretion by BECs incubated for 6 h in the absence (UT) or presence of the AC-activator forskolin (Fors) (50 μM) with or without NiCl₂ or PMB, or incubated with NiCl₂ or PMB in the absence of forskolin. *, p < 0.01 relative to UT BECs.

Figure 4. AC-3 Is responsible for Mediating E. coli–Induced cAMP Production in BECs

(A) RT-PCR of BECs using primers specific for the ten known mammalian AC isoforms. Only AC-3, AC-4, AC-6, and AC-7 mRNA was expressed. GAPDH-specific RT-PCR (lane G) was used as a loading control. M, marker; sAC, soluble AC. (B) RT-PCR of control-transfected BECs (Ctrl) and AC-3, AC-4, AC-6, or AC-7 KD BECs. GAPDH-specific RT-PCR was used as a loading control. (C and D) Intracellular cAMP production (C) and IL-6 secretion (D) by non-transfected BECs (NT), control-transfected BECs (Control), or AC-3, AC-4, AC-6, or AC-7 KD BECs left untreated (UT) or treated with E. coli (EC). *, p < 0.005 and **, p < 0.02 relative to respective UT values. (E and F) Intracellular cAMP production (E) and IL-6 secretion (F) by non-transfected BECs (NT), control-transfected BECs (Control), or AC-3 KD BECs were measured in the absence (UT) or presence of E. coli LPS, or presence of forskolin (Fors) without LPS. *, p < 0.003 relative to respective UT values. (G) BEC AC-3–specific Western blot before (UT) and after (EC) E. coli exposure for 1 h, or exposure to E. coli LPS for 6 h. An actin-specific Western blot was used as a loading control. (H) Densitometric analysis of AC-3–specific Western blots, using ImageJ software. (I) [Ca²⁺]_i tracing in AC-3 KD BECs before and after E. coli exposure. E. coli was added at time 0.

Figure 5. cAMP-Mediated Phosphorylation of CREB and Binding of CREB to CRE Oligonucleotides

(A and B) NF-κB nuclear translocation in control-transfected BECs and AC-3 KD BECs before and after E. coli (EC) exposure for 1 h (A) or 2 h (B). UT, untreated. (C and D) IL-6 message levels in non-transfected (NT), control-transfected (Ctrl), and AC-3 KD BECs before and after E. coli exposure for 1 h (C) or 6 h (D) as measured by RT-PCR. (E) Western blot of CREB phosphorylation levels (p-CREB) in UT BECs, and forskolin (Fors)– or calcium ionophore A23187 (A23187)–treated BECs. The treatment was for 1 h. (F) Western blot showing CREB phosphorylation of NT, Ctrl, and AC-3 KD BECs before and after 1 h E. coli exposure. (G) CREB binding to CRE site of the IL-6 promoter. Nuclear extracts of BECs exposed for 1 h to E. coli ORN103(pSH2) were incubated with biotinylated WT CRE oligonucleotides in the absence (Biotinylated WT CRE) or presence of specific (Biotinylated WT CRE + WT CRE) or non-specific (Biotinylated WT CRE + MT CRE) oligonucleotide competitors. ***, p < 0.0001. (H) Expression analysis of mRNA levels of various genes with CRE sites in their promoter. WT and AC-3 KD BECs were incubated for 1 h with E. coli ORN103(pSH2), and then total RNA was collected from untreated (UI) and bacteria-treated BECs (EC) and subjected to RT-PCR. GAPDH was used as a loading control.

Figure 6. The IL-6 Response to E. coli of BECs Is the Product of Two Separate Signaling Pathways

(A) IL-6 secretion response to E. coli ORN103(pSH2) by WT and AC-3 KD BECs in the absence or presence of pyrrolidine dithiocarbamate (PDTC). (B) IL-6 secretion response to UPEC CI5 by WT and AC-3 KD BECs. (C and D) IL-6 secretion responses by Mono Mac 6 cell line (C) and 16-HBE cell line (D). Cells were treated for 12 h with E. coli ORN103(pSH2) in the absence (EC) or presence of NiCl₂ or PKA inhibitor (PKI). *, p < 0.03 relative to untreated (UT) value. Identical results were obtained after 6 h of treatment with E. coli ORN103(pSH2). (E) Increased CREB phosphorylation in BECs in response to TLR2 and TLR3 ligands. Western blotting for phospho-CREB and total-CREB in UT BECs or BECs treated with either a TLR2 ligand (lipoteichoic acid) or a TLR3 ligand (polyinosine-polycytidylic acid).

Figure 7. The IL-6 Response of Primary Human BECs to UPEC Is Linked to [Ca²⁺]_i and cAMP Increase

(A) [Ca²⁺]_i tracing before and after exposure of primary human BECs to UPEC strain CI5. (B) [Ca²⁺]_i tracing of the primary human BECs pretreated with NiCl₂ for 30 min. UPEC was added at time 0. (C and D) Intracellular cAMP production (C), or IL-6 secretion (D) by the primary human BECs left untreated (UT) or treated with E. coli CI5 (UPEC) in the absence or presence of NiCl₂ (UPEC, NiCl₂), or treated with E. coli ORN103 (EC). *, p < 0.03 relative to UT value. **, p < 0.03 relative to UPEC (CI5)–treated value.

Figure 8. Proposed Model for TLR4 Signaling in BECs

The proposed rapidly induced second messenger– and CREB-mediated pathway (dark line) as well as the classical NF-κB (grey line) are shown. Both pathways are triggered by TLR4. P, phosphorylation; R and C, regulatory and catalytic subunits of PKA.