Proposed mechanism underlying 11ßHSD2 activity and lung tumorigenesis.

<p>11ßHSD2 inhibition leads to increased levels of tumor intracellular active glucocorticoid and activation of glucocorticoid receptors. The subsequent inhibition of the COX-2, ERK and mTOR pathways leads to suppression of lung tumorigenesis.</p

11βHSD2 inhibition suppressed lung tumorigenesis.

<p><b>A</b>. LLC tumor growth was significantly attenuated by 11βHSD2 inhibition with GA (**P < 0.01, n = 8 in each group). LLC cell suspensions (100 μl, 5 x 10⁵ cells) were injected subcutaneously into the flank of C57/B6 mouse (2 sites). The mice were sacrificed 18 days later and tumor growth (tumor weight from two sites) was evaluated. <b>B</b>. Kaplan-Meier survival curve indicated that 11βHSD2 inhibition with GA increased survival probability in mice with tail vein injections of LLC cells (100 μl of LLC cell suspensions containing 5 x 10⁵ cells). ***P < 0.001, n = 12 in control and n = 14 in GA group. In both models, GA was given at 10 mg/kg/day (i.p. injection) starting one day before LLC cell injections.</p

(A) The protein expression of human GPR40 (hGPR40) in the HEK293 cells transfected with human GPR40 cDNA construct.

<p>An anti-human GPR40 antibody was used for immunoblotting. Shown are representative stable clones expressing different levels of human GPR40. NT, non-transfected HEK293 cells, used as a negative control. (B) Quiescent hGPR40-transfected HEK293 cells were exposed to different concentrations of 11,12-EET or 14,15-EET for 15 min. 11,12-EET and 14,15-EET concentration-dependently induced ERK1/2 phosphorylation, respectively, in hGPR40-transfected HEK293 cells, compared with vehicle. The same immunoblot was stripped and re-probed with total ERK antibodies to ensure equal loading and transfer. Shown are representative blots of 3 separate experiments with similar results. *<i>p</i><0.05 compared with vehicle alone in hGPR40 transfected HEK293 cells.</p

Overexpression of human GPR40 in HEK293 cells enhanced release of soluble HB-EGF into conditioned media.

<p>Empty vector-transfected HEK293 cells (Vector) and human GPR40-transfected HEK293 cells (hGPR40) were rendered quiescent in serum-free medium and washed twice with PBS, followed by treatment with vehicle or 14,15-EET, respectively. The conditioned media were applied to a heparin-Sepharose column to purify heparin-binding proteins as described in Experimental Procedures and analyzed by immunoblotting with an antibody specific for HB-EGF. Shown is a representative blot from three separate experiments with similar results and the densitometry quantification of released soluble HB-EGF from the three experiments.</p

GPR40 expression in different cell lines.

<p>(A) The protein expression of GPR40 was most abundant in the renal tubule epithelial cell line LLCPKcl4 but very low in HEK293 cells, as revealed by immunoblotting a rabbit anti-human GPR40 antibody (Epitomics, Burlingame, CA) that also cross-reacts with monkey, porcine, and mouse GPR40. Shown was a representative blot from at least three separate experiments with similar results. (B) RT-PCR demonstrated that GPR40 mRNA expression was higher in LLCPKcl4 cells, with minimal expression in HEK293 cells.</p

Localization of GPR40 mRNA in mouse kidney.

<p>In situ hybridization showed that GPR40 mRNA is expressed in the renal cortical tubules. Note the branching indicating expression in cortical collecting duct.</p

11βHSD2 inhibition augmented corticosterone (CS)-induced COX-2 inhibition in lung cancer cells.

<p><b>A</b>. Immunoblotting indicated that 11βHSD2 was expressed in all investigated cell lines while COX-2 was detectable in some of them, including LLC, H1435 and A549 cells. <b>B</b>. Immunoblotting indicated that CS inhibited LLC cell COX-2 expression in a dose-dependent manner. <b>C</b>. CS (1μM)-induced LLC cell COX-2 inhibition was enhanced by 11βHSD2 inhibitor, GA. <b>D</b>. CS (1μM)-induced LLC cell COX-2 inhibition was enhanced by carbenoxolone (CBX, 10 μM), another 11βHSD2 inhibitor. <b>E</b>. CS (1μM)-induced H1435 cell COX-2 inhibition was enhanced by GA (10 μM). <b>F</b>. CS (1μM)-induced A549 cell COX-2 inhibition was enhanced by GA (10 μM).</p

11βHSD2 inhibition with GA increased lung corticosterone levels in association with attenuation of tumor COX-2 expression in KrasLA2 mice.

<p><b>A</b>. GA treatment markedly increased lung active corticosterone levels and decreased inactive 11-keto-corticosterone levels in KrasLA2 mice (20 weeks of age). *P <0.05 vs. control, n = 6. <b>B</b>. Lung tumor COX-2 expression was suppressed by 11ßHSD2 inhibition with GA. **P < 0.01 vs. control, n = 3. Immunostaining showed markedly decreased tumor COX-2 with GA treatment. Original magnification: x 250. <b>C</b>. GA treatment increased mannose receptor (MR, CD206) expressing macrophages in tumor, an indication of increased tumor levels of active corticosterone. ***P < 0.001 vs. control, n = 4. Original magnification: x 160.</p

11ßHSD2 expression in mouse and human lung tumors.

<p><b>A</b> and <b>B</b>. Representative photomicrographs indicated 11ßHSD2 expression in small airway and alveolar epithelial cells of athymic nude mice <b>(A)</b> and in A549 lung tumor from athymic nude mice <b>(B)</b>. <b>C</b>. Representative photomicrographs showed that 11ßHSD2 immunostaining was strong in lung adenocarcinoma and squamous cell carcinoma, moderate in papillary carcinoma and small cell lung cancer (SCLC), but very weak in uninvolved lung tissue. Original magnification: x 160 in all.</p

Effect of human GPR40 transfection on EET-induced cell proliferation.

<p>1.5 × 10⁵/well HEK293 cells transfected with hGPR40 or empty-vector were seeded into 24-well plates, respectively. 24 h later, the cells were made quiescent in serum-free medium, followed by stimulated with 5 μM 14,15-EET or vehicle alone in the presence of 0.5% FBS in the medium. Cell number per well in each treatment group was counted every day. Results were plotted as the cell number/well for the indicated durations. Each experimental data point represents triplicate wells from three different experiments. Human GPR40 overexpression in HEK293 cells significantly augmented 14,15-EET-stimulated cell proliferation, compared with empty vector-transfected HEK293 cells.</p