The anti-inflammatory agent bindarit acts as a modulator of fatty acid-binding protein 4 in human monocytic cells

Abstract

We investigated the cellular and molecular mechanisms by which bindarit, a small indazolic derivative with prominent anti-inflammatory effects, exerts its immunoregulatory activity in lipopolysaccharide (LPS) stimulated human monocytic cells. We found that bindarit differentially regulates the release of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), enhancing the release of IL-8 and reducing that of MCP-1. These effects specifically required a functional interaction between bindarit and fatty acid binding protein 4 (FABP4), a lipid chaperone that couples intracellular lipid mediators to their biological targets and signaling pathways. We further demonstrated that bindarit can directly interact with FABP4 by increasing its expression and nuclear localization, thus impacting on peroxisome proliferator-activated receptor γ (PPARγ) and LPS-dependent kinase signaling. Taken together, these findings suggest a potential key-role of FABP4 in the immunomodulatory activity of bindarit, and extend the spectrum of its possible therapeutic applications to FABP4 modulation.

Figure 1

Study of the immuno-modulatory activity of bindarit on LPS-stimulated monocytes. MM-6 cells were left untreated (−/−) or were treated with 100 ng/mL LPS (L) for 20 hours, in the absence (L/−) or presence of 300 μM bindarit (L/B). After treatment, IL-8 (a) and MCP-1 (b) levels were measured in cell supernatants by means of AlphaLISA. Values are means ± S.D. of 5 independent experiments, each performed in duplicate. Significance is shown as P value, calculated with an unpaired t-test. ***P < 0.001. (c) Representative immunoblot showing the expression profile of different lipid binding proteins in MM-6 cells left untreated (−/−) or treated with 100 ng/mL LPS (L) for 20 hours, in the absence (L/−) or presence of 300 μM bindarit (L/B). Protein lysates were subjected to immunoblotting following 10% SDS-PAGE against the antibody indicated on the right side. Actin was also probed with anti-actin antibody to confirm equal protein loading. Molecular weights (M.W.) of protein markers (in kDa) are shown on the left side. The original scans were manipulated to remove irrelevant lanes and/or background regions. Full-length and original blots are presented in supplementary information. (d) Bar graph of the densitometric analysis of 5 independent experiments. The results were normalized with respect to the actin signal and reported as percentage of increase (±S.D.) with respect to the normalized value of the treatment with LPS. Significance is shown as P value calculated using a one-sample t-test. ^*P < 0.05 vs LPS (set to 100%). Inhibition of the release of IL-8 (e) and MCP-1 (f) from LPS-stimulated monocytes by bindarit (B, 300 μM) and BMS309403 (I, 5 μM), used alone (B/− and −/I) or in combination (B/I). After treatment, chemokine content was analyzed in the supernatants by AlphaLISA and was expressed as percentage of inhibition of LPS-stimulated cells. Values are means ± S.D. of 5 independent experiments, each performed in duplicate. Significance is shown as P value, calculated using an unpaired t-test. ^###P < 0.001 vs LPS (set to 0%). ***P < 0.001.

Figure 2

In vitro and in silico studies of the physical interaction between human FABP4 and bindarit. Displacement of [³H]-arachidonic (a) and [³H]-oleic acid (b) from the binding site of human FABP4 by bindarit. Displacement curves were fit to a one-site model with Ki values of 19 μM and 60 μM for arachidonate and oleate, respectively. The graphed points represent the means ± S.D. of 2 independent experiments, each performed in triplicate. (c) Bindarit has a binding mode similar to that of ibuprofen in the active site of human FABP4. Grey: residue Phe57, involved in the binding of small molecules. (d) 2D plot representation of the bindarit’s interactions with amino acid residues in the fatty acid binding pocket of the human FABP4.

Figure 3

Effect of bindarit on the subcellular localization of FABP4 in MM-6 cells. (a) Indirect immunostaining of FABP4 in human monocytes. FABP4 was visualized by confocal immunofluorescence microscopy in MM-6 cells left untreated (−/−/−) or treated with 100 ng/mL LPS (L) for 20 hours, in the absence (L/−/−) or presence of 300 μM bindarit used alone (L/B/−) or in combination with 5 μM of BMS309403 (L/B/I). After each treatment, cells were fixed and stained with anti-FABP4 (green, middle panel) and with DAPI for nuclear labeling (cyan, top panels). Merged images are shown in the bottom panels. Images are representative of 2 independent experiments. (b) Ratios of fluorescence intensity between nucleus and cytosol of 20–28 cells under each condition were calculated as described in Materials and Methods, and are reported as mean ± S.D. Significance is shown as P value calculated using an unpaired t-test. ***P < 0.001.

Figure 4

Effect of pharmacological inhibition of PPARγ on the immuno-modulatory activity of bindarit. (a) Representative immunoblot showing the expression FABP4 in MM-6 cells left untreated (−/−) or treated with 100 ng/mL LPS (L) for 20 hours, in the absence (L/−) or presence of 300 μM bindarit (B) and 2 μM T0070907 (T), used alone or in combination (L/B− an L/B/T). Protein lysates were subjected to immunoblotting following 10% SDS-PAGE against the anti-FABP4 antibody. Actin was also probed with anti-actin antibody to confirm equal protein loading. Molecular weights (M.W.) of protein markers (in kDa) are shown on the left side. (b) Bar graph of the densitometric analysis of 5 independent experiments. The results were normalized with respect to the actin signal, and were reported as percentage of increase (±S.D.) with respect to the normalized value of the treatment with LPS. Significance is shown as P value calculated using a one-sample t-test. ^#P < 0.05 vs LPS (set to 100%). ^###P < 0.001 vs LPS. *P < 0.05. Inhibition of the production of IL-8 (c) and MCP-1 (d) from LPS-stimulated monocytes by bindarit (B, 300 μM) and T0070907 (T, 5 μM), used alone (B/− and −/I) or in combination (B/I). After treatment, chemokine contents were analyzed in the supernatants by AlphaLISA and were expressed as percentage of inhibition of LPS-stimulated cells. Values are means ± S.D. of five independent experiments, each performed in duplicate. Significance is shown as P value calculated using an unpaired t-test. ^###P < 0.001 vs LPS (set to 0%). *P < 0.05.

Figure 5

Impact of bindarit on LPS-induced activation of cellular kinases. MM-6 cells were left untreated (−/−/−) or treated with 100 ng/mL LPS (L) for 20 hours, in the absence (L/−/−) or presence of 300 μM bindarit (B), used alone (L/B/−) or in combination with 5 μM BMS309403 (L/B/I). Cell lysates containing 200 μg of total protein were analyzed for the relative levels of site-specific phosphorylation of selected kinases, by using the human phospho-mitogen-activated protein kinase array kit. (a) Representative images of phospho-kinase arrays for each treatment captured by C-DiGit blot scanner. Each kinase is spotted in duplicate. The pairs of dots in each corner (with the exception of the negative control pair at the lower left corner) are positive controls. Each pair of the most positive kinase dots is denoted by an ellipse, with the name of the corresponding kinases. (b) Graphical representation of the array signal intensities of AKT-2, p38α and p38γ. Below each kinase the specific phosphorylation site(s) detected is (are) reported. Relative spot phosphorylation was quantified by normalizing pixel density of the positive control to 100 and was expressed as a fraction (±S.D.) of the value of LPS-treated cells (n = 2). Significance is shown as P value calculated using a one-sample t-test. *P < 0.05. Effect of pharmacological inhibition of AKT-2 and p38α on the immuno-modulatory activity of bindarit. Inhibition of the release of IL-8 (c) and MCP-1 (d) from LPS-stimulated monocytes by bindarit (B, 300 μM), the AKT-2 inhibitor CCT128930 (C, 5 μM) and the p38α inhibitor JX-401 (J, 1 μM), used alone (B/−/−, −/C/− and −/−/J) or in combination (B/C/− and B/−/J). After treatment, chemokine content was measured in the supernatants by AlphaLISA and was expressed as percentage of inhibition of the values obtained in LPS-stimulated cells. Values are means ± S.D. of four independent experiments each performed in duplicate. Significance is shown as P value calculated using an unpaired t-test. ^#P < 0.05 vs LPS (set to 0%). ^###P < 0.001 vs LPS (set to 0%). *P < 0.05.

Figure 6

Proposed model for bindarit action. The potential mechanism of action of bindarit is presented. See text for details.