HGF modulates IL-6 production in LPS stimulated macrophages.

<p>BMM derived from C57BL6 mice were pretreated with or without 10 pg and 10 ng HGF for 24 hours and stimulated with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-6 levels were measured by ELISA. Results are representative of the mean (± SEM) of three independent experiment done in triplicate, * indicates <0.001.</p

Proposed mechanism of HGF-mediated suppression.

<p>The canonical signaling pathway of LPS-TLR engagement leads to NFκB dependent pro-inflammatory cytokine production through the interaction of CBP with NFκB. However, TLR signaling has also been shown to weakly activate alternative signaling through PI3K, resulting in phosphorylation and inactivation of GSK3β, subsequent sequestration of CBP from NFκB to phospho-CREB, and resultant anti-inflammatory (IL-10) production<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015384#pone.0015384-Martin1" target="_blank">[7]</a>. Our results show that the presence of HGF enhances the IL-10 pathway. We postulate that in order to resolve inflammation, the generation of IL-6 by LPS-TLR signaling leads to the production of HGF, ultimately leading to the inhibition of inflammation. Hence, we propose HGF acts as an internal rheostat for resolving acute phase inflammatory responses.</p

Deletion of the HGF receptor MET demonstrates a reversal in the effects of HGF on LPS stimulated BMM.

<p>BMM derived from either macrophage specific MET conditional knockout mice (MET^fl/fl:cre^lysZ+/−) or their wild type littermate controls (MET^fl/fl:cre^lysZ−/−) were pretreated with or without 1, 10 and 100 pg HGF for 24 hours and stimulated with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-6 levels were measured by ELISA. Results are representative of two independent experiments done in triplicate.</p

Treatment with HGF leads to increased GSK3β phosphorylation.

<p>(A) Cytoplasmic lysates prepared from BMM were incubated with 10 ng HGF for 24 hours prior to stimulation with LPS (1 µg/ml) for 15 minutes. The lysates were separated by SDS-PAGE and probed with a phospho-specific GSK3β antibody before re-probing for β-actin. (B) Densitometric analysis for phospho-GSK3β fold induction normalized to β-actin is shown for 3 separate experiments. Note that all experiments show induction greater than 1 when HGF is present.</p

HGF promotes the interaction of phosphorylated CREB with CBP along with an increased production of IL-10.

<p>(A) Whole cell lysates prepared from BMM were incubated overnight with HGF (10 ng) prior to stimulation LPS (1 µg/ml) for 15 minutes, then subjected to immunoprecipitation with a CBP antibody. The lysates were separated by SDS-PAGE and probed with a phospho-specific CREB antibody before re-probing for β-actin. (B) Densitometric analysis for phospho-CREB fold induction normalized to β-actin. Is shown for 3 separate experiments. Note that all experiments show induction greater than 1 when HGF is present. (C) BMM were pretreated with or without 1 µM of the MET kinase inhibitor (SU11274) for 2 h prior to incubation with HGF (1, 10 and 100 pg) for 24 hours followed by stimulation with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-10 levels were measured by ELISA. Results are representative of two independent experiments done in triplicate.</p

HGF prevents the nuclear translocation of phosphorylated p65.

<p>BMM were (A) untreated (B) stimulated with LPS (1 µg/ml) (C) treated with 10 pg HGF and stimulated with LPS (1 µg/ml) or (D) treated with the MET kinase inhibitor, SU11274, 10 pg HGF and stimulated with LPS (1 µg/ml). Cytospin preparations were then stained by immunoflourescence for phosphorylated p65 (Ser 276) and for nuclei with DAPI. The blue staining indicates nuclei, red staining indicates phosphorylated p65, and purple staining indicates colocalization of phospho-p65 within the nucleus.</p

A MET kinase inhibitor abrogates HGF suppression of IL-6.

<p>Using an optimal dose of the MET inhibitor SU11274 (1 µg), BMM were pretreated for 2 hours before an overnight incubation with 10 and 100 pg HGF and 24 hour stimulation with LPS. Results are representative of the mean (± SEM) of three independent experiments done in triplicate. *, p = 0.02 vs. the respective control group.</p

Rsu-1 levels in HCC.

<p>A) Graphical representation of the number of HCC cases positive, negative or moderately positive for Rsu-1 in HCC tissue array (24 cases/48 cores). B) Protein levels of Rsu-1 in HCC cell lines compared to human hepatocytes (HH). Most of the HCC cell lines show decrease in Rsu-1 protein. C) Successful overexpression of Rsu-GFP (fusion protein) in Hep3B cell line. GFP tagged ORF clone of Homo sapiens Rsu-1 (#RG203334, Origene) was transfected into Hep3B cell line and analyzed for Rsu-1 48 h after transfection. Since it is a GFP fused protein, the MW of Rsu-1 is ~fifty-five kd instead of twenty-nine kd (MW of GFP is ~twenty-six kd). D) GFP-Rsu-1 fusion protein associates with PINCH inside the cell. Overexpression of GFP-Rsu-1 in Hep3B cell line leads to association of GFP-Rsu-1 with PINCH. GFP was immunoprecipitated 48 h after transfection. GFP precipitates were probed with either GFP or PINCH. Presence of PINCH in GFP precipitates shows association of GFP-Rsu-1with PINCH.</p

ECM changes in PINCH DKO mice.

<p>A) Photomicrograph of a liver of 5 week old PINCH DKO mouse showing increased stellate cell activation as evident by αSMA stain (shown by arrows). Similar photomicrograph from a Control mouse of the same age shows minimal activation of stellate cells B) Photomicrograph of a liver of a 30 week PINCH DKO mouse showing increased ECM deposition as evident by reticulin stain. All the figures are 100X. The insert are of 200X magnification.</p

Liver regeneration kinetics in PINCH DKO mice.

<p>C) PINCH DKO mice show no termination defect. At Day 14 after partial hepatectomy, the percentage body/liver weight of the control and PINCH DKO mice reaches 100% of the original.</p