RGD peptides confer survival to hepatocytes via the β1-integrin-ILK-pAkt pathway

Inhibition of DNA fragmentation of isolated hepatocytes treated with anti-β1 antibodies, or RGD peptides. Freshly isolated hepatocytes were treated with normal hamster IgM (B), anti-β1 antibodies (C), or RGD peptides (E) and cultured on polyHEMA for 5h. Anti-Fas antibody-treated hepatocytes, which were cultured on collagen IV, served as a positive control (D). Lane A shows untreated hepatocytes. DNA fragmentation was analyzed by agarose gel electrophoresis and SYBRgreen staining.

Percentage of caspase 3 positive hepatocytes. Caspase 3 immunostaining of untreated (a) and RGD pretreated (b) hepatocytes. Freshly isolated hepatocytes were treated with different concentrations of (c) anti-β1 antibodies, (d) RGD peptides or with (e) cyclic RGD, pronectin F or scrambled peptides and cultured on polyHEMA for 18h. The percentage of caspase 3 positive hepatocytes was determined by calculating the number of caspase 3 positive hepatocytes per 100 hepatocytes. Data are expressed as means±SD of 5 independent experiments. *P<0.005 compared to the untreated hepatocytes. ^†P<0.001 compared to the untreated hepatocytes. [This figure appears in colour on the web.]

Percentage of phospho-Akt positive hepatocytes. Freshly isolated hepatocytes were treated with different concentrations of (a) anti-β1 antibodies or (b) RGD peptides, and cultured on polyHEMA for 18h. The hepatocytes were stained for phospho-Akt Ser 473. Data are expressed as means±SD of 5 independent experiments. *P<0.005 compared to the untreated hepatocytes. ^†P<0.001 compared to the untreated hepatocytes.

Integrin-linked kinase (ILK) is upregulated following integrin ligation in hepatocytes. (a) Western blot with anti-ILK shows equivalent amounts of the protein in each extract. (b) ILK assay using MBP as a substrate. The phosphorylation of the substrate by ILK is detected with [³²P]ATP. The ILK assay was performed with immunoprecipitation of cell extracts of hepatocytes that were either not treated (−) or treated with anti-beta 1 antibodies or RGD peptides for 30min. (c) Ratio ILK activity: ILK activity of hepatocytes, which were either not treated or pretreated with anti-β1 antibodies, RGD peptides, or scrambled (scr.) peptides for 30, 60min or 18h versus the ILK activity of freshly isolated hepatocytes.

Clustering of beta-1 integrins. (a) The presence of β1 integrins on hepatocytes in the liver. Binding of anti-β1-integrin moAbs (b and c) or RGD peptides (d) results in rapid redistribution of β1-integrins into clusters on the hepatocytes. Note the uniform distribution of β1 integrins on untreated cells (e).