Gene microarray analysis reveals a novel hypoxia signal transduction pathway in human hepatocellular carcinoma cells

The molecular details of hypoxia-induced cellular responses have been diffi cult to identify since there is as yet no known oxygen receptor. We used cD NA microarray technology to extend our studies pertaining to these molecula r details in human hepatocellular carcinoma (Hep3B) cells that produce eryt hropoietin (Epo) in response to hypoxia. Of approximately 1200 genes in the array, those associated with integrin-linked kinase (ILK), fibronectin pre cursor and glycogen synthase kinase-3 beta (GSK-3 beta) were markedly stimu lated after exposure of Hep3B cells to low oxygen (1%) for 6 h. Epo, HIF-1, and von Hippel-Lindau cDNAs were measured in parallel as markers of low ox ygen responses in Hep3B cells. ILK is a serine, threonine protein kinase th at interacts with the cytoplasmic domains of integrin beta1 and beta3. This interaction localizes ILK to focal adhesion plaques. ILK is stimulated by cell-fibronectin interaction as well as insulin. It is regulated in a phosp hatidylinositol 3-kinase dependent manner and can phosphorylate protein kin ase B (PKB/AKT) and GSK-3 beta. As a result of these and other activities I LK has been shown to affect anchorage-independent cell survival, cell cycle progression and tumorigenesis in nude mice. ILK has also been implicated i n the Wnt pathway and as a critical target in PTEN-dependent tumor therapie s. To our knowledge this is the first report implicating the ILK pathway in low oxygen responses. Other genes identified as a result of the microarray analysis not previously known to change as a result of low oxygen treatmen t were elongation factor-la, glycyl-tRNA synthetase, and laminin receptor p rotein-1. These findings were all corroborated by RT-PCR assays and in some instances Western blot analysis.