Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/ CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxiainducing factor-1␣ (HIF-1␣) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF-1␣ protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1␣ protein by recombinant Cyr61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl 2 treatment, both well known for induction of HIF-1␣. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1␣ up-regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1␣ protein accumulation. Blockage of HIF-1␣ activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1␣ strongly inhibited their invasion ability, suggesting that elevation in HIF-1␣ protein is vital for Cyr61-mediated gastric cancer cell invasion. In addition, several HIF-1␣-regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI-1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61-overexpressing cells. Transfection with dominant negative-HIF-1␣ to block HIF-1␣ activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1␣-dependent up-regulation of PAI-1.An emerging family of secreted, matrix-associated proteins encoded by immediate early genes that play various roles in angiogenesis and tumor growth is named the CCN family. Cysteine-rich 61 (Cyr61) 2 belongs to the CCN family of angiogenic regulators, which consist of Cyr61/CCN1, connective tissue growth factor (CTGF/CCN2), nephroblastoma overexpressed (Nov/CCN3), Wisp-1/elm1 (CCN4), Wisp-2/rCop1 (CCN5), and Wisp-3 (CCN6) (1-3). The CCN family shares a uniform modular structure, which mediates various cellular functions such as angiogenesis, cell proliferation, cell migration, and neoplastic transformation (4 -6).Cyr61 is a ligand that is encoded by an early response gene responsible for proangiogenesis. It regulates cell adhesion, ameliorates growth factor-stimulated DNA synthesis, and promotes neovascularization and tumor growth (7-9). Results from our own studies and investigations of other have demonstrated that Cyr61 expression is associated with advanced breast adenocarcinoma, pancreatic cancer, gliomas, and gastric adenoca...