Cooperation of actin-sequestering protein, thymosin β-4 and hypoxia inducible factor-1α in tumor cell migration

Abstract

Abstract. Cell migration plays an important role in many physiological and pathological processes, including tumor metastasis. Tumor cell migration is increased through the sequential induction of HIF-1· and VEGF under hypoxic conditions. Thymosin ß-4 (Tß4) is an actin-sequestering protein which controls cytoskeletal reorganization. Here, we investigated whether tumor cell migration could be cooperatively controlled by hypoxia and Tß4. Cell migration was measured by wound healing assay with scratching confluent monolayers of tumor cells. Cell migration was enhanced 18 h after scratching cells. In addition, we found that the expression of HIF-1·, VEGF-A isoform 164/120 and Tß4 was increased by scratching cells. Cell migration was decreased by the inhibition of Tß4 or HIF-1· expression with lentiviral shRNA of Tß4 or siRNA of HIF-1·, respectively. In contrast, cell migration was increased by the treatment with Tß4 proteins. The inhibitory effect of Tß4-shRNA or HIF-1·-siRNA was also attenuated by treatment with Tß4 proteins. Collectively, these findings suggest that Tß4 and HIF-1· cooperatively enhance tumor cell migration. IntroductionThysmosin-ß-4 (TB4), a small, naturally occurring 43-amino acid peptide, was initially isolated in 1981 from the thymus. TB4 is the most abundant member of the ß-thymosins, a family of highly conserved polar 5-kDa peptides (1). TB4 is the major G actin-sequestering molecule in mammalian cells (2,3) and can be also cross-linked to some proteins including fibrin and collagen increased in the sites of tissue damage (1,4,5). TB4 led to paclitaxel-resistance (6,7), tumor growth and metastasis (8,9).Cell migration plays a role in many physiological and pathological processes, including tumor metastasis (10). Cell migration requires the integration and temporal coordination of many different processes that occur in spatially distinct locations in the cell (11). Migration can be demonstrated as a multistep cycle including extension of a protrusion, formation of stable attachments near the leading edge of the protrusion, translocation of the cell body forward, release of adhesions and retraction at the cell rear (12,13). Spatially controlled polymerization of actin is at the origin of cell motility and actin networks continuously generated at the leading edge (14,15). The cellular factor that generates new filaments in a site-directed, signaling-controlled fashion is the Arp2/3 complex (16). Profilin, G-actin-binding protein is involved in motile processes mediated by actin polymerization. Actinsequestering protein, TB4, do not modify filament dynamics or the rate of movement but it is thought to buffer the free ATP-G-actin concentration (17). Tß4 proteins induce HIF-1· stabilization, through Erk activation (18). In the meanwhile, hypoxic condition enhances tumor cell migration through the sequential induction of . However, little is known about whether tumor cell migration could be cooperatively controlled by HIF-1· stabilization and Tß4 expression under normoxia.Here, we investigated wheth...