The transforming growth factor-beta (TGF-beta) family of cytokines regulates vascular development and inflammatory responses. We have recently shown that exposure of human umbilical vein endothelial cells (HUVECs) to hypoxia (1% O(2)) increases gene expression and bioactivation of TGF-beta2 and induces its downstream effectors, Smad proteins (Smads), to associate with DNA. In the present study, we show that hypoxia-induced TGF-beta2 gene expression is dependent on thrombospondin-1-mediated bioactivation of latent TGF-beta. Blocking TGF-beta2 but not TGF-beta1 in hypoxic endothelial cell cultures inhibited induction of the TGF-beta2 gene, indicating that an autocrine mechanism driven by bioactivation of TGF-beta2 leads to its gene expression in hypoxic HUVECs. Exposure of HUVECs to hypoxia resulted in phosphorylation and nuclear transportation of Smad2 and Smad3 proteins as well as stimulation of transcriptional activities of Smad3 and the transcription factor hypoxia-inducible factor-1alpha and culminated in up-regulation of TGF-beta2 gene expression. Autocrine regulation of TGF-beta2 production in hypoxia may involve cross-talk between Smad3 and HIF-1alpha signaling pathways, and could be an important mechanism by which endothelial cells respond to hypoxic stress.