Purpose: Previous work from the authors' laboratory has shown that Hsp27 is specifically upregulated after retinal ischemic preconditioning (IPC), and this upregulation acts as a key cytoprotective factor in preventing retinal ischemic damage. The regulatory mechanisms involved in the upregulation of Hsp27 after IPC are unknown. The purpose of this study was to explore the transcriptional events responsible for the upregulation of Hsp27 after IPC.
Methods: CoCl(2) was used to test for Hsp27 expression after hypoxic stimulus. The promoter and first intron regions of the human Hsp27 gene were cloned by PCR and characterized by deletion analysis by using a reporter assay. In vitro results were then applied to an in vivo model of retinal ischemia to determine whether CoCl(2) upregulates rHsp27 and protects the retina from ischemic injury.
Results: CoCl(2) upregulated Hsp27 in cultured retinal neurons. Promoter-intron reporter assays using various DNA deletion constructs indicated that several HIF-1 binding sites were necessary for CoCl(2)-induced expression of the Hsp27 gene. Furthermore, CoCl(2) upregulated Hsp27 in the rat retina and protected the rat retina from ischemic injury.
Conclusions: These data provide evidence that Hsp27 is regulated by hypoxic signaling through HIF-1 activation and support the idea that an early event in IPC is the activation of HIF-1. These findings are significant, because this is the first time HIF-1 activation has been associated with the protective effects of IPC and with Hsp27 upregulation.