Hypoxia-regulated neurotrophin-3 expression by multicopy hypoxia response elements reduces apoptosis in PC12 cells

Abstract

Abstract.We have previously reported that 5 copies of the hypoxia response element (HRE) can conditionally regulate brain-derived neurotrophic factor gene expression under hypoxic/ischemic conditions in mice. In the present study, we investigated the controlled expression of neurotrophin-3 (NT-3) by HRE under hypoxic conditions and determined the protective effects of conditionally expressed NT-3 on hypoxia-induced apoptosis in PC12 cells. Five copies of the HRE (5HRE) and the simian virus 40 minimal promoter (SV40mp) were employed to construct a cassette, and transfer of therapeutic gene, NT-3, into PC12 cells was achieved using a retroviral vector. Our results showed that the retroviral vector, pLNC-5HRE-NT3, was successfully constructed and transfected into PC12 cells. Compared with normal conditions, in which NT-3 was expressed at low levels, the expression of NT-3 significantly increased under hypoxic conditions in 5HRE-NT3 transgenic PC12 cells (P<0.05). By contrast, in NT-3 transgenic PC12 cells without HRE, we found no significant difference in NT-3 expression between the normoxic and hypoxic groups. The conditional adjustment of NT-3 expression by 5HRE significantly reduced apoptosis induced by hypoxia in 5HRE-NT3 transgenic PC12 cells (P<0.05) but not in 5HRE-enhanced green fluorescent protein (EGFP) transgenic PC12 cells and PC12 cells without gene transfer. In addition, the hypoxia-induced upregulation of both p38 and caspase-3 activities was suppressed in 5HRE-NT3 transgenic PC12 cells under hypoxic conditions (P<0.05). Taken together, these results demonstrate that 5HRE-SV40mp regulates NT-3 gene expression in response to hypoxia in PC12 cells. The data presented in this study may prove useful in future gene therapy studies for the treatment of ischemic diseases. IntroductionTo achieve the optimal effects of gene therapy, it is crucial to develop a strategy to conditionally express the therapeutic gene of interest and reduce any unexpected side-effects resulting from excessive expression of the exogenous gene (1,2). Previous studies have suggested that multiple copies of the hypoxia response element (HRE) can regulate the expression of therapeutic genes in response to hypoxia (3-6). The hypoxia inducible factor-1 (HIF-1) is a transcription factor that plays a key role in the regulation of gene expression under hypoxic conditions (7). HIF-1 is composed of 2 subunits, HIF-1α and HIF-1β. Under normoxic conditions, HIF-1α is rapidly degraded by the ubiquitin-proteasome pathway. However, under hypoxic conditions, HIF-1α becomes stabilized and forms an active HIF-1 heterodimer with HIF-1β. Through its binding to the HRE located in the regulatory domain of target genes, HIF-1α can upregulate the expression of several hypoxiarelated genes in response to hypoxia (8). Thus, HRE can regulate downstream gene expression according to the activity of HIF-1, which is affected by the oxygen concentration.Neurotrophin-3 (NT-3) not only activates its own specific orphan receptor, TrkC, but also activates TrkA and Trk...

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