HIF1 Mediates the in vivo Regulation of Lens Cell Proliferation by Oxygen

Abstract

Abstract: : Purpose: The lens exists in a hypoxic environment. Our previous studies show that exposing adult rats to 60% O₂ increases O₂ levels around the lens and promotes the proliferation of lens epithelial cells. HIF–1α is a transcription factor that accumulates during hypoxia and stimulates the transcription of genes that promote cell survival in hypoxic conditions. We used transgenic mice that over express stable (oxygen–independent) forms of HIF–1α in the lens to determine whether genes that are regulated by HIF1α suppress lens epithelial cell proliferation. Methods: We employed two lines of transgenic mice that use the keratin–14 promoter to over express mutated forms of human HIF1α in the lens. In K14–HIF1α–proline mice, two proline residues that are thought to be required for oxygen–dependent degradation are mutated. In K14–HIF1α–ΔODD mice, the entire oxygen–dependent degradation domain of HIF1α is deleted. Adult (7–8 month old) transgenic mice and their non–transgenic littermates were exposed to room air (21% O₂) or 60% O₂ for 3 days. One hour before sacrifice, BrdU (50 mg/kg) was injected i.p., animals were killed by CO₂ inhalation, lens epithelial whole mounts were dissected, fixed and stained with anti–BrdU antibody and TOTO–1 (to label all nuclei). The BrdU labeling index was determined using NIH Image software. HIF1α protein levels were determined by Western blotting. Results: In non–transgenic animals, the BrdU labeling index was 1% in room air and 2.5% in 60% O₂. In both K14–HIF1α transgenic lines the BrdU labeling index was 1% in room air and 1.7% in 60% O₂. The stimulation of proliferation by O₂ was significantly greater in the non–transgenic than in either strain of transgenic mice (p < 0.01). However, the BrdU labeling index in both strains of transgenic mice was significantly greater in 60% O₂ than in 21% O₂ (p < 0.01). On western blots, HIF 1α was detectable in lens epithelia of non–transgenic mice maintained in room air, but was undetectable after three days in 60% O₂. As expected, in K14–HIF1α–ΔODD mice, HIF1α levels were high and not affected by exposure to 60% O₂. Surprisingly, the proline mutant form of HIF1α decreased markedly when mice were exposed to 60% O₂. Conclusions:HIF1α contributes to the ability of hypoxia to inhibit lens cell proliferation.

Keywords: transcription factors • transgenics/knock-outs • proliferation