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J. Pack, P. R. Cammarata; Topotecan Prevents Induction of Hypoxia-Inducible Factor-1 in Human Lens Epithelial Cells: Implications for Hypoxia-Mediated Regulation of Extracellular Signal-Regulated Kinase (ERK). Invest. Ophthalmol. Vis. Sci. 2010;51(13):2631.
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Hypoxia-inducible factor-1α (HIF-1α) is expressed in the human lens and may be associated with mitochondrial protection against membrane permeability transition in human lens epithelium (HLE) via activation (i.e. phosphorylation) of ERK. It has been demonstrated in several other cell types that HIF-1α-activated ERK is inactivated through phosphate removal by mitogen-activated protein kinase phosphatase-1 (MKP-1). The molecular inter-relationship between HIF-1α levels, MKP-1 expression and pERK content is neither well characterized nor understood. We investigated the effect of the anti-cancer drug, Topotecan (Topo), a topoisomerase I inhibitor known to impede hypoxia-mediated HIF-1α induction, with the intent of determining resulting downstream influence on pERK quantity in cultured HLE-B3 cells.
Protein expression of HIF-1α, MKP-1, ERK and pERK was ascertained by Western blot analysis.
Hypoxia results in the robust induction of HIF-1α protein expression in a time-dependent manner. pERK levels are transient, peaking within 1 hr of hypoxia and diminishing thereafter, despite continuous application of hypoxia. Pretreatment of cells with Topo significantly diminished hypoxia-induced HIF-1α in a dose-dependent manner. Topo pretreatment also resulted in a sustained over-accumulation of hypoxia-mediated pERK. Under the same conditions, MKP-1 protein expression was markedly lowered.
MKP-1 is a hypoxia responsive protein, regulated by HIF-1α induction. HIF-1α-activated ERK, in human lens epithelial cells, is inactivated through dephosphorylation by MKP-1. Topotecan is a potent inhibitor of the HIF-1α subunit in hypoxic HLE-B3 cells, leading to decreased MKP-1 expression and over-accumulation of pERK. Taken together, the data is consistent with the fact that hypoxia induces HIF-1α levels resulting in elevated MKP-1 expression; thereby providing a level of homeostatic regulation on the amount of ERK phosphorylation. pERK accumulation is enhanced or reduced by MKP-1 suppression or MKP-1 expression, respectively. pERK has previously been shown to be a critical component in the lens epithelial cell mitoprotection pathway (Flynn et al., Am J Physiol. E589-E599, 2008). We are interested in determining whether this artificial enhancement of pERK via intervention by Topotecan, under hypoxic condition, provides enhanced mitoprotection upon reintroduction of oxygen.
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