Khaliullina H et al. (2016), Nutrient-Deprived Retinal Progenitors Prolifera...

XB-ART-52517

J Dev Biol 2016 Jun 01;42:. doi: 10.3390/jdb4020017.

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Nutrient-Deprived Retinal Progenitors Proliferate in Response to Hypoxia: Interaction of the HIF-1 and mTOR Pathway.

Khaliullina H , Love NK , Harris WA .

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At a cellular level, nutrients are sensed by the mechanistic Target of Rapamycin (mTOR). The response of cells to hypoxia is regulated via action of the oxygen sensor Hypoxia-Inducible Factor 1 (HIF-1). During development, injury and disease, tissues might face conditions of both low nutrient supply and low oxygen, yet it is not clear how cells adapt to both nutrient restriction and hypoxia, or how mTOR and HIF-1 interact in such conditions. Here we explore this question in vivo with respect to cell proliferation using the ciliary marginal zone (CMZ) of Xenopus. We found that both nutrient-deprivation and hypoxia cause retinal progenitors to decrease their proliferation, yet when nutrient-deprived progenitors are exposed to hypoxia there is an unexpected rise in cell proliferation. This increase, mediated by HIF-1 signalling, is dependent on glutaminolysis and reactivation of the mTOR pathway. We discuss how these findings in non-transformed tissue may also shed light on the ability of cancer cells in poorly vascularised solid tumours to proliferate.

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Species referenced: Xenopus
Genes referenced: arnt ccdc34 h3-3a hif1a mtor rps6
GO keywords: cell proliferation involved in compound eye morphogenesis [+]
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Phenotypes: Xl Wt + nutrient deprivation + hypoxia + Echinomycin [+]

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	Figure 1. Hypoxia affects proliferation of progenitors in the retinal stem cell niche in the opposite manner, dependent on the nutritional status. (A) Schematic representation of a cross section through a 38-stage Xenopus retina, indicating the position of the ciliary marginal zone (CMZ). (B–E) EdU incorporation measured after a 2 h EdU pulse in DAPI-stained retinas from animals that have been normal-fed (B,C) or nutrient-deprived for 24 h (D,E) incubated under normoxia (B,D) or hypoxia (C,E). Nutrient deprivation or hypoxia reduced the number of proliferating cells in the CMZ, whereas hypoxic nutrient-deprived retinas show an increase of proliferation in the CMZ. Scale bars = 50 µm. For each condition, between 10 and 15 retinas were quantified. (F) Quantification of the experiment performed in (B–E). Error bars represent standard deviations. Two hooked ends of horizontal lines indicate the two conditions compared, *** p-value < 0.001; n = 7. (G) Quantification of EdU-positive cells after an 1 h EdU incorporation in retinas from normal-fed (blue line) or nutrient-deprived (red line) animals after various times in hypoxia (time in minutes indicated on the x-axis). Error bars represent standard deviations of two independent experiments (n = 2). For each condition and time point, a minimum of 10 retinas was quantified.
	Figure 2. HIF-1 activity is required to induce re-initiation of proliferation in nutrient-deprived hypoxic retinas. (A) Western blot of retinas isolated from normal-fed or nutrient-deprived animals kept under normal oxygen concentrations or in hypoxia, probed for HIF-1α subunit and α-tubulin. HIF-1α is stabilized under hypoxia in both normal-fed and nutrient-deprived animals. At least 22 retinas were taken for each condition; all conditions in each experiment have the same number of retinas (n = 5). (B) Quantification of the Western blot performed in (A). Error bars represent standard deviations. Two hooked ends of horizontal lines indicate the two conditions compared, * p-value < 0.05, *** p-value < 0.001; n = 5. (C–H) EdU incorporation in DAPI-stained retinas from normal-fed animals (C–E) kept in normoxia (C), hypoxia (D) or incubated with Echinomycin under hypoxia (E) and in nutrient-deprived animals kept in normoxia (F), hypoxia (G) or incubated with Echinomycin under hypoxia (H). Nutrient-deprived retinas no longer resume their proliferation in hypoxia when function of HIF-1 is blocked by Echinomycin. Scale bars = 50 µm. In each condition, a minimum of 10 retinas was examined. (I) Quantification of the experiment performed in (C–H). Error bars represent standard deviations between three independent experiments. Two hooked ends of horizontal lines indicate the two conditions compared, * p-value < 0.05; n > 3. For each condition, between 10 and 15 retinas were quantified.
	Figure 3. Glutamine is essential for retinal progenitor cell proliferation. (A–E) EdU incorporation in DAPI-stained ex vivo retinal explants from normal-fed embryos maintained in L15 medium for 24 h (A), from ND embryos maintained in 1× MBS for 24 h (B) or from ND embryos re-fed for 24 h with L15 (C), L15 lacking glutamine (D) or L15 lacking glutamine with added glutamine (E). Removal of glutamine from L15 medium prevented the rescue in proliferation observed following re-feeding. (F) Quantification of the experiment performed in (A–F). Error bars are standard deviations between three independent experiments. Two hooked ends of horizontal lines indicate the two conditions compared, p-values < 0.01, * p-value < 0.001; scale bars = 20 μm; n = 3. Each experiment used six retinas per condition. These were from six different animals per condition (i.e., one retina from each animal).
	Figure 4. Block in glutaminolysis interferes with re-initiation of proliferation in nutrient-deprived hypoxic retinas. (A–F) EdU incorporation in DAPI-stained retinas from normal-fed animals (A–C) kept in normoxia (A), hypoxia (B) or incubated with BPTES under hypoxia (C) and in nutrient-deprived animals kept in normoxia (D), hypoxia (E) or incubated with BPTES under hypoxia (F). Nutrient-deprived retinas no longer resume their proliferation in hypoxia when glutaminolysis is blocked by BPTES. Scale bars = 50 µm. In each condition, a minimum of 8 retinas was observed. (E) Quantification of the experiment performed in (A–F). Error bars represent standard deviations between three independent experiments. Two hooked ends of horizontal lines indicate the two conditions compared, *** p-value < 0.001, n = 3. For each condition, between 8 and 17 retinas were quantified.
	Figure 5. Reactivation of mTOR signalling, mediated by HIF-1 function, is required when nutrient-deprived retinas increase proliferation in hypoxia. (A–D) Immunostaining for the mTOR target phospho-S6 (p-S6) in retinas from normal-fed (A–C) or nutrient-deprived (C–E) animals kept in normal oxygen concentration (A,D), under hypoxia (B,E) or under hypoxia and treated with Echinomycin (C,F). Incubation of nutrient-deprived retinas in hypoxia restores p-S6 staining in the CMZ. Echinomycin treatment interferes with the increase of p-S6 staining in the CMZ of nutrient-deprived hypoxic retinas. Scale bars = 50 µm; n = 3. In each condition, a minimum of 8 retinas was examined. (G) Western blot of retinas dissected from normal-fed or nutrient-deprived normoxic or hypoxic animals, probed for phospho-S6 and α-tubulin. At least 22 retinas were taken for each condition, and all conditions in each experiment have the same number of retinas (n = 10). (H) Quantification of the Western blot shown in (G). Phospho-S6 levels, normalized to tubulin, are significantly reduced upon nutrient deprivation, unaffected by hypoxia alone, and increase to 40% of control levels upon nutrient deprivation in hypoxia. In Western blot quantification, error bars represent standard deviations between 10 independent experiments. Two hooked ends of horizontal lines indicate the two conditions compared, * p-values < 0.001; n = 10. (I) Quantification of EdU incorporation in retinas from normal-fed or nutrient-deprived animals kept in normoxia, hypoxia or incubated with Rapamycin under hypoxia. Nutrient-deprived retinas no longer resume their proliferation in hypoxia when mTOR signalling is blocked by Rapamycin. Error bars represent standard deviations between two independent experiments. Two hooked ends of horizontal lines indicate the two conditions compared, * p-values < 0.001; n = 2. For each condition, between 7 and 15 retinas were quantified.

References [+] :

Agathocleous, Metabolism in physiological cell proliferation and differentiation. 2013, Pubmed