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. 2008 Oct 17;283(42):28074-80.
doi: 10.1074/jbc.M804203200. Epub 2008 Aug 14.

Metabolic stress induces the lysosomal degradation of neuropilin-1 but not neuropilin-2

Donggoo Bae  1 , Shaolei Lu, Cherie A Taglienti, Arthur M Mercurio
Affiliations

Metabolic stress induces the lysosomal degradation of neuropilin-1 but not neuropilin-2

Donggoo Bae et al. J Biol Chem. .

Abstract

The neuropilins-1 and -2 (NRP1 and NRP2) function as receptors for both the semaphorins and vascular endothelial growth factor. In addition to their contribution to the development of the nervous system, NRP1 and NRP2 have been implicated in angiogenesis and tumor progression. Given their importance to cancer and endothelial biology and their potential as therapeutic targets, an important issue that has not been addressed is the impact of metabolic stress conditions characteristic of the tumor microenvironment on their expression and function. Here, we demonstrate that hypoxia and nutrient deprivation stimulate the rapid loss of NRP1 expression in both endothelial and carcinoma cells. NRP2 expression, in contrast, is maintained under these conditions. The lysosomal inhibitors chloroquine and bafilomycin A1 prevented the loss of NRP1 expression, but proteasomal inhibitors had no effect. The hypothesis that NRP1 is degraded by autophagy is supported by the findings that its expression is lost rapidly in response to metabolic stress, prevented with 3-methyladenine and induced by rapamycin. Targeted depletion of NRP2 using small hairpin RNA revealed that NRP2 can function in the absence of NRP1 to mediate endothelial tube formation in hypoxia. Studies aimed at assessing NRP function and targeted therapy in cancer and angiogenesis should consider the impact of metabolic stress.

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Figures

FIGURE 1.
FIGURE 1.
Hypoxia suppresses NRP1 expression in carcinoma and endothelial cells. A, MDA-MB-231 and SUM159 breast carcinoma cells, PC-3 prostate carcinoma cells, and HUVECs were maintained at 37 °C in either 21% O2 or 0.1% O2 for 24 h. Protein extracts (50 μg) obtained from these cells were immunoblotted with the indicated antibodies (mouse anti-NRP1 and goat anti-hexokinase II (HK II; Santa Cruz Biotechnology) and rabbitβ-actin (Sigma-Aldrich). Similar results were observed in at least three independent experiments. B, SUM159 breast cancer cells were maintained in a range of oxygen tensions (0.3–5.0% O2) for 1 day, and the relative expression of NRP1 was compared with that in normoxia for each of these oxygen tensions.
FIGURE 2.
FIGURE 2.
NRP2 expression is not suppressed by hypoxia. A, protein extracts (50 μg) obtained from either SUM159 cells or HUVECs that had been maintained in normoxia or hypoxia (0.1% O2) for the indicated times were immunoblotted with a NRP2 Ab (goat anti-NRP2, R&D Systems). The blots were also probed with a β-actin Ab to assess protein loading. B, MDA-MB-453 breast carcinoma cells were transfected with either a mock, NRP1, or NRP2 expression plasmid. The cells were maintained in either normoxia or hypoxia (0.1% O2) for 24 h, and the expression of each NRP was assessed by immunoblotting. The bar graph depicts the relative expression of each NRP as determined by densitometry using β-actin as a control.
FIGURE 3.
FIGURE 3.
Cell surface expression of NRP1 is diminished in hypoxia. A, cell surface expression of the NRPs was assessed on HUVECs by flow cytometry using anti-BDCA-4 (NRP1) or anti-NRP2 (H300) Abs with fluorescein isothiocyanate (FITC)-labeled secondary Abs. Only live cells (DAPI– population) were analyzed. The data from multiple experiments were quantified and are shown in the bar graph. B, AP-conjugated, recombinant human Sema3F (for NRP2) and Sema3A (NRP1) proteins (provided by Dr. Alex Kolodkin (Johns Hopkins University) were allowed to interact with HUVECs in either normoxia (21% O2) or hypoxia (0.1% O2) for 24 h at 4 °C. AP alone was used for negative control. Bound semaphorins were visualized using an AP enzymatic assay (GenHunter) following the manufacturer's protocol. Inset, the expression of the NRPs under each of the above experimental conditions was assessed by immunoblotting as described in Figs. 1 and 2.
FIGURE 4.
FIGURE 4.
Hypoxia stimulates the lysosomal degradation of NRP1. A, HUVECs were maintained in either normoxia (21% O2) or hypoxia (0.1% O2) in the presence or absence of chloroquine (100 μm, Sigma-Aldrich) for the indicated times. The relative expression of NRP1, NRP2, and actin was assessed by immunoblotting and densitometry as described in Figs. 1 and 2. B, a similar experiment was performed using bafilomycin A1 (100 nm, Sigma-Aldrich). The data shown in the bar graph is one representative result from three independent experiments (±S.D.). C, cell surface expression of the NRPs was assessed on a live single-cell suspension of HUVECs in the presence or absence of chloroquine by flow cytometry as described in Fig. 3. The data from two experiments were quantified and are shown in the bar graph.
FIGURE 5.
FIGURE 5.
NRP1 is degraded by a mechanism characteristic of autophagy. A and B, the expression of NRP1 and NRP2 in HUVECs that had been maintained in PBS containing 4 g/liter glucose for the indicated times was assessed by immunoblotting (A) and by flow cytometry (B) as described in Fig. 3. C, HUVECs were maintained in either normal medium or PBS/glucose for 2 h in either the presence or absence of 3-methyladenine (3MA)(5 and 10 mm), and NRP expression was assessed by immunoblotting. D, HUVECs maintained in normal culture medium were treated with rapamycin (5 and 500 nm) for 24 h in normoxia, and NRP expression was assessed by immunoblotting.
FIGURE 6.
FIGURE 6.
NRP2 mediates endothelial cell tube formation in hypoxia. A, HUVECs (5 × 105 cells) were infected with either a green fluorescent protein shRNA or two different NRP2 shRNAs (5 × 106 lentiviruses/infection), and NRP1 and NRP2 expression was assayed at 72 h after infection by immunoblotting. B, at 72 h after infection, 104 cells were seeded onto preformed Matrigel (1 mg/ml), and photomicrographs (magnification, ×40) were taken after 24 h in hypoxia (0.5% O2) with an inverted-phase microscope. C, endothelial tube formation was quantified by determining the mean number of junctions formed per field (±S.D.). Significance was assessed by Student's t test by comparison with noninfected parental cells. D, NRP expression was assessed by immunoblotting before (normoxia) and after (hypoxia) the Matrigel assay.
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