Abstract
Recent studies have shown that various neural and embryonic stem cells cultured in 1-8% oxygen (O(2)), levels lower than those typically used in cell culture (20.9%), displayed increased rates of proliferation; however, the molecular mechanisms underlying these changes are largely undefined. In this study, using rigorously controlled O(2) levels, we found that neural stem cells (NSCs) from embryonic day 15 rat cortex increased their rate of proliferation and migration in 1% O(2) relative to 20% O(2) without changes in viability. We sought to identify molecular changes in NSCs grown in 1% O(2) that might account for these increases. In 1% O(2), levels of the hypoxia-inducible transcription factor HIF-1α were transiently increased. Reduced adherence of NSCs in 1% O(2) to basement membrane-coated plates was observed, and quantitative RT-PCR analysis confirmed that the levels of mRNA for an assortment of cell adhesion and extracellular matrix molecules were altered. Most notable was a 5-fold increase in matrix metalloproteinase (MMP)-9 mRNA. Specific inhibition of MMP-9 activity, verified using a fluorescent substrate assay, prevented the increase in proliferation and migration in 1% O(2). The canonical Wnt pathway was recently shown to be activated in stem cells in low O(2) via HIF-1α. Inhibition of Wnt signaling by DKK-1 also prevented the increase in proliferation, migration, and MMP-9 expression. Thus, MMP-9 is a key molecular effector, downstream of HIF-1α and Wnt activation, responsible for increased rates of NSC proliferation and migration in 1% O(2).
Highlights
A large body of work has described hypoxia-inducible transcription factors as key components of the O2-sensing machinery that responds to lowered O2 in cells and organisms [5, 10, 26, 27]
We identified matrix metalloproteinase (MMP)-9 as the molecular effector of increased neural stem cells (NSCs) proliferation and migration in 1% O2
We identified MMP-9 as a key molecular effector of increased NSC proliferation and migration in lowered O2
Summary
Reagents and Western Blotting—Reagents were purchased from Invitrogen unless otherwise noted. Cells in 6-well plates were washed in PBS for 5 min in the presence of the proteasome inhibitor MG132 (5 M) to stabilize the HIF-1␣ protein after which time RIPA buffer (100 l) with MG132 was added. QRT-PCR—Cells were seeded onto 10-cm plates (Corning Glass, Corning, NY) at 2000 cells/cm and grown for 3 days in vitro. Zymography and MMP-9 Activity Assay—For both assays, cells were seeded onto 6-well plates at ϳ150,000 cells/well in 2 ml of Neurobasal media supplemented as described above. Zymography was performed by loading 10 l of culture supernatant (in nondenaturing sample buffer) per well onto a Bio-Rad 1% gelatin gel. Digestion of gelatin was allowed to occur for 18 –20 h at 37 °C at which time the gel was rinsed and stained with 0.5% Coomassie Blue, 30% methanol, 10% acetic acid for 1 h. Statistical Analysis—Statistics were performed with a paired, type II, two-tailed t test (MS Excel, Microsoft, Redmond, WA) and repeated with one-way ANOVA (SPSS 16, IBM, Somers, NY) along with a post hoc Fisher’s least significant difference test (SPSS 16). *, p Ͻ 0.05; **, p Ͻ 0.01
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