Matrix Metalloproteinase (MMP)-9 Induced by Wnt Signaling Increases the Proliferation and Migration of Embryonic Neural Stem Cells at Low O2 Levels

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-1␣ 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-1␣. 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-1␣ and Wnt activation, responsible for increased rates of NSC proliferation and migration in 1% O 2 .A frequently overlooked variable for the in vitro expansion of stem/progenitor cells is the level of O 2 in cell culture (1-3). In fact, the level of O 2 (20.9% O 2 ) used for in vitro culture is hyperoxic for cells in vivo. The lung alveoli and bloodstream typically hold ϳ14% (4, 5) and 11.5% (6 -8) O 2 , respectively, and O 2 levels in various areas of the brain range from 0.1 to 9% (5, 7, 9 -12). Recent studies have shown that the use of lower O 2 levels in culture results in increased rates of proliferation of neural stem/progenitor cells (NSCs) 2 from various embryonic or adult brain regions (12-23), virally immortalized human NSCs (23), and embryonic stem (ES) cells (3,24,25) and that these O 2 levels can also influence the differentiation of NSCs (9 -16).A large body of work has described hypoxia-inducible transcription factors as key components of the O 2 -sensing machinery that responds to lowered O 2 in cells and organisms (5,10,26,27). It has been shown that levels of HIF-1␣ protein can vary significantly over physiological ranges of O 2 (28). Although HIF-1␣ is constitutively expressed, when O 2 levels are high, the protein is hydroxylated by a family of prolyl hydroxylase enzymes, catalyzing a modification that targets HIF-1␣ for proteasomal degradation. At lower O 2 levels, the HIF-1␣ protein undergoes less hydroxylation, is stabilized, and can then translocate to the nucleus where it activates several genes that modulate the cellular response to decreas...

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

“…It has been reported that culturing of a variety of healthy cell types under physioxia produced phenotypes closer to those observed in vivo [57][58][59][60] compared to cells cultured under air. Culture under physioxia enhanced clonal growth [61][62][63] and the generation of cytokines by blood mononuclear cells [59,63].…”

“…Culture under physioxia enhanced clonal growth [61][62][63] and the generation of cytokines by blood mononuclear cells [59,63]. Culturing stem cells under physioxia improved proliferation [58,60], whilst culturing stem-like cells under physioxia improved the development of stem cell characteristics [57], compared to culture under air.…”

Granulocyte/Macrophage Colony-stimulating Factor-dependent Dendritic Cells Restrain Lean Adipose Tissue Expansion

“…The primers/probes were ordered from PrimerBank. An RT2-Profiler PCR array for mouse extracellular matrix and adhesion molecules (PAMM-013Z, Qiagen) was used to quantify relative mRNA levels in adipose tissue (25). Data were analyzed by software on the SABiosciences Web site.…”

MiR-218 reverses high invasiveness of glioblastoma cells by targeting the oncogenic transcription factor LEF1

“…Increasing MMP-9 expression is associated with dysfunctions of Wnt signaling (19); therefore, we conceived a biological transport chain: the miR-218-intermediary molecule-MMP-9 axis. MMP-9 is a key molecule downstream of Wnt activation and is responsible for increased rates of neural stem cell (NSC) proliferation and migration in 1% O 2 (19). MMP-7 is also downstream effector in the Wnt pathway; therefore, we hypothesized that miR-218 can regulate the expression of MMP-9 and -7 by targeting transcription factors involved in the Wnt pathway.…”