Low Oxygen Modulates Multiple Signaling Pathways, Increasing Self-Renewal, While Decreasing Differentiation, Senescence, and Apoptosis in Stromal MIAMI Cells

Abstract

Human bone marrow multipotent mesenchymal stromal cell (hMSC) number decreases with aging. Subpopulations of hMSCs can differentiate into cells found in bone, vasculature, cartilage, gut, and other tissues and participate in their repair. Maintaining throughout adult life such cell subpopulations should help prevent or delay the onset of age-related degenerative conditions. Low oxygen tension, the physiological environment in progenitor cell-rich regions of the bone marrow microarchitecture, stimulates the self-renewal of marrow-isolated adult multilineage inducible (MIAMI) cells and expression of Sox2, Nanog, Oct4a nuclear accumulation, Notch intracellular domain, notch target genes, neuronal transcriptional repressor element 1 (RE1)-silencing transcription factor (REST), and hypoxia-inducible factor-1 alpha (HIF-1α), and additionally, by decreasing the expression of (i) the proapoptotic proteins, apoptosis-inducing factor (AIF) and Bak, and (ii) senescence-associated p53 expression and β-galactosidase activity. Furthermore, low oxygen increases canonical Wnt pathway signaling coreceptor Lrp5 expression, and PI3K/Akt pathway activation. Lrp5 inhibition decreases self-renewal marker Sox2 mRNA, Oct4a nuclear accumulation, and cell numbers. Wortmannin-mediated PI3K/Akt pathway inhibition leads to increased osteoblastic differentiation at both low and high oxygen tension. We demonstrate that low oxygen stimulates a complex signaling network involving PI3K/Akt, Notch, and canonical Wnt pathways, which mediate the observed increase in nuclear Oct4a and REST, with simultaneous decrease in p53, AIF, and Bak. Collectively, these pathway activations contribute to increased self-renewal with concomitant decreased differentiation, cell cycle arrest, apoptosis, and/or senescence in MIAMI cells. Importantly, the PI3K/Akt pathway plays a central mechanistic role in the oxygen tension-regulated self-renewal versus osteoblastic differentiation of progenitor cells.

FIG. 1.

Low oxygen tension increases key self-renewal transcription factors and LRP5. (A) Graph depicts relative fold change of mRNA expression of Wnt mediators and self-renewal markers (Nanog, hTeRT, Sox2, Oct4a, Oct4a/b) and Lrp5 in MIAMI cells grown for 7 days at low oxygen compared with cells kept at 21% O₂ for 7 days (dashed line set to 1). (B) Western blot of MIAMI cells after switch from 21% to 1% oxygen for 1 h. Specific antibodies to Nanog (top panel), Sox2 (second panel), and Oct4 (third panel) were used to detect proteins. (C) Western blot analysis shows increased levels of nuclear Oct4 (45 kDa) at 3% compared with 21% O₂. Coomassie blue stain (bottom panel) shown as loading control for (B) and (C). (D) DAPI-stained nuclei and merged Oct4-FITC/DAPI image of MIAMI cells at 3% (top) and 21% (bottom) oxygen (left). We confirmed the nuclear localization of Oct4 at 3% oxygen using confocal microscopy (right). MIAMI, marrow-isolated adult multilineage inducible. *P ≤ 0.05.

FIG. 2.

Low oxygen activates Notch signaling in MIAMI cells and increases REST and co-REST expression. (A) Relative fold change of Notch mediator (Notch2, Stat6, and Jagged1) mRNA levels after 7 days at 3% compared with 21% O₂ measured by RT-qPCR and normalized to housekeeping genes, EF1α and RPL13a. (B) Western blot analysis: lane 1 shows protein lysates from MIAMI cells grown at 21% O₂, lane 2 cells were grown at 3% O₂. Top panel shows HIF-1α protein, center panel shows NICD protein. (C) mRNA levels of REST and co-REST as measured by RT-qPCR from MIAMI cells grown at high and low oxygen for 7 days. (D) Protein levels of REST and co-REST were measured by western blot from the nuclear fraction of MIAMI cells grown at high and low oxygen for 7 days (top panel). Coomassie blue staining used as loading control (bottom panel). REST, repressor element-1-silencing transcription factor; RT-qPCR, quantitative real-time PCR. *P ≤ 0.05.

FIG. 3.

Low oxygen decreases markers of cell cycle arrest, apoptosis, and senescence. (A) Left panels show increased proapoptotic proteins; p53, AIF, and Bak in high (21% O₂) versus low oxygen (3% O₂). Right panels show no change in cell cycle proteins, p21 and p27, and apoptosis-related proteins, EndoG, Bax, Bid, Bcl-XL, CIAP-2, Mcl-1, and XIAP, at high versus low oxygen. Coomassie blue staining was used as loading control for western blots. (B) MIAMI cells were stained for senescence-associated β-galactosidase (SA-βgal) activity after 1 and 3 weeks at 3% O₂ versus 21% O₂. As early as after 1 week of culture, a blue stain was observed in a significant fraction of the cells exposed to 21% O₂ (48% ± 8% positive cells) compared with the cells exposed to 3% O₂ (5% ± 2% positive cells). Black arrows point to positive cells. After 3 weeks of culture, almost all of the cells exposed to 21% O₂ expressed SA-βgal (bottom). AIF, apoptosis-inducing factor.

FIG. 4.

Low oxygen tension stimulates the canonical Wnt pathway. Low oxygen increases (A) nuclear accumulation of β-catenin. (B) β-Catenin/TCF4 interactions: immunoprecipitation of 200 μg protein samples with TCF4, which were size separated and probed with β-catenin Abs in MIAMI cells grown at 3% oxygen (lane 1) and 21% oxygen (lane 2). Isotypic negative controls (normal rabbit serum); 3% samples (lane 3) and 21% (lane 4) Coomassie blue stain (bottom panel) as loading control. (C) CyclinD1 cytoplasmic (lanes 1–2) and nuclear proteins (lanes 3–4) at 3% and 21% oxygen. Coomassie stain is loading control. (D) Changes in the levels of cytoplasmic and nuclear β-catenin in response to oxygen and Wnt3a stimulation. Changes in the levels of cytoplasmic and nuclear Oct4 in response to oxygen and Wnt3a stimulation. (E) Low oxygen fails to stimulate Wnt3a secretion: ELISA from MIAMI cell-conditioned media taken from cells grown for 7 days at 3% and 21% oxygen. A media-only sample was run to determine baseline levels of Wnt3a. (F) Exogenous Wnt3a increased the cell number at low and high oxygen tension: total cell numbers of cells seeded at 100 cells/cm² and grown at 3% or 21% oxygen with or without rmWnt3a treatment for 7 days. **P ≤ 0.01 and ***P ≤ 0.001.

FIG. 5.

LRP5 knockdown by siLRP5 decreased mRNA of self-renewal markers and nuclear accumulation of Oct4a. (A) Western blot of LRP5 (top panel) from siLrp5-treated cells at 3% oxygen: Nontransfected cells (NT, lane 1). Cells electroporated with no siRNA (lane 2); siControl (siCo, lane 3); and siLRP5 (lane 4). Tubulin (bottom panel) is used as loading control. (B) RT-qPCR determined mRNA levels for Lrp5, Oct4a, Sox2, Nanog, Cyclin D1, and hTeRT in siLRP5 cells treated for 3 days compared with siControl-treated cells (siCo, set as 1 arbitrarily, *P ≤ 0.05 and **P ≤ 0.01). All genes normalized to EF1α and RPL13a. (C) ICF of cells treated with siControl or siLrp5 with anti-Oct4-FITC; DAPI-stained nucleus; images merged as indicated.

FIG. 6.

The Akt/PI3K pathway plays a central mechanistic role in the oxygen tension-regulated osteoblastic differentiation of MIAMI cells. (A) Akt was activated (phospho-Akt) by low oxygen after 30 min and p-Akt was back to the basal condition after 60 min. Akt did not change at 21% pO₂. Total Akt did not show any changes in both conditions. (B) MIAMI cells were cultured in the presence of expansion medium at 21% and 3% O₂ for 30 min in the presence and absence of wortmannin (PI3K/AKT inhibitor). (C) Wortmannin reverses the osteogenic inhibition induced by low oxygen. MIAMI cells were grown in 21% and 3% O₂ in the presence or in the absence of 100 nM wortmannin in osteogenic differentiation medium for 14 days. (a) MIAMI cells at 21% O₂ showed a high level of alkaline phosphatase. (b) In contrast, no alkaline phosphatase activity was detected in cells grown in the absence of the 100 nM PI3K/AKT inhibitor at 3% O₂. (c) 100 nM wortmannin did not have any effect on MIAMI cells grown in 21% O₂. (d) MIAMI cells grown at 3% O₂ in the presence of 100 nM wortmannin showed high alkaline phosphatase activity. (D) Left panels: the expression of Runx-2, collagen type I-α1, and osteopontin genes was inhibited in MIAMI cells exposed to osteogenic induction at low oxygen tension (3% O₂). (D) Right panels: Wortmannin (100 nM)-mediated inhibition of the Akt/PI3K pathway released the low oxygen tension-mediated inhibition of osteoblastic gene expression.

FIG. 7.

Oxygen tension modulates multiple signaling pathways regulating self-renewal, differentiation, senescence, and apoptosis in MIAMI cells. Diagrammatic representation of the pathways, targets, and biological responses modulated by oxygen tension in MIAMI stem cells, leading to functional changes. Color images available online at www.liebertpub.com/scd