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Severe hypoxia exerts parallel and cell-specific regulation of gene expression and alternative splicing in human mesenchymal stem cells.

Hu X, Wu R, Shehadeh LA, Zhou Q, Jiang C, Huang X, Zhang L, Gao F, Liu X, Yu H, Webster KA, Wang J - BMC Genomics (2014)

Bottom Line: Antioxidant genes, striated muscle genes and insulin/IGF-1 signaling intermediates were down-regulated.There was a coordinate induction of 9 out of 12 acidic keratins that along with other epithelial and cell adhesion markers implies a partial mesenchymal to epithelial transition.This is the first study to report hypoxia-regulation of AS in stem/progenitor cells and the first molecular genetic characterization of MSC in a hypoxia-induced quiescent immobile state.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cardiovascular Key Lab of Zhejiang Province, Department of Cardiology, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, P,R, China. kwebster@med.miami.edu.

ABSTRACT

Background: The endosteum of the bone marrow provides a specialized hypoxic niche that may serve to preserve the integrity, pluripotency, longevity and stemness of resident mesenchymal stem cells (MSCs). To explore the molecular genetic consequences of such a niche we subjected human (h) MSCs to a pO2 of 4 mmHg and analyzed global gene expression and alternative splicing (AS) by genome-exon microarray and RT-qPCR, and phenotype by western blot and immunostaining.

Results: Out of 446 genes differentially regulated by >2.5-fold, down-regulated genes outnumbered up-regulated genes by 243:203. Exon analyses revealed 60 hypoxia-regulated AS events with splice indices (SI) >1.0 from 53 genes and a correlation between high SI and degree of transcript regulation. Parallel analyses of a publicly available AS study on human umbilical vein endothelial cells (HUVECs) showed that there was a strong cell-specific component with only 11 genes commonly regulated in hMSCs and HUVECs and 17 common differentially spliced genes. Only 3 genes were differentially responsive to hypoxia at the gene (>2.0) and AS levels in both cell types. Functional assignments revealed unique profiles of gene expression with complex regulation of differentiation, extracellular matrix, intermediate filament and metabolic marker genes. Antioxidant genes, striated muscle genes and insulin/IGF-1 signaling intermediates were down-regulated. There was a coordinate induction of 9 out of 12 acidic keratins that along with other epithelial and cell adhesion markers implies a partial mesenchymal to epithelial transition.

Conclusions: We conclude that severe hypoxia confers a quiescent phenotype in hMSCs that is reflected by both the transcriptome profile and gene-specific changes of splicosome actions. The results reveal that severe hypoxia imposes markedly different patterns of gene regulation of MSCs compared with more moderate hypoxia. This is the first study to report hypoxia-regulation of AS in stem/progenitor cells and the first molecular genetic characterization of MSC in a hypoxia-induced quiescent immobile state.

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Western blot and quantification of phosphor-Akt expression in normoxic (N-MSC) and hypoxic (H-MSC) human MSCs (A and B). Western blot procedures are described in Methods. Akt-P-Thr308 quantification was by NIH image using total Akt as loading control; *p < 0.05, n = 3.
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Figure 6: Western blot and quantification of phosphor-Akt expression in normoxic (N-MSC) and hypoxic (H-MSC) human MSCs (A and B). Western blot procedures are described in Methods. Akt-P-Thr308 quantification was by NIH image using total Akt as loading control; *p < 0.05, n = 3.

Mentions: As discussed above, oxygen tensions above 15 mmHg enhance proliferative and migratory potential of MSCs while tensions below10 mmHg decrease proliferation [8-12,17-19]. In agreement with this we found that proliferation of hMSCs was decreased under a pO2 of 4 mmHg and the cultures became stationary after 3 days (data not shown). Enhanced proliferation under moderate hypoxia has been attributed to down-regulation of the p21-Ras pathway and increased activity of PI3-kinase-Akt while increased migration was attributed to increased expression of c-MET, VEGF, CXCR4 and CXCR1 (reviewed in [16]). Whereas we also observed increased expression of c-MET and VEGF-A we found no change in expression of CXCR receptors, increased expression of p21-Ras and decreased activity of the PI3-kinase-Akt pathway (see Tables 2 and 3). PI3-kinase-Akt is a central regulator of cell growth and survival. We found that the expression of both IGF-1 and PI3-kinase was significantly decreased under hypoxia while multiple IGF-1BPs were increased (Tables 2 and 3). To determine whether these changes were reflected by parallel changes in pathway activity we measured the phosphorylation of Akt-Thr308 after culture under normoxia or hypoxia. As shown in Figure 6, phosphor-Akt-Thr308 levels were significantly lower after hypoxia. This contrasts with the effects of moderate hypoxia where Akt phosphorylation is increased [26]. Other down-regulated transcripts related to growth and survival included senescence marker galactosidase beta-1-like (−3.0) and death associated kinase-2 (−2.4). There were also decreases of several interleukins and the cell migration cytokine CCL2, also known as MCP-1.


Severe hypoxia exerts parallel and cell-specific regulation of gene expression and alternative splicing in human mesenchymal stem cells.

Hu X, Wu R, Shehadeh LA, Zhou Q, Jiang C, Huang X, Zhang L, Gao F, Liu X, Yu H, Webster KA, Wang J - BMC Genomics (2014)

Western blot and quantification of phosphor-Akt expression in normoxic (N-MSC) and hypoxic (H-MSC) human MSCs (A and B). Western blot procedures are described in Methods. Akt-P-Thr308 quantification was by NIH image using total Akt as loading control; *p < 0.05, n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4234502&req=5

Figure 6: Western blot and quantification of phosphor-Akt expression in normoxic (N-MSC) and hypoxic (H-MSC) human MSCs (A and B). Western blot procedures are described in Methods. Akt-P-Thr308 quantification was by NIH image using total Akt as loading control; *p < 0.05, n = 3.
Mentions: As discussed above, oxygen tensions above 15 mmHg enhance proliferative and migratory potential of MSCs while tensions below10 mmHg decrease proliferation [8-12,17-19]. In agreement with this we found that proliferation of hMSCs was decreased under a pO2 of 4 mmHg and the cultures became stationary after 3 days (data not shown). Enhanced proliferation under moderate hypoxia has been attributed to down-regulation of the p21-Ras pathway and increased activity of PI3-kinase-Akt while increased migration was attributed to increased expression of c-MET, VEGF, CXCR4 and CXCR1 (reviewed in [16]). Whereas we also observed increased expression of c-MET and VEGF-A we found no change in expression of CXCR receptors, increased expression of p21-Ras and decreased activity of the PI3-kinase-Akt pathway (see Tables 2 and 3). PI3-kinase-Akt is a central regulator of cell growth and survival. We found that the expression of both IGF-1 and PI3-kinase was significantly decreased under hypoxia while multiple IGF-1BPs were increased (Tables 2 and 3). To determine whether these changes were reflected by parallel changes in pathway activity we measured the phosphorylation of Akt-Thr308 after culture under normoxia or hypoxia. As shown in Figure 6, phosphor-Akt-Thr308 levels were significantly lower after hypoxia. This contrasts with the effects of moderate hypoxia where Akt phosphorylation is increased [26]. Other down-regulated transcripts related to growth and survival included senescence marker galactosidase beta-1-like (−3.0) and death associated kinase-2 (−2.4). There were also decreases of several interleukins and the cell migration cytokine CCL2, also known as MCP-1.

Bottom Line: Antioxidant genes, striated muscle genes and insulin/IGF-1 signaling intermediates were down-regulated.There was a coordinate induction of 9 out of 12 acidic keratins that along with other epithelial and cell adhesion markers implies a partial mesenchymal to epithelial transition.This is the first study to report hypoxia-regulation of AS in stem/progenitor cells and the first molecular genetic characterization of MSC in a hypoxia-induced quiescent immobile state.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cardiovascular Key Lab of Zhejiang Province, Department of Cardiology, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, P,R, China. kwebster@med.miami.edu.

ABSTRACT

Background: The endosteum of the bone marrow provides a specialized hypoxic niche that may serve to preserve the integrity, pluripotency, longevity and stemness of resident mesenchymal stem cells (MSCs). To explore the molecular genetic consequences of such a niche we subjected human (h) MSCs to a pO2 of 4 mmHg and analyzed global gene expression and alternative splicing (AS) by genome-exon microarray and RT-qPCR, and phenotype by western blot and immunostaining.

Results: Out of 446 genes differentially regulated by >2.5-fold, down-regulated genes outnumbered up-regulated genes by 243:203. Exon analyses revealed 60 hypoxia-regulated AS events with splice indices (SI) >1.0 from 53 genes and a correlation between high SI and degree of transcript regulation. Parallel analyses of a publicly available AS study on human umbilical vein endothelial cells (HUVECs) showed that there was a strong cell-specific component with only 11 genes commonly regulated in hMSCs and HUVECs and 17 common differentially spliced genes. Only 3 genes were differentially responsive to hypoxia at the gene (>2.0) and AS levels in both cell types. Functional assignments revealed unique profiles of gene expression with complex regulation of differentiation, extracellular matrix, intermediate filament and metabolic marker genes. Antioxidant genes, striated muscle genes and insulin/IGF-1 signaling intermediates were down-regulated. There was a coordinate induction of 9 out of 12 acidic keratins that along with other epithelial and cell adhesion markers implies a partial mesenchymal to epithelial transition.

Conclusions: We conclude that severe hypoxia confers a quiescent phenotype in hMSCs that is reflected by both the transcriptome profile and gene-specific changes of splicosome actions. The results reveal that severe hypoxia imposes markedly different patterns of gene regulation of MSCs compared with more moderate hypoxia. This is the first study to report hypoxia-regulation of AS in stem/progenitor cells and the first molecular genetic characterization of MSC in a hypoxia-induced quiescent immobile state.

Show MeSH
Related in: MedlinePlus