Angiogenic CXC chemokine expression during differentiation of human mesenchymal stem cells towards the osteoblastic lineage

Abstract

The potential role of ELR(+) CXC chemokines in early events in bone repair was studied using human mesenchymal stem cells (hMSCs). Inflammation, which occurs in the initial phase of tissue healing in general, is critical to bone repair. Release of cytokines from infiltrating immune cells and injured bone can lead to recruitment of MSCs to the region of repair. CXC chemokines bearing the Glu-Leu-Arg (ELR) motif are also released by inflammatory cells and serve as angiogenic factors stimulating chemotaxis and proliferation of endothelial cells. hMSCs, induced to differentiate with osteogenic medium (OGM) containing ascorbate, beta-glycerophosphate (beta-GP), and dexamethasone (DEX), showed an increase in mRNA and protein secretion of the ELR(+) CXC chemokines CXCL8 and CXCL1. CXCL8 mRNA half-life studies reveal an increase in mRNA stability upon OGM stimulation. Increased expression and secretion is a result of DEX in OGM and is dose-dependent. Inhibition of the glucocorticoid receptor with mifepristone only partially inhibits DEX-stimulated CXCL8 expression indicating both glucocorticoid receptor dependent and independent pathways. Treatment with signal transduction inhibitors demonstrate that this expression is due to activation of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and is mediated through the G(alphai)-coupled receptors. Angiogenesis assays demonstrate that OGM-stimulated conditioned media containing secreted CXCL8 and CXCL1 can induce angiogenesis of human microvascular endothelial cells in an in vitro Matrigel assay.

Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin‐8), in human adult mesenchymal stem cells via the extracellular signal‐regulated kinase, p38 mitogen‐activated protein kinase, and NF‐κB pathways

“…Constitutive expression of CXCL8 mRNA has also been observed in human mesenchymal stem cells (hMSCs) [Majumdar et al, 1998[Majumdar et al, , 2000Kim et al, 2005], and we have shown induction of CXCL8 during differentiation of hMSCs towards the osteoblastic lineage [Bischoff et al, 2007]. While it has been noted that IL-1a stimulation of hMSC cultures can increase the steady-state mRNA levels of CXCL8 [Majumdar et al, 1998], it has not previously been systematically investigated if CXCL8 can be induced by hMSCs subjected to changes in extracellular proton concentration that would be expected during the initial phase of bone repair.…”

“…Stimulation of CXCL8 by the pro-inflammatory cytokines IL-1b and TNF-a has been shown to be regulated in large part through the NF-kB pathway. This pathway is sensitive to inhibition by glucocorticoids [McKay and Cidlowski, 1999;Bosscher et al, 2003], and we have previously shown that the IL-1b/TNF-a-stimulated CXCL8 expression is partially suppressed by DEX in hMSCs [Bischoff et al, 2007]. Thus, there appears to be a DEX-stimulated CXCL8 pathway either independent of the NF-kB signaling pathway involved in pro-inflammatory cytokine stimulation of CXCL8 or overcome when NF-kBmediated CXCL8 production is induced during hMSC differentiation.…”

“…For differentiation towards the osteoblastic lineage, hMSCs were treated every 3-4 days with osteogenic medium (OGM) which consisted of HMSCGM supplemented with 50 mM ascorbic acid-2-phosphate, 10 mM b-glycerophosphate, and 10 À7 M dexamethasone (Sigma-Aldrich, St. Louis, MO). We have previously shown that treatment of the hMSCs with OGM induces differentiation towards the osteogenic lineage as assayed by up-regulation of alkaline phosphatase (mRNA and activity) and bone sialoprotein (mRNA) and by von Kossa and alizarin red staining for mineralization [Bischoff et al, 2007].…”

“…Acidosis has been shown to activate NF-kB leading to CXCL8 expression in several cancer cell lines [Xu and Fidler, 2000;Shi et al, 2001;Karashima et al, 2003]. Dexamethasone is the medium component that is required for CXCL8 expression during differentiation of hMSCs towards the osteoblastic linage [Bischoff et al, 2007]. Stimulation of CXCL8 by the pro-inflammatory cytokines IL-1b and TNF-a has been shown to be regulated in large part through the NF-kB pathway.…”

“…In this manuscript, we present a novel observation that hMSCs can be stimulated by an acidic pH to express CXCL8 mRNA in both the multipotent state and when differentiated towards the osteogenic lineage. Since osteogenic differentiation results in expression of CXCL8 beginning at 5 days [Bischoff et al, 2007], we looked at early (3 day) and late (7 day) cultures to differentiate between CXCL8 expression due to osteogenic differentiation from Fig. 8.…”