Background: Bone marrow mesenchymal stem cells (MSCs) may mediate their beneficial effects by paracrine mechanisms. Recently, we reported that tumor necrosis factor-alpha (TNF-alpha) increased the release of vascular endothelial growth factor (VEGF) from human MSCs and augmented transforming growth factor-alpha (TGF-alpha)-stimulated VEGF secretion. However, it is unknown whether TNF-alpha stimulates VEGF production via TNF receptor 1 (TNFR1) or 2 (TNFR2) and the mechanism by which TNF-alpha augments TGF-alpha (a ligand of epidermal growth factor receptor, EGFR) stimulated VEGF production. We hypothesized that the ablation of TNFR2 would decrease TNF-alpha-stimulated and/or TGF-alpha- stimulated VEGF production via MEK-dependent mechanisms.
Methods: MSCs transfected with TNFR1, TNFR2, or GAPDH siRNA were stimulated with TNF-alpha and/or TGF-alpha for 24 h. VEGF levels in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA). A Western blot analysis was performed to measure the activation of MEK and ERK and the expression of TNFR1 and TNFR2.
Results: TNF-alpha or TGF-alpha increased VEGF secretion in cells transfected with GAPDH or TNFR1 siRNA. The combination of TNF-alpha and TGF-alpha increased VEGF production. TNF-alpha and/or TGF-alpha stimulation increased phospho-MEK and phospho-ERK in cells transfected with TNFR1 siRNA. Conversely, the effects of TNF-alpha and/or TGF-alpha on MSC VEGF production were significantly decreased, and MEK/ERK activation was negated in cells transfected TNFR2 siRNA.
Conclusion: TNFR2 plays a vital role in the effects of TNF-alpha and TGF-alpha on MSC VEGF production. The activation of MEK was implicated in this novel cross talk between TNFR2 and TGF-alpha-EGFR in regulating the production of VEGF in human MSCs.