MSCs transfected with TNFR1, TNFR2, or GAPDH siRNA were stimulated with TNF-α and/or TGF-α 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.
TNF-α or TGF-α increased VEGF secretion in cells transfected with GAPDH or TNFR1 siRNA. The combination of TNF-α and TGF-α increased VEGF production. TNF-α and/or TGF-α stimulation increased phospho-MEK and phospho-ERK in cells transfected with TNFR1 siRNA. Conversely, the effects of TNF-α and/or TGF-α on MSC VEGF production were significantly decreased, and MEK/ERK activation was negated in cells transfected TNFR2 siRNA.
TNFR2 plays a vital role in the effects of TNF-α and TGF-α on MSC VEGF production. The activation of MEK was implicated in this novel cross talk between TNFR2 and TGF-α–EGFR in regulating the production of VEGF in human MSCs.