Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling

Figure 1 CXCL5 chemokine induction in human mesenchymal stem cells treated with osteogenic medium or non-canonical Wnt5a. A: CXCL5 mRNA levels are induced at 7 d in complete osteogenic medium or medium containing 0.1 μmol/L dexamethasone. Conditioned medium containing non-canonical Wnt5a induces (B) mRNA expression and (C) protein secretion. All values are mean ± SD. ^aP < 0.05; ^bP < 0.01 vs HMSCGM-treated group. HMSCGM: Human mesenchymal stem cell growth medium; DEX: Dexamethasone; OGM: Osteogenic medium; L-cell: Conditioned medium from L-cells; Wnt5a: Conditioned medium from L-cells expressing Wnt5a.

Figure 2 Secreted frizzled-related protein-stimulated expression of CXCL5 and CXCL8 in human mesenchymal stem cells. A: sFRP1, an inhibitor of canonical and non-canonical Wnt signaling, induces expression of CXCL5 mRNA. Dkk1, an inhibitor of canonical Wnt signaling does not induce CXCL5; B: All four of the sFRPs stimulated CXCL5 protein secretion as determined by ELISA analysis of cell supernatants. sFRP1 induces expression of CXCL8 mRNA in a dose- (C) and time- (D) dependent manner with maximum expression at 150 ng/mL and 7 d post-treatment. All values are mean ± SD. ^aP < 0.05; ^bP < 0.01 vs HMSCGM-treated group (A, B), vs untreated (C), or 3-d CXCL8 mRNA levels (D). HMSCGM: Human mesenchymal stem cell growth medium; sFRP: Secreted frizzled-related protein; Dkk1: Dickkopf-related protein 1.

Figure 3 Phosphorylated state of mitogen-activated protein kinase p44/42 extracellular-signal-regulated kinases in response to secreted frizzled-related protein 1 stimulation. Western Blot time-course analysis of pan and phosphorylated states of the MAPK p44/42 ERK proteins (A); The ratio of phospho/pan p44/42 ERK reaches a maximum at 5 min in the presence of OGM and sFRP1 (B); whereas, in OGM alone the maximal ratio level is not reached until 10 min post stimulation (representative experiment). sFRP1: Secreted frizzled-related protein 1; MAPK: Mitogen-activated protein kinase; ERK: Extracellular-signal-regulated kinases; OGM: Osteogenic medium.

Figure 4 Modulation of secreted frizzled-related protein-stimulated induction of CXCL8 mRNA. sFRP1/2-stimulated CXCL8 mRNA expression is suppressed in the presence of the PLC inhibitor U73122 suggesting that signaling is via the Wnt/calcium non-canonical pathway (values are mean ± SD). ^aP < 0.05 vs HMSCGM-treated levels. sFRP1/2: Secreted frizzled-related protein 1/2; PLC: Phospholipase C.

Figure 5 Effect of siRNA directed to frizzled receptors or receptor tyrosine kinase-like orphan receptor-2 on secreted frizzled-related protein-stimulated CXCL8 mRNA levels. sFRP-stimulated CXCL8 mRNA expression is inhibited in the presence of siRNAs directed toward non-canonical (A) Fzd2 and (B) Fzd5 or (C) the non-canonical Frizzled co-receptor RoR2. All values are mean ± SD. ^aP < 0.05 vs untreated siControl levels for each graph. sFRP1-3: Secreted frizzled-related protein 1-3; siFzd2/5: siRNA to the non-canonical frizzled receptor 2/5; RoR2: Receptor tyrosine kinase-like orphan receptor-2; siRoR2: siRNA to RoR2; siControl: Scrambled control siRNA.