EGFR signaling mediates the chemotactic effect of PTH on mesenchymal progenitors.

<p>(A) Conditioned media from PTH-treated UMR 106-01 cells increased the phosphorylation of EGFR in mesenchymal progenitors. In this experiment, mesenchymal progenitors were treated with conditioned media for 5 min and then lysed for Western blot. (B) The enhanced phosphorylation of Akt and p38MAPK in mesenchymal progenitors by conditioned media from PTH-treated UMR106-01 cells is dependent on the EGFR pathway. Mesenchymal progenitors were pre-incubated with either DMSO or gefitinib (10 µM, GEF) for 30 min followed by addition of conditioned media to the culture. Cell lysates were collected 5 min later for Western blot analyses. (C) The EGFR inhibitor PD153035 (10 µM, PD) was added to both the upper and lower wells of the chemotaxis assay and partially blocked the PTH-induced chemotactic activity of conditioned media from UMR 106-01 cells. ***: p<0.001 vs. DMSO CON; &: p<0.01 vs. DMSO PTH. (D) An EGFR neutralizing antibody (4 µg/ml) was mixed with mesenchymal progenitors before the chemotaxis assay and suppressed the migration of mesenchymal progenitors towards conditioned media from PTH-treated UMR 106-01 cells. IgG: isotype control. **: p<0.01; ***: p<0.001 vs. CON; $: p<0.05; #: p<0.001 vs. PTH. (E) qRT-PCR demonstrates the knockdown of EGFR mRNA levels in mesenchymal progenitors by siRNAs. ***: p<0.001 vs. MOCK. (F) Immunoblotting reveals that the EGFR protein level was dramatically decreased in mesenchymal progenitors transfected with siRNAs for EGFR. (G) Blocking of EGFR expression in mesenchymal progenitors by siRNAs abolished the chemotactic migration of these cells toward conditioned media from PTH-treated UMR 106-01 cells. ***: p<0.001 vs. mock CON; #: p<0.001 vs. mock PTH.