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. 2020 Jun 16;11(24):2327–2344. doi: 10.18632/oncotarget.27634

Figure 5. Increase in phospho-p38 / total p38 MAPK follows decline in ARSB and activation of Wnt signaling.

Figure 5

(A) Exposure of the prostate stromal cells to the Wnt inhibitor IWP-2 blocked the increase in phospho-p38/total p38 MAPK ratio in the epithelial cells, when they were exposed to spent media from the stromal cells (SCM) in 1:1 combination with the epithelial cell media (p < 0.001, n = 3). (B) In the prostate epithelial cells, exposure to prostate stromal cell spent media (SCM; 1:1 with epithelial cell media), Wnt3A (1 ng/ml × 24 h), and PHPS1 (30 μM) × 24 h increased the phospho-p38 / total p38 MAPK ratio (p < 0.001; n = 3). Effects of exposure to the SCM and Wnt3A at this concentration were similar. (C) Addition of DKK-3 capture antibody (0.2 ug/ml × 24 h; AF1118) to epithelial cell media reduced the effect of DKK3 and increased Wnt signaling, leading to increased phospho-p38/total p38 MAPK ratio (p < 0.001; n = 3). (D) Exposure to mithramycin (250 nM x 24 h) or to SR11302 (5 μM × 24 h) had no effect on the increase in phospho-p38/total p38 MAPK. In contrast, SB23580 (10 μM × 24 h) totally blocked the increase (p < 0.001; n = 3). (E) JW67 (4 mg/ml × 24 h), an inhibitor of canonical Wnt signaling, and Y27632 (10 μM × 24 h), an inhibitor of Rho kinase, did not block the increases induced by prostate stromal cell spent media (SCM) and ARSB silencing. However, NSC23766 (10 μM × 24 h), which inhibits Rac-1 GTPase, blocked the increase in phospho-p38/total p38 MAPK, indicating a requirement for Rac-1 GTPase (p < 0.001; n = 3). (F) Similarly, in HepG2 cells, NSC23766, the inhibitor of Rac-1 GTPase, blocked the increase in phospho-p38/total p38 MAPK, which followed treatment with the prostate stromal cell spent media (SCM) and ARSB silencing (p < 0.001; n = 3). (G) Also, in PC-3 cells, NSC23766, the inhibitor of Rac-1 GTPase, blocked the increase in phospho-p38/total p38 MAPK induced by spent cell media (SCM) and ARSB silencing (p < 0.001; n = 3). [ARSB = arylsulfatase B; CHST = chondroitin sulfotransferase; DKK = Dickkopf Wnt signaling pathway inhibitor; JW67 = β-catenin nuclear translocation inhibitor; N.D. = no difference; NSC23766 = Rac-1 GTPase inhibitor; PHPS1 = SHP2 inhibitor = phenylhydrazonopyrazolone sulfonate; SB203580 = p38 MAPK inhibitor; SR11302 = AP-1 inhibitor; Y27632 = Rho kinase inhibitor; SCM = stromal cell spent media; si = siRNA]. *** for p < 0.001 greater than control; ### for p < 0.001less than epithelial control].