4A). PDGF-BB significantly induced the translocation of SMO from intracellular compartments to the plasma membrane (arrows; Fig 4A, middle). This process appears to be PKA dependent, because it was effectively attenuated by the PKA inhibitor, H-89. Similar results were obtained when we employed KMCH-1 cells transiently transfected with a plasmid expressing GFP-tagged human SMO and analyzed GFP-SMO localized at the plasma membrane by TIRF microscopy26 (Fig. 4B). Moreover, PDGF-BB derived from cocultured LX-2 cells also induced SMO trafficking, as assessed by TIRF microscopy (Supporting Fig. 4A). As a further indicator for Hh-signaling activation,

we examined the effect of PDGF-BB on GLI2 nuclear translocation in KMCH-1 cells by immunoblotting analysis (Fig 4C). PDGF-BB treatment increased click here GLI2 abundance in nuclear protein

extracts, an effect that, again, was attenuated by the PKA inhibitor, H-89. Consistent with these results, KMCH-1 cells transiently transfected with a GLI reporter construct displayed GLI activation upon PDGF-BB treatment. The SMO inhibitor, cyclopamine, effectively blocked PDGF-BB-mediated GLI activation (Fig. 4D, upper). Likewise, stable scrambled KMCH cells also displayed PDGF-BB-induced GLI activation, whereas no PDGF-BB effect was observed in shSMO KMCH-1 cells (Fig. 4D, lower). Because PKA function is cAMP dependent, 37 we measured the effect of PDGF-BB on intracellular cAMP Resveratrol levels (Supporting Fig. 4B). Indeed, PDGF-BB-treated MLN0128 order cells rapidly displayed significant increases of cAMP levels, as compared to controls, an effect that was blocked by the PDGFR(-β)

inhibitor, imatinib. Thus, PDGF-BB appears to promote Hh-signaling–dependent cytoprotection by inducing cAMP/PKA-mediated SMO trafficking to the plasma membrane. To further confirm the PDGF-BB-stimulated, SMO-dependent gene regulation, we identified 67 target genes to be commonly up-regulated (50 genes) or down-regulated (17 genes) by both SHH and PDGF-BB in a cyclopamine-dependent manner in KMCH-1 cells via an Affymetrix U133 Plus 2.0 GeneChip analysis (Table 1). To determine whether the proapoptotic in vitro effect of Hh-signaling inhibition by cyclopamine observed in cocultures would be translatable to an in vivo model, we employed a syngeneic rat orthotopic CCA model (BDEneu malignant cells injected into the liver of male Fischer 344 rats). Like human CCA, the BDEneu cells also express TRAIL in vivo.28-30 We confirmed that BDEneu cells express mRNA of members of the Hh-signaling pathway (i.e., SHH, IHH, DHH, PTCH1, SMO, and GLI 1-3) (Supporting Fig. 5). This rodent model of CCA also duplicates the desmoplasia characteristic of the human disease, with numerous α-SMA-positive MFBs present in the stromal tumor microenvironment (Fig. 5A).