The aggregate findings suggest that Notch signaling interfaces with fibrogenic signals that are transduced by TGF-β and the Hh pathway in multipotent liver progenitor cells. This is particularly intriguing because both TGF-β and Hh signaling promote epithelial-to-mesenchymal transitions in developing embryos,[34] and Hh has been proven to stimulate epithelial-to-mesenchymal–like transitions in both adult HSCs and progenitor cells.[8,

35] Having confirmed that DAPT performed as anticipated in Notch-responsive liver progenitor cells, we evaluated its actions in HSCs. For these studies, primary murine HSCs were cultured for 4 days to induce MF transdifferentiation and then treated with DAPT for an additional 3 days. As in 603B cells (Fig. 4), MFs/HSCs showed DAPT-inhibited expression of Notch-2, Jagged-1, and several Notch target

gene (Hey1, XL184 cost Hey2, and HeyL) mRNAs (Fig. http://www.selleckchem.com/products/pf-06463922.html 5A). IHC confirmed that mRNA suppression was accompanied by decreased protein expression (Fig. 5E). Blocking Notch signaling in MFs/HSCs also repressed typical MF-associated genes (α-SMA, collagen, and TGF-β) and Hh target genes that are known to be expressed by MFs/HSCs (Gli2, Ptc, and Sonic Hedgehog [Shh]; Fig. 5B). In contrast, mRNA levels of various epithelial genes (bone morphogenic protein-7, desmoplakin, E-cadherin, AFP, HNF-4α, and Krt19) and Q-HSC markers (peroxisome proliferator-activated receptor gamma [PPAR-γ] and GFAP) were up-regulated (Fig. 5C). Immunocytochemistry confirmed the DAPT-induced reversion of MFs/HSCs to a more quiescent phenotype, showing decreased staining for α-SMA and Ki67 (proliferation marker) and increased Oil Red O staining, indicative of neutral lipid accumulation (Fig. 5F). Interestingly, when Notch signaling was inhibited and MFs/HSCs reverted to a more quiescent phenotype, mRNA expression of delta-like 1 homolog, a Notch-related gene that marks liver progenitors,[36] and mRNAs

encoding other progenitor cell markers (e.g., Nanog, octamer-binding transcription factor 4 [Oct4], and FN14) were down-regulated (Fig. 5D). selleck chemicals llc Thus, Notch signaling is activated during culture-induced primary MF/HSC transdifferentiation, and this permits the cells to acquire a more mesenchymal phenotype with progenitor-like features. This process parallels activation-associated induction of Hh signaling and might be regulated by cross-talk between the Notch and Hh pathways, because HSCs require Hh signaling to become MFs.[8, 31] To further examine possible cross-talk between Notch and Hh signaling, the two Notch-responsive cell types (603B and primary MFs/HSCs) were treated with an Hh-signaling antagonist (GDC-0449). GDC-0449 directly interacts with and inhibits the Hh coreceptor, Smoothened.[37] Earlier work has proven that GDC-0449 recapitulates the effect of Smoothened gene knockdown in MFs/HSCs, with both approaches inhibiting canonical Hh signaling, thereby blocking the nuclear localization and transcriptional activation of Gli DNA-binding proteins.