Role for Hedgehog signaling in hepatic stellate cell activation and viability

Abstract

Hepatic stellate cells (HSC) have a complex phenotype that includes both neural and myofibroblastic features. The Hedgehog (Hh) pathway has been shown to direct the fate of neural and myofibroblastic cells during embryogenesis and during tissue remodeling in adults. Therefore, we hypothesized that Hh signaling may regulate the fate of HSC in adults. In this study, we find that freshly isolated stellate cells from adult PatchedlacZ transgenic mice exhibit b-galactosidase activity, indicating Hh pathway activity. Transcripts of Hh ligands, the Hh pathway receptor, and Hh-regulated transcription factors are expressed by stellate cells from mice, rats, and humans. Transfection experiments in a cell line using a Hh-inducible luciferase reporter demonstrate constitutive Hh pathway activity. Moreover, neutralizing antibodies to Hh increase apoptosis, while viability is restored by treatment with Hh ligand. In vitro treatment of primary stellate cells with cyclopamine (Cyc), a pharmacologic inhibitor of the Hh pathway, inhibits activation and slightly decreases cell survival, while a single injection of Cyc into healthy adult mice reduces activation of HSC by more than 50% without producing obvious liver damage. Our findings reveal a novel mechanism, namely the Hh pathway, that regulates the activation and viability of HSC.

Sonic hedgehog is an autocrine viability factor for myofibroblastic hepatic stellate cells

“…Adding Shh neutralizing antibodies to primary rat HSC increased their inherent caspase activity. These findings extend and confirm data derived from earlier studies of clonal HSC lines, in which Shh antibodies increased cellular apoptosis and recombinant Shh stimulated growth [29]. The present study also demonstrates that proliferative actions of PDGF-BB, a major HSC mitogen [45,46], are mediated via the Hh pathway because three distinct approaches that abrogate Hh signaling each significantly inhibited PDGF-related increases in HSC incorporation of BrdU.…”

“…Although Hh ligands are not known to regulate liver development during embryogenesis [24], Hh pathway activity has been demonstrated in some cholangiocarcinomas [25] and hepatocellular carcinomas [26][27][28]. We reported that clonal HSC lines derived from cirrhotic rat liver express mRNA for Hh ligands [29]. However, the significance of this finding is uncertain.…”

“…Apoptotic activity was assayed in parallel using the Apo-ONE Homogeneous Caspase 3/7 Apoptosis Assay (Promega, Madison, WI), according to the vendor's instructions [29]. A FLUOstar OPTIMA microplate reader (BMG Labtech; Durham, NC) was used for all absorbance, luminescence and fluorescence measurements…”

“…To determine the bioactivity of Shh in HSC culture medium, NIH 3T3 cells in 12-well plates (70,000 cells/well) were transiently transfected with a 9x-Gli-BS-Luciferase reporter construct that contained nine Gli-binding sites upstream of a luciferase reporter driven by a thymidine kinase promoter (provided by W. Chen, Duke U [29]), using Fugene 6 (Roche, Indianapolis, IN). HSC conditioned medium were add to transfected cells 24 later.…”

The niche of stellate cells within rat liver

“…9 Moreover, HSCs possess signaling pathways required for stem cell functions such as hedgehog and ␤-catenin-dependent Wnt signaling. 10,11 Similar to hematopoietic stem cells, quiescent HSCs are resistant against CD95-mediated apoptosis and proliferate in response to CD95 ligand. [12][13][14][15] Thus, there is growing evidence that stellate cells are stem/progenitor cells of the vertebrate liver.…”