Smoothened is a master regulator of adult liver repair
Gregory A. Michelotti, Guanhua Xie, Marzena Swiderska, Steve S. Choi, Gamze Karaca, Leandi Krüger, Richard Premont, Liu Yang, Wing-Kin Syn, Daniel Metzger, Anna Mae Diehl
Gregory A. Michelotti, Guanhua Xie, Marzena Swiderska, Steve S. Choi, Gamze Karaca, Leandi Krüger, Richard Premont, Liu Yang, Wing-Kin Syn, Daniel Metzger, Anna Mae Diehl
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Research Article Hepatology

Smoothened is a master regulator of adult liver repair

Abstract

When regenerative processes cannot keep pace with cell death, functional epithelia are replaced by scar. Scarring is characterized by both excessive accumulation of fibrous matrix and persistent outgrowth of cell types that accumulate transiently during successful wound healing, including myofibroblasts (MFs) and progenitors. This suggests that signaling that normally directs these cells to repair injured epithelia is deregulated. To evaluate this possibility, we examined liver repair during different types of liver injury after Smoothened (SMO), an obligate intermediate in the Hedgehog (Hh) signaling pathway, was conditionally deleted in cells expressing the MF-associated gene, αSMA. Surprisingly, blocking canonical Hh signaling in MFs not only inhibited liver fibrosis but also prevented accumulation of liver progenitors. Hh-sensitive, hepatic stellate cells (HSCs) were identified as the source of both MFs and progenitors by lineage-tracing studies in 3 other strains of mice, coupled with analysis of highly pure HSC preparations using flow cytometry, immunofluorescence confocal microscopy, RT-PCR, and in situ hybridization. The results identify SMO as a master regulator of hepatic epithelial regeneration based on its ability to promote mesenchymal-to-epithelial transitions in a subpopulation of HSC-derived MFs with features of multipotent progenitors.

Authors

Gregory A. Michelotti, Guanhua Xie, Marzena Swiderska, Steve S. Choi, Gamze Karaca, Leandi Krüger, Richard Premont, Liu Yang, Wing-Kin Syn, Daniel Metzger, Anna Mae Diehl

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Figure 6

Epithelial and stromal cells are progeny of cells that express markers of HSCs.

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Epithelial and stromal cells are progeny of cells that express markers o...
(A) αSMA-Cre-ERT2 x ROSA-Stop-flox-YFP mice were subjected to sham surgery or 14-day BDL and treated with either vehicle or TMX. Immunohistochemistry for YFP was performed, and representative images are shown (original magnification, ×40 [i–iv]). Boxed regions show increased magnification of representative YFP staining of ductular cells in periportal areas (v; arrows) and hepatocytic and stromal cells in midzonal areas (vi; black and red arrows, respectively; original magnification, ×100 [v and vi]). Vehicle-treated controls are also shown (ii and iii, respectively). (B) Wild-type, ROSA-Stop-flox-YFP (no Cre), and αSMA-Cre-ERT2/ROSA-Stop-flox-YFP (DTG-YFP) mice were subjected to sham surgery or BDL and treated with vehicle or TMX. Primary hepatocytes were isolated 14 days after BDL and analyzed for direct YFP fluorescence by flow cytometry. The percentages of YFP-positive cells relative to WT control are indicated. (C) Hepatocytes were isolated from ROSA-Stop-flox-YFP and DTG-YFP mice; DNA was analyzed by PCR to examine rearrangement of the ROSA26 locus (gray triangles depict LoxP sites). A common forward primer was used with 2 downstream primers specific for the indicated regions (PGK Neo, EYFP; Supplemental Table 3). Cre-mediated recombination was quantified as described in Figure 1 and shown below each lane. (D) Lineage tracing was also performed in GFAP-Cre-ERTM x ROSA-Stop-flox-YFP mice subjected to 14-day BDL injury and treated with vehicle or TMX. Immunohistochemistry for hepatic YFP was performed, and representative images are shown of YFP+ cells in ductular (arrowhead), stromal (red arrow), and hepatocytic (black arrows) regions (original magnification, ×40 [top]; ×100 [bottom]).