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Smoothened is a master regulator of adult liver repair
Gregory A. Michelotti, … , Daniel Metzger, Anna Mae Diehl
Gregory A. Michelotti, … , Daniel Metzger, Anna Mae Diehl
Published April 8, 2013
Citation Information: J Clin Invest. 2013;123(6):2380-2394. https://doi.org/10.1172/JCI66904.
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Research Article Hepatology

Smoothened is a master regulator of adult liver repair

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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 1

Conditional disruption of SMO in αSMA+ cells inhibits Hh signaling in MFs.

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Conditional disruption of SMO in αSMA+ cells inhibits Hh signaling in MF...
(A) HSCs were isolated from Smo-flox mice. AdGFP or AdCre (MOI 25) were added on culture day 4, and cells were harvested 3 days later. Effects on gene expression were assessed by qRT-PCR. Results are normalized to day 0 HSCs. *P < 0.05. Inset shows representative Western blot for expression of Cre and tubulin. (B) Oligonucleotides for the floxed SMO allele (refs. 56, 63; Supplemental Table 3) were used to distinguish the unrearranged allele (Smoc; 1.7 Kb) from the Cre-recombined null allele (Smon; 0.5 Kb). Genomic DNA was isolated from livers of DTG mice treated either with vehicle (VEH, n = 3) or TMX (n = 4) from day 4–10 after BDL, and Smon and Smoc amplicons were amplified. Quantification of the amplicons was performed by densitometry to assess the efficiency of Cre-mediated recombination. Results are expressed as Smon/Smon + Smoc x 100, shown below each lane. (C) Total RNA was isolated from livers of vehicle- and TMX-treated DTG mice 14 days after sham surgery or BDL. SMO expression was assessed by qRT-PCR, and results are expressed as fold over sham plus vehicle control. *P < 0.05. (D) Representative immunohistochemistry for hepatic GLI2. GLI2+ cells in vehicle-treated and TMX-treated DTG mice were quantified in sham and BDL groups and represented as number of positive cells per field (n = 11/group; original magnification, ×20 [Sham]; ×63 [BDL]). *P < 0.05, **P < 0.01. (E) Total RNA was isolated from livers of vehicle- and TMX-treated DTG mice 14 days after BDL and analyzed by qRT-PCR for expression of Hh-regulated genes. Results are expressed as fold over vehicle-treated group. *P < 0.05.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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