Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease

Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (gt;99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMOdependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness

Widespread GLI expression in human donor and cirrhotic liver.

<p><b>(A)</b> Frozen (4 μm) human donor (<i>n</i> = 5), and cirrhotic liver sections [ALD (<i>n</i> = 6), NASH (<i>n</i> = 3), PBC (n = 1)] were screened for GLI2 (red) expression by immunofluorescence. Representative images taken at 5x or 40x (insets) objective shown. DAPI, blue. <b>(B)</b> qRT-PCR for <i>GLI1</i> and <i>GLI3</i> transcript in human donor or ALD samples. Mean±S.E.M. Significant (*) difference between means (One-sided student t-test, **<i>p</i><0.005). Western blot for full-length GLI1 protein (>150 kDa) in donor (Don) or ALD patient samples. Densitometry analysis with GLI1 normalised to GAPDH (Image J). Mean±S.E.M; **<i>p</i> = 0.0093 (Two-sided student t-test). <b>(C)</b> Nuclear GLI2 (green) expression in EpCAM⁺ (red) LPCs in donor, ALD, PBC and NASH liver. <b>(D)</b> Nuclear GLI2 (green) expression demonstrated within CD31⁺ (red) ECs, CK18⁺ (red) hepatocytes, CD45⁺ (red) leukocytes and vimentin⁺ (red) HSCs/myofibroblasts, in ALD. 63x objective. <b>(E)</b> Maximum intensity projection illustrating close physical association between EpCAM⁺ LPCs (green) and vimentin⁺ HSCs/myofibroblasts (red), both of which express GLI2 (grey), in ALD tissue. Arrows indicate myofibroblasts directly contacting LPCs. Confocal microscopy, 63x objective. Quantitation (%) of EpCAM⁺ GLI2⁺ cells and vimentin⁺ GLI2⁺ cells within the same FOV (<i>n</i> = 3 ALD samples, 8 FOV/sample).</p

Changes to transcript expression in two liver progenitor cell lines following GLI inhibition.

<p>Heat map representing changes to gene transcript levels in BMOL1.2 (<i>n</i> = 3) or BMOL-TAT (<i>n</i> = 3) lines following GANT61 (10 μM) 8 h treatment. Log₂ intensity scale shown. Downregulated genes (blue) are shown above the yellow dashed line, with upregulated genes (red) below. Grey indicates no data.</p

A very minimal population of human vimentin⁺ HSCs/myofibroblasts express a primary cilium, with none detected on CD31⁺ endothelial cells.

<p>Human ALD liver tissue was examined for the expression of primary cilia (α-acetylated tubulin, green; γ-tubulin, red) by vimentin⁺ (grey) HSCs/myofibroblasts <b>(A)</b> or CD31+ (grey) ECs <b>(C)</b>. <b>(A)</b> The majority of vimentin⁺ cells were Pc^-ve in the tissues examined. Representative image shown, displaying absence of Pc on vimentin⁺ cells. To confirm this result, ciliary protein Arl13b (green) was co-stained with vimentin (grey). Rare Arl13b ciliary structures (arrow) co-localised with vimentin⁺ cells. Final panel in A illustrates rare Pc⁺ (α-acetylated tubulin, green; γ-tubulin, red) vimentin⁺ (grey) HSCs/myofibroblasts, at the cirrhotic interface. <b>(B)</b> Number of vimentin⁺ Pc⁺ cells or vimentin⁺ Pc^neg cells per FOV (<i>n</i> = 3 ALD samples, 8 FOV/sample). <b>(C)</b> No Pc were detected on CD31⁺ cells in the tissues examined (ALD <i>n</i> = 3, 8 FOV/sample). Representative image shown. All images obtained using confocal microscopy, 63x objective. DAPI, blue. White arrows illustrate Pc. * Non-specific liver autofluorescence.</p

Gene changes in BMOL1.2 cells following GANT61 treatment.

<p>Gene changes in BMOL1.2 cells following GANT61 treatment.</p

Inhibition of GLI signaling reduces the viability of liver progenitor cell lines.

<p>LPC lines BMOL1.2 <b>(A, C, E, G)</b> and BMOL-TAT <b>(B, D, F, H)</b> were grown over 72 h in the presence of signaling inhibitors for GLI (GANT61; <b>A, B</b>), Notch (DAPT; <b>C,D</b>) c-Met (SGX523; <b>E, F</b>), Wnt (XAV939; <b>G, H</b>) signaling and matching [DMSO]. Viability was determined by ALAMAR Blue assays, and an interaction between time and dose was assessed using two-way ANOVA followed by Tukey’s multiple comparisons test (α = 0.001) (Prism, GraphPad). **** <i>p</i><0.001.</p

Common gene changes in BMOL1.2 and BMOL-TAT cells following GANT61 treatment.

<p>Common gene changes in BMOL1.2 and BMOL-TAT cells following GANT61 treatment.</p

Gene changes in BMOL-TAT cells following GANT61 treatment.

<p>Gene changes in BMOL-TAT cells following GANT61 treatment.</p