The response to DDC liver injury is blunted in Irs2^−/− mice

We examined how Irs2 deletion affected the chronic liver-injury response in adult mice during 0.1% DDC feeding, in which paracrine Fgf7 signaling plays a central role in coordinating the LPC response and epithelial repair (Fig 1A). Transient induction of Irs2 occurred in wild-type (WT) livers on day 7 of the DDC diet (Fig 1B), correlating with a peak in liver injury as judged by serum aspartate/alanine transaminase (AST/ALT) activity (Fig 1C). No differences in serum bilirubin or liver tissue bile acid levels were observed between the two groups (Fig 1D), and initial induction of AST/ALT was comparable on day 7, suggesting the cholestatic effects of the DDC diet were equivalent in WT and Irs2^−/− mice. However, past day 7, serum transaminases were reduced in controls (days 14–21), whereas in the Irs2^−/− mice, they remained significantly elevated (Fig 1C), suggesting a role for Irs2 in the attenuation of liver damage.

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Fig 1. Irs2 is required for the amelioration of liver injury during DDC feeding.

(A) Schematic showing DDC feeding protocol (upper panel) and the cellular impact of chronic DDC feeding on the liver (lower panel): Hepatocellular damage is accompanied by expansion of bipotent LPCs and fibrotic stroma in periportal tracts, where cells are exposed to high concentrations of insulin. Portal vein (P), Hepatic artery (A), and Central vein (C). (B) Whole-liver mRNA levels of Irs2 in WT and Irs2^−/− mice during a time course of DDC feeding based on RT-qPCR analysis (n = 3–8). Expression data are presented in RUs normalized to ribosomal protein L19 (Rpl19). (C) Liver injury measured by serum transaminase levels AST/ALT during a time course of DDC feeding in WT and Irs2^−/− mice (n = 4). (D) Equivalent levels of cholestasis in WT and Irs2-deficient mice during DDC injury. Left—serum levels of TBIL during a time course of DDC feeding (n = 3). Right—Whole-liver tissue bile acid levels prior to (Cont) and after 21 days of DDC feeding (DDC) (n = 4). Data information: underlying data are available in S1 Data. Values plotted as mean + SEM: Two-way ANOVA was used to compare means. Significance P values were calculated using Bonferroni test *P < 0.05, **P <0.01, and ***P < 0.001. Dotted lines indicate statistically significant decrease with time. ALT, alanine transaminase; AST, aspartate transaminase; Cont, control; DDC, 3.5-diethoxycarbonyl-1.4-dihydrocollidine; Fgf7, fibroblast growth factor 7; Irs2, insulin receptor substrate 2; LPC, liver progenitor cell; mRNA, messenger RNA; RT-qPCR, reverse transcriptase-quantitative PCR; RU, relative unit; TBIL, total bilirubin; WT, wild type.

https://doi.org/10.1371/journal.pbio.2006972.g001

Fgf7 expression and LPC expansion are impaired in Irs2^−/− mice

We observed robust Fgf7 activation in WT livers (days 14–21), which coincided with the significant reductions in AST/ALT recorded at these time points (Figs 2A and 1B), consistent with the proregenerative role played by Fgf7 in the response to DDC injury. Fgf7-expressing stromal cells surrounded duct-like structures in periportal tracts that stained positive for LPC markers including epithelial cell adhesion molecule (EpCAM) and osteopontin (secreted phosphoprotein 1, Spp1) (Fig 2B). In Irs2^−/− mice, we observed a striking failure to induce Fgf7 during DDC feeding (Fig 2A). We also observed modest but significant down-regulation of Fgf10 but no change in Fgf22 (S1 Fig), suggesting other stromal Fgfr2-IIIb ligands were also affected. Failure to induce Fgf7 expression in Irs2^−/− livers occurred in parallel with a delay in the overall increase in hepatocyte nuclear factor 4-alpha (HNF4α)− NPCs (S2A and S2B Fig), as well as a specific delay in the induction of LPC genes EpCAM and osteopontin/Spp1 (Fig 2A) and decreased ductular immunostaining in periportal areas (Fig 2B). Proliferative expansion of cells expressing osteopontin/Spp1 was also diminished (day 14), as judged by Ki67 immunostaining (Fig 2C), and ducts containing LPCs appeared disorganized (Fig 2B).

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Fig 2. Fgf7 induction and the LPC response are impaired in Irs2^−/− mice during DDC injury.

(A) RT-qPCR analysis of indicated genes in whole-liver mRNA from WT and Irs2^−/− mice during a time course of DDC feeding (n = 6–8). (B) Immunofluorescence staining for Fgf7-expressing stroma and EpCAM-Osteopontin/Spp1-expressing LPC ducts in livers of WT and Irs2^−/− mice after 14 days of DDC feeding. Dotted lines indicate portal vein (H = Hoechst). (C) Quantification of LPC proliferation in livers of WT and Irs2^−/− mice on DDC day 14. (left) Spp1/Ki67 immunofluorescence staining highlighting proliferation of cells within ducts (arrow heads). Dotted lines indicate portal vein. (Right) Quantification by INcell analysis of Spp1 cell proliferation (n = 6, total of 6.47 × 10⁴ Spp+ cells analyzed). Data information: underlying data are available in S1 Data. Values plotted as mean + SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. (A) Two-way ANOVA was used to compare means. Significance P values were calculated using Tukey's multiple comparison test. (C) Unpaired Student t test. DDC, 3.5-diethoxycarbonyl-1.4-dihydrocollidine; EpCAM, epithelial cell adhesion molecule; Fgf7, fibroblast growth factor 7; Irs2, insulin receptor substrate 2; LPC, liver progenitor cell; mRNA, messenger RNA; RT-qPCR, reverse transcriptase-quantitative PCR; RU, relative unit; Spp1, secreted phosphoprotein 1; WT, wild type.

https://doi.org/10.1371/journal.pbio.2006972.g002

Parenchymal cell depletion was exacerbated in Irs2^−/− mice during DDC feeding, based on quantification of HNF4α+ hepatocyte nuclei (S2A and S2C Fig). This was characterized by failure to sustain numbers of so-called "small" hepatocytes (nuclear area < 75 μm²), parenchymal cells attributed with the greatest regenerative potential (S2D Fig) [26,27]. Using an original methodology to estimate ploidy (described in Materials and methods), we analyzed changes in hepatocyte DNA content in whole-liver tissue sections during DDC injury (S3A and S3B Fig). We found that the population of small hepatocytes with approximately 2n DNA content (2c) was increased in the WT group during the later stages of DDC injury (days 14–21, 1.6-fold P = 0.0335), consistent with a regenerative response (S3C Fig). During the same period, 2c hepatocytes declined in numbers in the livers of Irs2^−/− mice, indicating a failure to maintain parenchymal tissue homeostasis during chronic injury.

Altered profile of stromal activation in Irs2^−/− mice highlights aberrant response by Fgf7-expressing mesenchymal cells

In rodent and human livers, Fgf7 is expressed by HSCs during injury [18]. However, in the DDC mouse model, resident Thy1+ PFs have also been identified as important Fgf7-expressing mesenchymal cells and first responders to cholestatic liver damage [4,19]. Profiling of stromal gene expression in WT mice during DDC injury revealed sustained induction of PF/myofibroblast genes Thy1, Elastin, Vimentin, and alpha-smooth muscle actin (αSma/Acta2), together with profibrogenic gene Connective tissue growth factor (Ctgf) (Fig 3A), whereas in Irs2^−/− mice, the activation profile of these genes was either blunted (Thy1, Elastin), less sustained (Acta2), or down-regulated from day 7 (Vimentin, Ctgf, and Thy1), suggesting a negative impact on the PF/myofibroblast population within the activated stroma. Interestingly, we also observed a decline in expression of the HSC marker glial fibrillary acidic protein (Gfap) from day 7 (Fig 3A), coincident with significant reductions in mesenchymal marker Vimentin and in Ctgf, a regulator of epithelial mesenchymal transition. From day 7, a significant decrease in Gfap immunostaining in Irs2^−/− livers was also observed (Fig 3B and 3C). This reduction in Gfap+ cells was apparent throughout the parenchyma and in periportal areas where it colocalized with Fgf7-expressing stroma (Fig 3C).

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Fig 3. The stromal response to DDC injury in Irs2^−/− mice is characterized by loss of HSCs and blunted activation of PF/myofibroblast gene expression.

(A) RT-qPCR analysis of whole-liver mRNA during a time course of DDC injury using a panel of genes associated with Fgf7-expressing stromal cells: PFs (Thy1, Elastin), myofibroblasts (Acta2, Vimentin, Ctgf), and HSCs (Gfap). In livers of Irs2^−/− mice, we observed impaired or less sustained induction of PF/myofibroblast markers (Thy1, Elastin, Acta1) combined with a tendency for all genes (except Elastin) to be down-regulated from day 7 in parallel with loss of Gfap (n = 3–8). (B) Gfap+ HSCs were depleted in Irs2^−/− mice during DDC injury. Quantification of Gfap immunostaining in the livers of WT and Irs2^−/− mice during the time course of DDC injury (n = 3–4). (C) Loss of Gfap+ HSCs in Irs2^−/− mice parallels the failure to induce Fgf7 in periportal areas. Confocal immunofluorescence images showing reduced Gfap/Fgf7 double staining in Irs2^−/− mice after 14 days of DDC feeding (day 14). Data information: underlying data are available in S1 Data. Data are presented as mean + SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. (A) Two-way ANOVA was used to compare means. Significance P values were calculated using Tukey's multiple comparison test. (B) Unpaired Student t test. Acta1, alpha-smooth muscle actin; Ctgf, Connective tissue growth factor; DDC, 3.5-diethoxycarbonyl-1.4-dihydrocollidine; Fgf7, fibroblast growth factor 7; Gfap, glial fibrillary acidic protein; HSC, hepatic stellate cell; Irs2, insulin receptor substrate 2; mRNA, messenger RNA; PF, portal fibroblast; RT-qPCR, reverse transcriptase-quantitative PCR; RU, relative unit; WT, wild type.

https://doi.org/10.1371/journal.pbio.2006972.g003

In contrast with the results of messenger RNA (mRNA) analysis (Fig 3A), immunostaining for Thy1, Elastin, and αSMA tended towards increased expression in Irs2^−/− livers on day 21, while mesenchymal intermediate filament protein vimentin was similarly induced (S4 Fig). Thy1+ stroma were observed in close contact with Spp1+/Epcam+ LPCs in both WT and Irs2^−/− mice as previously described [19] (S5A Fig), and a similar relationship between αSma-expressing myofibroblasts and Spp1+ LPCs was also apparent in both groups (S5B Fig), indicating that failure to induce Fgf7 was not due to the lack of myofibroblast-like cells in the LPC niche of Irs2^−/− livers per se. In contrast, lack of contact between Gfap+ HSCs and LPCs was observed in Irs2^−/− livers (S5B Fig). Hence, failure to induce Fgf7 coincided with an overall loss of Gfap+ HSCs and loss of association between HSCs and LPCs in portal tracts, whereas we were unable to confirm that stromal PFs were negatively influenced by Irs2 deletion. However, increased Thy1 staining in Irs2^−/− livers was partially attributable to the increase in markers of bone-marrow–derived cells; cd45/protein tyrosine phosphatase receptor type C gene encoding CD45 cell surface antigen (Ptprc) and cd117/Proto-oncogene c-kit (Kit), on day 7, which coincided with early activation of matrix remodeling factors (tissue inhibitor of metalloproteinase 1 [Timp1] and Matrix metallopeptidase 9 [Mmp9]) and MYC proto-oncogene (cMyc) and a dramatic early peak in transforming growth factor beta (Tgfβ)—a master regulator of fibrogenesis and mobilizer of circulating populations of collagen-expressing fibrocytes [28] (S6A Fig). Consistent with this, we also observed greater Thy1/Cd45 colocalization in Irs2^−/− livers on day 21 (S6B Fig), suggesting more extensive incorporation of bone-marrow–derived cells into the stromal niche. Furthermore, the numbers of Cd3+ T cells were also increased in Irs2^−/− livers on day 21 (S6C Fig), suggesting that Thy1+ cells from the bone marrow, whose expression of Fgf7 is lower than that of resident Thy1+ fibroblast populations [4], played a more significant role in the altered stromal response to DDC injury in Irs2^−/− mice.

Survival of Fgf7-expressing HSCs is Irs2 dependent

We assessed the survival of Fgf7-expressing stroma during the early phase of DDC injury, when Irs2 was maximally expressed in WT livers (day 7) and before significant loss of Gfap+ HSCs was observed. Analysis of cleaved caspase 3 (c-Casp3) staining revealed a 3.0-fold (P = 0.0395) increase in apoptosis in Fgf7-expressing cells in Irs2^−/− livers at this time, confirming accelerated loss of this subpopulation within the fibrotic stroma during injury (Fig 4A). In order to test the hypothesis that IRS2 promoted survival of Fgf7-expressing stroma, we performed stable knockdown of IRS2 in the human HSC (hHSC) cell line LX-2 using lentiviral short hairpin RNA (shRNA) (Fig 4B and 4C). Silencing of IRS2 had no direct impact on LX-2 cell viability, Fgf7 expression, or fibrogenic gene expression under standard culture conditions (S7 Fig). However, exposure to cytotoxic alkylating agent mitomycin C (MitoC) resulted in cell-cycle arrest and apoptotic cell death, during which IRS2 promoted survival (Fig 4B) by significantly reducing P53 expression and caspase 3 cleavage (Fig 4C). We confirmed a positive role for IRS2 in protecting HSCs from apoptosis by treating primary hHSC cultures with an allosteric inhibitor of IRS proteins (NT-157). NT-157 triggers proteasomal depletion of IRS1/IRS2 [29], which resulted in dramatic activation of P53/caspase 3 cleavage and apoptosis in hHSCs (Fig 4D). We therefore concluded that IRS2 plays an indirect role in driving Fgf7 expression during DDC injury by protecting HSCs from apoptosis.

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Fig 4. Irs2 depletion leads to reduced survival of Fgf7-expressing stroma and human HSCs.

(A) Representative immunofluorescence images and graphical quantification of cleaved caspase 3 staining in Fgf7-expressing stroma in livers of WT and Irs2^−/− mice after 7 days of DDC feeding (n = 3), duplicates analyzed for each animal, mean total area 33.9 mm² per section. Dotted lines indicate portal vein. (B–C) IRS2 protects hHSC-derived LX-2 cells from apoptotic cell death following MitoC treatment in vitro. LX-2 cells were stably transduced with sh-luc or sh-IRS2 and treated with MitoC. (C) Representative phase-contrast images showing reduced survival of sh-IRS2 cells at 72 h post-MitoC treatment (above). Graph below shows results of MTT cell-viability analysis, also performed at 72 h, confirming reduced survival (n = 3). (D) Western blot analysis with graphical quantitation of indicated relative protein expression levels (n = 3–5). (D) Inhibition of IRS proteins in primary hHSCs activates apoptotic cell death. Cells were treated with NT-157 (5 μM) or Control (DMSO) for 72 h. Phase-contrast images showing dramatic cell death induced by NT-157 treatment (left) and western blot (representative of n = 4) showing cleaved caspase and P53 activation corresponded with reduced IRS2 levels. Data information: underlying data are available in S1 Data. Data are presented as mean + SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. (A) Unpaired Student t test. (B–C) Paired Student t test. c-CASP3, cleaved caspase 3; Cont, control; Fgf7, fibroblast growth factor 7; Gfap, glial fibrillary acidic protein; hHSC, human HSC; HSC, hepatic stellate cell; Irs2, insulin receptor substrate 2; MitoC, mitomycin C; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NT-157, allosteric inhibitor of IRS1 and 2; RU, relative unit; sh-IRS2, shRNA-targeting IRS2; sh-luc, control luciferase; shRNA, short hairpin RNA; WT, wild type.

https://doi.org/10.1371/journal.pbio.2006972.g004

Irs2 promotes epithelial sensitivity to Fgf7 and hepatic re-epithelialization during injury

In addition to the defect in stromal Fgf7 expression, Irs2 deletion also had a negative impact on sensitivity to Fgf7 in epithelial cells. Stromal Fgf7 drives re-epithelialization during skin wound healing, signaling exclusively to cells expressing the epithelial isoform of Fgfr2 (Fgfr2-IIIb), which is highly expressed by LPCs during DDC feeding [19]. In Irs2^−/− livers, we noted a sharp decline of Fgfr2-IIIb expression in the latter stages of DDC feeding (days 14–21), which contrasted with the steady increase in Fgfr2-IIIb observed in the WT group (Fig 5A). In WT mice, we observed increased antibody staining for Fgfr2 in periportal ducts surrounded by Fgf7+ stroma (Fig 5B). No such induction was observed in Irs2^−/− livers, consistent with a failure of FGFR2-IIIb expression in LPCs. We confirmed that diminished Fgfr2-IIIb expression resulted in reduced Fgf7-sensitivity in injured Irs2^−/− livers by testing the signaling response to intraperitoneal (i.p.) injection of recombinant Fgf7 (rFgf7) protein. In WT mice, rFgf7 robustly activated Fgf7/Fgfr2 signaling as assessed by western blot, demonstrating increased in vivo phosphorylation of extracellular signal-regulated kinases (ERK)1/2 in whole-liver lysates (Fig 5C). This response to rFgf7 was abrogated in the Irs2^−/− livers, indicating that they were refractory to Fgf7. Taken together, our findings suggested that Irs2 played a dual role in (i) promoting stromal expression of Fgf7 and (ii) sustaining sensitivity of the hepatic epithelia to Fgf7 during chronic DDC liver injury by promoting Fgfr2-IIIb expression (Fig 5D). Interestingly, the timing of Fgfr2-IIIb loss in Irs2^−/− mice (days 14–21) corresponded with a period during which the parenchymal architecture failed to re-epithelialize, as judged by quantitative analysis of β-catenin immunostaining in hepatocytes (Fig 5E), suggesting that in the later stages of DDC injury, Irs2 was required upstream of Fgf7–Fgfr2-IIIb signaling to driving adaptive epithelial remodeling.

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Fig 5. Irs2 is required to sustain hepatic Fgfr2-IIIb expression and re-epithelialization during DDC injury.

(A) RT-qPCR analysis of Fgfr2-IIIb expression in whole-liver mRNA from WT and Irs2^−/− mice during a time course of DDC feeding (n = 6–8). (B) Immunofluorescence images of WT and Irs2^−/− livers after 14 and 21 days of DDC feeding. Induction of Fgfr2 occurred within duct-like structures in periportal areas (*) surrounded by Fgf7-expressing stroma (s). The failure to sustain Fgf7 expression in Irs2^−/− mice was paralleled by concomitant failure to induce Fgfr2 in LPCs (arrows). (C) The livers of Irs2^−/− mice are refractory to exogenous Fgf7 stimulation following DDC injury. Western blot analysis of whole-liver extracts from DDC-treated WT and Irs2^−/− mice (day 21). 30 minutes prior to being killed, mice were administered rFgf7 or vehicle by i.p. injection. P-Erk levels relative to total Erk were quantified and are shown in the graph above (n = 4–7), and representative images of blots are shown below. (D) Schematic summarizing the observed negative impact of Irs2 deletion on Fgf7 signaling between the hepatic stroma and LPCs during DDC injury. P = portal vein, A = hepatic artery, C = central vein. (E) Immunohistochemical staining for β-catenin in WT and Irs2^−/− liver parenchyma during a time course of DDC injury. Modal pixel intensity was graphed and used to quantify the dramatic remodeling of β-catenin staining (days 0–14) and reestablishment of epithelial architecture in livers of WT but not Irs2^−/− mice in the latter period of the diet (days 14–21) (n = 6–9). Data information: underlying data are available in S1 Data. Data are presented as mean + SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. (A and E) Two-way ANOVA was used to compare means. Significance P values were calculated using Bonferroni test. (C) One-way ANOVA was used to compare means. Significance P values were calculated using Tukey’s test. DDC, 3.5-diethoxycarbonyl-1.4-dihydrocollidine; Erk, extracellular signal-regulated kinase; Fgfr2-IIIb, Fgf7 ligand and receptor; Fgf7, fibroblast growth factor 7; i.p., intraperitoneal; Irs2, insulin receptor substrate 2; LPC, liver progenitor cell; mRNA, messenger RNA; P-Erk, phosphorylated Erk (Tyrosine 204); RT-qPCR, reverse transcriptase-quantitative PCR; RU, relative unit; WT, wild type.

https://doi.org/10.1371/journal.pbio.2006972.g005

Insulin/IRS2 signaling and FGF7 synergize to promote FGFR2-IIIb expression and the emergence of hepatocytic epithelia in bipotent human HepaRG cells

We examined the possibility that IRS2 played a direct role in regulating LPC sensitivity to FGF7 and epithelialization using a human model of bipotent adult liver progenitor cells (HepaRG) [30]. Upon reaching confluence, HepaRG cells spontaneously differentiate in media supplemented with insulin to produce phase-bright epithelial “islands” of hepatocyte-like cells (Fig 6A and S8 Fig). Within these “islands,” cells express hepatocyte genes such as albumin, HNF4α, cytochrome P450 3A4 (CYP3A4), and apolipoprotein A2 (APOA2) (S8 Fig). Using this model, we found that IRS2 was induced prior to FGFR2-IIIb and hepatocyte-specific genes such as APOA2 (Fig 6B). Using a human IRS2-promoter construct to visualize the pattern of IRS2 expression via green fluorescent protein (pIRS2-GFP), we also demonstrated colocalization of IRS2 with albumin and FGFR2 in the early stages of hepatocyte island formation (Fig 6C). These data were consistent with an early role for IRS2 in the patterning of FGF7 sensitivity during LPC differentiation.

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Fig 6. Insulin/IRS2 signaling promotes epithelialization and sensitivity to FGF7 in vitro.

(A) Schematic showing differentiation of bipotent HepaRG human LPCs in vitro produces epithelial “islands” containing hepatocyte-like cells. (B) RT-qPCR time course of HepaRG differentiation showing early induction of IRS2, and FGFR2-IIIb prior to hepatocyte differentiation (indicated by APOA2). (C) Colocalization of Alb, FGFR2, and IRS2 by immunostaining within hepatocyte islands during the early stages of HepaRG differentiation (day 14). IRS2 expression was visualized by stable transduction with a human IRS2 promoter (pIRS2) GFP reporter. Dotted line delimits “island.” (D–E) IRS2 is necessary for FGFR2-IIIb expression during HepaRG differentiation: (D) phase-contrast images of differentiated cells after stable silencing for IRS2 using shRNA lentivirus (sh-IRS2). Arrowheads highlight “islands” observed in control cultures expressing sh-scram but not in sh-IRS2 cells. Specificity of the knockdown phenotype was confirmed by a “rescue” in which Lenti-mIrs2 was constitutively expressed in sh-IRS2 cells. (E) RT-qPCR analysis of FGFR2-IIIb expression (n = 3). (F–G) Insulin/IRS2 is required for functional sensitivity to rFGF7 in HepaRG cells and is necessary for feed-forward induction of FGFR2-IIIb by FGF7 ligand. (F) Phase-contrast and immunofluorescence images of HepaRG cells (day 15) cultured with rhFGF7 for 11 days, showing an increase in epithelial islands positive for Alb/HNF4α and E-cad immunostaining and pAPOA2-GFP reporter expression (H = Hoechst). (G) RT-qPCR analysis for indicated genes following long-term (11-day) rhFGF7 treatment in the presence (+) or absence (−) of supplemented insulin in control (sh-luc) or IRS2 knockdown cells (sh-IRS2) (n = 3). Data information: underlying data are available in S1 Data. Data are presented as mean + SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. (E and G) One-way ANOVA was used to compare means. Significance P values were calculated using Newman–Keuls multiple comparison test. Alb, albumin; APOA2, apolipoprotein A2; Cont, control; E-cad, E-cadherin; EF1, Human elongation factor-1 alpha promoter; FGF7, fibroblast growth factor 7; FGFR2, fibroblast growth factor receptor 2; GFP, green fluorescent protein; HNF4α, hepatocyte nuclear factor 4-alpha; IRS2, insulin receptor substrate 2; Lenti-mIrs2, murine Irs2 transgene not targeted by human-gene–silencing construct; mRNA, messenger RNA; pAPOA2, human APOA2 promoter; pIRS2, IRS2 promoter; rFgf7, recombinant Fgf7; rhFGF7, recombinant human FGF7; RT-qPCR, reverse transcriptase-quantitative PCR; RU, relative unit; sh-IRS2, shRNA-targeting IRS2; sh-luc, control luciferase; shRNA, short hairpin RNA; sh-scram, scrambled shRNA.

https://doi.org/10.1371/journal.pbio.2006972.g006

Removal of insulin from the media (S8B and S8C Fig) or stable silencing of IRS2 (Fig 6D, S8D–S8F Fig) resulted in a striking failure to generate hepatocytic epithelia within the cultures, together with concomitant loss of FGFR2-IIIb expression (Fig 6E). Importantly, the formation of epithelial islands and FGFR2-IIIb were restored by exogenous expression of a mouse Irs2 not targeted by the human IRS2 shRNA construct (Fig 6D and 6E); hence, we confirmed a cell-autonomous role for IRS2 in promoting FGFR2-IIIb expression and differentiation in LPC-like cells.

Culturing of HepaRG cells with recombinant human FGF7 (rhFGF7) accelerated the formation of islands and increased hepatocyte differentiation, as determined by increased albumin/HNF4α and E-cadherin (E-cad) immunostaining, human APOA2-promoter (pAPOA2)-GFP reporter activity (Fig 6F), and analysis of hepatocyte-specific gene expression (Fig 6G). This occurred downstream of a rapid (3–6 h) and sustained induction in osteopontin/SPP1 (S9 Fig), consistent with our in vivo observation that osteopontin/Spp1 and Fgf7 were both impaired in Irs2^−/− livers during injury (Fig 2). Importantly, the ability of rhFGF7 to promote expression of osteopontin/SPP1, as well as hepatocyte-specific genes such as albumin and APOA2, was both insulin and IRS2 dependent (Fig 6G), revealing synergy between these two pathways in driving the differentiation of bipotent progenitor cells. Stable silencing of IRS2 or omission of supplemented insulin from the medium was sufficient to abrogate the positive long-term effects of rhFGF7 on the hepatogenic program, thus demonstrating a functional requirement for insulin/IRS2 upstream of paracrine FGF7 signaling. Interestingly, rhFGF7 treatment also increased FGFR2-IIIb expression, highlighting the potential for positive feedback between ligand and receptor—an effect that was also insulin/IRS2 dependent (Fig 6G) and hence reinforced the conclusion that insulin/IRS2 signaling in LPC-like cells primed FGF7 sensitivity.

Our data supported the hypothesis that insulin/IRS2 signaling was functionally upstream of FGF7-mediated epithelialization of LPCs and highlighted a cell-intrinsic role for IRS2 in promoting sensitivity to paracrine FGF7 produced by the fibrotic stroma. However, they also revealed that loss of stromal FGF7 could by itself be sufficient to explain a decline in FGFR2-IIIb expression and epithelialization in a non-cell–autonomous manner as a consequence of reduced positive feedback between ligand and receptor.

Insulin/IRS2 resistance in fibrotic stroma limits hepatocyte differentiation and FGFR2-IIIb expression in cocultured LPCs

Our results highlighted the potential for a paracrine feed-forward loop in which FGF7 drove LPC differentiation and FGFR2-IIIb expression, potentiating both epithelialization and FGF7 sensitivity in an insulin/IRS2-dependent manner. To test this hypothesis further and to more rigorously address the role of insulin/IRS2 signaling in the fibrotic stroma, we performed coculture experiments with hHSCs and HepaRG cells, seeding the two cell types together in order to model the intimate heterotypic cell–cell interactions observed between LPCs and their stromal niche in periportal tracts during DDC injury (Fig 7A). LX-2 control (sh-luc) and IRS2-knockdown (sh-IRS2) cells were used to simulate "normal" versus "IRS2-deficient" fibrotic stroma and gauge how stromal insulin/IGF-1 resistance influenced the LPC response in a non-cell–autonomous manner.

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Fig 7. Heterotypic cell–cell crosstalk between HSCs and LPCs is IRS2 dependent.

(A) Schematic illustrating the HSC/LPC coculture model designed to test whether IRS2 expression in the stromal cell compartment was required for heterotypic interactions leading to increased FGFR2-IIIb expression and APOA2 induction in adjacent HepaRG cells. Control (shRNA-luc) or IRS2-deficient (shRNA-IRS2) LX-2 cells were seeded for a day prior to addition of HepaRG cells as indicated, and the resulting cocultures were then studied for 2–14 days. (B) RT-qPCR time course of gene expression in the HSC/LPC coculture system showing stromal IRS2 expression was required for sustained FGF7 expression and induction of FGFR2-IIIb (n = 3). (C) RT-qPCR analysis of control (sh-luc) and IRS2-deficient (sh-IRS2) LX-2 cocultures performed in the presence (+) or absence (−) of supplemented insulin (n = 3). (D) Coculture immunostaining (day 14) highlighting localized FGFR2 expression (arrows) at sites of contact between HepaRG and HSC stroma (marked by phalloidin). Data information: underlying data are available in S1 Data. Data are presented as mean + SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. (B) Two-way ANOVA was used to compare means. Significance P values were calculated using Tukey's multiple comparison test. (C) Two-way ANOVA was used to compare means. Significance P values were calculated using Sidak's multiple comparison test. APOA2, apolipoprotein A2; Fgfr2-IIIb, Fgf7 receptor; Fgf7, fibroblast growth factor 7; H, Hoechst; HSC, hepatic stellate cell; Irs2, insulin receptor substrate 2; LPC, liver progenitor cell; mRNA, messenger RNA; RT-qPCR, reverse transcriptase-quantitative PCR; RU, relative unit; shIRS2, shRNA-targeting IRS2; sh-luc, control luciferase; shRNA, short hairpin RNA.

https://doi.org/10.1371/journal.pbio.2006972.g007

Consistent with the hypothesis that FGF7 was indirectly regulated by stromal IRS2, we observed similar levels of FGF7 in the early stages of LX-2/HepaRG coculture (Fig 7B). However, while FGF7 expression was sustained in cocultures containing normal LX-2 stroma, a significant long-term decline was observed in those that contained IRS2-deficient LX-2 (Fig 7B), demonstrating an indirect role for stromal IRS2 in promoting FGF7 expression in coculture. The decline FGF7 expression in the IRS2-deficient stromal cocultures also coincided with a striking failure to induce FGFR2-IIIb on day 14 (Fig 7B). Interestingly, we also observed blunted induction of FGF10 within cocultures containing IRS2-deficient stroma (days 10–14), whereas FGF22 expression was unaffected (S10A Fig). These data reinforced the notion that FGFR2-IIIb ligands such as FGF7/FGF10 were required to drive expression of their epithelial receptor in LPCs both in vivo and in vitro.

Using the coculture method, we also found that FGFR2-IIIb and APOA2 induction by LPCs required insulin signaling via IRS2 within the fibrotic stroma. Knockdown of IRS2 in LX-2 cells completely abrogated the ability of insulin to potentiate epithelial sensitivity to FGF7 and hepatocyte differentiation within the cocultures, as judged by loss of time-dependent induction of FGFR2-IIIb and APOA2, respectively (Fig 7C). These results demonstrated a requirement for stromal IRS2 in the non-cell–autonomous potentiation of FGF7 target genes in HepaRG cells. Moreover, we observed localized expression of FGFR2 in HepaRG at sites of contact with LX-2 cells, suggesting regulation of LPC differentiation by the stroma at close range, consistent with the short-rage paracrine actions of FGF7 (Fig 7D).

In order to further test whether paracrine FGF7 signaling was downstream of IRS2-dependent stromal–epithelial crosstalk in hHSC/HepaRG cocultures, we used primary hHSCs, which expressed higher levels of endogenous FGF7 than LX-2 (Fig 8). Interestingly, we found that both FGF7 and IRS2 expression were significantly increased in hHSCs following MitoC-induced cell-cycle arrest (Fig 8A). This observation provided us with a useful means to modulate endogenous FGF7 expression prior to coculturing the cells with HepaRG. Consistent with the increase in FGF7 expression, we observed a dramatic enhancement of APOA2 expression in HepaRG cocultures using hHSCs pretreated with MitoC (Fig 8B). Moreover, inclusion in the medium of a competitive FGF7 inhibitor, consisting of a chimeric rFGFR2-IIIb extracellular domain/Human IgG1 crystallizable fragment (Fc) domain protein (rhFGFR2-IIIb-Fc), significantly inhibited APOA2 expression by HepaRG, confirming APOA2 as a downstream target of paracrine FGF7 and demonstrating a role for FGF7 signaling in driving hepatocyte differentiation in coculture.

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Fig 8. Paracrine FGF7 signaling by HSCs is activated during cell-cycle arrest.

(A) RT-qPCR analysis of primary hHSC cultures treated with vehicle (cont) or MitoC 72 h prior to harvesting. FGF7 and IRS2 expression were enhanced by MitoC (n = 3). (B) MitoC treatment of hHSCs increased paracrine FGF7 signaling. Cont/MitoC-treated hHSCs were cocultured with HepaRG cells for 14 days in the presence/absence of the rhFGFR2-IIIb-Fc fusion protein (rhIIIb-Fc) used to block Fgf7 binding its receptor. Cocultures were then analyzed by RT-qPCR for APOA2 expression (n = 3) as a functional readout of hepatocyte differentiation and FGF7 target gene activity. (C) HNF4α/Vimentin immunostaining of MitoC-treated hHSC/HepaRG cocultures (day 14). HepaRG differentiation (HNF4α+) was highly localized to sites of contact between hHSCs (Vimentin+). Graph shows INcell analysis comparing the percentage of HNF4α+ cells in contact (vimentin+) or remote from sites of contact with hHSCs (vimentin−) (n = 4). Total of 6.69 × 10⁵ cells analyzed. (D–F) Functional bioassay for FGF7 signaling reveals a requirement for direct coculturing of hHSCs with target cells. (D) RT-qPCR showing the time course response of HepaRG cells (day 13) to a single dose of vehicle (cont) or rhFGF7. Changes in APOA2 expression at 48 h were used as a biological readout for FGF7 activity in E–F. (E) Conditioned medium from MitoC-treated hHSCs was insufficient to illicit an APOA2 response at 48 h, whereas (F) direct addition of hHSCs to HepaRG cultures activated APOA2 expression in an FGF7-dependent manner, as judged by the ability of rhFGFR2-IIIb-Fc to block induction. Importantly, this effect was conditional upon prior treatment of hHSCs with MitoC, thus confirming the positive effect of mitotic inhibition on the ability of hHSCs to communicate with LPCs via FGF7. Data information: underlying data are available in S1 Data. Data are presented as mean + SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. (A) Paired Student t test. (B, E, F) One-way ANOVA was used to compare means. Significance P values were calculated using Tukey's multiple comparison test. (D) Two-way ANOVA was used to compare means. Significance P values were calculated using Tukey's multiple comparison test. APOA2, apolipoprotein A2; cont, control; Fgfr2-IIIb, Fgf7 receptor; Fgf7, fibroblast growth factor 7; hHSC, human HSC; HNF4α, hepatocyte nuclear factor 4-alpha; HSC, hepatic stellate cell; Irs2, insulin receptor substrate 2; LPC, liver progenitor cell; MitoC, mitomycin C; mRNA, messenger RNA; rhFGFR2-IIIb-Fc or rhIIIb-Fc, recombinant protein comprising human Fgfr2-IIIb extracellular domain fused to Human IgG1 Fc domain used to inhibit FGF7 binding its receptor; rhFGF7, recombinant human Fgf7; RT-qPCR, reverse transcriptase-quantitative PCR; RU, relative unit.

https://doi.org/10.1371/journal.pbio.2006972.g008

The diffusion of FGF7 is highly restricted because of its affinity for heparin-sulphate proteoglycans in extracellular matrix and cell membranes [23]. Consistent with this short-range mode of action, we found that hepatocyte differentiation within the cocultures (judged by HNF4α immunostaining) was spatially limited to sites of contact between HepaRG and vimentin+ hHSCs (Fig 8C). To explore the properties of the HSC signal driving differentiation within the cocultures further, we developed a short-term bioassay using APOA2 expression as a functional readout for FGF7 activity. Stimulation of HepaRG monocultures with a single dose of rhFGF7 rapidly induced APOA2 at 48 h (Fig 8D). Hence, we collected conditioned medium from MitoC-treated hHSCs and added it to HepaRG cells; however, no induction of APOA2 was observed, suggesting the factor was either labile or unavailable in the cellular supernatant because of cell/matrix binding (Fig 8E). Using a similar approach, we were able to confirm that direct contact between HepaRG and hHSCs (achieved by short-term 48 h coculture) was sufficient to induce APOA2 expression in an FGF7-dependent manner, an effect that was blocked by inclusion of the competitive FGF7 inhibitor (Fig 8F). Importantly, the ability of hHSCs to induce APOA2 within 48 h in this model was conditional upon the pretreatment of hHSCs with MitoC, confirming that paracrine FGF7 signaling in hHSCs was enhanced by mitotic inactivation.

Taken together, these data demonstrated a positive role for paracrine FGF7 signaling by human HSC in supporting FGF7 sensitivity and hepatocyte differentiation in adjacent epithelial progenitors.

IRS2 deficiency promotes stromal activation at the expense of epithelial repair

The stromal injury response precedes epithelial repair. In the liver, this is characterized by HSC-derived myofibroblast proliferation and the laying down of extracellular matrix, which is followed by cell-cycle exit, fibrogenic reversion, and apoptotic cell clearance that accompanies tissue repair. The equilibrium within the fibrotic stroma between activated and reverted HSCs is therefore central to the regulation of the stromal injury response and has important consequences for liver fibrosis and scarring in the context of chronic injury.

Our data showed that both FGF7 and IRS2 expression were induced in hHSCs upon triggering of cell-cycle arrest using MitoC (Fig 8A). MitoC treatment was also accompanied by a phenotypic shift in hHSCs and LX-2s consistent with fibrogenic reversion (Fig 9A), as judged by loss of Collagen 1A1 (COL1A1) expression and activation of antiapoptotic heat shock 70 kDa protein 1B (HSPA1B), previously identified as a biomarker of HSC reversion [31]. Importantly, the down-regulation of myofibroblast marker protein αSMA upon cell-cycle exit was delayed in LX-2 cells in which IRS2 was silenced (Fig 9B), suggesting impaired transition from an activated to “reverted” state. These in vitro data, which suggested that IRS2 could participate in the transition of HSCs to a “reverted” phenotype, were consistent with the in vivo observation that DDC-treated Irs2^−/− mice had increased periportal and perisinusoidal fibrosis, judged by Sirius Red collagen morphometry and αSMA immunostaining (Fig 9C).

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Fig 9. Loss of IRS2 promotes stromal activation and inhibits HSC reversion.

(A–B) Cell-cycle arrest induced by MitoC leads to fibrogenic reversion of hHSCs, which was delayed by IRS2 silencing. (A) RT-qPCR analysis of LX-2 cells and primary hHSC cultures treated with vehicle (cont) or MitoC for 72 h prior to harvesting showing decreased COL1A1 expression together with concomitant increase in HSC reversion marker HSPA1B (n = 3). (B) Western blot analysis of αSMA protein down-regulation in cont (sh-luc) and silenced (sh-IRS2) LX-2 cells during MitoC treatment (72 h) (n = 4–5). (C) Hepatic fibrosis after 21 days of DDC treatment was exacerbated in Irs2^−/− mice. (Left) Representative immunohistochemical images of control (WT) and Irs2^−/− livers for myofibroblast marker αSMA together with Picro Sirius Red histochemistry used for collagen morphometry; quantified in graph (right) (n = 8). Red arrowheads highlight pattern of increased perisinusoidal fibrosis that was more pronounced in Irs2^−/− livers. (D) RT-qPCR analysis of cont (sh-luc) and IRS2-deficient (sh-IRS2) LX-2 cocultures with HepaRG cells performed in the presence (+) or absence (−) of supplemented insulin. Fibrogenic gene expression was analyzed at 2 days and 14 days after seeding the two cell types together (n = 3). Data information: underlying data are available in S1 Data. Data are presented as mean + SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. (A) Paired Student t test. (B and D) Two-way ANOVA was used to compare means. Significance P values were calculated using Sidak's multiple comparison test. (C) Unpaired Student t test. Acta2, gene encoding αSMA; AU, arbitrary unit; COL1A1, Collagen 1A1; cont, control; hHSC, human HSC; HSC, hepatic stellate cell; HSPA1B, heat shock 70 kDa protein 1B; Irs2, insulin receptor substrate 2; MitoC, mitomycin C; mRNA, messenger RNA; pHSC, primary human HSC; RT-qPCR, reverse transcriptase-quantitative PCR; RU, relative unit; shIRS2, shRNA-targeting IRS2; sh-luc, control luciferase; shRNA, short hairpin RNA; WT, wild type; αSMA, alpha-smooth muscle actin.

https://doi.org/10.1371/journal.pbio.2006972.g009

We therefore returned to the LX-2/HepaRG cocultures to assess changes in fibrogenic markers. Using this method, we found that upon coculture with LPC-like cells, control (sh-luc) LX-2 cells underwent a process of time-dependent fibrogenic reversion, during which fibrillar collagen (COL1A1) expression was significantly down-regulated and αSMA/ACTA2 dampened (Fig 9D). In striking contrast to this, we found that silencing of IRS2 in LX-2 cells abrogated their ability to suppress COL1A1 in coculture, and we instead observed a dramatic induction of nonfibrillar type III collagen (COL3A1) indicative of an activated stromal injury response. Time-dependent switching from myofibroblast marker THY1 to LPC-associated SPP1 was also dampened by silencing of IRS2 in the stroma (S10B Fig), and the numbers of GFP-expressing LX-2 cells at the end of the cocultures was increased (S10C Fig). Interestingly, unlike LPC and epithelial repair genes (SPP1/APOA2/FGFR2-IIIb), whose expression in coculture was both insulin and IRS2 dependent (Fig 7C and S10 Fig), changes in fibrogenic/myofibroblast genes COL1A1, COL3A1, ACTA2, and THY1 (Fig 9D and S10 Fig) were largely unaffected by omission of insulin from the medium, suggesting the ability of IRS2 to restrain activation of the fibrogenic stroma was insulin independent. We concluded that silencing of IRS2 in HSCs prolonged their fibrogenic activation in coculture while slowing fibrogenic reversion by MitoC and reducing the survival of mitotically inactivated cells in which FGF7 expression was increased. Thus, IRS2 signaling in the fibrotic stroma served as a switch between states of stromal activation and fibrogenic reversion that potentiated heterotypic paracrine signaling between the two cell types and drove epithelial FGF7 sensitivity in LPCs. Together with our results demonstrating a cell-intrinsic role for IRS2 in LPCs in promoting FGF7 sensitivity, we proposed a model in which IRS2 exerted multiple proregenerative effects in both stromal and LPC cell compartments that favored epithelial repair over fibrogenesis during chronic liver injury (Fig 10).

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Fig 10. IRS2 promotes epithelial repair in the liver by potentiating HSC–LPC crosstalk.

Model summarizing the major findings of our study in stromal (red) and epithelial (blue) cell compartments. IRS2 promoted reversion of activated (a) fibrogenic stroma and survival of inactivated (i) cells expressing high levels of FGF7. In progenitors, IRS2 was required for differentiation and FGFR2-IIIb expression—priming sensitivity to FGF7. Epithelial sensitivity to FGF7 was then further increased by the ability of the FGF7 ligand to increase FGFR2-IIIb expression, resulting in a feed-forward paracrine loop capable of sustaining epithelial repair by potentiating heterotypic cell–cell communication. Fgfr2-IIIb, Fgf7 receptor; Fgf7, fibroblast growth factor 7; Irs2, insulin receptor substrate 2; HSC, hepatic stellate cell; LPC, liver progenitor cell.

https://doi.org/10.1371/journal.pbio.2006972.g010

Ethics statement

Mice were housed in the facility of the CIPF (Valencia, Spain), which is registered as an experimental animal Breeding, User, and Supplier Center (reg. no. ES 46 250 0001 002) under current applicable Spanish regulations (RD 53/2013). Animals were kept in ventilated racks with microisolators under specific-pathogen–free (SPF) conditions and handled by accredited personnel and treated in accordance with EU legislation and welfare guidelines. Procedures were approved by the CIPF "Comite Ético de Experimentación Animal" (CEEA) and the ministry for "Agricultura, pesca, alimentacion y agua" of the Generalitat Valenciana (Valencia, Spain), under the authorization code 2014/VSC/PEA/00062 tipo 2. Mice were killed by fentanil/pentobarbital overdose followed by cervical dislocation.

Animal experimentation

WT and Irs2^−/− C57BL/6 littermates (females aged 10–12 weeks, fasting blood glucose < 100 mg/dL) were fed a modified LabDiet (5015) containing 0.1% DDC (TestDiet, St. Louis, MO, USA). Animals were killed at intervals up to 21 days during the light cycle. FGF7 stimulation was performed by i.p. injection of (0.3 mg/kg) recombinant mouse KGF (Taper BioLegend, San Diego, CA, USA).

Liver injury and cholestasis markers

Serum transaminases were measured by ELISA according to manufacturer’s instructions (MilliporeSigma, St. Louis, MO, USA), and serum total bilirubin (TBIL) was measured by colorimetric assay according to the Jendrassik-Grof method (MilliporeSigma). Bile acids were measured by metabolomic analysis. Briefly, samples were extracted from whole-liver tissue using the metanol-chloroform-H₂O method [48] and analyzed on a 600 MHz NMR spectrometer equipped with a cryoprobe. Metabolites were identified with the aid of the spectral databases HMBD [49] and BMRB [50] and quantified using MestreNova8.

Cell culture

HepaRG cells (kind gift of Anne Corlu and Cristiane Guillouzo) were passaged and differentiated as previously described [30] using Williams E medium (10% fetal calf serum, 50 μM hydrocortisone hemisuccinate, 0.88 μM insulin, and 2 mM L-glutamine and penicillin-streptomycin). DMSO (2%) was added from day 14. Insulin supplement was omitted from the medium where indicated 24 h postplating. rhFGF7 (50 ng/ml; Cell Guidance Systems, St. Louis, MO, USA) or rhFGFR2-IIIb Fc Chimera (100 ng/ml; R&D Systems, Minneapolis, MN, USA) were added to media where indicated every 48 h. Cryopreserved primary hHSCs were obtained from a healthy 15-year-old Caucasian female donor using a previously described isolation method [51] (Innoprot, Derio, Spain). LX-2 cells (kind gift of Scott Friedman) and primary hHSCs were cultured in DMEM (2% fetal calf serum), and primary HSCs were used within 5 passages. LX-2/HSCs were inactivated for 3 h with 1mg/ml MitoC (MilliporeSigma) before seeding onto 0.1%-gelatin–coated plates. After 24 h, activated/inactivated HSC cultures were switched to HepaRG medium until the end of the experiments. Stable cell lines were generated by lentiviral transduction (vectors summarized in S1 Table) using a multiplicity of infection of 0.5–20 TU/cell in the presence of 8 μM polybrene. Infections were performed 16 h postplating for 6 h in serum-free medium. Where applicable, cells were selected for 7 days in media containing 2.5 μg/ml puromycin.

Cell viability

MTT assay: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (5 mg/ml, MilliporeSigma) was added to media in a 1:5 ratio and incubated in the dark for 4 h. Resulting formazan crystals were resuspended in DMSO, and optical density (OD) was measured at 570 nm using an automated microplate reader (Victor; PerkinElmer, Waltham, MA, USA).

Fluorescence immunostaining

Fresh frozen liver tissue sections (6 μm, mounted in OCT, MilliporeSigma) or cells were fixed using 4% paraformaldehyde–PBS, washed, and permeabilized with 0.5% Triton-X100 before blocking (1% BSA, 5% horse serum, 0.2% Triton-X100). Primary antibodies (S2 Table) were incubated over night at 4 °C. Alexa-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) were applied for 1 h together with 5 μg/ml Hoechst 33342 prior to mounting. Confocal images were obtained using a Leica TCS-SP6 (Leica, Wetzlar, Germany). Fluorescence image analysis was performed using INCell Analyzer 1000 (GE Healthcare, Chicago, IL, USA) as outlined below.

Immunohistochemistry

Formalin-fixed paraffin-embedded tissue sections (4 μm) were processed automatically using the Autostainer Link 48 (Agilent, Santa Clara, CA, USA) with EnVision FLEX reagents according to manufacturers’ instructions (Agilent). Antigen retrieval was performed using high-pH target retrieval solution (Agilent). Antibodies are provided in S2 Table.

Collagen morphometry

Collagen was visualized in PFE liver sections using the Picro Sirius Red technique (Abcam, Cambridge, UK) according to manufacturer’s instructions. Following imaging, threshold analysis was performed using FIJI software to quantify staining in a total area comprising >10 portal fields per animal.

Image analysis

Fluorescence immunostained cells/cryosections were analyzed using the “INcell 1000” imaging platform and software (GE Healthcare) where indicated. Individual cells were identified by Hoechst staining. Threshold analysis was used to determine the expression of the following markers: pAPOA2-GFP, albumin, CYP3A4, HNF4α, Ki67, Spp1, and Vimentin. For replicate well/tissue sections, at least 20 fields were randomly analyzed for each replicate. PFE tissue sections were imaged using Pannoramic Viewer (3DHISTEC) and analyzed with FIJI software; β-catenin staining was quantified using FIJI (histogram function) to calculate modal pixel intensity.

Hepatocyte ploidy

Interpolation of DNA content in HNF4α/Hoechst-stained liver sections was performed using a methodology developed by our laboratory using INcell 1000 (GF Healthcare) that enabled in situ approximation of hepatocyte ploidy (S3 Fig). DNA content was calculated for all circular hepatocyte nuclei (nuclear elongation value > 0.8) as a combined function of Hoechst nuclear fluorescence intensity (DNA density) and nuclear volume (calculated as a spherical function of nuclear radius). Hepatocyte ploidy was then calibrated using circular HNF4α− NPC cell population within the tissue as a 2n control. The 2c hepatocyte population was defined as the population of HNF4α+ nuclei within the 19.99 μm²–34.99 μm² nuclear size range with a nuclear circularity index >0.8.

Immunoblotting

Tissues were lysed in RIPA buffer containing complete protease and phosphatase inhibitors (Roche, Basel, Switzerland). Protein (20 μg/well) was separated using standard SDS-PAGE. Transferred PVDF membranes were blocked (TBS–Tween 3% BSA) and probed with primary antibodies (S2 Table).

RT-qPCR

Total RNA was extracted using RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) with DNaseI Digestion. Liver tissue was preprocessed using Trizol (MilliporeSigma). For experiments using cells, RNA from duplicate wells were analyzed. First-strand synthesis was performed using EcoDry Premix (Takara, Kusatsu, Japan), and real time-PCR was carried out in LightCycler 480 II (Roche) using SYBR PreMix ExTaq (Mi RNaseH Plus, Takara). Reverse transcriptase-quantitative PCR (RT-qPCR) was performed in triplicates for all RNAs analyzed. Primers are listed in S3 Table. Relative gene expression was calculated by normalization to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in mouse liver and to Ribosomal Protein L19 (RPL19) in human cell lines.

Statistics and data processing

For experiments using animals, values of n reflect the number of animals per cohort. Cohort size was based on previous studies and expertise using the Irs2^−/− model. For nonanimal experiments, values of n reflect the number of independent experiments performed. When possible, automated methods of image analysis were employed to quantify immunostainings using the “INcell 1000” imaging platform and software (GE Healthcare). Statistical analyses were performed with Prism version 7.00 for Windows, GraphPad Software, La Jolla, CA, USA, www.graphpad.com. One-way ANOVA was used to compare multiple means with one variable. Multiple comparisons were obtained applying Tukey’s test. Ordinary two-way ANOVA was used to analyze data sets with several variables and Bonferroni’s test for multiple comparisons. When two data sets were compared, unpaired Student t test was used. Results were represented as mean + SEM considering statistical significance: *P < 0.05, **P < 0.01, and ***P < 0.001.