ECM gene expression parallels the induction of Wnts and their transducers during early liver injury

In response to toxicant exposure, inflammation and wound healing programs are dynamically controlled to promote liver regeneration. HSCs are key factors for liver regeneration, as their early activation may be beneficial to the tissue by providing ECM for scaffolding to support the parenchyma to regenerate and reestablish tissue integrity (Borthwick et al. 1832; Lachowski et al. 2019; Duarte et al. 2015). Despite the known role of Wnts in promoting liver regeneration, less is known about Wnts as coordinating factors for HSC function during early wound healing responses in the liver. We first asked if morphogen expression is associated with acute toxicant-induced liver damage and the early fibrogenic process in vivo, (Fig. 1A). CCl4-treated animals displayed elevated hepatocyte injury, including increased plasma aspartate aminotransferase (AST) activity; peak injury occurred 24 h following CCl4 challenge but remained elevated after 72 h (Fig. 1B). Hepatic expression of fibrogenic genes (col1a1 and a-sma) were elevated by 72 h, but not 24 h, following acute CCl4 challenge (Fig. 1C), consistent with prior reports (Das et al. 2014). Because hepatic injury is associated with a dynamic production of inflammatory mediators, we next confirmed the expression of TGF-β, CTGF, and CCL-2 in the liver. Expression of tgf-β and ccl-2 were all increased in the hepatic tissue (Fig. 1D). Because HSCs express select Fzd receptors (Brenner et al. 2009), we next asked if Wnts and their transducers are upregulated during early liver injury. Expression of canonical wnt3a, non-canonical wnt5a, and their transducers (fzd1, fzd2, fzd7, and lrp-6 co-receptor) were increased by 72 h after acute CCl4 compared to control mice (Fig. 1E & F). To determine if the expression pattern of Wnt machinery was unique to acute liver injury and/or may be persistently upregulated in a chronic model of liver injury, we next profiled these genes in a model of chronic liver disease characterized by established liver injury and fibrosis (Supplemental Fig. 1). Hepatic expression of Wnt machinery (wnt3a/wnt5a, and transducers fzd1, fzd2, fzd7, and lrp6) were increased after chronic CCl4 compared to control mice (Supplemental Fig. 1). Taken together, these data provide evidence that during early liver injury, heightened mediators of inflammation and ECM deposition are associated with increased Wnt production and expression of Wnt transducers on HSCs which is maintained in the chronic state. These data set the precedence that Wnt3a and Wnt5a may promote the initiation of the fibrogenic events and wound healing responses in the liver.

Fig. 1

Expression of Wnt machinery correlates with early fibrotic changes during acute liver injury in mice. A) Mice were given a single dose of olive oil or CCl4 and liver tissues were harvested 24 h and 72 h later. B) AST activity was measured in plasma. C) Expression of ECM mRNA (col1a1 and α-sma) was detected in mouse liver by qRT-PCR. Expression of (D) tgf-β, ctgf, mcp-1, (E) wnt3a and wnt5a, and (F) fzd1, fzd2, fzd7, and lrp6 mRNA was detected in mouse liver by qRT-PCR. N = 4–5 mice per group. *P < 0.05, **P < 0.01

Expression of Wnts and Fzd reveal a hepatocyte-HSC axis during acute liver injury

While there is a robust increase in expression of Wnt machinery and ECM markers in hepatic tissue during early liver injury, the cellular source of Wnts and the hepatic localization of their transducers remain unclear. While multiple cell types in the liver, including hepatocytes, Kupffer cells, and HSCs express certain Fzd receptors, including Fzd2 and Fzd7 (Zeng et al. 2007), it has yet to be determined if the expression of the transducers of Wnts are associated with the transdifferentiation state of mHSCs during acute liver injury. The expression of an HSC activation marker, desmin, was used to determine the spatial localization of HSCs in the lobule and activation state following acute CCl4 exposure in the liver (Fig. 2A). Desmin-positive cells increased the expression of the transducers of Wnts, including Fzd2 and Fzd7,72 h after CCl4 injection (Fig. 2A). Expression of Wnt3a was increased 72 h after acute CCl4, predominately in the mid-zone region of the lobule while Wnt5a was increased by 24 h in periportal and midzonal regions (Fig. 2B). To confirm if hepatocytes are a source of Wnt3a following acute liver injury, we made use of an hepatocyte-specific marker, HNF4-α. HNF4-α is crucial for the functional differentiation of hepatocytes and its expression is dysregulated during liver regeneration following acute injury (Zhou et al. 2020). 72 h after CCl4, HNF4-α-positive midzonal hepatocytes express Wnt3a (Fig. 2B). Highly proliferative PCNA-positive cells, including cells morphologically representing hepatocytes and HSCs, are dynamically present in different zones 24 h and 72 h post-CCl4; notably, PCNA-positive cells, morphologically appearing to be hepatocytes, have a trending increase in Wnt5a expression at 24 h. (Fig. 2B). Taken together, these data indicate that transducers of Wnts are expressed and upregulated by activated myofibroblasts following acute liver injury. Additionally, these data further provide evidence that hepatocytes are a cellular source of Wnts.

Fig. 2

Desmin-positive cells increase Fzd2/7 while proliferating hepatocytes increase Wnt3a and Wnt5a following acute liver injury. WT C57BL/6 J mice were challenged with CCl4 or OO and tissues were harvested up to 72 h later. A) Frozen liver sections were immunostained with Fzd2 or Fzd7 (green) or Desmin (red); images were quantified using Image J and co-localization of Fzd2 or Fzd7 and Desmin was determined by quantifying yellow pixels in Desmin-positive cells. B) Integrated density of Wnt3a (red) or Wnt5a (red) expression in liver tissues. White boxes denote magnified regions demonstrating co-localized expression. N = 4–5 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001

Proinflammatory cytokines stimulate Wnt3a and Wnt5a production by hepatocytes and Kupffer cells in vitro

While proliferating hepatocytes increase Wnt3a and Wnt5a expression following acute liver injury, these data do not exclude the possibility that other liver cells, including Kupffer cells, are another source of Wnts in this model. Because the production of proinflammatory cytokines coordinate wound healing responses following liver injury, we next asked if inflammatory cytokines are causal factors for Wnt production by Kupffer cells and hepatocytes. Consistent with prior reports, the expression of proinflammatory cytokines, including tumor necrosis factor alpha (tnf-a) and interleukin-6 (il-6), were increased 24 h after CCl4 injection, while expression of interleukin-10 (il-10) was increased by 72 h (Fig. 3A). Given our observation of robust cytokine expression in the liver after CCl4 in vivo, we next challenged immortalized mouse hepatocytes (AML12) and immortalized mouse Kupffer Cells(ImKCs) with exogenous TNF-α and IL-6 (10 ng/mL) for 24 h. TNF-α and IL-6 increased both Wnt3a and Wnt5a expression in AML12 and ImKCs compared to basal. Interestingly, IL-6 further increased the expression of Wnt3a in ImKCs and Wnt5a in AML12 cells compared to TNFα (Fig. 3B/C). Taken together, these data suggest that during acute liver injury, inflammatory processes and specifically, pro-inflammatory cytokines, contribute to morphogen production by both hepatocytes and Kupffer cells. Importantly, these data highlight the complexity of Wnt signaling networks in the liver, demonstrating that Wnts likely coordinate paracrine signaling networks throughout the wound healing process.

Fig. 3

Proinflammatory cytokine mediators are increased following acute liver injury and stimulate Wnt3a and Wnt5a production by hepatocytes and Kupffer cells. WT C57BL/6 J mice were challenged with CCl4 or OO and tissues were harvested up to 72 h later. A) Expression of proinflammatory marker mRNA (tnfα, il-6, and il-10) was detected in mHSCs by qRT-PCR. B) AML12 cells and C) ImKCs were cultured in the presence or absence of tnf-α or il-6 for 24 h then fixed on glass coverslips and immunostained for Wnt3a and Wnt5a. Immunofluorescence was quantified using ImageJ; relative expression is denoted as arbitrary units of density. Data are from four independent experiments N = 3–4 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Spontaneous activation of primary mouse hepatic stellate cells are associated with increased expression of ECM genes and Wnt transducers

While Wnt and Fzd expression is dynamically increased following CCl4 -induced liver injury, it remains unclear if canonical wnt signaling is an early driver of fibrotic gene reprogramming in HSCs. Therefore, to better define the causal role of canonical Wnt signaling, we next characterized Wnt machinery in a model of spontaneous HSC activation (Das et al. 2014). To better understand the role of β-catenin signaling in HSCs specifically, primary mouse HSCs (mHSCs) were plated on plastic culture plates for ten days to monitor spontaneous activation (Fig. 4A). Freshly isolated mHSCs retain their quiescent phenotype when cultured on plastic for 2 days, displaying a distinct stellate cell morphology with abundant lipid droplets (Supplemental Fig. 2). When cultured on plastic for a period of ten days, we find that mHSCs spontaneously start activating by day 3 and are fully activated by day 10 as characterized by their large nuclei accompanied with their large polygonal shape and loss of lipid droplets. Gene expression profiling paralleled morphologic outcomes of activation; expression of col1a1 and α-sma was increased by day 5, which was further increased by day 10 (Fig. 4B). We next determined if the expression of Wnt transducers and LRP6 co-receptor is associated with the activation state of HSCs. Spontaneous activation of mHSCs on day 7 increased expression of fzd1, fzd2, and fzd7, but not lrp6 (Fig. 4C). Because canonical Wnt signaling induces a proliferative response and morphological changes through nuclear translocation of cytosolic β-catenin and activation of TCF/LEF-dependent transcription, we examined the expression of β-catenin in day 2 (quiescent), day 5 (early activation), and day 10 (activated) mHSCs by immunofluorescence. An increase in a known HSC activation marker, desmin, and β-catenin expression is evident in day 10 mHSCs compared to their quiescent state (Day 0.5) (Fig. 4D). Interestingly, spontaneous activation of mHSCs revealed an increase in expression of wnt5a, but not wnt3a (Fig. 4E), which may partially explain the observed increase in β-catenin expression. Taken together, these data suggest that expression of the transducers of Wnts and β-catenin parallel the activation state of primary mHSCs in vitro, suggesting that canonical Wnt signaling may promote transdifferentiation of mHSCs in culture.

Fig. 4

Spontaneous activation of mouse hepatic stellate cells is associated with increased expression of ECM genes, Wnt transducers, and β-catenin. A) Primary mouse hepatic stellate cells were isolated from livers of Wild-type C57BL/6 J mice and cultured for up to 10 days. B) Expression of activation marker mRNA (col1a1 and α-sma) was detected in mHSCs by qRT-PCR. C) Expression of Wnt transducer mRNA (fzd1, fzd2, fzd7, and lrp6) was detected in mHSCs by qRT-PCR. Data are expressed as mHSCs cultured to day 7 and normalized to day 0.5. Data are significantly different from day 0.5, #P < 0.05. D) mHSCs were fixed on glass coverslips and immunostained for β-catenin and Desmin. Immunofluorescence was quantified using ImageJ; relative expression is denoted as arbitrary units of density. E) Expression of wnt3a and wnt5a mRNA was determined by qRT-PCR. Data are from three independent experiments, N = 5–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Wnt3a and Wnt5a accelerate spontaneous activation of mHSCs

Having observed that the transducers of Wnts are increased while mHSCs activate in culture, we next wanted to determine if exogenous canonical Wnt3a and noncanonical Wnt5a are causative factors for mHSC transdifferentiation in vitro. Accordingly, primary mHSCs were challenged with exogenous Wnt3a, Wnt5a, or a major profibrotic factor, TGF-β, as a positive control (10 ng/mL) on day 5 of culture for 48 h; cells were then harvested on day 7 (Fig. 5A). Exogenous TGF-β, Wnt3a, and Wnt5a increased both col1a1 mRNA and α-SMA protein in mHSCs compared to untreated, spontaneously activated day 7 cells (Fig. 5B/C), suggesting that both Wnt3a and Wnt5a can accelerate spontaneous mHSC activation in culture. Expression of β-catenin and desmin were increased in activated day 7 mHSCs, which was further increased by TGF- β and Wnt3a, but not Wnt5a (Fig. 5D/E). In day 7 cells, β-catenin expression is intensified along the plasma membrane and in the nuclei. Importantly, the nuclear localization of β-catenin (colocalization with DAPI, red arrows) was noticeably increased by Wnt3a (Fig. 5D/E). Expression of axin2, a target gene of TCF/LEF-dependent transcription and a marker for β-catenin translocation to the nucleus, was increased by Wnt3a, but not Wnt5a or TGF- β (Fig. 5F). During transdifferentiation, HSCs will express paracrine factors that are associated with the fibrotic process via ECM synthesis (CTGF), and composition (MMPs and TIMPs) (Decaens et al. 2008). Accordingly, expression of ctgf mRNA was increased by TGF- β and Wnt3a, while exogenous treatment to mHSCs did not impact mmp9, mmp13, timp1, or cyclind1 mRNA expression (Fig. 5F), suggesting that the Wnts are canonically signaling through active β-catenin and not influencing cell cycle regulation at the time points measured (Batsali et al. 2017). Making use of a GSK3β inhibitor (CHIR 99021) to release β-catenin from the destruction complex, we find that mHSCs upregulated fibrogenic factors (α-sma, col1a1, ctgf) compared to culture-induced activated cells at day 7, further confirming a role for non-canonical β-catenin activation and early HSC transdifferentiation (supplemental Fig. 4). Importantly, while our data previously show that mHSCs produce wnt5a, but not wnt3a, as they spontaneously activate in culture (Fig. 4E), and given the selected timepoint of our exogenous cell treatments, we next determined if the expression of these Wnts differ between day 5 and day 7. Accordingly, expression of both wnt3a and wnt5a did not change between days 5 and 7 (Fig. 5G), indicating that the production of Wnts (specifically Wnt5a) is not influencing the accelerated transdifferentiation of mHSCs in an autocrine manner. These data demonstrate that exogenous Wnts, via canonical β-catenin signaling, can influence the fibrogenic program of mHSCs in vitro, suggesting they are key morphogenic factors promoting early fibrotic processes in the liver.

Fig. 5

Exogenous Wnt3a and Wnt5a accelerate the spontaneous activation of mouse hepatic stellate cells. A) Primary mouse hepatic stellate cells were isolated then cultured in the presence or absence of 10 ng/mL of exogenous TGF-β, Wnt3a, and Wnt5a from day 5 to day 7. B) Expression of col1a1 mRNA was detected in mHSCs by qRT-PCR. C) mHSC lysates were prepared and proteins separated by SDS-PAGE. α-SMA and HSC70 (loading control) were measured by Western blot. D) β-catenin and Desmin expression was analyzed by immunofluorescence in cells following TGF-β, Wnt3a, and Wnt5a treatment. Red arrows highlight nuclear localization of β-catenin. E) β-catenin expression and nuclear localization, and Desmin expression by immunofluorescence was quantified using ImageJ software. F) Expression of axin2, ctgf, cyclind1, mmp9, mmp13, and timp1mRNA and G) wnt3a and wnt5a mRNA was detected by qRT-PCR. N = 5–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001

LRP6 Inhibition by DKK-1 prevents Wnt-induced HSC activation

It is known that canonical Wnt signaling occurs in a LRP6-dependent manner (Logan and Nusse 2004) and to delineate the canonical and noncanonical actions of Wnts, we hypothesize that obstruction of Fzd-mediated signaling via the co-receptor LRP6 will attenuate HSC activation. Primary mHSCs were challenged with exogenous TGF- β, Wnt3a, and Wnt5a for 48 h, in the presence or absence of the LRP6 co-receptor antagonist DKK-1 (Fig. 6A). LRP6 inhibition via DKK-1 attenuated Wnt3a- and Wnt5a-dependent expression of col1a1 mRNA (Fig. 6B) and α-SMA protein expression (Fig. 6C). Furthermore, to investigate if nuclear localization of active β-catenin is suppressed via DKK-1, we analyzed the expression of β-catenin-dependent gene axin2. Our data show that Wnt3a increased the expression of axin2, which was attenuated by pre-treatment of DKK-1 (Fig. 6D). Additional markers of HSC transdifferentiation, including ctgf, mmp9 and mmp13, trended the expression profile of axin2, but were not statistically significant. Our data show that with the addition of DKK-1, there is no change in Wnt production in mHSCs, specifically wnt5a (Fig. 6E). Taken together, these data demonstrate that early HSC activation in vitro occurs via canonical Wnt signaling and indicates that Wnts, via the action of LRP6, may be important physiologic contributors to promote wound healing in the liver.

Fig. 6

HSC activation, via LRP6, occurs in a canonical Wnt-dependent manner. A) Primary mouse HSCs were then cultured in the presence or absence of TGF-β, Wnt3a, and Wnt5a and the LRP-6 antagonist DKK-1 (10 ng/mL) from day 5 to day 7. B) Expression of col1a1 mRNA was determined by qRT-PCR. C) mHSC lysates were prepared and proteins separated by SDS-PAGE. α-SMA expression and HSC70 (loading control) were measured by Western Blot. α-SMA was normalized to HSC70 as a loading control and quantified using ImageJ software. D) Expression of axin2, mmp9, and mmp13 mRNA; and E) wnt3a and wnt5a mRNA was determined in mHSCs by qRT-PCR. Data are from three independent experiments. N = 3–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001