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Two-signal requirement for growth-promoting function of Yap in hepatocytes

  1. Tian Su
  2. Tanya Bondar
  3. Xu Zhou
  4. Cuiling Zhang
  5. Hang He
  6. Ruslan Medzhitov  Is a corresponding author
  1. Howard Hughes Medical Institute, Yale University School of Medicine, United States
  2. College of Life Sciences, Peking University, China
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Cite this article as: eLife 2015;4:e02948 doi: 10.7554/eLife.02948

Abstract

The transcriptional coactivator Yes-associated protein (Yap) promotes proliferation and inhibits apoptosis, suggesting that Yap functions as an oncogene. Most oncogenes, however, require a combination of at least two signals to promote proliferation. In this study, we present evidence that Yap activation is insufficient to promote growth in the otherwise normal tissue. Using a mosaic mouse model, we demonstrate that Yap overexpression in a fraction of hepatocytes does not lead to their clonal expansion, as proliferation is counterbalanced by increased apoptosis. To shift the activity of Yap towards growth, a second signal provided by tissue damage or inflammation is required. In response to liver injury, Yap drives clonal expansion, suppresses hepatocyte differentiation, and promotes a progenitor phenotype. These results suggest that Yap activation is insufficient to promote growth in the absence of a second signal thus coordinating tissue homeostasis and repair.

https://doi.org/10.7554/eLife.02948.001

eLife digest

As we grow up, the organs in our body tend to stop growing and then remain roughly the same size for the rest of our lives. This is possible because of control systems that determine how often the cells within the organ can divide and when they should die. If these controls fail, the cells may divide rapidly and not die when they should, which can cause tumors to grow. In healthy cells, the proteins that promote cell division and/or prevent cell death are strictly controlled—usually by at least two different ‘on’ signals—so that they are only active at specific times.

Yap is one such protein that promotes cell division and inhibits programmed cell death. Previous studies have reported that artificially increasing the levels of Yap in cells is sufficient to make tumors grow in a seemingly unrestrained fashion. This suggests that only one signal is required to over-activate Yap. However, it is also possible that these experiments were carried out under conditions where a second unknown growth-promoting signal was also present.

Here, Su, Bondar et al. genetically altered mice to produce more Yap in some, but not all, cells in the liver. This revealed that when surrounded by normal cells, the high levels of Yap in individual cells do not lead to excessive liver growth. The cells do divide more, but this is balanced by an increase in the numbers of cells that die.

However, if the liver is injured, the high levels of Yap can keep the cells in a stem cell-like state and cause them to grow and divide excessively. This helps the liver to recover, and once the recovery is complete, the cells that produce more Yap are killed. Su, Bondar et al. suggest that the excessive cell growth observed in previous studies may be due to the researchers unintentionally simulating the conditions of inflammation by increasing the levels of Yap in all the cells.

These findings reveal that Yap needs to be activated by both a signal from within the cell and a second signal from other cells—caused by injury or inflammation—to promote the growth of tumors.

https://doi.org/10.7554/eLife.02948.002

Introduction

The transcriptional coactivator Yap is an evolutionarily conserved mediator of cell fate decisions such as proliferation, differentiation, and survival (Yu and Guan, 2013). Together with a related protein Taz, Yap is the central target of the Hippo pathway, a growth-suppressive network of kinase complexes that inactivates Yap and Taz (Hong and Guan, 2012). The Hippo pathway has been implicated in organ size control, as overexpression of Yap or inactivation of the Hippo components leads to tissue overgrowth and organomegaly in Drosophila and mouse models (Camargo et al., 2007; Dong et al., 2007; Lin et al., 2013). These effects are largely mediated by Yap-TEAD complexes that activate transcription of genes promoting cell proliferation and inhibiting apoptosis (Wu et al., 2008; Zhang et al., 2008; Zhao et al., 2008). Yap is interconnected with RTK (Reddy and Irvine, 2013), GPCR (Yu et al., 2012), PI3K (Fan et al., 2013), Wnt (Bernascone and Martin-Belmonte, 2013), and TGF-beta (Ferrigno et al., 2002; Attisano and Wrana, 2013; Mullen, 2014) signaling, and Yap co-regulates transcription by interacting with Smads (Ferrigno et al., 2002; Beyer et al., 2013), TCF/LEF (Konsavage and Yochum, 2013), Tbx5 (Beyer et al., 2013), Runx2 (Zaidi et al., 2004), FoxO1 (Shao et al., 2014), and p73 (Strano et al., 2001), among others (Barry and Camargo, 2013).

The intestinal epithelium in both Drosophila (Karpowicz et al., 2010; Ren et al., 2010; Shaw et al., 2010; Staley and Irvine, 2010) and mice does not depend on Yap for homeostatic tissue turnover (Zhou et al., 2011), but it does respond very strongly to Yap overexpression (Barry et al., 2013) and requires Yap for tissue repair (Cai et al., 2010). Thus, in some tissues Yap may be dispensable for homeostasis but required specifically in response to injury. The liver is one of the organs most responsive to excessive Yap activity. Transgenic overexpression of Yap or inactivation of its upstream negative regulators causes a dramatic increase in liver size, hepatocyte proliferation, progenitor cell expansion, and tumorigenesis (Camargo et al., 2007; Dong et al., 2007; Lee et al., 2010; Lu et al., 2010; Zhang et al., 2010; Kowalik et al., 2011; Zheng et al., 2011). In contrast, deletion of Yap in the liver leads to defects in bile duct formation but no apparent defects in hepatocyte number and function (Bai et al., 2012), suggesting that Yap may be dispensable for hepatocyte homeostasis. However, whether its function is required for hepatocyte homeostasis and response to injury remains to be established (Yu et al., 2014).

Yap activation promotes proliferation, survival, stemness, and tumor development in mouse models (Camargo et al., 2007; Dong et al., 2007; Barry and Camargo, 2013) and is commonly observed in human cancers (Fernandez et al., 2009; Wang et al., 2009). Collectively, these data suggest that hyperactivation of Yap abrogates organ size control mechanisms and drives tumorigenesis in a seemingly unrestrained fashion. However, growth-promoting pathways are normally safeguarded by tumor-suppressive mechanisms (Hahn and Weinberg, 2002). For example, c-myc hyperactivation sensitizes cells to apoptosis (Evan et al., 1992), oncogenic Ras induces senescence (Serrano et al., 1997), and overexpression of Bcl-2 inhibits cell proliferation (O'Reilly et al., 1996). Whether or not Yap activity is subject to a similar tumor-suppressive regulation is currently unclear. While Yap is known to interact with p73 and promote apoptosis in response to DNA damage in vitro (Strano et al., 2001; Lapi et al., 2008), there is no evidence that Yap can induce apoptosis in vivo.

Control of cell fate decisions at the tissue level is poorly understood, but it is likely to involve cell contact-dependent regulation. Yap activity is regulated by adherens and tight junctions, cell polarity complexes, and the actin cytoskeleton (Boggiano and Fehon, 2012). At high cell densities, Yap is either directly recruited to intercellular junctions or undergoes phosphorylation and cytosolic retention via the Hippo pathway, which itself is also regulated in a cell contact-dependent manner. This feature makes Yap competent to direct cell fate decisions depending on cell density and architecture. Thus, cell environment may be a major determinant of the outcome of Yap activation. Moreover, evidence from Drosophila (Chen et al., 2012) and mammalian cell culture (Norman et al., 2012) suggest that the outcome of Yap activation depends on Yap activity in neighboring cells. The regulation of Yap activity by such mechanism has not been characterized in mammalian tissues in vivo.

Here, we describe a mosaic model of Yap activation in the mouse liver, where a high level of Yap expression is induced in a fraction of hepatocytes surrounded by normal tissue. We present evidence that a high level of Yap in this context is insufficient to drive clonal expansion, as increased proliferation of Yap-overexpressing hepatocytes is balanced by their increased susceptibility to apoptosis. Yap-mediated clonal expansion requires a second signal provided by disruption of tissue homeostasis, such as injury or inflammation. Upon returning to homeostasis, excessive Yap-overexpressing cells are eliminated by apoptosis.

Results

Generation of mosaic Yap mouse model

To study the role of cell environment in the regulation of the mammalian Hippo pathway, we generated a conditional mosaic mouse model of Yap overexpression (YapKI mice). We used Yap1 mutant S112A which has enhanced nuclear localization and has been previously shown to cause a several-fold increase in liver size when overexpressed (Schlegelmilch et al., 2011; Zhang et al., 2011) in the entire organ. A targeting construct driving expression of the Yap1S112A-IRES-GFP downstream of a floxed transcriptional STOP cassete was knocked into the Gt(ROSA)26Sor (Rosa26) locus. Upon expression of Cre recombinase, the transcriptional STOP cassette is removed, allowing expression of Yap together with GFP (YapKI mice; Figure 1—figure supplement 1).

We used the Rosa26-IRES-GFP targeting construct which is expressed bimodally (Bondar and Medzhitov, 2010) (likely due to epigenetic regulation of the locus), generating GFPlow and GFPhigh populations in all cells of hematopoietic lineages (Bondar and Medzhitov, 2010) and solid tissues tested (liver, pancreas, muscle, intestine; Figure 1—figure supplement 2 and data not shown). Both populations have a recombined STOP cassette, but the gene of interest is expressed above physiological level only in GFPhigh cells. GFPlow cells serve as an internal control population of the identical genetic background for the GFPhigh cells. In the absence of selective pressure, the ratio of GFPlow to GFPhigh cells remains constant throughout life. Thus, crossing YapKI mice to Albumin-Cre (Alb-Cre) yields mosaic Yap expression in the hepatic lineage, where individual cells overexpressing Yap can be traced by GFP fluorescence (YapKIAlb-Cre mice). Similar to the previously reported Rosa26-DNp53 mice (Bondar and Medzhitov, 2010), the proportion of GFPlow to GFPhigh cells remained stable in the blood of YapKICreER mice upon tamoxifen induction for many months (Figure 1—figure supplement 2B–D). In the colons of YapKIVillin-Cre mice, crypts were either entirely GFPhigh or GFPlow (GFPlow appearing as GFP− due to limited sensitivity of GFP detection by tissue immunostaining). There were no crypts that have mixed populations, as would be expected if cells randomly switched expression level (Figure 1—figure supplement 2E). Collectively, these data illustrate stable inheritance of the Rosa26 allele expression level.

First, we verified the correlation of Yap and GFP expression by immunofluorescence staining of the YapKIAlb-Cre liver sections. Indeed, Yap and GFP in hepatocytes displayed mosaic and overlapping expression pattern, with cytosolic as well as nuclear Yap localization (Figure 1A). Utilizing the GFP marker, we tested the recombination efficiency by flow cytometry. More than 90% of hepatocytes were GFP-positive, indicating high deletion efficiency of the STOP cassette (Figure 1B). Recombination was also confirmed by genomic qPCR (Figure 1—figure supplement 3). The hepatocytes formed two distinct populations differing in GFP brightness. To determine the level of Yap activity in these cells, we sorted each of these populations by FACS. Since GFP fluorescence varied 10-fold within the GFPlow population, we further subdivided it into GFPlow and GFPmed-low populations (Figure 1B). The exogenous transcripts of Yap are highly expressed only in the GFPhigh population, but not in the GFPlow and GFPmed-low populations. The small amount of the exogenous Yap mRNA in the GFPlow and GFPmed-low populations is insignificant due to a potential negative feedback mechanism that reduces the endogenous Yap expression, resulting in a similar level of total Yap mRNA compared to that of wild-type (WT) (Figure 1—figure supplement 4). Additionally, CTGF, a known Yap downstream target gene (Zhao et al., 2008), was significantly upregulated only in the GFPhigh population, whereas its expression remained unchanged in GFPlow and GFPmed-low populations, indicating normal levels of Yap activity in these cells (Figure 1C). Thus, GFP and Yap protein levels correlate in YapKIAlb-Cre mice, and Yap is overexpressed and has higher activity specifically in the GFPhigh hepatocytes (to which we will refer as Yaphigh cells).

Figure 1 with 4 supplements see all
YapKIAlb-Cre strain is a hepatocyte-specific mosaic mouse model of Yap activation.

(A) Detection of exogenous Yap and GFP by immunofluorescent staining of YapKIAlb-Cre liver sections. (B) Flow cytometric analysis of GFP fluorescence in primary hepatocytes. The plots are gated on hepatocytes by forward and side scatter and on live cells by excluding 7-AAD positive events. (C) Expression of Yap and CTGF was determined by qPCR in primary hepatocyte populations sorted based on GFP levels as shown in B.

https://doi.org/10.7554/eLife.02948.003

Yap activation in hepatocytes does not lead to clonal expansion at steady state

The first question we addressed is whether liver size is affected by mosaic Yap overexpression. Previous studies showed that hyperactivation of Yap in the entire liver leads to rapid and progressive hepatomegaly: liver weight increases fivefold in 1 month in Yap transgenic mice (Camargo et al., 2007; Dong et al., 2007) and fourfold by 3 months of age in Stk3, Stk4 (Mst1/2) double null mice (Lu et al., 2010; Song et al., 2010) (where Yap is activated due to an absence of upstream negative regulators). However, the liver/body weight ratio of the mosaic YapKIAlb-Cre mice was only slightly elevated (Figure 2A). Hepatocyte numbers (Figure 2B) and liver morphology (Figure 2C) also remained normal. Moreover, Yaphigh cells did not have a growth advantage over the WT neighbors, since neither the average size of Yaphigh cells clusters in the liver sections (Figure 2—figure supplement 1) nor the average frequency of Yaphigh hepatocytes increased over time (Figure 2D).

Figure 2 with 4 supplements see all
Yap overexpression in hepatocytes does not induce hepatomegaly or clonal expansion at steady state.

(A) Liver/body weight ratios of 1- to 3-month-old YapKIAlb-Cre mice (KI) and littermate controls (WT), n ≥ 11. ****p ≤ 0.0001. (B) Total hepatocyte numbers were determined by quantitative flow cytometry of primary hepatocytes isolated from 1- to 3-month-old mice of the indicated genotypes, n ≥ 14. (C) A representative image of H&E staining performed on liver sections of 6-week-old control (WT) and YapKIAlb-Cre mice (KI). (D) Primary hepatocytes were isolated by collagenase perfusion from YapKIAlb-Cre mice of indicated age groups, and percentage of GFPhigh hepatocytes was determined by flow cytometry; n ≥ 11. (E) Mice were injected with BrdU for 3 consecutive days and liver sections were stained with BrdU and GFP antibodies. Percent of BrdU+ hepatocytes was quantified in liver sections of the controls (WT) and within Yaplow (GFP−) and Yaphigh (GFP+) populations of the YapKIAlb-Cre livers. n ≥ 8. (F) TUNEL-positive nuclei were quantified on liver sections of control (WT) and YapKIAlb-Cre (KI) mice. 3 mice were used for each group and 4 images were taken for each mouse. Each dot represents cell count from each image. **p ≤ 0.01.

https://doi.org/10.7554/eLife.02948.008

These results were unexpected, considering the dramatic increase in hepatocyte proliferation and survival driven by Yap in the previously published whole-liver transgenic models (Camargo et al., 2007; Dong et al., 2007). We therefore tested whether Yap influenced proliferation and apoptosis in YapKIAlb-Cre liver. Indeed, more Yaphigh hepatocytes incorporated BrdU than their GFPlow neighbors or hepatocytes in WT livers (Figure 2E). Meanwhile, TUNEL staining indicated more apoptosis in the YapKIAlb-Cre livers (Figure 2F). It is noteworthy that only a minor fraction of the Yaphigh cells proliferated, and over 95% remained quiescent throughout the 3-day BrdU labeling period. These data suggest that increased Yap levels sensitize hepatocytes to both mitogenic and apoptotic signals but are insufficient to drive cell expansion, resulting in slightly increased cell turnover without net growth advantage.

This insufficiency was not due to an overall lack of Yap activation, as Yap overexpression induced a significant change in gene expression in our model. Sorted WT, GFPlow and GFPhigh hepatocytes were compared by RNA-sequencing analysis. Gene expression of GFPlow cells isolated from YapKIAlb-Cre livers was similar to that of the WT hepatocytes, whereas GFPhigh hepatocytes showed robust transcriptional changes: 821 genes were significantly up-regulated (p ≤ 0.005 and fold change ≥2) in Yaphigh hepatocytes compared to WT hepatocytes (Figure 2—figure supplement 2A). 37% of these genes overlapped with genes upregulated in the previously characterized Yap overexpression model (Dong et al., 2007) (Figure 2—figure supplements 2B, 3A–B), further validating our model. Dong et al (Dong et al., 2007) used an ApoE promoter which is not restricted to hepatocytes, and assayed the whole liver that includes stromal, endothelial and immune cells with microarray, whereas we assayed sorted hepatocytes in a model with hepatocyte-specific Yap induction with RNA-sequencing. Thus, a complete overlap in gene profile would not be expected. Yap-induced genes showed an enrichment in cell adhesion, extracellular matrix (ECM), and cell–cell junction functional groups (Figure 2—figure supplement 2C), according to DAVID functional cluster analysis (Huang et al., 2009a, 2009b). In contrast, the cell cycle category was not significantly enriched, likely because only a small fraction of all Yaphigh hepatocytes were proliferating, as evidenced by BrdU labeling.

It is well known that pro-growth effects of Yap overexpression are mediated through TEAD (Wu et al., 2008; Zhang et al., 2008; Zhao et al., 2008), and the dominant-negative TEAD mutant largely suppresses effects of Yap transgene in the liver (Liu-Chittenden et al., 2012). To evaluate how many of the Yap-induced genes are potentially regulated directly by Yap-TEAD complexes, we compared these genes to genes with TEAD binding sites identified previously by ChIP-seq (ENCODE Project Consortium, 2011). Indeed, over 25% of all Yap-activated genes are associated with TEAD compared to only 9% of all genes in the genome. Moreover, a large fraction of genes induced in Yaphigh hepatocytes and related to cell adhesion (33%), ECM (36%), and cell–cell junctions (36%) contain TEAD binding regions (Figure 2—figure supplement 2D). The gene expression data shown here suggest that inability of Yap to drive cell expansion in our model is not due to lack of transcriptionally competent Yap-TEAD complexes.

To test whether mosaic Yap activation can lead to clonal expansion in previously characterized mouse models, we injected Stk4−/−;Stk3flox/flox mice with a low dose of adenovirus expressing Cre and GFP, and compared patterns of BrdU incorporation and GFP+ hepatocytes 5 and 35 days later. At both time points, we could only observe single GFP+ hepatocytes, BrdU negative (Figure 2—figure supplement 4A). Semi-quantitative PCR of the recombined Stk3 (∆Mst2) allele also did not show an increase at 35 days, and liver/body mass ratio remained normal (Figure 2—figure supplement 4B,C).

Taken together, these data suggest that clonal activation of Yap in hepatocytes is not sufficient to overrule growth restricting mechanisms at steady state, but is sufficient to regulate transcription of a large number of genes related to extracellular environment.

Yap promotes hepatocyte proliferation in response to injury

Previous studies demonstrated that Yap is activated during liver injury (Bai et al., 2012; Wang et al., 2012; Anakk et al., 2013) and protects mice from oxidative stress-induced damage (Wu et al., 2013). We therefore tested whether Yap induction provides selective advantage during liver injury. We chose carbon tetrachloride (CCl4) as a damaging agent due to its localized effects around the central vein (CV) (Weber et al., 2003).

We first characterized the kinetics of liver damage after a single injection of CCl4. Blood ALT levels in YapKIAlb-Cre and WT mice rose to similar levels, indicating comparable hepatocyte damage (Figure 3—figure supplement 1). By day 4 the acute damage phase ceased completely (Figure 3—figure supplement 1) and very few hepatocytes incorporated BrdU (Figure 3—figure supplement 2). We therefore chose day 4 to study the outcome of the tissue repair response.

In response to CCl4 damage Yaphigh cells expanded dramatically within 4 days in the pericentral area, comprising over 80% of all hepatocytes in this zone (Figure 3A,B). These pericentral Yaphigh cells incorporated significantly more BrdU during days 1–3 after CCl4 administration compared to Yaplow hepatocytes in the same location (Figure 3C). In contrast, Yaphigh cells proliferated much less in the CV-distal areas than in the pericentral area (Figure 3C). In fact, the BrdU index outside the pericentral zone did not differ between Yaphigh and Yaplow hepatocytes within the same liver. Even though Yaphigh cells expanded locally at the injury sites, the effect was strong enough to significantly increase total percentage of Yaphigh cells in the liver (Figure 3D). Nevertheless, this expansion of Yaphigh cells did not overrule normal liver size control, as the total number of hepatocytes was similar in YapKIAlb-Cre and WT mice (Figure 3—figure supplement 3). Liver/body weight ratios in both groups also returned to steady-state levels within a month after undergoing a similar increase in response to CCl4, (likely due to the infiltration of immune cells during early phase of tissue repair) (Figure 3—figure supplements 3–5). These results indicate that Yaphigh cells undergo expansion in response to local tissue injury signals.

Figure 3 with 5 supplements see all
Injury induces proliferation and clonal expansion of Yap-overexpressing hepatocytes.

(A) Liver sections of control (WT) and YapKIAlb-Cre (KI) mice isolated at steady state (SS) or on days 4 and 14 after CCl4 treatment were stained with antibodies to GFP and BrdU. BrdU was injected in all groups for 3 consecutive days before harvesting the livers. (B) The percentage of Yaphigh (GFP+) cells among all hepatocytes was quantified within the pericentral zone (‘damage site’) and outside the pericentral zone (‘outside’) and in the liver sections of YapKIAlb-Cre mice on day 4 after CCl4 treatment. 3–4 mice were used for each group and 3–4 images were taken for each mouse. Each dot represents cell count from each image. ****p ≤ 0.0001. (C) The percentages of BrdU+ hepatocytes among GFP− and GFP+ hepatocytes were quantified outside the CV zone (‘outside’) and around CV (‘damage site’) in liver sections of YapKIAlb-Cre mice on day 4 after CCl4 treatment. 3–4 mice were used for each group and 3–4 images were taken for each mouse. Each dot represents a cell count from each image. ****p ≤ 0.0001. (D) Hepatocytes were isolated by collagenase perfusion from 1- to 2-month-old YapKIAlb-Cre mice at steady state (SS), at day 4 (D4) and day 14 (D14) after CCl4 treatment, or after a 6-day LPS treatment (LPS), and percentage of GFPhigh cells determined by flow cytometry.

https://doi.org/10.7554/eLife.02948.013

To investigate whether Yap activity in hepatocytes is required for tissue repair, we generated Yap conditional knockout mice and crossed them to the AlbCre strain to obtain hepatocyte-specific Yap knockout Yap1flox/flox;AlbCre (YapCKOAlb-Cre) mice (Figure 4—figure supplements 1, 2). These mice were treated with CCl4 using the same protocol as described above. A similar increase in blood ALT level in YapCKOAlb-Cre and WT mice indicated comparable liver damage in the first 2 days (Figure 4—figure supplement 3). By day 4, ALT levels returned to baseline (Figure 4—figure supplement 3), normal pericentral morphology was restored, and expression of the CV-proximal marker glutamine synthase was reestablished in both groups (Figure 4A). However, YapCKOAlb-Cre hepatocytes proliferated significantly less than their WT counterparts throughout the repair phase, as shown by BrdU labeling on days 1–3 (Figure 4B,C). Consistent with the defective regeneration, YapCKOAlb-Cre livers displayed abnormal collagen deposition around the CV (Figure 4D). Interestingly, the steady-state liver/body mass ratio (Figure 4—figure supplement 4) and BrdU incorporation rate (Figure 4—figure supplement 5) were higher in the YapCKOAlb-Cre mice than in the WT mice, suggesting that Yap is not required for homeostatic hepatocyte proliferation.

Figure 4 with 5 supplements see all
Yap function in hepatocytes is required for tissue repair.

(A) Expression of glutamine synthase detected by immunofluorescent staining of littermate controls (WT) and YapCKOAlb-Cre YapCKO livers on day 4 after CCl4 treatment. CV, central vein. (B) BrdU staining illustrating lack of hepatocyte proliferation in Yap-deficient hepatocytes in response to CCl4-induced injury. The results are quantified in Figure 4C. (C) Total BrdU+ hepatocytes in WT, YapKIAlb-Cre (KI) and YapCKOAlb-Cre (CKO) mice were quantified outside the CV zone (‘outside’) and around CV (‘damage site’) in liver sections on day 4 after CCl4 treatment. 3–4 mice were used for each group and 3–4 images were taken from each mouse. Each dot represents a cell count from each image. (D) Excessive collagen deposition in YapCKOAlb-Cre mice on day 4 after CCl4 treatment revealed by Sirius Red staining.

https://doi.org/10.7554/eLife.02948.019

Thus, while Yap is not required for liver development and hepatocyte proliferation under normal conditions, Yap function is required for hepatocyte proliferation during tissue repair. Additionally, hepatocytes with a higher level of Yap have growth advantage during the early repair phase.

Yap promotes hepatocyte proliferation in response to inflammation

Given differential effects of Yap on hepatocyte proliferation during homeostasis vs injury, we wondered how growth control mechanisms might differ under these conditions. Tissue injury is accompanied by inflammation, which can promote proliferation by mechanisms distinct from homeostasis. To test whether Yap cooperates with inflammatory signals to drive cell cycle progression, we measured hepatocyte BrdU incorporation in YapKIAlb-Cre mice after 6 continuous days of intraperitoneal injections of LPS. This chronic LPS treatment induced a massive expansion of Yaphigh hepatocytes, similar to what was seen after CCl4 treatment (Figure 3D). Consistently, the BrdU incorporation index indicated that Yaphigh hepatocytes proliferated significantly more than hepatocytes in the WT livers (Figure 5A,B). YapKIAlb-Cre livers displayed significant hyperplasia, as visualized by increased hepatocyte density (Figure 5C) and measured by total number of hepatocytes per liver (Figure 5D).

Figure 5 with 2 supplements see all
Inflammation induces proliferation of Yap-overexpressing hepatocytes.

(A) Control (WT) or YapKIAlb-Cre mice were coinjected with BrdU and LPS for 6 days. Percentage of BrdU+ hepatocytes was quantified. 4–6 mice were used for each group and 3–4 images were taken for each mouse. Each dot represents cell count from each image. (B) A representative image of chronic (6xLPS) LPS injected livers used for the quantification in A. Arrows indicate BrdU+ hepatocyte nuclei. (C) H&E representative images of the chronic LPS-treated livers. (D) Hepatocyte numbers of the chronic LPS-treated livers was measured by quantitative flow cytometry.

https://doi.org/10.7554/eLife.02948.025

We then tested whether IL-6, an inflammatory cytokine induced by LPS and by liver injury, can cooperate with Yap in promoting hepatocyte growth. When IL-6 was produced by LLC cells growing as subcutaneous tumors in YapKIAlb-Cre mice, it caused expansion of Yaphigh hepatocytes in the livers of mice that had the highest levels of systemic IL-6 (Figure 5—figure supplement 1A,B), suggesting that IL-6 may cooperate with Yap to promote hepatocyte proliferation.

Yap upregulates progenitor markers and represses hepatocyte differentiation in response to injury

4 days after CCl4 treatment in WT mice, normal liver tissue morphology was restored around the CV. In contrast, we observed high cell density, strong cytoplasmic eosinophilia, round to ovoid nuclei, and increased nuclear/cytoplasmic ratio in the pericentral zones of livers from CCl4-treated YapKIAlb-Cre mice (Figure 6A). These morphological changes are often seen in hepatocellular carcinomas (HCC) and are associated with dedifferentiation (Chang et al., 2010; Balani et al., 2013). Immunofluorescent staining indicated that these cells were GFP+ and therefore originated from Yaphigh hepatocytes (Figure 3A). To determine whether Yap induction affects differentiation status of hepatocytes, we compared the gene expression of hepatocytes sorted from YapKIAlb-Cre mice at steady state and 4 days after CCl4 damage. The majority of liver-specific transcripts (Pan et al., 2013) (which roughly represent hepatocyte differentiation state) were strongly repressed in Yaphigh hepatocytes from CCl4-treated mice, whereas WT and Yaplow hepatocytes showed minimal alterations and no bias towards decreased expression (Figure 6B). Immunofluorescent staining and Western blot for hepatocyte-specific gene glutamine synthase also showed a strong downregulation of expression in YapKIAlb-Cre livers after CCl4 damage (Figure 6C,D). Downregulation of other hepatocyte-specific genes (acute phase protein Orm1 and urea cycle component Asl) was also confirmed by qPCR (Figure 6—figure supplement 1). To further verify RNA-sequencing data and evaluate levels of Yap activation in our model, we performed qPCR analysis of the genes previously shown to be induced in Yap-Tg liver by Pan's group (Dong et al., 2007) and found comparable effects (Figure 6—figure supplement 1).

Figure 6 with 1 supplement see all
Yap activation cooperates with tissue injury to repress hepatocyte differentiation and promote progenitor phenotype.

(A) H&E staining of liver sections of control (WT) and YapKIAlb-Cre (KI) mice isolated on day 4 after CCL4 treatment. (B) Heatmap of RNA-sequencing data comparing expression of liver specific genes (see ‘Materials and methods’). Hepatocytes were sorted from wild-type livers (WT) or from YapKIAlb-Cre livers based on GFP levels (Lo and Hi), at steady state (SS) or on day 4 after CCl4 treatment (CCl4). The fold change is calculated between the indicated samples and WT in steady state. Data represent the mean of the duplicates. (C) Liver sections of control (WT) and YapKIAlb-Cre (KI) mice isolated on days 4 CCL4 treatment were stained with antibodies to glutamine synthase. (D) Glutamine synthase (GS), Yap and beta-actin protein levels in whole-liver protein lysates prepared on day 4 after CCl4 treatment were determined by Western blotting. Higher migrating band in the middle panel corresponds to the exogenous Yap (due to the in-frame triple flag tag). (E and F) Heatmaps showing the RNA-sequencing data for genes enriched in embryonic tissues (E) or progenitor markers (F). Hepatocytes were sorted from wild type livers (WT) or from YapKIAlb-Cre livers based on GFP levels (Lo and Hi), at steady state (SS) or on day 4 after CCl4 treatment (CCl4). The fold change is calculated between the indicated samples and wild type in steady state. (G and H) Flow cytometric analysis of primary hepatocytes isolated by collagenase perfusion from CCl4-treated control (WT) or YapKIAlb-Cre (KI) mice. (G) Representative flow cytometry plots gated of CD45 CD31 population. The numbers on the plots indicate the percentages of the gated population (EpCam+ progenitors). (H) The results from (G) were combined with the total hepatocyte numbers to calculate the number of hepatic EpCam+ progenitors per liver.

https://doi.org/10.7554/eLife.02948.028

The transcriptional repression was not global: a number of genes expressed predominantly during embryogenesis (Pan et al., 2013) were induced in Yaphigh hepatocytes, and this effect was strongly enhanced by injury (Figure 6E). In addition, several hepatocyte progenitor markers were upregulated in Yaphigh cells after injury, and some of them were already elevated in the steady state (Figure 6F). FACS staining of liver cells isolated on day 4 post-CCl4 confirmed expansion of CD45 CD31 EpCam+ hepatic progenitors in YapKI;AlbCre livers (Figure 6G,H). These data are in line with the recent report by Yimlamai et al. (2014).

Altogether, these data suggest that Yap cooperates with injury signals to repress hepatocyte differentiation and promote a progenitor phenotype.

Excessive Yaphigh cells are eliminated during repair resolution phase

The expansion of Yaphigh cells induced by CCl4 did not persist, and by day 14 after the treatment, the proportion of Yaphigh cells per liver returned to the average value of the untreated group (Figure 3D). Yaphigh cells formed a ring of only 1–2 cell layers around CVs, in contrast to multiple layers observed on day 4 (Figure 3A), indicating that the Yaphigh cell pool underwent contraction. TUNEL staining suggested that the contraction was mediated by apoptosis: the number of TUNEL positive cells was markedly elevated in the YapKIAlb-Cre livers specifically around the CV on day 4 after CCl4 treatment (Figure 7A). Accordingly, RNA sequencing revealed a number of pro-apoptotic genes (e.g., Bok, Bax, NGFRAP1, and Tnfrsf10b (Dr5)) (Figure 7B) upregulated as a consequence of Yap overexpression and further induced after damage. We verified by qPCR that Dr5 gene expression was strongly upregulated in the Yaphigh cells after CCl4 (Figure 7C,D). Indeed, Yaphigh cells sorted from CCl4-treated livers were more sensitive to killing mediated by TRAIL in vitro, compared to WT or GFPlow counterparts (Figure 7E), suggesting that Yap activation sensitizes hepatocytes to Dr5-mediated cell death. This higher sensitivity to cell death was induced by a combination of injury and Yap overexpression, as it was not observed neither in Yaphigh hepatocytes isolated from untreated mice nor in WT hepatocytes from CCl4 livers.

Yap sensitizes hepatocytes to TRAIL-mediated apoptosis.

(A) Cell death as reflected by TUNEL-positive cell numbers quantified outside the CV zone (‘outside’) and around CV (‘damage site’) in liver sections of YapKIAlb-Cre mice on day 4 after CCl4 treatment. 3–4 mice were used for each group and 3–4 images were taken from each mouse. Each dot represents cell count from each image. (B) RPKM data from RNA-sequencing illustrating expression of apoptosis-related genes in WT or Yaphigh (Hi) hepatocytes sorted from untreated (SS) livers or on day 4 after CCl4 treatment (CCl4). (C and D) Dr5 mRNA levels in WT and YapKIAlb-Cre livers (KI) at indicated time points after CCl4 treatment were determined by qPCR in whole liver extracts (C), or in primary hepatocytes sorted by flow cytometry based on GFP levels from steady-state livers (SS) or on day 4 after CCl4 treatment (CCL4) (D). 2–3 mice were used for each group. *p ≤ 0.05. (E) Primary hepatocytes sorted as in (D) were cultured on collagen-coated plates with or without TRAIL, and cell viability measured by CellTiter-Blue the next day. 3–4 mice were used for each group and 3–4 wells were seeded with hepatocytes from each mouse. Each dot represents reading from each well.

https://doi.org/10.7554/eLife.02948.030

In summary, the expanded population of Yaphigh cells contracts to baseline at later stages of the tissue injury response. This process is likely mediated, in part, by sensitization of Yaphigh cells to TRAIL-induced apoptosis via upregulation of Dr5.

Discussion

Yap has emerged as a central mediator of signals promoting proliferation, survival, and stemness (Barry and Camargo, 2013). Not surprisingly, an expanding number of pathways restricting the pro-growth potential of Yap have been identified, including the Hippo pathway, the actin cytoskeleton and cell junction complexes (Boggiano and Fehon, 2012), C/EBPa, and Trib2 (Wang et al., 2013). Despite these multifactorial control mechanisms, mere overexpression of Yap in the whole liver in mouse models leads to hyperplasia, organomegaly, dedifferentiation, and cancer (Camargo et al., 2007; Dong et al., 2007; Schlegelmilch et al., 2011), raising a question as to whether some layer of homeostatic growth control may be lacking in such models. Organ-wide Yap activation in the liver does occur during embryonic development (Septer et al., 2012) and adaptive hepatomegaly (Kowalik et al., 2011). Interestingly, these are also examples of physiologically relevant settings of liver size increase. Transgenic Yap overexpression in the entire liver may thus be viewed as a model of these physiological scenarios. In contrast, during response to a localized injury, only the cells within the damage site need to proliferate, and Yap activation in this case should conform to the physiological organ size limits. Our study suggests that under these circumstances, activation of a pro-growth gene program by Yap requires an additional local signal (e.g., growth factor, cytokine, or release of contact inhibition) provided by the injury. Our preliminary data implicate IL-6 as at least one of such signals (Figure 5—figure supplement 1). Proto-oncogenes typically require two signals in order to induce proliferation. In case of Yap, which acts as a sensor of cell density, its overexpression mimics a condition in which a cell has lost its contacts with the neighbors. This in itself should not induce proliferation unless cell-extrinsic signals of tissue damage such as inflammatory cytokines are also present. The mechanism of cooperation between activation of Yap and injury/inflammatory signals may occur at the level of gene expression, as many proliferation genes have TEAD as well as NFkB binding sites (Figure 5—figure supplement 2). While it is also possible that cooperation is mediated by increased Yap stability or recruitment of TEAD, this possibility is less likely as we observed induction of high number of TEAD-dependent genes in Yap-overexpressing hepatocytes whereas proliferation genes were not induced.

Our data suggest that the tissue environment imposes a selective pressure on cells that activate Yap. The mosaic model of Yap induction described here reveals selective pressures that determine the fate of Yaphigh hepatocytes: no growth advantage at steady state, positive selection during early stages of tissue injury, and negative selection at later stages when injury induced signals subside. The mechanisms opposing clonal expansion of Yaphigh hepatocytes under homeostatic conditions may have many components, but one implicated by our data is apoptosis. Pro-apoptotic effects of Yap have been extensively documented in vitro (Strano et al., 2001; Lapi et al., 2008; Tomlinson et al., 2010), and increased apoptosis has been reported in Mst1/2-deficient mouse livers (Lu et al., 2010), but to our knowledge there has been no evidence of Yap promoting cell death in vivo. We show that Yap activation in the mouse liver leads to increased apoptosis in vivo and sensitization to TRAIL measured ex vivo. The induction of proliferation or apoptosis by Yap is modulated by tissue damage and inflammatory signals. This is analogous to the well-characterized functions of c-myc, which is also known induce proliferation or apoptosis, depending on growth factor availability (Evan et al., 1992; Harrington et al., 1994). These examples may represent a general principle of mitogenic pathway design, aimed at elimination of aberrant cells.

YapKIAlb-Cre mice display many features described in other in vivo models of Yap induction, including increased proliferation, dedifferentiation, and activation of TEAD target genes. However, the unexpected phenotype unique to YapKIAlb-Cre mice is that the pro-growth effects of Yap are only manifested in a state of altered homeostasis, such as tissue injury or inflammation. These differences are unlikely to be due to an insufficient level of Yap expression, as a large number of genes are highly induced by Yap under steady-state conditions in YapKIAlb-Cre mice. One relevant difference may be mosaic vs tissue-wide Yap activation. We show that mosaic deletion of Mst2 in Mst1−/− background does not lead to clonal expansion or liver size increase. Furthermore, in another mouse model recently reported by Camargo et al. (Yimlamai et al., 2014) while this manuscript was under consideration, mosaic Yap activation in hepatocytes did not induce proliferation of clonal expansion in the first weeks. Instead, it promoted hepatocyte dedifferentiation into progenitors, which then expanded. Taken together, these results suggest that activation of Yap in hepatic progenitor cells is sufficient for clonal expansion but in mature hepatocytes it requires the second signal to drive proliferation. Another possible cause of different phenotypes may lie in different levels of inflammation between our mouse model and the whole liver transgenics. Of note, livers with a hepatocyte-specific ablation of Mst1/2 or Sav (both of which result in Yap activation) have elevated expression of immune response genes, including TNFa and IL-6 (Lu et al., 2010). Many variables may contribute to differences in systemic inflammation. One likely contributor is the intestinal microbiome composition, which varies greatly across mouse facilities (Bleich and Hansen, 2012) and can induce inflammatory responses in the liver (Henao-Mejia et al., 2012). Another source of inflammation in case of whole-liver Yap overexpression may be the disruption of systemic metabolism due to Yap-mediated repression of liver-specific genes ([Yimlamai et al., 2014] and our RNA-sequencing analysis). As liver-specific genes are still expressed at normal levels in a large fraction of hepatocytes (Yaplow cells), systemic metabolism is not compromised in YapKIAlb-Cre mice. It is therefore possible that the level of systemic inflammation is low in our mice, and this is why they require an exogenous inflammatory signal to enable Yap pro-growth effects. Inflammation is an essential component of carcinogenesis (Ben-Neriah and Karin, 2011) and anti-inflammatory treatments can inhibit aberrant proliferation and delay tumor development (Pribluda et al., 2013). Inflammation can promote tumor development through multiple mechanisms (Ben-Neriah and Karin, 2011), including provision of a permissive signal for proliferation in the settings of disturbed homeostasis (Bondar and Medzhitov, 2013; Pribluda et al., 2013).

Our RNA-sequencing analysis has revealed two important functions of Yap in hepatocytes: first, the largest category of Yap-regulated genes is related to ECM and cell adhesion. These transcriptional targets of Yap appear to be the most common across many cell types and contexts of Yap activation; for example, the gene widely used as a hallmark of Yap activation is a matricellular growth factor CTGF. Since loss of cell contacts is known to activate Yap, it makes sense that Yap induces a restorative transcriptional program. Second, Yap strongly suppresses liver-specific genes in response to injury. Hepatocytes are known to downregulate liver-specific transcripts as they enter the cell cycle during liver regeneration (Ito et al., 1991). Proliferation and tissue-specific activities are often segregated between progenitor and differentiated cells, especially in tissues with high cell turnover. This suggests that extensive proliferation may be generally incompatible with specialized tissue functions.

In conclusion, tissue remodeling, cell cycle progression, and repression of tissue-specific functions all need to be orchestrated in response to injury, and our findings suggest Yap as a key coordinator of these activities during tissue repair. Furthermore, our data highlight the role of tissue microenvironment in the outcome of Yap activation and argue that during homeostasis, growth-promoting function of Yap in differentiated cells requires an additional signal, which may be provided by inflammation or injury.

Materials and methods

Generation of YapKI and YapCKO mice

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To generate the YapKI and YapCKO lines, targeting vectors were designed and constructed as shown in Figure 1—figure supplement 1A and Figure 4—figure supplement 1. NM-001171147 mouse Yap1 isoform with introduced S112A mutation (corresponding to S127A of the human protein) was used to generate YapKI allele. After the successful construction of the targeting vectors, the purified plasmid DNA was linearized and transfected into the C57BL/6-derived Bruce4 ES cells. Correct recombination and replacement of the DNA were verified by Southern blots. Selected clones were expanded and submitted to the Yale Transgene Facility for injections into BALB/c blastocysts to generate chimeric mice. In case of the YapCKO line, mice with the germline-transmitted Yap floxed allele were then crossed to Flp-deleter transgenic mice (ENCODE Project Consortium, 2011) to remove the neomycin cassette.

Mouse treatments

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Animals were maintained at the Yale Animal Resources Center. All animal experiments were performed with approval by the Institutional Animal Care and Use Committee of Yale University. All mice were on C57BL/6 background. AlbCre mice were from Jackson Laboratory. For BrdU staining, daily intraperitoneal (i.p.) injections of BrdU (Sigma, St. Louis, MO; 100 μg/mouse) were performed for 3 days before liver dissection. Liver damage was induced by i.p. injection of CCl4 (Sigma, 1 μl/g, diluted 1:10 with peanut oil). For the LLC-IL-6 experiment, 1 million LLC cells stably expressing mouse IL-6 or empty vector were injected into each flank of 1- to 1.5-month-old YapKIAlb-Cre mice. 2–3 weeks later when tumors reached approximately 1 cm in diameter, the mice were eye-bled for IL-6 ELISA and sacrificed for hepatocyte cell preparation. For the in vivo deletion of Mst2flox/flox allele, AdCMVCre-RSV-GFP adenovirus from KeraFast was amplified in 293A cells, purified by ViraPur kit, dialized against PBS and injected into the tail vein of the 1- to 2-month-old Mst1−/− Mst2flox/flox mice.

Immunofluorescence microscopy

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Liver lobes were fixed in 4% PFA overnight and embedded in paraffin. Sections were deparaffinized and heated for 20 min at 95°C for antigen retrieval in Tris-EDTA buffer pH 9.

TUNEL staining was performed using In Situ Cell Death Detection Kit (Roche, Germany).

For GFP, Yap, and GS immunofluorescence staining, samples were then blocked in TBS-T with 3% BSA for 2 hr (staining buffer), incubated with the primary antibody overnight and with the secondary antibody for 1 hr. For BrdU staining, after the described steps, the samples were fixed in 4% PFA in PBS for 20 min at RT. DNA was then denatured in 2 N HCl for 30 min at 37°C. After neutralization with 0.1 M sodium tetraborate, the sections were stained with mouse anti-BrdU antibodies for 2 hr and visualized with flourophore-conjugated anti-mouse secondary antibodies. The slides were mounted in Vectashield anti-fade media containing Hoescht dye to counterstain the nuclei. 20–30 20× tiled images were obtained using Zeiss Axioplan microscope and AxioVision software. Cells were counted manually in AxioVison software. Only hepatocytes (as determined by morphology and GFP expression where applicable) were scored for BrdU quantification.

Hepatocyte FACS

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Primary hepatocytes were prepared by collagenase perfusion at Yale Liver Center. Hepatocytes were washed twice with FACS buffer (2 mM EDTA, 2% FBS in PBS), stained with 7-Aminoactinomycin D (7AAD) or propidium iodide, and analyzed on BD Accuri or LSRII, or sorted on MoFlo cytometers. Samples were gated on live hepatocytes based on forward and side scatter and live dye exclusion.

RNA sequencing analysis

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Duplicate hepatocyte samples were prepared in several independent experiments. Each of the duplicates contained material pooled from multiple mice. Hepatocytes were isolated by flow cytometry, and RNA was extracted using RNeasy kit (Qiagen, Germany). 1–10 μg of each sample was submitted to the Yale Center of Genome Analysis.

Sequencing libraries were constructed and were sequenced by Illumina Hiseq 2500 with 76 bp single-end reads, which generated 20 million raw reads per sample on average. After removing the low-quality reads (around 0.18% of all reads) and low-quality portions (Q value <30) of each of the raw reads, the RNA-sequencing data for each sample were mapped to the GRCm38/mm10 mouse reference first. The GRCm38 reference was downloaded from the Mouse Genome Informatics (MGI). The mapping was performed using TopHat v2.0.8, allowing two mismatches. The percentages of mapped reads were 71.16% on average (from 70.00% to 73.79%). After mapping, Cufflinks v2.0.2 was applied to assemble and quantify the transcripts and discover the differentially expressed genes, with the annotated gene information from the MGI. The gene expression values were calculated for each sample based on the number of fragments per kilobase of exon per million reads mapped (FPKM). The significance of differential expression of genes was detected using Cuffdiff for all comparisons of every two samples.

To select differentially expressed genes, the average of two biological replicates was compared between the conditions examined. To select genes with significant activation or repression in any comparison, the p-value of 0.005 was used together with a fold-change threshold of twofold. A pseudocount of 0.01 was added to RPKM value of each gene as a sequencing background, to avoid inflated fold change caused by lowly expressed genes.

Gene functional analysis

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Gene functional analysis was performed with the DAVID functional annotation tools (Huang et al., 2009a, 2009b) (http://david.abcc.ncifcrf.gov). The p-value was adjusted with Benjamini methods for multiple hypothesis testing.

Analysis of tissue-specific genes

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Tissue-specific genes were obtained from Pattern Gene Database (Pan et al., 2013) (http://bioinf.xmu.edu.cn/PaGenBase). Genes expressed specifically (only in one tissue) and selectively (only in a group of samples) were pooled together as the tissue-specific genes in our analysis. Liver-specific genes are identified from mouse liver sample, and mouse embryo specific genes are identified from mouse day 9.5 embryo sample.

Transcription factor binding analysis

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Genome-wide binding of Tead4 (identified by ChIP-sequencing) was retrieved from the ENCODE Project Consortium (2011). The uniform peaks determined in all available samples (HepG2 cell line, K562 cell line, and hESC) were combined, and genes whose TSS is within 10 kb distance from any Tead4 uniform peak were defined as Tead4-associated genes. Hg19 genome annotation was downloaded from the UCSC database (Karolchik et al., 2014).

TRAIL sensitivity assay

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Primary hepatocytes were plated in collagen-coated wells in Williams' E medium (WEM) supplemented with 5% FCS, 10 mM HEPES, 2 mM L-glutamine, 10 mM penicillin/streptomycin, 8 μg/ml gentamicin, 100 μg/ml chloramphenicol, 100 nM dexamethasone, and 1 nM Insulin. 4 hr after plating, the medium was changed to the supplemented WEM without chloramphenicol and dexamethasone. On the second day cells were treated with recombinant TRAIL (R&D Systems, Minneapolis, MN) overnight. Viable cell number was measured by the CellTiter-Blue assay (Sigma) according to the manufacturer protocol.

Western blotting

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Whole liver tissue was snap frozen in liquid nitrogen, homogenized, and resuspended in TNT buffer (0.1 M Tris-HCL pH 7.5, 150 mM NaCl, 0.1% Tween20) supplemented with the Complete protease inhibitors (Roche) and cleared by centrifugation at 100×g. Protein concentration in the supernatants was normalized using Bradford reagent. 15 μg of the extracts were separated on a gradient SDS-PAGE gel and transferred to PVDF membrane. After blocking with 3% BSA in TBST buffer, the membranes were probed with antibodies to Yap (Cell Signaling, Beverly, MA), glutamine synthase (Genscript, Piscataway, NJ), and beta-actin (Sigma).

ALT test

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ALT tests were performed using ALT activity assay (Sigma) according to the manufacturer's protocol.

Real-time PCR

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RNA was extracted with RNA-bee, and cDNA synthesis was performed using SMART MMLV reverse transcriptase according to the manufacturers' protocols. Real time PCR was performed in triplicates using Quanta SYBR reagent on C1000 Thermal Cycler (BioRad, Hercules, CA). Primer sequences are listed in the Supplementary file1.

Liver genomic PCR

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Three 1 mm3 pieces of liver tissue per mouse were digested with Pronase overnight, and genomic DNA isolated by NaCl/ethanol precipitation. The three DNA samples for each mouse were pooled and used at fourfold dilutions to amplify the Rag2 or recombined Stk3flox/flox alleles using Taqman polymerase.

Enzyme linked immunosorbent assay (ELISA)

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Paired antibodies against IL-6 were purchased from BD Biosciences to perform ELISA.

Statistical analysis

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All statistical analysis was performed by two tailed unpaired Student's t test, unless specified otherwise.

Accession numbers

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The RNA sequencing data have been deposited to GEO database under the accession number GSE65207.

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Decision letter

  1. Alejandro Sánchez Alvarado
    Reviewing Editor; Howard Hughes Medical Institute, Stowers Institute for Medical Research, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “Two-signal requirement for growth-promoting function of Yap” for consideration at eLife. Your article has been favorably evaluated by Tadatsugu Taniguchi (Senior editor), Alejandro Sánchez Alvarado (Reviewing editor), and 4 reviewers, one of whom, Sergei Grivennikov, has agreed to share his identity.

The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

This timely and interesting manuscript reports intriguing and important findings:

1) That high clonal expression of the Yap oncogene in the mouse liver does not cause hepatomegaly and overgrowth as seen with expression of Yap across the entire liver.

2) That in addition to Yap activation, another signal from inflammation or injury provides the context for which Yap may promote growth.

By limiting the expression of Yap to a small number of cells, this study reveals a new level of complexity for the role of Yap in tissue homeostasis that has been thus far so unappreciated. The studies reported in this manuscript begin to reveal that additional cues are necessary for Yap to realize its full pro-proliferative power, and identifies built-in pro-apoptotic restrictive mechanisms. Overall, the manuscript nicely demonstrates the link between acute injury and liver cell repair responses in the YAP mosaic overexpression mice. Additionally, the interplay between proliferation and cell death is laid out clearly. Although the link between inflammation/injury and full blown YAP pathway activation is still somewhat underdeveloped, the concept is of enough novelty that it will be beneficial for this rather unique model, data, and concept describing YAP biology be published.

Before this manuscript can be accepted for publication in eLife, there are, however, a number of issues that can be largely addressed via text modification, better explanations and sometimes inclusion of data the authors claim that they already have but did not include into the current manuscript.

Major points:

1) For systemic treatment of LPS, it would be nice to see cytokine profiles in the liver in WT vs. YAPKI mice, etc. The authors discuss at length about possible cooperation of NF-kB and TEAD as one of the possible mechanisms of cooperation between YAP overexpression and inflammation. They also mention TNF as one of the cytokines. However, the authors claim that one of the links is IL-6 activated by LPS in myeloid cells in an NF-kB dependent manner and acting on hepatocytes in an NF-KB independent fashion, likely via a STAT3 dependent manner. Il-6 data should be shown, as it is one of the novel and clearly mechanistic aspects of this study. If the data are not readily available, the authors should consider removing this line of mechanistic argumentation from the text.

2) Did the authors stain for markers of senescence in addition to markers of proliferation and apoptosis with clonal Yap overexpression? The failure of oncogene overexpression to promote growth is usually associated with apoptosis and/or senescence; this could also explain lack of clonal expansion.

3) The authors quantify BrdU and TUNEL but show minimal images used for quantification. In mosaic studies, there are often “border” issues. Is there any pattern of proliferating or dying Yap high cells (center of clone, on the border)? Is there an increase in proliferation or death in cells adjacent to Yap clones? The authors should show images that reveal or rule out patterns or make a text statement addressing this.

4) The authors should discuss and interpret their findings in light of the Yimlamai paper. One possibility (among many) is that the Su et al. model results in less Yap activation than what was published in the literature, either due to different expression levels or protein activities. This interpretation would be consistent with the lower induction of several Yap target genes reported in the present manuscript compared to what was previously reported. For example, H19 and AFP were barely induced in the Su et al. model, but significantly induced in the Dong et al. (2007) model. Given the Yimlamai et al. paper, it would be difficult for the authors to definitively draw the conclusion that mosaic Yap activation does not yield clonal expansion. What they can conclude is that under a condition in which Yap overexpression can drive the expression of at least some of the target genes, it is insufficient to drive clonal expansion.

5) Related to point #5 above, the authors did not provide details of the YAP cDNA that was used to make their Yap112A mice. Since there are 8 different isoforms of YAP that differ in activities, it is important to know which isoform was used in the paper. This is obviously important given the discrepancies with prior published observations by other laboratories.

6) Figure 3–figure supplement 3A: While ALT rise to the same levels, the temporal pattern is different (see also Figure 4–figure supplement 3). Is this indeed the case reproducibly? How can this be explained seeing that most of the liver in the KI mice is practically WT?

7) The authors in some instances overstate their own findings and should rephrase misleading and inappropriate statements made throughout the manuscript regarding published literature. Some (not all) examples are:

They comment that Yap deletion does not markedly affect liver size or function. Previous literature (including Zhang et al., 2010, Developmental Cell) showed Yap knockout in the liver produced pale and enlarged livers with a number of defects.

The discussion of Drosophila scrib clones is inappropriate. scrib loss across an entire organ results in neoplastic overgrowth of slow-growing tissue while elimination of clones occurs through cell competition when clones are out-competed by faster growing wild-type tissue. This is a very different context than oncogene overexpression being constrained and should not be invoked to justify the authors' model.

In describing Figure 7, they write “RNA sequencing revealed a number of pro-apoptotic genes… induced by Yap.” As worded, this suggests that Yap directly induces these genes. However, the authors have not shown direct induction of these genes by Yap, they have only shown that there is a relationship.

They state that their findings “argue that growth-promoting function of Yap requires a signal derived from loss of tissue homeostasis such as inflammation or injury.” Do they mean Yap cannot promote growth in the absence of injury in all contexts? They should rephrase their language to indicate their findings argue for their specific context.

https://doi.org/10.7554/eLife.02948.032

Author response

1) For systemic treatment of LPS, it would be nice to see cytokine profiles in the liver in WT vs. YAPKI mice, etc. The authors discuss at length about possible cooperation of NF-kB and TEAD as one of the possible mechanisms of cooperation between YAP overexpression and inflammation. They also mention TNF as one of the cytokines.

We address the question of the cytokine expression levels here. We will address whether Yaphigh hepatocytes are more responsive to these cytokines in the second part of this question.

We have measured the levels of TNFa, IL-1b, IL-6 and CCL2 in whole livers of WT and YapKI mice at steady state and after chronic LPS treatment (Author response image 1, top left panel). YapKI livers had elevated IL-1b at steady state, and CCL2 at steady state and in chronic LPS condition. To determine whether this increase was hepatocyte-intrinsic, we looked and IL-1b and CCL2 levels in sorted hepatocytes samples devoid of contaminating stromal (CD38-Thy1.2-) and immune cells (CD45-) at steady state and after CCl4 treatment (Author response image 1, top right panel). CCL2 levels were increased in the GFPhigh hepatocytes, confirming cell-intrinsic effects of Yap on activation of this gene. This is also consistent with our RNA-seq data.

IL-1b was not elevated in GFPhigh YapKI hepatocytes (Author response image 1, top right panel), thus its increased levels in the whole YapKI liver are likely due to increased number or activation of accessory cells (please also see our response to the major point 3, regarding accessory cell proliferation). Similarly, we did not see a significant increase in IL-6 or TNFa in the mosaic YapKI livers (Author response image 1, top left panel). It was only observed in the YapKI livers with a high percentage of GFPhigh cells(Author response image 1, bottom left panel). Of note, purified hepatocytes from livers with high percentage of Yap high cells did not have elevated IL-6 levels (Author response image 1, bottom right panel). These results strongly suggest that IL-6 is induced in the liver stromal cells by Yap-high hepatocytes.

Immune and stromal cells rather than hepatocytes are the major source of pro-inflammatory cytokines in the liver (Hu et al., 2014, Science). Hepatocyte-specific ablation of Mst1/2 leads to increased levels of hepatic macrophages and increased IL-6 levels (Lu et al., 2010, PNAS). Thus, we propose that activation of Yap in hepatocytes leads to high expression of CCL2 (and other chemokines, according to our RNA-seq) that recruit accessory cells which then produce pro-inflammatory cytokines.

Author response image 1

Expression of pro-inflammatory genes in YapKI livers.

However, the authors claim that one of the links is IL-6 activated by LPS in myeloid cells in an NF-kB dependent manner and acting on hepatocytes in an NF-KB independent fashion, likely via a STAT3 dependent manner. Il-6 data should be shown, as it is one of the novel and clearly mechanistic aspects of this study. If the data are not readily available, the authors should consider removing this line of mechanistic argumentation from the text.

Since Yaphigh and Yaplow hepatocytes are intermixed in the same environment, the question is whether Yaphigh hepatocytes are more responsive to pro-inflammatory cytokines. We now present a new experiment implicating IL-6 in expansion of Yaphigh hepatocytes. When IL-6 was produced by LLC cells growing as subcutaneous tumors in YapKI mice, it caused expansion of Yaphigh hepatocytes in the livers of mice that had highest levels of systemic IL-6 (new Figure 5–figure supplement 1). However, neither recombinant IL-6 nor co-injected IL-1a + TNFa could induce the expansion (data not shown). The amount of recombinant IL-6 (6 ug/mouse for 6 days) may not have been high enough to induce the expansion. To repeat the experiment with even higher dose of IL-6 would be too costly (this experiment required $1100 for IL-6). Alternatively, IL-6 alone is not sufficient to cause the expansion and may cooperate with another factor secreted by the tumor (note that tumor not secreting IL-6 does not induce expansion of Yaphigh hepatocytes (Figure 5–figure supplement 1).

2) Did the authors stain for markers of senescence in addition to markers of proliferation and apoptosis with clonal Yap overexpression? The failure of oncogene overexpression to promote growth is usually associated with apoptosis and/or senescence; this could also explain lack of clonal expansion.

p16 is the most consistent marker of senescence across cell types and conditions. We were not able to detect cdkn2a transcripts by RNA-seq in YapKI hepatocytes, and neither p16 nor Arf by PCR in YapKI hepatocytes and whole liver samples. Most of other markers associated with senescence are also induced in other conditions of stress such as apoptosis (phospho-H2AX), transient cell cycle arrest (p21), and inflammation (most of the SASP genes). Analysis of other genes that have been implicated in senescence in various contexts is presented in Author response image 2.

Author response image 2

Expression of senescence-related genes in YapKI livers. ND, not detectable.

While p21 and p15 were not increased, p57, which has been implicated in senescence of HCC (Giovannini et al., 2012, American Journal of Pathology), was upregulated in YapKI background. Thus, we cannot exclude a contribution of growth arrest/senescence to limiting clonal expansion of YapKI cells. However, since p57 is not downregulated in CCL4 damage condition, the growth advantage of Yap-high hepatocytes in injured liver is unlikely to be mediated by overcoming p57-mediated growth arrest/senescence.

Finally, senescent hepatocytes can get cleared by immune cells (Kang et al., 2011, Nature). Thus it is possible that senescent Yap- high hepatocytes at steady state are not detectable due to their rapid clearance, similar to apoptotic bodies.

The failure of oncogene overexpression to promote growth is usually associated with apoptosis and/or senescence; this could also explain lack of clonal expansion.

3) The authors quantify BrdU and TUNEL but show minimal images used for quantification. In mosaic studies, there are often “border” issues. Is there any pattern of proliferating or dying Yap high cells (center of clone, on the border)? Is there an increase in proliferation or death in cells adjacent to Yap clones? The authors should show images that reveal or rule out patterns or make a text statement addressing this.

This is a very interesting point. We did not observe any obvious “border” patterns of cell death, but this may be due to technical limitations. Dead cells are rapidly cleared from the tissue, unless cell death is synchronous and massive. Because apoptotic cells are extremely rare in a steady state tissue, we could not determine if there was a pattern to cell death distribution.

We do not see increased proliferation of the Yap-low hepatocytes near Yap-high hepatocytes. We note that the GFP-high clusters (we cannot definitively call them clones) in the liver parenchyma (zone 2) are dispersed and irregular in shape, as if the Yap-high cells ‘prefer’ to border with the Yap-low cells rather than with each other. We also note that Yap-high hepatocytes are under-represented near the portal vein (zone 1) comparing to the intermediate zone 2 and the peri-central zone 3 in steady state. Clusters in zone 3 at steady state never encircle the entire CV area in a way it is occurs upon CCl4 damage.

There are higher numbers of stromal BrdU+ cells next to Yap-high hepatocytes. Several images to illustrate are presented in Author response image 3. We see increased number of stellate cells and macrophages in YapKI livers by FACS, and this increase is proportional to the number of Yap-high hepatocytes (data not shown). This is consistent with increased number of F4/80+ cells in Mst1/2 and Sav1 mutant mice (Lu et al., 2010, PNAS). In accord, M-CSF is upregulated in Yap-high hepatocytes and also in other published transgenic liver Yap models. We also find increased production of chemokines by Yap-high cells

Author response image 3

Stromal cells proliferate near Yaphigh hepatocytes

4) The authors should discuss and interpret their findings in light of the Yimlamai paper. One possibility (among many) is that the Su et al. model results in less Yap activation than what was published in the literature, either due to different expression levels or protein activities. This interpretation would be consistent with the lower induction of several Yap target genes reported in the present manuscript compared to what was previously reported. For example, H19 and AFP were barely induced in the Su et al. model, but significantly induced in the Dong et al. (2007) model.

We agree with the reviewer that difference in Yap expression level and activity may contribute to the different gene expression patterns described in Dong et al. (2007) and our work. It is worth mentioning that we observed 556 genes that were induced in our model but were not induced in Dong et al., 2007 (Figure 2–figure supplement 2), which might argue that the difference is not simply in lower expression level in our model, but more likely to do with the mosaic expression versus uniform expression of Yap. We think both types of data are important as they highlight different aspects of Yap biology.

Given the Yimlamai et al. paper, it would be difficult for the authors to definitively draw the conclusion that mosaic Yap activation does not yield clonal expansion. What they can conclude is that under a condition in which Yap overexpression can drive the expression of at least some of the target genes, it is insufficient to drive clonal expansion.

We focused on the role of Yap in mature hepatocytes whereas Yimlamai paper is focused on progenitor population, but in many ways Yimlamai paper is consistent with our findings. The data in Figure 2A of the Yimlamai paper shows that when Yap is activated in single hepatocytes, no clonal expansion occurs in the first 2 weeks. The expansion is evident only at 4 and 8 weeks, when the cells acquire progenitor morphology. As the authors state in the article: ”One week after YAP induction… many of these clones were still composed of single cells, indicating that cell division is not necessary for the initiation of dedifferentiation (Figure 2C)”; “YAP activation does not simply lead to hyperplastic response as no enrichment was found when compared to a gene signature derived from livers recovering from partial hepatectomy”.

Thus, in Yimlamai model, hepatocytes overexpressing Yap do not expand until they are reprogrammed into progenitors. It is entirely consistent with our finding that Yap cannot drive clonal expansion of differentiated hepatocytes. However, we agree that Yimlamai paper provides an example of clonal expansion driven by Yap at steady state (in progenitors), and we included discussion of that point and modified statements about two-signal requirement accordingly. Of note, Yimlamai study also does not show increased liver size.

We did see enrichment of Yap-high cells within hepatic progenitors at steady state, which was further promoted by CCl4. It would be hard to compare the degree of progenitor expansion between our models, as clonal expansion is not quantified in the Yimlamai paper. From the representative images, it does look more dramatic in their paper. This difference in the degree of progenitor expansion could be due to differences in Yap isoforms or level of expression between our models.

Further validating the observations made in the YapKI mice, we now show that mosaic Yap activation is insufficient for clonal expansion in another model of Yap activation in hepatocytes: mosaic deletion of Mst2flox/flox allele on Mst1-/- background (new Figure 2–Figure Supplement 4).

5) Related to point #5 above, the authors did not provide details of the YAP cDNA that was used to make their Yap112A mice. Since there are 8 different isoforms of YAP that differ in activities, it is important to know which isoform was used in the paper. This is obviously important given the discrepancies with prior published observations by other laboratories.

We used the longest mouse Yap variant (NM_001171147, NP_001164618, 488 aa). This isoform has all the domains present in human Yap. We introduced S112A mutation, which in the mouse protein is equivalent to the aa 127 in human Yap1.

6) Figure 3figure supplement3A: While ALT rise to the same levels, the temporal pattern is different (see also Figure 4figure supplement 3). Is this indeed the case reproducibly?

Yes, the temporal difference is reproducible. See another repeat (Author response image 4) showing that the kinetics is very similar to the results shown in Figure 3–figure supplement 1.

Author response image 4

An independent repeat of the ALT levels in the blood of WT and YapKI littermates after CCl4 injection.

How can this be explained seeing that most of the liver in the KI mice is practically WT?

CCl4 affects hepatocytes around CV that express cytochrome P4502E1 which converts CCl4 to toxic metabolites. There is an increased number of Yaphigh hepatocytes near CVs, even though their overall numbers are small. Differences in the temporal pattern may be caused by several factors:

A) Yaphigh hepatocytes express lower levels of P4502E1, thus the level of the primary CCl4-induced necrosis is likely to be lower in YapKI livers;

B) YapKI livers have increased number of Kupffer cells, which may enhance the inflammation-dependent damage (Kiso et al., 2012, Biological and Pharmaceutical Bulletin) as well as consequent inflammation-dependent repair;

C) Yaphigh hepatocytes may have different cell death thresholds in response to CCl4 metabolites or proinflammatory cytokines;

D) Yaphigh hepatocytes may have different responsiveness to pro-survival and mitogenic signals (again including proinflammatory cytokines).

All these differences are likely to contribute at least partially to the altered kinetics, and it is difficult to take into account all of them. However, despite the different kinetics, the degree of damage is similar between the WT and YapKI mice, as judged by the peak ALT levels and our histology analysis of liver sections on days 1 and 2 after CCl4. To avoid difficulties in interpretation, we have focused our analysis on day 4 when the phase of damage has ended in all genotypes.

7) The authors in some instances overstate their own findings and should rephrase misleading and inappropriate statements made throughout the manuscript regarding published literature. Some (not all) examples are:

They comment that Yap deletion does not markedly affect liver size or function. Previous literature (including Zhang et al., 2010, Developmental Cell) showed Yap knockout in the liver produced pale and enlarged livers with a number of defects.

We apologize for not describing the phenotype of YapKO livers to reflect these points properly. What we meant to say was that YapKO liver size is increased, not decreased, and hepatocytes are able to proliferate in the absence of Yap in vivo at steady state no less than in the normal liver. The defects in YapKO livers arise from bile duct epithelium, leading to cholestasis and to hepatocyte necrosis as a consequence. Homeostatic hepatocyte proliferation in vivo does not require Yap, and is even slightly elevated in YapKO livers (most likely, due to bile acid-induced damage). We have now rephrased this statement to avoid misinterpretations; the changes in the text are highlighted.

The discussion of Drosophila scrib clones is inappropriate. scrib loss across an entire organ results in neoplastic overgrowth of slow-growing tissue while elimination of clones occurs through cell competition when clones are out-competed by faster growing wild-type tissue. This is a very different context than oncogene overexpression being constrained and should not be invoked to justify the authors' model.

We have deleted this discussion and will not invoke it to justify our conclusions. However, we’d like to clarify this point: While our data do not prove that the growth Yaphigh hepatocytes is restrained by cell competition, we mention this as one of the possible mechanisms. We are not aware that tissue made entirely of scrib mutant cells is slow growing and in contrary, several publications suggest that Scrib mutant tissue analyzed at the same developmental stage as controls is enlarged, suggesting faster growth (Bilder et al., 2000, Science; Chen et al., 2012, PNAS; Norman et al., 2012, Journal of Cell Science; Brumby et al., 2003, EMBO). Scrib mutant clones are slow-growing when their overproliferation is counterbalanced by increased apoptosis induced by the wild type cells (Brumby et al., 2003, EMBO). Scrib is a negative regulator of the Yap homolog Ykie, and Scrib competitive interactions define cell fate via Ykie levels, leading to apoptosis or proliferation (Chen, et al., 2012, PNAS). Mammalian scrib mutant cells are also eliminated by cell competition (Norman et al., 2012, Journal of Cell Science).

In describing Figure 7, they write “RNA sequencing revealed a number of pro-apoptotic genes… induced by Yap.“ As worded, this suggests that Yap directly induces these genes. However, the authors have not shown direct induction of these genes by Yap, they have only shown that there is a relationship.

We agree. This is now rephrased to “Accordingly, RNA sequencing revealed a number of pro-apoptotic genes… induced as a consequence of Yap overexpression.”

They state that their findings “argue that growth-promoting function of Yap requires a signal derived from loss of tissue homeostasis such as inflammation or injury.” Do they mean Yap cannot promote growth in the absence of injury in all contexts? They should rephrase their language to indicate their findings argue for their specific context.

We have rephrased it to: “during homeostasis, growth-promoting function of Yap in differentiated cells requires additional signal, which may be provided by inflammation or injury”.

https://doi.org/10.7554/eLife.02948.033

Article and author information

Author details

  1. Tian Su

    Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, United States
    Contribution
    TS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Tanya Bondar
    Competing interests
    The authors declare that no competing interests exist.
  2. Tanya Bondar

    Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, United States
    Contribution
    TB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Tian Su
    Competing interests
    The authors declare that no competing interests exist.
  3. Xu Zhou

    Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, United States
    Contribution
    XZ, Performed bioinformatic analysis of RNA-seq data to identify enrichment in transcription factor binding sites in the promoters and enhancers of Yap-regulated genes under normal conditions and in response to liver damage
    Competing interests
    The authors declare that no competing interests exist.
  4. Cuiling Zhang

    Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, United States
    Contribution
    CZ, Performed experiments with MST1/MST2 deficient mice, CCL4 treated mice, as well as experiments with mice injected with IL-6 transfected LLCs
    Competing interests
    The authors declare that no competing interests exist.
  5. Hang He

    Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, China
    Contribution
    HH, Processed raw RNA-seq data, Performed bioinformatic and statistical analysis
    Competing interests
    The authors declare that no competing interests exist.
  6. Ruslan Medzhitov

    Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, United States
    Contribution
    RM, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    ruslan.medzhitov@yale.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

Howard Hughes Medical Institute (HHMI)

  • Ruslan Medzhitov

National Cancer Institute (NCI) (CA157461)

  • Tian Su
  • Tanya Bondar
  • Xu Zhou
  • Cuiling Zhang
  • Ruslan Medzhitov

National Institute of Allergy and Infectious Diseases (NIAID) (AI046688)

  • Tanya Bondar
  • Xu Zhou
  • Cuiling Zhang
  • Ruslan Medzhitov

Jane Coffin Childs Foundation

  • Xu Zhou

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Kathy Harry from Yale Liver Center for primary hepatocyte preparation, Gouzel Tokmoulina for assistance with hepatocyte sorting, Timothy Nottoli for help with generation of transgenic mice, Charles Anicelli, Sophie Cronin, and Steven Zhu for technical assistance with mouse treatments and colony maintenance, Raj Chovatiya for the critical reading of the manuscript, and all members of the Medzhitov lab for helpful discussions. This work was supported by the Howard Hughes Medical Institute and a grant from NIH to RM, a postdoctoral fellowship from the Jane Coffin Childs Foundation to XZ, and by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Number P30KD034989 (Yale Liver Center).

Ethics

Animal experimentation: All animal experiments were performed with approval by the Institutional Animal Care and Use Committee of Yale University (protocol # 2014-08006).

Reviewing Editor

  1. Alejandro Sánchez Alvarado, Howard Hughes Medical Institute, Stowers Institute for Medical Research, United States

Publication history

  1. Received: March 29, 2014
  2. Accepted: February 9, 2015
  3. Accepted Manuscript published: February 10, 2015 (version 1)
  4. Version of Record published: March 18, 2015 (version 2)

Copyright

© 2015, Su et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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