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Lymphocyte-Specific Protein-1 Suppresses Xenobiotic-Induced Constitutive Androstane Receptor and Subsequent Yes-Associated Protein–Activated Hepatocyte Proliferation

Open AccessPublished:April 03, 2022DOI:https://doi.org/10.1016/j.ajpath.2022.03.010
      Activation of constitutive androstane receptor (CAR) transcription factor by xenobiotics promotes hepatocellular proliferation, promotes hypertrophy without liver injury, and induces drug metabolism genes. Previous work demonstrated that lymphocyte-specific protein-1 (LSP1), an F-actin binding protein and gene involved in human hepatocellular carcinoma, suppresses hepatocellular proliferation after partial hepatectomy. The current study investigated the role of LSP1 in liver enlargement induced by chemical mitogens, a regenerative process independent of tissue loss. 1,4-Bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a direct CAR ligand and strong chemical mitogen, was administered to global Lsp1 knockout and hepatocyte-specific Lsp1 transgenic (TG) mice and measured cell proliferation, hypertrophy, and expression of CAR-dependent drug metabolism genes. TG livers displayed a significant decrease in Ki-67 labeling and liver/body weight ratios compared with wild type on day 2. Surprisingly, this was reversed by day 5, due to hepatocyte hypertrophy. There was no difference in CAR-regulated drug metabolism genes between wild type and TG. TG livers displayed increased Yes-associated protein (YAP) phosphorylation, decreased nuclear YAP, and direct interaction between LSP1 and YAP, suggesting LSP1 suppresses TCPOBOP-driven hepatocellular proliferation, but not hepatocyte volume, through YAP. Conversely, loss of LSP1 led to increased hepatocellular proliferation on days 2, 5, and 7. LSP1 selectively suppresses CAR-induced hepatocellular proliferation, but not drug metabolism, through the interaction of LSP1 with YAP, supporting the role of LSP1 as a selective growth suppressor.
      Constitutive androstane receptor (CAR), a xenobiotic-recognizing nuclear receptor activated by several chemicals and drugs, including 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and phenobarbital, is traditionally known for its essential role in inducing drug-metabolizing enzymes.
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      Given the previous findings of LSP1 acting as a suppressor of hepatocellular proliferation, hepatocyte-specific Lsp1 transgenic mice as well as global Lsp1 knockout mice were treated with TCPOBOP. Whether LSP1 could regulate CAR-driven TCPOBOP-stimulated proliferation and hypertrophy, and/or alter CAR-regulated drug metabolism, was investigated. The current findings indicate that LSP1 specifically regulates hepatocellular proliferation in this model, likely by regulating Yes-associated protein (YAP) localization. It does not, however, affect TCPOBOP- and CAR-associated hepatocyte hypertrophy or induction of drug-metabolizing enzymes.

      Materials and Methods

       Animals and TCPOBOP Treatment

      Male, 129svJ [genetic control strain for Lsp1 knockout (KO) mice], global Lsp1 KO,
      • Jongstra-Bilen J.
      • Misener V.L.
      • Wang C.
      • Ginzberg H.
      • Auerbach A.
      • Joyner A.L.
      • Downey G.P.
      • Jongstra J.
      LSP1 modulates leukocyte populations in resting and inflamed peritoneum.
      hepatocyte-specific Lsp1 transgenic (TG) mice,
      • Koral K.
      • Haynes M.
      • Bowen W.C.
      • Orr A.
      • Mars W.
      • Michalopoulos G.K.
      Lymphocyte-specific protein-1 controls sorafenib sensitivity and hepatocellular proliferation through extracellular signal-regulated kinase 1/2 activation.
      and C57 (genetic control strain for Lsp1 TG mice) (n = 3 to 6) between 15 and 20 weeks old were administered 3 mg/kg of TCPOBOP in corn oil by oral gavage. The Lsp1 transgene was generated in C57 mice and is expressed specifically in hepatocytes under the albumin promoter and α-fetoprotein enhancer.
      • Koral K.
      • Haynes M.
      • Bowen W.C.
      • Orr A.
      • Mars W.
      • Michalopoulos G.K.
      Lymphocyte-specific protein-1 controls sorafenib sensitivity and hepatocellular proliferation through extracellular signal-regulated kinase 1/2 activation.
      Livers were harvested at various time points [days 0 (untreated), 1, 2, 3, 5, and 7] following TCPOBOP administration. Liver tissue was processed for Western blot analysis, immunohistochemistry, real-time quantitative PCR, and RNA sequencing. Wild-type (WT) C57 and Lsp1 TG mice (n = 3) were given TCPOBOP; and on day 2 following TCPOBOP, livers were perfused using collagenase, as previously described.
      • Novicki D.L.
      • Rosenberg M.R.
      • Michalopoulos G.
      Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes.
      The hepatocytes and nonparenchymal cells were separated by centrifugation and used for Western blot analysis, real-time quantitative PCR, and RNA sequencing. Studies were performed according to the guidelines published by the NIH in the Guide for the Care and Use of Laboratory Animals
      Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council
      Guide for the Care and Use of Laboratory Animals.
      and were approved by the University of Pittsburgh's Institutional Animal Care and Use Committee.

       Immunohistochemistry

      Liver tissue was fixed in formalin and embedded in paraffin. Sections (4 μm thick) were stained with Ki-67 antibody (Cell Signaling Technologies, Danvers, MA; number 12202), as previously described.
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      • Bowen W.C.
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      • Tseng G.C.
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      Mechanisms of hepatocyte growth factor-mediated and epidermal growth factor-mediated signaling in transdifferentiation of rat hepatocytes to biliary epithelium.
      Biotinylated secondary antibody, goat anti-rabbit, was purchased from Millipore (St. Louis, MO; number AP187B).

       Immunofluorescence

      Frozen liver tissue embedded in OCT was sectioned at 5 μm and fixed with 2% paraformaldehyde for 1 hour. Fixed tissue was incubated with total YAP antibody (number 4912; Cell Signaling Technologies) in 0.5% bovine serum albumin for 1 hour. Following primary antibody incubation, sections were incubated with donkey anti-rabbit secondary antibody conjugated to Cy3 (Jackson ImmunoResearch Labs, West Grove, PA) for 1 hour. DAPI stain was used to visualize the nuclei. Images were taken at ×200 magnification on the Olympus (Tokyo, Japan) Fluoview 1000-2 confocal microscope in the Center for Biologic Imaging at the University of Pittsburgh.

       Protein Isolation and Western Blot Analysis

      Protein isolation was performed using frozen liver tissue in radioimmunoprecipitation assay buffer with proteinase and phosphatase inhibitors. Pooled protein lysates were utilized for Western blot analysis. Antibodies utilized for Western blot analysis include the following: from Cell Signaling Technologies: cyclin D1 (number 2978), phosphorylated ERK (p44/42; number 9101), total ERK (number 4696), phosphorylated YAP (S127; number 4911), phosphorylated YAP (S397; number 13619), total YAP (number 14074), p16 (number 80772), p27 (number 3686), glyceraldehyde-3-phosphate dehydrogenase (number 5174), phosphorylated epidermal growth factor receptor (EGFR) 846 (number 2231), phosphorylated EGFR 1068 (number 3777), total EGFR (number 4267), phosphorylated Met 1349 (number 3121), and total Met (number 8191). The antibody used for p21 was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX; sc6246). Total CAR (number PA5-95066) was purchased from Thermo Fisher Scientific (Waltham, MA).

       Immunoprecipitation

      Immunoprecipitation was performed using 500 μg of whole liver lysate in 500 μL of radioimmunoprecipitation assay buffer incubated with 10 μL of primary antibody overnight with end-over-end mixing at 4°C. Protein A/G agarose beads (Santa Cruz Biotechnology, Inc.) were added to the lysate the following day and incubated overnight with end-over-end mixing at 4°C. Pellet was collected by centrifugation at 1000 × g for 5 minutes and washed 3 times in radioimmunoprecipitation assay buffer with Halt protease and phosphatase inhibitors (Thermo Fisher Scientific) added. Following the final wash, 30 μL of 4× sample buffer was added to the pellet, and the samples were subsequently incubated at 95°C for 10 minutes and then run on a NuPAGE 4% to 12% Bis-Tris gel (Thermo Fisher Scientific).

       Real-Time Quantitative PCR

      Total RNA was isolated from frozen liver tissue using Trizol (Sigma, St. Louis, MO) following the manufacturer's protocol. RNA was utilized to produce cDNA using the First Strand Superscript Kit (Thermo Fisher Scientific). Real-time quantitative PCR analysis was performed using SYBR Green (Thermo Fisher Scientific) to determine mRNA levels of various targets following TCPOBOP administration. PCR primers used in this study are included in Table 1.
      Table 1List of Forward and Reverse Primers Used for Quantitative RT-PCR
      Gene symbolForward primer sequenceReverse primer sequence
      Cyb2b105′-GGCACTGGAGAAATCAATCAAC-3′5′-CTTGGGCTATTGGGAGGAAA-3′
      Cyp2c555′-AATGATCTGGGGGTGATTTTCAG-3′5′-GCGATCCTCGATGCTCCTC-3′

       RNA Sequencing

      RNA isolated from frozen liver tissue (time 0, day 2, and day 5) and WT and TG hepatocytes was sent to Novogene Corp., LTD (Beijing, China) for RNA sequencing. Ingenuity pathway analysis (IPA; Ingenuity Systems, Redwood City, CA) was utilized to analyze genes that were significantly altered by TCPOBOP treatment. For analysis of TCPOBOP-related genes, lists of significantly up-regulated and down-regulated genes from day 2 compared with time 0 as well as the corresponding fold changes were uploaded and analyzed by IPA. Gene sets that were significantly up-regulated and down-regulated on day 2 in the WT versus the Lsp1 TG isolated hepatocytes and day 5 in the WT129 versus the Lsp1 KO liver tissue (with a 1.5-fold cutoff) were uploaded to the IPA software version 22.0 (Qiagen, Redwood City, CA). IPA analysis was utilized to predict canonical signaling pathways and upstream regulators that were altered because of changes in downstream gene expression. DAVID software version 6.8 (Frederick National Laboratory, Frederick, MD) was utilized to determine biological processes (Gene Ontology terms) that were enriched because of TCPOBOP compared with the Mus musculus reference gene list.

       Statistical Analysis

      Data are presented as the means ± SEM. All statistical analyses were performed using the unpaired t-test and GraphPad Prism software version 9.3.0 (San Diego, CA). Statistically significant differences were defined at P < 0.05. All experiments were performed at least three times, and each time point contains data from at least three independent animals.

      Results

       LSP1 Expression Suppresses TCPOBOP-Driven Hepatocellular Proliferation but Not Hypertrophy

      In Lsp1 transgenic mice, which express the transgene in hepatocytes under the albumin promoter and α-fetoprotein enhancer, there was decreased liver/body weight ratios on day 2 after administration of TCPOBOP in comparison to WT mice (5.8% to 6.9%, respectively). However, by day 5, the liver/body weight ratios of the TG animals were significantly higher than those of the WT mice (8.9% to 8%, respectively) (Figure 1, A and B ). Hepatocellular proliferation driven by CAR activation was measured by Ki-67 immunohistochemistry. Following TCPOBOP administration, TG livers exhibited significantly less Ki-67–positive hepatocytes compared with WT hepatocytes on day 2 (20% versus 50%; P = 0.003), day 3 (P = 0.03), and day 5 (P = 0.0007) (Figure 1, C and D). Because hepatocyte proliferation did not account for the increased liver/body weight ratios in the TG at days 5 and 7, and TCPOBOP is known to not only promote hepatocellular proliferation but hypertrophy as well, the average volume of hepatocytes in both the WT and TG animals was measured following TCPOBOP treatment. TG hepatocytes were approximately twice the size of WT hepatocytes on day 5 and 35% larger on day 7 (Figure 1, D and E). We conclude that enhanced LSP1 expression in TG mice suppresses hepatocellular proliferation but not hypertrophy in response to TCPOBOP administration.
      Figure thumbnail gr1
      Figure 1Effect of lymphocyte-specific protein-1 (LSP1) expression on 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP)–driven hepatocellular proliferation and hypertrophy. A: Liver weight (LW)/body weight (BW) ratios of wild-type (WT) and Lsp1 transgenic (TG) animals. B: Liver/body weight ratios of WT and Lsp1 TG animals normalized to time 0. C: Percentage of Ki-67–positive hepatocytes in WT and Lsp1 TG livers after TCPOBOP. D: Representative images of Ki-67 immunohistochemistry. E: Average hepatocyte volume (number of hepatocytes per unit area). n = at least 3 animals per time point (B). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bar = 200 μm (D). Original magnification, ×200 (D).

       Increased LSP1 Expression Does Not Suppress CAR Expression or Its Drug Metabolism Functions in Response to TCPOBOP

      To determine whether the suppression of proliferation in Lsp1 TG livers after TCPOBOP treatment is due to decreased CAR expression and activation, CAR expression was measured by Western blot analysis as well as the ability of CAR to activate downstream transcriptional targets using IPA analysis of RNA-sequencing data. Comparing the differentially expressed genes of both the WT and TG on day 2 versus time 0, a significant activation of downstream CAR target genes, including the ones associated with drug metabolism in both the WT and TG livers (WT activation z-score, 5.771; TG activation z-score, 5.466) was observed (Figure 2A). Activation of typical CAR target genes, Cyp2b10 and Cyp2c55,
      • Tojima H.
      • Kakizaki S.
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      Ligand dependent hepatic gene expression profiles of nuclear receptors CAR and PXR.
      by quantitative RT-PCR was confirmed in both Lsp1 TG and WT livers (Figure 2, B and C). There was no suppression in CAR expression between the WT and TG in the same dates, as evidenced by Western blot analysis of all date time points; which was elevated relative to control (Figure 2D). Because Lsp1 TG mice did not display suppressed levels of CAR expression nor downstream activation of target genes in response to TCPOBOP, the study showed that the observed decrease in proliferation in TG livers is not due to decreased CAR activation.
      Figure thumbnail gr2
      Figure 2Constitutive androstane receptor CAR) expression and activity is unaffected by lymphocyte-specific protein-1 (LSP1) expression. A: Ingenuity pathway analysis of RNA-sequencing data, demonstrating activation of CAR target genes in both wild-type (WT; top panel) and Lsp1 transgenic (TG; bottom panel) groups on day 2 after 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) treatment in comparison to time 0. Quantitative RT-PCR data of CAR target genes. Number next to gene indicates number of isoforms. B: Cyp2b10 in liver. C: Cyp2c55 in liver. D: Western blot analysis of total CAR expression in WT and TG liver whole cell lysates. Samples are pooled from three independent livers. Genes marked with an asterisk indicate that multiple identifiers exist in the data set file map to a single gene in the Global Molecular Network. n = 3 (D). ∗P < 0.05, ∗∗∗P ≤ 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

       Increased Cell Cycle Inhibitor Expression, Decreased Nuclear YAP, and Increased YAP Interaction with LSP1 in Lsp1 TG Livers following TCPOBOP

      Because CAR activation and its ability to regulate drug metabolism genes is not suppressed by LSP1 expression, the role of LSP1 in regulating cell cycle progression after TCPOBOP administration was analyzed next. Western blot analyses demonstrated increased p16 (days 2, 3, and 5), p21 (all days), and p27 (days 2, 3, and 5) in the Lsp1 TG livers in comparison to those in the WT controls (Figure 3A), suggesting inhibition of the cell cycle. Because LSP1 inhibits ERK activation in liver after hepatectomy,
      • Koral K.
      • Paranjpe S.
      • Bowen W.C.
      • Mars W.
      • Luo J.
      • Michalopoulos G.K.
      Leukocyte-specific protein 1: a novel regulator of hepatocellular proliferation and migration deleted in human hepatocellular carcinoma.
      LSP1 overexpression in hepatocytes was confirmed to lead to decreased ERK phosphorylation following TCPOBOP treatment at days 2, 3, 5, and 7 (Figure 3A). Examination of YAP and phosphorylated YAP by Western blot analysis showed no changes in total YAP but an increased phosphorylation of YAP at S397 at days 1, 2, 3, and 5, which is associated with cytoplasmic retention of YAP (Figure 3B). Immunofluorescence staining corroborated the loss of nuclear YAP in the TG hepatocytes on day 2 after TCPOBOP administration (Figure 3D). Immunoprecipitation of total YAP in WT and Lsp1 TG livers following TCPOBOP treatment demonstrated an interaction between YAP and LSP1 that increased in the TG animals at day 2 after TCPOBOP (Figure 3C). LSP1 overexpression is cytoplasmic.
      • Koral K.
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      Leukocyte-specific protein 1: a novel regulator of hepatocellular proliferation and migration deleted in human hepatocellular carcinoma.
      The findings in Figure 3 demonstrate that LSP1 may sequester YAP in the cytoplasm, preventing its translocation to the nucleus and subsequent activation of proliferation. Lsp1 TG livers displayed suppressed EGFR activation in response to TCPOBOP, as evidenced by decreased phosphorylation of EGFR at the 845 and 1068 tyrosine sites compared with that in the WT (Supplemental Figure S1). There was no effect on the activation of MET, the hepatocyte growth factor receptor. Thus, the study shows that LSP1 expression leads to decreased hepatocellular proliferation induced by TCPOBOP, in part by causing increased expression of cell cycle inhibitor pathways, as well as decreased YAP translocation to the nucleus.
      Figure thumbnail gr3
      Figure 3Lymphocyte-specific protein-1 (LSP1) expression results in increased expression of cell cycle inhibitors and decreased YAP in the nucleus. A: Western blot (WB) analysis of wild-type (WT) and Lsp1 transgenic (TG) whole liver lysates following 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) for cyclin D1, phosphorylated extracellular signal-regulated kinase (pERK), total ERK, p16, p21, and p27. Ponceau S staining was used as a loading control. Each lane is a pooled sample. B: Western blot analysis for phosphorylated YAP (pYAP; S127) and pYAP (S397), with total YAP, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. C: Immunoprecipitation (IP) of total YAP in WT and Lsp1 TG livers at time (T) 0 and day (D) 2 after TCPOBOP treatment. Top panels: Western blot analysis: total YAP. Bottom panels: Western blot analysis: total LSP1. IgG: control. Compared with the C57 control mice, there is enhanced coprecipitation of LSP1 protein with YAP at day 2 after TCPOBOP. D: Immunofluorescence of WT and Lsp1 TG livers for total YAP (red). DAPI (nuclei) and merge of DAPI and total YAP. Insets: Magnified images from boxed areas to demonstrate YAP staining in the hepatocyte nuclei. n = at least 3 (A). Scale bar = 100 μm (D). Original magnifications, ×200 (Dmain images); ×400 (D, insets). exp., exposure.

       Lsp1 Transgenic Hepatocytes, but Not the Nonparenchymal Cells, Display Decreased Proliferation and Increased Cell Cycle Inhibitor Expression

      Although Lsp1 TG preparations from whole livers displayed decreased cell cycle progression in response to treatment with TCPOBOP, cyclin D1 expression levels were increased in whole TG livers on days 2 to 7 compared with those in the WT (Figure 3A). In addition, IPA analysis of RNA-sequencing data from the Lsp1 TG and WT livers did not show any alterations in cell cycle–related canonical pathways between the two groups. These results were unexpected because Lsp1 TG livers displayed decreased proliferation of hepatocytes by Ki-67 immunohistochemistry at those time points (Figure 1, C and D). Because TG animals only express the Lsp1 transgene in hepatocytes, both WT and Lsp1 TG animals were treated with TCPOBOP and hepatocytes and nonparenchymal cells (NPCs) were isolated from these livers on day 2 after TCPOBOP (the day of the most enhanced cyclin D1 expression in TG animals) (Figure 3A), to determine whether NPCs affected the results. As shown in Figure 4, enrichment analysis of RNA-sequencing data using both the DAVID database and IPA demonstrated that, following TCPOBOP administration, Lsp1 TG hepatocytes displayed decreased activation of DNA replication and cell cycle–related pathways, such as cell cycle control of chromosomal replication, DNA repair, DNA replication, and the nuclear excision repair pathway, compared with that in WT hepatocytes (Table 2 and Figure 4A). Further analyses demonstrated that in response to TCPOBOP, Lsp1 TG hepatocytes displayed decreased activation of forkhead box M1 (Foxm1), E2F transcription factor 1 (E2f1), and Yap1 protein downstream pathways (Figure 4B and Supplemental Figure S2), as well as increased activation of the p16 (Cdkn2a) activated downstream pathways (Figure 4B), compared with those in WT hepatocytes. RNA-sequencing predicted certain upstream regulators to be activated in Lsp1 TG versus those in WT hepatocytes such as cell cycle inhibitors p16 (Cdkn2a), p21 (Cdkn1a), p53 (Tp53), as well as Hnf4α and Rb. On the other hand, it predicted transcription factors involved in activating cell cycle progression, such as Foxm1, to be significantly attenuated in the Lsp1 TG hepatocytes versus those in WT (activation z-score, −2.412; P value of overlap, 1.02 × 10−5) (Table 3). RNA-sequencing also predicted inhibition of the upstream regulator Yap1 in Lsp1 TG hepatocytes (activation z-score, −2.053; P value of overlap, 7.00 × 10−9) (Figure 4B and Table 3). Yap1 is necessary to promote hepatocellular proliferation in response to TCPOBOP expression.
      • Bhushan B.
      • Molina L.
      • Koral K.
      • Stoops J.W.
      • Mars W.M.
      • Banerjee S.
      • Orr A.
      • Paranjpe S.
      • Monga S.P.
      • Locker J.
      • Michalopoulos G.K.
      YAP is crucial for CAR-driven hepatocyte proliferation, but not for induction of drug metabolism genes in mice.
      As indicated in Figure 5A, RNA levels of the cell cycle–related genes, aurora kinase B (Aurkb; P = 0.026) and cyclin E1 (Ccne1; P = 0.027), were significantly decreased in the TG hepatocytes. To demonstrate that these differences occur also at the protein level, Western blot analyses on both Lsp1 TG and WT hepatocytes and NPCs were performed, which demonstrated that cyclin D1 expression was decreased in TG compared with that in the WT hepatocytes. However, cyclin D1 levels were high in both TG and control (C57) NPC populations (Figure 5B). Expression of cell cycle inhibitors, p21 and p27,
      • Abbastabar M.
      • Kheyrollah M.
      • Azizian K.
      • Bagherlou N.
      • Tehrani S.S.
      • Maniati M.
      • Karimian A.
      Multiple functions of p27 in cell cycle, apoptosis, epigenetic modification and transcriptional regulation for the control of cell growth: a double-edged sword protein.
      was elevated in the TG hepatocyte but not the NPCs. There was also a hepatocyte-specific increase in the phosphorylation of YAP at S127 and S397 (Figure 5, B and C). The cell cycle inhibitor p16 was increased only in NPCs but not in hepatocytes (Figure 5B). These data show that LSP1 expression inhibits proliferation of hepatocytes but not NPCs in response to TCPOBOP through increased expression of cell cycle inhibitors and decreased activation of pathways involved in cell division through a YAP-dependent mechanism.
      Figure thumbnail gr4
      Figure 4Lsp1 transgenic (TG) hepatocytes exhibit decreased cell cycle activation and YAP activation after 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) treatment. A: Ingenuity pathway analysis (IPA)–based canonical signaling pathways predicted to be changed in Lsp1 TG hepatocytes versus wild-type (WT) hepatocytes on day 2 after TCPOBOP. B: IPA of RNA-sequencing data displaying inhibition of Foxm1 and YAP1 and activation of p16 in Lsp1 TG hepatocytes versus WT hepatocytes on day 2 after TCPOBOP. The number next to the genes indicates the number of isoforms. n = 3 per time point and strain (B). LPS, lipopolysaccharide; NER, nuclear excision repair; RXR, retinoid X receptor.
      Table 2Enrichment Analysis Utilizing the DAVID Database Displaying Biological Processes (GO Terms) that Differ between Lsp1 TG and WT Hepatocytes Isolated on Day 2 after TCPOBOP
      GO term (biological processes)Genes, NP value
      DNA replication328.40 × 10−14
      Cell cycle756.10 × 10−12
      Metabolic process543.90 × 10−8
      Cellular amino acid biosynthetic process112.00 × 10−7
      Oxidation-reduction process668.10 × 10−7
      Cellular response to DNA damage stimulus426.20 × 10−5
      DNA unwinding involved in DNA replication67.20 × 10−5
      DNA repair341.00 × 10−4
      Cell division381.10 × 10−4
      DNA replication initiation81.50 × 10−4
      Lipid metabolic process432.10 × 10−4
      Mitotic nuclear division302.20 × 10−4
      GO, Gene Ontology; TCPOBOP, 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene; TG, transgenic; WT, wild type.
      Table 3IPA of RNA-Sequencing Data Demonstrating Upstream Regulators Predicted to be Changed in Lsp1 TG versus WT Hepatocytes at Day 2 after TCPOBOP
      Upstream regulatorPredicted activation stateActivation z-scoreP value of overlap
      p16 (Cdkn2a)Activated4.7192.25 × 10−8
      RbActivated3.9403.72 × 10−8
      p53Activated3.0062.14 × 10−10
      Hnf4aActivated2.7634.46 × 10−10
      p21 (Cdkn1a)Activated2.6502.45 × 10−18
      Erbb2Inhibited−5.1888.03 × 10−13
      E2f1Inhibited−3.6428.33 × 10−8
      Nrf2Inhibited−3.1754.56 × 10−4
      MycInhibited−3.1251.22 × 10−3
      Foxm1Inhibited−2.4121.02 × 10−5
      E2f3Inhibited−2.3823.43 × 10−5
      HGFInhibited−2.2765.02 × 10−9
      YapInhibited−2.0537.00 × 10−5
      IPA, ingenuity pathway analysis; TCPOBOP, 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene; TG, transgenic; WT, wild type.
      Figure thumbnail gr5
      Figure 5Lymphocyte-specific protein-1 (LSP1) expression in hepatocytes leads to increased activation of cell cycle inhibitors and increased YAP phosphorylation. A: RNA-sequencing data from wild-type (WT) and transgenic (TG) hepatocytes (Heps) on day 2 after 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) for aurora kinase B (P = 0.026) and cyclin E1 (P = 0.027). B: Western blot analyses of cyclin D1, p16, p21, p27, phosphorylated YAP (pYAP) S127, pYAP S397, total YAP, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in WT hepatocytes and nonparenchymal cells (NPCs) and Lsp1 TG hepatocytes and NPCs isolated from livers on day 2 following TCPOBOP. Ponceau S staining was used as a secondary loading control. Each lane represents hepatocytes and NPCs isolated from one mouse. C: Quantification of Western blot analysis of pYAP S127/total YAP (P = 0.0003) and pYAP S397/total YAP (P = 0.074). n = 3 (B). ∗P < 0.05, ∗∗∗P < 0.001.

       Lsp1 KO Mice Exhibit Increased Hepatocellular Proliferation following TCPOBOP Administration

      Because enhanced LSP1 expression in TG mice led to decreased hepatocellular proliferation in response to treatment with TCPOBOP, global Lsp1 KO animals were treated with TCPOBOP to determine whether loss of LSP1 expression would conversely result in increased cell cycle progression. After normalizing liver/body weight ratios to the baseline ratios, Lsp1 KO mice displayed significantly increased liver/body weight ratios compared with control mice at all time points following TCPOBOP treatment to both groups, except for day 3 (Figure 6, A and B ). In addition, the percentage of Ki-67–positive hepatocytes was significantly increased in the Lsp1 KO on days 2 and 5 compared with WT [day 2: WT versus KO, 22% versus 48% (P = 0.0097); day 5: WT versus KO, 9.6% versus 21% (P = 0.033)] (Figure 6, C and D). Hepatocellular volume was also significantly increased by 20% on day 5 in comparison to WT (P = 0.005) (Figure 6E). Therefore, loss of LSP1 protein expression in KO mice led to increased hepatocellular proliferation in response to TCPOBOP administration. Altered patterns of EGFR phosphorylation were also observed in the KO mice, with enhanced phosphorylation at days 1 and 2 at site 845, and days 5 and 7 for site 1068, after TCPOBOP (Supplemental Figure S3).
      Figure thumbnail gr6
      Figure 6Loss of lymphocyte-specific protein-1 (LSP1) expression leads to increased 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP)–driven hepatocellular proliferation. A: Liver weight (LW)/body weight (BW) ratios of wild-type (WT) and Lsp1 knockout (KO) animals. B: Liver/body weight ratios of WT and Lsp1 KO animals normalized to time 0. C: Percentage of Ki-67–positive hepatocytes in WT and Lsp1 KO livers after TCPOBOP. D: Representative images of Ki-67 immunohistochemistry. E: Average hepatocyte volume (number of hepatocytes per unit area). n = at least 3 animals per time point (B). ∗P < 0.05, ∗∗∗P < 0.001. Scale bar = 200 μm (D). Original magnification, ×200 (D).

       CAR Expression and Role in Drug Metabolism Remains Functional in the Absence of LSP1 in Response to TCPOBOP

      Both the KO and WT livers displayed robust activation of CAR pathways related to drug metabolism to a similar degree and increased activation of CAR target genes on day 2 following TCPOBOP treatment (WT: activation z-score, 5.697; P value of overlap, 1.52 × 10−36; KO: activation z-score, 6.044; P value of overlap, 8.46 × 10−28) (Figure 7A). Western blot analysis of total CAR expression was performed. Although there were changes in CAR expression in both KO and WT mice through the days after TCPOBOP treatment, there was no difference between the KO and WT livers at any time points following TCPOBOP (Figure 7B). Therefore, the data indicate that the increased hepatocyte proliferation measured in the KO is not due to increased CAR activation per se or its ability to activate downstream target genes involved in drug metabolism.
      Figure thumbnail gr7
      Figure 7Lsp1 knockout (KO) mice display no change in constitutive androstane receptor (CAR) activation but have increased cell cycle activation in response to 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). A: Ingenuity pathway analysis of RNA-sequencing data of CAR target genes in both wild-type (WT; top panel) and Lsp1 KO (bottom panel) livers on day 2 after TCPOBOP compared with time 0. The number next to the genes indicates the number of isoforms. B: Western blot analysis of total CAR expression in WT and KO liver whole cell lysates with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. Samples were pooled from three independent livers. Genes marked with an asterisk indicate that multiple identifiers exist in the data set file map to a single gene in the Global Molecular Network. n = 3 (B).

       Loss of LSP1 Leads to Increased Expression of Cell Cycle Related Genes

      To understand how loss of LSP1 expression leads to increased hepatocyte proliferation in response to TCPOBOP, the study utilized both enrichment analysis using DAVID as well as IPA on RNA-sequencing data from day 5 after TCPOBOP to determine the biological processes and canonical pathways altered in the KO versus WT. The biological processes most altered in the KO were related to the cell cycle as well as mitotic nuclear division, cell division, and DNA replication (Table 4). The canonical pathways predicted to be most activated in the KO livers on day 5 following TCPOBOP administration were kinetochore metaphase signaling pathway (activation z-score, 2.887; P value of overlap, 4.86 × 10−14) and cell cycle control of chromosomal replication (activation z-score, 4.123; P value of overlap, 3.02 × 10−8) (Figure 8A). Upstream regulators inhibited in the KO livers in comparison to WT on day 5 were the cell cycle inhibitors, p16 (Cdkn2a; activation z-score, −3.502; P value of overlap, 1.85 × 10−6) and p21 (Cdkn1a; activation z-score, −2.331; P value of overlap, 6.20 × 10−15) (Table 5 and Supplemental Figure S4). Conversely, factors that promote cell cycle progression were activated in the KO versus WT mice on day 5, which included Foxm1 (activation z-score, 4.063; P value of overlap, 1.41 × 10−15), cyclin D1 (Ccnd1; activation z-score, 4.382; P value of overlap, 1.02 × 10−23), and E2f (activation z-score, 4.555; P value of overlap, 2.01 × 10−12) (Table 5 and Supplemental Figures S5–S7). Interestingly, the transcription factor that displayed the greatest level of activation in the KO on day 5 after TCPOBOP was Tbx2 (activation z-score, 5.253; P value of overlap, 5.81 × 10−29), which has been shown to promote proliferation through the ERK signaling pathway.
      • Liu X.
      • Miao Z.
      • Wang Z.
      • Zhao T.
      • Xu Y.
      • Song Y.
      • Huang J.
      • Zhang J.
      • Xu H.
      • Wu J.
      • Xu H.
      TBX2 overexpression promotes proliferation and invasion through epithelial-mesenchymal transition and ERK signaling pathway.
      Western blot analyses did not show changes in most cell cycle suppressor genes; however, total YAP expression and nuclear localization as measured by immunofluorescence was increased in the KO livers in comparison to control livers on day 5 after TCPOBOP (Figure 8, B and C). These data show that LSP1 loss leads to increased hepatocellular proliferation following TCPOBOP due to increased activation of pathways involved in cell cycle progression, including YAP.
      Table 4Enrichment Analysis Utilizing the DAVID Database Displaying Biological Processes (GO Terms) that Differ between Lsp1 KO and WT Livers Isolated on Day 5
      GO term (biological processes)Genes, nP value
      Cell cycle1134.70 × 10−22
      Mitotic nuclear division591.50 × 10−14
      Cell division703.80 × 10−14
      DNA replication285.90 × 10−8
      Cell adhesion653.80 × 10−7
      Chromosome segregation212.00 × 10−6
      Cellular response to DNA damage stimulus563.20 × 10−6
      Response to mechanical stimulus173.90 × 10−6
      Mitotic chromosome condensation88.60 × 10−6
      DNA repair441.70 × 10−5
      DNA replication initiation101.70 × 10−5
      Protein localization to kinetochore71.70 × 10−5
      GO, Gene Ontology; KO, knockout; WT, wild type.
      Figure thumbnail gr8
      Figure 8Increased cell cycle activation on day 5 following 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) in Lsp1 knockout (KO) livers. A: Ingenuity pathway analysis–based canonical signaling pathways predicted to be changed in Lsp1 KO versus wild-type (WT) livers on day 5 after TCPOBOP. B: Western blot analyses of phosphorylated extracellular signal-regulated kinase (pERK), total ERK, p21, phosphorylated YAP (pYAP) S127, pYAP S397, total YAP, p27, and cyclin D1 in WT and Lsp1 KO whole liver lysates following TCPOBOP. Each lane contains a pooled sample. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), loading control. C: Immunofluorescence of WT and Lsp1 KO livers for total YAP (red) on day 5. DAPI (nuclei) and merge of DAPI and total YAP. Insets: Magnified images from boxed areas to demonstrate YAP staining in the hepatocytes. n = 3 (B). Scale bar = 100 μm (C). Original magnifications, ×200 (C, main images); ×400 (C, insets).
      Table 5IPA of RNA-Sequencing Data Demonstrating Upstream Regulators Predicted to be Changed in Lsp1 KO versus WT Livers on Day 5 after TCPOBOP Treatment
      Upstream regulatorPredicted activation stateActivation z-scoreP value of overlap
      Tbx2Activated5.2535.81 × 10−29
      RabI6Activated5.1128.70 × 10−19
      E2fActivated4.5552.01 × 10−12
      Cyclin D1 (ccnd1)Activated4.3821.02 × 10−23
      Foxm1Activated4.0631.41 × 10−15
      HgfActivated3.7201.80 × 10−18
      Erbb2Activated2.9253.74 × 10−28
      p53Inhibited−4.4883.95 × 10−21
      Rbl2Inhibited−3.7805.01 × 10−9
      p16 (Cdkn2a)Inhibited−3.6643.04 × 10−17
      Smarcb1Inhibited−3.6049.66 × 10−15
      RbInhibited−3.4476.44 × 10−13
      p21 (Cdkn1a)Inhibited−3.2871.27 × 10−33
      IPA, ingenuity pathway analysis; KO, knockout; TCPOBOP, 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene; WT, wild type.

      Discussion

      Despite multiple studies demonstrating the association of LSP1 with hepatocyte growth suppression, its precise mechanism of action remains elusive. Original studies related to the discovery of LSP1 and its functions showed that in lymphocytes LSP1 is an F-actin binding protein connecting the ERK pathway to the cytoskeleton.
      • Harrison R.E.
      • Sikorski B.A.
      • Jongstra J.
      Leukocyte-specific protein 1 targets the ERK/MAP kinase scaffold protein KSR and MEK1 and ERK2 to the actin cytoskeleton.
      Recent studies have also shown that modulation of the cytoskeleton activates pathways related to mechanotransduction at the level of individual cells, resulting in a variety of effects on pathways related to cell proliferation, including migration of YAP1 to the nucleus or inactivation of YAP1 by phosphorylation, leading to its degradation.
      • Wagh K.
      • Ishikawa M.
      • Garcia D.A.
      • Stavreva D.A.
      • Upadhyaya A.
      • Hager G.L.
      Mechanical regulation of transcription: recent advances.
      ,
      • Totaro A.
      • Panciera T.
      • Piccolo S.
      YAP/TAZ upstream signals and downstream responses.
      The findings from the current study provide another example of the important role played by cytoskeleton proteins in different aspects of cell proliferation and differentiation. LSP1 binds to F-actin in hepatocytes,
      • Koral K.
      • Paranjpe S.
      • Bowen W.C.
      • Mars W.
      • Luo J.
      • Michalopoulos G.K.
      Leukocyte-specific protein 1: a novel regulator of hepatocellular proliferation and migration deleted in human hepatocellular carcinoma.
      regulates phosphorylation of ERK,
      • Harrison R.E.
      • Sikorski B.A.
      • Jongstra J.
      Leukocyte-specific protein 1 targets the ERK/MAP kinase scaffold protein KSR and MEK1 and ERK2 to the actin cytoskeleton.
      ,
      • Koral K.
      • Haynes M.
      • Bowen W.C.
      • Orr A.
      • Mars W.
      • Michalopoulos G.K.
      Lymphocyte-specific protein-1 controls sorafenib sensitivity and hepatocellular proliferation through extracellular signal-regulated kinase 1/2 activation.
      and binds to MEK1 and ERK2 via kinase suppressor of Ras to target this complex to the cytoskeleton. The current study demonstrates the complexity of the regulation exerted on hepatocyte functions by LSP1. LSP1 is associated with F-actin and cytoskeletal regulation in a variety of cells besides hepatocytes.
      • Jongstra-Bilen J.
      • Janmey P.A.
      • Hartwig J.H.
      • Galea S.
      • Jongstra J.
      The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain.
      • Jongstra-Bilen J.
      • Jongstra J.
      Leukocyte-specific protein 1 (LSP1): a regulator of leukocyte emigration in inflammation.
      • Cervero P.
      • Wiesner C.
      • Bouissou A.
      • Poincloux R.
      • Linder S.
      Lymphocyte-specific protein 1 regulates mechanosensory oscillation of podosomes and actin isoform-based actomyosin symmetry breaking.
      • Scharinger K.
      • Maxeiner S.
      • Schalla C.
      • Rutten S.
      • Zenke M.
      • Sechi A.
      LSP1-myosin1e bimolecular complex regulates focal adhesion dynamics and cell migration.
      It acts as a growth suppressor of hepatocyte proliferation not only in relation to liver regeneration
      • Koral K.
      • Paranjpe S.
      • Bowen W.C.
      • Mars W.
      • Luo J.
      • Michalopoulos G.K.
      Leukocyte-specific protein 1: a novel regulator of hepatocellular proliferation and migration deleted in human hepatocellular carcinoma.
      ,
      • Koral K.
      • Haynes M.
      • Bowen W.C.
      • Orr A.
      • Mars W.
      • Michalopoulos G.K.
      Lymphocyte-specific protein-1 controls sorafenib sensitivity and hepatocellular proliferation through extracellular signal-regulated kinase 1/2 activation.
      but also, as demonstrated in this work, in xenobiotic-induced hepatocyte proliferation. The effects of xenobiotics (as exemplified by TCPOBOP) on hepatocyte proliferation and liver size are an important issue related to regulation of therapeutic levels of drugs in clinical pharmacology.
      The direct association of LSP1 with YAP seen herein is surprising; however, several recent studies have shown that regulation of YAP by the Hippo pathway is directly connected to structural and functional aspects of the cytoskeleton. Mechanotransduction has a direct impact on YAP activation or inactivation,
      • Wagh K.
      • Ishikawa M.
      • Garcia D.A.
      • Stavreva D.A.
      • Upadhyaya A.
      • Hager G.L.
      Mechanical regulation of transcription: recent advances.
      ,
      • Totaro A.
      • Panciera T.
      • Piccolo S.
      YAP/TAZ upstream signals and downstream responses.
      ,
      • Croci O.
      • De Fazio S.
      • Biagioni F.
      • Donato E.
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      • Doni M.
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      • Biancotto C.
      • Verrecchia A.
      • Olivero D.
      • Amati B.
      • Campaner S.
      Transcriptional integration of mitogenic and mechanical signals by Myc and YAP.
      ,
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      • Hajduch M.
      • Sanz-Garcia A.
      • Pugno N.M.
      • Stokin G.B.
      • Forte G.
      YAP regulates cell mechanics by controlling focal adhesion assembly.
      and YAP is involved in anchoring the actin cytoskeleton to the plasma membrane during the formation of focal adhesions to enable cell spreading.
      • Nardone G.
      • Oliver-De La Cruz J.
      • Vrbsky J.
      • Martini C.
      • Pribyl J.
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      • Pesl M.
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      • Pagliari S.
      • Martino F.
      • Maceckova Z.
      • Hajduch M.
      • Sanz-Garcia A.
      • Pugno N.M.
      • Stokin G.B.
      • Forte G.
      YAP regulates cell mechanics by controlling focal adhesion assembly.
      Fascin1, an F-actin bundling protein, has recently been demonstrated to also mediate YAP activation due to changes in the extracellular matrix in the context of cholangiocarcinoma development.
      • Pocaterra A.
      • Scattolin G.
      • Romani P.
      • Ament C.
      • Ribback S.
      • Chen X.
      • Evert M.
      • Calvisi D.F.
      • Dupont S.
      Fascin1 empowers YAP mechanotransduction and promotes cholangiocarcinoma development.
      Therefore, actin binding proteins and the cytoskeleton have an important function in modulating YAP activity, with LSP1 a newly identified and important modulator in this system.
      Multiple studies have shown that CAR activation is associated with the following: i) proliferation of hepatocytes in the absence of liver injury, ii) induction of enzymes involved in drug metabolism, and iii) increases in overall liver mass. Although induction of enzymes associated with drug metabolism is directly controlled by CAR, the concomitant effects on hepatocyte proliferation and expansion of liver mass are elicited by CAR through recruitment of other regulators and signaling pathways directly associated with hepatocyte proliferation. Inhibition of receptor tyrosine kinases (MET and EGFR) dramatically decreases hepatocyte proliferation following activation of CAR by TCPOBOP.
      • Bhushan B.
      • Stoops J.W.
      • Mars W.M.
      • Orr A.
      • Bowen W.C.
      • Paranjpe S.
      • Michalopoulos G.K.
      TCPOBOP-induced hepatomegaly and hepatocyte proliferation are attenuated by combined disruption of MET and EGFR signaling.
      ,
      • Donthamsetty S.
      • Bhave V.S.
      • Kliment C.S.
      • Bowen W.C.
      • Mars W.M.
      • Bell A.W.
      • Stewart R.E.
      • Orr A.
      • Wu C.
      • Michalopoulos G.K.
      Excessive hepatomegaly of mice with hepatocyte-targeted elimination of integrin linked kinase following treatment with 1,4-bis [2-(3,5-dichaloropyridyloxy)] benzene.
      The current study demonstrates that LSP1 also regulates activation of EGFR, linking EGFR activation to regulation by mechanotransduction (Supplemental Figures S1 and S3). These studies need to be explored further. A similar effect was seen in YAP KO mice.
      • Bhushan B.
      • Stoops J.W.
      • Mars W.M.
      • Orr A.
      • Bowen W.C.
      • Paranjpe S.
      • Michalopoulos G.K.
      TCPOBOP-induced hepatomegaly and hepatocyte proliferation are attenuated by combined disruption of MET and EGFR signaling.
      ,
      • Bhushan B.
      • Molina L.
      • Koral K.
      • Stoops J.W.
      • Mars W.M.
      • Banerjee S.
      • Orr A.
      • Paranjpe S.
      • Monga S.P.
      • Locker J.
      • Michalopoulos G.K.
      YAP is crucial for CAR-driven hepatocyte proliferation, but not for induction of drug metabolism genes in mice.
      The findings in the current work add to the overall role of LSP1 by demonstrating that direct binding of LSP1 to YAP is a mechanism by which LSP1, in TG mice, prevents YAP from moving to the nucleus, subsequently limiting its ability to interact with CAR and thus affecting CAR-induced hepatocyte proliferation. Combined, these results provide a background for further investigation that may explain the documented role of LSP1 in breast and urinary bladder cancers.
      • Zeng S.X.
      • Zhu Y.
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      • et al.
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      Interestingly, LSP1 overexpression did not inhibit TCPOBOP-driven hepatocyte hypertrophy,
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      • Ehninger A.
      • Offner S.
      • Dubey C.
      • Huang W.
      • Moore D.D.
      • Trumpp A.
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      CAR, the continuously advancing receptor, in drug metabolism and disease.
      • Huang W.
      • Zhang J.
      • Washington M.
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      • Moore D.D.
      Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor.
      which led to increased liver/body weight ratios in the TG mice in the absence of increased proliferation. Increased hepatocyte hypertrophy is associated with enhanced expression of drug metabolism enzymes along with increased organelle size; however, the current results indicate that LSP1 does not affect CAR-driven drug metabolism.
      • Tian J.
      • Huang H.
      • Hoffman B.
      • Liebermann D.A.
      • Ledda-Columbano G.M.
      • Columbano A.
      • Locker J.
      Gadd45beta is an inducible coactivator of transcription that facilitates rapid liver growth in mice.
      Other studies have also demonstrated that CAR-driven proliferation and hypertrophy can be driven by different mechanisms. Gadd45b-KO mice display increased hepatocyte proliferation in response to TCPOBOP without increased hypertrophy, leading to smaller liver weights in the KO versus WT mice.
      • Tian J.
      • Huang H.
      • Hoffman B.
      • Liebermann D.A.
      • Ledda-Columbano G.M.
      • Columbano A.
      • Locker J.
      Gadd45beta is an inducible coactivator of transcription that facilitates rapid liver growth in mice.
      In addition, YAP-KO mice show decreased proliferation but no discernible difference in hepatocellular volume following TCPOBOP administration.
      • Bhushan B.
      • Molina L.
      • Koral K.
      • Stoops J.W.
      • Mars W.M.
      • Banerjee S.
      • Orr A.
      • Paranjpe S.
      • Monga S.P.
      • Locker J.
      • Michalopoulos G.K.
      YAP is crucial for CAR-driven hepatocyte proliferation, but not for induction of drug metabolism genes in mice.
      Therefore, signaling mechanisms controlling TCPOBOP-driven hepatocellular proliferation differ from those governing hypertrophy.
      Lsp1 TG hepatocytes displayed inhibition of the YAP signaling pathway, which is corroborated by previous findings that loss of YAP expression in the hepatocytes leads to decreased proliferation after TCPOBOP.
      • Bhushan B.
      • Molina L.
      • Koral K.
      • Stoops J.W.
      • Mars W.M.
      • Banerjee S.
      • Orr A.
      • Paranjpe S.
      • Monga S.P.
      • Locker J.
      • Michalopoulos G.K.
      YAP is crucial for CAR-driven hepatocyte proliferation, but not for induction of drug metabolism genes in mice.
      LSP1 expression is down-regulated when YAP is overexpressed in the MCF10A human breast epithelial cell line.
      • Hao Y.
      • Chun A.
      • Cheung K.
      • Rashidi B.
      • Yang X.
      Tumor suppressor LATS1 is a negative regulator of oncogene YAP.
      This finding suggests a reciprocal regulation of functionality between YAP and CAR in specific tissues and circumstances. Lsp1 TG hepatocytes also displayed decreased activation of Foxm1 signaling, which has been shown to facilitate TCPOBOP-driven proliferation by regulating cell cycle related genes, such as cyclins, cdc25 phosphatases, and regulators of mitosis, such as aurora kinase B.
      • Costa R.H.
      • Kalinichenko V.V.
      • Tan Y.
      • Wang I.C.
      The CAR nuclear receptor and hepatocyte proliferation.
      In addition to its role in promoting transcription of cell cycle progressing genes, Foxm1 also inhibits expression of cell cycle inhibitors, such as p21,
      • Costa R.H.
      • Kalinichenko V.V.
      • Tan Y.
      • Wang I.C.
      The CAR nuclear receptor and hepatocyte proliferation.
      which is up-regulated in the Lsp1 TG hepatocytes after TCPOBOP. The current data demonstrate that Foxm1 is involved in the signaling mechanisms by which LSP1 affects hepatocyte proliferation.
      These results demonstrate that the effect of global loss of LSP1 in transgenic animals was opposite to that observed in the WT animals. Lsp1 KO livers displayed decreased induction of cell cycle inhibitor pathways, including p16 and p21. In addition, YAP translocation to the nucleus was enhanced in the absence of LSP1 expression in response to TCPOBOP. YAP activation and deactivation are affected by changes in the cytoskeleton.
      • Sun S.
      • Irvine K.D.
      Cellular organization and cytoskeletal regulation of the hippo signaling network.
      Therefore, LSP1 may affect YAP signaling through modulation of the actin cytoskeleton. Even though the liver responses of KO mice were symmetrically different from those of TG mice, the possibility that the global elimination of LSP1 may have caused changes in extrahepatic sites that may have affected the liver responses shown in this study cannot be entirely ruled out. Expression of LSP1 has been primarily examined in lymphoid cells.
      • Harrison R.E.
      • Sikorski B.A.
      • Jongstra J.
      Leukocyte-specific protein 1 targets the ERK/MAP kinase scaffold protein KSR and MEK1 and ERK2 to the actin cytoskeleton.
      LSP1 expression is also seen in bladder and breast in the context of broader studies, where LSP1 is present along with many other unrelated genes.
      • Zeng S.X.
      • Zhu Y.
      • Ma A.H.
      • Yu W.
      • Zhang H.
      • Lin T.Y.
      • Shi W.
      • Tepper C.G.
      • Henderson P.T.
      • Airhart S.
      • Guo J.M.
      • Xu C.L.
      • deVere White R.W.
      • Pan C.X.
      The phosphatidylinositol 3-kinase pathway as a potential therapeutic target in bladder cancer.
      • Mulligan A.M.
      • Couch F.J.
      • Barrowdale D.
      • Domchek S.M.
      • Eccles D.
      • Nevanlinna H.
      • et al.
      Common breast cancer susceptibility alleles are associated with tumour subtypes in BRCA1 and BRCA2 mutation carriers: results from the Consortium of Investigators of Modifiers of BRCA1/2.
      • Tang J.
      • Li H.
      • Luo J.
      • Mei H.
      • Peng L.
      • Li X.
      The LSP1 rs3817198 T > C polymorphism contributes to increased breast cancer risk: a meta-analysis of twelve studies.
      • Nickels S.
      • Truong T.
      • Hein R.
      • Stevens K.
      • Buck K.
      • Behrens S.
      • et al.
      Evidence of gene-environment interactions between common breast cancer susceptibility loci and established environmental risk factors.
      In none of these studies, however, was there any analysis of global signaling changes that may affect liver-specific gene expression. This issue may be addressed with detailed evaluation of LSP1 function in other tissues.
      Overall, the current study demonstrated that LSP1 is a protein with many functionalities. Its effects on ERK phosphorylation and activation have been demonstrated in many other studies. In normal liver, LSP1 is critical in regulating mitogenic effects through EGFR and MET.
      • Koral K.
      • Paranjpe S.
      • Bowen W.C.
      • Mars W.
      • Luo J.
      • Michalopoulos G.K.
      Leukocyte-specific protein 1: a novel regulator of hepatocellular proliferation and migration deleted in human hepatocellular carcinoma.
      Its effects on the Hippo/YAP pathway have not been previously described, and the findings presented in this study demonstrate that LSP1 should be considered as a complex regulator causing decreased mitogenic responses through both receptor tyrosine kinases (MET and EGFR) and the YAP/Hippo pathway. In view of the F-actin binding of LSP1
      • Jongstra-Bilen J.
      • Janmey P.A.
      • Hartwig J.H.
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      • Jongstra J.
      The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain.
      and its effects on the cytoskeleton, LSP1 is an important contributor to the mechanosensory effects of regulation of cell proliferation and function. Because LSP1 is involved in breast,
      • Mulligan A.M.
      • Couch F.J.
      • Barrowdale D.
      • Domchek S.M.
      • Eccles D.
      • Nevanlinna H.
      • et al.
      Common breast cancer susceptibility alleles are associated with tumour subtypes in BRCA1 and BRCA2 mutation carriers: results from the Consortium of Investigators of Modifiers of BRCA1/2.
      • Tang J.
      • Li H.
      • Luo J.
      • Mei H.
      • Peng L.
      • Li X.
      The LSP1 rs3817198 T > C polymorphism contributes to increased breast cancer risk: a meta-analysis of twelve studies.
      • Nickels S.
      • Truong T.
      • Hein R.
      • Stevens K.
      • Buck K.
      • Behrens S.
      • et al.
      Evidence of gene-environment interactions between common breast cancer susceptibility loci and established environmental risk factors.
      bladder,
      • Zeng S.X.
      • Zhu Y.
      • Ma A.H.
      • Yu W.
      • Zhang H.
      • Lin T.Y.
      • Shi W.
      • Tepper C.G.
      • Henderson P.T.
      • Airhart S.
      • Guo J.M.
      • Xu C.L.
      • deVere White R.W.
      • Pan C.X.
      The phosphatidylinositol 3-kinase pathway as a potential therapeutic target in bladder cancer.
      and liver
      • Nalesnik M.A.
      • Tseng G.
      • Ding Y.
      • Xiang G.S.
      • Zheng Z.L.
      • Yu Y.
      • Marsh J.W.
      • Michalopoulos G.K.
      • Luo J.H.
      Gene deletions and amplifications in human hepatocellular carcinomas: correlation with hepatocyte growth regulation.
      ,
      • Koral K.
      • Paranjpe S.
      • Bowen W.C.
      • Mars W.
      • Luo J.
      • Michalopoulos G.K.
      Leukocyte-specific protein 1: a novel regulator of hepatocellular proliferation and migration deleted in human hepatocellular carcinoma.
      ,
      • Koral K.
      • Haynes M.
      • Bowen W.C.
      • Orr A.
      • Mars W.
      • Michalopoulos G.K.
      Lymphocyte-specific protein-1 controls sorafenib sensitivity and hepatocellular proliferation through extracellular signal-regulated kinase 1/2 activation.
      ,
      • Zhang H.
      • Wang Y.
      • Liu Z.
      • Yao B.
      • Dou C.
      • Xu M.
      • Li Q.
      • Jia Y.
      • Wu S.
      • Tu K.
      • Liu Q.
      Lymphocyte-specific protein 1 inhibits the growth of hepatocellular carcinoma by suppressing ERK1/2 phosphorylation.
      ,
      • Lubecka K.
      • Flower K.
      • Beetch M.
      • Qiu J.
      • Kurzava L.
      • Buvala H.
      • Ruhayel A.
      • Gawrieh S.
      • Liangpunsakul S.
      • Gonzalez T.
      • McCabe G.
      • Chalasani N.
      • Flanagan J.M.
      • Stefanska B.
      Loci-specific differences in blood DNA methylation in HBV-negative populations at risk for hepatocellular carcinoma development.
      cancers, understanding how LSP1 functions to suppress hepatocellular proliferation can provide new avenues of investigation to develop novel therapeutics.

      Author Contributions

      All authors had access to all of the data and have approved the final article.

      Supplemental Data

      • Supplemental Figure S1

        Western blot analyses of wild-type (WT) and Lsp1 transgenic (TG) livers after 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) treatment. Western blot analyses of WT and Lsp1 TG whole liver lysates for phosphorylated epidermal growth factor receptor (pEGFR) Tyr845, pEGFR Tyr1046, pEGFR Tyr1068, pEGFR Tyr1173, total EGFR, phosphorylated MET (pMET) Tyr1349, and total MET. Ponceau staining indicates similar loading. Each lane is a pooled sample. n = 3.

      • Supplemental Figure S2

        Ingenuity pathway analysis (IPA) and Western blot analysis of wild-type (WT) C57 and Lsp1 transgenic (TG) hepatocytes (Heps) and nonparenchymal cells (NPCs) after 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) treatment. A: IPA of RNA-sequencing data displaying inhibition of E2f1 in Lsp1 TG hepatocytes versus WT hepatocytes on day 2 after TCPOBOP. The number next to the genes indicates the number of isoforms. B: Western blot analyses of WT C57 and Lsp1 TG hepatocytes and NPCs on day 2 after TCPOBOP treatment for phosphorylated MET (pMET) Tyr1349, total MET, phosphorylated epidermal growth factor receptor (pEGFR) Tyr1068, total EGFR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control). Each lane represents hepatocytes or NPCs from one mouse. Activation z-score >0 indicates pathway enhancement. Activation z-score <0 indicates pathway decrease. n = 3 per time point and strain (A).

      • Supplemental Figure S3

        Western blot analyses of wild-type (WT) 129 and Lsp1 knockout (KO) livers after 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) treatment. Western blot analyses of WT 129 and Lsp1 KO whole liver lysates for phosphorylated epidermal growth factor receptor (pEGFR) Tyr845, pEGFR Tyr1046, pEGFR Tyr1068, pEGFR Tyr1173, total EGFR, phosphorylated MET (pMET) Tyr1349, and total MET. Ponceau staining indicates similar loading. Each lane is a pooled sample. n = 3.

      • Supplemental Figure S4

        Ingenuity pathway analysis (IPA) of wild-type (WT) 129 and Lsp1 knockout (KO) livers after 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) treatment. IPA of RNA-sequencing data from WT 129 and Lsp1 KO livers on day 5 after TCPOBOP treatment, demonstrating inhibition of the p16 (A) and p21 (B) pathways in the KO livers. Activation z-score >0 indicates pathway enhancement. Activation z-score <0 indicates pathway decrease. The number next to the genes indicates the number of isoforms.

      • Supplemental Figure S5

        Activation of Foxm1 pathway in Lsp1 knockout (KO) livers on day 5 following 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene administration. Ingenuity pathway analysis of RNA-sequencing data displaying activation of Foxm1 in Lsp1 KO versus wild-type livers on day 5 based on the profile of downstream gene expression. Activation z-score >0 indicates pathway enhancement. Activation z-score <0 indicates pathway decrease. The number next to the genes indicates the number of isoforms.

      • Supplemental Figure S6

        Activation of cyclin D1 pathway in Lsp1 knockout (KO) livers on day 5 following 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) treatment. Ingenuity pathway analysis of RNA-sequencing data displaying increased cyclin D1 pathway activation in Lsp1 KO mice on day 5 after TCPOBOP based on the profile of downstream gene expression. Activation z-score >0 indicates pathway enhancement. Activation z-score <0 indicates pathway decrease. The number next to the genes indicates the number of isoforms.

      • Supplemental Figure S7

        Activation of E2f1 pathway in Lsp1 knockout (KO) livers on day 5 following 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) administration. Ingenuity pathway analysis of RNA-sequencing data displaying activation of E2f1 in Lsp1 KO versus wild-type livers on day 5 based on the profile of downstream gene expression. Activation z-score >0 indicates pathway enhancement. Activation z-score <0 indicates pathway decrease. The number next to the genes indicates the number of isoforms.

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