Thrombospondin-1 is a novel negative regulator of liver regeneration after partial hepatectomy through transforming growth factor-beta1 activation in mice

Authors

  • Hiromitsu Hayashi,

    1. Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, OH
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  • Keiko Sakai,

    1. Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, OH
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  • Hideo Baba,

    1. Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
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  • Takao Sakai

    Corresponding author
    1. Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, OH
    2. Orthopedic and Rheumatologic Research Center, Cleveland Clinic, Cleveland, OH
    • Department of Biomedical Engineering/ND20, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195
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    • fax: +01-216-444-9198


  • Potential conflict of interest: Nothing to report.

Abstract

The matricellular protein, thrombospondin-1 (TSP-1), is prominently expressed during tissue repair. TSP-1 binds to matrix components, proteases, cytokines, and growth factors and activates intracellular signals through its multiple domains. TSP-1 converts latent transforming growth factor-beta1 (TGF-β1) complexes into their biologically active form. TGF-β plays significant roles in cell-cycle regulation, modulation of differentiation, and induction of apoptosis. Although TGF-β1 is a major inhibitor of proliferation in cultured hepatocytes, the functional requirement of TGF-β1 during liver regeneration remains to be defined in vivo. We generated a TSP-1-deficient mouse model of a partial hepatectomy (PH) and explored TSP-1 induction, progression of liver regeneration, and TGF-β-mediated signaling during the repair process after hepatectomy. We show here that TSP-1-mediated TGF-β1 activation plays an important role in suppressing hepatocyte proliferation. TSP-1 expression was induced in endothelial cells (ECs) as an immediate early gene in response to PH. TSP-1 deficiency resulted in significantly reduced TGF-β/Smad signaling and accelerated hepatocyte proliferation through down-regulation of p21 protein expression. TSP-1 induced in ECs by reactive oxygen species (ROS) modulated TGF-β/Smad signaling and proliferation in hepatocytes in vitro, suggesting that the immediately and transiently produced ROS in the regenerating liver were the responsible factor for TSP-1 induction. Conclusions: We have identified TSP-1 as an inhibitory element in regulating liver regeneration by TGF-β1 activation. Our work defines TSP-1 as a novel immediate early gene that could be a potential therapeutic target to accelerate liver regeneration. (HEPATOLOGY 2011)

Cell proliferation is part of the wound-healing response and plays a central role in regeneration after tissue damage. It is crucial to advance our understanding of the molecular mechanisms underlying tissue regeneration and to develop a novel strategy to enhance the regenerative process. Such knowledge, in turn, would yield clinical benefits, such as decreased morbidity and mortality. Partial hepatectomy (PH) is a well-established model system in rodents for studying the molecular mechanisms of liver regeneration. PH triggers activation of the immediate early genes (i.e., genes that are rapidly, but transiently, activated) within approximately the first 4 hours,1 and thereby hepatocytes reenter the cell-division cycle. Immediate early genes encode proteins that regulate later phases in G1 and play an important role in cell growth in the regenerating liver.1, 2 The process of liver regeneration after hepatectomy is coordinated by both pro- and antiproliferative factors. Transforming growth factor-beta1 (TGF-β1) is a potent inhibitor of mitogen-stimulated DNA synthesis in cultured hepatocytes.3 Therefore, it has been thought that TGF-β1 is a potent candidate to limit or stop liver regeneration after PH hepatectomy.4 Because TGF-β is synthesized and secreted as a latent complex, the important step in regulating its biological activity is the conversion of the latent form into the active one. However, the contribution of TGF-β to the liver's regenerative response after PH hepatectomy is still poorly understood. TGF-β1 messenger RNA (mRNA) induction occurs within 4 hours, and levels of TGF-β1 remain elevated until 72 hours after PH hepatectomy.5, 6 In sharp contrast, in the model of complete lack of TGF-β signaling using hepatocyte-specific TGF-β type II receptor knockout mice, the lack of TGF-β signaling does not result in prolonged hepatocyte proliferation; rather, only transiently up-regulated proliferation of hepatocytes is shown in the later phase after hepatectomy, with a peak at ∼36 hours.7 These differences raise an open question about whether locally activated TGF-β1 is indeed essential for the inhibition of hepatocyte proliferation in vivo. Furthermore, the time course of locally activated TGF-β1 and its activation mechanism after PH hepatectomy still remain largely unknown.

The matricellular protein, thrombospondin-1 (TSP-1), was first shown as a component of the α-granule in platelets and can act as a major activator of latent TGF-β1.8, 9 TSP-1 is induced in response to tissue damage or stress and plays a role as a transient component of extracellular matrix during tissue repair.8, 10, 11 However, the roles of TSP-1 and of TSP-1/TGF-β1 interdependence during liver regeneration have not yet been addressed. We hypothesize that the initiation of local TGF-β activation occurs much earlier after PH hepatectomy, and TSP-1 plays a critical role in this process. Here, using a TSP-1-deficient mouse model, we investigated whether TSP-1 would be a suitable molecular target for accelerating liver regeneration after PH.

Abbreviations

BrdU, 5-bromo-2-deoxyuridine; CD, cluster of differentiation; CM, conditioned media; ECs, endothelial cells; Erk1/2, extracellular signal-related kinase 1 and 2; HSC, hepatic stellate cell; HUVEC, human umbilical vein endothelial cell; ICC, immunocytochemistry; IF, immunofluorescence; IHC, immunohistochemical; MDA, malondialdehyde; mRNA, messenger RNA; NAC, N-acetylcysteine; PAI-1, plasminogen activator inhibitor-1; PECAM-1, platelet/endothelial cell adhesion molecule-1; PCR, polymerase chain reaction; PH, partial hepatectomy; PI3K, phosphatidylinositide 3-kinase; (PI3K ROS, reactive oxygen species; α-SMA, alpha smooth muscle actin; STAT3, signal transducer and activator of transcription 3; TSP-1, thrombospondin-1; TGF-β1, transforming growth factor-β1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VEGFR, vascular endothelial growth factor-A receptor; WT, wild type.

Materials and Methods

Mutant Mice and Animal Studies.

TSP-1-null mice were kindly provided by Dr. Jack Lawler (Beth Israel Deaconess Medical Center, Boston, MA).12 Male wild-type (WT) and TSP-1-null mice, at 8-12 weeks old (C57BL/6 background), were used for the experiments. The two anterior lobes (i.e., median and left lateral lobes), which comprise 70% of liver weight, were resected, whereas the caudate and right lobes were left intact. This study was approved by the institutional animal care and use committee.

Immunostaining and Western Blotting.

For histological analyses, liver samples (the same lobe from each mouse) were either directly frozen in OCT compound (Tissue-Tek; Sakura Finetek, Tokyo, Japan) or fixed overnight in 4% paraformaldehyde in phosphate-buffered saline (pH 7.2) and dehydrated in a graded alcohol series and embedded in paraffin. Then, the materials were sectioned at a thickness of 5 μm. Immunofluorescence (IF) and immunohistochemical (IHC) staining was performed as described previously.13 The negative control staining was performed without the addition of primary antibody. Immunostained slides were viewed under a Leica DM 5500B microscopic system (Leica Microsystems, Buffalo Grove, IL). A minimum of 10 different images were randomly selected, and the data shown are representative of the results observed. Western blotting analysis was performed as described previously.13 The same lobe from each mouse was used for protein isolation and subsequent analysis. ImageJ software (version 1.40) was used for densitometric analysis.

Assessment of 5-Bromo-2-Deoxyuridine Incorporation.

Mice received an intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdU; 100 mg/kg; Roche Applied Science, Indianapolis, IN) 2 hours before sacrifice. Six random visual high-power fields (0.64 mm2 per field) per mouse were evaluated to determine the number of BrdU-positive nuclei in hepatocytes and nonparenchymal cells. Nonparenchymal cells were defined as cells with smaller, irregularly shaped nuclei, compared with larger, circular nuclei of hepatocytes, as previously described.14 All BrdU-positive cells, from both cell types, were summed at each time point.

Assessment of Apoptotic Index.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis was performed using an in situ apoptosis detection kit (Roche). Six visual high-power fields (0.64 mm2 per field) per mouse were evaluated to determine the number of TUNEL-positive nuclei.

Antibodies.

The antibodies used for analyses are summarized in Supporting Table 1. The amount of active and total TGF-β1 in liver samples was determined using an enzyme-linked immunosorbent assay kit (Quantikine TGF-β1 Immunoassay; R&D Systems, Inc., Minneapolis, MN), according to the manufacturer's instructions.

Real-time Polymerase Chain Reaction.

Real-time polymerase chain reaction (PCR) was performed as described previously.13 The primers used are summarized in Supporting Table 2.

Lipid Peroxidation Assay.

Liver tissue content of malondialdehyde (MDA) was measured by the thiobarbituric acid reduction method using a commercially available kit (#10009055; Cayman Chemical, Ann Arbor, MI). Values were obtained after 30-minute incubation at 90°C under acidic conditions.

In Vitro Assay Using Human Umbilical Vein Endothelial Cells and Mouse Primary Hepatocytes.

Human umbilical vein endothelial cells (HUVECs) were used at passages 3-6. For analysis of reactive oxygen species (ROS), H2O2 (Thermo Fisher Scientific, Waltham, MA) and N-acetylcysteine (NAC; Calbiochem, San Diego, CA) were used as an ROS inducer and an ROS scavenger, respectively. To examine the effects of H2O2 on TSP-1 expression, HUVECs were seeded on 0.1% gelatin-coated culture plates and incubated overnight. Without change of medium, H2O2 was applied at final concentrations of 0.01, 0.05, and 0.1 mM and incubated for 10 minutes. For immunocytochemistry (ICC), HUVECs were plated into Lab-Tek Permanox slides precoated with 0.1% gelatin and incubated overnight. Then, the cells, with or without pretreatment with 30 mM of NAC for 60 minutes, were treated with 0.1 mM of H2O2 for 10 minutes.

To examine the effects of HUVEC-derived TSP-1 on TGF-β/Smad signaling and proliferation in primary hepatocyte cultures, primary hepatocytes were isolated from 8- to 12-week-old adult WT mouse livers using collagenase perfusion as previously described.15 Isolated hepatocytes were plated on type I collagen (10-μg/mL)-coated dishes in Williams' E medium, supplemented with 5 μg/mL of insulin, 5 μg/mL of transferrin, 10 ng/mL of endothelial growth factor, 10−5 M aprotinin, 10−5 M of dexamethasone, 10−3 M of nicotinamide, and 10% fetal bovine serum and incubated at 37°C for 24 hours. To examine the effect of HUVEC-derived TSP-1 on TGF-β/Smad signaling in hepatocytes, the conditioned media from HUVECs (treated with 1.0 mM of H2O2 for 2 hours) were added to primary hepatocytes with or without pretreatment of 5 μM of LSKL or SLLK peptide (GenScript, Piscataway, NJ),16, 17 cultured for an additional 4 hours, and the cells were used for the analysis. To examine the effect of HUVEC-derived TSP-1 on hepatocyte proliferation, the conditioned media from HUVECs were added to primary hepatocytes, cultured for an additional 24 hours, and the cells were used for the analysis.

Data Presentation and Statistical Analysis.

All experiments were performed in triplicate, and the data shown are representative of results consistently observed. Data are expressed as the mean ± standard deviation. Data analysis was performed with SPSS 12.0.1 for Windows (SPSS, Inc., Chicago, IL). Statistical analyses were performed using the Student's t test or analysis of variance, followed by Bonferroni's multiple comparison tests, when appropriate. A P value of <0.05 was considered significant.

Results

PH Induces an Immediate and Prominent Induction of TSP-1 mRNA and Protein in the Regenerating Liver.

An intact liver in adult mice expresses nearly undetectable levels of TSP-1 mRNA.12 We first determined whether PH could trigger TSP-1 induction in the regenerating liver. TSP-1 mRNA was immediately induced, with a peak at 3 hours after hepatectomy, in WT mice by real-time PCR (Fig. 1A). TSP-1 protein was also induced, reaching a peak at ∼6 hours (Fig. 1B). Those mRNA and protein levels returned to basal levels by 24 hours (Fig. 1A,B). Thus, PH induced immediate and transient TSP-1 expression in the initial phase of liver regeneration. Secondary minor inductions of TSP-1 mRNA and protein were found to peak at 48 and 72 hours, respectively (Fig. 1A,B).

Figure 1.

An immediate and significant induction of TSP-1 mRNA and protein in response to PH. (A) Real-time PCR analysis of TSP-1 mRNA expression after 70% PH (n = 8 per time point) versus sham operation (n = 3 per time point). Data were normalized to the amount of 18S ribosomal RNA serving as the internal control. *P < 0.05 versus sham-operated mice; **P < 0.01 versus sham-operated mice. (B) Western blotting analysis of TSP-1 protein expression in the regenerating liver. Heat shock cognate protein 70 (HSC70) served as a loading control. (C) Double IF staining of TSP-1 (red) and GPIIb/IIIa (green) at 0 hours in WT (TSP1+/+) and TSP-1-null (TSP1−/−) liver. Note that the distribution of TSP-1 in the intact WT liver shows only in platelets, as evidenced by colocalization of TSP-1 with the platelet marker, GPIIb/IIIa (yellowish dots in the merged image). Scale bar = 50 μm. (D) IF staining for TSP-1 in WT liver at 0, 6, and 72 hours after PH hepatectomy. Scale bar = 50 μm.

We next determined the cellular source of TSP-1 by immunostaining. In the intact liver, the expression of TSP-1 protein was detectable only in platelets with GPIIb/IIIa expression by double IF staining (Fig. 1C). The tissue distribution of TSP-1 protein localized in the sinusoid at 6 and 72 hours after PH hepatectomy (Fig. 1D), suggesting that cells localized in the sinusoid (e.g., endothelial cells [ECs], Kupffer cells, and hepatic stellate cells; HSCs) are responsible for newly synthesized TSP-1 in the regenerating liver. Double IF staining revealed that TSP-1 protein predominantly colocalized with platelet/endothelial cell adhesion molecule-1 (PECAM-1)/cluster of differentiation (CD)31 (an EC marker) at 6 hours in the regenerating liver (Fig. 2A). In contrast, TSP-1 protein at 6 hours did not colocalize with either F4/80 (a Kupffer cell marker) or alpha smooth muscle actin (α-SMA; a marker for myofibroblasts, such as activated HSCs) (Fig. 2A). The activation peak of HSCs is at 72 hours after PH hepatectomy,18 and many α-SMA-positive cells were observed (Supporting Fig. 1). At 72 hours, however, TSP-1 protein did colocalize with PECAM-1/CD31 and α-SMA, but not with F4/80 (Fig. 2B). Indeed, it is known that activated HSCs express TSP-1 and thereby activate the TGF-β-signaling pathway in vitro.19 These results suggest that ECs are the major source of TSP-1 expression in the initial phase at 6 hours, whereas ECs and activated HSCs participate in secondary TSP-1 expression at 72 hours. As noted above, immediate early genes are genes that are rapidly, but transiently (within approximately the first 4 hours), activated in response to hepatectomy.1, 2 Thus, TSP-1 produced by ECs is a novel candidate immediate early gene in the initial response to PH.

Figure 2.

Tissue distribution of TSP-1 protein in the regenerating liver. (A and B) TSP-1 expression at 6 (A) and 72 hours (B) after PH. Double IF staining for TSP-1/PECAM-1 (CD31), TSP-1/F4/80, and TSP-1/α-SMA at 6 and 72 hours in WT mice (TSP-1, red; PECAM-1 [CD31], F4/80, and α-SMA, green). Scale bar = 25 μm.

TSP-1 Deficiency Accelerates a Liver Regeneration After PH, but Does Not Affect the Termination Phase.

Because immediate early genes play a significant role in the regulation of cell growth in the regenerating liver,1, 2 we next examined the involvement of TSP-1 in the control of liver regeneration. The rates of recovery of liver mass and of cell proliferation after PH hepatectomy were compared between WT and TSP-1-null mice. TSP-1-null mice showed significantly faster recovery of liver:body-weight ratio from day 1 to day 7 after surgery, compared with controls (P < 0.05 at 24, 48, and 168 hours and P < 0.01 at 72 hours; Fig. 3A). However, no excess liver mass had been gained at day 14 in TSP-1-null mice, compared with controls. Next, cell proliferation was evaluated using a BrdU incorporation assay (a marker for the S phase of the cell cycle). The proliferation peaks of hepatocytes and nonparenchymal cells after PH occurred at ∼36-48 and 72 hours, respectively.2, 4, 14 Although only a few BrdU-positive hepatocytes were detectable at 24 hours in WT mice, TSP-1-null mice showed a significantly increased number of BrdU-positive hepatocytes (8-fold over controls) (P < 0.01; Fig. 3B,C). The number of BrdU-positive nonparenchymal cells in TSP-1-null mice significantly increased (2-fold) at 72 hours, compared with controls (P < 0.01; Fig. 3C). Total proliferative activity (of hepatocytes and nonparenchymal cells) in TSP-1-null mice was significantly higher at 24 and 72 hours, compared with controls (P < 0.01 in both; Fig. 3C).

Figure 3.

Accelerated liver regeneration with down-regulation of p21 protein expression in TSP-1-null mice after PH. (A) Assessment of restoration of liver mass. Liver:body-weight ratio was measured after PH (n = 10 per time point in each group). *P < 0.05 versus TSP-1+/+ mice; **P < 0.01 versus TSP-1+/+ mice. (B and C) Assessment of BrdU incorporation in the regenerating liver. (B) IHC of BrdU in the regenerating liver. Arrowheads indicate BrdU-positive hepatocyte nuclei (brown) at 24 hours. Scale bar = 50 μm. (C) The number of BrdU-positive hepatocytes, nonparenchymal cells, and all positive cells (n = 10 per time points in each group). **P < 0.01 versus TSP-1+/+ mice. (D) Real-time PCR analysis of cyclin A2 (Ccna2) and cyclin D1 (Ccnd1) mRNA expression in the regenerating liver (n = 10 per time point in each group). *P < 0.05 versus TSP-1+/+ mice. (E) Assessment of p21 protein expression in the regenerating liver. Left panels: western blotting analysis of p21 protein expression in WT versus TSP-1-null liver. HSC70 was used as a loading control. Right panel: densitometric analysis of p21 protein expression (n = 3). Each p21 intensity was normalized to HSC70, then the intensity of WT mice at 0 hours was set to 1. *P < 0.05 versus TSP-1+/+ mice. HSC70, heat shock cognate protein 70.

Cyclins are required for cell-cycle progression. The mRNA levels of cyclin A2 (Ccna2) and cyclin D1 (Ccnd1) increase and peak in S phase and early to mid G1 phase, respectively. Expression levels of Ccna2 mRNA in TSP-1-null mice were significantly higher at 24 (2.3-fold) and 72 hours (1.5-fold), compared with controls (P < 0.05 in both; Fig. 3D). Although Ccnd1 mRNA levels increased and peaked at 48 hours in both WT and TSP-1-null mice, there was no significant difference between them (Fig. 3D). The cyclin-dependent kinase inhibitor, p21, plays a critical role in the inhibition of hepatocyte proliferation at the G1/S transition of the cell cycle in vivo.20 Induction levels of p21 protein in TSP-1-null mice significantly diminished at 12 and 24 hours, compared with controls (70% less than that of controls, both at 12 and 24 hours; P < 0.05 in both), whereas p21 showed at similar levels at 48 hours in WT and TSP-1-null liver (Fig. 3E). These results suggest that TSP-1 is a negative regulator of liver regeneration after PH, and that TSP-1 deficiency accelerates the S-phase entry of hepatocytes by down-regulation of p21 protein expression. However, TSP-1 does not affect the termination phase of liver regeneration after PH.

TGF-β/Smad Signaling Is Activated by TSP-1 in Response to PH.

To address the possible mechanisms underlying this accelerated liver regeneration in TSP-1-null mice, we examined TGF-β/Smad signaling. TGF-β1 mRNA levels in both WT and TSP-1-null mice increased after hepatectomy by real-time PCR, and those levels in TSP-1-null mice were significantly up-regulated at 3 and 6 hours, compared with controls (P < 0.05 at 3 hours and P < 0.01 at 6 hours; Fig. 4A). In sharp contrast, the levels of active TGF-β1 in TSP-1-null liver were significantly lower than controls at 6 hours after PH, whereas the levels of total TGF-β1 did not show any significant differences between them (Fig. 4B). Furthermore, the levels of phosphorylated Smad2 (pSmad2, C-terminal Ser465/467) protein, as a downstream mediator of active TGF-β1, significantly diminished at 6 and 12 hours in TSP-1-null mice, compared with controls (to 16% at 6 hours and 69% at 12 hours versus controls, respectively; P < 0.01 at 6 hours and P < 0.05 at 12 hours), as determined by western blotting (Fig. 4C). Using IF staining, we confirmed the significantly decreased number of nuclear localized pSmad2-positive cells at 6 hours in TSP-1-null mice, compared with controls (P < 0.01; Fig. 4D). A secondary, minor induction of pSmad2 at 72 hours was also significantly attenuated in TSP-1-null mice, compared with controls (Fig. 4C).

Figure 4.

Significantly decreased TGF-β/Smad signal transduction and cell death in TSP-1-null mice after PH. (A) Real-time PCR analysis of TGF-β1 mRNA expression after PH (n = 8 per time point in each group). *P < 0.05 versus TSP-1+/+ mice; **P < 0.01 versus TSP-1+/+ mice. (B) Levels of active and total TGF-β1 in WT and TSP-1-null liver at 6 hours after PH (n = 6 per time point in each group). **P < 0.01 versus TSP-1+/+ mice. (C-E) Effects of TSP-1 deficiency on pSmad2 expression in the regenerating liver. (C) Upper panels: western blotting analysis of pSmad2 and total Smad2 in WT and TSP-1-null liver. Lower panel: densitometric analysis of pSmad2 protein expression (n = 3). Each pSmad2 intensity was normalized to total Smad2, then the intensity of WT mice at 0 hours was set to 1. *P < 0.05 versus TSP-1+/+ mice; **P < 0.01 versus TSP-1+/+ mice. (D) Assessment of pSmad2 nuclear localization. Left panel: IF staining for pSmad2 at 6 hours in WT and TSP-1-null liver. Right panel: analysis of pSmad2-positive nuclei (n = 5 in each group). Arrowheads indicate pSmad2 (red)/ DAPI (blue) double-positive nuclei (purple). **P < 0.01 versus TSP-1+/+ mice. Scale bar = 25 μm. (E) Real-time PCR analysis of PAI-1 mRNA expression in WT and TSP-1-null liver after PH (n = 8 per time point in each group) and in WT liver after sham operation (n = 3 per time point). **P < 0.01 versus TSP-1+/+ mice. (F) Assessment of TUNEL-positive cell death in the regenerating liver. Left panel: TUNEL staining at 6 hours after PH in WT and TSP-1-null liver. Right panel: analysis of TUNEL-positive cells (n = 5 per time point in each group). Arrowheads indicate TUNEL (red)/DAPI (blue) double-positive nuclei (purple). **P < 0.01 versus TSP-1+/+ mice. Scale bar = 50 μm. DAPI, 4′,6-diamidino-2-phenylindole.

Plasminogen activator inhibitor-1 (PAI-1) is one of the downstream targets of TGF-β1 in hepatocytes.21 Although intense inductions of PAI-1 mRNA at 6 hours after hepatectomy were observed in both WT mice and TSP-1-null mice by real-time PCR, the induction level in TSP-1-null mice was significantly diminished (to 37% of controls; P < 0.05 at 6 hours) (Fig. 4E).

Cell death is also implicated as a mechanism of TGF-β-mediated cell-growth inhibition. TUNEL-positive cells, as a marker for cell death, are immediately and transiently detectable after hepatectomy.22 We determined whether deficiency in TSP-1 affected cell death in the regenerating liver. Although the number of TUNEL-positive cells in WT liver transiently increased at 6 hours after hepatectomy, TSP-1-null liver showed a significant reduction, compared with controls (P < 0.05 at 6 hours; Fig. 4F).

These results suggest that TSP-1-mediated active TGF-β1 plays a pivotal role in TGF-β/Smad signal transduction after PH.

Deficiency in TSP-1 Accelerates STAT3 and PI3K/Akt Signals, Not Extracellular Signal-Related Kinase 1 And 2 Signal, in the Early Phase After PH.

There is in vitro evidence that TSP-1 down-regulates phosphorylated Akt (Ser473) expression through its receptor, CD47, in HUVECs.23 Indeed, signaling pathways, such as phosphatidylinositide 3-kinase (PI3K)/Akt, signal transducer and activator of transcription 3 (STAT3), and extracellular signal-related kinase 1 and 2 (Erk1/2), are important for cell survival and/or proliferation after PH hepatectomy.24 Therefore, we next examined whether the deficiency in TSP-1 affected the activation of these signaling pathways in the early phases posthepatectomy. TSP-1-null mice showed earlier, more intense phosphorylation of STAT3 (Tyr705) (6-fold at 1 hour; P < 0.01) and Akt (Ser473) (4.2-fold at 1 hour; P < 0.01) in the early stage after PH hepatectomy, compared with controls, as determined by western blotting (Fig. 5). In contrast, levels of phosphorylated Erk1/2 did not show any remarkable differences between the two groups (Fig. 5).

Figure 5.

TSP-1 deficiency enhances STAT3 and PI3K/Akt, but not Erk1/2 signal, in the early phase after PH. Upper panels: western blotting analysis of STAT3, PI3K/Akt, and Erk1/2 signals. HSC70 served as a loading control. Lower panels: densitometric analysis of phosphorylated protein expression after PH. Each pSTAT3, pAkt, and pErk1/2 intensity was normalized to HSC70, then the intensity of WT mice at 0 hours was set to 1. *P < 0.05 versus TSP-1+/+ mice; **P < 0.01 versus TSP-1+/+ mice.

TSP-1 Induction in ECs Is Associated With ROS.

Although our findings show that TSP-1 plays a potential role as a negative regulator in the regenerating liver, the mechanism of TSP-1 induction in ECs in response to PH hepatectomy remains unknown. There is a line of evidence that ROS are produced in the regenerating liver after PH hepatectomy.22, 25 In WT mice, levels of tissue content of MDA as a lipid peroxidation marker for ROS generation were significantly increased at both 3 and 6 hours and returned to basal levels by 12 hours after hepatectomy (P < 0.05 in both; Fig. 6A). Next, to determine whether ROS could induce TSP-1 expression in ECs, we performed an in vitro study using HUVECs with the potent ROS inducer, H2O2. In HUVECs, treatment with H2O2 induced TSP-1 protein expression in a dose-dependent manner (Fig. 6B-D). Furthermore, this induction was inhibited by pretreatment with 30 mM of NAC, a scavenger of ROS (Fig. 6B-D). Thus, these results indicate that oxidative stress is one factor responsible for TSP-1 induction in ECs.

Figure 6.

TSP-1 induction in ECs by ROS. (A) Assessment for levels of MDA after 70% PH (n = 5) and sham operation (n = 3). *P < 0.05 versus sham-operated mice. (B-D) TSP-1 protein expression by H2O2, a potent ROS inducer, in HUVECs. (B) Western blotting analysis of TSP-1 after treatment of HUVECs with H2O2. β-tubulin served as a loading control. (C) Densitometric analysis of TSP-1 expression from three independent experiments. Each TSP-1 intensity was normalized to β-tubulin, then the intensity of control was set to 1. Note that the TSP-1 protein expression levels after treatment with 0.05 and 0.1 mM of H2O2 are significantly higher versus controls, whereas the induction of TSP-1 by treatment with 0.1 mM of H2O2 is significantly inhibited with a pretreatment using 30 mM of NAC. *P < 0.05; **P < 0.01. (D) ICC for TSP-1 protein in HUVECs after treatment with H2O2 (TSP-1, green; DAPI, blue). Note that HUVECs after treatment with 0.1 mM of H2O2 express TSP-1 in their cytoplasm (arrowheads), whereas the induction of TSP-1 is inhibited by pretreatment using 30 mM of NAC. Scale bar = 50 μm. (E and F) Assessment of pSmad2 nuclear localization in primary hepatocytes. (E) IF staining for pSmad2 with or without ROS-treated CM from HUVECs (HUVEC CM). Scale bar = 25 μm. (F) Effect of TSP-1-inhibitory peptide LSKL on pSmad2 induction. Error bars represent standard deviation (n = 5 in each group; field = 0.15 mm2). SLLK, control peptide. **P < 0.01. DAPI, 4′,6-diamidino-2-phenylindole.

To further determine whether HUVEC-derived TSP-1 could modulate TGF-β/Smad signaling and proliferation in hepatocytes in vitro, we isolated primary hepatocytes from adult WT mice.15 The treatment of conditioned media from HUVECs with primary hepatocytes actually induced pSmad2 (Fig. 6E). Furthermore, the pretreatment of primary hepatocytes with TSP-1-inhibitory peptide LSKL16, 17 significantly suppressed conditioned media (CM)-induced pSmad2 expression, whereas the control peptide, SLLK, showed no effects (Fig. 6F). It is known that primary hepatocytes lack the ability to proliferate, even though such cells in vivo readily replicate and/or synthesize DNA after PH.26 Although a few proliferative primary hepatocytes were found by Ki67 immunostaining in culture, the treatment of CM from HUVECs with primary hepatocytes significantly reduced the number of Ki67-positive cells (Supporting Fig. 2).

Discussion

In the present study, we have demonstrated the following (Fig. 7): (1) TSP-1 is induced in ECs as an immediate early gene by ROS and participates in TGF-β signal transduction in the initial response to PH and (2) TSP-1 deficiency results in the significant reduction of TGF-β/Smad signal, and this could cause the accelerated S-phase entry of hepatocytes by down-regulation of p21 protein expression. Thus, this is the first study providing compelling evidence that local TGF-β activation machinery plays an important role in inhibiting liver regeneration after PH hepatectomy.

Figure 7.

Schematic illustration of the role of TSP-1 in the regenerating liver. In WT mice, newly synthesized ROS in response to PH stimulate ECs to express TSP-1. TSP-1 induced by ECs converts latent TGF-β1 into its active form. Active TGF-β1 suppresses cell-cycle progression in hepatocytes at the G1/S checkpoint. In contrast, TSP-1 deficiency decreases active TGF-β1 levels, which, in turn, results in the acceleration of liver regeneration.

Our study supports the notion that oxidative stress is one factor responsible for TSP-1 induction in the regenerating liver. TSP-1 is the most likely candidate protein induced by oxidative stress in proteomic analysis using brain ECs.27 These findings imply that ECs initially sense locally produced ROS in response to tissue damage, and that the subsequent induction of TSP-1 in these cells after initiates tissue remodeling. Indeed, our results revealed that EC-derived TSP-1 can modulate TGF-β/Smad signaling and proliferation in hepatocytes. ECs represent the largest population of nonparenchymal cells in the liver. Identification of the functional role of immediate early genes provides the clues for understanding the molecular bases of liver regeneration. One recent study documented that Id-1, a vascular endothelial growth factor-A receptor (VEGFR)-2-mediated transcriptional factor, was induced in ECs at ∼48 hours after hepatectomy; Id-1, in turn, promoted hepatocyte proliferation.28 There has, as yet, been no report implicating ECs in earlier stages of the regenerating liver (within 24 hours). We have identified TSP-1 as a novel immediate early gene derived from ECs, showing that the expression level of TSP-1 was immediately up-regulated and returned to basal levels by 24 hours in response to PH hepatectomy. Our findings and the previous report28 suggest that ECs may play two distinct roles in hepatocyte proliferation after PH hepatectomy: One is an antiproliferative role by activating the TSP-1/TGF-β1 axis within 24 hours, and the other is a proproliferative role by activating VEGFR-2 after 24 hours. This finding is consistent with the evidence that TSP-1 inhibits the activation of VEGFR-2 through its receptor, CD47, in ECs,23 and suggests that the reduction of TSP-1 expression may be required for the functional shift in ECs from an anti- to a proproliferative role in hepatocytes. Microvascular rearrangement is important for tissue remodeling, and the antiangiogenic action is one of the well-recognized functions of TSP-1.29 However, the expression of CD31 mRNA for monitoring angiogenesis did not show any significant difference between WT and TSP-1-null mice at 24, 48, and 72 hours after PH hepatectomy (Hayashi H, and Sakai T; unpublished data), suggesting that TSP-1 does not affect vascularization during liver regeneration after PH hepatectomy.

TGF-β1 is known to be a potent inhibitor of mitogen-stimulated DNA synthesis in cultured hepatocytes.3 p21 is important for inhibiting hepatocyte proliferation in vivo, especially at the G1/S transition of the cell cycle,20 and the expression of p21 is up-regulated by TGF-β1.30 There is evidence that TGF-β1 mRNA induction occurs within 4 hours and remains elevated until 72 hours after PH hepatectomy.5, 6 In contrast, we found the only limited activation of TGF-β signaling in an earlier phase (within 24 hours), with a peak at ∼12 hours. It is known that TGF-β is secreted as latent forms and they are converted into active TGF-β in response to injury. There are several mechanisms for activation, such as by proteases, integrins (e.g., αvβ6 and αvβ8), and TSP-1, all of which are likely to be tissue specific.31 Whereas the complete lack of TGF-β-mediated signal in hepatocyte-specific TGF-β type II receptor knockout mice accelerates hepatocyte proliferation in the later phase (∼36-48 hours) after hepatectomy,7 the role of TGF-β signaling in the earlier phase (within 24 hours) remains to be elucidated. Our present findings provide compelling evidence that locally activated TGF-β1 mediated by TSP-1 as an immediate early gene is critical in the early phase (within 24 hours) post PH posthepatectomy to initiate the inhibitory effect on hepatocyte proliferation, and this TGF-β signaling has a functional link to the G1/S-phase transition by modulating p21 protein expression. A major downstream target of TGF-β1, PAI-1,21 is a negative regulator of liver regeneration, and PAI-1-null mice show acceleration of liver regeneration after Fas-mediated massive hepatocyte death.32 The significant down-regulation of PAI-1 expression in our TSP-1-null liver may be implicated in the accelerated hepatocyte proliferation after PH hepatectomy. However, our TSP-1-null model did not show any obvious differences in the termination phase of liver regeneration, compared with controls, such as the TGF-β type II receptor knockout mice model.7 Although the molecular mechanisms underlying the termination of liver regeneration remain to be elucidated,4 our and other findings suggest that the orchestrating interactions among positive and negative regulators in hepatocyte proliferation would be critical for the termination of liver regeneration.4, 24

Active TGF-β1 induces hepatocyte cell death. STAT3- and PI3K/Akt-signaling pathways are crucial for cell survival (i.e., antiapoptosis) in the acute phase after PH. Our signaling data using TSP-1-null mice are consistent with previous findings showing that STAT3- and PI3K/Akt-signaling pathways, but not the Erk1/2 pathway, play a protective role against TGF-β-induced apoptosis in hepatocyte cell lines.33, 34 Several in vitro studies have reported that TSP-1 down-regulates phosphorylated Akt expression in retina35 and ECs.23 Another in vitro study showed that the lack of TSP-1 in retinal ECs results in up-regulation of phosphorylated Akt expression, but not phosphorylated Erk1/2.36 Because TSP-1 is a multidomain and multifunctional matricellular protein, our data and these findings suggest that TSP-1 modulates not only TGF-β signal, but also cell survival signals, such as STAT3 and PI3K/Akt signals, through its multidomain.

In the clinical setting, no established therapeutic strategies to accelerate liver regeneration have been available, to date. The inhibition of TSP-1 function attenuates locally activated TGF-β1 signals and thereby accelerates hepatocyte proliferation; hence, TSP-1 could be a novel therapeutic target for accelerating liver regeneration after PH.

Acknowledgements

The authors thank Dr. Jack Lawler for TSP-1-null mice, Drs. Koichi Matsuzaki and Deane Mosher for antibodies, and Diskin Erik and Dr. Judy Drazba (Imaging Core, Lerner Research Institute, Cleveland, OH) for IF microscopic analyses. The authors are also grateful to Dr. Jo Adams for assistance with TSP-1 immunostaining experiments and scientific discussions.

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