Tissue inhibitor of metalloproteinase 1 (TIMP-1) deficiency exacerbates carbon tetrachloride-induced liver injury and fibrosis in mice: involvement of hepatocyte STAT3 in TIMP-1 production
© Wang et al; licensee BioMed Central Ltd. 2011
Received: 10 January 2011
Accepted: 4 April 2011
Published: 4 April 2011
Tissue inhibitor of metalloproteinase 1 (TIMP-1), which is thought to be produced mainly by activated hepatic stellate cells and Kupffer cells in the liver, plays a pivotal role in matrix remodeling during liver injury and repair; while the effect of TIMP-1 on hepatocellular damage remains obscure.
Hepatic expression of TIMP-1 mRNA and protein was up-regulated both in acute and chronic liver injury induced by carbon tetrachloride (CCl4). Compared with wild-type mice, TIMP-1 knockout mice were more susceptible to CCl4-induced acute and chronic liver injury, as shown by higher levels of serum alanine aminotransferase (ALT), greater number of apoptotic hepatocytes, and more extended necroinflammatory foci. TIMP-1 knockout mice also displayed greater degree of liver fibrosis after chronic CCl4 injection when compared with wild-type mice. In vitro treatment with TIMP-1 inhibited cycloheximide-induced cell death of primary mouse hepatocytes. Finally, up-regulation of TIMP-1 in the liver and serum after chronic CCl4 treatment was markedly diminished in hepatocyte-specific signal transducer and activator of transcription 3 (STAT3) knockout mice. In vitro treatment with interleukin-6 stimulated TIMP-1 production in primary mouse hepatocytes, but to a lesser extent in STAT3-deficient hepatocytes.
TIMP-1 plays an important role in protecting against acute and chronic liver injury and subsequently inhibiting liver fibrosis induced by CCl4. In addition to activated stellate cells and Kupffer cells, hepatocytes are also responsible for TIMP-1 production during liver injury via a STAT3-dependent manner.
Chronic liver fibrosis induced by viral hepatitis, alcohol abuse, and nonalcoholic steatohepatitis is a major cause of morbidity and mortality worldwide . The progression of liver disease can be defined as an alteration of hepatic parenchyma characterized by two major events: injury and regeneration. The initial cause of the injury determines the loss of hepatocytes including apoptosis and necrosis followed by inflammatory response . Consequently, the loss of tissues or liver injury leads viable hepatocytes to re-enter the cell cycle and divide by mitosis, to replace the lost or damaged hepatocytes . During these wound healing processes, the extracellular matrix (ECM) also undergoes a process of remodeling stimulated by persisting inflammatory injury, which may result in abnormal collagen deposition . This microenvironment alteration responsible for hepatocyte damage and ECM remodeling is highly complex and its mechanisms are not fully understood. It seems that all types of liver cells and a variety of soluble factors are involved in the process of ECM remodeling, contributing to hepatocyte injury, inflammation, fibrosis and liver regeneration [4–8].
Matrix metalloproteinases (MMPs) and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) play an important role in inducing and preventing the degradation of the ECM, respectively . Many studies have shown that MMPs and TIMPs play a pivotal role in matrix remodeling during hepatic injury and repair [5, 8, 10–12]. Among them, TIMP-1 is a widely expressed, and secreted protein that plays a critical role in tissue remodeling via inhibiting members of a large family of MMPs . TIMP-1 has been suggested to be a serum marker for liver fibrosis, and the expression is induced during liver injury . In addition, TIMP-1 also plays an important role in promoting liver fibrosis [15–17] but inhibiting liver regeneration . The profibrogenic effects of TIMP-1 are thought to be mediated via preventing collagen degradation through inhibition of MMPs and protecting against activated hepatic stellate cell (HSC) death [17–20]. It is believed that activated HSCs and Kupffer cells are the major sources for TIMP-1 production during liver injury . Although early studies also showed TIMP-1 mRNA and protein expression are up-regulated by inflammatory cytokines in rat hepatocytes [22, 23], the precise roles of TIMP-1 produced by hepatocytes in liver injury remain largely unknown. In this study, we found that TIMP-1-deficient (TIMP-1-/-) mice were more susceptible to CCl4-induced liver injury and fibrosis, suggesting the protective feature of TIMP-1 in liver injury. Moreover, in vitro experiments showed that TIMP-1 directly protected against cycloheximide-induced hepatocyte death. Lastly, we provided evidence suggesting that hepatocytes also contribute to TIMP-1 production during chronic liver injury, which is controlled by STAT3.
Up-regulation of TIMP-1 in acute and chronic liver injury after CCl4 exposure
TIMP-1-/- mice are more susceptible to acute liver injury induced by CCl4 administration
TIMP-1-/- mice are more susceptible to CCl4-induce chronic liver injury and fibrosis
TIMP-1 directly protects against hepatocyte death in vitro
IL-6 up-regulates TIMP-1 mRNA and protein in primary cultured hepatocytes via a STAT3-dependent manner
Deletion of STAT3 in hepatocytes reduces hepatic and serum levels of TIMP-1 after chronic CCl4 treatment
Although the profibrotic effect of TIMP-1 mainly produced by HSCs has been well documented [15, 19], we reveal here for the first time an unexpected hepatoprotective feature of TIMP-1 and contribution of hepatocytes to TIMP-1 production during CCl4-induced liver injury.
Hepatoprotection of TIMP-1: dual roles of TIMP-1 in liver fibrosis
TIMP-1-/- mice were more susceptible to hepatocelluar damage induced by CCl4 treatment, suggesting that TIMP-1 plays a hepatoprotective role during liver injury. Such hepatoprotection is mediated, at least in part, via directly inhibiting hepatocyte death as TIMP-1 treatment prevented cycloheximide-induced hepatocyte damage (Figure 5). At the present, the mechanism underlying TIMP-1 hepatoprotection remains unknown. TIMP-1 is a survival factor for many cell types dependent and/or independent of the MMP-inhibitory activity . For example, TIMP-1 inhibits HSC apoptosis via MMP inhibition , while the anti-apopotic effect of TIMP-1 on human breast carcinoma cells does not require MMP inhibition . Further studies will be required to investigate the mechanism underlying the anti-apoptotic effect of TIMP-1 on hepatocytes.
TIMP-1 has been suggested as a profibrogenic factor to promote liver fibrosis as liver-specific TIMP-1 transgenic mice were resistant to fibrosis resolution  and TIMP-1 neutralizing antibody inhibited liver fibrosis . However, surprisingly, TIMP-1-/- mice developed greater fibrosis compared with wild-type mice after CCl4 challenge (Figure 3). As TIMP-1 protects against HSC death  and hepatocyte apoptosis (Figure 5), we speculate that TIMP-1 may have dual roles in liver fibrosis: stimulating liver fibrosis via promoting HSC survival and inhibiting liver fibrosis via preventing liver injury. The final effect of TIMP-1 on liver fibrosis is determined by the balance between these stimulatory and inhibitory effects. Deletion of TIMP-1 may reduce liver fibrosis through abolishing the profibrogenic effect of TIMP-1, but may also accelerate liver fibrosis by increasing liver injury. Acceleration of liver fibrosis by increased liver injury in TIMP-1-/- mice may dominate over the profibrogenic effect of TIMP-1 on liver fibrosis, leading to greater liver fibrosis in TIMP-1-/- mice after CCl4 treatment.
Hepatocytes contribute to TIMP-1 production during liver injury: controlled by STAT3
Expression of TIMP-1 is induced in the liver during liver injury. It is generally believed that activated HSCs and Kupffer cells are the major source of TIMP-1 production as strong TIMP-1 immunostaining was detected in activated HSCs and Kupffer cells . Our findings here suggest that hepatocytes also contribute significantly to TIMP-1 production that is controlled by STAT3. As shown in Figure 7, serum and hepatic levels of TIMP-1 were lower in STAT3Hep-/- mice, suggesting that activation of STAT3 in hepatocytes plays an important role in induction of TIMP-1 during liver injury. This induction is likely due to the direct stimulation of TIMP-1 production in hepatocytes by STAT3 as in vitro IL-6 treatment induced TIMP-1 production in cultured hepatocytes [23, 29] and such induction was diminished in STAT3-deficient hepatocytes (Figure 6). In addition, STAT3 binding sites were found on TIMP-1 promoter , providing a molecular basis for STAT3-mediated induction of TIMP-1. Finally, the conclusive evidence for contribution of hepatocytes to TIMP-1 production is that TIMP-1 was stained strongly in hepatocytes from the livers of mice with acute and chronic CCl4 treatment. Collectively, these findings suggest that in addition to HSCs and Kupffer cells, hepatocytes are also a source for TIMP-1 production which is controlled partially by STAT3 during chronic liver injury.
In summary, our observations collectively identify newly hepatoprotective role of TIMP-1 in a positive feedback manner during liver injury, which is regulated by IL-6/STAT3 signaling pathway. TIMP-1 plays dual roles in regulating liver fibrosis by inhibiting liver fibrosis via protecting against liver injury or by promoting liver fibrosis via protecting against HSC death.
Materials and methods
Eight- to ten-week-old male TIMP-1-/- mice and their wild-type control C57BL/6 mice were purchased from the Jackson laboratory (Bar Harbor, Maine). Hepatocyte-specific STAT3 knockout mice (AlbCre+/-STAT3flox/flox)(STAT3Hep-/-) and their littermate wild-type controls (AlbCre-STAT3flox/flox) were described previously . All animal experiments were approved by the Institutional Animal Care and Use Committee of the NIAAA.
CCl4-induced liver injury
For acute CCl4-induced liver injury, mice were injected (i.p) with a single dose of CCl4 (2 ml/kg body weight of 10% CCl4 dissolved in olive oil). For chronic CCl4 studies, mice received CCl4 injection (2 ml/kg body weight of 10% CCl4) 3 times a week for up to 4 weeks. Control groups were treated with vehicle (100% olive oil, 2 ml/kg). In chronic studies, the mice were sacrificed at different time points after the last injection of chronic CCl4 treatment.
Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) were determined using a chemistry analyzer (PROCHEM-V; Barrow-in-Furness, UK). Serum TIMP-1 levels were assessed by Quantikine enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN).
Formalin-fixed liver samples were processed, and paraffin-embedded liver tissue sections were stained with hematoxylin and eosin (H&E). Liver fibrosis was determined by Sirius Red staining for collagens or immunohistochemical staining for activated HSCs with anti-α-smooth muscle actin (α-SMA) (Dako, Carpinteria, CA), and were quantified by digital imaging with NIH Scion Image and Adobe Photoshop (San Jose, CA). Expression of TIMP-1 in the liver was measured by immunohistochemical staining with anti-TIMP-1 antibody (R&D Systems).
Hepatocyte apoptosis was detected by using an Apoptag Apoptosis Detection Kit (Chemicon International, Temecula, CA) as previously described .
Real Time PCR
Total RNA was purified from about 30 mg liver samples according to the manufacturer (Qiagen, Valencia, CA) and then 1 μg mRNA was reverse-transcribed to cDNA using a High Capacity cDNA Reverse Transcription kit (Invitrogen, Carlsbad, CA). The cDNA template was diluted 1:5 and amplified in real-time PCR using iTaq SYBR Green Supermix (Bio-rad, Hercules CA). An initial denaturation at 95°C for 3 min was followed with PCR cycling: 95°C (15 sec), and 58°C (30 sec) for 40 cycles. Relative mRNA levels were calculated by means of 2-ΔΔCT (ΔΔCT = difference of crossing points of test samples and respective control samples as extracted from amplification curves by the LightCycler software) after normalization to 18S expression used as an internal standard. Fold inductions of analyzed mRNA expression were normalized on 18S RNA expression. The sequences of primers were described previously .
Liver homogenates were prepared in RIPA buffer (50 mM Tris; 1% NP40; 0.25% Deoxycholic acid sodium salt; 150 mM NaCl; 1 mM EGTA) containing 1 mM Na3VO4 and a protease inhibitor cocktail (Sigma, St. Louis, MO). Protein concentrations were quantified with a detergent compatible protein assay kit (Bio-Rad Laboratories) according to the manufacture's manual. Fifty μg of total protein extracts were denatured in Laemmli buffer containing 5% β-mercaptoethanol, then loaded and separated by gel electrophoresis on a 7% Bis-Tris gel (Invitrogen). Primary antibody was incubated at 4°C overnight under shaking conditions. Immunoreactive bands were visualized on nitrocellulose membranes using alkaline-phosphotase-linked anti-mouse or rabbit antibody and the ECF detection system with a PhosphorImager (GE Healthcare, Piscataway, NJ). Mouse monoclonal anti-α-SMA antibody was obtained from Sigma-Aldrich. Mouse monoclonal anti-GAPDH antibody was obtained from Cell Signaling Technology (Danvers, MA).
Hepatocyte culture and treatment
Mouse hepatocytes were isolated by in situ collagenase perfusion method . Hepatocytes (2 × 105 cells/per well) were cultured in 6-well plates with serum-free medium and treated with IL-6 (50 ng/ml), followed by the measurement of TIMP-1 protein in culture medium. The hepatocytes were also cultured in medium containing 5% serum and treated with cycloheximide (100 μM) (Sigma) in the presence or absence of recombinant murine TIMP-1 (100 ng/ml or 200 ng/ml). Cycloheximide was used to induce hepatocyte apoptosis. Hepatocyte death was quantified by measuring the activity of released AST in culture medium.
Data are expressed as means ± SEM (N = 5-12 in each group). Student t test was performed to compare values from 2 groups. To compare values obtained from three or more groups, 1-factor analysis of variance (ANOVA) was used, followed by Tukey's post hoc test. Statistical significance was taken at the P < 0.05 level.
List of Abbreviations
signal transducer and activator of transcription
- STAT3Hep-/- mice:
hepatocyte-specific STAT3 knockout mice
tissue inhibitor of metalloproteinase
This work was supported in part by the intramural program of NIAAA, NIH (B Gao) and in part by the Natural Science Foundation of China (H Wang, No. 30973467/H1611).
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