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Retinol dehydrogenase 13 deficiency diminishes carbon tetrachloride-induced liver fibrosis in mice
Accepted Manuscript Title: Retinol dehydrogenase 13 deficiency diminishes carbon tetrachloride-induced liver fibrosis in mice Author: Xiaofang Cui Suying Dang Yan Wang Yan Chen Jia Zhou Chunling Shen Ying Kuang Jian Fei Lungen Lu Zhugang Wang PII: DOI: Reference:
S0378-4274(16)33304-5 http://dx.doi.org/doi:10.1016/j.toxlet.2016.11.010 TOXLET 9639
To appear in:
Toxicology Letters
Received date: Revised date: Accepted date:
20-10-2015 11-11-2016 15-11-2016
Please cite this article as: Cui, Xiaofang, Dang, Suying, Wang, Yan, Chen, Yan, Zhou, Jia, Shen, Chunling, Kuang, Ying, Fei, Jian, Lu, Lungen, Wang, Zhugang, Retinol dehydrogenase 13 deficiency diminishes carbon tetrachloride-induced liver fibrosis in mice.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2016.11.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Retinol dehydrogenase 13 deficiency diminishes carbon tetrachloride-induced liver fibrosis in mice
Xiaofang Cuia,b , Suying Danga, Yan Wangc, Yan Chena, Jia Zhoua, Chunling Shena,d, Ying Kuangd, Jian Feid, Lungen Luc* and Zhugang Wanga,b,d*
a
State Key Laboratory of Medical Genomics, Research Center for Experimental
Medicine, Shanghai Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (SJTUSM), b
Model Organism Division, E-Institutes of Shanghai Jiao Tong Universities School of
Medicine (SJTUSM), Shanghai 200025, China; c
Department of Gastroenterology, Shanghai First People’s Hospital Affiliated to
SJTUSM, Shanghai 200080, China; d
Shanghai Research Center for Model Organisms,
*
Corresponding authors. E-mail addresses: [email protected] (Z. Wang) or
[email protected] (L. Lu)
Highlights Retinol dehydrogenase 13 (RDH13) is a mitochondrion-localized member of the short-chain dehydrogenases/reductases (SDRs) superfamily that participates in metabolism of some compound.Rdh13 mRNA is most highly expressed in mouse liver. Rdh13 deficiency reduces the extent of liver injury and fibrosis, reduces hepatic stellate cell
(HSC)
activation,
metalloproteinase1(TIMP-1)
attenuates
collagen
andtransforming
I(II)
,
growth
tissue
inhibitor
factor
of
beta1
(Tgf-β1)expression.The resultsindicate an important role of Rdh13 and suggest
RDH13 as a possible new therapeutic target for CCl4-induced fibrosis.
1
Abstract Retinol dehydrogenase 13 (RDH13) is a mitochondrion-localized member of the short-chain dehydrogenases/reductases (SDRs) superfamily that participates in metabolism of some compound. Rdh13 mRNA is most highly expressed in mouse liver. Rdh13 deficiency reduces the extent of liver injury and fibrosis, reduces hepatic stellate cell (HSC) activation, attenuates collagen I (II) , tissue inhibitor of
metalloproteinase 1 (TIMP-1) and transforming growth factor beta 1 (Tgf-β1) expression. The results indicate an important role of Rdh13 and suggest RDH13 as a possible new therapeutic target for CCl4-induced fibrosis. Key words: Retinol dehydrogenase 13, Liver fibrosis, Knockout, Hepatic stellate cells, Carbon tetrachloride Introduction Retinol dehydrogenase 13 (RDH13) is a recently identified member of the short-chain dehydrogenases/reductases (SDRs) superfamily that participate in metabolism of prostaglandins, retinoids, steroids and aliphatic alcohols (Haeseleer et al., 2002; Levi et al., 2012; Oppermann et al., 2003). RDH13 shares sequence similarity with RDH11, RDH12 and RDH14 proteins which have been characterized as microsomal proteins that recognize retinoids and medium-chain aldehydes as substrates, with NADP+/NADPH as the preferred cofactors (Belyaeva et al., 2005). It has been demonstrated that RDH13 is localized on the outer side of the inner mitochondrial membrane and exhibits a wide tissue distribution. Purified RDH13 is catalytically active and recognizes retinoids as substrates, strongly prefers NADPH over NADH as a cofactor, and has a much greater catalytic efficiency as a reductase than as a dehydrogenase (Belyaeva et al., 2008). Our previous work show that Rdh13 plays an important role in retinal light damage which is consistent with Rdh11 and Rdh12 in mice (Wang et al., 2012). Thus, RDH13 is also considered to participate in the retinoid metabolism. The liver is the most important organ in the body involved in retinoid storage and metabolism, and also an important target organ for retinoid actions. Within the liver, 2
both hepatocytes and HSCs are importantly involved in retinoid metabolism (Shirakami et al., 2012). Retinoic acid is synthesized in the liver and can interact with retinoid receptors which control expression of a large number of genes involved in hepatic processes. Altered retinoid metabolism and the accompanying dysregulation of retinoid signaling in the liver contribute to hepatic disease including fatty liver, liver fibrosis, cirrhosis and hepatocellular carcinoma (Shirakami et al., 2012). This is related to HSCs, which store more than 50% of all retinoid present in the body and contribute significantly to the development of hepatic disease when they undergo a process of cellular activation. HSC activation results in the loss of HSC retinoid stores and changes in extracellular matrix deposition leading to the onset of liver fibrosis. Within the liver, like in other tissues, retinol is converted to retinoic acid via two oxidative steps. Retinol dehydrogenases (RDHs) catalyze the first enzymatic step which is the oxidation of retinol to retinaldehyde. Several RDHs including RDH5, RDH10, RDH11, RDH1 and RDH13 are expressed in the liver and exhibit RDH activity in vitro (Shirakami et al., 2012). But the physiological role of each of them in retinoic acid synthesis and liver disease remains unclear. Our previous work showed that Rdh13 mRNA is most highly expressed in mouse liver, which is 3-10 times that of other tissues. This suggests possible importance of Rdh13 in the retinoid signaling and hepatic pathology. Recently, we investigated the function of Rdh13 in liver and acute liver injury by using Rdh13-deficient mice. We found that there was no obvious difference in the serum biochemistry and the liver histology between wild-type and Rdh13-deficient mice under normal condition, and Rdh13-deficient mice showed reduced liver injury and hepatocyte proliferation compared to wild-type mice following carbon tetrachloride administration. Here, we use Rdh13 knockout mice to investigate the role of Rdh13 in liver fibrosis after repeated CCl4 administration. The results indicate that Rdh13 deficiency reduces the extent of liver injury and liver fibrosis, reduces HSC activation, attenuates collagen I(II), and TIMP-1 expression after chronic CCl4 treatment. Our findings indicate an important role of Rdh13 in liver disease in vivo and suggest RDH13 as a possible new therapeutic target for the prevention and treatment of hepatic fibrosis in 3
chronic liver disease.
Materials and methods Animals WT and Rdh13 KO mice (8-10-week-old) with a mixed genetic background of C57BL/6 and 129/Sv were used in the study. The generation of Rdh13 KO mice has been described previously (Wang et al., 2012). Mice were allowed free access to food and water, and were housed under specific pathogen-free (SPF) conditions at a constant room temperature of 22-24 °C with a 12 h light-dark cycle during the study. All animal experiments were approved by the Animal Use and Care Committee of Shanghai Jiao Tong University School of Medicine. CCl4-induced liver injury CCl4 was used to induce liver injury and liver fibrosis. For acute CCl4-induced liver injury, a single dose of CCl4 (1.0 ml/kg body weight of 20% CCl4 diluted in olive oil) was administered intraperitoneally to WT and Rdh13 KO mice (n=4 per group for each time point, 2 male and 2 female mice). Animals were sacrificed 0, 24 and 72 h after CCl4 injection. In the chronic injury model, WT and Rdh13 KO mice (n=10 per group,5 male and 5 female mice) were administered intraperitoneally with the same dose of CCl4 as acute liver injury twice per week for 4 weeks. Animals were sacrificed three days after the final CCl4 injection. At the time of sacrifice, mice were anesthetized and liver samples were harvested, rapidly washed with ice-cold phosphate buffered saline (PBS) and dissected into smaller pieces. Liver tissues from different lobes were fixed in 10% neutral formalin for 24 h and then embedded in paraffin for histological examination. Other parts were snap-frozen in liquid nitrogen and stored at -80 °C for RNA and protein analysis. Histological analysis Formalin-fixed liver samples were processed, and paraffin-embedded liver tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined by immunohistochemistry. Liver fibrosis was assessed by means of Masson’s trichrome staining for hepatic collagen deposition. Fibrosis deposition was quantified and 4
analyzed by digital imaging with a Zeiss Axioplan 2 imaging analysis system. Immunohistochemistry Formalin-fixed paraffin embedded liver sections (5 μm) were deparaffinized with xylene, hydrated in decreasing concentrations of ethanol. Protein epitopes were unmasked by citrate buffer incubation at 92-98 °C for 15 minutes. After heat treatment, the sections were allowed to cool at room temperature. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide in PBS for 10 minutes. Sections were incubated with serum-containing blocking reagents (Vector Laboratories, lnc. Burlingame, CA) for 1 h, then incubated with primary antibody overnight at 4 °C. The staining procedure was performed according to the manufacturer’s instructions of VECTASTAINABC system (Vector Labs). Finally, sections were dehydrated and preserved using permount mounting medium and glass cover-slips. Rabbit anti-collagen I (II) (Abcam), rabbit anti-α-SMA (Abcam) and rabbit anti-TIMP-1 (Proteintech Group) antibodies were used. RNA isolation and quantitative real-time PCR Total RNA was isolated from frozen liver tissues using Trizol reagent according to the manufacturer’s protocol (Invitrogen). RNA concentration and purity were measured by ultraviolet absorbance. RNA (1 μg) was reverse-transcribed using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Quantitative PCR was carried out with SYBR Green real-time PCR Master Mix (Takara). The reactions were performed on an Applied Biosystems 7900HT real-time PCR System, and the cycling parameters were as follows: 95 °C for 5 min and then 42 cycles of 95 °C for 15 sec, 58 °C for 15 sec and 72 °C for 25 sec, followed by a melting curve analysis. Samples were run in triplicate, and relative expression values were normalized to housekeeping gene Gapdh and calculated by the formula: 2-∆∆Ct. The primer sequences were shown in Table S1. Western blotting Frozen liver tissues were homogenized in NETN buffer (0.5% Nonidet P-40, 1 mM EDTA, 100 mM NaCl, and 20 mM Tris (pH 8.0)) containing complete Mini EDTA-free Protease Inhibitor Cocktail (Roche) with a homogenizer and sonicator at 5
4 °C. The samples were centrifuged at 12,000 rpm at 4 °C for 30 min. The supernatants were transferred to new tubes and quantified by Bio-Rad DC Protein Assay. Equal amounts of proteins were denatured, separated on 10% SDS-PAGE and transferred to nitrocellulose transfer membranes (Bio-Rad) which were blocked with 5% non-fat milk (diluted in PBS) for 1 h at room temperature. The membranes were incubated with the primary antibodies overnight at 4 °C under shaking conditions, washed
three
times
with
PBST,
and
then
incubated
with
appropriate
fluorescence-conjugated secondary antibodies for 2 h at room temperature. Finally, the membranes were washed and scanned with the Odyssey Infrared Imaging System (LI-COR Biotechnology). Antibodies of Collagen I (II), α-SMA, PDGF, TGF-β1, TIMP-1 (Abcam) were used and Gapdh was a loading control in this study. Statistical analyses Statistics were performed using the SPSS statistical software package. Quantitative data were expressed as mean ± standard deviation (SD). Comparisons between two groups were performed by the two-tailed unpaired Student’s t-test. P values less than 0.05 were considered to be statistically significant.
Results Rdh13-deficient mice are less susceptible to CCl4-induced chronic liver injury and fibrosis After 4 weeks of CCl4 injection, serum biochemistry of the mice was detected to evaluate the liver function, including ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), ALB (albumin)，TP (total protein) , T-BIL (total bilirubin), T-CHO (total cholesterol) and TG (triglyceride). All the values of serum biochemistry were increased in both Rdh13 KO and WT mice compared to those of the mice at the basal condition, indicating the liver function of the mice changed from liver injury. However, although the values of KO mice were lower than those of WT mice, there was no significant difference in the serum biochemistry between Rdh13 KO and WT mice (Fig. S2). Hepatic histology of the mice administrated for 4 weeks of CCl4 was assessed by H&E staining. The results 6
reveal that there was a dramatic exacerbation of liver damage in WT mice, with widespread foci of necrotic cells, not only in the pericentral region, but also in the midzonal and periportal regions. While necro-inflammatory liver damage in Rdh13 KO mice was significantly slighter than that in WT mice, with foci located predominantly in the pericentral region (Fig. 1A). The results from the TUNEL analysis indicated the amount of apoptotic cells in the liver sections from KO mice was much less than that from WT mice (KO vs. WT, 2% vs. 3%) (Fig. 1B, Fig. 1C). As expected, 4 weeks of CCl4 treatment caused hepatic fibrosis in the livers of the mice. Liver fibrosis in the mice was observed through Masson’s trichrome staining. The results reveal that there was obvious bridging fibrosis, extending into the lobule, branched and irregular fibrosis in WT mice. While liver fibrosis in Rdh13 KO mice was significantly reduced compared to WT mice and only portal and sinusoid fibrosis was found. The Masson’s trichrome-positive area in Rdh13 KO mice was significantly reduced compared to WT mice (KO vs. WT, 10.1% vs. 14.1%) (Fig. 2A & 2B). Rdh13 deficiency inhibits the activation and sustain of hepatic stellate cells after CCl4 administration in mice The activated hepatic stellate cells are the primary source of increased extracellular matrix in chronic liver injury, α-smooth muscle actin (α-SMA) is a well known marker of HSC activation (Friedman, 2008a, b; Reeves and Friedman, 2002). The expression of α-SMA at different time point after CCl4 administration was detected by western-blotting. The results show that the expression of α-SMA dramatically increased in both WT and Rdh13 KO mouse livers at 72 hours after acute CCl4 treatment. However, this increase in Rdh13 KO mice livers was markedly less than that in WT mouse livers. After 31 days of chronic CCl4 treatment, a-SMA protein increased than that at 72 h in WT mice, while it did not show an obvious increase compared to that at 72 h in Rdh13 KO mice (Fig. 3B). Moreover, the α-SMA-positive cells in the livers of WT detected by immunohistochemistry were more than those of Rdh13 KO mice (Fig. 3A) after 4 weeks of CCl4 administration. All these data suggest that Rdh13 deficiency inhibits the activation and sustain of hepatic stellate cells after CCl4 administration in mice. 7
Rdh13 deficiency inhibits induction of Collagen I (II) synthesis after CCl4 administration Collagen I (II) is the predominant component of newly synthesized extracellular matrix associated with liver fibrosis (Weiler-Normann et al., 2007). To determine the mRNA and protein expression of Collagen I (II) in the liver of the mice during the course of acute and chronic liver injury, real-time PCR, immunohistochemistry and Western blotting were performed at different time point after CCl4 administration The results show the expression of collagen I (II) after CCl4 administration increased with the time of treatment in the liver of both WT and Rdh13 KO mice, but the extent of collagen increment in Rdh13 KO mice was significantly lower than that in WT mice (Fig. 4A-4C). Rdh13 deficiency inhibits TIMP-1 expression in CCl4-induced liver fibrosis TIMP-1 plays a critical role in preventing the degradation of the extracellular matrix (ECM) via inhibiting members of a large family of matrix metalloproteinases and protecting activated HSCs from death (Gieling et al., 2008). The effect of Rdh13 deficiency on hepatic expression of TIMP-1 was evaluated by real-time PCR, western blotting and immunohistochemical staining. As shown in Figure 5A and 5B, hepatic TIMP-1 mRNA and protein after CCl4 administration increased in the liver of both WT and Rdh13 KO mice, but the increments in Rdh13 KO mice were significantly less than that in WT mice at both 3 days and 31 days after CCl4 administration. Similar results were observed by immunohistochemical staining (Fig. 5C). Nevertheless, the results show that chronic CCl4 treatment induced marked TIMP-1 expression predominantly around necroinflammatory areas and few in hepatocytes in Rdh13-deficient mice, while TIMP-1 expression was mainly in hepatocytes and nonparenchymal cells in WT mice (Fig. 5C). These results could also account for the reduced fibrosis in Rdh13 KO mice. Effect of Rdh13 deficiency on the expression of profibrotic molecules HSC activation is viewed as a two-stage process consisting of initiation and perpetuation (Inagaki and Okazaki, 2007). TGF-β1 is the main cytokines that activate 8
HSCs in liver fibrosis, and TGF-β1 is a well-known profibrotic cytokine released by damaged hepatocytes, endothelial cells, Kupffer cells, and platelets (Iredale, 2007). To further establish the role of Rdh13 in experimental liver fibrosis, we determined the expression of the pivotal fibrosis-related molecule Tgf-β1 in the livers of Rdh13-deficient mice and WT mice after CCl4 administration. Hepatic expression of TGF-β1 mRNA increased significantly after CCl4 exposure, whereas such an induction decreased markedly in deficient mice (Fig.6A).Western blot analysis showed that TGF-β1 protein levels were present at mildly lower levels in Rdh13 KO mice 4 weeks after repeated CCl4 injection compared to WT mice, but no significant difference was noted between WT and deficient mice after CCl4-induced acute liver injury (Fig.6B). Taken together, these data suggest that loss of Rdh13 inhibits HSC activation and profibrogenic molecules expression, eventually reducing liver fibrosis in mice with chronic CCl4 injury.
Discussion The data presented in this study firstly demonstrate that Rdh13 deficient mice developed reduced chronic liver injury and fibrosis compared with wild-type mice after repeated exposure to CCl4, a widely used model of toxin-induced hepatic parenchymal damage. CCl4 toxicity results from liver microsomal metabolism of CCl4, leading to the generation of the toxin trichloromethyl radical that induces hepatocyte necrosis and apoptosis. The results in local inflammation subsequently trigger fibrogenic cell activation (Lafdil et al., 2009; Weiler-Normann et al., 2007). Hepatic fibrosis is the results of repeated cycles of liver injury coupled to incomplete resolution of a normal wound healing response (Friedman, 2008b). Successful injury repair involves an orderly progression of inflammation, angiogenesis, tissue formation, and ECM remodeling (Hantash et al., 2008). But during chronic liver fibrosis, this cycle is interrupted mid-sequence and then is re-initiated, which results in excessive ECM deposition and contributes to eventual hepatic dysfunction related to fibrosis. RDH is a subfamily of SDRs, and performs critical oxidation-reduction reactions during the 9
retinoid cycle (Oppermann et al., 2003). Our recent work showed that the expression level of Rdh13 mRNA in liver is significantly higher than in other tissues (unpublished). The liver is the most important organ for the storage and metabolism of retinol (vitamin A) (Shirakami et al., 2012). Hepatic fibrosis is a dynamic process resulting from liver tissue injury. It is an active, potentially reversible process originating from wound-healing responses to chronic liver injury. The principal effector of hepatic fibrogenesis is now widely recognized as the hepatic stellate cells (Reeves and Friedman, 2002). HSCs are usually quiescent cells, but in response to liver injury they undergo a process of activation in which they convert from a quiescent condition to a myofibroblastic phenotype. One of the prominent characteristic features of HSC activation is the loss of retinol (Friedman, 2008c; Natarajan et al., 2005; Shirakami et al., 2012). Exogenous retinol treatments have been considered as a form of anti-fibrotic therapy (Murakami et al., 2011; Sato et al., 2008). Therefore, we demonstrate here that RDH13, as a member of the short-chain dehydrogenases/reductases (SDRs) superfamily, is involved in liver fibrosis, which may be through affecting the retinoid metabolism. Our results show that the liver damage in Rdh13 KO mice was significantly slighter than in WT mice, and the amount of activated HSCs in Rdh13 KO mice was much less than that of wild-type mice during the chronic CCl4 treatment. These findings suggest that Rdh13 deficiency shows a protective effect against chronic liver injury and fibrosis. But the exact role of Rdh13 in retinoid metabolism and the mechanism of Rdh13 in liver fibrogenesis in vivo need more investigations. Liver fibrosis is characterized by excessive deposition of extracellular matrix proteins. During fibrosis progression, activated HSCs synthesize extracellular matrix components, particularly collagen I, and extracellular matrix remodeling enzymes such as tissue inhibitor of matrix metalloprotease (TIMP) (Bataller and Brenner, 2005; Friedman, 2000). TIMP-1 plays a pivotal role in liver fibrogenesis by regulating matrix degradation. It inhibits collagenases and protects newly synthesized collagen from immediate degradation (Benyon and Arthur, 2001; Gieling et al., 2008). To evaluate the role of Rdh13 in liver fibrosis, we compared the expression of Collagen I 10
(II) and TIMP-1 in the liver of the mice during the course of acute and chronic liver injury. The results show the mRNA and protein levels of collagen I (II) and TIMP-1 significantly increased during liver fibrogenesis in wild-type liver, while the extent of increment of collagen I (II) and TIMP-1 in Rdh13 KO mice was much less (Fig. 5A and 5B). To support the possible role of Rdh13 deficiency as a suppressor of chronic fibrosis, quantitative RT-PCR and Western blot analysis showed that expression of TGF-β1，a pluripotent immunomodulatory and classic fibrogenic factor that largely regulates HSCs transition to myofibroblast-like cells(Breitkopf et al., 2006; Inagaki and Okazaki, 2007), was decreased in the KO mice. These findings further suggest less HSC activation, less ECM deposition, and attenuated ECM degradation inhibition are the causes of reduced liver fibrosis in Rdh13 deficient mice than wild-type mice. Studies have revealed that some SDRs that possess RDH activity include 11-cis-RDH (RDH5), RoDH4, RL-HSD, retSDR1, RDH10,RDH11, RoDH1, RoDH2, CRAD1, CRAD2, CRAD3, 17β-HSD9, RDH1, RRD are expressed in the liver (Shirakami et al., 2012). There has not been rigorous genetic study as to the possible physiological importance of each of these SDRs in retinoic acid synthesis and in liver function. Using Rdh13 knockout mice, our study reveals that Rdh13 deficiency plays a protective role in chronic liver injury and attenuates the development of hepatic fibrosis, which shed a light on the function of RDH13 in liver disease in vivo and suggests RDH13 as a possible new therapeutic target for the prevention and treatment of CCl4 induced hepatic fibrosis.
Acknowledgments This work was supported by National Natural Science Foundation of China (No: 81270518), Science and Technology Commission of Shanghai Municipality (No: 10411955300 and 09XD1403200), Shanghai Municipal Health Bureau (No: XBR2011012).
11
References Bataller, R., Brenner, D.A., 2005. Liver fibrosis. J CLIN INVEST 115, 209-218. Belyaeva, O.V., Korkina, O.V., Stetsenko, A.V., Kedishvili, N.Y., 2008. Human retinol
dehydrogenase
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dehydrogenase/reductase with a retinaldehyde reductase activity. FEBS J 275, 138-147. Belyaeva, O.V., Korkina, O.V., Stetsenko, A.V., Kim, T., Nelson, P.S., Kedishvili, N.Y., 2005. Biochemical properties of purified human retinol dehydrogenase 12 (RDH12): catalytic efficiency toward retinoids and C9 aldehydes and effects of cellular retinol-binding protein type I (CRBPI) and cellular retinaldehyde-binding protein (CRALBP) on the oxidation and reduction of retinoids. BIOCHEMISTRY-US 44, 7035-7047. Benyon, R.C., Arthur, M.J., 2001. Extracellular matrix degradation and the role of hepatic stellate cells. SEMIN LIVER DIS 21, 373-384. Breitkopf, K., Godoy, P., Ciuclan, L., Singer, M.V., Dooley, S., 2006. TGF-beta/Smad signaling in the injured liver. Z GASTROENTEROL 44, 57-66. Friedman, S.L., 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J BIOL CHEM 275, 2247-2250. Friedman, S.L., 2008a. Hepatic fibrosis -- overview. TOXICOLOGY 254, 120-129. Friedman,
S.L.,
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Mechanisms
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GASTROENTEROLOGY 134, 1655-1669. Friedman, S.L., 2008c. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. PHYSIOL REV 88, 125-172. Gieling, R.G., Burt, A.D., Mann, D.A., 2008. Fibrosis and cirrhosis reversibility molecular mechanisms. CLIN LIVER DIS 12, 915-937. Haeseleer, F., Jang, G.F., Imanishi, Y., Driessen, C.A., Matsumura, M., Nelson, P.S., Palczewski, K., 2002. Dual-substrate specificity short chain retinol dehydrogenases from the vertebrate retina. J BIOL CHEM 277, 45537-45546. Hantash, B.M., Zhao, L., Knowles, J.A., Lorenz, H.P., 2008. Adult and fetal wound healing. Front Biosci 13, 51-61. 12
Inagaki, Y., Okazaki, I., 2007. Emerging insights into Transforming growth factor beta Smad signal in hepatic fibrogenesis. GUT 56, 284-292. Iredale, J.P., 2007. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J CLIN INVEST 117, 539-548. Lafdil, F., Chobert, M.N., Deveaux, V., Zafrani, E.S., Mavier, P., Nakano, T., Laperche, Y., Brouillet, A., 2009. Growth arrest-specific protein 6 deficiency impairs liver tissue repair after acute toxic hepatitis in mice. J HEPATOL 51, 55-66. Levi, L., Ziv, T., Admon, A., Levavi-Sivan, B., Lubzens, E., 2012. Insight into molecular pathways of retinal metabolism, associated with vitellogenesis in zebrafish. Am J Physiol Endocrinol Metab 302, E626-E644. Murakami, K., Kaji, T., Shimono, R., Hayashida, Y., Matsufuji, H., Tsuyama, S., Maezono, R., Kosai, K., Takamatsu, H., 2011. Therapeutic effects of vitamin A on experimental cholestatic rats with hepatic fibrosis. PEDIATR SURG INT 27, 863-870. Natarajan, S.K., Thomas, S., Ramachandran, A., Pulimood, A.B., Balasubramanian, K.A., 2005. Retinoid metabolism during development of liver cirrhosis. ARCH BIOCHEM BIOPHYS 443, 93-100. Oppermann, U., Filling, C., Hult, M., Shafqat, N., Wu, X., Lindh, M., Shafqat, J., Nordling, E., Kallberg, Y., Persson, B., Jornvall, H., 2003. Short-chain dehydrogenases/reductases (SDR): the 2002 update. Chem Biol Interact 143-144, 247-253. Reeves, H.L., Friedman, S.L., 2002. Activation of hepatic stellate cells--a key issue in liver fibrosis. Front Biosci 7, d808-d826. Sato, Y., Murase, K., Kato, J., Kobune, M., Sato, T., Kawano, Y., Takimoto, R., Takada, K., Miyanishi, K., Matsunaga, T., Takayama, T., Niitsu, Y., 2008. Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. NAT BIOTECHNOL 26, 431-442. Shirakami, Y., Lee, S.A., Clugston, R.D., Blaner, W.S., 2012. Hepatic metabolism of retinoids and disease associations. Biochim Biophys Acta 1821, 124-136. Wang, H., Cui, X., Gu, Q., Chen, Y., Zhou, J., Kuang, Y., Wang, Z., Xu, X., 2012. 13
Retinol dehydrogenase 13 protects the mouse retina from acute light damage. MOL VIS 18, 1021-1030. Weiler-Normann, C., Herkel, J., Lohse, A.W., 2007. Mouse models of liver fibrosis. Z GASTROENTEROL 45, 43-50.
Figure legends Figure 1.
Rdh13-deficient mice show less liver injury than wild-type mice after
4-week-CCl4 treatment. (A) Liver sections of Rdh13 knockout (KO) and wide-type (WT) mice were stained with haematoxylin and eosin (H&E) solution. Original magnification, ×100. (B) In situ fluorescence TUNEL assay on liver frozen sections shows hepatocyte nuclei (Blue, DAPI) and apoptotic cells (Green, FITC). (C) Quantitative analysis of apoptotic cells in the liver upon CCl4 treatment for the indicated time points (**p<0.01). Figure 2.
Rdh13-deficient mice show less liver fibrosis than wild-type after post
4-week-CCl4 treatment. (A)Histopathological detection of collagen in liver by measns of Masson ’s trichrome. Original magnification, ×100. (B) Masson’s trichrome collagen staining was semi-quantified for hepatic fibrosis by a Zeiss Axioplan 2 imaging analysis system. (*p<0.05). Figure 3. α-SMA expression decreased in Rdh13 knockout mice after CCl4 exposure. Western Blot analysis (A) and immunohistochemical staining (B) of α-SMA protein in the liver tissues during development of CCl4 induced fibrosis
in
wide-type
(WT)
and
Rdh13
knockout
(KO)
mice.
liver
Original
magnification×100. Images are representative of n = 4 to 10 per experimental group. Figure 4.
CollagenⅠ（Ⅱ）increased much more in wild-type (WT) mice than
that in Rdh13 knockout mice following CCl4-induced liver fibrosis. (A) The hepatic mRNA levels of CollagenⅠin wide-type (WT) and Rdh13 knockout (KO) mice during liver fibrosis were measured by quantitative real-time reverse transcription PCR. Data represent means ± SD, n= 3 per experimental group, (*P＜ 0.05, **P＜ 0.01 WT vs. KO). (B) Western Blot analysis of Collagen Ⅰ（Ⅱ） performed on protein (100 μg) extracted from WT and Rdh13 knockout liver tissues at 14
various time points following CCl4 injection. (C) Hepatic immunohistochemical staining of collagen Ⅰ（Ⅱ）with a polyclonal rabbit anti-murine collagen typeⅠ antibody in WT and knockout mice. Original magnification, ×200． Figure 5.
Rdh13 deficiency attenuates hepatic Timp-1 mRNA and protein
increment following CCl4-induced chronic liver injury in mice. (A) The hepatic mRNA levels of Timp-1 for wide-type (WT) and Rdh13 knockout (KO) mice after CCl4 exposure were measured by quantitative real-time reverse transcription PCR. Data represent means ± SD, n = 3 per experimental group, (**P＜ 0.01, WT vs. KO). (B) Western Blot analysis of hepatic Timp-1 protein levels at various time points following CCl4 injection. (C) Wide-type (WT) and Rdh13 knockout (KO) mice were treated with CCl4 for 4 weeks. Liver tissues were collected for immunohistochemical staining with anti-Timp-1 antibody. Representative pictures are shown. Original magnification, ×200． Figure 6. Effect of Rdh13 knockout on hepatic Tgf-β1 expression in mice with acute liver injury and liver fibrosis mice induced by CCl4. (A) The hepatic mRNA levels Tgf-β1 for wide-type (WT) and Rdh13 knockout (KO) mice during liver fibrosis were measured by quantitative real-time reverse transcription PCR. Data represent means ± SD, n = 3 per experimental group, (**P＜ 0.01 WT vs. KO). (B) Western Blot analysis of Tgf-β1 protein levels in the liver tissues from wild-type mice and Rdh13 knockout at various time points following CCl4 injection.
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Figure 5
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S0378-4274(16)33304-5 http://dx.doi.org/doi:10.1016/j.toxlet.2016.11.010 TOXLET 9639
To appear in:
Toxicology Letters
Received date: Revised date: Accepted date:
20-10-2015 11-11-2016 15-11-2016
Please cite this article as: Cui, Xiaofang, Dang, Suying, Wang, Yan, Chen, Yan, Zhou, Jia, Shen, Chunling, Kuang, Ying, Fei, Jian, Lu, Lungen, Wang, Zhugang, Retinol dehydrogenase 13 deficiency diminishes carbon tetrachloride-induced liver fibrosis in mice.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2016.11.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Retinol dehydrogenase 13 deficiency diminishes carbon tetrachloride-induced liver fibrosis in mice
Xiaofang Cuia,b , Suying Danga, Yan Wangc, Yan Chena, Jia Zhoua, Chunling Shena,d, Ying Kuangd, Jian Feid, Lungen Luc* and Zhugang Wanga,b,d*
a
State Key Laboratory of Medical Genomics, Research Center for Experimental
Medicine, Shanghai Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (SJTUSM), b
Model Organism Division, E-Institutes of Shanghai Jiao Tong Universities School of
Medicine (SJTUSM), Shanghai 200025, China; c
Department of Gastroenterology, Shanghai First People’s Hospital Affiliated to
SJTUSM, Shanghai 200080, China; d
Shanghai Research Center for Model Organisms,
*
Corresponding authors. E-mail addresses: [email protected] (Z. Wang) or
[email protected] (L. Lu)
Highlights Retinol dehydrogenase 13 (RDH13) is a mitochondrion-localized member of the short-chain dehydrogenases/reductases (SDRs) superfamily that participates in metabolism of some compound.Rdh13 mRNA is most highly expressed in mouse liver. Rdh13 deficiency reduces the extent of liver injury and fibrosis, reduces hepatic stellate cell
(HSC)
activation,
metalloproteinase1(TIMP-1)
attenuates
collagen
andtransforming
I(II)
,
growth
tissue
inhibitor
factor
of
beta1
(Tgf-β1)expression.The resultsindicate an important role of Rdh13 and suggest
RDH13 as a possible new therapeutic target for CCl4-induced fibrosis.
1
Abstract Retinol dehydrogenase 13 (RDH13) is a mitochondrion-localized member of the short-chain dehydrogenases/reductases (SDRs) superfamily that participates in metabolism of some compound. Rdh13 mRNA is most highly expressed in mouse liver. Rdh13 deficiency reduces the extent of liver injury and fibrosis, reduces hepatic stellate cell (HSC) activation, attenuates collagen I (II) , tissue inhibitor of
metalloproteinase 1 (TIMP-1) and transforming growth factor beta 1 (Tgf-β1) expression. The results indicate an important role of Rdh13 and suggest RDH13 as a possible new therapeutic target for CCl4-induced fibrosis. Key words: Retinol dehydrogenase 13, Liver fibrosis, Knockout, Hepatic stellate cells, Carbon tetrachloride Introduction Retinol dehydrogenase 13 (RDH13) is a recently identified member of the short-chain dehydrogenases/reductases (SDRs) superfamily that participate in metabolism of prostaglandins, retinoids, steroids and aliphatic alcohols (Haeseleer et al., 2002; Levi et al., 2012; Oppermann et al., 2003). RDH13 shares sequence similarity with RDH11, RDH12 and RDH14 proteins which have been characterized as microsomal proteins that recognize retinoids and medium-chain aldehydes as substrates, with NADP+/NADPH as the preferred cofactors (Belyaeva et al., 2005). It has been demonstrated that RDH13 is localized on the outer side of the inner mitochondrial membrane and exhibits a wide tissue distribution. Purified RDH13 is catalytically active and recognizes retinoids as substrates, strongly prefers NADPH over NADH as a cofactor, and has a much greater catalytic efficiency as a reductase than as a dehydrogenase (Belyaeva et al., 2008). Our previous work show that Rdh13 plays an important role in retinal light damage which is consistent with Rdh11 and Rdh12 in mice (Wang et al., 2012). Thus, RDH13 is also considered to participate in the retinoid metabolism. The liver is the most important organ in the body involved in retinoid storage and metabolism, and also an important target organ for retinoid actions. Within the liver, 2
both hepatocytes and HSCs are importantly involved in retinoid metabolism (Shirakami et al., 2012). Retinoic acid is synthesized in the liver and can interact with retinoid receptors which control expression of a large number of genes involved in hepatic processes. Altered retinoid metabolism and the accompanying dysregulation of retinoid signaling in the liver contribute to hepatic disease including fatty liver, liver fibrosis, cirrhosis and hepatocellular carcinoma (Shirakami et al., 2012). This is related to HSCs, which store more than 50% of all retinoid present in the body and contribute significantly to the development of hepatic disease when they undergo a process of cellular activation. HSC activation results in the loss of HSC retinoid stores and changes in extracellular matrix deposition leading to the onset of liver fibrosis. Within the liver, like in other tissues, retinol is converted to retinoic acid via two oxidative steps. Retinol dehydrogenases (RDHs) catalyze the first enzymatic step which is the oxidation of retinol to retinaldehyde. Several RDHs including RDH5, RDH10, RDH11, RDH1 and RDH13 are expressed in the liver and exhibit RDH activity in vitro (Shirakami et al., 2012). But the physiological role of each of them in retinoic acid synthesis and liver disease remains unclear. Our previous work showed that Rdh13 mRNA is most highly expressed in mouse liver, which is 3-10 times that of other tissues. This suggests possible importance of Rdh13 in the retinoid signaling and hepatic pathology. Recently, we investigated the function of Rdh13 in liver and acute liver injury by using Rdh13-deficient mice. We found that there was no obvious difference in the serum biochemistry and the liver histology between wild-type and Rdh13-deficient mice under normal condition, and Rdh13-deficient mice showed reduced liver injury and hepatocyte proliferation compared to wild-type mice following carbon tetrachloride administration. Here, we use Rdh13 knockout mice to investigate the role of Rdh13 in liver fibrosis after repeated CCl4 administration. The results indicate that Rdh13 deficiency reduces the extent of liver injury and liver fibrosis, reduces HSC activation, attenuates collagen I(II), and TIMP-1 expression after chronic CCl4 treatment. Our findings indicate an important role of Rdh13 in liver disease in vivo and suggest RDH13 as a possible new therapeutic target for the prevention and treatment of hepatic fibrosis in 3
chronic liver disease.
Materials and methods Animals WT and Rdh13 KO mice (8-10-week-old) with a mixed genetic background of C57BL/6 and 129/Sv were used in the study. The generation of Rdh13 KO mice has been described previously (Wang et al., 2012). Mice were allowed free access to food and water, and were housed under specific pathogen-free (SPF) conditions at a constant room temperature of 22-24 °C with a 12 h light-dark cycle during the study. All animal experiments were approved by the Animal Use and Care Committee of Shanghai Jiao Tong University School of Medicine. CCl4-induced liver injury CCl4 was used to induce liver injury and liver fibrosis. For acute CCl4-induced liver injury, a single dose of CCl4 (1.0 ml/kg body weight of 20% CCl4 diluted in olive oil) was administered intraperitoneally to WT and Rdh13 KO mice (n=4 per group for each time point, 2 male and 2 female mice). Animals were sacrificed 0, 24 and 72 h after CCl4 injection. In the chronic injury model, WT and Rdh13 KO mice (n=10 per group,5 male and 5 female mice) were administered intraperitoneally with the same dose of CCl4 as acute liver injury twice per week for 4 weeks. Animals were sacrificed three days after the final CCl4 injection. At the time of sacrifice, mice were anesthetized and liver samples were harvested, rapidly washed with ice-cold phosphate buffered saline (PBS) and dissected into smaller pieces. Liver tissues from different lobes were fixed in 10% neutral formalin for 24 h and then embedded in paraffin for histological examination. Other parts were snap-frozen in liquid nitrogen and stored at -80 °C for RNA and protein analysis. Histological analysis Formalin-fixed liver samples were processed, and paraffin-embedded liver tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined by immunohistochemistry. Liver fibrosis was assessed by means of Masson’s trichrome staining for hepatic collagen deposition. Fibrosis deposition was quantified and 4
analyzed by digital imaging with a Zeiss Axioplan 2 imaging analysis system. Immunohistochemistry Formalin-fixed paraffin embedded liver sections (5 μm) were deparaffinized with xylene, hydrated in decreasing concentrations of ethanol. Protein epitopes were unmasked by citrate buffer incubation at 92-98 °C for 15 minutes. After heat treatment, the sections were allowed to cool at room temperature. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide in PBS for 10 minutes. Sections were incubated with serum-containing blocking reagents (Vector Laboratories, lnc. Burlingame, CA) for 1 h, then incubated with primary antibody overnight at 4 °C. The staining procedure was performed according to the manufacturer’s instructions of VECTASTAINABC system (Vector Labs). Finally, sections were dehydrated and preserved using permount mounting medium and glass cover-slips. Rabbit anti-collagen I (II) (Abcam), rabbit anti-α-SMA (Abcam) and rabbit anti-TIMP-1 (Proteintech Group) antibodies were used. RNA isolation and quantitative real-time PCR Total RNA was isolated from frozen liver tissues using Trizol reagent according to the manufacturer’s protocol (Invitrogen). RNA concentration and purity were measured by ultraviolet absorbance. RNA (1 μg) was reverse-transcribed using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Quantitative PCR was carried out with SYBR Green real-time PCR Master Mix (Takara). The reactions were performed on an Applied Biosystems 7900HT real-time PCR System, and the cycling parameters were as follows: 95 °C for 5 min and then 42 cycles of 95 °C for 15 sec, 58 °C for 15 sec and 72 °C for 25 sec, followed by a melting curve analysis. Samples were run in triplicate, and relative expression values were normalized to housekeeping gene Gapdh and calculated by the formula: 2-∆∆Ct. The primer sequences were shown in Table S1. Western blotting Frozen liver tissues were homogenized in NETN buffer (0.5% Nonidet P-40, 1 mM EDTA, 100 mM NaCl, and 20 mM Tris (pH 8.0)) containing complete Mini EDTA-free Protease Inhibitor Cocktail (Roche) with a homogenizer and sonicator at 5
4 °C. The samples were centrifuged at 12,000 rpm at 4 °C for 30 min. The supernatants were transferred to new tubes and quantified by Bio-Rad DC Protein Assay. Equal amounts of proteins were denatured, separated on 10% SDS-PAGE and transferred to nitrocellulose transfer membranes (Bio-Rad) which were blocked with 5% non-fat milk (diluted in PBS) for 1 h at room temperature. The membranes were incubated with the primary antibodies overnight at 4 °C under shaking conditions, washed
three
times
with
PBST,
and
then
incubated
with
appropriate
fluorescence-conjugated secondary antibodies for 2 h at room temperature. Finally, the membranes were washed and scanned with the Odyssey Infrared Imaging System (LI-COR Biotechnology). Antibodies of Collagen I (II), α-SMA, PDGF, TGF-β1, TIMP-1 (Abcam) were used and Gapdh was a loading control in this study. Statistical analyses Statistics were performed using the SPSS statistical software package. Quantitative data were expressed as mean ± standard deviation (SD). Comparisons between two groups were performed by the two-tailed unpaired Student’s t-test. P values less than 0.05 were considered to be statistically significant.
Results Rdh13-deficient mice are less susceptible to CCl4-induced chronic liver injury and fibrosis After 4 weeks of CCl4 injection, serum biochemistry of the mice was detected to evaluate the liver function, including ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), ALB (albumin)，TP (total protein) , T-BIL (total bilirubin), T-CHO (total cholesterol) and TG (triglyceride). All the values of serum biochemistry were increased in both Rdh13 KO and WT mice compared to those of the mice at the basal condition, indicating the liver function of the mice changed from liver injury. However, although the values of KO mice were lower than those of WT mice, there was no significant difference in the serum biochemistry between Rdh13 KO and WT mice (Fig. S2). Hepatic histology of the mice administrated for 4 weeks of CCl4 was assessed by H&E staining. The results 6
reveal that there was a dramatic exacerbation of liver damage in WT mice, with widespread foci of necrotic cells, not only in the pericentral region, but also in the midzonal and periportal regions. While necro-inflammatory liver damage in Rdh13 KO mice was significantly slighter than that in WT mice, with foci located predominantly in the pericentral region (Fig. 1A). The results from the TUNEL analysis indicated the amount of apoptotic cells in the liver sections from KO mice was much less than that from WT mice (KO vs. WT, 2% vs. 3%) (Fig. 1B, Fig. 1C). As expected, 4 weeks of CCl4 treatment caused hepatic fibrosis in the livers of the mice. Liver fibrosis in the mice was observed through Masson’s trichrome staining. The results reveal that there was obvious bridging fibrosis, extending into the lobule, branched and irregular fibrosis in WT mice. While liver fibrosis in Rdh13 KO mice was significantly reduced compared to WT mice and only portal and sinusoid fibrosis was found. The Masson’s trichrome-positive area in Rdh13 KO mice was significantly reduced compared to WT mice (KO vs. WT, 10.1% vs. 14.1%) (Fig. 2A & 2B). Rdh13 deficiency inhibits the activation and sustain of hepatic stellate cells after CCl4 administration in mice The activated hepatic stellate cells are the primary source of increased extracellular matrix in chronic liver injury, α-smooth muscle actin (α-SMA) is a well known marker of HSC activation (Friedman, 2008a, b; Reeves and Friedman, 2002). The expression of α-SMA at different time point after CCl4 administration was detected by western-blotting. The results show that the expression of α-SMA dramatically increased in both WT and Rdh13 KO mouse livers at 72 hours after acute CCl4 treatment. However, this increase in Rdh13 KO mice livers was markedly less than that in WT mouse livers. After 31 days of chronic CCl4 treatment, a-SMA protein increased than that at 72 h in WT mice, while it did not show an obvious increase compared to that at 72 h in Rdh13 KO mice (Fig. 3B). Moreover, the α-SMA-positive cells in the livers of WT detected by immunohistochemistry were more than those of Rdh13 KO mice (Fig. 3A) after 4 weeks of CCl4 administration. All these data suggest that Rdh13 deficiency inhibits the activation and sustain of hepatic stellate cells after CCl4 administration in mice. 7
Rdh13 deficiency inhibits induction of Collagen I (II) synthesis after CCl4 administration Collagen I (II) is the predominant component of newly synthesized extracellular matrix associated with liver fibrosis (Weiler-Normann et al., 2007). To determine the mRNA and protein expression of Collagen I (II) in the liver of the mice during the course of acute and chronic liver injury, real-time PCR, immunohistochemistry and Western blotting were performed at different time point after CCl4 administration The results show the expression of collagen I (II) after CCl4 administration increased with the time of treatment in the liver of both WT and Rdh13 KO mice, but the extent of collagen increment in Rdh13 KO mice was significantly lower than that in WT mice (Fig. 4A-4C). Rdh13 deficiency inhibits TIMP-1 expression in CCl4-induced liver fibrosis TIMP-1 plays a critical role in preventing the degradation of the extracellular matrix (ECM) via inhibiting members of a large family of matrix metalloproteinases and protecting activated HSCs from death (Gieling et al., 2008). The effect of Rdh13 deficiency on hepatic expression of TIMP-1 was evaluated by real-time PCR, western blotting and immunohistochemical staining. As shown in Figure 5A and 5B, hepatic TIMP-1 mRNA and protein after CCl4 administration increased in the liver of both WT and Rdh13 KO mice, but the increments in Rdh13 KO mice were significantly less than that in WT mice at both 3 days and 31 days after CCl4 administration. Similar results were observed by immunohistochemical staining (Fig. 5C). Nevertheless, the results show that chronic CCl4 treatment induced marked TIMP-1 expression predominantly around necroinflammatory areas and few in hepatocytes in Rdh13-deficient mice, while TIMP-1 expression was mainly in hepatocytes and nonparenchymal cells in WT mice (Fig. 5C). These results could also account for the reduced fibrosis in Rdh13 KO mice. Effect of Rdh13 deficiency on the expression of profibrotic molecules HSC activation is viewed as a two-stage process consisting of initiation and perpetuation (Inagaki and Okazaki, 2007). TGF-β1 is the main cytokines that activate 8
HSCs in liver fibrosis, and TGF-β1 is a well-known profibrotic cytokine released by damaged hepatocytes, endothelial cells, Kupffer cells, and platelets (Iredale, 2007). To further establish the role of Rdh13 in experimental liver fibrosis, we determined the expression of the pivotal fibrosis-related molecule Tgf-β1 in the livers of Rdh13-deficient mice and WT mice after CCl4 administration. Hepatic expression of TGF-β1 mRNA increased significantly after CCl4 exposure, whereas such an induction decreased markedly in deficient mice (Fig.6A).Western blot analysis showed that TGF-β1 protein levels were present at mildly lower levels in Rdh13 KO mice 4 weeks after repeated CCl4 injection compared to WT mice, but no significant difference was noted between WT and deficient mice after CCl4-induced acute liver injury (Fig.6B). Taken together, these data suggest that loss of Rdh13 inhibits HSC activation and profibrogenic molecules expression, eventually reducing liver fibrosis in mice with chronic CCl4 injury.
Discussion The data presented in this study firstly demonstrate that Rdh13 deficient mice developed reduced chronic liver injury and fibrosis compared with wild-type mice after repeated exposure to CCl4, a widely used model of toxin-induced hepatic parenchymal damage. CCl4 toxicity results from liver microsomal metabolism of CCl4, leading to the generation of the toxin trichloromethyl radical that induces hepatocyte necrosis and apoptosis. The results in local inflammation subsequently trigger fibrogenic cell activation (Lafdil et al., 2009; Weiler-Normann et al., 2007). Hepatic fibrosis is the results of repeated cycles of liver injury coupled to incomplete resolution of a normal wound healing response (Friedman, 2008b). Successful injury repair involves an orderly progression of inflammation, angiogenesis, tissue formation, and ECM remodeling (Hantash et al., 2008). But during chronic liver fibrosis, this cycle is interrupted mid-sequence and then is re-initiated, which results in excessive ECM deposition and contributes to eventual hepatic dysfunction related to fibrosis. RDH is a subfamily of SDRs, and performs critical oxidation-reduction reactions during the 9
retinoid cycle (Oppermann et al., 2003). Our recent work showed that the expression level of Rdh13 mRNA in liver is significantly higher than in other tissues (unpublished). The liver is the most important organ for the storage and metabolism of retinol (vitamin A) (Shirakami et al., 2012). Hepatic fibrosis is a dynamic process resulting from liver tissue injury. It is an active, potentially reversible process originating from wound-healing responses to chronic liver injury. The principal effector of hepatic fibrogenesis is now widely recognized as the hepatic stellate cells (Reeves and Friedman, 2002). HSCs are usually quiescent cells, but in response to liver injury they undergo a process of activation in which they convert from a quiescent condition to a myofibroblastic phenotype. One of the prominent characteristic features of HSC activation is the loss of retinol (Friedman, 2008c; Natarajan et al., 2005; Shirakami et al., 2012). Exogenous retinol treatments have been considered as a form of anti-fibrotic therapy (Murakami et al., 2011; Sato et al., 2008). Therefore, we demonstrate here that RDH13, as a member of the short-chain dehydrogenases/reductases (SDRs) superfamily, is involved in liver fibrosis, which may be through affecting the retinoid metabolism. Our results show that the liver damage in Rdh13 KO mice was significantly slighter than in WT mice, and the amount of activated HSCs in Rdh13 KO mice was much less than that of wild-type mice during the chronic CCl4 treatment. These findings suggest that Rdh13 deficiency shows a protective effect against chronic liver injury and fibrosis. But the exact role of Rdh13 in retinoid metabolism and the mechanism of Rdh13 in liver fibrogenesis in vivo need more investigations. Liver fibrosis is characterized by excessive deposition of extracellular matrix proteins. During fibrosis progression, activated HSCs synthesize extracellular matrix components, particularly collagen I, and extracellular matrix remodeling enzymes such as tissue inhibitor of matrix metalloprotease (TIMP) (Bataller and Brenner, 2005; Friedman, 2000). TIMP-1 plays a pivotal role in liver fibrogenesis by regulating matrix degradation. It inhibits collagenases and protects newly synthesized collagen from immediate degradation (Benyon and Arthur, 2001; Gieling et al., 2008). To evaluate the role of Rdh13 in liver fibrosis, we compared the expression of Collagen I 10
(II) and TIMP-1 in the liver of the mice during the course of acute and chronic liver injury. The results show the mRNA and protein levels of collagen I (II) and TIMP-1 significantly increased during liver fibrogenesis in wild-type liver, while the extent of increment of collagen I (II) and TIMP-1 in Rdh13 KO mice was much less (Fig. 5A and 5B). To support the possible role of Rdh13 deficiency as a suppressor of chronic fibrosis, quantitative RT-PCR and Western blot analysis showed that expression of TGF-β1，a pluripotent immunomodulatory and classic fibrogenic factor that largely regulates HSCs transition to myofibroblast-like cells(Breitkopf et al., 2006; Inagaki and Okazaki, 2007), was decreased in the KO mice. These findings further suggest less HSC activation, less ECM deposition, and attenuated ECM degradation inhibition are the causes of reduced liver fibrosis in Rdh13 deficient mice than wild-type mice. Studies have revealed that some SDRs that possess RDH activity include 11-cis-RDH (RDH5), RoDH4, RL-HSD, retSDR1, RDH10,RDH11, RoDH1, RoDH2, CRAD1, CRAD2, CRAD3, 17β-HSD9, RDH1, RRD are expressed in the liver (Shirakami et al., 2012). There has not been rigorous genetic study as to the possible physiological importance of each of these SDRs in retinoic acid synthesis and in liver function. Using Rdh13 knockout mice, our study reveals that Rdh13 deficiency plays a protective role in chronic liver injury and attenuates the development of hepatic fibrosis, which shed a light on the function of RDH13 in liver disease in vivo and suggests RDH13 as a possible new therapeutic target for the prevention and treatment of CCl4 induced hepatic fibrosis.
Acknowledgments This work was supported by National Natural Science Foundation of China (No: 81270518), Science and Technology Commission of Shanghai Municipality (No: 10411955300 and 09XD1403200), Shanghai Municipal Health Bureau (No: XBR2011012).
11
References Bataller, R., Brenner, D.A., 2005. Liver fibrosis. J CLIN INVEST 115, 209-218. Belyaeva, O.V., Korkina, O.V., Stetsenko, A.V., Kedishvili, N.Y., 2008. Human retinol
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dehydrogenase/reductase with a retinaldehyde reductase activity. FEBS J 275, 138-147. Belyaeva, O.V., Korkina, O.V., Stetsenko, A.V., Kim, T., Nelson, P.S., Kedishvili, N.Y., 2005. Biochemical properties of purified human retinol dehydrogenase 12 (RDH12): catalytic efficiency toward retinoids and C9 aldehydes and effects of cellular retinol-binding protein type I (CRBPI) and cellular retinaldehyde-binding protein (CRALBP) on the oxidation and reduction of retinoids. BIOCHEMISTRY-US 44, 7035-7047. Benyon, R.C., Arthur, M.J., 2001. Extracellular matrix degradation and the role of hepatic stellate cells. SEMIN LIVER DIS 21, 373-384. Breitkopf, K., Godoy, P., Ciuclan, L., Singer, M.V., Dooley, S., 2006. TGF-beta/Smad signaling in the injured liver. Z GASTROENTEROL 44, 57-66. Friedman, S.L., 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J BIOL CHEM 275, 2247-2250. Friedman, S.L., 2008a. Hepatic fibrosis -- overview. TOXICOLOGY 254, 120-129. Friedman,
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GASTROENTEROLOGY 134, 1655-1669. Friedman, S.L., 2008c. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. PHYSIOL REV 88, 125-172. Gieling, R.G., Burt, A.D., Mann, D.A., 2008. Fibrosis and cirrhosis reversibility molecular mechanisms. CLIN LIVER DIS 12, 915-937. Haeseleer, F., Jang, G.F., Imanishi, Y., Driessen, C.A., Matsumura, M., Nelson, P.S., Palczewski, K., 2002. Dual-substrate specificity short chain retinol dehydrogenases from the vertebrate retina. J BIOL CHEM 277, 45537-45546. Hantash, B.M., Zhao, L., Knowles, J.A., Lorenz, H.P., 2008. Adult and fetal wound healing. Front Biosci 13, 51-61. 12
Inagaki, Y., Okazaki, I., 2007. Emerging insights into Transforming growth factor beta Smad signal in hepatic fibrogenesis. GUT 56, 284-292. Iredale, J.P., 2007. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J CLIN INVEST 117, 539-548. Lafdil, F., Chobert, M.N., Deveaux, V., Zafrani, E.S., Mavier, P., Nakano, T., Laperche, Y., Brouillet, A., 2009. Growth arrest-specific protein 6 deficiency impairs liver tissue repair after acute toxic hepatitis in mice. J HEPATOL 51, 55-66. Levi, L., Ziv, T., Admon, A., Levavi-Sivan, B., Lubzens, E., 2012. Insight into molecular pathways of retinal metabolism, associated with vitellogenesis in zebrafish. Am J Physiol Endocrinol Metab 302, E626-E644. Murakami, K., Kaji, T., Shimono, R., Hayashida, Y., Matsufuji, H., Tsuyama, S., Maezono, R., Kosai, K., Takamatsu, H., 2011. Therapeutic effects of vitamin A on experimental cholestatic rats with hepatic fibrosis. PEDIATR SURG INT 27, 863-870. Natarajan, S.K., Thomas, S., Ramachandran, A., Pulimood, A.B., Balasubramanian, K.A., 2005. Retinoid metabolism during development of liver cirrhosis. ARCH BIOCHEM BIOPHYS 443, 93-100. Oppermann, U., Filling, C., Hult, M., Shafqat, N., Wu, X., Lindh, M., Shafqat, J., Nordling, E., Kallberg, Y., Persson, B., Jornvall, H., 2003. Short-chain dehydrogenases/reductases (SDR): the 2002 update. Chem Biol Interact 143-144, 247-253. Reeves, H.L., Friedman, S.L., 2002. Activation of hepatic stellate cells--a key issue in liver fibrosis. Front Biosci 7, d808-d826. Sato, Y., Murase, K., Kato, J., Kobune, M., Sato, T., Kawano, Y., Takimoto, R., Takada, K., Miyanishi, K., Matsunaga, T., Takayama, T., Niitsu, Y., 2008. Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. NAT BIOTECHNOL 26, 431-442. Shirakami, Y., Lee, S.A., Clugston, R.D., Blaner, W.S., 2012. Hepatic metabolism of retinoids and disease associations. Biochim Biophys Acta 1821, 124-136. Wang, H., Cui, X., Gu, Q., Chen, Y., Zhou, J., Kuang, Y., Wang, Z., Xu, X., 2012. 13
Retinol dehydrogenase 13 protects the mouse retina from acute light damage. MOL VIS 18, 1021-1030. Weiler-Normann, C., Herkel, J., Lohse, A.W., 2007. Mouse models of liver fibrosis. Z GASTROENTEROL 45, 43-50.
Figure legends Figure 1.
Rdh13-deficient mice show less liver injury than wild-type mice after
4-week-CCl4 treatment. (A) Liver sections of Rdh13 knockout (KO) and wide-type (WT) mice were stained with haematoxylin and eosin (H&E) solution. Original magnification, ×100. (B) In situ fluorescence TUNEL assay on liver frozen sections shows hepatocyte nuclei (Blue, DAPI) and apoptotic cells (Green, FITC). (C) Quantitative analysis of apoptotic cells in the liver upon CCl4 treatment for the indicated time points (**p<0.01). Figure 2.
Rdh13-deficient mice show less liver fibrosis than wild-type after post
4-week-CCl4 treatment. (A)Histopathological detection of collagen in liver by measns of Masson ’s trichrome. Original magnification, ×100. (B) Masson’s trichrome collagen staining was semi-quantified for hepatic fibrosis by a Zeiss Axioplan 2 imaging analysis system. (*p<0.05). Figure 3. α-SMA expression decreased in Rdh13 knockout mice after CCl4 exposure. Western Blot analysis (A) and immunohistochemical staining (B) of α-SMA protein in the liver tissues during development of CCl4 induced fibrosis
in
wide-type
(WT)
and
Rdh13
knockout
(KO)
mice.
liver
Original
magnification×100. Images are representative of n = 4 to 10 per experimental group. Figure 4.
CollagenⅠ（Ⅱ）increased much more in wild-type (WT) mice than
that in Rdh13 knockout mice following CCl4-induced liver fibrosis. (A) The hepatic mRNA levels of CollagenⅠin wide-type (WT) and Rdh13 knockout (KO) mice during liver fibrosis were measured by quantitative real-time reverse transcription PCR. Data represent means ± SD, n= 3 per experimental group, (*P＜ 0.05, **P＜ 0.01 WT vs. KO). (B) Western Blot analysis of Collagen Ⅰ（Ⅱ） performed on protein (100 μg) extracted from WT and Rdh13 knockout liver tissues at 14
various time points following CCl4 injection. (C) Hepatic immunohistochemical staining of collagen Ⅰ（Ⅱ）with a polyclonal rabbit anti-murine collagen typeⅠ antibody in WT and knockout mice. Original magnification, ×200． Figure 5.
Rdh13 deficiency attenuates hepatic Timp-1 mRNA and protein
increment following CCl4-induced chronic liver injury in mice. (A) The hepatic mRNA levels of Timp-1 for wide-type (WT) and Rdh13 knockout (KO) mice after CCl4 exposure were measured by quantitative real-time reverse transcription PCR. Data represent means ± SD, n = 3 per experimental group, (**P＜ 0.01, WT vs. KO). (B) Western Blot analysis of hepatic Timp-1 protein levels at various time points following CCl4 injection. (C) Wide-type (WT) and Rdh13 knockout (KO) mice were treated with CCl4 for 4 weeks. Liver tissues were collected for immunohistochemical staining with anti-Timp-1 antibody. Representative pictures are shown. Original magnification, ×200． Figure 6. Effect of Rdh13 knockout on hepatic Tgf-β1 expression in mice with acute liver injury and liver fibrosis mice induced by CCl4. (A) The hepatic mRNA levels Tgf-β1 for wide-type (WT) and Rdh13 knockout (KO) mice during liver fibrosis were measured by quantitative real-time reverse transcription PCR. Data represent means ± SD, n = 3 per experimental group, (**P＜ 0.01 WT vs. KO). (B) Western Blot analysis of Tgf-β1 protein levels in the liver tissues from wild-type mice and Rdh13 knockout at various time points following CCl4 injection.
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