Elsevier

Life Sciences

Volume 221, 15 March 2019, Pages 293-300
Life Sciences

Cathepsin L-deficiency enhances liver regeneration after partial hepatectomy

https://doi.org/10.1016/j.lfs.2019.02.040Get rights and content

Abstract

Aim

Cathepsin L (Ctsl) plays a pivotal role in lysosomal and autophagic proteolysis. Previous investigations revealed that partial hepatectomy (PH) decreases biosynthesis of cathepsins in liver, followed by suppression of lysosomal and autophagic proteolysis during liver regeneration. Conversely, it was reported that autophagy-deficiency suppressed liver regeneration. Thus, the purpose of this study is to determine if Ctsl deficiency affects liver regeneration after PH.

Methods

70% of PH was performed in male Ctsl-deficient mice (Ctsl−/−) and wild-type littermates (Ctsl +/+) after PH. Mice were sacrificed and wet weight of the whole remaining liver was measured. Bromodeoxyuridine (BrdU)-immunostaining of liver sections was performed. Expression of cyclin D1, p62, LC-3, Nrf2, cleaved-Notch1, Hes1 was evaluated by western blot analysis. NQO1 mRNA expression was measured by realtime-PCR.

Results

After a 70% of PH, the liver mass was significantly restored within 5 days in Ctsl−/− mice compared to wild-type. Ctsl-deficiency enhanced the increases in both the rate of BrdU-positive cells and cyclin D1 expression after PH more than wild-type mice. On the other hand, Ctsl-deficiency upregulated p62, cleaved-Notch1 and Hes1 expression after PH. Moreover, the protein level of Nrf2 in the nucleus and mRNA expression of NQO1 in the liver after PH was also up-regulated in Ctsl−/− mice.

Conclusions

These findings suggest that accumulation of p62 due to loss of Ctsl plays an important role in liver regeneration through activation of Nrf2-Notch1 signaling. Taken together, Ctsl might be a new therapeutic target on disorder of liver regeneration.

Introduction

Liver regeneration proceeds through a series of complex signaling pathways [1]. Although hepatocytes rarely divide under normal circumstances, the liver has a remarkable ability to regenerate after surgical removal or after liver injury. Understanding the hepatic regenerative process has clinical interest, since the effectiveness of many treatments for chronic liver diseases, such as resection of tumors and donor liver transplantation, is dependent upon efficient liver regeneration. Post-hepatectomy liver insufficiency is one of the main problems associated with major hepatic resection in cirrhotic livers. Thus, liver resection in cirrhotic patients is a risky therapeutic decision. Accordingly, it is necessary to identify new therapeutic targets to stimulate liver regeneration.

Lysosomes play an essential role in protein turnover by degrading exogenous and endogenous proteins and supplying cells with amino acids necessary for protein synthesis or as an energy source [2]. Lysosomal proteolysis is caused mainly by cysteine proteinases such as cathepsin B, H, and L [2]. Cathepsin L (Ctsl) expressed in almost all types of eukaryotic cells, is primarily involved in turnover and degradation of intracellular proteins [2]. Hepatocytes also synthesize and secrete large amounts of Ctsl. It is well known that lysosomal proteolytic activities are inversely correlated with cell growth [3]. Lysosomal proteolysis is lower in transformed cells than in parent cells. Further insight into the relationship between lysosomal proteolysis and cell growth has been obtained from the studies using regenerating liver [4]. In regenerating liver, decreased protein degradation rather than increased protein synthesis, has been shown to be the main cause of the rapid increase in cell mass after hepatectomy. Lysosomal and autophagic proteolysis in regenerating liver is suppressed principally through a suppression of cathepsins expression [5]. As previous investigations have shown, the down-regulation of Ctsl activity by pharmacological inhibitors suppressed autophagic membrane turnover and the resulting Ctsl deficiency enhanced accumulation of the selective autophagic substrate p62 [6,7].

Autophagy is a catabolic process which degrades long-lived proteins and cellular organelles such as mitochondria and endoplasmic reticulum (ER) for an alternative energy source during nutrient deprivation [8]. Autophagosomes engulf organelles and then fuse with lysosomes to form mature autolysosomes, in which the sequestered proteins are digested into amino acids by lysosomal enzymes. Selective recruitment of p62 into the autophagosome is then degraded [9]. Thus, impaired autophagy caused accumulation of p62. Recent evidence demonstrated that the accumulation of p62 due to autophagy-deficiency plays a pivotal role in hepatoma development via persistent activation of nuclear factor erythroid 2–related factor 2 (Nrf2) [10]. The Nrf2–Keap1 system is one of the major cellular defense mechanisms against oxidative stress. It was reported that the accumulation of p62 promotes the stabilization of Nrf2 followed by transcriptional activation of Nrf2 target genes encoding antioxidant proteins. Previous reports demonstrated that liver regeneration after partial hepatectomy was down-regulated in Nrf2 null mice [11]. It was suggested that activation of Nrf2 plays a pivotal role in the regenerative response after partial hepatectomy. Therefore, in this study, we hypothesize that Ctsl-deficiency enhances liver regeneration after PH with an up-regulation of p62-associated Nrf2 signaling.

Section snippets

Animals and experimental design in an in vivo model

Male Cathepsin L−/− (Ctsl−/−) [12] and wild-type littermates mice 8 weeks after birth were housed under specific pathogen-free conditions. All animals received humane care in compliance with the experimental protocol approved by the Committee of Laboratory Animals according to institutional guidelines (permit no. 290176). A 70% partial hepatectomy (PH) or sham procedure was performed on male Ctsl−/− and wild-type littermates. Mice were sacrificed 1, 6, 12, 24, 48, 120 h after PH and wet weight

Liver regeneration after partial hepatectomy is up-regulated in Ctsl −/− mice

In this study, we first investigated the differences in liver regeneration after 70% PH between wild-type mice and Cts−/− mice. All mice survived after PH. Regenerated liver mass at 5 days after PH in wild-type mice was 74% of total liver weight predicted by resected-liver weight; however, liver mass was increased to 89% of predicted total liver in Ctsl−/− mice. Restoration of liver weight 5 days after PH in Ctsl−/− mice was significantly higher than wild-type mice (p=0.008) (Fig. 1A). To

Discussion

The liver is one of the most complex organs, playing an important role in digestion, detoxification, blood sugar regulation, and fat metabolism. Liver regeneration induced by surgical injury is an orchestrated response. Here, we found that Ctsl deficiency accelerates liver regeneration after 70% PH (Fig. 1A). After PH, Ctsl deficiency accelerated cellular proliferation resulting in tissue remodeling and restoration to normal hepatic mass during shorter period than wild type mice (Fig. 1B).

Conclusion

In summary, the potentiation of Nrf2 signaling promotes liver regeneration after PH through an accumulation of p62 in Ctsl deficiency. Suppression of cathepsin expression after PH is fundamental to liver regeneration. It is indicated that Ctsl is involved specifically in the degradation of autophagosomal membrane but not essential for autophagic function during liver regeneration after PH. Thus, Ctsl regulation might be a new therapeutic target for disorders of liver regeneration.

The following

Financial support

This work was supported in part by Grant-in-Aid (No.26461020 to SY, No.16H05293 to SW, and No.15K09023 to KI) and High Technology Research Center Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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