Elsevier

Journal of Hepatology

Volume 57, Issue 4, October 2012, Pages 844-851
Journal of Hepatology

Research Article
A hedgehog survival pathway in ‘undead’ lipotoxic hepatocytes

https://doi.org/10.1016/j.jhep.2012.05.011Get rights and content

Background & Aims

Ballooned hepatocytes in non-alcoholic steatohepatitis (NASH) generate sonic hedgehog (SHH). This observation is consistent with a cellular phenotype in which the cell death program has been initiated but cannot be executed. Our aim was to determine whether ballooned hepatocytes have potentially disabled the cell death execution machinery, and if so, can their functional biology be modeled in vitro.

Methods

Immunohistochemistry was performed on human NASH specimens. In vitro studies were performed using HuH-7 cells with shRNA targeted knockdown of caspase 9 (shC9 cells) or primary hepatocytes from caspase 3−/− mice.

Results

Ballooned hepatocytes in NASH display diminished expression of caspase 9. This phenotype was modeled using shC9 cells; these cells were resistant to lipoapoptosis by palmitate (PA) or lysophosphatidylcholine (LPC) despite lipid droplet formation. During lipid loading by either PA or LPC, shC9 cells activate JNK which induces SHH expression via AP-1. An autocrine hedgehog survival signaling pathway was further delineated in both shC9 and caspase 3−/− cells during lipotoxic stress.

Conclusions

Ballooned hepatocytes in NASH downregulate caspase 9, a pivotal caspase executing the mitochondrial pathway of apoptosis. Hepatocytes engineered to reduce caspase 9 expression are resistant to lipoapoptosis, in part, due to a hedgehog autocrine survival signaling pathway.

Introduction

Human hepatocellular injury is ubiquitous due to the prevalence of steatohepatitis syndromes, viral hepatitis, immune mediated insults, genetic diseases, etc. [1]. In these hepatic diseases, hepatocyte fate is often modeled as a binary process – cells either survive or die to be replaced by liver regenerative processes [2]. Yet, cells may resist cell death by downregulating or inhibiting cell death programs, thereby surviving in an altered state. This new cell fate does not merely reflect an escape from death, but rather results in the formation of a unique, functional cell phenotype. The cell phenotype of non-lethal cell injury has been best characterized in model systems where it has been termed the ‘undead cell’ [3], [4]. Because cell death by proapoptotic stimuli requires activation of executioner caspases [5], the undead cell can be engineered by deletion of these downstream caspases. For example, in Drosophila melanogaster genetic deletion of the executioner caspase DrICE or Dcp-1 results in a cell type in which otherwise toxic stimuli activate alternative cell signaling cascades producing signals which enhance tissue remodeling [3], [4], [6].

A candidate undead cell in the liver is the ballooned hepatocyte in non-alcoholic steatohepatitis (NASH). Ballooned hepatocytes are thought to be important in the pathogenesis of NASH and the presence of such cells is used to score disease severity [7], [8]. Little is known about ballooned hepatocytes other than they are enlarged cells which have lost cell polarity, frequently contain Mallory–Denk bodies, store neutral triglycerides, contain oxidized phospholipids, no longer stain for cytokeratin 18, and generate the ligand sonic hedgehog [8], [9], [10]. The seminal work by Diehl and colleagues demonstrating sonic hedgehog (SHH) generation by ballooned hepatocytes suggests this cell may, in fact, be a functional cell with an altered phenotype [8], [11]. For example, in D. melanogaster retinal cells in which the cell death program is initiated but cannot be executed also generate SHH [6]. The mechanisms by which undead cells continue to evade cell death remain elusive, but given the potency of SHH as a survival factor, it is possible that this ligand serves as an autocrine survival factor.

Herein, we demonstrate that ballooned cells have reduced expression of a potent downstream caspase, caspase 9, suggesting they may not efficiently execute cell death pathways. To understand the functional implications of this observation, we modeled lipotoxic stress in HuH-7 cells in which caspase 9 had been knocked down by short hairpin RNA technology (shC9 cells). Knockdown of caspase 9 protected hepatocytes from lipotoxicity by the saturated free fatty acid (FFA), palmitate (PA) or the phospholipid lysophosphatidylcholine (LPC). Interestingly, PA or LPC treated shC9, but not wild type cells, generate sonic hedgehog by a c-Jun-N-terminal kinase (JNK)-dependent pathway. Blockade of hedgehog signaling resulted in cell death of shC9 cells following exposure to lipotoxic agents. Similar observations were confirmed in primary caspase 3−/− mouse hepatocytes. We speculate that these engineered cells model the functional phenotype of ballooned hepatocytes, albeit not the specific morphology, and, in turn, support the concept of ballooned hepatocytes as an altered, but functional cell population which has escaped cell death.

Section snippets

Immunohistochemistry (IHC)

Formalin-fixed, paraffin-embedded liver sections (5 μm thick) from three patients with non-alcoholic steatohepatitis (NASH) were obtained following approval by the Institutional Review Board (IRB), Mayo Clinic, Rochester MN, USA. In conjunction with the clinical core of P30DK084576, the sections were pre-selected by an experienced hepatopathologist for their abundance of ballooned hepatocytes. These sections were deparaffinized in xylene and rehydrated through graded alcohols. After tissue

Ballooned hepatocytes in NASH display reduced caspase 9 expression

Loss of executioner caspase function is sufficient to prevent cell death in developmental systems. Therefore, we performed immunohistochemistry in human NASH specimens for caspase 9, a potent executioner caspase. Ballooned hepatocytes, but not neighboring hepatocytes, displayed diminished caspase 9 expression (Fig. 1A). In contrast, expression of the upstream caspase 2 was readily identified in ballooned hepatocytes (Fig. 1A). Caspase 2 has a long prodomain similar to caspase 9, and therefore,

Discussion

We posited that the ballooned hepatocyte in NASH represents an altered cellular phenotype which has escaped cell death analogous to the ‘undead cell’ in developmental models. Consistent with this concept, we observed a reduction of caspase 9 protein expression in ballooned hepatocytes. We sought to model this phenotype in vitro and explore its functional biology. Our results indicate that during lipotoxic stress by PA or LPC: (1) targeted knockdown of caspase 9 or genetic deficiency of caspase

Financial support

This work was supported by NIH Grants DK41876 to G.J.G., the optical microscopy and clinical cores of P30DK084567, and the Mayo Foundation.

Conflict of interest

The underlying research reported in the study was funded by the NIH Institutes of Health.

Acknowledgements

This work was supported by NIH Grant DK41876 and the optical and microscopy cores of P30DK084567 and the Mayo Foundation. We thank Drs. Christian D. Fingas, Maria E. Guicciardi, Justin L. Mott, and Scott H. Kaufmann for helpful discussions; Dr. Harmeet Malhi for reading the manuscript and providing insightful comments and suggestions; Eugene W. Krueger for aiding the studies employing contrast microscopy; and Courtney N. Hoover for her excellent secretarial assistance.

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