Elsevier

Hepatology Research

Volume 28, Issue 3, March 2004, Pages 140-145
Hepatology Research

Expression of cytosolic and membrane associated tissue transglutaminase in rat hepatic stellate cells and its upregulation during transdifferentiation to myofibroblasts in culture

https://doi.org/10.1016/j.hepres.2003.11.004Get rights and content

Abstract

Transdifferentiation of hepatic stellate cells (HSC) to collagen producing myofibroblasts (MFB) is a principal event in liver fibrogenesis.

In our studies we investigated if tissue transglutaminase (tTG) from these cell types may play a role in liver fibrosis. Separation of cytosol and membrane components showed membrane associated tTG and during transdifferentiation an upregulation of total tTG on mRNA and protein level was found, but no modulation during stimulation with TGF-β1. In HSC and fully differentiated MFB a significant amount of the total tTG synthesised during transdifferentiation is found to be membrane-associated whereas the remaining portion is cytosol-associated and only very little is found within the extracellular matrix (ECM).

The data implicate that tTG in this cell type seems to play an important role in liver fibrogenesis.

Introduction

Tissue transglutaminase (tTG) catalyses in a Ca2+-dependent fashion the acyl-transfer of peptide-bound glutamine residues to primary amines of polyamines like putrescine or spermidine and to the ε-amino groups of peptide-bound lysine residues resulting in covalent and irreversible cross-links between or within proteins [1]. Structurally it is a monomeric globular protein of 77 kDa which exists in both extracellular and intracellular locations. Intracellularly tTG seems to be involved in apoptotic processes [2], [3], [4], [5]. Matrix and cell surface associated tTG was shown to be important for stabilisation of the extracellular matrix (ECM) [6], [7] and in promoting fibronectin assembly mediated by α1β5 integrin [8]. Furthermore, cell surface tTG has been suggested to be involved in the fixation of the large latent TGF-β complex to the ECM immediately after secretion of this cytokine [9]. TGF-β, a key cytokine in liver fibrogenesis, is secreted by hepatic stellate cells (HSC) in a latent form as a large latent TGF-β complex [10] which contains a high molecular weight glycoprotein (125–210 kDa), termed latent TGF-β binding protein 1 (LTBP-1). LTBP-1 is covalently linked to the TGF-β precursor, named latency associated peptide (LAP, 75 kDa) [11], which noncovalently binds the mature active TGF-β dimer. Thus, TGF-β might be stored in its inactive form in the ECM until activation. Studies with peritoneal macrophages and tumour cell lines indicate that tTG mediated covalent binding of the large latent TGF-β complex to the ECM is a prerequisite for the activation of TGF-β [12], [13]. The transdifferentiation of HSC to ECM-producing myofibroblasts (MFB) is a crucial event in hepatic fibrogenesis which is stimulated by TGF-β [14], [15]. We investigated whether this process or TGF-β stimulation entailed modulations of tTG on mRNA and protein level in these cell types in turn suggesting a functional role of this enzyme during HSC activation.

Section snippets

Isolation and culture of HSC

HSC were isolated from normal rat livers (1-year-old male Sprague–Dawley rats, weighing 500–700 g) by pronase–collagenase perfusion technique, as described previously [16]. The purified cells were seeded and cultured in Dulbecco’s modified Eagle’s medium (Bio Whittaker Europe, Verviers, Belgium), supplemented with 10% fetal calf serum (Seromed, Berlin, Germany), glutamine and antibiotics. Cell purity was determined to be at least 95% and the plating efficiency varied between 40 and 70%. Fully

Results

tTG expression was monitored during transdifferentiation of cultured HSC between days 2 and 7 of primary culture. After subculturing, MFB were lysed on day 4. Northern blot analysis was performed with total RNA from transdifferentiating cells and revealed a graduate upregulation of tTG mRNA with time and the most prominent increase occured between days 4 and 7 (Fig. 1). Western blotting of cell lysates was performed with 10 μg total protein on each lane and showed similar results: basal

Discussion

The principal effector cell of liver fibrosis is widely recognised to be HSC. Independently from the cause of injury these cells undergo an activation process leading to a myofibroblast-like phenotype synthesising and secreting a broad array of ECM-components. We investigated if tTG expression is altered during this process and thus might be relevant for liver fibrogenesis.

As surface and matrix associated tTG are not easily detectable by antibodies, possibly due to masking of the active-site

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    Present address: Central Laboratories, Institute of Clinical Chemistry, Center of Clinic-Theoretical Medicine I, University Hospital Eppendorf, 20251 Hamburg, Germany.

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