L(59) TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice.

Mitsuko Hara,Tomokazu Matsuura,Daniel B Rifkin,Yuta Yamazaki,Soichi Kojima,Ikuyo Inoue,Scott L Friedman,Akiko Kirita

doi:10.1186/s13069-015-0034-9

Abstract

BackgroundHepatic fibrosis, which is the excessive accumulation of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs), develops to cirrhosis over several decades. There are no validated biomarkers that can non-invasively monitor excessive production of ECM (i.e., fibrogenesis). Transforming growth factor (TGF)-β, a key driver of fibrogenesis, is produced as an inactive latent complex, in which active TGF-β is enveloped by its pro-peptide, the latency-associated protein (LAP). Thus, active TGF-β must be released from the complex for binding to its receptor and inducing ECM synthesis. We recently reported that during the pathogenesis of liver fibrosis, plasma kallikrein (PLK) activates TGF-β by cleavage between R58 and L59 residues within LAP and that one of its by-products, the N-terminal side LAP degradation products ending at residue R58 (R58 LAP-DPs), can be detected mainly around activated HSCs by specific antibodies against R58 cleavage edges and functions as a footprint of PLK-dependent TGF-β activation. Here, we describe a sandwich enzyme-linked immunosorbent assay (ELISA) that detects the other by-products, the C-terminal side LAP-DPs starting from residue L59 (L59 LAP-DPs). We demonstrated that the L59 LAP-DPs are a potentially novel blood biomarker reflecting hepatic fibrogenesis.ResultsWe established a specific sandwich ELISA to quantify L59 LAP-DPs as low as 2 pM and measured L59 LAP-DP levels in the culture media of a human activated HSC line, TWNT-4 cells. L59 LAP-DPs could be detected in their media, and after treatment of TWNT-4 cells with a TGF-β receptor kinase inhibitor, SB431542, a simultaneous reduction was observed in both L59 LAP-DP levels in the culture media and the mRNA expression levels of collagen type (I) α1. In carbon tetrachloride- and bile duct ligation-induced liver fibrosis models in mice, plasma L59 LAP-DP levels increased prior to increase of hepatic hydroxyproline (HDP) contents and well correlated with α-smooth muscle actin (αSMA) expression in liver tissues. At this time, αSMA-positive cells as well as R58 LAP-DPs were seen in their liver tissues.ConclusionsL59 LAP-DPs reflect PLK-dependent TGF-β activation and the increase in αSMA-positive activated HSCs in liver injury, thereby serving as a novel blood biomarker for liver fibrogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13069-015-0034-9) contains supplementary material, which is available to authorized users.

Highlights

Hepatic fibrosis, which is the excessive accumulation of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs), develops to cirrhosis over several decades
We describe a sandwich enzyme-linked immunosorbent assay (ELISA) using the specific antibody against L59 latency-associated protein (LAP)-DPs and examine whether L59 LAP degradation product (LAP-DP) can be used as a blood biomarker reflecting plasma kallikrein (PLK)-dependent Transforming growth factor (TGF)-β activation that correlates with activated HSCs and whether they can be used to monitor liver fibrogenesis in mice
LAP-DPs To quantify the levels of L59 LAP-DPs in a culture medium of collagen-producing cells including HSCs or blood from animal models with fibrogenesis, we established a sandwich ELISA using a combination of L59 and commercially available anti-LAP antibodies (Fig. 1a) and examined the specificity of the ELISA for L59 LAP-DPs initially in test tube reactions

Summary

Introduction

Hepatic fibrosis, which is the excessive accumulation of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs), develops to cirrhosis over several decades. Transforming growth factor (TGF)-β, a key driver of fibrogenesis, is produced as an inactive latent complex, in which active TGF-β is enveloped by its pro-peptide, the latency-associated protein (LAP). The common pathology resulting from chronic liver diseases regardless of etiology, is characterized as the excessive deposition of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs) in injured tissue [1, 2]. After processing at R278-A279 by a furin-like protease, the cleaved LAP still remains non-covalently associated with the active TGF-β, forming a small latent complex and preventing active TGF-β from binding to its cognate receptors. Resultant active TGF-β binds to cognate serine/threonine kinase signaling receptors and via the Smad signaling pathway stimulates the expression of target genes, including ECM proteins and TGF-β itself, in an autocrine manner [5, 8]

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