Expression of ADAMTS metalloproteinases in the retinal pigment epithelium derived cell line ARPE-19: transcriptional regulation by TNFα

https://doi.org/10.1016/S0167-4781(03)00047-2Get rights and content

Abstract

ADAMTS (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type I motifs) are multidomain proteins with demonstrated metalloproteinase functionality and have potential roles in embryonic development, angiogenesis and cartilage degradation. We present here investigations of ADAMTS expression in an ocular cell type, ARPE-19, with a view to implicating them in retinal matrix turnover. Expression analysis was undertaken using a combination of reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting experiments, which together detected the expression of mRNAs for several ADAMTS proteins, all of which have active site motifs characteristic of matrix metalloproteases (MMPs). These included ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS5, ADAMTS6, ADAMTS7 and ADAMTS9. The expression of mRNA isoforms for ADAMTS7 and ADAMTS9 were also detected. Following stimulation with TNFα, ADAMTS1, ADAMTS6 and both ADAMTS9 transcripts expressed in ARPE-19 cells showed a potent upregulation. The expression of ADAMTS genes in ARPE-19 cells and the transcriptional stimulation of some family members by TNFα may implicate them in inflammatory eye disease and the compromise of retinal matrix structure, which is evident in age-related macular degeneration (ARMD) and other retinal pathologies.

Introduction

The retinal pigment epithelium (RPE) is a highly specialised cell layer that plays an important role in the regulation and development of the photoreceptors in the vertebrate retina. It is a polarised monolayer of cells with distinct apical projections that are closely associated with photoreceptor outer segments and is responsible for secretion of matrix constituents into the interphotoreceptor space. The basolateral aspect of the RPE cells lies adjacent to Bruch's membrane, which is a complex and highly specialised extracellular matrix lying between the outer retina and the choriocapillaris. Bruch's membrane is believed to be composed of a five-layered structure, which contains collagens type I, III, IV and V and numerous proteoglycan species with both chondroitin sulfate and heparan sulfate chains. The basement membrane components laminin and fibronectin are also present [1].

The turnover of matrix in the retina bordering the apical interphotoreceptor matrix (IPM) and the basolateral aspect of RPE cells (Bruch's membrane) is thought to be mediated by matrix metalloproteinases (MMPs) and their cognate inhibitors the TIMPs (Tissue Inhibitor of Metalloproteinases). The role that the balance between MMPs and TIMPs plays in pathological aspects of matrix turnover has been documented both with respect to the IPM and Bruch's membrane [2], [3], [4], [5]. Recently, a family of metalloproteinase enzymes have been described, termed ADAMTS (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type I motifs), which show some similarity in domain structure to snake venom metalloproteinases. ADAMTS-type metalloproteinases have an MMP-like catalytic domain with associated latency domain, a disintegrin-like domain, a cysteine-rich domain and several distinct domains with homology to thrombospondin. Several are known to be secreted enzymes. These ADAMTS proteins have several substrates within the ECM such as pro-collagens [6] and proteoglycans [7]. In common with MMPs, some ADAMTS family members show potent inhibition by Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) [8].

TNFα is known to play a role in the development of retinal neovascularisation [9] and in RPE cell migration [10], which are features of age-related macular degeneration (ARMD) and other retinal disorders such as proliferative vitreoretinopathy (PVR) and diabetic vitreoretinopathy (DVR). Two members of the ADAMTS family, ADAMTS1 and ADAMTS8 (METH-1 and METH2), have been implicated in the mechanism of neovascularisation [11] and a homologue of ADAMTS9, gon1, is known to promote cell migration in C. elegans [12]. We therefore propose that members of the ADAMTS family may be involved in vascularisation and cell migration in the retina and that their expression may be modulated by TNFα. To explore this hypothesis, we have analysed transcription of ADAMTS1–9 in the ARPE19 cell line [13] and quantified expression of these genes after stimulation with the pro-inflammatory cytokine TNFα.

Section snippets

Cell culture

ARPE-19 retinal pigment epithelial cells (ATCC number CRL-2302, passage less than 30) were grown in a 1:1 mixture of DMEM/HamsF12 supplemented with 2.5 mM l-glutamine and 10% FBS. Confluent cells were grown in the absence of serum for 24 h prior to addition of TNFα (R&D Systems) at 5 and 50 ng/ml. TNFα was not added to control flasks. Total RNA was extracted after 2, 6, 24 and 48 h further incubation.

RNA preparation

At each time point, the monolayers were washed once with PBS and total RNA was extracted using

Detection of known ADAMTS mRNA sequences by RT-PCR

RT-PCR was used to amplify a panel of ADAMTS cDNAs from unstimulated ARPE-19 cells and from ARPE-19 cells stimulated with TNFα, both harvested 24 h after stimulation. Amplicons of the expected size were obtained using primers designed to amplify ADAMTS1, ADAMTS3 and ADAMTS5 regardless of TNFα stimulation (Fig. 1A). The mRNA for ADAMTS2 and ADAMTS6 were very weakly detected in extracts of unstimulated ARPE-19 cells but were readily detectable in cells stimulated with TNFα (Fig. 1B). DNA sequence

Discussion

Transcription of several ADAMTS genes in the ARPE19 cell line is described here for the first time, adding to the extensive literature on metalloproteinases expressed by retinal cell types [20], [21], [22], [23]. We have also described potential splice variants of both ADAMTS7 and ADAMTS9 and demonstrated by RT-PCR amplification that the splice variants are co-expressed in the ARPE-19 cells. Northern blot analysis has been used to show that the expression of three of the ADAMTS genes, namely

Acknowledgments

This study was supported by the Royal Victoria Infirmary Special Trustees, the Royal Victoria Infirmary, Newcastle-upon-Tyne, UK, by grants to MPC and NM, and by PPP Medical Healthcare Trust, by grants to MDB and NM.

References (42)

  • K. Kuno et al.

    Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motif as an inflammation associated gene

    J. Biol. Chem.

    (1997)
  • K. Kuno et al.

    ADAMTS-1 cleaves a cartilage proteoglycan, aggrecan

    FEBS Lett.

    (2000)
  • B.E. Jones et al.

    Characterization and novel activation of 72-kDa metalloproteinase in retinal interphotoreceptor matrix and Y-79 cell culture medium

    Exp. Eye Res.

    (1994)
  • J.G. Hollyfield et al.

    Chondroitin sulfate proteoglycan core proteins in the interphotoreceptor matrix: a comparative study using biochemical and immunohistochemical analysis

    Exp. Eye Res.

    (1999)
  • F. Vazquez et al.

    METH-1, a human ortholog of ADAMTS-1, and METH-2 are members of a new family of proteins with angio-inhibitory activity

    J. Biol. Chem.

    (1999)
  • J.C. Rodriguez-Manzaneque et al.

    Characterization of METH-1/ADAMTS1 processing reveals two distinct active forms

    J. Biol. Chem.

    (2000)
  • R.J. Fernandes et al.

    Procollagen II amino propeptide processing by ADAMTS-3-Insights on dermatosparaxis

    J. Biol. Chem.

    (2001)
  • T.L. Hurskainen et al.

    ADAM-TS5, ADAM-TS6, and ADAM-TS7, novel members of a new family of zinc metalloproteases. General features and genomic distribution of the ADAM- TS family

    J. Biol. Chem.

    (1999)
  • A. Colige et al.

    Human Ehlers-Danlos syndrome type VIIC and bovine dermatosparaxis are caused by mutations in the procollagen IN- proteinase gene

    Am. J. Hum. Genet.

    (1999)
  • I. Abbaszade et al.

    Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family

    J. Biol. Chem.

    (1999)
  • A. Colige et al.

    Cloning and characterization of ADAMTS-14, a novel ADAMTS displaying high homology with ADAMTS-2 and ADAMTS-3

    J. Biol. Chem.

    (2002)

    • Cited by (47)

    • Modulatory effect of interleukin-1αpression of structural matrix proteins, MMPs and TIMPs in human cardiac myofibroblasts: Role of p38 MAP kinase

      2010, Matrix Biology
      Citation Excerpt :

      In chondrosarcoma cells, hypoxia has no modulatory effect, whereas IL-1 reduces ADAMTS1 expression (Kalinski et al., 2007); findings in agreement with our results in CMF. In contrast, proinflammatory cytokines have been reported to increase ADAMTS1 expression in some other cell types (Ng et al., 2006; Bevitt et al., 2003). Hence transcriptional regulation of ADAMTS1 is highly dependent on the cell type studied, as well as on the stimulus applied.

    • ADAMTS1 is a unique hypoxic early response gene expressed by endothelial cells

      2009, Journal of Biological Chemistry
      Citation Excerpt :

      Ng et al. (28) reported that interleukin-1β (IL-1β) increased ADAMTS1 mRNA levels in human decidual stromal cells. Bevitt et al. (29) reported that ADAMTS1 was induced by TNFα stimulation in ARPE. Cross et al. (30) reported that ADAMTS1 mRNA was increased by IL-1β and TNF in cultured astrocytes; however, Kalinski et al. (31) reported that ADAMTS1 mRNA was decreased by IL-1β in the human chondrosarcoma cell line C3842.

    • Ranibizumab Modifies the Expression of Metalloproteinases and Their Tissue Inhibitors in Peripheral Blood Mononuclear Cells in Patients with Exudative Age-Related Macular Degeneration

      2024, Journal of Clinical Medicine

    • M6a Mrna Methylation Analysis Provides Novel Insights into Pigmentation in Sheep Skin

      2023, SSRN

    • Expression Profiling of ADAMTS (L) Superfamily of Genes in Various Human Eye Tissues

      2023, Iranian Journal of Public Health

    • m<sup>6</sup>A mRNA Methylation Analysis Provides Novel Insights into Pigmentation in Sheep Skin

      2023, Epigenetics
    View all citing articles on Scopus
    View full text
    Copyright © 2003 Elsevier Science B.V. All rights reserved.