Identification of cell surface receptors for osteoprotegerin using human cell microarrays: Unravelling the molecular mechanisms of pulmonary arterial hypertension

Background

Pulmonary arterial hypertension (PAH) is a fatal lung disease characterised by progressive vascular remodelling, a key component of which is the proliferation and migration of pulmonary arterial smooth muscle cells. There are currently no therapies that reverse the vascular remodelling although some therapies do alleviate some symptoms of the disease. The molecular mechanisms of PAH are not thoroughly understood, however, it has been demonstrated that the secreted glycoprotein, osteoprotegerin (OPG) is elevated in patients. In vitro, OPG induces proliferation and migration of pulmonary arterial smooth muscle cells and, in animal models, inhibition of OPG can prevent and reverse the disease.

 

Aim of this study

An academic research group, led by Dr Allan Lawrie at the University of Sheffield, UK, sought to identify the human cell surface receptors for OPG using the Retrogenix cell microarray technology.

Identification of key receptors using human cell microarrays

A total of 2,054 plasma membrane proteins expressed in Retrogenix’s cell microarray were screened for interactions with OPG. Binding was detected using a polyclonal anti-OPG antibody, uncovering 19 potential hits on an initial (primary) screen. The cDNA for all these hits were respotted onto fresh slides and a second screen then performed. This confirmed four interactions that were both specific and reproducible:

  • IL1RAcP
  • Fas
  • TMPRSS11D
  • GAP43

The known interaction between OPG and syndecan 1 was used as a positive control throughout the study. The project was undertaken by Retrogenix scientists at the laboratory in Whaley Bridge, UK, with results delivered within six weeks.

Next steps

Following the human cell microarray screen, Dr Lawrie’s group then used co-immunprecipitation to confirm the OPG interactions with IL1RAcP and Fas in pulmonary arterial smooth muscle cells. Fas RNA and protein expression was also found to be elevated in patient tissues. Further work has since shown that neutralisation of Fas reduces OPG-induced cell proliferation (paper in preparation).

Impact

Identifying and confirming the receptor interaction for OPG sheds light on the signalling mechanisms through which OPG induces proliferation of pulmonary arterial smooth muscle cells. This highlights the potential therapeutic value in targeting OPG as a strategy for tackling pulmonary arterial hypertension and providing relief to patients suffering from this serious condition.

This study, performed in 2012, used a panel of 2,054 clones to rapidly identify the key receptor for an important ligand. Despite the already high success rates of the cell microarray technology at the time, rapid developments have continued at Retrogenix leading to two major expansions – first to include around 3,500 plasma membrane proteins and now to cover over 4,300. More than doubling the capabilities of the screening tool is providing even greater opportunities to accurately identify cell surface receptors and targets. As such, the Retrogenix technology is routinely used by both industry and academia to accelerate medical research, overcoming the traditional frustrations of elucidating the binding partners for key ligands implicated in disease.

 

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References:

  • Dawson S, Pickworth J, Rothman AMK, Iremonger J, Arnold N, Francis S, Lawrie A. ‘T5 OPG regulates pulmonary arterial smooth muscle cell proliferation and the expression of PAH-associated genes via Fas.’ Thorax (2014) 69:A2-A3