Abnormal Trafficking of Endogenously Expressed BMPR2 Mutant Allelic Products in Patients with Heritable Pulmonary Arterial Hypertension

Andrea L Frump,Rizwan Hamid,Jonathan W Lowery,Eric D Austin,Mark De Caestecker,Philip Michael Bauer

doi:10.1371/journal.pone.0080319

Andrea L Frump, Rizwan Hamid + Show 4 more

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https://doi.org/10.1371/journal.pone.0080319

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Abstract

More than 200 heterozygous mutations in the type 2 BMP receptor gene, BMPR2, have been identified in patients with Heritable Pulmonary Arterial Hypertension (HPAH). More severe clinical outcomes occur in patients with BMPR2 mutations by-passing nonsense-mediated mRNA decay (NMD negative mutations). These comprise 40% of HPAH mutations and are predicted to express BMPR2 mutant products. However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described. Here, we characterize the expression and trafficking of an HPAH-associated NMD negative BMPR2 mutation that results in an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2) in HPAH patient-derived lymphocytes and in pulmonary endothelial cells (PECs) from mice carrying the same in-frame deletion of Exon 2 (Bmpr2 ΔEx2/+ mice). The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum. Moreover, chemical chaperones 4-PBA and TUDCA partially restore cell surface expression of Bmpr2ΔEx2 in PECs, suggesting that the mutant product is mis-folded. We also show that PECs from Bmpr2 ΔEx2/+ mice have defects in the BMP-induced Smad1/5/8 and Id1 signaling axis, and that addition of chemical chaperones restores expression of the Smad1/5/8 target Id1. These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention. Partial correction of this folding defect and restoration of defective BMP signaling using chemical chaperones suggests that protein-folding agents could be used therapeutically in patients with these NMD negative BMPR2 mutations.

Highlights

Despite modern vasodilator treatments, patients with Pulmonary Arterial Hypertension (PAH) only have a 50% 5year survival[1]
For these studies we evaluated BMPR2 expression in lymphocytes derived from an Heritable Pulmonary Arterial Hypertension (HPAH) patient from Vanderbilt PAH Family 108 (F108) who carry a splice site mutation predicted to result in an in-frame deletion of BMPR2 EXON2, which encodes residues 26-82 of the 1038 full length BMPR2 protein[30]
We provide the first evidence that an NMD negative BMPR2 mutation found in patients with HPAH is expressed endogenously, and that this mutant protein product is mis-folded and incorrectly trafficked to the cell surface

Summary

Introduction

Patients with Pulmonary Arterial Hypertension (PAH) only have a 50% 5year survival[1]. The mechanism by which defects in the BMP signaling contribute to HPAH remains elusive, BMP-signaling defects associated with mutations in BMPR2 have been implicated in abnormal pulmonary vascular cell proliferation, remodeling and vascular tone [8,9,10,11,12,13,14] These diverse roles in regulating the pulmonary vasculature and the defects in this pathway detected in patients with HPAH, suggest that strategies to correct BMP signaling defects may have long-term disease modifying effects. Drugs that promote read-through of pre-termination codons associated with BMPR2 mutation that result in non-sense mediated decay of the mutant mRNA product, increase expression of functional BMPR2 mutant products in cells from patients with HPAH [16,17] In these studies we take an alternative approach to correct BMP signaling defects by correcting abnormal trafficking of a BMPR2 mutant protein product

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