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

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.

Genotype‐Phenotype Effects of Bmpr2 Mutations on Disease Severity in Mouse Models of Pulmonary Hypertension

“…ΔEx2/+ mouse mutation corresponds to an in-frame, extracellular domain, NMD− BMPR2ΔEx2 mutation also described in patients with HPAH, 10,16,20 these findings support our hypothesis that Bmpr2 +/− and Bmpr2 ΔEx2/+ mice model the molecular effects of NMD+ and NMD− BMPR2 mutations, respectively, in patients with HPAH. Our data also show that the level of expression of Bmpr2 in mouse lungs is lower than that in cultured PECs, suggesting that PECs, which constitute only a fraction of the total cells in whole lung, express higher levels of Bmpr2 than the other cellular compartments.…”

“…We compared the severity of experimentally induced pulmonary hypertension (PH) in mice on otherwise identical genetic backgrounds carrying one of two well-characterized heterozygous, splice-site, germ line Bmpr2 mutations that model the effects of known NMD+ and NMD− BMPR2 mutations in patients with HPAH: the out-of-frame Bmpr2ΔEx4-5 mutation [16][17][18][19] and the in-frame, extracellular-domain Bmpr2ΔEx2 mutation. 10,16,20 Previous studies have shown that mice carrying the Bmpr2 ΔEx4-5/+ mutation (which we refer to as Bmpr2 ΔEx2/+ -mutant mice. These studies suggest that the two mutations might confer differential susceptibility to PH in response to chronic hypoxia.…”

“…This simple hypothesis is confounded by the fact that some of these NMD− mutations, for example mis-sense mutations in the C-terminal cytoplasmic tail domain of BMPR2, may have only minor effects on BMPR2 function, 9 while others, including mis-sense and in-frame deletions in the extracellular domain of BMPR2, may have more profound effects on cellular function resulting from protein misfolding and retention in the endoplasmic reticulum (ER). [10][11][12] Despite this, there is some clinical evidence to support the hypothesis that HPAH patients with NMD− BMPR2 mutations have more severe disease than those with NMD+ mutations. Austin et al 13 evaluated disease penetrance and survival in HPAH patients in whom the NMD status was determined in cultured, patient-derived lymphoblasts.…”

Constitutive negative regulation in the processing of the anti-Müllerian hormone receptor II

“…Of these regulatory mechanisms, modifications related to the biosynthetic maturation of TGFb-SF receptors have been relatively less studied. The importance of regulation of such receptors at early stages of folding, maturation and trafficking is exemplified by the molecular fate of receptors carrying genetic disease-related mutations, which induce the intracellular retention of AMHRII and the type-II BMP receptor (BMPRII, also known as BMPR2), and abolish the signaling response to their respective ligands (Belville et al, 2009;Faure et al, 1996;Frump et al, 2013;Li et al, 2010;Sobolewski et al, 2008).…”

KCNK3: new gene target for pulmonary hypertension?

“…Similarly, the next step for PAH management may be the development of causal mutation-specific therapies. Frump and colleagues have demonstrated that it is possible to restore the TGF-b activity in cells carrying an in-frame deletion of exon 2 of the BMPR2 gene by improving trafficking of the mutated protein using chemical chaperones [28]. These authors speculated that chemical chaperones could be beneficial for all carriers of a missense BMPR2 mutation or an in-frame large rearrangement which affects the trafficking of the protein to the cell surface.…”