A Molecular Mechanism for Therapeutic Effects of cGMP-elevating Agents in Pulmonary Arterial Hypertension

Raphaela Schwappacher,Ana Kilic,Baktybek Kojonazarov,Michaela Lang,Thuan Diep,Shunhui Zhuang,Thomas Gawlowski,Ralph T Schermuly,Alexander Pfeifer,Gerry R Boss,Renate B Pilz

doi:10.1074/jbc.m113.458729

Abstract

Pulmonary arterial hypertension (PAH) is a progressive, usually fatal disease with abnormal vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) from PAH patients are hyperproliferative and apoptosis-resistant and demonstrate decreased signaling in response to bone morphogenetic proteins (BMPs). Cyclic GMP-elevating agents are beneficial in PAH, but their mechanism(s) of action are incompletely understood. Here we show that BMP signaling via Smad1/5/8 requires cGMP-dependent protein kinase isotype I (PKGI) to maintain PASMCs in a differentiated, low proliferative state. BMP cooperation with cGMP/PKGI was crucial for transcription of contractile genes and suppression of pro-proliferative and anti-apoptotic genes. Lungs from mice with low or absent PKGI (Prkg1(+/-) and Prkg1(-/-) mice) exhibited impaired BMP signaling, decreased contractile gene expression, and abnormal vascular remodeling. Conversely, cGMP stimulation of PKGI restored defective BMP signaling in rats with hypoxia-induced PAH, consistent with cGMP-elevating agents reversing vascular remodeling in this PAH model. Our results provide a mechanism for the therapeutic effects of cGMP-elevating agents in PAH and suggest that combining them with BMP mimetics may provide a novel, disease-modifying approach to PAH therapy.

Highlights

Pulmonary arterial hypertension (PAH) is characterized by abnormal vascular remodeling and impaired bone morphogenetic proteins (BMPs)/Smad signaling
PKGI Facilitates BMP/Smad Signaling in smooth muscle cells (SMCs)—We studied interactions between BMP and cGMP signaling in primary human pulmonary artery smooth muscle cells (PASMCs), immortalized rat PASMCs, and immortalized SMC precursors (C3H/10T1/2 cells)
Using pharmacological and genetic approaches, we found that BMP/Smad and cGMP/PKGI signaling pathways cooperate in PASMCs to regulate expression of multiple genes involved in proliferation, apoptosis, and maintenance of a differentiated, contractile phenotype (Fig. 5D)

Summary

Background

Pulmonary arterial hypertension (PAH) is characterized by abnormal vascular remodeling and impaired BMP/Smad signaling. Results: cGMP/PKGI are required for the anti-proliferative and pro-differentiation effects of BMP in pulmonary artery smooth muscle cells (PASMCs) in vivo. CGMP and BMP Signaling in Pulmonary Hypertension from the receptor complex and associates with activated Smads; the PKGI-Smad complex translocates to the nucleus, where it recruits the transcriptional co-factor TFII-I to enhance Smad-dependent transcription from a BMP response elementcontaining reporter gene (11). We demonstrate functional consequences of cGMP/BMP cross-talk in SMCs: cGMP/PKGI and BMP/Smad cooperatively regulate PASMC growth, survival, and differentiation; defective Smad phosphorylation and Smad downstream signaling are observed in mice with reduced PKG expression, and PKG activation in a rat model of PAH improves Smad signaling

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION