Microtubule Arrangement and FKBP51 Localization is Altered in BMPR2‐Deficient Pulmonary Artery Endothelial Cells Isolated from a Rat Model of PAH

Abstract

Pulmonary arterial hypertension (PAH) is an incurable vasoconstrictive pathology of which there are limited palliative treatments. The hallmarks of PAH include vascular smooth muscle cell proliferation and endothelial dysfunction in the small pulmonary arteries. Endothelial cytoskeletal defects have been implicated in BMPR2‐associated PAH, and BMPR2‐silencing alters cytoskeletal arrangement in pulmonary artery endothelial cells (PAECs). Indeed, targeting cytoskeletal proteins, particularly microtubules, may be a novel approach to PAH treatment as microtubule disruption has been efficacious in the treatment of other inflammatory and proliferative diseases. FK506‐binding protein 51 (FKBP51), a peptidyl‐prolyl isomerase member of the immunophilin family, is a known regulator of the glucocorticoid receptor and its expression is upregulated upon glucocorticoid receptor activation. Furthermore, FKBP51 is a binding partner to Hsp90 and stabilizes microtubules in PAECs. Here, we show microtubule disruption and altered FKBP51 distribution in PAECs from a rat model of PAH. PAECs isolated from the Sugen/hypoxia rat model of PAH (PAH PAECs) or normoxic control cells were gifted from the University of South Alabama. The PAH PAECs have lower BMPR2 gene expression than normoxic controls, as shown by real‐time PCR. These cells also demonstrate decreased BMPR2 signaling compared to normoxic control PAECs, as demonstrated by lower ID‐1 expression levels upon BMP4 or BMP6 stimulation. Immunocytochemistry studies of α‐tubulin indicate microtubule disorganization in PAH PAECs, as compared to the normoxic control PAECs, which demonstrate normal perinuclear distribution of interphase microtubules. Immunocytochemical staining of FKBP51, using a novel antibody developed by our laboratory, reveals a microtubule‐like distribution of FKBP51 in normoxic control PAECs. Upon treatment with the synthetic glucocorticoid dexamethasone (DEX), FKBP51 is predominantly observed in the nucleus of normoxic control PAECs, with reduced cytosolic organization and distribution. PAH PAECs, however, showed diffuse cytoplasmic staining of FKBP51, with no change in distribution upon DEX treatment. Western blot analysis of PAEC nuclear fractions also demonstrated greater protein levels of FKBP51 upon DEX treatment, compared to DEX‐treated PAH PAECs. Taken together, these data indicate that microtubule rearrangement is evident in BMPR2‐deficient PAH PAECs, and FKBP51 dysregulation may play a role in cytoskeletal abnormalities as well as the disruption of metabolic homeostasis in these cells.Support or Funding InformationSupported by NIH1R15HL137135‐01A1