Faculty Collaboration Database

Rap1A Modulates Store-Operated Calcium Entry in the Lung Endothelium: A Novel Mechanism Controlling NFAT-Mediated Vascular Inflammation and Permeability. Arterioscler Thromb Vasc Biol 2024 Nov;44(11):2271-2287

Date

09/26/2024

Pubmed ID

39324266

Pubmed Central ID

PMC11495542

DOI

10.1161/ATVBAHA.124.321458

Scopus ID

2-s2.0-85206451357 (requires institutional sign-in at Scopus site)   12 Citations

Abstract

BACKGROUND: Store-operated calcium entry mediated by STIM (stromal interaction molecule)-1-Orai1 (calcium release-activated calcium modulator 1) is essential in endothelial cell (EC) functions, affecting signaling, NFAT (nuclear factor for activated T cells)-induced transcription, and metabolic programs. While the small GTPase Rap1 (Ras-proximate-1) isoforms, including the predominant Rap1B, are known for their role in cadherin-mediated adhesion, EC deletion of Rap1A after birth uniquely disrupts lung endothelial barrier function. Here, we elucidate the specific mechanisms by which Rap1A modulates lung vascular integrity and inflammation.

METHODS: The role of EC Rap1A in lung inflammation and permeability was examined using in vitro and in vivo approaches.

RESULTS: We explored Ca²⁺ signaling in human ECs following siRNA-mediated knockdown of Rap1A or Rap1B. Rap1A knockdown, unlike Rap1B, significantly increased store-operated calcium entry in response to a GPCR (G-protein-coupled receptor) agonist, ATP (500 µmol/L), or thapsigargin (250 nmol/L). This enhancement was attenuated by Orai1 channel blockers 10 μmol/L BTP2 (N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide), 10 μmol/L GSK-7975A, and 5 μmol/L Gd³⁺. Whole-cell patch clamp measurements revealed enhanced Ca²⁺ release-activated Ca²⁺ current density in siRap1A ECs. Rap1A depletion in ECs led to increased NFAT1 nuclear translocation and activity and elevated levels of proinflammatory cytokines (CXCL1 [C-X-C motif chemokine ligand 1], CXCL11 [C-X-C motif chemokine 11], CCL5 [chemokine (C-C motif) ligand 5], and IL-6 [interleukin-6]). Notably, reducing Orai1 expression in siRap1A ECs normalized store-operated calcium entry, NFAT activity, and endothelial hyperpermeability in vitro. EC-specific Rap1A knockout (Rap1A^iΔEC) mice displayed an inflammatory lung phenotype with increased lung permeability and inflammation markers, along with higher Orai1 expression. Delivery of siRNA against Orai1 to lung endothelium using lipid nanoparticles effectively normalized Orai1 levels in lung ECs, consequently reducing hyperpermeability and inflammation in Rap1A^iΔEC mice.

CONCLUSIONS: Our findings uncover a novel role of Rap1A in regulating Orai1-mediated Ca²⁺ entry and expression, crucial for NFAT-mediated transcription and endothelial inflammation. This study distinguishes the unique function of Rap1A from that of the predominant Rap1B isoform and highlights the importance of normalizing Orai1 expression in maintaining lung vascular integrity and modulating endothelial functions.

Author List

Kosuru R, Romito O, Sharma GP, Ferraresso F, Ghadrdoost Nakhchi B, Yang K, Mammoto T, Mammoto A, Kastrup CJ, Zhang DX, Goldspink PH, Trebak M, Chrzanowska M

Authors

Magdalena Chrzanowska PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
Christian Kastrup PhD, MS Professor in the Surgery department at Medical College of Wisconsin
Akiko Mammoto PhD, MD Professor in the Pediatrics department at Medical College of Wisconsin
Guru Prasad Sharma Research Scientist I in the Radiation Oncology department at Medical College of Wisconsin
Kai Yang PhD Assistant Professor in the Data Science Institute department at Medical College of Wisconsin
David X. Zhang PhD Associate Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Calcium
Calcium Signaling
Capillary Permeability
Cells, Cultured
Disease Models, Animal
Endothelial Cells
Human Umbilical Vein Endothelial Cells
Humans
Lung
Male
Mice
Mice, Inbred C57BL
Mice, Knockout
NFATC Transcription Factors
ORAI1 Protein
Pneumonia
RNA Interference
Stromal Interaction Molecule 1
rap GTP-Binding Proteins
rap1 GTP-Binding Proteins