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Loss of ERG in human lung endothelial cells leads to release of pro-inflammatory and pro-fibrotic factors.

ABSTRACT: : Although vascular dysfunction is a hallmark of chronic aging-associated diseases, including idiopathic pulmonary fibrosis, the role of the pulmonary vasculature to lung repair versus lung fibrosis is not fully understood. We identified the endothelial transcription factor ETS-related gene (ERG) as an orchestrator of vascular homeostasis and repair following lung injury. To evaluate whether loss of endothelial ERG influences the activation of neighboring naïve lung fibroblasts, we collected the conditioned media (CM) generated by control- and ERG-silenced human lung endothelial cells (ECs) and we applied them to normal human lung fibroblasts. We found that CM from ERG-silenced human lung ECs strongly promoted human lung fibroblast activation and enhanced the effect of the fibrogenic mediator TGF. In support of these results, analysis of CM using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) revealed increased secretion of numerous pro-inflammatory and pro-fibrogenic mediators by ERG-silenced human lung ECs in comparison to control-silenced human lung ECs.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Lung Endothelial Cell

DISEASE(S): Pulmonary Fibrosis

SUBMITTER: Benjamin Madden

LAB HEAD: Nunzia Caporarello

PROVIDER: PXD033847 | Pride | 2022-06-24

REPOSITORIES: Pride

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			Action	DRS
	Eclipse_Core_2020Nov23_Tschum_TMT.mgf	Mgf
	Eclipse_Core_2020Nov23_Tschum_TMT.mzML	Mzml
	Eclipse_Core_2020Nov23_Tschum_TMT.mzid.gz	Mzid
	Eclipse_Core_2020Nov23_Tschum_TMT.raw	Raw
	checksum.txt	Txt

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Project description:Background: Palmdelphin (PALMD), a protein of unknown function, belongs to the family of Paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms (SNPs) in the PALMD locus, which reduce its expression, are strong risk factors for development of calcific aortic valve stenosis (CAVS) and for severity of the disease. Methods: Public database screening and immunodetection of PALMD showed dominant expression in endothelial cells (ECs). Mass spectrometry allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in siRNA silenced EC cultures, in knockout mice and in human valve samples and EC cultures from patients. RNA sequencing on siRNA silenced cells and transcript arrays on valve samples from a Swedish SNP rs7543130 patient cohort informed about gene regulatory changes. Results: ECs express the cytoplasmic PALMD-KKVI splice variant, which was shown to exist in complex with RAN GTPase activating protein1 (RANGAP1). RANGAP1 regulates the activity of the GTPase RAN and thereby, nucleocytoplasmic shuttling via Exportin1 (XPO1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, providing a mechanism for the gene regulatory changes established in PALMD-deficiency. Gene ontology analysis showed enrichment of actin and RHO GTPAse related terms in PALMD-silenced ECs. In accordance, PALMD-silenced ECs showed multiple changes in adhesive and migratory properties and cells were softer. In particular, PALMD-deficient ECs failed to form a perinuclear actin cap when exposed to flow. The perinuclear actin cap is known to provide protection against mechanical stress. Lack of the actin cap correlated with tilting of the nuclear long axis relative to the cell body, which was established in PALMD-deficient ECs, mice and patient valve samples. The loss of the actin cap may contribute to further dysregulated gene transcription eventually promoting calcific precipitations and inflammation in CAVS. Conclusions: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex changes the subcellular localization of essential nucleocytoplasmic shuttling components, thereby instigating the loss of actin-dependent nuclear resilience, which in turn promotes further deterioration of gene expression patterns predisposing to the establishment of CAVS.

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Loss of ERG in human lung endothelial cells leads to release of pro-inflammatory and pro-fibrotic factors.

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