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Integrative phosphoproteomic analysis reveal hemostatic-endothelial signaling interplay

ABSTRACT: The vascular endothelial cell (EC) monolayer plays a crucial part in maintaining hemostasis. An extensive array of G protein-coupled receptors (GPCRs) allows ECs to dynamically act on key hemostatic stimuli such as thrombin and histamine. The impact of these individual stimuli on EC signal transduction has been the subject of various studies, but insight into discordant and concordant EC signaling between different GPCRs remain limited. Using mass spectrometry- based phosphoproteomics, we delineated the unbiased signaling response for histamine stimulation in the endothelium and observed highly similar responses between different protease activated receptors (PAR1-4). Finally, we compare both histamine and PAR agonists, indicating uniquely activated proteins between both stimuli, but a general overlapping activation of cell-junction and actin cytoskeletal proteins. Thus, we provide an integrative phosphoproteomic analysis of histamine and PAR agonist in the endothelium that highlights the endothelial response programs that are at the bases of regulating hemostasis.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Endothelial Colony Forming Cell, Endothelial Cell

SUBMITTER: Stijn Groten

LAB HEAD: Arie J Hoogendijk

PROVIDER: PXD052627 | Pride | 2024-11-27

REPOSITORIES: Pride

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Project description:Endometrial cancer (EC) is the most common cancer of female reproductive organs. Because some low-grade ECs might also experience tumor recurrence after surgery and a worse prognosis, the study of alterations related to EC pathogenesis of the disease might help to get insights into underlying mechanisms involved in EC development and metastasis and identify novel markers associated to the disease. Here, low C1GALT1 protein expression levels were associated to a more aggressive phenotype of EC. Then, we aimed at investigating the role of C1GALT1 in EC progression by quantitative proteomics. ECC-1 cells were used as endometrioid EC model, and the effect of C1GALT1 depletion was analyzed using C1GALT1 specific short hairpin RNAs (shRNA) in comparison to SCRAMBLE shRNA. SILAC and mass spectrometry analysis were performed to identify and quantify dysregulated proteins associated with C1GALT1 depletion in the cell extract and secretome proteome of shC1GALT1 and SCRAMBLE ECC-1 cells. Out of 5208 proteins identified and quantified by LC-MS/MS, 76 and 143 proteins showed dysregulation (fold-change ≥1.5 or ≤0.67) in shC1GALT1 ECC-1 cells’ extracts and secretome, respectively. Nine dysregulated proteins were selected for validation by orthogonal techniques, confirming their dysregulation upon C1GALT1 depletion. Bioinformatics analyses pointed out to an increase in pathways and dysregulated proteins that associated with more aggressive phenotype. This finding was corroborated by loss-of-function cell-based assays. A higher proliferation, invasion, migration, colony formation and angiogenesis capacity of C1GALT1 depleted cells was observed. Finally, the negative protein expression correlation found by proteomics between C1GALT1 and LGALS3 was confirmed by IHC in actual EC samples, suggesting C1GALT1 stably depleted ECC-1 cells mimic the aggressive phenotype of EC cells and might be useful for the identification and validation of potential markers in aggressive ECs.

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	1_BE_E1_N1_TiO2.raw	Raw
	1_BE_E1_N3_TiO1.raw	Raw
	1_BE_E1_N3_TiO2.raw	Raw
	1_BE_E2_N3_TiO1.raw	Raw

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Integrative phosphoproteomic analysis reveal hemostatic-endothelial signaling interplay

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