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Phosphoproteomic profiling of lipopolysaccharide stimulated toll-like receptor pathways in immortalized mouse macrophages

ABSTRACT: Toll-like receptors (TLRs) are present on the membranes of immune cells such as dendritic cells, macrophages, and natural killer cells. It is essential for the recognition of microbiological components. They trigger both innate and adaptive immune responses against pathogenic microorganisms. Among TLRs, TLR4 is one of the well-studied receptors that recognizes lipopolysaccharide (LPS) and initiates the TRIF- and MyD88-dependent signaling cascades. Regulators in TLR4 signaling, including protein kinases, play a key role in innate and adaptive immune responses. Protein phosphorylation on serine, threonine, and tyrosine is essential regulatory post-translational modification (PTM) because it serves as a signal transduction mechanism enabling the cells to connect extracellular signals to the regulation of multiple biological processes. Although phosphoproteins in TLR4 signaling pathways have been extensively investigated by mass spectrometry using data-dependent acquisition (DDA), the phosphoproteins that initiate TLR4 signaling cascades remain poorly understood due to technical limitations. We utilized data-independent acquisition (DIA) to get comprehensive information on phosphorylation inside LPS-stimulated TLR4 signaling pathways in macrophages.

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ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Macrophage

SUBMITTER: Aleksandra Nita-Lazar

LAB HEAD: Aleksandra Nita-Lazar

PROVIDER: PXD064064 | Pride | 2025-09-30

REPOSITORIES: Pride

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Project description:Melanoma cells from three surgical specimens of nodular melanoma were grown as anchorage-independent melanospheres in stem cell bFGF(+)EGF(+) medium and as adherent monolayer cultures in the presence of serum. RNA from melanospheres (DMBC2s, DMBC8s, DMBC10s) and their adherent monolayer counterparts (DMBC2a, DMBC8a, DMBC10a) were hybridized to the dual-color Agilent human genome array G4450A. A transcriptome profile was generated to explore the molecules governing phenotypes of melanospheres and monolayers. We demonstrated that melanospheres contained more pigmented cells which was consistent with higher expression of MITF and MITF-dependent genes responsible for differentiation, including TYR, TYRP1, MLANA and DCT. Expression of MITF-dependent BIRC7, BCL2 and BCL2A1 was reduced in monolayers, thus limiting the anti-apoptotic protection. The enhanced activity of MITF shown as the elevated expression of 74 MITF-dependent genes in melanospheres, identified MITF as a central regulator of this phenotype. Importantly, our study revealed that MITF and MITF-dependent genes were expressed in melanospheres at similar levels as in original tumors, much higher than in monolayers. The reduced MITF level in monolayers might be partially explained by suppression of the Wnt/beta-catenin pathway. Differential expression of Wnt/beta-catenin pathway components and target genes in melanospheres and monolayers points to strong influence of the microenvironment on the functional outcome of Wnt/beta-catenin pathway in melanoma, including differentiation, survival, proliferation and invasiveness. Silencing of the Wnt inhibitor DKK1 in monolayers increased the percentage of clonogenic cells. In summary, melanospheres more accurately mirrored the morphology, heterogeneity and gene expression profiles of original tumors than monolayers. Our study demonstrates the feasibility of utilizing melanospheres to unravel the molecular pathways sustaining melanoma heterogeneity and potentially to select compounds with anticancer activities.

			Action	DRS
	1st_DDA_phospho_0_1.raw	Raw
	1st_DDA_phospho_0_2.raw	Raw
	1st_DDA_phospho_0_3.raw	Raw
	1st_DDA_phospho_0_4.raw	Raw
	1st_DDA_phospho_0_5.raw	Raw

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Phosphoproteomic profiling of lipopolysaccharide stimulated toll-like receptor pathways in immortalized mouse macrophages

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