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. 2024 Jul 31;25(15):8363.
doi: 10.3390/ijms25158363.

Functional Insights into the Sphingolipids C1P, S1P, and SPC in Human Fibroblast-like Synoviocytes by Proteomic Analysis

Thomas Timm  1 , Christiane Hild  2 , Gerhard Liebisch  3 , Markus Rickert  2 , Guenter Lochnit  1 , Juergen Steinmeyer  2
Affiliations

Functional Insights into the Sphingolipids C1P, S1P, and SPC in Human Fibroblast-like Synoviocytes by Proteomic Analysis

Thomas Timm et al. Int J Mol Sci. .

Abstract

The (patho)physiological function of the sphingolipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and sphingosylphosphorylcholine (SPC) in articular joints during osteoarthritis (OA) is largely unknown. Therefore, we investigated the influence of these lipids on protein expression by fibroblast-like synoviocytes (FLSs) from OA knees. Cultured human FLSs (n = 7) were treated with 1 of 3 lipid species-C1P, S1P, or SPC-IL-1β, or with vehicle. The expression of individual proteins was determined by tandem mass tag peptide labeling followed by high-resolution electrospray ionization (ESI) mass spectrometry after liquid chromatographic separation (LC-MS/MS/MS). The mRNA levels of selected proteins were analyzed using RT-PCR. The 3sphingolipids were quantified in the SF of 18 OA patients using LC-MS/MS. A total of 4930 proteins were determined using multiplex MS, of which 136, 9, 1, and 0 were regulated both reproducibly and significantly by IL-1β, C1P, S1P, and SPC, respectively. In the presence of IL-1ß, all 3 sphingolipids exerted ancillary effects. Only low SF levels of C1P and SPC were found. In conclusion, the 3 lipid species regulated proteins that have not been described in OA. Our results indicate that charged multivesicular body protein 1b, metal cation symporter ZIP14, glutamine-fructose-6-P transaminase, metallothionein-1F and -2A, ferritin, and prosaposin are particularly interesting proteins due to their potential to affect inflammatory, anabolic, catabolic, and apoptotic mechanisms.

Keywords: ceramide-1-phosphate; functions; inflammation; lysosphingomyelin; mass spectrometry; osteoarthritis; proteomics; sphingolipid; sphingosine-1-phosphate; sphingosylphosphorylcholine.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Venn diagram of proteins that are reproducibly regulated by (A) C1P (red), C1P in the presence of IL-1ß (yellow), and IL-1ß alone (green); (B) S1P (blue), S1P in the presence of IL-1ß (orange), and IL-1ß alone (green); (C) SPC in the presence of IL-1ß (violet) and IL-1ß alone (green); and (D) IL-1ß in the presence of C1P (yellow), S1P (orange), or SPC (violet). The number of proteins that can be reproducibly regulated is shown. In Tables S1 and S2, the AR of each protein is listed together with the results of the statistical analysis.
Figure 2
Figure 2
C1P and S1P reproducibly and significantly regulate the level of proteins in FLSs. Protein levels of (A) stromelysin, charged multivesicular body protein 1b, metal cation symporter ZIP14; (B) cPLA2, cytochrome c oxidase subunit 1, SOD; and (C) long-chain-fatty-acid–CoA ligase 4, ICAM-1, and glutamine fructose-6-Ptransaminase were quantified by MS in duplicate in the 7 biological replicates. The dot plots represent the data obtained from the resulting 14 replicates and illustrate the x-fold abundance of the proteins in the treated FLS cells in comparison to that of only vehicle-treated controls (which are normalized to 1 and shown as a dotted line). The mean value ± SD is represented by lines within each figure (n = 7). * 0.05 ≥ p > 0.01; ** 0.01 ≥ p > 0.001; *** p ≥ 0.001.
Figure 3
Figure 3
The biological processes of FLS being altered by C1P. The 9 proteins were reproducibly upregulated by more than 1.2-fold by C1P in FLS during 48 h of treatment, and Table S1 provides further data on these proteins. The Go Slim categories for proteins were generated by Proteome Discoverer 2.5 software using the Gene Ontology (GO) database.
Figure 4
Figure 4
The molecular functions of FLS being altered by C1P. The 9 proteins were reproducibly upregulated by more than 1.2-fold by C1P in FLS during 48 h of treatment, and Table S1 provides further data on these proteins. The Go Slim categories for proteins were generated by Proteome Discoverer 2.5 software using the Gene Ontology (GO) database.
Figure 5
Figure 5
The cellular localization of 9 proteins being regulated by C1P. The 9 proteins were reproducibly upregulated by more than 1.2-fold by C1P in FLS during 48 h of treatment, and Table S1 provides further data on these proteins. The Go Slim categories for proteins were generated by Proteome Discoverer 2.5 software using the Gene Ontology (GO) database.
Figure 6
Figure 6
Levels of C1P 16:0 ad SPC 18:1;O2 in knee SF of patients with early-stage or late-stage knee OA. Sphingolipids were quantified by LC-MS/MS in extracts of SF obtained from 8 patients with eOA (black circle) and 10 patients with lOA (blue open circle). Data presented indicate mean ± SD of lipid concentration in SF (N = 8 or 10).
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