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Role of Calprotectin as a Modulator of the IL27-Mediated Proinflammatory Effect on Endothelial Cells.

Dorosz SA, Ginolhac A, Kähne T, Naumann M, Sauter T, Salsmann A, Bueb JL - Mediators Inflamm. (2015)

Bottom Line: However, regarding cytokine IL27, the controversial current knowledge about its inflammatory role and the involved regulatory elements requires clarification.A qPCR-based screening demonstrated high IL27-mediated gene expression of IL7, IL15, CXCL10, and CXCL11.Furthermore, we showed evidence for STAT1 involvement in this process.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Research Unit, University of Luxembourg, 162a Avenue de la Faïencerie, 1511 Luxembourg City, Luxembourg.

ABSTRACT
An underlying endothelial dysfunction plays a fundamental role in the pathogenesis of cardiovascular events and is the central feature of atherosclerosis. The protein-based communication between leukocytes and inflamed endothelial cells leading to diapedesis has been largely investigated and several key players such as IL6, TNFα, or the damage associated molecular pattern molecule (DAMP) calprotectin are now well identified. However, regarding cytokine IL27, the controversial current knowledge about its inflammatory role and the involved regulatory elements requires clarification. Therefore, we examined the inflammatory impact of IL27 on primary endothelial cells and the potentially modulatory effect of calprotectin on both transcriptome and proteome levels. A qPCR-based screening demonstrated high IL27-mediated gene expression of IL7, IL15, CXCL10, and CXCL11. Calprotectin time-dependent downregulatory effects were observed on IL27-induced IL15 and CXCL10 gene expression. A mass spectrometry-based approach of IL27 ± calprotectin cell stimulation enlightened a calprotectin modulatory role in the expression of 28 proteins, mostly involved in the mechanism of leukocyte transmigration. Furthermore, we showed evidence for STAT1 involvement in this process. Our findings provide new evidence about the IL27-dependent proinflammatory signaling which may be under the control of calprotectin and highlight the need for further investigations on molecules which might have antiatherosclerotic functions.

No MeSH data available.


Related in: MedlinePlus

Label-free quantified proteins represented as relative protein levels. The calprotectin significant modulatory effects on mutual proteins in HUVECs stimulated with IL27 (30 ng/mL) ± calprotectin (1 μg/mL) are shown for 6, 12, and 24 h (n = 9). Significance is indicated by q-value: ∗q < 0.15, ∗∗q < 0.10, and ∗∗∗q < 0.01. Significance is indicated for each stimulation by stars and between IL27 and IL27 + calprotectin stimulation by stars with a line. AHSG, alpha-2-HS-glycoprotein; ARFGAP1, ADP-ribosylation factor GTPase-activating protein 1; BASP1, brain acid soluble protein 1; CANX, calnexin; COPE, coatomer subunit epsilon; FERMT3, fermitin family homolog 3; GBP1, interferon-induced guanylate binding protein 1; GNB2, guanine nucleotide-binding protein, subunit beta-2; GPI, glucose-6-phosphate isomerase; GSTP1, glutathione S-transferase P; H2B1C, histone H2B type 1; HMOX2, heme oxygenase 2; KP2, importin subunit alpha-1; NID1, nidogen-1; NMT1, glycylpeptide N-tetradecanoyl transferase 1; PDLIM5, PDZ and LIM domain protein 5; PEBP1, phosphatidylethanolamine-binding protein 1; PECAM1, platelet/endothelial cell adhesion molecule; PSMA7, proteasome subunit alpha type-7; RPL30, 60S ribosomal protein L30; S100A9, S100 calcium binding protein A9; SRSF7, serine/arginine-rich splicing factor 7; STAT, signal transducers and activators of transcription; TPM1, tropomyosin 1; WARS, tryptophan tRNA ligase, cytoplasmic; YBX3, Y-box-binding protein 3.
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fig5: Label-free quantified proteins represented as relative protein levels. The calprotectin significant modulatory effects on mutual proteins in HUVECs stimulated with IL27 (30 ng/mL) ± calprotectin (1 μg/mL) are shown for 6, 12, and 24 h (n = 9). Significance is indicated by q-value: ∗q < 0.15, ∗∗q < 0.10, and ∗∗∗q < 0.01. Significance is indicated for each stimulation by stars and between IL27 and IL27 + calprotectin stimulation by stars with a line. AHSG, alpha-2-HS-glycoprotein; ARFGAP1, ADP-ribosylation factor GTPase-activating protein 1; BASP1, brain acid soluble protein 1; CANX, calnexin; COPE, coatomer subunit epsilon; FERMT3, fermitin family homolog 3; GBP1, interferon-induced guanylate binding protein 1; GNB2, guanine nucleotide-binding protein, subunit beta-2; GPI, glucose-6-phosphate isomerase; GSTP1, glutathione S-transferase P; H2B1C, histone H2B type 1; HMOX2, heme oxygenase 2; KP2, importin subunit alpha-1; NID1, nidogen-1; NMT1, glycylpeptide N-tetradecanoyl transferase 1; PDLIM5, PDZ and LIM domain protein 5; PEBP1, phosphatidylethanolamine-binding protein 1; PECAM1, platelet/endothelial cell adhesion molecule; PSMA7, proteasome subunit alpha type-7; RPL30, 60S ribosomal protein L30; S100A9, S100 calcium binding protein A9; SRSF7, serine/arginine-rich splicing factor 7; STAT, signal transducers and activators of transcription; TPM1, tropomyosin 1; WARS, tryptophan tRNA ligase, cytoplasmic; YBX3, Y-box-binding protein 3.

Mentions: To investigate the IL27-induced protein expression in HUVECs and to examine the potential regulatory role of calprotectin in this process, we used a label-free MS-based proteomic approach. HUVECs were stimulated with IL27 (30 ng/mL) ± calprotectin (1 μg/mL) for 6, 12, and 24 h. In total, the number of unique proteins detected for each time point was 1061 at 6 h, 994 at 12 h, and 886 at 24 h (Table 1). As shown in Figure S4 by a representation of q-values over p values derived from proteome data, a significance cut-off threshold corresponding to q-value <0.15 could be chosen. The data indicated the following unique differentially regulated proteins detected for each time step: 11 at 6 h, 143 at 12 h, and 193 at 24 h (Table 1). Furthermore, a time-dependent increase for significant expressed proteins for IL27- and calprotectin-stimulated HUVECs was observed. Interestingly, the costimulation revealed already highest protein expression number at 12 h suggesting a calprotectin modulatory role in IL27-mediated protein expression (Table 1). As shown in Figure 5, calprotectin time-dependent modulatory effects on protein expression were observed on 28 unique proteins. Figure 6 summarizes qualitatively the modulatory role of calprotectin in IL27-mediated protein expression. While calprotectin potentialized the IL27-dependent expression of NMT1 (12 h) and STAT1 (24 h) and upregulated STAT3 (6 h), it decreased the expression of GBP1 and WARS at 24 h and downregulated STAT1 at 6 h. Furthermore, calprotectin prevented the IL27-mediated downregulation of NID1 and TPM1, as well as the upregulation of 14 proteins: ARFGAP1, BASP1, CANX, COPE, GNB2, GP1, GSTP1, HMOX2, PKM, PEBP1, PDLIM5, RPL30, PSMA7, and YBX3. The results also showed that the costimulation of HUVECs with IL27 + calprotectin induced the downregulation of 5 proteins (FERMT3, H2B1C, KP2, PECAM1, and SRSF7) whereas 2 proteins (AHSG and S100A9) were upregulated. However, none of the proteins corresponding to the genes induced by IL27 and regulated by calprotectin were differentially expressed. These findings emphasize at the protein level the role played by calprotectin in the regulation of IL27-dependent protein expression.


Role of Calprotectin as a Modulator of the IL27-Mediated Proinflammatory Effect on Endothelial Cells.

Dorosz SA, Ginolhac A, Kähne T, Naumann M, Sauter T, Salsmann A, Bueb JL - Mediators Inflamm. (2015)

Label-free quantified proteins represented as relative protein levels. The calprotectin significant modulatory effects on mutual proteins in HUVECs stimulated with IL27 (30 ng/mL) ± calprotectin (1 μg/mL) are shown for 6, 12, and 24 h (n = 9). Significance is indicated by q-value: ∗q < 0.15, ∗∗q < 0.10, and ∗∗∗q < 0.01. Significance is indicated for each stimulation by stars and between IL27 and IL27 + calprotectin stimulation by stars with a line. AHSG, alpha-2-HS-glycoprotein; ARFGAP1, ADP-ribosylation factor GTPase-activating protein 1; BASP1, brain acid soluble protein 1; CANX, calnexin; COPE, coatomer subunit epsilon; FERMT3, fermitin family homolog 3; GBP1, interferon-induced guanylate binding protein 1; GNB2, guanine nucleotide-binding protein, subunit beta-2; GPI, glucose-6-phosphate isomerase; GSTP1, glutathione S-transferase P; H2B1C, histone H2B type 1; HMOX2, heme oxygenase 2; KP2, importin subunit alpha-1; NID1, nidogen-1; NMT1, glycylpeptide N-tetradecanoyl transferase 1; PDLIM5, PDZ and LIM domain protein 5; PEBP1, phosphatidylethanolamine-binding protein 1; PECAM1, platelet/endothelial cell adhesion molecule; PSMA7, proteasome subunit alpha type-7; RPL30, 60S ribosomal protein L30; S100A9, S100 calcium binding protein A9; SRSF7, serine/arginine-rich splicing factor 7; STAT, signal transducers and activators of transcription; TPM1, tropomyosin 1; WARS, tryptophan tRNA ligase, cytoplasmic; YBX3, Y-box-binding protein 3.
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Related In: Results  -  Collection

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fig5: Label-free quantified proteins represented as relative protein levels. The calprotectin significant modulatory effects on mutual proteins in HUVECs stimulated with IL27 (30 ng/mL) ± calprotectin (1 μg/mL) are shown for 6, 12, and 24 h (n = 9). Significance is indicated by q-value: ∗q < 0.15, ∗∗q < 0.10, and ∗∗∗q < 0.01. Significance is indicated for each stimulation by stars and between IL27 and IL27 + calprotectin stimulation by stars with a line. AHSG, alpha-2-HS-glycoprotein; ARFGAP1, ADP-ribosylation factor GTPase-activating protein 1; BASP1, brain acid soluble protein 1; CANX, calnexin; COPE, coatomer subunit epsilon; FERMT3, fermitin family homolog 3; GBP1, interferon-induced guanylate binding protein 1; GNB2, guanine nucleotide-binding protein, subunit beta-2; GPI, glucose-6-phosphate isomerase; GSTP1, glutathione S-transferase P; H2B1C, histone H2B type 1; HMOX2, heme oxygenase 2; KP2, importin subunit alpha-1; NID1, nidogen-1; NMT1, glycylpeptide N-tetradecanoyl transferase 1; PDLIM5, PDZ and LIM domain protein 5; PEBP1, phosphatidylethanolamine-binding protein 1; PECAM1, platelet/endothelial cell adhesion molecule; PSMA7, proteasome subunit alpha type-7; RPL30, 60S ribosomal protein L30; S100A9, S100 calcium binding protein A9; SRSF7, serine/arginine-rich splicing factor 7; STAT, signal transducers and activators of transcription; TPM1, tropomyosin 1; WARS, tryptophan tRNA ligase, cytoplasmic; YBX3, Y-box-binding protein 3.
Mentions: To investigate the IL27-induced protein expression in HUVECs and to examine the potential regulatory role of calprotectin in this process, we used a label-free MS-based proteomic approach. HUVECs were stimulated with IL27 (30 ng/mL) ± calprotectin (1 μg/mL) for 6, 12, and 24 h. In total, the number of unique proteins detected for each time point was 1061 at 6 h, 994 at 12 h, and 886 at 24 h (Table 1). As shown in Figure S4 by a representation of q-values over p values derived from proteome data, a significance cut-off threshold corresponding to q-value <0.15 could be chosen. The data indicated the following unique differentially regulated proteins detected for each time step: 11 at 6 h, 143 at 12 h, and 193 at 24 h (Table 1). Furthermore, a time-dependent increase for significant expressed proteins for IL27- and calprotectin-stimulated HUVECs was observed. Interestingly, the costimulation revealed already highest protein expression number at 12 h suggesting a calprotectin modulatory role in IL27-mediated protein expression (Table 1). As shown in Figure 5, calprotectin time-dependent modulatory effects on protein expression were observed on 28 unique proteins. Figure 6 summarizes qualitatively the modulatory role of calprotectin in IL27-mediated protein expression. While calprotectin potentialized the IL27-dependent expression of NMT1 (12 h) and STAT1 (24 h) and upregulated STAT3 (6 h), it decreased the expression of GBP1 and WARS at 24 h and downregulated STAT1 at 6 h. Furthermore, calprotectin prevented the IL27-mediated downregulation of NID1 and TPM1, as well as the upregulation of 14 proteins: ARFGAP1, BASP1, CANX, COPE, GNB2, GP1, GSTP1, HMOX2, PKM, PEBP1, PDLIM5, RPL30, PSMA7, and YBX3. The results also showed that the costimulation of HUVECs with IL27 + calprotectin induced the downregulation of 5 proteins (FERMT3, H2B1C, KP2, PECAM1, and SRSF7) whereas 2 proteins (AHSG and S100A9) were upregulated. However, none of the proteins corresponding to the genes induced by IL27 and regulated by calprotectin were differentially expressed. These findings emphasize at the protein level the role played by calprotectin in the regulation of IL27-dependent protein expression.

Bottom Line: However, regarding cytokine IL27, the controversial current knowledge about its inflammatory role and the involved regulatory elements requires clarification.A qPCR-based screening demonstrated high IL27-mediated gene expression of IL7, IL15, CXCL10, and CXCL11.Furthermore, we showed evidence for STAT1 involvement in this process.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Research Unit, University of Luxembourg, 162a Avenue de la Faïencerie, 1511 Luxembourg City, Luxembourg.

ABSTRACT
An underlying endothelial dysfunction plays a fundamental role in the pathogenesis of cardiovascular events and is the central feature of atherosclerosis. The protein-based communication between leukocytes and inflamed endothelial cells leading to diapedesis has been largely investigated and several key players such as IL6, TNFα, or the damage associated molecular pattern molecule (DAMP) calprotectin are now well identified. However, regarding cytokine IL27, the controversial current knowledge about its inflammatory role and the involved regulatory elements requires clarification. Therefore, we examined the inflammatory impact of IL27 on primary endothelial cells and the potentially modulatory effect of calprotectin on both transcriptome and proteome levels. A qPCR-based screening demonstrated high IL27-mediated gene expression of IL7, IL15, CXCL10, and CXCL11. Calprotectin time-dependent downregulatory effects were observed on IL27-induced IL15 and CXCL10 gene expression. A mass spectrometry-based approach of IL27 ± calprotectin cell stimulation enlightened a calprotectin modulatory role in the expression of 28 proteins, mostly involved in the mechanism of leukocyte transmigration. Furthermore, we showed evidence for STAT1 involvement in this process. Our findings provide new evidence about the IL27-dependent proinflammatory signaling which may be under the control of calprotectin and highlight the need for further investigations on molecules which might have antiatherosclerotic functions.

No MeSH data available.


Related in: MedlinePlus