Transcription and translation of human F11R gene are required for an initial step of atherogenesis induced by inflammatory cytokines

Abstract

Background: The F11 Receptor (F11R; aka JAM-A, JAM-1) is a cell adhesion protein present constitutively on the membrane surface of circulating platelets and within tight junctions of endothelial cells (ECs). Previous reports demonstrated that exposure of ECs to pro-inflammatory cytokines causes insertion of F11R molecules into the luminal surface of ECs, ensuing with homologous interactions between F11R molecules of platelets and ECs, and a resultant adhesion of platelets to the inflamed ECs. The main new finding of the present report is that the first step in this chain of events is the de-novo transcription and translation of F11R molecules, induced in ECs by exposure to inflammatory cytokines.

Methods: The experimental approach utilized isolated, washed human platelet suspensions and cultured human venous endothelial cells (HUVEC) and human arterial endothelial cells (HAEC) exposed to the proinflammatory cytokines TNF-alpha and/or IFN-gamma, for examination of the ability of human platelets to adhere to the inflamed ECs thru the F11R. Our strategy was based on testing the effects of the following inhibitors on this activity: general mRNA synthesis inhibitors, inhibitors of the NF-kappaB and JAK/STAT pathways, and small interfering F11R-mRNA (siRNAs) to specifically silence the F11R gene.

Results: Treatment of inflamed ECs with the inhibitors actinomycin, parthenolide or with AG-480 resulted in complete blockade of F11R- mRNA expression, indicating the involvement of NF-kappaB and JAK/STAT pathways in this induction. Transfection of ECs with F11R siRNAs caused complete inhibition of the cytokine-induced upregulation of F11R mRNA and inhibition of detection of the newly- translated F11R molecules in cytokine-inflamed ECs. The functional consequence of the inhibition of F11R transcription and translation was the significant blockade of the adhesion of human platelets to inflamed ECs.

Conclusion: These results prove that de novo synthesis of F11R in ECs is required for the adhesion of platelets to inflamed ECs. Because platelet adhesion to an inflamed endothelium is crucial for plaque formation in non-denuded blood vessels, we conclude that the de-novo translation of F11R is a crucial early step in the initiation of atherogenesis, leading to atherosclerosis, heart attacks and stroke.

Figure 1

Expression of F11R mRNA in human aortic endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC) exposed to proinflammatory cytokines TNFα and/or IFNγ: time course. Real-time PCR was performed in cultured HAEC (top panels) treated for 0, 3, 6, 12, and 24 hrs with TNFα (100 u/mL) and/or IFNγ (200 u/mL), and in cultured HUVEC (bottom panels) treated for 0,4,8,12, and 24 hrs with TNFα (100 u/mL) and/or IFNγ (200 u/mL). Real-time PCR was performed three times in triplicate for each time point. Values represent the mean ± SEM. *P < 0.05 indicates the level of significance determined at a specific interval of time of cytokine- treatment of ECs in comparison to the zero time points.

Figure 2

The expression of F11R mRNA in human endothelial cells (ECs) exposed to proinflammatory cytokines TNFα and IFNγ: dose response. Endothelial cells, HAEC and HUVEC in culture, were treated with different concentrations of TNFα (0.5 to 1000 pM; 1 to 2000 units) and IFNγ (0.1 - 100 nM; 3.4 - 3448 units/ml) for 12 hrs at 37°C. Real-time PCR was performed three times in triplicate for each time point. Values represent the mean ± SEM. * P < 0.05. Significant differences in F11R mRNA observed at the indicated concentrations of cytokines in comparison to levels of F11R mRNA measured in the absence of cytokines.

Figure 3

De novo expression of F11R mRNA in inflamed endothelial cells: blockade of F11R mRNA expression in endothelial cells treated with TNFα and IFNγ by the RNA synthesis inhibitor, actinomycin. Confluent monolayers of HUVEC were maintained under Untreated conditions, or pretreated with the RNA synthesis inhibitor, actinomycin D (ActD) (5 μg/mL), in growth supplement-free media for 1 hr at 37°C. The response of HUVEC maintained in the presence of ActD alone is shown in histogram labeled ActD. The response of HUVEC treated with TNFα alone(100 u/ml) is shown in Figure 3a, and the response of HUVEC treated with IFNγ alone(200 u/ml) for 24 hrs is shown in Figure 3b. The response of HUVEC pretreated with ActD prior to 24 hr exposure to either TNFα (100 u/mL) or IFNγ (200 u/mL), is shown in the histograms labeled TNFα & ActD (see Figure 3a) or IFNγ & ActD (see Figure 3b). The F11R mRNA levels were measured by Real-Time PCR in triplicate for each condition. Values are the mean ± SEM. * P < 0.05 significant differences in F11R mRNA observed between cells exposed to TNFα or IFNγ alone vs ECs treated (or not treated) with ActD alone or ECs treated with ActD followed by their exposure to either TNFα or IFNγ.

Figure 4

Upregulation of F11R mRNA expression by TNFα and INFγ in endothelial cells: inhibition by the NF-kB blocker and JAK protein kinase inhibitor. Panels (a) and (b). Confluent monolayers of HUVEC were pretreated (or Untreated) for 1 hr at 37°C with the NF-kB inhibitor, parthenolide (50 μM, final concentration), added to growth supplement-free media. The proinflammatory cytokines, TNFα (100 u/mL) or IFNγ (200 u/ml), were added to the media, and the cells were incubated at 37°C for an additional 24 hrs (see TNFα & Parthenolide in Figure 4a, and IFNγ & Parthenolide in Figure 4b). The response of cells exposed only to TNFα alone (100 u/ml) is shown in the histogram displayed in Figure 4a, and the response of cells exposed only to IFNγ alone is shown in Figure 4b. The F11R mRNA levels were measured by Real-time PCR performed in triplicate for each condition. Values are the mean ± SEM. * P < 0.05 level of significance observed between ECs exposed to TNFα or IFNγ alone vs ECs not exposed to TNFα/INFγ or ECs previously treated with parthenolide followed by their exposure to cytokines. Figure 4c demonstrates the upregulation of F11R mRNA in endothelial cells by IFNγ and its inhibition by the JAK protein kinase inhibitor, AG-490. Confluent monolayers of HUVEC were either Untreated or treated with the JAK protein kinase inhibitor AG-490 (50 μM) alone (AG 490) added to growth supplement-free media and incubated for 1 hr at 37°C. The response of cells that were exposed to the cytokine IFNγ alone is depicted in the histogram IFNγ. The response of cells that were pretreated with AG 490 for 1 hr followed by their exposure to IFNγ (200 u/mL) for an additional 24 hrs is depicted in histogram labeled IFNγ & AG-490. The F11R mRNA levels were measured by Real-time PCR performed in three separate experiments, in triplicate, for each condition. Values are the mean ± SEM. * P < 0.05 significance differences in F11R mRNA in ECs exposed to IFNγ alone vs untreated ECs or ECs treated with AG-490 alone or ECs previously treated with AG-490 followed by their exposure to IFNγ

Figure 5

F11R protein expression in endothelial cells treated with TNFα and INFβ. (a). Immunoblotting: HAEC or HUVEC cells were treated with TNFα (100 u/mL), IFNγ (200 u/mL) or TNFα (100 u/mL) and IFNγ (200 u/mL) for 24 hrs. Collected media and cell lysates were examined for the presence of the F11R protein by SDS-PAGE (10%) followed by immunoblotting utilizing antibodies against F11R and tubulin (protein loading control, 50 kDa). (b). Quantitation of immunoblots - cell lysates. Enhanced expression of the F11R protein in cytokine-treated human aortic endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC). Quantitation of the F11R protein in cell lysates of the TNFα and/or IFNγ-treated HUVEC and HAEC, as detailed in the legend of Figure 5a. Immunoblots derived, following SDS-PAGE, were immunostained utilizing an F11R antibody. The level of the immunostained F11R protein band, of 37 kDa, was normalized to tubulin, the loading protein control, of 50 kDa. Values represent the mean ± SEM. * P < 0.05. (c). Quantitation of immunoblots - cell media. Quantitation of the F11R protein detected in the cell culture media of TNFα and/or IFNγ-treated HUVEC and HAEC (as detailed in the legend of Figure 5a), normalized to input volume. Values represent the mean ± SEM. * P < 0.05.

Figure 6

Expression of the F11R protein in inflamed endothelial cells: silencing of the F11R gene in HAEC and HUVEC using F11R siRNA. (a). Immunoblots demonstrate the detection of the F11R protein retained in cells (cell lysates) and released into the media of inflamed HAEC and HUVEC. Both aortic and umbilical vein endothelial cells were transfected with either the control, non-targeting siRNA or by the specific F11R targeting siRNA (as detailed in the Material and Methods section). Subsequently, the cells were treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs, followed by SDS-PAGE and immunoblotting utilizing F11R antibody (arrows point to F11R), and tubulin, as the protein loading control, of 50 kDa. Lanes 1 and 3 depict the F11R protein as detected in cytokine-treated HAEC or HUVEC transfected with the nontargeting siRNA. Lanes 2 and 4 depict the F11R protein as detected in cytokine-treated HAEC and HUVEC transfected with the specific targeting F11R siRNA.(b). Quantitation of immunoblots of the immunostained F11R protein, detected in the cell culture media of HAEC and HUVEC endothelial cells transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, followed by the exposure of transfected HAEC and HUVEC to a combination of the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. The values for F11R were normalized to tubulin levels by dividing the integrated density of the specific band by the integrative density of the tubulin band. ANOVA statistical analysis was performed on the normalized values. All values are the average of three immunoblots ± SEM. (c). Quantitation of the immunostained F11R protein within the cell lysates of HAEC and HUVEC transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, and further treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. F11R-immunostained protein bands were quantified by normalization to tubulin using image J. The F11R values were normalized to tubulin. ANOVA was performed on the normalized value (n = 3). Values depict the mean ± SEM, * p < 0.005.

Figure 7

Blockade of platelet adhesion to inflamed human aortic (HAEC) and human umbilical endothelial vein endothelial cells (HUVEC) by F11R siRNA: inhibition by silencing of the F11R gene. Transfection of HUVEC and HAEC was conducted by using either the non-targeting siRNA or the F11R targeting F11R siRNA, as detailed in the Material and Methods section. Following transfection, both HAEC and HUVEC were pretreated with a combination of cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. Afterwards, suspensions of either non-activated or collagen-activated platelets (as detailed in the Material and Methods section) were applied unto monolayers of the inflamed ECs, and the adhesion of platelets to the cytokine-treated ECs was monitored. The values represent the adjusted means ± SEM for the number of platelets bound to the ECs/per well from 5 separate experiments. * P < 0.05.