Identification of DKK-1 as a novel mediator of statin effects in human endothelial cells

Abstract

This study shows that DKK-1, a member of the Dickkopf family and a regulator of the Wnt pathways, represents a novel target of statins which, through the inhibition of HMG-CoA reductase and of non-steroidal isoprenoid intermediates, exert extra-beneficial effect in preventing atherosclerosis beyond their effect on the lipid profile. We found that atorvastatin downregulates DKK-1 protein (-88.3 ± 4.1%) and mRNA expression (-90 ± 4.2%) through the inhibition of Cdc42, Rho and Rac geranylgeranylated proteins. Further, a combined approach based on the integration of label-free quantitative mass spectrometry based-proteomics and gene silencing allowed us to demonstrate that DKK-1 itself mediates, at least in part, statin effects on human endothelial cells. Indeed, DKK-1 is responsible for the regulation of the 21% of the statin-modulated proteins, which include, among others, clusterin/apoJ, plasminogen activator inhibitor type 1 (PAI-1), myristoylated alanine-rich C-kinase substrate (MARCKS), and pentraxin 3 (PTX3). The Gene Ontology enrichment annotation revealed that DKK-1 is also a potential mediator of the extracellular matrix organization, platelet activation and response to wounding processes induced by statin. Finally, we found that plasma level of DKK-1 from cholesterol-fed rabbits treated with atorvastatin (2.5 mg/kg/day for 8 weeks) was lower (-42 ± 23%) than that of control animals. Thus, DKK-1 is not only a target of statin but it directly regulates the expression of molecules involved in a plethora of biological functions, thus expanding its role, which has been so far restricted mainly to cancer.

Figure 1

Atorvastatin reduces DKK-1 antigen release (a) and mRNA (b) in human endothelial cells. Data are expressed as mean ± SEM of 3 individual experiments performed in duplicate. AU, arbitrary units. *p < 0.001 vs control cells; ^#p < 0.05 vs control cells.

Figure 2

DKK-1 downregulation occurs via geranylgeranylation inhibition. DKK-1 antigen was analysed in HUVECs treated with atorvastatin alone and in combination with mevalonate (a), GGPP and FPP (b), or squalene (c), and in HUVECs treated with the prenyltransferases inhibitors, GGTI-286 and FTI-277 (d). Values are the mean ± SEM of 3 individual experiments. *p < 0.01 vs control cells; ^#p < 0.01 vs statin-treated cells.

Figure 3

DDK1 release is regulated by geranylgeranylated proteins. DKK-1 antigen was analysed in HUVECs treated with the Rho-associated protein kinase inhibitors, Y-27632 (a) and H-1152 (b), the Rac1/2 inhibitor, NSC23766 (c), and the Cdc42 inhibitor, ML-141 (d). Values are the mean ± SEM of 3 individual experiments. *p < 0.05 vs control cells.

Figure 4

DKK-1 gene knockdown reduces DKK-1 in HUVECs. HUVECs were transfected with negative siRNA (siNEG) or with DKK-1 siRNA (siDKK-1) and then analysed for DKK-1 antigen release (a) and DKK-1 mRNA levels (b). Values are the mean ± SEM of 3 individual experiments. *p < 0.01 vs. control siRNA-treated cells. (c) Comparison of the effects of DKK-1 silencing and statin treatment on endothelial cell secretome using a Venn diagram generated with the differentially abundant proteins.

Figure 5

Validation of proteomics results regarding PAI-1 on endothelial cells treated with atorvastatin (Atorva) or by DKK-1 silencing as described in the Material and methods section (n = 3). Effect on PAI-1 mRNA levels exerted by siDKK-1 (a) or atorvastatin (b). PAI-1 was evaluated at antigen levels by ELISA in cells treated with siDKK-1 (c), atorvastatin (d) or recombinant DKK-1 (e). The data are expressed as ng/ml of PAI-1. Values are the mean ± SEM of 3 individual experiments.*p < 0.05 vs siNEG or control cells.

Figure 6

Validation of proteomics results regarding MMP-2 and MARCKS on endothelial cells treated with atorvastatin or by DKK-1 silencing. Effect on MMP-2 and MARCKS mRNA levels exerted by siDKK-1 (a,c) or atorvastatin (b,d). Values are the mean ± SEM of 3 individual experiments.*p < 0.05 vs siNEG or control cells.

Figure 7

Validation of proteomics results regarding clusterin (CLU) and PTX3 on endothelial cells treated with atorvastatin or by DKK-1 silencing. (a) Effect of treatment with atorvastatin (a) and siDKK-1 on CLU antigen release. (c) Effect on PTX3 antigen exerted by siDKK-1. Values are the mean ± SEM of 3 individual experiments. *p < 0.05 vs siNEG-treated cells.

Figure 8

Gene ontology analysis of secreted proteins modulated by DKK-1 silencing in the cellular component and molecular function categories visualised with STRING. (a) Within the cellular component category the GO term related to Extracellular region resulted overrepresented (purple circles). (b) In the molecular function category the GO term protein binding was associated with the highest number of proteins (red circles). Detailed results are reported in Supplementary Table S3.

Figure 9

Biological processes of secreted proteins modulated by DKK-1 silencing visualised with STRING. Considering the biological processes category the enriched terms were response to wounding (green circles), extracellular matrix organization (red circles) and platelet activation (blue circles). Detailed results are reported in Supplementary Table S3.