Protein kinase CK2 inhibition suppresses neointima formation via a proline-rich homeodomain-dependent mechanism

Abstract

Neointimal hyperplasia is a product of VSMC replication and consequent accumulation within the blood vessel wall. In this study, we determined whether inhibition of protein kinase CK2 and the resultant stabilisation of proline-rich homeodomain (PRH) could suppress VSMC proliferation. Both silencing and pharmacological inhibition of CK2 with K66 antagonised replication of isolated VSMCs. SiRNA-induced knockdown as well as ectopic overexpression of proline-rich homeodomain indicated that PRH disrupts cell cycle progression. Mutation of CK2 phosphorylation sites Ser¹⁶³ and Ser¹⁷⁷ within the PRH homeodomain enabled prolonged cell cycle arrest by PRH. Concomitant knockdown of PRH and inhibition of CK2 with K66 indicated that the anti-proliferative action of K66 required the presence of PRH. Both K66 and adenovirus-mediated gene transfer of S163C:S177C PRH impaired neointima formation in human saphenous vein organ cultures. Importantly, neither intervention had notable effects on cell cycle progression, cell survival or migration in cultured endothelial cells.

Keywords: Atherosclerosis; CK2; In-stent restenosis; PRH; Vascular smooth muscle; Vein graft.

Graphical abstract

Fig. 1

Inhibition and silencing of protein kinase CK2 reduced proliferation of cultured VSMCs. Quiesced rat aortic VSMCs were stimulated with either 20 ng/ml PDGF-BB and 20 ng/ml bFGF, or 500 ng/ml Wnt-4, or remained serum-starved, in the presence of a CK2 inhibitor (a, 1 μM TBB; b, 10 μM K66) or vehicle control (0.1% DMSO). Proliferation was assessed by quantification of BrdU incorporation after 24 h. * indicates significant difference compared to serum-starved – 0.1% DMSO, n = 3. † indicates significant difference compared to PDGF & bFGF – 0.1% DMSO, n = 3. ‡ indicates significant difference compared to Wnt-4 – 0.1% DMSO, n = 3. $ indicates significant difference compared to serum-starved – 0.1% DMSO and serum-starved - 1 μM TBB/10 μM K66, n = 3, p < 0.05, ANOVA and Student Newman Keuls post hoc test. c, Quiesced human saphenous vein VSMCs were stimulated with 20 ng/ml PDGF-BB and 20 ng/ml bFGF, or serum starved, for 24 h. Representative Western blot for CK2 substrates with a consensus pS/pTDXE motif, n = 3. Grey arrows indicate quantified bands. d, Quiesced human saphenous vein VSMCs were stimulated with 20 ng/ml PDGF-BB and 20 ng/ml bFGF in the presence of 10 μM K66 or 0.1% DMSO, and proliferation assessed by quantification of EdU incorporation after 24 h. * indicates significant difference compared to 0.1% DMSO, n = 5, p < 0.05, Students t-test. e, Quantification of migration using the scratch wound assay in VSMCs cultured in the presence of CK2 inhibitor (10 μM K66) or vehicle control (0.1% DMSO), n = 4. f, Rat aortic VSMCs were stimulated with 20 ng/ml PDGF-BB and 20 ng/ml bFGF in the presence of 10 μM K66 or 0.1% DMSO for 24 h and then immunocytochemistry for cleaved caspase-3 performed to quantify apoptosis. 200 ng/ml Fas ligand was used a positive control. * indicates significant difference compared to serum-starved control, n = 5, p < 0.05, ANOVA and Student Newman Keuls post hoc test. NS denotes not significant. g, Quiesced rat aortic VSMCs were subjected to nucleofection with either Allstars negative control, or CK2α & α’ siRNAs, then stimulated with both 20 ng/ml PDGF-BB and 20 ng/ml bFGF. Proliferation was assessed via quantification of BrdU incorporation between 48 and 72 h post-introduction of siRNAs. * indicates significant difference compared to Allstars Negative Control, n = 3, p < 0.05, Students t-test. Western blots show validation of silencing of CK2α & α′ catalytic subunits, representative of n = 3.

Fig. 2

PRH inhibited VSMC proliferation. a, Quiescent rat aortic VSMCs were nucleofected with either eGFP, or eGFP and wild-type PRH expression vectors – co-transfection with eGFP enabled identification of cells expressing the transgene. Cells were stimulated with 10% FCS culture medium and proliferation assessed via quantification of BrdU incorporation between 24 and 48 h post-transfection. * indicates significant difference compared to eGFP, n = 7, p < 0.05, Students t-test. b, Serum-starved rat aortic VSMCs were subjected to nucleofection with either Allstars negative control or PRH siRNAs; cells were nucleofected twice, 72 h apart, to achieve significant depletion of PRH protein. Cells were stimulated with 20 ng/ml PDGF-BB and 20 ng/ml bFGF, and proliferation was assessed between 48 and 72 h post-transfection in the presence of either DMSO (vehicle control) or 10 μM K66. * indicates significant difference compared to Allstars Neg. Control - DMSO, † indicates significant difference compared to Allstars Neg. Control - 10 μM K66, n = 3, p < 0.05, ANOVA and Student Newman Keuls post hoc test. NS denotes not significant. Western blot shows validation of silencing of PRH, representative of n = 3. c, Quantification of apoptosis by immunofluorescence for cleaved caspase-3 48 h post-introduction of either eGFP, or eGFP and wild-type PRH expression vectors in rat aortic VSMCs. Fas ligand was used as a positive control. * indicates significant difference compared to eGFP and eGFP + wild-type PRH, n = 3, p < 0.05, ANOVA and Student Newman Keuls post hoc test. NS denotes not significant. d, Asynchronous rat aortic VSMCs over-expressing either wild-type PRH or S163C:S177C PRH were stimulated with 10% FCS culture medium, and proliferation quantified over a 96-h time course. * indicates significant difference compared to eGFP within the same time-frame, n = 3, p < 0.05, ANOVA and Student Newman Keuls post hoc test.

Fig. 3

Effect of inhibition of protein kinase CK2 and overexpression of PRH on proliferation and apoptosis of HSaVECs. HSaVECs were cultured in endothelial cell growth medium. a, Quantification of proliferation by fluorescent labelling of incorporated EdU in HSaVECs treated with CK2 inhibitor (10 μM K66) or vehicle control (0.1% DMSO) for 24 h. * indicates significant difference compared to 0.1% DMSO, n = 3, p < 0.05, Students t-test. b, Quantification of apoptosis by immunocytochemistry for cleaved caspase-3 in HSaVECs treated with 10 μM K66 or 0.1% DMSO for 24 h. Fas ligand was used as a positive control. * indicates significant difference compared to 0.1% DMSO and 10 μM K66, n = 3, p < 0.05, ANOVA and Student Newman Keuls post hoc test. NS denotes not significant. c, Migration was quantified by scratch wound assay in HSaVECs treated with 10 μM K66 or 0.1% DMSO for 24 h. NS denotes not significant, n = 3. Quantification of proliferation by fluorescent labelling of incorporated EdU (d), apoptosis by immunocytochemistry for cleaved caspase-3 (e) or migration by scratch wound assay (f) in HSaVECs overexpressing PRH, n = 3. NS denotes not significant, Students t-test.

Fig. 4

Enhanced CK2 activity as a result of neointima formation. Segments of human saphenous vein were subjected to organ for 0 or 14 days, n = 6, and subjected to immunofluorescence for phosphorylated CK2 substrates. a, vein segment at day 0 incubated with anti-phosphorylated CK2 substrate antibody, b, segment after 14 days of cultured incubated with anti-phosphorylated CK2 substrate antibody. c, segment after 14 days of cultured incubated with non-immune rabbit IgG (negative control). Phosphorylated CK2 protein (green) and nuclei are blue (DAPI). Scale bars represents 10 μm and apply to all panels. Dashed lines indicate intimal:medial boundaries. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Treatment of human saphenous organ cultures with 10 μM K66 retarded neointima formation. Segments of human saphenous vein were subjected to organ culture in the presence of BrdU, and 10 μM K66 or vehicle control (0.1% DMSO) for 14 days, n = 6. a, EVG-stained sections of human saphenous vein organ cultures. b, Immunohistochemistry for intimal (upper panels) and medial (lower panels) α-smooth muscle cell actin (brown). c, Proliferation detected by immunofluorescence for incorporated BrdU (green). Nuclei are blue (DAPI) in lower panels. Arrows indicate positive cells. Scale bars represents 10 μm and apply to all panels. Dashed lines indicate intimal:medial boundaries. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Accumulation of PRH and depletion of phospho-PRH in human saphenous vein organ cultures treated with 10 μM K66. Segments of human saphenous vein were subjected to organ culture in the presence of either 10 μM K66 or vehicle control (0.1% DMSO) for 14 days. a, Immunofluorescence for PRH protein (green). Nuclei are blue (DAPI) in lower panels. Arrows indicate positive cells. b, Immunohistochemistry for phospho-PRH protein (brown). Nuclei are-blue (haematoxylin). Scale bars represent 10 μm and apply to all panels. Dashed lines indicate intimal:medial boundaries. c, Quantification of PRH protein in whole tissue lysates by Western blotting; normalisation by β-actin. * indicates significant difference compared to 0.1% DMSO, n = 5, p < 0.05, one sample t-test. d, Representative Western blot for PRH protein in whole tissue lysates. e, Quiesced human saphenous vein VSMCs were stimulated with 20 ng/ml PDGF-BB and 20 ng/ml bFGF for 24 h in the presence of either 10 μM K66 or 0.1% DMSO. Representative Western blot for phospho-PRH protein. f, Quantification of phospho-PRH protein by Western blotting; normalisation by in-gel stain-free band. * indicates significant difference compared to 0.1% DMSO, n = 3, p < 0.05, one sample t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Adenovirus-mediated gene transfer of S163C:S177C PRH retarded neointima formation. Segments of human saphenous vein were subjected to organ culture in the presence of BrdU, with or without infection with Ad-β-galactosidase or Ad-c-myc-tagged-S163CS177C-PRH for 14 days, n = 5. a, EVG-stained sections of human saphenous vein organ cultures. b, Immunohistochemistry for c-myc-tagged protein (brown). c, Immunohistochemistry for α-smooth muscle cell actin (brown). d, Immunofluorescence for incorporated BrdU (green). Nuclei are blue (DAPI) in lower panels. Scale bars represents 10 μm and apply to all panels. Dashed lines indicate intimal:medial boundaries. Arrows indicate positive cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)