Page 1 - Y. Vascular biology

Expression and function of an IL-12R type in

the human aortic smooth muscle cells

Heesu Lee, Sang Won Kang
1 Department of Life Science, Ewha Womans University, Seoul 03760, Republic of Korea;

BACKGROUND AIM

VSMCs are known to play important roles throughout all stages IL12Rβ2 expression in immune cells is well known, but its
of atherosclerosis. Especially, VSMCs proliferation and migration expression in vascular cells is unclear. Because endothelial cells
are important for the progression of arteriosclerosis. Also, are almost denuded or detached in the balloon injury model,
VSMCs proliferation contributes to in-stent restenosis. Balloon VSMCs are considered a source of IL12Rβ2, but it is still
injury model is a model that mimics this phenomenon. In the uncertain. Therefore, this data shows whether VSMCs express
previous study, proteomics was performed in the balloon-injured IL12Rβ2 and how IL12Rβ2 expression level increases
rat carotid artery to identify signaling molecules organizing the METHOD
VSMC hyperplasia. The differential proteomics analysis revealed
the protein that the expressions are changed within 3 days post - Cell culture and Transfection
injury. One of which was Interleukin-12 receptor β2 (IL12Rβ2). - Quantitative real time PCR
IL12Rβ2 expression increased during 7 days post injury. IL12Rβ2 - Reverse transcription-PCR
is one of Interleukin-12 family receptor, and its ligand is - Immunoprecipitation(IP) and Immunoblotting (IB)
Interleukin 12 (IL-12) or Interleukin 35 (IL-35).
RESULTS 293T 293T
VCAM-1 mRNA expression (Fold chnage) 6 IL12Rb2 mRNA expression (Fold change) IB : Flag IB : Flag
pre-treated
si-IL12Rb2
Vector
Vector
HASMC 10 8 8 6 si-CON siRNA : control IL12Rb2 IL12Rb2 IL12Rb2 siRNA : control IL12Rb2 IL12Rb2 IL12Rb2
TNF-α (10ng/mL) : 0 3 5 10 30 (min) DNA : DNA :
IκB 4 4 IP : Flag IL12Rb2 IP : IL12Rb2 IL12Rb2
Tubulin 2 2 Flag Flag
0
TNF-α (10ng/mL) : 0 4 8 12 24 (hours) 0 IB IL12Rb2 IB IL12Rb2
IFN-γ : - + + Tubulin HASMC Tubulin HASMC
Figure 2. IL12Rβ2 mRNA expression in HASMCs is upregulated by IFN-γ.
VCAM-1 mRNA expression (Fold chnage) (50ng/ml) for 48 hours. IB : Flag
6
IL12Rβ2 mRNA expression level was measured by qRT-PCR. (n=3) (**P<0.005).
HASMC 5 (A) Time course of IL12Rβ2 mRNA expression following treatment with indicated cytokines. siRNA : control IL12Rb2 siRNA : control IL12Rb2
(B) IL12Rβ2 mRNA expression level was measured by qRT-PCR. HASMCs were transfected
IL-1β (10ng/mL) : 0 5 10 15 30 60 (min) 4 with siIL12Rβ2 using Lipofectamine® RNAiMAX Reagent for 6 hours and then treated with IFN-γ Virus : Vector IL12Rb2 IL12Rb2 Virus : Vector IL12Rb2 IL12Rb2
IκB 3 Full length primer Primer 1 Primer 2 IP : Flag IB : Flag IP : IL12Rb2
Exon : 1
Tubulin 2 1 variant 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 IL12Rb2 IL12Rb2
Flag
Flag
variant 2
0
IL-1β (10ng/mL) : 0 4 8 12 24 (hours) variant 3 IB IL12Rb2 IB IL12Rb2
variant 4 Tubulin Tubulin
HASMC 5 * variant 5 Figure 4. Exogenous IL12Rβ2 expression on 293T and HASMCs.
variant 6
293T cells were overexpressed with the empty vector (pQ) or vector encoding human
IFN-γ (50ng/mL) : 0 5 10 15 30 60 (min) 4 variant 7 IL12Rβ2 with Flag tag using turbofect reagent for 24 hours and then transfected with
pSTAT1 * U937 HASMC siIL12Rβ2 using Lipojet reagent for 48 hours. The immunoprecipitated (A) Flag or (B)
(Y701) T-bet mRNA expression (Fold chnage) 6 3 ** IFN-γ : - - + U937 U937 IL12Rβ2 proteins were subjected to the immunoblotting.
HASMCs were overexpressed with human IL12Rβ2 with Flag tag encoding retrovirus for 24
STAT1 2 IL12Rb2 colony : 2 primer 1 18 (bp) colony : 2 primer 2 18 hour and then transfected with siIL12Rβ2 using Lipofectamine®RNAiMAX reagent for 48
13
13
hours. The immunoprecipitated (C) Flag or (D) IL12Rβ2 proteins were subjected to the
Tubulin 1 0 β-actin (bp) 500 400 immunoblotting. HASMC HASMC
400
300
200
300
IFN-γ (50ng/mL) : 0 24 48 72 (hours) HASMC+IFN-γ HASMC virus : vec IL12Rb2-Flag Virus : vec IL12Rb2-Flag
Figure 1. Effect of pro-inflammatory cytokines on HASMCs primer 1 siRNA : control IL12Rb2 PDGF-BB : 0 5 10 30 0 5 10 30 (min) IL-12: 0 5 10 30 0 5 10 30 (min)
HASMCs were treated with (A) TNF-α (10ng/ml) or (B) IL-1β (10ng/ml) for the indicated colony : 4 8 13 14 16 pSTAT4
time. IκB and tubulin were analyzed by immunoblotting. Tubulin is loading control. (bp) 500 pTyr (Y693)
(C) After the serum starvation, HASMCs were treated with IFN-γ (50ng/ml) for the 400
300
indicated time. STAT1, phospho-STAT1, and tubulin were analyzed by immunoblotting. IL12Rb2(G465D) IL12Rb2(G465D) PDGFR STAT4
Tubulin is loading control.
The fold change of VCAM-1 mRNA levels in (D) TNF-α (10ng/ml) or (E) IL-β (10ng/ml) 0, 4, HASMC+IFN-γ Vector IL12Rb2 IL12Rb2 pErk 1/2 Flag
8, 12, 24 hours treated HASMCs. Time course of mRNA expression measured by qRT- primer 2 Virus :
PCR. colony : 4 8 13 14 16 Tubulin
(F) The fold change of T-bet mRNA levels in IFN-γ (50ng/ml) 0, 24, 48, 72 hours treated (bp) 400 IL12Rb2 Erk 2
HASMCs. Time course of mRNA expression measured by qRT-PCR. (n=3) (*P<0.05, 300
200
**P<0.005). Tubulin Flag HASMC
Virus : vec IL12Rb2-Flag
IL12Rb2 pSTAT1 0 5 10 30 (min)
IL-35 : 0 5 10 30
IL12Rb2 mRNA expression (Fold chnage) 10 8 6 4 TNFα IL12Rb2 mRNA expression (Fold chnage) 10 8 6 4 (A) The transcription variant and isoform of IL12Rβ2. Figure 5. IL12Rb2 affects signaling in HASMC. STAT1
12
12
Tubulin
(Y701)
Figure 3. IL12Rβ2 transcription variant 1 is expressed in HASMCs
**
IFNγ
IL12
IL1β
(B) RT-PCR analysis of IL12Rβ2 mRNA expressed in U937, HASMCs, and IFN-γ (50ng/ml)
48hr treated HASMCs. β-actin is an endogenous control gene.
Cloning of IL12Rβ2 full length PCR product in (C) U937 and (D) IFN-γ treated HASMCs was
Flag
performed. Colony PCR to separate the IL12Rβ2 transcriptions variant based on primer from
Tubulin
(A).
(E) Sequencing data of IL12Rβ2 full length in IFN-γ treated HASMCs.
2
2
retrovirus for 24 hour and then transfected with siIL12Rβ2 using Lipofectamine® RNAiMAX
hour and then, after starvation, treated with (A) PDGF-BB (25ng/ml), (B) IL-12 (10ng/ml) or (C)
0
0
Reagent for 48 hours. The protein expression was detected by immunoblotting.
IL-35 (10ng/ml) for the times indicated. pTyr, PDGFR, pERK, ERK2, pSTAT4, STAT4,
0 24 48 72 (hours) IFN-γ (50ng/mL) : 0 24 48 72 (hours) (F) HASMCs were overexpressed with IL12Rβ2 wild type or variant G465D encoding HASMCs were overexpressed with human IL12Rb2 with Flag tag encoding retrovirus for 24
pSTAT1, STAT1, Flag, IL12Rβ2 and tubulin were analyzed by immunoblotting. Tubulin is
loading control.
CONCLUSION REFERENCES
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but increase IL-35 signal in VSMCs. Involved in SMC Phenotypic Change: OLR1 as a SMC Receptor
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of VSMCs via cell assay. Therefore, this study suggest a novel [4] de Paus, R.A., et al., Differential expression and function of
role of IL12Rβ2 in VSMCs and proposes IL12Rβ2 may be human IL-12Rbeta2 polymorphic variants. Mol Immunol, 2013.
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