Janus Kinase 3, a Novel Regulator for Smooth Muscle Proliferation and Vascular Remodeling

Abstract

Objective: Vascular remodeling because of smooth muscle cell (SMC) proliferation is a common process occurring in several vascular diseases, such as atherosclerosis, aortic aneurysm, post-transplant vasculopathy, restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completely understood. The objective of this study is to determine the role and mechanism of Janus kinase 3 (JAK3) in vascular remodeling and SMC proliferation.

Approach and results: Platelet-derived growth factor-BB, an SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by short hairpin RNA, inhibits the SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Mechanistically, JAK3 promotes the phosphorylation of signal transducer and activator of transcription 3 and c-Jun N-terminal kinase in SMC, 2 signaling pathways known to be critical for SMC proliferation and vascular remodeling. Blockade of these 2 signaling pathways by their inhibitors impeded the JAK3-mediated SMC proliferation. In vivo, knockdown of JAK3 attenuates injury-induced neointima formation with attenuated neointimal SMC proliferation. Knockdown of JAK3 also induces neointimal SMC apoptosis in rat carotid artery balloon injury model.

Conclusions: Our results demonstrate that JAK3 mediates SMC proliferation and survival during injury-induced vascular remodeling, which provides a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases.

Keywords: Janus kinase 3; apoptosis; interleukin; smooth muscle proliferation; vascular remodeling.

Figure 1. JAK3 was upregulated and activated by platelet-derived growth factor (PDGF)-BB in SMCs

A, JAK3 mRNA expression was dose-dependently induced by PDGF-BB. Rat aortic SMCs were treated with PDGF-BB (20 ng/ml) for 8 hours. JAK3 mRNA levels were detected by qRT-PCR. B, JAK3, phospho-JAK3 (pJAK3), and proliferating cell nuclear antigen (PCNA) protein was dose-dependently induced by PDGF-BB. SMCs were treated with PDGF-BB (20 ng/ml) for 24 hours. JAK3, pJAK3 and PCNA protein were detected by Western blot. C, Quantification of the protein levels shown in B by normalizing to α-Tubulin. D. JAK3, pJAK3, and PCNA protein was time-dependently induced by PDGF-BB (20 ng/ml). E, Quantification of JAK3, pJAK3, and PCNA protein level shown in D by normalizing to α-Tubulin. *P < 0.05 vs vehicle-treated cells (Ctrl), n=3.

Figure 2. p38 MAPK, ERk1/2, and PI3K/Akt signaling regulated PDGF-BB-induced JAK3 expression or activation

A PDGF-BB (20 ng/ml) time-dependently induced JAK3 activation during the initial treatment. B, Quantification of pJAK3 level shown in A by normalizing to α-Tubulin. C, Blockade of either PI3K/Akt or ERk1/2 signaling by their pathway-specific inhibitors attenuated PDGF-BB-induced JAK3 activation. Rat aortic SMCs were pre-treated with pathway-specific inhibitor SB203580 (p38 MAPK), LY294002 (PI3K/Akt), or U0126 (ERk1/2) for an hour followed by PDGF-BB induction for another hour. JAK3 phosphorylation was detected by Western blot. D, Quantification of pJAK3 levels shown in C by normalizing to α-Tubulin. E, The effect of pathway inhibitors on PDGF-BB-induced JAK3 and PCNA expression. SMCs were treated with pathway inhibitors the same as in C followed by 24 hours of PDGF-BB treatment. JAK3 and PCNA expression was detected by Western blot. F-G, Quantification of the JAK3 (F) and PCNA (G) levels shown in E by normalizing to α-Tubulin. *P < 0.05 vs vehicle-treated cells (Ctrl or -); ^#P <0.05 vs PDGF-BB-treated cells without inhibitors (−), n=3.

Figure 3. JAK3 was essential for SMC proliferation in vitro

Cell proliferation was measured by EdU assay as described in Method. A, Knockdown of JAK3 by adenovirus-expressed shRNA (Ad-shJAK3) blocked platelet-derived growth factor (PDGF)-BB-induced SMC proliferation. B, Knockdown of JAK3 decreased PDGF-BB-induced proliferating cell nuclear antigen (PCNA) protein expression. C, Quantification of JAK3 and PCNA protein expression shown in B by normalizing to α-Tubulin level. *P < 0.05 vs scramble shRNA (Ad-shScr)-transduced cells; ^#P < 0.05 vs Ad-shScr-transduced cells with PDGF-BB treatment (n=3). D, Forced expression of JAK3 by adenoviral vector (Ad-JAK3) stimulated SMC proliferation. E, Forced expression of JAK3 induced PCNA protein expression. F, Quantification of JAK3 and PCNA protein expression shown in E by normalizing to α-Tubulin level. *P<0.05 vs control group (Ad-GFP) (n=3). G, JAK3 selective inhibitor, Janex-1, blocked PDGF-BB-induced SMC proliferation. H, Janex-1 decreased PDGF-BB-induced PCNA protein expression. I, Quantification of the PCNA protein expression shown in H by normalizing to α-Tubulin level. *P < 0.05 vs vehicle-treated cells (−); ^#P <0.05 vs PDGF-BB-treated cells without Janex-1 (−), n=3.

Figure 4. Blockade of JAK3 expression suppressed injury-induced neointima formation and attenuated SMC proliferation in vivo

A, JAK3 and pJAK3 levels in neointimal SMCs of balloon-injured rat carotid arteries as measured by immunohistochemistry staining (shown in Supplementary Figure II) and quantified by calibrating their staining intensity to the mean signal in uninjured vessels (Ctrl, set as 1). B, JAK3, pJAK3 and PCNA protein expression in the injured arteries was detected by Western blot. C, Quantification of JAK3, pJAK3 and PCNA protein expression shown in B by normalizing to α-Tubulin level. *P < 0.05 vs uninjured arteries (Ctrl), n=5. D-E, Knockdown of JAK3 by shRNA (Ad-shJAK3) blocked neointima formation following balloon injury, as shown by hematoxylin and eosin (HE) and Elastica van Gieson (VG) staining, respectively. F-G, Quantification of the neointima area (F) and intima/media ratio (G). *P < 0.05 vs control adenovirus (Ad-GFP)-treated arteries, n=5. H, Knockdown of JAK3 attenuated the PCNA expression in balloon-injured arteries. I, Quantification of the PCNA level shown in H by calibrating its staining intensity to the mean signal in uninjured arteries (Ctrl, set as 1). *P < 0.05 vs uninjured arteries (Ctrl); ^#P < 0.05 vs control adenovirus (Ad-GFP)-treated arteries, n=5.

Figure 5. JAK3 mediated PDGF-BB function in activating signal transducer and activator of transcription 3 (STAT3) and c-Jun N-terminal kinase (JNK)

A, Janex-1 attenuated PDGF-BB-induced phosphorylation of STAT3 and JNK. B and C, Quantification of pSTAT3 and pJNK protein expression shown in A by normalizing to α-Tubulin level. *P < 0.05 vs vehicle-treated cells (−); ^#P < 0.05 vs PDGF-BB-treated groups for each individual protein, respectively, n=3. D, Knockdown of JAK3 by shRNA (Ad-shJAK3) attenuated PDGF-BB-induced STAT3 and JNK phosphorylation. E and F, Quantification of pJAK3, pSTAT3 and pJNK levels shown in D by normalizing to α-Tubulin level, respectively. *P < 0.05 vs scramble shRNA (Ad-shScr)-transduced cells; ^#P < 0.05 vs Ad-shScr-transduced cells with PDGF-BB, for each individual protein, respectively, n=3.

Figure 6. Blockade of STAT3 or JNK activity attenuated JAK3-induced SMC proliferation

A, STAT3 inhibitor S3I-201 blocked JAK3-induced phosphorylation of STAT3. B, Quantification of the pSTAT3 level shown in A by normalizing to the α-Tubulin level. C, JNK inhibitor SP600125 blocked JAK3-induced JNK phosphorylation. D, Quantification of the pJNK level in C by normalizing to the α-Tubulin level. E, Blockade of STAT3 and/or JNK signaling by their inhibitors inhibited JAK3-induced SMC proliferation as measured by EdU assay. F, Blockade of STAT3 and/or JNK signaling by their inhibitors inhibited JAK3-induced PCNA expression. G, Quantification of PCNA expression shown in F by normalizing to the α-Tubulin level. Combined use of the two inhibitors achieved a greater inhibition of cell proliferation (E) and PCNA expression (F) than the individual inhibitor. *P < 0.05 vs Ad-GFP group within each panel; ^# P < 0.05 vs Ad-JAK3-treated group within each panel; ^$P < 0.05 vs individual inhibitor-treated cells in E and G; n=3.