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p21WAF1 Is Required for Interleukin-16-Induced Migration and Invasion of Vascular Smooth Muscle Cells via the p38MAPK/Sp-1/MMP-9 Pathway.

Park SL, Hwang B, Lee SY, Kim WT, Choi YH, Chang YC, Kim WJ, Moon SK - PLoS ONE (2015)

Bottom Line: Among the relevant signaling pathways examined, only p38MAPK phosphorylation was significantly stimulated in IL-16-treated VSMCs.The IL-16 induced cell-cycle-inhibitor p21WAF1 expression in VSMCs, but had no effect on the expression levels of other cell-cycle negative regulators.These results could provide that IL-16 is a new target in the treatment of vascular diseases such as atherosclerosis and re-stenosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Nutrition, Chung-Ang University, Ansung 456-756, Korea.

ABSTRACT
Interleukin-16 (IL-16) is a lymphocyte chemoattractant factor well known for its role in immune responses, but its role in vascular disease is unknown. Here, we explored the novel physiological function of IL-16 in vascular smooth muscle cells (VSMCs). The expression of IL-16 and its receptor CD4 was observed in VSMCs. Treatment with IL-16 enhanced the migration and invasion by VSMCs without altering the proliferative potential. IL-16 induced MMP-9 expression via the binding activity of transcription factors NF-κB, AP-1, and Sp-1 motifs in VSMCs. Among the relevant signaling pathways examined, only p38MAPK phosphorylation was significantly stimulated in IL-16-treated VSMCs. Treatment with p38MAPK inhibitor SB203580 prevented the IL-16-induced migration and invasion of VSMCs. SB203580 treatment inhibited the MMP-9 expression and activation of Sp-1 binding in IL-16-treated VSMCs, and siRNA knockdown of CD4 expression blocked the induction of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation stimulated by IL-16. The IL-16 induced cell-cycle-inhibitor p21WAF1 expression in VSMCs, but had no effect on the expression levels of other cell-cycle negative regulators. Finally, blockage of p21WAF1 function with specific siRNA abolished the IL-16-induced elevation of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation in VSMCs. Taken together, p21WAF1 was required for the induction of p38MAPK-mediated MMP-9 expression via activation of the Sp-1 binding motif, which led to migration and invasion of VSMCs interacting with IL-16/CD4. These results could provide that IL-16 is a new target in the treatment of vascular diseases such as atherosclerosis and re-stenosis.

No MeSH data available.


Related in: MedlinePlus

Involvement of p21WAF1 in the induction of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activity in IL-16-stimulated VSMCs.VSMCs were transfected with either si-p21-1 or scramble siRNA, and stimulated with IL-16 (50 ng/ml) for 24 h. (A, B) Indicated cells were examined via wound-healing migration and invasion assay. Scale bars represent 400 μm (wound-healing) and 100 μm (invasion). *P < 0.01 compared with IL-16 treatment. (C) Expression level of MMP-9 was determined by zymography and immunoblot analysis using culture medium and cell lysates. For the phosphorylation level of p38MAPK, indicated transfected cells were cultured with IL-16 for 10 min. Immunoblot was performed with antibody specific for p38MAPK using cell lysates. *P < 0.01 compared with IL-16 treatment. (D) Transfected cells were incubated with IL-16 (50 ng/ml) for 24 h. Nuclear extracts were acquired from indicated cells, then subjected to EMSA for an evaluation of the binding activity of the Sp-1 motif using radiolabeled oligonucleotide probes. One picomole of unlabeled Sp-1 was loaded as a negative control (competitor). *P < 0.01 compared with IL-16 treatment. (E) Effect of knockdown of p21WAF1 gene in VSMCs. VSMCs were transfected with either si-p21-1 or scramble siRNA. The efficacy of silencing the p21WAF1 gene was assessed by immunoblot. GAPDH was employed as a loading control. Results are reported as the means ± SE from three triplicate experiments. *P < 0.01 compared with control.
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pone.0142153.g007: Involvement of p21WAF1 in the induction of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activity in IL-16-stimulated VSMCs.VSMCs were transfected with either si-p21-1 or scramble siRNA, and stimulated with IL-16 (50 ng/ml) for 24 h. (A, B) Indicated cells were examined via wound-healing migration and invasion assay. Scale bars represent 400 μm (wound-healing) and 100 μm (invasion). *P < 0.01 compared with IL-16 treatment. (C) Expression level of MMP-9 was determined by zymography and immunoblot analysis using culture medium and cell lysates. For the phosphorylation level of p38MAPK, indicated transfected cells were cultured with IL-16 for 10 min. Immunoblot was performed with antibody specific for p38MAPK using cell lysates. *P < 0.01 compared with IL-16 treatment. (D) Transfected cells were incubated with IL-16 (50 ng/ml) for 24 h. Nuclear extracts were acquired from indicated cells, then subjected to EMSA for an evaluation of the binding activity of the Sp-1 motif using radiolabeled oligonucleotide probes. One picomole of unlabeled Sp-1 was loaded as a negative control (competitor). *P < 0.01 compared with IL-16 treatment. (E) Effect of knockdown of p21WAF1 gene in VSMCs. VSMCs were transfected with either si-p21-1 or scramble siRNA. The efficacy of silencing the p21WAF1 gene was assessed by immunoblot. GAPDH was employed as a loading control. Results are reported as the means ± SE from three triplicate experiments. *P < 0.01 compared with control.

Mentions: We finally investigated whether p21WAF1 regulates the IL-16-mediated VSMC responses by introducing a knockdown of the p21WAF1 expression using the siRNA system (Fig 7E). Transfection of VSMCs with p21WAF1-specific siRNA (si-p21-1) blocked the increased migration and invasion in response to IL-16 (Fig 7A and 7B). Knockdown of p21WAF1 also significantly diminished the induction of p38MAPK phosphorylation in IL-16-treated VSMCs (Fig 7C). Moreover, IL-16-induced MMP-9 expression in VSMCs was abolished by the transfection of si-p21-1 (Fig 7C). To further investigate the detailed regulatory mechanism of MMP-9, as induced by IL-16, we used EMSA to focus on the transcriptional regulation. As shown in Fig 7D, VSMCs transfected with si-p21-1 reversed the enhanced binding activation of Sp-1 that had been induced by IL-16. Finally, similar results were found in VSMCs transfected with another design of p21WAF1 siRNA (si-p21-2) (S2A–S2E Fig). Based on these results, p21WAF1 might contribute to migration and invasion through p38MAPK-associated MMP-9 expression by enhancing the activation of the Sp-1 binding motif in IL-16-treated VSMCs.


p21WAF1 Is Required for Interleukin-16-Induced Migration and Invasion of Vascular Smooth Muscle Cells via the p38MAPK/Sp-1/MMP-9 Pathway.

Park SL, Hwang B, Lee SY, Kim WT, Choi YH, Chang YC, Kim WJ, Moon SK - PLoS ONE (2015)

Involvement of p21WAF1 in the induction of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activity in IL-16-stimulated VSMCs.VSMCs were transfected with either si-p21-1 or scramble siRNA, and stimulated with IL-16 (50 ng/ml) for 24 h. (A, B) Indicated cells were examined via wound-healing migration and invasion assay. Scale bars represent 400 μm (wound-healing) and 100 μm (invasion). *P < 0.01 compared with IL-16 treatment. (C) Expression level of MMP-9 was determined by zymography and immunoblot analysis using culture medium and cell lysates. For the phosphorylation level of p38MAPK, indicated transfected cells were cultured with IL-16 for 10 min. Immunoblot was performed with antibody specific for p38MAPK using cell lysates. *P < 0.01 compared with IL-16 treatment. (D) Transfected cells were incubated with IL-16 (50 ng/ml) for 24 h. Nuclear extracts were acquired from indicated cells, then subjected to EMSA for an evaluation of the binding activity of the Sp-1 motif using radiolabeled oligonucleotide probes. One picomole of unlabeled Sp-1 was loaded as a negative control (competitor). *P < 0.01 compared with IL-16 treatment. (E) Effect of knockdown of p21WAF1 gene in VSMCs. VSMCs were transfected with either si-p21-1 or scramble siRNA. The efficacy of silencing the p21WAF1 gene was assessed by immunoblot. GAPDH was employed as a loading control. Results are reported as the means ± SE from three triplicate experiments. *P < 0.01 compared with control.
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pone.0142153.g007: Involvement of p21WAF1 in the induction of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activity in IL-16-stimulated VSMCs.VSMCs were transfected with either si-p21-1 or scramble siRNA, and stimulated with IL-16 (50 ng/ml) for 24 h. (A, B) Indicated cells were examined via wound-healing migration and invasion assay. Scale bars represent 400 μm (wound-healing) and 100 μm (invasion). *P < 0.01 compared with IL-16 treatment. (C) Expression level of MMP-9 was determined by zymography and immunoblot analysis using culture medium and cell lysates. For the phosphorylation level of p38MAPK, indicated transfected cells were cultured with IL-16 for 10 min. Immunoblot was performed with antibody specific for p38MAPK using cell lysates. *P < 0.01 compared with IL-16 treatment. (D) Transfected cells were incubated with IL-16 (50 ng/ml) for 24 h. Nuclear extracts were acquired from indicated cells, then subjected to EMSA for an evaluation of the binding activity of the Sp-1 motif using radiolabeled oligonucleotide probes. One picomole of unlabeled Sp-1 was loaded as a negative control (competitor). *P < 0.01 compared with IL-16 treatment. (E) Effect of knockdown of p21WAF1 gene in VSMCs. VSMCs were transfected with either si-p21-1 or scramble siRNA. The efficacy of silencing the p21WAF1 gene was assessed by immunoblot. GAPDH was employed as a loading control. Results are reported as the means ± SE from three triplicate experiments. *P < 0.01 compared with control.
Mentions: We finally investigated whether p21WAF1 regulates the IL-16-mediated VSMC responses by introducing a knockdown of the p21WAF1 expression using the siRNA system (Fig 7E). Transfection of VSMCs with p21WAF1-specific siRNA (si-p21-1) blocked the increased migration and invasion in response to IL-16 (Fig 7A and 7B). Knockdown of p21WAF1 also significantly diminished the induction of p38MAPK phosphorylation in IL-16-treated VSMCs (Fig 7C). Moreover, IL-16-induced MMP-9 expression in VSMCs was abolished by the transfection of si-p21-1 (Fig 7C). To further investigate the detailed regulatory mechanism of MMP-9, as induced by IL-16, we used EMSA to focus on the transcriptional regulation. As shown in Fig 7D, VSMCs transfected with si-p21-1 reversed the enhanced binding activation of Sp-1 that had been induced by IL-16. Finally, similar results were found in VSMCs transfected with another design of p21WAF1 siRNA (si-p21-2) (S2A–S2E Fig). Based on these results, p21WAF1 might contribute to migration and invasion through p38MAPK-associated MMP-9 expression by enhancing the activation of the Sp-1 binding motif in IL-16-treated VSMCs.

Bottom Line: Among the relevant signaling pathways examined, only p38MAPK phosphorylation was significantly stimulated in IL-16-treated VSMCs.The IL-16 induced cell-cycle-inhibitor p21WAF1 expression in VSMCs, but had no effect on the expression levels of other cell-cycle negative regulators.These results could provide that IL-16 is a new target in the treatment of vascular diseases such as atherosclerosis and re-stenosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Nutrition, Chung-Ang University, Ansung 456-756, Korea.

ABSTRACT
Interleukin-16 (IL-16) is a lymphocyte chemoattractant factor well known for its role in immune responses, but its role in vascular disease is unknown. Here, we explored the novel physiological function of IL-16 in vascular smooth muscle cells (VSMCs). The expression of IL-16 and its receptor CD4 was observed in VSMCs. Treatment with IL-16 enhanced the migration and invasion by VSMCs without altering the proliferative potential. IL-16 induced MMP-9 expression via the binding activity of transcription factors NF-κB, AP-1, and Sp-1 motifs in VSMCs. Among the relevant signaling pathways examined, only p38MAPK phosphorylation was significantly stimulated in IL-16-treated VSMCs. Treatment with p38MAPK inhibitor SB203580 prevented the IL-16-induced migration and invasion of VSMCs. SB203580 treatment inhibited the MMP-9 expression and activation of Sp-1 binding in IL-16-treated VSMCs, and siRNA knockdown of CD4 expression blocked the induction of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation stimulated by IL-16. The IL-16 induced cell-cycle-inhibitor p21WAF1 expression in VSMCs, but had no effect on the expression levels of other cell-cycle negative regulators. Finally, blockage of p21WAF1 function with specific siRNA abolished the IL-16-induced elevation of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation in VSMCs. Taken together, p21WAF1 was required for the induction of p38MAPK-mediated MMP-9 expression via activation of the Sp-1 binding motif, which led to migration and invasion of VSMCs interacting with IL-16/CD4. These results could provide that IL-16 is a new target in the treatment of vascular diseases such as atherosclerosis and re-stenosis.

No MeSH data available.


Related in: MedlinePlus