Stat3-dependent acute Rantes production in vascular smooth muscle cells modulates inflammation following arterial injury in mice
Jason C. Kovacic, … , Cynthia St. Hilaire, Manfred Boehm
Jason C. Kovacic, … , Cynthia St. Hilaire, Manfred Boehm
Published December 28, 2009
Citation Information: J Clin Invest. 2010;120(1):303-314. https://doi.org/10.1172/JCI40364.
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Stat3-dependent acute Rantes production in vascular smooth muscle cells modulates inflammation following arterial injury in mice

Abstract

Inflammation is a key component of arterial injury, with VSMC proliferation and neointimal formation serving as the final outcomes of this process. However, the acute events transpiring immediately after arterial injury that establish the blueprint for this inflammatory program are largely unknown. We therefore studied these events in mice and found that immediately following arterial injury, medial VSMCs upregulated Rantes in an acute manner dependent on Stat3 and NF-κB (p65 subunit). This led to early T cell and macrophage recruitment, processes also under the regulation of the cyclin-dependent kinase inhibitor p21Cip1. Unique to VSMCs, Rantes production was initiated by Tnf-α, but not by Il-6/gp130. This Rantes production was dependent on the binding of a p65/Stat3 complex to NF-κB–binding sites within the Rantes promoter, with shRNA knockdown of either Stat3 or p65 markedly attenuating Rantes production. In vivo, acute NF-κB and Stat3 activation in medial VSMCs was identified, with acute Rantes production after injury substantially reduced in Tnfa–/– mice compared with controls. Finally, we generated mice with SMC-specific conditional Stat3 deficiency and confirmed the Stat3 dependence of acute Rantes production by VSMCs. Together, these observations unify inflammatory events after vascular injury, demonstrating that VSMCs orchestrate the arterial inflammatory response program via acute Rantes production and subsequent inflammatory cell recruitment.

Authors

Jason C. Kovacic, Rohit Gupta, Angela C. Lee, Mingchao Ma, Fang Fang, Claire N. Tolbert, Avram D. Walts, Leilani E. Beltran, Hong San, Guibin Chen, Cynthia St. Hilaire, Manfred Boehm

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Figure 5

Stat3 and p65 coassociate and regulate Rantes expression in VSMCs.

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Stat3 and p65 coassociate and regulate Rantes expression in VSMCs. (A) V...
(A) VSMCs were transfected with reporter plasmids containing the native Rantes promoter (Co) or mutated plasmids (M1 or M2) designed to abolish putative NF-κB–binding site activity. Location and sequence of putative NF-κB–binding sites and mutations are indicated. Comparisons are Co p21–/– versus Co WT, or M1 or M2 versus Co for WT and p21–/–. pGL2 indicates WT VSMCs transfected with pGL2 plasmid containing no promoter. n = 3 for all groups; **P < 0.005, ***P < 0.001, ****P < 0.0001. (B) shRNA p65Kd in p21 VSMCs reduced mRNA expression of p65 (left panel, n = 3 both groups; **P < 0.01) and Rantes (right panel, n = 4 both groups; *P < 0.05) compared with Co shRNA. (C) Tnf-α treatment for 4 hours increased Rantes mRNA expression in WT Co VSMCs (n = 6 for unstimulated, n = 7 for stimulated; P < 0.0005), while p65Kd and Stat3Kd VSMCs exhibited attenuated Tnf-α–induced Rantes mRNA expression compared with Tnf-α–treated Co VSMCs (p65Kd: n = 3, *P < 0.05; Stat3Kd: n = 4, **P < 0.005). Data are shown as mean + SEM. (D) IP followed by WB in WT Co and Stat3Kd VSMCs (input protein levels as indicated). IP with anti-Stat3 then WB with anti-p65 (upper left) and IP with anti-p65 then WB with anti-Stat3 (lower left) revealed coassociation of p65 and Stat3. IP with anti-p65 then WB with anti-p21Cip1 (upper right) revealed coassociation of p65 with p21Cip1, which was virtually abolished in Stat3Kd VSMCs.