The major findings of this study are that Bcr kinase activation by AngII inhibits PPARγ activation, and that AngII induced NF-κB activation occurs in part via Bcr kinase activation and subsequent inhibition of PPARγ activation. These data suggest that Bcr inhibits PPARγ activation via phosphorylation of S82. Furthermore, to our knowledge this is the first report to show that activation of Bcr kinase plays an important role in arterial proliferative disease in vivo. AngII is an inflammatory mediator that activates NF-κB, a key component of inflammation. Previously reported data show that crosstalk between NF-κB and PPARγ is important in the pro-inflammatory effects of NF-κB.36
Specifically, NF-κB has been shown to block PPARγ ligand-induced transactivation in adipocytes. Our data using VSMC and previous reports 30, 37
show the converse, that PPARγ inhibits NF-κB activity. Therefore we propose that crosstalk between NF-κB and PPARγ, which are regulated by Bcr kinase, is important in regulating VSMC inflammatory gene expression. Given our novel findings that Bcr kinase inhibits PPARγ transcriptional activation and enhances NF-κB coupled with our finding that PPARγ inhibits NF-κB activation we believe that Bcr acts as a set point mechanism that regulates the sensitivity of VSMC to inflammatory stimuli.
In this study we demonstrate that Bcr is a major regulator of SMC that sits at the cross roads of inflammation and proliferation (). Our findings that Bcr inhibits PPARγ transcriptional activation () and that knockdown of Bcr with Bcr siRNA or DN-Bcr reverses AngII inhibition of PPARγ () demonstrate that Bcr is a positive regulator of AngII mediated inflammation. We also found that both Bcr siRNA and DN-Bcr block AngII mediated NF-κB activation () demonstrating that Bcr regulation of NF-κB is a key component of Bcr’s regulation of inflammation. Furthermore both our in vitro and in vivo studies showing that knock down or absence of Bcr reduces AngII/PDGF induced [3
H] thymidine incorporation and reduces cell growth and intimal thickening (, , and Supplemental Figure III
) demonstrate that Bcr regulates proliferation.
Our data suggest that the pro-inflammatory and proliferative effects of Bcr are mediated, at least in part, by inhibition of PPARγ and suggest that AngII-mediated Bcr kinase activation inhibits PPARγ by phosphorylation of S82. Our demonstration of nuclear localization of Bcr in VSMC is consistent with this concept. It remains unclear whether there are distinct differences between nuclear and cytoplasmic Bcr. Interestingly, Bcr contains a putative nuclear localization signal at amino acid 802–819 (http://myhits.isb-sib.ch/cgi-bin/motif_scan
), but the functional consequence of this domain needs further investigation.
Overexpression of Bcr can inhibit PPARγ activation without showing any ERK1/2 activation, suggesting that Bcr inhibited PPARγ activation in an ERK1/2-independent manner. PPARγ plays an important role in regulating inflammation. PPARγ is a negative regulator of macrophage activation 30
and PPARγ agonists have been demonstrated to inhibit the production of monocyte inflammatory cytokines.10
The PPARγ agonist 15d-PGJ2
has been shown to inhibit transcription factors including NF-κB.30
The anti-proliferative effect of PPARγ has several possible mechanisms. One is a direct result of its anti-inflammatory effect as cytokines and chemokines may promote lesion progression in a paracrine fashion.11
In addition, the PPARγ agonist troglitazone has been shown to inhibit bFGF induced DNA synthesis in VSMC and to inhibit intimal proliferation in a rat aortic balloon injury model.13
Troglitazone was shown to inhibit c-fos induction and to inhibit transactivation of the serum response element that regulates c-fos expression, but the exact inhibitory target of PPARγ agonists against inflammation and proliferation remains unclear.
Our results do not exclude the possibility of an effect of Bcr on inflammation and proliferation that is independent of PPARγ (). Indeed as noted, we did find a phosphorylated protein around 60 kD that correlated well with Bcr kinase activation induced by AngII. Future studies will focus on PPARγ independent effects of Bcr signaling.
In conclusion, our data suggest that Bcr is an important regulator of inflammation and proliferation in VSMC and that Bcr plays a key role in arterial proliferative disease. This effect of Bcr is mediated in part by inhibition of PPARγ transcriptional activation via phosphorylation of PPARγ by Bcr.