Abstract
We showed previously that the small molecule indirubin-3'-monoxime (I3MO) prevents vascular smooth muscle cell (VSMC) proliferation by selectively inhibiting signal transducer and activator of transcription 3 (STAT3). Looking for the underlying upstream molecular mechanism, we here reveal the important role of reactive oxygen species (ROS) for PDGF-induced STAT3 activation in VSMC. We show that neither NADPH-dependent oxidases (Noxes) nor mitochondria, but rather 12/15-lipoxygenase (12/15-LO) are pivotal ROS sources involved in the redox-regulated signal transduction from PDGFR to STAT3. Accordingly, pharmacological and genetic interference with 12/15-LO activity selectively inhibited PDGF-induced Src activation and STAT3 phosphorylation. I3MO is able to blunt PDGF-induced ROS and 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) production, indicating an inhibitory action of I3MO on 12/15-LO and consequently on STAT3. We identify 12/15-LO as a hitherto unrecognized signaling hub in PDGF-triggered STAT3 activation and show for the first time a negative impact of I3MO on 12/15-LO.
Highlights
The small molecule indirubin-3Ј-monoxime (I3MO) inhibits activation of signal transducer and activator of transcription 3 (STAT3) in vascular smooth muscle cells, with an unresolved mechanism
To test whether STAT3 can be activated by reactive oxygen species (ROS), we exposed vascular smooth muscle cell (VSMC) to exogenous H2O2 for 10 min, a time that leads to STAT3 activation in response to Platelet-derived growth factor (PDGF) [1]
diphenyleneiodonium chloride (DPI)-treated cells exhibited the same signaling pattern as those treated with I3MO upon PDGF stimulation: STAT3 phosphorylation at Tyr705 was inhibited without an effect on the phosphorylation and activation of Akt and ERK1/2 kinases (Fig. 1, b and c)
Summary
The small molecule indirubin-3Ј-monoxime (I3MO) inhibits activation of STAT3 in vascular smooth muscle cells, with an unresolved mechanism. Results: Activation of 12/15-lipoxygenase (LO) is crucial for PDGF-induced Src and STAT3 activation and is impaired by I3MO. Conclusion: I3MO interferes with PDGFR-Src-STAT3 signaling via impaired 12/15-LO activation. We showed previously that the small molecule indirubin-3monoxime (I3MO) prevents vascular smooth muscle cell (VSMC) proliferation by selectively inhibiting signal transducer and activator of transcription 3 (STAT3). Looking for the underlying upstream molecular mechanism, we here reveal the important role of reactive oxygen species (ROS) for PDGF-induced STAT3 activation in VSMC. We show that neither NADPHdependent oxidases (Noxes) nor mitochondria, but rather 12/ 15-lipoxygenase (12/15-LO) are pivotal ROS sources involved in the redox-regulated signal transduction from PDGFR to STAT3. Pharmacological and genetic interference with 12/15-LO activity selectively inhibited PDGF-induced Src activation and STAT3 phosphorylation. We identify 12/15-LO as a hitherto unrecognized signaling hub in PDGFtriggered STAT3 activation and show for the first time a negative impact of I3MO on 12/15-LO
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