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C-Src/Jak2/PDGFR/PKCδ-dependent MMP-9 induction is required for thrombin-stimulated rat brain astrocytes migration
Chih-Chung Lin1, I-Ta Lee, Pei-Ling Chi
1Department of Anesthetics, Chang Gung Memorial Hospital at Lin-Kou and College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan.
Abstract:
Among matrix metalloproteinases (MMPs), MMP-9 has been observed in patients with brain inflammatory diseases and may contribute to the pathology of brain diseases. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) were not completely understood. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 in RBA-1 cells and cells migration which were attenuated by pretreatment with the inhibitor of receptor tyrosine kinase (Genistein), c-Src (PP1), Jak2 (AG490), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), PKCs (Ro318220), PKCδ (Rottlerin), or NF-κB (Bay11-7082) and transfection with siRNA of c-Src, PDGFR, Akt, PKCδ, ATF2, p65, IKKα, or IKKβ. In addition, thrombin-stimulated c-Src, Jak2, or PDGFR phosphorylation was inhibited by a thrombin inhibitor (PPACK), PP1, AG490, or AG1296. Thrombin further stimulated c-Src and PDGFR complex formation in RBA-1 cells. Thrombin also stimulated Akt and PKCδ phosphorylation and PKCδ translocation which were reduced by PPACK, PP1, AG490, AG1296, or LY294002. We further observed that thrombin markedly stimulated ATF2 or IκBα phosphorylation and NF-κB p65 translocation which were inhibited by Rottlerin or LY294002. Finally, thrombin stimulated in vivo binding of p65 to the MMP-9 promoter, which was reduced by pretreatment with Rottlerin or LY294002. These results concluded that in RBA-1 cells, thrombin activated a c-Src/Jak2/PDGFR/PI3K/Akt/PKCδ pathway, which in turn triggered ATF2 and NF-κB activation and ultimately induced MMP-9 expression associated with cell migration.
Insights
Thrombin induces matrix metalloproteinase-9 (MMP-9) expression and cell migration in rat brain astrocytes via a signaling pathway involving c-Src, Jak2, PDGFR, PI3K, Akt, and PKCδ, leading to ATF2 and NF-κB activation.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Matrix metalloproteinases (MMPs), specifically MMP-9, are implicated in brain inflammatory diseases.
- Thrombin is a known regulator of MMP-9 expression and cell migration, but its precise mechanisms in brain astrocytes are unclear.
Purpose of the Study:
- To elucidate the signaling pathways through which thrombin induces MMP-9 expression and cell migration in rat brain astrocytes (RBA-1 cells).
Main Methods:
- Utilized inhibitors for various kinases (receptor tyrosine kinase, c-Src, Jak2, PDGFR, PI3K, Akt, PKCs) and NF-κB.
- Employed small interfering RNA (siRNA) for key signaling molecules.
- Assessed protein phosphorylation, complex formation, translocation, and DNA binding activity.
- Measured pro-form MMP-9 expression and cell migration.
Main Results:
- Thrombin induced pro-form MMP-9 expression and cell migration in RBA-1 cells.
- This induction was attenuated by inhibitors and siRNA targeting c-Src, PDGFR, Akt, PKCδ, ATF2, p65, IKKα, and IKKβ.
- Thrombin activated a signaling cascade involving c-Src, Jak2, PDGFR, PI3K, Akt, and PKCδ, leading to ATF2 and NF-κB activation.
- Thrombin stimulated p65 binding to the MMP-9 promoter.
Conclusions:
- Thrombin activates a specific signaling pathway (c-Src/Jak2/PDGFR/PI3K/Akt/PKCδ) in rat brain astrocytes.
- This pathway culminates in the activation of ATF2 and NF-κB, ultimately driving MMP-9 expression and associated cell migration.
