Tissue Plasminogen Activator Activates NF-κB through a Pathway Involving Annexin A2/CD11b and Integrin-Linked Kinase

Abstract

NF-kB activation is central to the initiation and progression of inflammation, which contributes to the pathogenesis of CKD. Tissue plasminogen activator (tPA) modulates the NF-kB pathway, but the underlying mechanism remains unknown. We investigated the role of tPA signaling in macrophage NF-kB activation and found that tPA activated NF-kB in a time-and dose-dependent manner. tPA also induced the expression of the NF-kB-dependent chemokines IP-10 and MIP-1a. The protease-independent action of tPA required its membrane receptor, annexin A2. tPA induced the aggregation and interaction of annexin A2 with integrin CD11b, and ablation of CD11b or administration of anti-CD11b neutralizing antibody abolished the effect of tPA. Knockdown of the downstream effector of CD11b, integrin-linked kinase, or disruption of its engagement with CD11b also blocked tPA-induced NF-kB signaling. In vivo, tPA-knockout mice had reduced NF-kB signaling, fewer renal macrophages, and less collagen deposition than their counterparts. Taken together, these data suggest that tPA activates the NF-kB pathway in macrophages through a signaling pathway involving annexin A2/CD11b-mediated integrin-linked kinase.

Tissue-type Plasminogen Activator (tPA) Promotes M1 Macrophage Survival through p90 Ribosomal S6 Kinase (RSK) and p38 Mitogen-activated Protein Kinase (MAPK) Pathway

“…Mouse recombinant IFN-␥ was supplied by Peprotech (Rocky Hill, NJ). Mouse monoclonal anti-LRP-1 (11H4) antibody was prepared as previously described (11,12 Cell Culture-Mouse macrophages J774.A1 were purchased from ATCC and maintained as previously described (14). Primary bone marrow-derived macrophages were prepared and maintained as previously reported (19).…”

“…Animal Model-Homozygous tPA knock-out (KO) and wildtype (WT) mice on C57BL/6 background were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained as previously described (8,10,12,14). Macrophage-specific LRP-1 KO mice (LysMCre ϩ LRP flox/flox ) were generated by crossbreeding LRP-1 floxed mice on C57BL/6 background (provided by Drs.…”

“…The animal protocol was approved by the Institutional Animal Care and Use Committee at the Penn State University College of Medicine. Unilateral ureteral obstruction (UUO) was performed in 20 -22 g male mice (3-5 mice per group) using established procedures (8,10,12,14).…”

“…Flow Cytometry-Single-cell suspensions from the whole kidneys were prepared as preciously described (14,19). The cells were stained with antibodies against CD45, CD11b, F4/80, CD206, TNF␣, arginase 1, IL-1␤, and cleaved caspase-3, followed by flow cytometry analysis using LSR II SORP machine (BD Biosciences, San Diego, CA) and FlowJo 7.6.1 software (Tree Star Inc., Ashland, OR).…”

“…Increasing evidence indicates that tPA modulates inflammation in various disease models (14 -20). Our recent work demonstrated that tPA promotes macrophage accumulation and renal inflammation, and induces NF-B-dependent chemokine expression in a CKD model (14,19). Notably, increased macrophage accumulation * This work was supported, in whole or in part, by National Institutes of Health in the obstructed kidneys is accompanied by the concomitant up-regulation of tPA and its receptor LRP-1 (10,11), suggesting that tPA signaling may play an important role in the modulation of the fate and polarity of the differentiated macrophages during chronic kidney disease.…”