Essential Role of Cofilin-1 in Regulating Thrombin-induced RelA/p65 Nuclear Translocation and Intercellular Adhesion Molecule 1 (ICAM-1) Expression in Endothelial Cells

Abstract

The nuclear factor B (NF-B)2 represents a ubiquitously expressed family of transcription factor participating in various biological effects ranging from immune, inflammatory, and stress-induced responses to cell fate decisions such as proliferation, differentiation, apoptosis, and tumorigenesis (1, 2). The mammalian NF-B family is comprised of five members: RelA (p65), RelB, c-Rel, NF-B1 (p50 and its precursor p105), and NF-B2 (p52 and its precursor p100). A characteristic feature of these proteins is the presence of a conserved N-terminal 300-amino acid Rel homology domain that contains nuclear localization signal and is involved in dimerization, sequencespecific DNA binding, and interaction with inhibitory IB proteins. A distinguishing feature of RelA, RelB, and c-Rel from p50 and p52 is the possession of a transactivation domain within the C-terminal region. Typically, NF-B exists as a heterodimer of p50 and RelA/p65 subunits associated with IB␣, the prototype of a family of inhibitory proteins IBs that keeps NF-B in the cytoplasm by virtue of masking the nuclear localization signal of RelA/p65 (3, 4). Activation of NF-B requires phosphorylation of IB␣ on two specific serine residues (Ser 32 and Ser 36 ) by a macromolecular cytoplasmic IB kinase (IKK) complex composed of the catalytic subunits IKK␣ and IKK␤ and the regulatory subunit NEMO/IKK␥ (5, 6). Phosphorylation triggers the ubiquitination of IB␣ by the E3-SCF␤-TrCP ubiquitin ligase, which in turn marks it for degradation by the 26 S proteasome (7,8). The unleashed NF-B migrates to the nucleus to activate transcription of target genes including intercellular adhesion molecule-1 (ICAM-1) (9 -14), an inducible endothelial adhesive protein that serves as a ligand for ␤ 2 -integrins (CD11/ CD18) present on the surface of leukocytes (15-17). Interaction of ICAM-1 with ␤ 2 -integrins enables polymorphonuclear leukocytes to adhere firmly and stably to the vascular endothelium and to migrate across the endothelial barrier (18 -20). We have shown that RelA/p65 is an essential regulator of endothelial ICAM-1 following stimulation of protease-activated receptor-1 by thrombin, a serine protease released during intravascular coagulation initiated by tissue injury or sepsis (21,22). A key signal mediating RelA/p65 activation by thrombin involves stimulation of the small GTPase RhoA and its effector Rhoassociated kinase (23,24). Activated RhoA/ROCK leads to activation of IKK␤, which in turn mediates the release of RelA/p65 for its nuclear uptake and binding to the ICAM-1 promoter, secondary to phosphorylation and degradation of IB␣ (24).

Regulation of Rela/p65 and Endothelial Cell Inflammation by Proline-Rich Tyrosine Kinase 2

“…Given the involvement of Pyk2 and its downstream kinase c-Src in activating RhoA/ ROCK pathway (19,45,46), it is possible that Pyk2 promotes nuclear translocation and, consequently, DNA binding of RelA/ p65 by causing alterations in the actin cytoskeleton. Consistent with this possibility, inhibition of Pyk2 by tyrphostin A9 attenuates thrombin-induced Ser3 phosphorylation and, thereby, inactivation of cofilin1 (K.M.B., unpublished results), an actin-binding protein that occupies a central position in Rho-actin pathway mediating RelA/p65 nuclear translocation (43). However, in view of the reports that Pyk2 can also function downstream of RhoA/ROCK (47,48), the other possibility that RhoA/ROCK engages Pyk2 to mediate the above responses cannot be excluded.…”

“…These results suggest that Pyk2 may also be involved in facilitating the translocation of released RelA/ p65 to the nucleus. We recently found that dynamic changes in actin cytoskeleton induced by thrombin via RhoA/Rho-associated kinase (ROCK)/Cofilin pathway are necessary for nuclear translocation of RelA/p65 (43,44). Given the involvement of Pyk2 and its downstream kinase c-Src in activating RhoA/ ROCK pathway (19,45,46), it is possible that Pyk2 promotes nuclear translocation and, consequently, DNA binding of RelA/ p65 by causing alterations in the actin cytoskeleton.…”

The Anti-Inflammatory Effect of Ripasudil (K-115), a Rho Kinase (ROCK) Inhibitor, on Endotoxin-Induced Uveitis in Rats

“…Moreover, because NF-jB response elements are present in the promoters of TNF-a, IL-6, MCP-1, and ICAM-1 genes in the inflammatory response, [39][40][41] and ripasudil significantly inhibits the levels of these proinflammatory cytokines, it is reasonable to postulate that ripasudil regulates the expression of these proteins by inhibiting NF-jB activation. Several previous studies have reported that ROCK inhibition suppressed LPSinduced activation of NF-jB in several cells and tissues.…”

Endothelin-1 contributes to increased NFATc3 activation by chronic hypoxia in pulmonary arteries

“…39,72,74). In addition, actin cytoskeleton polymerization has been implicated in the transport of transcription factors to the nucleus (26). Actin cytoskeleton dynamics are regulated by many signaling pathways.…”