Involvement of small GTPase RhoA in the regulation of superoxide production in BV2 cells in response to fibrillar Aß peptides.

Fibrillar amyloid-beta (fAß) peptide causes neuronal cell death, which is known as Alzheimer's disease. One of the mechanisms for neuronal cell death is the activation of microglia which releases toxic compounds like reactive oxygen species (ROS) in response to fAß. We observed that fAß rather than soluble form blocked BV2 cell proliferation of microglial cell line BV2, while N-acetyl-l-cysteine (NAC), a scavenger of superoxide, prevented the cells from death, suggesting that cell death is induced by ROS. Indeed, both fAß1-42 and fAß25-35 induced superoxide production in BV2 cells. fAß25-35 produced superoxide, although fAß25-35 is not phagocytosed into BV2 cells. Thus, superoxide production by fAß does not seem to be dependent on phagocytosis of fAß. Herein we studied how fAß produces superoxide in BV2. Transfection of dominant negative (DN) RhoA (N19) cDNA plasmid, small hairpin (sh)-RhoA forming plasmid, and Y27632, an inhibitor of Rho-kinase, abrogated the superoxide formation in BV2 cells stimulated by fAß. Furthermore, fAß elevated GTP-RhoA level as well as Rac1 and Cdc42. Tat-C3 toxin, sh-RhoA, and Y27632 inhibited the phosphorylation of p47(PHOX). Moreover, peritoneal macrophages from p47(PHOX) (-/-) knockout mouse could not produce superoxide in response to fAß. These results suggest that RhoA closely engages in the regulation of superoxide production induced by fAß through phosphorylation of p47(PHOX) in microglial BV2 cells.

Interaction of microglia and amyloid-beta through beta2-integrin is regulated by RhoA.

Amyloid-beta (Abeta) is one of the main factors to cause Alzheimer's disease. Although fibrillar Abeta (fAbeta) activates microglial cells that release toxic compounds to induce partial neuronal death, the mechanism of interaction between Abeta and microglia remains unclear. Therefore, we examined the interaction of microglial cells (BV2) and fAbeta on a gelatin-precoated plate. The binding was markedly enhanced by RhoA inactivation using Tat-C3, dominant negative RhoA, and si-RhoA. To identify the receptor for fAbeta, we tested various antibodies to mask receptors. Among them, anti-beta2-integrin antibody mostly suppressed cell binding to fAbeta. The incremental binding of cells induced by RhoA inhibition was also blocked by addition of anti-beta2-integrin antibody. These results suggest that RhoA inhibition stimulates beta2-integrin-mediated cell interaction to fAbeta.

Transforming growth factor-beta1 regulates macrophage migration via RhoA.

Brief treatment with transforming growth factor (TGF)-beta1 stimulated the migration of macrophages, whereas long-term exposure decreased their migration. Cell migration stimulated by TGF-beta1 was markedly inhibited by 10 mug/mL Tat-C3 exoenzyme. TGF-beta1 increased mRNA and protein levels of macrophage inflammatory protein (MIP)-1alpha in the initial period, and these effects also were inhibited by 10 mug/mL Tat-C3 and a dominant-negative (DN)-RhoA (N19RhoA). Cycloheximide, actinomycin D, and antibodies against MIP-1alpha and monocyte chemoattractant protein-1 (MCP-1) abolished the stimulation of cell migration by TGF-beta1. These findings suggest that migration of these cells is regulated directly and indirectly via the expression of chemokines such as MIP-1alpha and MCP-1 mediated by RhoA in response to TGF-beta1. TGF-beta1 activated RhoA in the initial period, and thereafter inactivated them, suggesting that the inactivation of RhoA may be the cause of the reduced cell migration in response to TGF-beta1 at later times. We therefore attempted to elucidate the molecular mechanism of the inactivation of RhoA by TGF-beta1. First, TGF-beta1 phosphorylated RhoA via protein kinase A, leading to inactivation of RhoA. Second, wild-type p190 Rho GTPase activating protein (p190RhoGAP) reduced and DN-p190RhoGAP reversed the reduction of cell migration induced by TGF-beta, suggesting that it inactivated RhoA via p190 Rho GAP.