by Mi-Ae Kang, Su-Young Yun, and Jonghwa Won Rosmarinic acid inhibits Ca2+-dependent pathways of T-cell antigen receptor-mediated signaling by inhibiting the PLC-γ1 and Itk activity by Mi-Ae Kang, Su-Young Yun, and Jonghwa Won Blood Volume 101(9):3534-3542 May 1, 2003 ©2003 by American Society of Hematology

NF-AT reporter activity but not AP-1 is inhibited by RosA NF-AT reporter activity but not AP-1 is inhibited by RosA.(A-B) Jurkat cells (2 × 106) were transfected with NF-AT (A) or AP-1 (B) reporter. NF-AT reporter activity but not AP-1 is inhibited by RosA.(A-B) Jurkat cells (2 × 106) were transfected with NF-AT (A) or AP-1 (B) reporter. Twenty-four hours after transfection, Jurkat cells were preincubated in the presence or absence of RosA (30 μM) for 2 hours and then stimulated with immobilized anti-CD3 mAb (UCHT1, 5 μg/mL) (░) or PMA (5 ng/mL) and ionomycin (0.5 μg/mL) (▨) for 16 hours. In the RosA-treated group, RosA was present throughout the whole 16 hours of incubation. Jurkat cells without any stimulation are marked as open bars (■). Shown are the relative light units (RLU), indicated as the average RLU and standard error of 2 independent experiments. Mi-Ae Kang et al. Blood 2003;101:3534-3542 ©2003 by American Society of Hematology

RosA suppresses TCR-induced elevation of intracellular Ca2+ and IP3 production in Jurkat T cells.(A) Reduced TCR-induced elevation of intracellular Ca2+release by RosA. RosA suppresses TCR-induced elevation of intracellular Ca2+ and IP3 production in Jurkat T cells.(A) Reduced TCR-induced elevation of intracellular Ca2+release by RosA. Jurkat cells were loaded with the indicator dye fura-2/am for 45 minutes, as described in “Materials and methods.” Fura-2–loaded Jurkat cells (5 × 106 cells/3 mL/sample) were preincubated in the absence or presence of various concentrations of RosA for 15 minutes and stimulated with anti-CD3 mAb (UCHT1) followed by cross-linking with antimouse IgG antibody. The arrows indicate time points for the addition of anti-CD3 mAb and antimouse IgG antibody. The Rmax/Rmin ratios of Ca2+ are representative of 4 independent experiments. (B) Inhibition of TCR-induced IP3 production by RosA. Jurkat cells were preincubated with or without the indicated concentrations of RosA. RosA-treated Jurkat T cells were either unstimulated or stimulated as indicated earlier for 3 minutes. The amount of IP3 was quantified using a competitive [3H] IP3 binding assay kit. The graph shows the average and the standard error of at least 3 independent experiments. Mi-Ae Kang et al. Blood 2003;101:3534-3542 ©2003 by American Society of Hematology

RosA inhibits TCR-induced tyrosine phosphorylation of PLC-γ1 RosA inhibits TCR-induced tyrosine phosphorylation of PLC-γ1.Jurkat cells (5 × 107) were preincubated with 30 μM (A) or 3 to 15 μM RosA (B) for 4 hours and then either unstimulated or stimulated with OKT3 ascites (1:200) and OKT3–cross-linking antimouse IgG... RosA inhibits TCR-induced tyrosine phosphorylation of PLC-γ1.Jurkat cells (5 × 107) were preincubated with 30 μM (A) or 3 to 15 μM RosA (B) for 4 hours and then either unstimulated or stimulated with OKT3 ascites (1:200) and OKT3–cross-linking antimouse IgG antibody (40 μg/mL) at 37°C for 2 minutes. PLC-γ1 was immunoprecipitated with anti–PLC-γ1 mAb (5 μg/mL), and tyrosine phosphorylation (PTyr) status was determined by blotting with antiphosphotyrosine mAb (0.5 μg/mL). After stripping, the nitrocellulose membrane was reblotted with rabbit polyclonal anti–PLC-γ1 antibody (1 μg/mL) to confirm equal precipitation of PLC-γ1. Results represent 1 of 4 independent experiments. Mi-Ae Kang et al. Blood 2003;101:3534-3542 ©2003 by American Society of Hematology

Lck kinase activity is not inhibited by RosA Lck kinase activity is not inhibited by RosA.(A) The effect of RosA on TCR-induced activation of Lck kinase activity. Lck kinase activity is not inhibited by RosA.(A) The effect of RosA on TCR-induced activation of Lck kinase activity. Lck was immunoprecipitated by anti-Lck mAb (5 μg/mL) from Jurkat cell lysates and analyzed for its ability to phosphorylate enolase (top panel). Lck kinase assay was performed as described in “Materials and methods.” Autophosphorylation status (middle panel) and equivalent immunoprecipitation of Lck (bottom panel) was confirmed by Western blotting using antiphosphotyrosine antibody and anti-Lck mAb (1 μg/mL), respectively. (B) The effect of RosA on OKT3/4-induced Lck activation. After preincubation with or without RosA, Jurkat cells were harvested and activated with OKT3/4 for 2 minutes. Jurkat cell lysates (2 × 106 cells per lane) were subjected to 12% SDS-PAGE, transferred to the nitrocellulose membrane, and blotted with anti-Lck mAb (1 μg/mL). Lck isoforms p56 and p59 are indicated by arrows. Results are representative of 2 separate experiments. Mi-Ae Kang et al. Blood 2003;101:3534-3542 ©2003 by American Society of Hematology

RosA does not interfere with the TCR-induced tyrosine phosphorylation of the TCR ζ-chain and ζ-chain–associated ZAP-70.(A) The effect of RosA on phosphorylation status of ζ-chain and ζ-chain–associated ZAP-70. RosA does not interfere with the TCR-induced tyrosine phosphorylation of the TCR ζ-chain and ζ-chain–associated ZAP-70.(A) The effect of RosA on phosphorylation status of ζ-chain and ζ-chain–associated ZAP-70. Jurkat cells were preincubated with or without RosA (30 μM) for 4 hours and stimulated for 3 minutes. Jurkat cells (5 × 107) were then lysed, immunoprecipitated with anti-TCR ζ-chain mAb (5 μg/mL), and separated by 12% SDS-PAGE. The tyrosine phosphorylation status of the ζ-chain and ζ-chain–associated ZAP-70 was verified by Western blot using antiphosphotyrosine mAb (top panel). The same membrane was reprobed with anti-TCR ζ-chain mAb (1 μg/mL) (bottom panel) to confirm the equal immunoprecipitation of the ζ-chain. (B) The effect of RosA on the tyrosine phosphorylation of ZAP-70. ZAP-70 was immunoprecipitated using anti–ZAP-70 mAb (5 μg/mL) and probed with antiphosphotyrosine mAb (top panel). The same blot was stripped and reprobed with anti–ZAP-70 mAb (bottom panel) (1 μg/mL). Results represent one of 3 independent experiments. Mi-Ae Kang et al. Blood 2003;101:3534-3542 ©2003 by American Society of Hematology

TCR-induced tyrosine phosphorylation of SLP-76 and LAT, and the association of LAT with SLP76 and Grb2 are not inhibited by RosA treatment.Jurkat cells were preincubated with 30 μM RosA for 4 hours and stimulated for the indicated periods of time (A) or 2 m... TCR-induced tyrosine phosphorylation of SLP-76 and LAT, and the association of LAT with SLP76 and Grb2 are not inhibited by RosA treatment.Jurkat cells were preincubated with 30 μM RosA for 4 hours and stimulated for the indicated periods of time (A) or 2 minutes (B). Cells were then lysed, precleared with rabbit serum agarose, and immunoprecipitated with rabbit polyclonal anti–SLP-76 (5 μg/mL) (A) or rabbit polyclonal anti-LAT antibodies (5 μg/mL) (B), respectively. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine mAb (0.5 μg/mL) (top panels) or specific antibodies (0.5 μg/mL) (middle and bottom panels) as indicated. Representative data from more than 3 independent experiments are shown. Mi-Ae Kang et al. Blood 2003;101:3534-3542 ©2003 by American Society of Hematology

RosA does not suppress the TCR-induced tyrosine/threonine phosphorylation of Erk.Jurkat cells were preincubated with the indicated concentrations of RosA for 4 hours and kept unstimulated or stimulated for 2 minutes as indicated in “Materials and methods.” ... RosA does not suppress the TCR-induced tyrosine/threonine phosphorylation of Erk.Jurkat cells were preincubated with the indicated concentrations of RosA for 4 hours and kept unstimulated or stimulated for 2 minutes as indicated in “Materials and methods.” Cells approximately of 2 × 106 were lysed in lysis buffer and resolved by 12% SDS-PAGE. The blot was probed with antiphospho-Erk1/2 mAb or rabbit polyclonal anti-ERK1/2 antibody. Erk1/2 of p44 and p42 are indicated by arrows. Results are representative of 2 separate experiments. Mi-Ae Kang et al. Blood 2003;101:3534-3542 ©2003 by American Society of Hematology

RosA inhibits the TCR-induced tyrosine phosphorylation and subsequent activation of Itk.(A) RosA inhibits TCR-induced tyrosine phosphorylation of Itk. RosA inhibits the TCR-induced tyrosine phosphorylation and subsequent activation of Itk.(A) RosA inhibits TCR-induced tyrosine phosphorylation of Itk. Jurkat cells were preincubated with the indicated concentrations of RosA for 4 hours and stimulated for 3 minutes as described in “Materials and methods.” Supernatants of TCR-stimulated Jurkat cell lysates (5 × 107) were precleared with mouse serum agarose and immunoprecipitated with anti-Itk mAb (5 μg/mL). Immunoprecipitates were resolved by 12% SDS-PAGE, and the blot was subsequently probed with antiphosphotyrosine mAb (0.5 μg/mL) or rabbit polyclonal anti-Itk antibody (1 μg/mL). Representative data from more than 2 similar experiments are shown. (B) RosA inhibits TCR-induced activation of Itk kinase activity. Jurkat cells were left unstimulated or stimulated with OKT3 cross-linking and then lysed. Total cell lysates were precipitated with anti-Itk mAb. Itk immunoprecipitates were divided into 2 parts, one for in vitro kinase assay (top panel) and the other for Western blotting (bottom panel). Itk kinase assay was performed as described in “Materials and methods.” Equivalent immunoprecipitation of Itk (bottom panel) was confirmed by Western blotting. Data are representative of 2 independent experiments. Mi-Ae Kang et al. Blood 2003;101:3534-3542 ©2003 by American Society of Hematology

RosA inhibits TCR-induced intracellular calcium mobilization and the tyrosine phosphorylation of Itk but not ZAP-70 in human peripheral blood lymphocytes.Human PBLs from healthy volunteers were isolated as described in “Materials and methods.” (A) hPBLs wer... RosA inhibits TCR-induced intracellular calcium mobilization and the tyrosine phosphorylation of Itk but not ZAP-70 in human peripheral blood lymphocytes.Human PBLs from healthy volunteers were isolated as described in “Materials and methods.” (A) hPBLs were loaded with calcium-sensitive dye fura-2/am and then treated with varying concentrations of RosA for 15 minutes. Cells were stimulated, as indicated by arrows, with OKT3 ascites (1:1000) and cross-linking antibodies, and results are representative of 2 separate experiments. (B-C) hPBLs were pretreated with indicated concentrations of RosA for 4 hours and then either left unstimulated or stimulated for 3 minutes with OKT3. hPBLs were either subjected to immunoprecipitation with the rabbit polyclonal anti-Itk (B) or anti–ZAP-70 mAb (C), separated on SDS-PAGE, and immunoblotted with antiphosphotyrosine mAb (top panels). The membranes were stripped and reblotted with anti-Itk or ZAP-70 mAb (bottom panels). Mi-Ae Kang et al. Blood 2003;101:3534-3542 ©2003 by American Society of Hematology

Schematic diagram of the TCR-signaling pathway suppressed by RosA treatment.The shaded area indicates the route of the TCR-signaling pathway that is affected by RosA treatment. Schematic diagram of the TCR-signaling pathway suppressed by RosA treatment.The shaded area indicates the route of the TCR-signaling pathway that is affected by RosA treatment. Thin lines indicate protein-protein interactions and arrows indicate the directions of signaling. *Denotes the activated status and Y denotes tyrosine phosphorylation. The figure schematically depicts 2 pathways, which are RosA susceptible, namely, the Ca2+-dependent pathway and the RosA-resistant, Ca2+-independent pathway. Mi-Ae Kang et al. Blood 2003;101:3534-3542 ©2003 by American Society of Hematology