Kinin B2 Receptor-Coupled Signal Transduction in Human Cultured Keratinocytes  Maria A. Vidal, Angel Astroza, Carola E. Matus, Pamela Ehrenfeld, Francisca Pavicic, Tamara Sanchez, Christian Salem, Jaime Figueroa, Miguel Concha, Carlos B. Gonzalez, Carlos D. Figueroa  Journal of Investigative Dermatology  Volume 124, Issue 1, Pages 178-186 (January 2005) DOI: 10.1111/j.0022-202X.2004.23518.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Human keratinocytes express immunoreactive kinin B2 receptors (B2R). (a, b) Frozen sections of normal human skin immunostained with the anti-B2R antisera mixture. Biotin–streptavidin–peroxidase ethylcarbazole system. (d) Visualization of kinin B2R in cultured human keratinocytes by immunofluorescence. (c, e) Controls in which the anti-B2R antibody was replaced by non-immune serum. Scale bar=20 μm (a); 7 μm (b); 10 μm (c–e). Journal of Investigative Dermatology 2005 124, 178-186DOI: (10.1111/j.0022-202X.2004.23518.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Lys-bradykinin (LBK) induces a sustained 42/44 mitogen-activated protein kinase (MAPK) phosphorylation. Keratinocytes were incubated with various concentrations of LBK for 5 min (a) or with 10−8 M LBK for different periods of time (b). Cell proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes that were immunoblotted with anti-phosphorylated MAPK antibodies (P-MAPK, phosphorylated MAPK). Antibodies were stripped and the membrane was incubated with anti-total MAPK (T-MAPK) antibodies (phosphorylated and non-phosphorylated, T-MAPK). AU, arbitrary units. Blots are representative of four independent experiments (n=4). Journal of Investigative Dermatology 2005 124, 178-186DOI: (10.1111/j.0022-202X.2004.23518.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The 42/44 mitogen-activated protein kinase (MAPK) phosphorylation induced by Lys-bradykinin (LBK) is mediated by B2 receptors. (a) Keratinocytes were pre-incubated with 10−6 M of the kinin B2 antagonist (HOE140), or the kinin B1 antagonists des[Arg9]Leu8-BK (ANT B1(a)) and des[Arg9]-Leu8-LBK (ANT B1(b)) for 30 min and then stimulated with 10−8 M LBK. (b) Representative experiment using 10−8 M of the B1 agonist des[Arg9]-LBK for the same periods of time used in Figure 2. (c) Effect of HOE140 and B1 antagonists on the phosphorylation induced by des[Arg9]-LBK (LDBK). Values represent the mean±SEM (n=4). *p<0.01. Journal of Investigative Dermatology 2005 124, 178-186DOI: (10.1111/j.0022-202X.2004.23518.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Lys-bradykinin (LBK) produces nuclear translocation of phosphorylated 42/44 mitogen-activated protein kinase (MAPK). Cells were grown to subconfluence and then stimulated with 100 nM LBK for 5–30 min, fixed, permeabilized, and incubated with anti-P-MAPK (phosphorylated MAPK) antibodies and with fluorescein-labeled F(ab′)2 anti-mouse immunoglobulin G. Scale bar=25 μm. Journal of Investigative Dermatology 2005 124, 178-186DOI: (10.1111/j.0022-202X.2004.23518.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 42/44 mitogen-activated protein kinase (MAPK) phosphorylation is mediated by protein kinase C (PKC) and mitogen-activated protein kinase kinase (MEK). Cells were stimulated with 10−8 M Lys-bradykinin (LBK) after pre-incubation with the PKC inhibitor GF109203X (1 μM, for 30 min), the phorbolester phorbol 12-myristate 13-acetate (PMA) (500 nM for 24 h), and the MEK inhibitor PD98059 (50 μM for 30 min). Additionally, keratinocytes were incubated directly with 50 nM PMA for 15 min. Values represent the mean±SEM (n=3). *p<0.05 and **p<0.01, with respect to LBK-stimulated cells. Journal of Investigative Dermatology 2005 124, 178-186DOI: (10.1111/j.0022-202X.2004.23518.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of 1-butanol, brefeldin A, and wortmannin on 42/44 mitogen-activated protein kinase (MAPK) phosphorylation. Keratinocytes were stimulated with 10−8 M Lys-bradykinin (LBK) after 30 min of pre-incubation with 2% v/v 1-butanol, 10 μg per mL brefeldin A (BFA), and 50 nM wortmannin. Values represent the mean±SEM (n=3). *p<0.05, with respect to LBK-stimulated cells. Journal of Investigative Dermatology 2005 124, 178-186DOI: (10.1111/j.0022-202X.2004.23518.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Lys-bradykinin (LBK) induces a partial epidermal growth factor receptor (EGFR) transactivation. (a) Keratinocytes were first pre-treated with various concentrations of AG1478 for 30 min and then stimulated with 5 ng per mL of EGF for 5 min. (b) In a separate set of experiments, keratinocytes were pre-treated with 1 μM AG1478 for 30 min and then stimulated with 10−8 M LBK. Values represent the mean±SEM (n=3). *p<0.01. Journal of Investigative Dermatology 2005 124, 178-186DOI: (10.1111/j.0022-202X.2004.23518.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Lys-bradykinin (LBK) does not induce keratinocyte proliferation. Cells were grown on 96-well plates and stimulated with various doses of LBK (open circles) or epidermal growth factor (EGF) (closed circles). The 5-bromo-2′-deoxyuridine (BrdU) incorporation rate was determined by a colorimetric cell proliferation immunoassay and measuring absorbance at 450 nm. Values represent the mean±SEM (n=4). *p<0.001; **p<0.0001. Journal of Investigative Dermatology 2005 124, 178-186DOI: (10.1111/j.0022-202X.2004.23518.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Lys-bradykinin (LBK) activates nuclear factor-κ B (NF-κB) inducing its translocation to the nucleus. (a) Cells were grown to subconfluence and then stimulated with 100 nM LBK, fixed, permeabilized, and incubated with an anti-p65 NF-κB subunit antibody and a fluorescein-labeled F(ab′)2 anti-rabbit immunoglobulin G. Scale bar=25 μm. (b) Nuclear (N) and cytoplasmic (C) fractions, isolated from untreated and LBK-stimulated cells, were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and immunoprinted with anti-p65 antibody. Values represent the mean±SEM (n=3). *p<0.01. Journal of Investigative Dermatology 2005 124, 178-186DOI: (10.1111/j.0022-202X.2004.23518.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 Lys-bradykinin (LBK) induces an early expression of c-Fos. (a) Keratinocytes were incubated for various periods of time with 10−8 M LBK. Cell proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose and immunoprinted with anti-c-Fos antibody. Values represent the mean±SEM (n=3). (b) In parallel experiments, cells were fixed, permeabilized, and immunostained with the anti-c-Fos antibody and the biotin/streptavidin–peroxidase ethylcarbazole system. Scale bar=25 μm. Journal of Investigative Dermatology 2005 124, 178-186DOI: (10.1111/j.0022-202X.2004.23518.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 11 Lys-bradykinin (LBK) stimulates a moderated (pro)filaggrin synthesis. (a) LBK (10−7 M) was used as a single dose and in the presence of ANT B1(a) (the B1 receptor antagonist des[Arg9]Leu8-BK, 10−6 M) or HOE140 (the B2R antagonist, 10−6 M). (b) As positive controls, keratinocytes were stimulated with anti-CD40 monoclonal antibody, M89; immunoglobulin (Ig) G1 is an isotype-matched mouse IgG used as a control for M89. To ensure that an equal amount of protein is present in each lane, the nitrocellulose membranes were stained with Coomassie blue. Blots are representative of three independent experiments and values represent the mean±SEM (n=3). *p<0.05, with respect to untreated cells and HOE140; **p<0.001, with respect to IgG1. Journal of Investigative Dermatology 2005 124, 178-186DOI: (10.1111/j.0022-202X.2004.23518.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions