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First published online January 27, 2006
doi: 10.1242/10.1242/jcs.02772

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The ß2-adrenergic receptor activates pro-migratory and pro-proliferative pathways in dermal fibroblasts via divergent mechanisms

Department of Dermatology, University of California, Davis, TB 192, One Shields Avenue, CA 95616, USA

View larger version (28K): [in a new window]   Fig. 1. Dermal fibroblasts were plated onto collagen-coated glass coverslips at a concentration of 125 cells/mm2 in FM for 3-6 hours at 37°C. Migration experiments were performed in FM in the presence or absence of ß-AR agonist (10 nM-100 µM). The migration of each single cell was monitored over a 1-hour period. The speed and distance traveled, at a concentration of 1 µM ß-AR agonist, are represented graphically in A and B, respectively. The ß-AR-mediated dose-dependent increase in distance traveled is represented graphically in C (ß-AR agonist 10 nM-100 µM). The data are representative of three independent experiments with three different fibroblast strains (n=50). Values plotted are mean ± s.e.m. *P<0.01 between ß-AR agonist and controls. Cells were starved of growth factors in DMEM for 16 hours. 1-2x107 cells were either left un-treated or treated with 1 µM ß-AR agonist in DMEM for 10 minutes at 37°C. After treatment, cell lysates were prepared and the EGFR was immunoprecipitated. EGFR antibody-associated proteins were electrophoresed on two separate 10% polyacrylamide gels at the same time and transferred to membranes. Membranes were immunoblotted with either an EGFR antibody (EGFR WB) or an anti-phosphotyrosine antibody (PY WB; D). Gels were aligned to allow correct identification of the EGFR protein. The data shown are representative of three independent experiments from three separate cell strains. Three blots from three separate experiments were scanned for EGFR tyrosine phosphorylation and densitometry was performed using a gel plotting macro in NIH Image 1.62. Data was averaged, statistically analyzed and represented graphically (Fig. 1E). Values plotted are mean ± s.e.m. (n=3). *P<0.01 between ß-AR agonist and control.

View larger version (19K): [in a new window]   Fig. 2. Dermal fibroblasts were starved of growth factors in DMEM for 16 hours as described. 1-2x106 cells were either left un-treated or treated with 1 µM ß-AR agonist in DMEM for 5-60 minutes at 37°C. After treatment, cell lysates were prepared, electrophoresed on 10% polyacrylamide gels and transferred to membranes. Membranes were immunoblotted with either an anti-phospho ERK antibody or an anti-ERK antibody (A). Three blots from three separate experiments were scanned for P-ERK and densitometry performed using a gel plotting macro in NIH Image 1.62. Data was averaged, statistically analyzed and represented graphically (B). Values plotted are means ± s.e.m. (n=3). *P<0.01 between ß-AR agonist and controls (0). The data shown are representative of three independent experiments from three separate cell strains.

View larger version (35K): [in a new window]   Fig. 3. Dermal fibroblasts were starved of growth factors in DMEM for 16 hours as described. 1-2x106 cells were pre-incubated with either DMEM alone (0, 5-60 minutes ISO) or DMEM containing either 10 µM AG1478 (A,B) for 90 minutes or 10 µM PP2 for 6 hours at 37°C (C,D). Cells were either untreated (control, 0, 5-60 minutes ISO) or stimulated with DMEM containing inhibitor and 1 µM ß-AR agonist for 5-60 minutes at 37°C, unless otherwise noted. After treatment, cell lysates from each experiment were prepared and electrophoresed on the same 10% polyacrylamide gels and transferred to membranes. Membranes were immunoblotted with either an anti-ERK antibody, a anti-phospho ERK antibody (P-ERK) an anti-EGFR antibody (EGFR) or an anti-phosphotyrosine antibody (PY). Three blots from separate AG1478 or PP2 experiments were scanned for p-ERK or PY and densitometry performed using a gel plotting macro in NIH Image 1.62. Data was averaged, statistically analyzed and represented graphically (B,D,F). Values plotted are means ± s.e.m. (n=3). *P<0.01 between conditions and controls. # no significant difference between AG1478/control and AG1478/ß-AR agonist or PP2/control and PP2/ß-AR agonist. The data shown are representative of three independent experiments from three separate cell strains.

View larger version (20K): [in a new window]   Fig. 4. Dermal fibroblasts were plated onto collagen-coated glass coverslips in FM as described and pre-treated with 10 µM PP2 for 6 hours at 37°C. The migration of each single cell was monitored over a 1-hour period in FM in the presence or absence of 1 µM ß-AR agonist, as described. The speed and distance traveled are represented graphically in A and B, respectively. The data are representative of three independent experiments with three different fibroblast strains (n=50). Values plotted are means ± s.e.m. *P<0.01 between ß-AR agonist and controls.

View larger version (12K): [in a new window]   Fig. 5. 5x104 dermal fibroblasts were plated per well in FM in a 12-well plate in triplicate and either untreated (A) or pre-treated with 50 µM sp-cAMP for 30 minutes (B, sp-cAMP alone, +, medium dashed line) or 50 µM rp-cAMP for 30 minutes (C, rp-cAMP alone, +, medium dashed line) prior to the addition of FM alone (control, , solid line in A-C) or FM containing 1 µM ß-AR agonist alone (, short dashed line in A-C), or ß-AR agonist and sp-cAMP (x, medium dashed line in B) or rp-cAMP (x, long dashed line in C). Cells were harvested and counted on days 2, 4, 6, 8. The data are representative of three independent experiments with three different fibroblast strains. Values plotted are mean ± s.e.m. *P<0.01 between ß-AR agonist and controls.

View larger version (40K): [in a new window]   Fig. 6. Fibroblast-embedded collagen gels were prepared and cast in the absence (control) or presence of 10 µM ß-AR agonist, 50 µM rp-cAMP, or both 10 µM ß-AR agonist and 50 µM rp-cAMP. The diameters of the collagen gels were measured after 24 hours and the percentage collagen gel contraction calculated; maximum contraction (1.5 cm) was considered as 100%. Values represent mean ± s.e.m. (n=6). *P<0.01 between ß-AR agonist and controls, #P<0.05 between rp-cAMP/ß-AR agonist and ß-AR agonist. Results were combined from three independent experiments from at three dermal fibroblast strains.

View larger version (87K): [in a new window]   Fig. 7. Sterile coverslips were coated with collagen and cells plated in FM. Cells were untreated (A), treated with 1 µM ß-AR agonist for 15 minutes (B), treated with 50 µM sp-cAMP (C) or rp-cAMP (E) for 45 minutes or pre-treated with either 50 µM sp-cAMP (D) or rp-cAMP (F) for 30 minutes prior to the addition of 1 µM ß-AR agonist for 15 minutes in FM. Cells were fixed and stained for actin (green) and vinculin (red). Bar, 20 µM

View larger version (16K): [in a new window]   Fig. 8. A diagrammatic representation of the divergent signaling mechanisms activated by ß2-AR. A simplified diagram is shown outlining the divergent mechanisms and interactive pathways occurring upon ß2-AR activation in dermal fibroblasts.

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