1. The Proteinase-Activated Receptor-2 Mediates Phagocytosis in a Rho-Dependent Manner in Human Keratinocytes 
Glynis Scott, Sonya Leopardi, Lorelle Parker, Laura Babiarz, Miri Seiberg, Rujiing Han 
Journal of Investigative Dermatology 
Volume 121, Issue 3, Pages (September 2003)
DOI: /j x
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
PAR-2-mediated phagocytosis is Rho dependent in human keratinocytes. (A)-(C) Human keratinocytes were treated with Toxin B (18 h; A), C3 exoenzyme (4 d; B), or Y27632 (18 h; C), and uptake of Nile-Red-conjugated microspheres was analyzed following incubation with the microspheres for 18 h. There is a concentration-dependent decrease in phagocytosis under all three conditions. Asterisks indicate a statistically significant decrease (p<0.002) determined using the Student's t test. Results represent the average of three experiments performed in duplicate ± SEM. (D) Representative photograph of keratinocytes treated with Y27632 (18 h), C3 exoenzyme (4 d), or vehicle control following an 18 h incubation with Nile-Red-conjugated microspheres. Approximately 50% of the untreated (control) cells contain clusters of phagocytized beads in a perinuclear distribution (arrow). In contrast, Y and C3-treated cells show many cells with no ingested microspheres, and a reduction in the number of microspheres in positive cells. Bar: 15 μm. (E) Keratinocytes were infected with adenovirus vectors expressing either constitutively active (V12Rho) or dominant negative (N17Rho) Rho recombinant proteins for 48 h. During the last 18 h cells were incubated with Nile-Red-conjugated microspheres. Sixty-eight percent of cells expressing V12Rho had ingested microspheres compared with empty vector expressing cells; cells expressing N17Rho had reduced (26%) phagocytosis of beads compared with empty vector expressing cells. Asterisks indicate a statistically significant change (p<0.002) determined using the Student's t test. Results represent the average of three experiments performed in duplicate ±SEM. (F) Keratinocytes were treated with either ISLLRG-NH2 or SLIGRL-NH2 (50 μM) for 5 min in the presence or absence of Y27632 (5 μM). Nile Red microspheres were added for a 4 h incubation in the presence of Y Stimulation of PAR-2 by SLIGRL-NH2 resulted in an increase in microsphere uptake that was abrogated by the presence of the Rho kinase inhibitor Y Results represent the average of four experiments, each performed in duplicate, ±SEM.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Toxin B, C3 exoenzyme, and Y27632 do not diminish keratinocyte viability. Human keratinocytes were treated with Toxin B (18 h; A), C3 exoenzyme (4 d; B) or Y27632 (18 h; C), and cell viability was analyzed using an MTT assay. There was no decrease in cell viability following treatment with toxins or Y Each bar represents the averaged results of triplicate wells ±SEM.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
PAR-2 stimulation activates Rho in HaCaT. (A) HaCaT were serum starved and treated with the inactive peptide ISLLRG-NH2 (I) or the PAR-2-specific activating peptide SLIGRL-NH2 (S) at 50 μM, and activated Rho was affinity purified from lysates using GST-Rhotekin and resolved by 15% SDS-PAGE. The total amount of Rho protein is shown in the lower gel. Densitometry normalized for sample loading shows a rapid induction of Rho activation at 5 min (1.2-fold) and 10 min (1.3-fold), which increased to 1.6-fold over control cells by 15 min. The blot is representative of two experiments; densitometry data are the average results of two experiments. (B) Serum-starved HaCaT were treated with trypsin (500 units per ml) and GTP-Rho was affinity purified from cell lysates with GST-Rhotekin and resolved by 15% SDS-PAGE. The total amount of Rho protein is shown in the lower gel. Densitometry normalized for sample loading shows an increase in GTP-Rho at 2 min by 1.8-fold over control levels with a peak at 5 min (1.9-fold over control levels). The blot is representative of two experiments; densitometry data are the average results of two experiments. (C) Serum-starved HaCaT were treated with activating peptide SLIGRL-NH2 (S) or inactive peptide ISLLRG-NH2 (I) for up to 72 h and GTP-Rho was affinity purified from cell lysates. The total amount of Rho protein is shown in the lower gel. Densitometry normalized for sample loading shows an increase in GTP-Rho with PAR-2 activation at 5 min and 30 min (1.6-fold and 1.4-fold, respectively). GTP-Rho returned to control levels at 1 h. Prolonged exposure of cells to activating peptides (24 h) resulted in a PAR-2-mediated decrease in GTP-Rho (2.2-fold). The blot is representative of two experiments; densitometry data are the average results of two experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Rho activation has no effect on PAR-2 activity levels. Total protease activity (A) and PAR-2 specific protease activity (B) were analyzed from culture supernatants of HaCaT treated with Y27632 (1 μM or 5 μM) or Toxin B (1 ng per ml or 10 ng per ml) for 18 h. There was a slight (0.13-fold) increase in PAR-2-specific protease activity at the 1 μM and 5 μM dose of Y27632 compared with untreated cells, which was not interpreted to be significant. Each bar represents the averaged results of six wells ±SD. Shown are the averaged results of two experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
PAR-2 increases cAMP in HaCaT. (A) Serum-starved HaCaT were treated with ISLLRG-NH2 or SLIGRL-NH2 at the indicated concentrations for 1 min, and cAMP levels were analyzed in cell lysates. cAMP was increased 4.5-fold in cells treated with activating peptides (50 μM) compared with control cells. At 100 μM and 200 μM of SLIGRL-NH2, cAMP increased 9.2-fold and 35-fold, respectively, compared with ISLLRG-NH2-treated cells. Asterisks indicate a statistically significant increase (p-value between 0.05 and 0.002) determined using the Student's t test. Results represent the averaged results of three experiments ± SEM. (B) To assess the time course of the cAMP response serum-starved HaCaT were treated with 50 μM ISLLRG-NH2 or SLIGRL-NH2 and cAMP levels were analyzed in cell lysates. At 15 s a 1.8-fold increase in cAMP was seen in cells treated with SLIGRL-NH2. cAMP levels peaked at 1 min (4.6-fold increase) and were still elevated at 10 min (1.6-fold over control levels). Asterisks indicate a statistically significant increase (p-value between 0.05 and 0.002). Results represent the averaged results of three experiments ±SEM.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
PAR-2-dependent Rho activation is independent of PKA. HaCaT were serum starved for 1 h in the presence or absence of the PKA inhibitors Rp-8-Cl-cAMPS (Rp8) or H-89 and were treated with either ISLLRG-NH2 or SLIGRL-NH2 (100 μM) for 5 min; GTP-Rho was affinity purified from cell lysates and resolved by 15% SDS-PAGE. Densitometry normalized for sample loading showed a 2.8-fold increase in GTP-Rho in SLIGRL-NH2-treated samples compared with ISLLRG-NH2-treated samples. Treatment with Rp-8-Cl-cAMPS or H-89 in the presence of inactive peptide resulted in Rho activation by 1.5- and 2.7-fold, respectively, indicating that the PKA pathway is inhibitory to Rho activation in HaCaT. Pretreatment of cells with PKA inhibitors did not eliminate PAR-2-dependent Rho activation.
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
PAR-2-mediated Rho activation is pertussis toxin insensitive. (A) HaCaT were treated with pertussis toxin (PT) for 18 h (100 ng per ml), serum starved for 1 h, and treated with either ISLLRG-NH2 or SLIGRL-NH2 (100 μM) for 5 min; GTP-Rho was affinity purified from cell lysates and resolved by 15% SDS-PAGE. Densitometry normalized for sample loading showed a 2.5-fold increase in GTP-Rho in cells treated with SLIGRL-NH2 (S) compared with cells treated with ISLLRG-NH2 (I). In cells pretreated with pertussis toxin prior to the addition of SLIGRL-NH2, a 3-fold increase in GTP-Rho was seen indicating that PAR-2-dependent Rho activation is pertussis toxin insensitive. (B) HaCaT were treated with pertussis toxin for 18 h (100 ng per ml), serum starved for 1 h, and treated with trypsin (500 units per ml) for 2 min or 5 min; GTP-Rho was affinity purified from cell lysates and resolved by 15% SDS-PAGE. Densitometry normalized for sample loading showed a 3-fold increase in GTP-Rho in cells treated with trypsin for 2 min and 5 min compared with control cells. Pretreatment with pertussis toxin did not diminish Rho activation induced by trypsin.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions