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 529-541 (September 2003) DOI: 10.1046/j.1523-1747.2003.12427.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

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, Y27632- 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 Y27632. 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 Y27632. Results represent the average of four experiments, each performed in duplicate, ±SEM. Journal of Investigative Dermatology 2003 121, 529-541DOI: (10.1046/j.1523-1747.2003.12427.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

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 Y27632. Each bar represents the averaged results of triplicate wells ±SEM. Journal of Investigative Dermatology 2003 121, 529-541DOI: (10.1046/j.1523-1747.2003.12427.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

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 2003 121, 529-541DOI: (10.1046/j.1523-1747.2003.12427.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

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 2003 121, 529-541DOI: (10.1046/j.1523-1747.2003.12427.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

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 2003 121, 529-541DOI: (10.1046/j.1523-1747.2003.12427.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

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 2003 121, 529-541DOI: (10.1046/j.1523-1747.2003.12427.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

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 2003 121, 529-541DOI: (10.1046/j.1523-1747.2003.12427.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions