Pertussis Toxin-sensitive Secretory Phospholipase A2 Expression and Motility in Activated Primary Human Keratinocytes  Krystyna E. Rys-Sikora, Alice P. Pentland, Raymond L. Konger  Journal of Investigative Dermatology  Volume 120, Issue 1, Pages 86-95 (January 2003) DOI: 10.1046/j.1523-1747.2003.12001.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The effect of PTX holoenzyme or B-oligomer pretreatment on PGE2levels in nonconfluent primary human keratinocytes cultures. (a) Nonconfluent keratinocytes were treated with vehicle (control) or PTX holotoxin (PTX; 20 ng per ml) or B-oligomer (20 ng per ml) for 24 h. Supernatants were then removed and PGE2 levels were determined by enzyme-linked immunosorbent assay. Values are mean±SD of cumulative (24 h) PGE2 production normalized to total cellular protein. Each condition was performed in triplicate and these results are representative of five identical experiments using keratinocyte preparations from different individuals. (b) Cultures were pretreated for 24 h with increasing concentrations (0–20 ng per ml) of PTX holoenzyme (v) or B-oligomer (h). PGE2 levels were assayed as described above. Results are presented as percentage of inhibition of PGE2 production in comparison with control condition. Results are expressed as mean±SD of a representative experiment of three identical experiments, with each condition performed in triplicate. Journal of Investigative Dermatology 2003 120, 86-95DOI: (10.1046/j.1523-1747.2003.12001.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Evaluation of ADP ribosylation activities in nonconfluent cultures pretreated with increasing concentration of PTX holotoxin and B-oligomer. (a) A representative autoradiogram of 12% sodium dodecyl sulfate–polyacrylamide gel resolving the products of an ADP ribosylation reactions using membranes prepared by nonconfluent keratinocytes cultures that were pretreated for 24 h with 0.02–20 ng per ml PTX or B-oligomer as described in Materials and Methods. Catalytic activity is represented by the ability of holoezyme or B-oligomer pretreatment to inhibit further PTX-catalyzed [32P]ADP ribosylation of Giα subunits in isolated membranes. A single labeled band of the same apparent molecular weight as ADP-ribosylated Giα subunit (41 kDa) was observed. This figure represents duplicate reactions for each condition from a single individual. (b) Dose–response curves demonstrating the relative ability of PTX holoenzyme (v) and B-oligomer (h) to inhibit ADP ribosylation were generated by densitometric analysis of scanned gels. [32P]-labeled 41 kDa bands were quantitated by volume integration of phosphoimager scans. Relative densitometry units for duplicate determinations of pretreatment and control were averaged and percent inhibition of PTX-catalyzed ADP ribosylation was calculated by the formula: [100-(sample mean÷control mean)]×100. Values represent the mean±SD of results derived from two separate individuals done in duplicate. Journal of Investigative Dermatology 2003 120, 86-95DOI: (10.1046/j.1523-1747.2003.12001.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Assessment of COX-2 activity in nonconfluent keratinocyte cultures pretreated with increasing concentrations of PTX. Nonconfluent keratinocyte cultures were treated for 24 h with vehicle (v), 0.2 nM PTX (h), 2.0 nM PTX (σ), or 20 nM PTX (υ). These pretreated cultures were then pulsed with increasing concentrations of exogenously added AA (1–30 μM) or vehicle for 15 min. Reactions were stopped by rapid cooling and removal of supernatants. Aliquots of supernatants were taken for PGE2 analysis. Each condition was performed in triplicate and the results reported are mean ± SEM of two experiments. Journal of Investigative Dermatology 2003 120, 86-95DOI: (10.1046/j.1523-1747.2003.12001.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 A comparison of sPLA2, COX-2, cPLA2, and COX-1 protein levels in total cell lysates isolated from either control or PTX-treated nonconfluent cultures. Equivalent amounts of total cell lysates isolated from nonconfluent cultures that were pretreated for 24 h with either vehicle (control) or 20 nM PTX were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose and probed with antibody of interest as described in methods. Western blot analysis was performed in duplicate on three different individual skin samples; representative results are shown. Journal of Investigative Dermatology 2003 120, 86-95DOI: (10.1046/j.1523-1747.2003.12001.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The effects of PTX, SC-58236, and 12-epi-scalaradial, on nonconfluent keratinocyte morphology. Nonconfluent keratinocyte cultures were treated for 24 h with vehicle (a,c), 20 nM PTX (b,d), 20 nM SC-58236 (e), or 5 μM 12-epi-scalaradial (f). Phase contrast photographs of nonconfluent keratinocytes treated with vehicle alone (a) or PTX (b) are shown. To visualize changes better in cellular morphology and extent of actin-containing structures, cultures were fixed and stained with rhodamine phallodin as discussed in methods. Nonconfluent keratinocytes treated with vehicle alone (a,c) are highly elongated and contain lamellipodia (short arrow), membrane ruffling (arrowhead), and intercellular processes (long arrow). PTX-treated keratinocytes (b,d) are rounded and devoid of lamellipodia, ruffling, and intercellular processes. Keratinocytes treated with SC-58236 (e) are similar to vehicle-treated cells in appearance. Lamellipodia (short arrow), membrane ruffling (arrowhead), and intercellular processes (long arrow) are present. Whereas, 12-epi-scalaradial (f)-treated cells are rounded and have blunted lamellipodia (short arrows) and significant loss of intercellular processes. Data are representative of three different experiments using skin samples from three different individuals. Scale bar=50 μm. Journal of Investigative Dermatology 2003 120, 86-95DOI: (10.1046/j.1523-1747.2003.12001.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The effect of PTX treatment on keratinocyte motility using a colliodal gold particle motility assay. Nonconfluent keratinocyte cultures were treated for 24 h with either vehicle (control) or 20 nM PTX. Cells were then harvested and plated on to chamber slides coated with colloidal gold particles and collagen as described in Materials and Methods. After 18 h incubation, slides were fixed and photographed under dark field optics. Dark regions in the bright substratum correspond to the areas of motility, quantification of these areas was performed as discussed in Materials and Methods. Photographs of phagokinetic tracks of keratinocytes and frequency distribution of area cleared after vehicle (a,c) or PTX (b,d) pretreatment are shown, respectively (n=100). These findings are representative of three independent experiments using skin samples from different individuals. The effect of PTX treatment on keratinocyte motility was statistically different from vehicle treatment (p<0.05, Student's t test). Scale bar=100 μm. Journal of Investigative Dermatology 2003 120, 86-95DOI: (10.1046/j.1523-1747.2003.12001.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Measurement of keratinocytes motility after 12-epi-scalaradial or SC8236 treatment. Motility of nonconfluent keratinocytes after 24 h pretreatment with either vehicle (a), 5 μM 12epi-scalaradial (b), or 20 nM SC-58236 (c) was evaluated using the colloidal gold particle motility assay and quantified as discussed in Materials and Methods (n=100). The frequency distribution for areas cleared by keratinocytes after corresponding treatment are shown. Similar results were obtained in three independent experiments. The effect of 12-epi-scalaradial treatment on keratinocyte motility was statistically different (p<0.05, Student's t test) from vehicle treatment (p<0.05, Student's t test) and SC-58236 treatment (p<0.05, Student's t test). Journal of Investigative Dermatology 2003 120, 86-95DOI: (10.1046/j.1523-1747.2003.12001.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions