Basal Keratinocytes from Uninvolved Psoriatic Skin Exhibit Accelerated Spreading and Focal Adhesion Kinase Responsiveness to Fibronectin Guofen Chen,

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Basal Keratinocytes from Uninvolved Psoriatic Skin Exhibit Accelerated Spreading and Focal Adhesion Kinase Responsiveness to Fibronectin Guofen Chen, Thomas S. McCormick, Craig Hammerberg, Shauna Ryder-Diggs, Seth R. Stevens, Kevin D. Cooper Journal of Investigative Dermatology Volume 117, Issue 6, Pages 1538-1545 (December 2001) DOI: 10.1046/j.0022-202x.2001.01535.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Enhanced spreading of basal keratinocytes from psoriatic uninvolved relative to normal epidermis on fibronectin but not collagen-laminin. (A) Fresh epidermal cells from psoriatic uninvolved (pso-uni) and normal epidermis were incubated on either fibronectin- or collagen-laminin (Co + La) coated slides for 1 h. The pattern elicited by staining with rhodamine-phalloidin, K1/K10+, and vimentin+ is shown in keratinocytes obtained from normal skin on fibronectin (a) and on collagen-laminin (b), and in keratinocytes obtained from psoriatic uninvolved skin on fibronectin (c) and on collagen-laminin (d). Whereas K1/K10 - vimentin- round cells without condensed actin at the periphery dominate in cultures of normal epidermal cells or psoriatic uninvolved epidermal cells in collagen-laminin, the majority of psoriatic uninvolved K1/K10− vimentin− cells exhibit spreading as defined by our criteria detailed in Methods (c). (B) The percentages of rhodamine-phalloidin+, K1/K10−, and vimentin− spreading cells are shown as open bars for normals, and a hatched bar for basal keratinocytes from psoriatic uninvolved skin. Cumulative data (n = 10–12) are expressed as mean ±SEM. Journal of Investigative Dermatology 2001 117, 1538-1545DOI: (10.1046/j.0022-202x.2001.01535.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 α5β1 and αVβ1 integrins mediate psoriatic uninvolved basal keratinocyte spreading responses. Fresh epidermal cell suspensions were incubated with anti-α5, αV and β1 antibodies at 4°C for 1 h before incubation on fibronectin at 37°C for 1 h. After staining for phalloidin and vimentin, the rhodamine-phalloidin+ and vimentin− spreading cells were counted. The rhodamine-phalloidin+ and vimentin− spreading cells following anti-integrin blocking antibody treatment are expressed as a percentage of isotype control. Data are shown as the mean ± SEM, n = 2–3. Journal of Investigative Dermatology 2001 117, 1538-1545DOI: (10.1046/j.0022-202x.2001.01535.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Enhanced FAK expression by psoriatic uninvolved (pso-uni) basal keratinocytes on fibronectin (FN) but not collagen-laminin (Co + La). (A) Fresh epidermal cells from psoriatic uninvolved and normal epidermis were incubated on either fibronectin- or collagen-laminin-coated slides for 1 h. After staining for FAK and phalloidin, FAK positive staining cells were visualized as spread cells with bright green fluorescent points within cytoplasmic extensions and at the condensations of actin microfilaments stained with rhodamine-phalloidin. (a) Psoriatic uninvolved epidermal cells on fibronectin-coated slides; (b) psoriatic uninvolved epidermal cells on collagen-laminin-coated slides; (c) normal epidermal cells on fibronectin-coated slides. (B) Cumulative data from FAK positive staining cell numbers are shown as open bars for epidermal cells obtained from normal subjects and hatched bars for epidermal cells obtained from psoriatic uninvolved patients. Bars represent mean ±SEM, n = 3. Journal of Investigative Dermatology 2001 117, 1538-1545DOI: (10.1046/j.0022-202x.2001.01535.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Elevated levels of phosphoryalated FAK in epidermal cells of psoriatic uninvolved skin compared to normal skin. (A) Western blot analysis of FAK and phosphorylated FAK. Equal amounts of protein (50 µg) of epidermal cell lysates from psoriatic uninvolved (pso-uni) and normal skin, and a FAK positive control (10 µl) were separated by SDS-PAGE, transblotted, immunoblotted with anti-FAK monoclonal antibody (upper panel), and reprobed by anti-PY20 (lower panel). Densitometric quantitation and densities of FAK phosphotyrosine are shown in the open bar for normal and the hatched bar for psoriatic uninvolved epidermal cells. Data are expressed as mean ± SEM, n = 2 (normal), n = 4 (pso-uni), p =0.01. (B) FAK was immunoprecipitated from equal protein (500 µg) amounts from each normal and psoriatic uninvolved epidermal cell lysate. The immunoprecipitated FAK was then separated by SDS-PAGE, transblotted, and immunoblotted with antiphosphotyrosine monoclonal antibody 4G10. Densitometric quantitation and densities of FAK phosphotyrosine are shown in the open bar for normal and the hatched bar for psoriatic uninvolved epidermal cells. Data are expressed as mean ± SEM, n = 2 (normal), n = 4 (pso-uni), p =0.02. Journal of Investigative Dermatology 2001 117, 1538-1545DOI: (10.1046/j.0022-202x.2001.01535.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions