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Relationship Between Cell-Associated Matrix Metalloproteinase 9 and Psoriatic Keratinocyte Growth  Nathalie Buisson-Legendre, Hervé Emonard, Philippe.

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Presentation on theme: "Relationship Between Cell-Associated Matrix Metalloproteinase 9 and Psoriatic Keratinocyte Growth  Nathalie Buisson-Legendre, Hervé Emonard, Philippe."— Presentation transcript:

1 Relationship Between Cell-Associated Matrix Metalloproteinase 9 and Psoriatic Keratinocyte Growth 
Nathalie Buisson-Legendre, Hervé Emonard, Philippe Bernard, William Hornebeck  Journal of Investigative Dermatology  Volume 115, Issue 2, Pages (August 2000) DOI: /j x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Growth rate of first passage keratinocytes from two psoriatic patients and two healthy individuals. ▪, Psoriatic patient, aged 38 y; ▵, psoriatic patient, aged 35 y; ♦, healthy individual, aged 30 y; ○, healthy individual, aged 47 y. Cell counts represent the mean ± SEM of three triplicate samples. *Significantly different from both control values at p < 0.01; **p < Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 MMP-9 production by keratinocytes as a function of cell growth. (A) Gelatin zymography: lane 1, conditioned medium from control keratinocytes (age 30 y), 3 d of culture; lane 2, conditioned medium from psoriatic keratinocytes (age 38 y), 3 d of culture. (B) ELISA: ▴, conditioned medium from control keratinocytes (age 30 y); ⋄, conditioned medium from psoriatic keratinocytes (age 38 y). Data are means of three separate determinations. Bars represent SEM values. Similar differences between control and psoriatic keratinocytes have been obtained with two other pairs. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Influence of batimastat and MMP-9 blocking antibody on the proliferation of psoriatic keratinocytes (78-y-old). Keratinocytes were seeded at 1 × 104 cell density and effectors were added at day 1 of culture: ♦, cell growth in the absence of added effector; ▵, cell growth in the presence of MMP-9 blocking antibody (100 ng per ml); ▪, cell growth in the presence of batimastat (100 nM). Data are means of three separate determinations; bars represent SEM values. *Significantly different from control at p < 0.01; **p < Batimastat and MMP-9 blocking antibody also induced significant differences in cell growth using two other psoriatic keratinocyte strains. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Effect of truncated ceramides (C2 or C6 ceramides) and bacterial Smase on normal and psoriatic keratinocyte growth. Cells were seeded at 1 × 104 density and treated on day 2 of culture with Smase or ceramide. On day 4 of culture, after 48 h of treatment with ceramide or Smase, cell number was determined by the crystal violet assay. Significantly different from control (absence of treatment) at *p < 0.02; **p < 0.01; ***p < Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Increased production of MMP-9 and TIMP-1 following treatment of psoriatic keratinocytes with bacterial Smase. (A) MMP-9; (B) TIMP-1. Cells seeded at 1 × 104 density were treated on day 2 of culture with Smase (100 mU per ml). MMP-9 and TIMP-1 secreted levels were determined by ELISA following 48 h of culture and expressed as pg per cell: 1, psoriatic patient (38-y-old), lesional skin; 2, psoriatic patient (78-y-old), lesional skin; 3, psoriatic patient (35-y-old), lesional skin; 4, psoriatic patient (35-y-old), nonlesional skin. (A) MMP-9: control; in presence of Smase; inset, gelatin zymography (from patient 1). (B) TIMP-1: ▪, control; m, in presence of Smase; inset, gelatin reverse zymography (from patient 1). Data are means of three separate determinations. Significantly different from control at *p < 0.01; at **p < Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Presence and regulation of MMP-9 in cell membrane extracts of normal and psoriatic keratinocytes. (A) Normal (lanes 1 and 2) or psoriatic (lanes 3 and 4) keratinocytes were either treated with bacterial Smase (100 mU per ml) (lanes 2 and 4) or left untreated (lanes 1 and 3). (B) Control keratinocytes (lane 1) were treated with 100 mU per ml of Smase alone (lane 2) or in combination with 5 μM of batimastat (lane 3). After 24 h of treatment, the aqueous phase of the membrane extract of each set of keratinocytes was analyzed by gelatin zymography. (C) The electrophoretic position of pro-MMP-9 was identified by Western blotting of 10 ng purified pro-MMP-9 (lane 2). The aqueous phase of membrane extracts from psoriatic keratinocytes was also subjected to gelatin zymography (lane 1) or Western blotting after electrophoretic separation under reducing (lane 3) or nonreducing (lane 4) conditions. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Influence of TIMP-1 on keratinocyte growth. Psoriatic (Ps) as well as control (C) keratinocytes were seeded at a density of 104 cells per dish. On day 2 of culture, they were treated with 5 μg per ml anti-TIMP-1 antibody or left untreated. Cell proliferation was analyzed at 48 h (A) or 72 h (B). Significantly different from control at *p < 0.01; **p < Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 8 Presence of TIMP-1 in cell membrane extracts of psoriatic keratinocytes. The aqueous phase of membrane extracts from 1 × 105 psoriatic keratinocytes treated with 100 mU per ml of Smase for 48 h was incubated with gelatin beads. Both bound and unbound fractions were analyzed by gelatin reverse zymography. Lane a, MMP-2-TIMP-2 complex; lane b, bound fraction (note the presence of both MMP-9 and TIMP-1 in the gel); lane c, unbound fraction. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

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