Differentiated Keratinocyte-Releasable Stratifin (14-3-3 Sigma) Stimulates MMP-1 Expression in Dermal Fibroblasts Aziz Ghahary, Yvonne Marcoux, Feridoun.

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Differentiated Keratinocyte-Releasable Stratifin (14-3-3 Sigma) Stimulates MMP-1 Expression in Dermal Fibroblasts Aziz Ghahary, Yvonne Marcoux, Feridoun Karimi-Busheri, Yunyaun Li, Edward E. Tredget, Ruhangiz T. Kilani, Eugene Lam, Michael Weinfeld Journal of Investigative Dermatology Volume 124, Issue 1, Pages 170-177 (January 2005) DOI: 10.1111/j.0022-202X.2004.23521.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Keratinocyte-conditioned medium (KCM) increases collagenase mRNA expression in dermal fibroblasts. Total RNA from either fibroblasts (F/F) or fibroblasts (F/K) co-cultured with keratinocytes for different time points was extracted and used to evaluate the expression of collagenase mRNA by northern analysis (panel A). In panel B, two different strains of keratinocytes (K1 and K2) and fibroblasts (FI and F2) were cultured individually and their conditioned media were collected and passed through a Centricon filter with a 30 kDa cut-off. Fibroblasts were then treated with either unfiltrated whole conditioned medium (C), the corresponding filtrate (F), or retentate (R) for 24 h. Cells were then harvested and extracted RNA was used to evaluate the expression of collagenase mRNA by northern analysis. Panel C shows the pattern of 18S and 28S ethidium bromide-stained ribosomal RNA used as a loading control of the same blot seen in panel B. Journal of Investigative Dermatology 2005 124, 170-177DOI: (10.1111/j.0022-202X.2004.23521.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Recombinant 14-3-3 sigma (14-3-3σ) protein increases the expression of collagenase mRNA in dermal fibroblasts. Cells were treated with either nothing (C) or various concentrations (0.25, 0.5, 1.0, and 2.5 μg per mL) of our recombinant 14-3-3σ for 24 h and the expression of collagenase mRNA and 18S ribosomal RNA (loading control) was assessed by Northern analysis (panel A). To determine the lasting effect of 14-3-3σ on collagenase expression, fibroblasts were treated with either nothing (C) or 2.5 μg per mL of 14-3-3σ for 24 h. The medium was replaced with fresh medium without 14-3-3σ and cells were then harvested at 0, 3, 6, 12, 24, 36, 48, and 72 h post 14-3-3σ removal. The expression of collagenase mRNA in dermal fibroblasts was then evaluated by northern analysis. The pattern of 18S ribosomal RNA is also shown as a loading control. Journal of Investigative Dermatology 2005 124, 170-177DOI: (10.1111/j.0022-202X.2004.23521.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Differentiated keratinocytes release a greater level of 14-3-3 sigma (14-3-3σ) into conditioned medium. To evaluate intra- and extracellular forms of 14-3-3σ protein in differentiated keratinocytes, cells (0.5 × 106 cells per well) were grown in keratinocyte serum-free medium (KSFM) for 48 h and conditioned medium was replaced with test medium. The protein contents of conditioned media collected on day 0, 2, 4, 6, 8, and 10 and corresponding cell extracts were determined by western blot analysis. Panels A and B show the patterns of intracellular form 14-3-3σ and β-actin, respectively. The pattern of the releasable form of 14-3-3σ is shown in panel C. Journal of Investigative Dermatology 2005 124, 170-177DOI: (10.1111/j.0022-202X.2004.23521.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Time-dependent expression of 14-3-3 sigma (14-3-3σ) and involucrin mRNA in keratinocytes. Keratinocytes were cultured in keratinocyte serum-free medium (KSFM) for 48 h and medium was replaced with test medium. Keratinocytes were then harvested on day 0, 1, 2, 3, 4, 5, 14, 18, and 20. Total RNA was extracted and analyzed by northern analysis. The blot was initially hybridized with cDNA specific for 14-3-3σ and subsequently with cDNA for involucrin and 18S ribosomal RNA used as a control for RNA loading (panel A). In another set of experiments, keratinocytes were cultured in KSFM for 48 h and medium was replaced with test medium. The conditioned media collected every 48 h up to 20 d were then used to treat fibroblasts for 24 h (day 2–20). Panel B, total RNA from cells that received either fresh medium (C), fresh medium plus 1 μg per mL of 14-3-3σ protein (K), keratinocyte-conditioned medium (KCM) collected at different days (day 2–20), or conditioned medium from proliferating keratinocyte grown in KSFM plus additive for 48 h (P). The RNA was extracted and analyzed for collagenase mRNA expression. The blot was subsequently re-hybridized with cDNA specific for 18S ribosomal RNA and used as a RNA loading control. Journal of Investigative Dermatology 2005 124, 170-177DOI: (10.1111/j.0022-202X.2004.23521.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunohistochemical detection of 14-3-3 sigma (14-3-3σ) and involucrin in cultured keratinocytes. Panels A and B show the phase contrast microscopic appearance of keratinocytes grown in test medium to induce differentiation on day 5 (A) and day 9 (B). Scale bar=200 μm. Panels C and D show the low magnification of 14-3-3σ (panel C) and involucrin (panel D) of cultured keratinocytes on day 9. Scale bar=500 μm, whereas panels E–H represent a high magnification of corresponding areas related to the red (panels E and F) and green (panels G and H) rectangular insets shown in panels C and D. Journal of Investigative Dermatology 2005 124, 170-177DOI: (10.1111/j.0022-202X.2004.23521.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of 14-3-3 sigma (14-3-3σ) mRNA in keratinocytes correlates with collagenase mRNA expression in dermal fibroblasts grown in a co-culture system. To demonstrate the expression of 14-3-3σ mRNA in keratinocytes (K/F) and correlate that with collagenase expression in dermal fibroblasts (F/K), a keratinocyte/fibroblast co-culture system was established. Cells were then individually harvested and the expression of 14-3-3σ and collagenase mRNA detected by northern analysis was compared with those of cells grown in a mono-culture system (panel A). The pattern of 18S ribosomal RNA obtained from re-hybridization of the same blot used as an RNA loading control (panel B). Journal of Investigative Dermatology 2005 124, 170-177DOI: (10.1111/j.0022-202X.2004.23521.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions