TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

Abstract

The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial-stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin.

Keywords: Dermis; Keratinocytes; MMP-10; NIKS; TCDD; TIMP-1.

Figure 1. TCDD results in intercellular spaces and a diffuse basement membrane morphology in organotypic cultures of human keratinocytes

NIKS were grown in organotypic culture in the presence of DMSO (upper panel) or 10 nM TCDD (lower panel) for 18 days. Cultures were then fixed, paraffin embedded, sectioned and stained with hematoxylin and eosin. Black bars designate the cornified layer while arrows designate intercellular spaces. White scale bars each represent 50 µm.

Figure 2. TCDD treatment increases the expression of MMP-10 in organotypic cultures of keratinocytes

(A) NIKS were grown in organotypic culture in the presence of DMSO or 10nM TCDD. Total RNA was isolated on day 18 post-treatment. Relative expression level of MMP-1, MMP-2, MMP-3 MMP-9 and MMP-10 mRNA were determined by Q-PCR analysis. MMP transcript levels were normalized to the level of cyclophilin-A transcript, and expressed as fold induction of the level found in day 18 DMSO-treated organotypic cultures for each gene. Data shown are mean ± SEM from a single experiment performed in triplicate and are representative of at least 3 independent experiments. *** P<0.001 by t-test. (B,C) NIKS cells or (D,E) dermises lacking keratinocytes were grown in organotypic culture in the presence of DMSO or 10 nM TCDD. Eighteen days post-treatment, cultures were sectioned and stained using a monoclonal antibody against human MMP-10 (green). Sections were counterstained with Hoechst to visualize the nuclei (blue). Scale bars equal 50 µm.

Figure 3. TCDD increases expression of MMP-10 in monolayer cultures of keratinocytes

(A) NIKS cells were grown without feeder layers in 6 well plates and treated with DMSO. The relative expression level of MMP-10 mRNA was determined by Q-PCR at day 6, 8, 12 and 18 post-treatment. Values were normalized to cyclophilin-A, then expressed as fold induction of the level found in day 6 DMSO treated cells. Data shown are means ± SEM from a single experiment performed in triplicate and are representative of at least 3 independent experiments. (B) The relative expression level of MMP-10 mRNA was calculated by Q-PCR for NIKS treated with DMSO control, or 10nM TCDD, at 12 days post-treatment. The levels of MMP-10, were normalized to cyclophilin-A, then expressed as fold induction over day 6 DMSO treated cells. Data shown are means ± SEM from a single experiment performed in triplicate and are representative of at least 3 independent experiments. *P<0.05 by t-test (C) Whole cell lysates of NIKS treated with DMSO or 10 nM TCDD for 12 days (left panel) or 12 hr conditioned media from these cultures (right panel) were resolved by SDS-polyacrylamide gel electrophoresis and probed using a monoclonal antibody against human recombinant MMP-10 (R&D systems, Minneapolis, MN).

Figure 4. NIKS^TIMP-1 exhibits elevated TIMP-1 protein expression and activity

(A) Diagram of K14/TIMP-1/pUB/BS vector. NIKS cells transfected with either empty vector (NIKS^EV) or K14/TIMP-1/pUB/BS (NIKS^TIMP-1) were grown without feeder layers in 60 mm dishes and conditioned with 3:1 phenol red-free HAM’s F-12:HyQ DMEM/High Modified medium for 24 hr. (B) TIMP-1 protein expression was quantified by ELISA. (C) TIMP-1 activity was determined using a fluorescent substrate assay. Results were standardized to the cell number present at time of conditioned medium collection, and are expressed as fold induction of the EV control cells. (D) NIKS^TIMP-1 shows greater inhibition of MMP-10 activity than unmodified NIKS. Data shown are means ± SEM from 3 independent experiments. *** P<0.05 by t-test.

Figure 5. TIMP-1 overexpressing keratinocytes show no change in tissue architecture following TCDD treatment in organotypic culture

NIKS^EV (A,C) and NIKS^TIMP-1 (B,D) cells were grown in organotypic culture in the presence of DMSO (A,B) or 10nM TCDD (C,D) for 10 days. Cultures were then fixed, paraffin embedded, sectioned and stained with hematoxylin and eosin. White dashed lines designate the boundary between the stratum corneum and stratum granulosum. Black bars indicate the cornified layer. The arrow highlights separation of the dermis from basal layer. The arrow head designates intercellular spaces. White scale bars each equal 50 µm.