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Genetic deficiency or pharmacological inhibition of cyclooxygenase-1 or -2 induces mouse keratinocyte differentiation in vitro and in vivo¹

Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA; and
^* Myriad Pharmaceuticals, Inc., Salt Lake City, Utah, USA

²Correspondence: National Institute of Environmental Health Sciences, M.D. C4-09, PO Box 12233, Research Triangle Park, NC 27709, USA. E-mail: langenb1{at}niehs.nih.gov

SPECIFIC AIMS

In mouse skin, genetic deficiency of COX-1 or COX-2 is associated with premature terminal differentiation of epidermal keratinocytes and reduced tumorigenesis. COX-2-selective and dual COX inhibitors have been shown to reduce skin tumorigenesis in some mouse models. The mechanisms by which these compounds act are not well understood. Our aim was to use in vivo and in vitro approaches to determine whether selective COX inhibitors like COX deficiency could cause premature terminal differentiation of keratinocytes. By correlating the results of COX deficiency and COX inhibitors, we can better identify the COX-dependent mechanisms by which inhibition of COX activity suppresses epidermal tumors.

PRINCIPAL FINDINGS

1. Celecoxib, a COX-2-selective inhibitor, and SC-560, a COX-1-selective inhibitor, induce epidermal keratinocyte differentiation in vivo
To determine whether selective inhibition of the COX isoforms in the epidermis would induce keratinocyte differentiation as observed in COX-deficient mice, we treated mice with TPA (4 µg/mouse) three times/wk or TPA plus daily doses of celecoxib or SC-560 (both at 500 µg/mouse) topically for 2 wk. Figure 1 shows representative skin sections from control and COX inhibitor-treated mice, as well as the percentage of the basal epidermal cells expressing K1. Skin from the mice in all treatment groups was hyperplastic, but the number of nucleated cell layers was significantly reduced in celecoxib- and SC-560-treated epidermis, averaging 4.68 ± 0.18 in the celecoxib and 4.80 ± 0.10 in SC-560-treated skin vs. 6.38 ± 0.03 in the TPA-only treatment. In mice treated with TPA only, K1 expression in basal cells was only 3% of the total basal cells (K1-positive cells are stained brown; Fig. 1A, D ). Treatment with SC-560 and celecoxib increased the number of basal cells staining for K1 to 15% and 16.5%, respectively, (Fig. 1B-D ). To determine whether the COX inhibitors inhibited prostaglandin biosynthesis, we measured the PGE₂ levels in the skin of the TPA- and TPA + COX inhibitor-treated mice. In TPA-treated skin where COX-1 is the major isoform expressed, SC-560 inhibited PGE₂ production by 80% and celecoxib inhibited 50% of the PGE₂ produced (Fig. 1E ).

View larger version (69K): [in this window] [in a new window]   Figure 1. Effect of selective COX inhibitors on keratin 1 (K1) expression in mouse skin. Wild-type mice were treated topically with 4 µg TPA five times with or without daily administration of COX inhibitors (500 µg/mouse). Skin samples were taken 16–18 h after the last treatment and processed for histology. K1 expression was detected by immunohistochemistry (K1-positive cells are stained brown). In mice treated with acetone-TPA (A), there were few basal epidermal cells staining positive for K1, whereas in celecoxib- (B) or SC-560-treated skin (C) the number of basal epidermal cells expressing K1 was increased. D) Bar chart shows results obtained when K1-positive basal cells were counted and expressed as a % of total number of basal cells (mean±SE). E) The amount of PGE2 (mean±SD) was determined in skin. Celecoxib and SC-560 induced K1 expression and reduced PGE2 biosynthesis significantly. The experiment was conducted with at least 2 mice per group and repeated once with similar results. *Significant difference between control and treatment. *P 0.05, **P 0.01.

2. Deficiency of COX-1 or COX-2 leads to altered expression of K1 and involucrin in mouse primary keratinocytes
To determine whether differentiation was altered in primary keratinocytes from COX-deficient cells, K1 and involucrin (an early and a late marker of keratinocyte differentiation, respectively) were analyzed. In attached confluent keratinocytes, K1 expression was increased fivefold in COX-1^–/– and COX-2^–/– cells compared with the respective wild-type keratinocytes; involucrin was increased 50% in the COX-1^–/– cells and increased threefold in the COX-2-deficient keratinocytes. In the spontaneously detached (differentiated) keratinocytes, K1 was increased in COX-1- and COX-2-deficient cells, but to a greater extent in the COX-2^–/– cells. Involucrin was not altered in COX-1-deficient cells and increased threefold in the COX-2^–/– cells. Although COX-1 contributes only a low level of PGE₂ in cultured cells, both forms of COX have a role in keratinocyte differentiation.

3. COX inhibitors lead to increased K1 expression but no change in apoptosis of primary mouse keratinocytes
The effects of COX inhibitors on cells in vitro were determined by exposing wild-type primary keratinocyte cultures to celecoxib (10 µm), SC-560 (10 µm), and indomethacin (50 µm). Whole cell homogenates from the attached keratinocytes were subjected to Western blot analysis. Celecoxib and indomethacin both increased K1 expression in attached keratinocytes. However, possibly because of the low COX-1 expression in attached keratinocytes, SC-560 did not alter K1 levels significantly (Fig. 2 A). Densitometric analysis of the K1 bands showed a time-dependent increase of K1 after exposure to celecoxib and indomethacin, with only minimal changes observable at 24 h and further increases at 48 h (Fig. 2B ). These results suggest that K1 expression was increased in keratinocyte cultures primarily by the pharmacological inhibition of COX-2.

View larger version (21K): [in this window] [in a new window]   Figure 2. Effects of COX inhibitors on K1 expression, cell numbers, and apoptosis of primary mouse keratinocytes. Cells grown for 5 days were exposed to celecoxib (10 µM), SC-560 (10 µM) or indomethacin (50 µM). Attached cells were harvested 24 or 48 h after treatment. A) K1 expression was determined in whole cell homogenates by Western analysis. B) Densitometric analysis of the signal intensities of the K1 bands at 24 and 48 h; bars = control (black), SC-560 (gray), indomethacin (hatched), celecoxib (white). Data are means ± SD. C) The effect of celecoxib, indomethacin, and SC-560 on the number (triplicate cultures) of attached primary keratinocytes per culture. D) The effect of COX deficiency and COX inhibition on apoptosis in primary keratinocytes (attached and detached combined). % apoptosis obtained by FACS analysis of annexin V-FITC and propidium iodide-stained cells is shown. UV was used as a positive control. Experiment was performed using duplicate samples and repeated with similar results. C, D) Data = mean ± SE. *Significant difference between control and treatment. *P 0.05, **P 0.01.

Because differentiated cells detach from the culture dish and COX inhibitors induced differentiation, the effect of the inhibitors on cell numbers was determined. Celecoxib and indomethacin reduced the number of attached cells compared with vehicle controls in a time-dependent manner (Fig. 2C ). At 24 h after treatment, only celecoxib reduced the number of cells significantly. However, 48 h after treatment there was a 50% reduction of attached keratinocytes in celecoxib- and indomethacin-treated cultures (Fig. 2C ) and increased numbers of detached cells (data not shown). SC-560 treatment at 10 µm did not have a significant effect on the number of attached keratinocytes at either time (Fig. 2C ).

As COX inhibitors have been shown to induce apoptosis in cultured cells, we hypothesized that increased apoptosis and altered differentiation could be a possible mechanism by which the COX inhibitors reduced attached keratinocyte numbers. We determined the effects of the COX inhibitors on keratinocyte apoptosis in vitro. The number of apoptotic keratinocytes in UV-treated cultures (positive control) increased by 2.5 fold compared with control cultures (Fig. 2D ). No differences in the level of apoptosis were observed in vehicle and inhibitor-treated groups or between wild-type and COX-deficient keratinocytes (Fig. 2D ). Thus, increased apoptosis was not a significant effect of the COX inhibitors or COX deficiency on primary keratinocytes in culture.

CONCLUSIONS AND SIGNIFICANCE

Our results indicate that the pharmacological inhibition and genetic deficiency of COX-1 or COX-2 have similar effects on keratinocyte differentiation. Several mechanisms have been proposed to account for the anti-tumorigenic activity of NSAIDs, and the data reported here suggest that the induction of terminal differentiation may be an important mechanism by which NSAIDs could alter epidermal physiology and reduce skin tumorigenesis.

Our findings that celecoxib induced differentiation, but not apoptosis, in cultured keratinocytes is unique in that celecoxib and other COX inhibitors have been shown to primarily induce apoptosis in a variety of cell types in culture. In agreement with the current findings, we earlier showed that in the mouse initiation/promotion model, apoptosis was not significantly elevated in the skin or papillomas from the COX-deficient mice, whereas premature terminal differentiation was more evident in skin and papillomas of these mice. Thus, altered keratinocyte differentiation may be an important mechanism by which either inhibition or absence of COX activity leads to reduced tumor formation.

This study therefore provides the first evidence, that pharmacological inhibition of either COX isoform causes premature terminal differentiation of keratinocytes. Increased differentiation is an important mechanism of reduction of tumorigenesis since keratinocytes that have entered the program of differentiation lose their proliferative capacity. As depicted in Fig. 3 , reduction of prostaglandins achieved by the absence of COX or COX-selective inhibition causes accelerated differentiation, which leads to reduced skin tumorigenesis.

View larger version (21K): [in this window] [in a new window]   Figure 3. Summary of mechanisms by which deficiency or inhibition of COX may lead to increased differentiation and reduced skin tumorigenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1192fje

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