Curcumin Suppresses AP1 Transcription Factor-dependent Differentiation and Activates Apoptosis in Human Epidermal Keratinocytes

Sivaprakasam Balasubramanian,Richard L Eckert

doi:10.1074/jbc.m606003200

Sivaprakasam Balasubramanian, Richard L Eckert

Open Access

https://doi.org/10.1074/jbc.m606003200

Copy DOI

Abstract

The diet-derived cancer preventive agent, curcumin, inhibits skin cancer cell proliferation and tumor formation. However, its effect on normal human keratinocyte differentiation, proliferation, and apoptosis has not been adequately studied. Involucrin (hINV) is a marker of keratinocyte differentiation and a useful model for the study of chemopreventive agent action. We show that curcumin suppresses the differentiation agent-dependent activation of hINV gene expression and that an AP1 transcription factor DNA binding site in the hINV gene is required for this regulation. A protein kinase C, Ras, MEKK1, MEK3 signaling cascade controls hINV expression by regulating AP1 factor level. Curcumin treatment inhibits the novel protein kinase C-, Ras-, and MEKK1-dependent activation of hINV promoter activity and reduces the differentiation agent-dependent increase in AP1 factor level and DNA binding. This reduction requires proteasome function. In addition, curcumin treatment reduces cell number, which is associated with a reduced cyclin and cdk1 levels. Curcumin treatment also suppresses the Bcl-xL level, leading to reduced mitochondrial membrane potential and increased cleavage of procaspases and poly(ADP-ribose) polymerase. These studies provide important insights regarding the mechanism whereby curcumin acts as a chemopreventive agent in normal human epidermis.

Highlights

Administration of 0.2–1% curcumin in the mouse diet is sufficient to achieve this response [7,8,9]
Our results suggest that curcumin treatment inhibits keratinocyte proliferation and TPA-dependent activation of involucrin gene expression and enhances keratinocyte apoptosis
To study the impact of curcumin on keratinocyte differentiation, we selected a gene, involucrin, which is expressed in the differentiated suprabasal epidermal layers [20]

Summary

EXPERIMENTAL PROCEDURES

Chemicals and Reagents—Phorbol ester (TPA), Me2SO, and curcumin were obtained from Sigma. Mouse monoclonal anti-caspase 8 (#9746) and rabbit polyclonal antibodies specific for caspase 9 (#9502) and caspase 3 (#9665) were purchased from Cell Signaling (Beverly, MA). Preparation of Cell Extracts and Immunoblot Analysis—Seventy percent confluent human keratinocytes were treated with 50 ng/ml TPA in the absence or presence of 20 ␮M curcumin. The cells were treated for 24 h with 50 ng of TPA/ml or 20 ␮M curcumin, and extracts were prepared for assay of luciferase activity. Proteasome Activity Assay—Subconfluent keratinocyte cul- tored the impact of curcumin treatment on activity of contures, maintained in 35-mm dishes, were treated with 20 ␮M cur- structs encoding point mutations at the AP1 and EBS-2 sites. As cumin prior to preparation of total cell extracts according the shown, TPA treatment increased pINV-241 and manufacturer’s instructions (Proteasome Activity kit APT280, pINV-241(EBS-2m) activity, and this response was suppressed.

RESULTS

DISCUSSION