Involvement of PKC in Increased Expression of Beta-defensins in Keratinocyte

Objectives: Human beta-defensin (hBDs) belong to a group of antimicrobial peptides which are mainly expressed in epithelial cells, including oral and skin keratinocytes. Protein kinase C (PKC) regulates keratinocyte differentiation. We have previously reported upregulation of hBDs during keratinocyte differentiation. (Abiko, Y et al. J.Dermatol Sci, 2003). The present study investigated whether PKC activation induced increased expression of hBDs, and PKC inhibited increased expression of hBDs during keratinocyte differentiation. Methods: HaCaT cells, keratinocyte cell lines, were grown in keratinocyte basal medium (KBM) (modified MCDB153, 0.03mM Ca2+). The cells were treated with 100nM phorbol 12-myristate 13-acetate (PMA, PKC activator) for 4h and 24h. In order to stimulate the keratinocyte differentiation, cells were grown in KBM containing 1.8mM Ca2+ for 4days. The cells were daily treated with 1uM GF109203X compound (PKC inhibior) up to 4 days. After 16hr, 2 and 4 days, RNA was isolated from each dish. A quantitative RT-PCR assay was performed TaqMan probes to observe mRNA level of hBD-1~3 and keratinocyte differentiation markers (involucrin, karatin 10 and keratin 14). The statistic significance of the expression was analyzed using Student't t-test (n=5). Results: hBD-2 and -3 mRNA levels were significantly increased by the stimulation with PMA. hBD-1 and -3 mRNA levels were significantly increased during keratinocyte differentiation. PKC inhibitor abolished the increased expression of hBD-1, whereas the inhibitor did not abolish the increased expression of hBD-3. Conclusion: The present study indicates that PKC may be involved in increased expression of hBDs.