Increased and Decreased Expression of Beta-defensins Alter Keratinocyte Differentiation

Objective: Human beta-defensins (hBDs) belong to a group of antimicrobial peptides expressed mainly in epithelial cells, including oral and skin keratinocytes. The present study investigated whether increased or decreased expression of hBDs in keratinocytes induces their differentiation. Methods: KB cells and HaCaT cells, keratinocyte cell lines, were grown in DMEM supplemented with 10% fetal bovine serum. For generating KB cells stably transfected with hBDs, the Flp-InTM system (Invitrogen, CA) was used. In order to generate knockdown of hBDs in HaCaT cells, siRNA inserts of hBD-1, -2 and -3 were designed using website named siRNA target finder (Ambion, TX). Synthetic oligos of hBD-1, -2 and -3 were inserted into Hind III and BamHI sites of pSilencerTM 4.1-CMV hygro vector (Ambion, TX). The plasmids were transfected into HaCaT cells using lipofection method (EffectenTM, Qiagen, Germany). Increased and Silencing transcription of hBDs cells was confirmed by RT-PCR and quantitative RT-PCR using TagMan probes(Applied Biosystem, CA). Expression levels of keratin 10, 14 and involucrin were observed by quantitative RT-PCR. Cell growth was evaluated by XTT assays (Sigma-Aldrich, MO). The results were compared between hBDs-KB cells or sihBDs-HaCaT and control cells. The statistical significance of the difference was analyzed using the Student t-test and Scheffes test. Results: The hBDs-KB cells showed higher level of keratin 10 than the control cells. The sihBDs-HaCaT showed lower level of keratin 10 and higher level of keratin 14 than the control cells. The cell growth rate of KB-hBDs was significantly lower than that of control cells (p<0.05). The cell growth rate of sihBDs (-1, -2 and -3)-HaCaT was significantly higher than that of control cells (p<0.0001). Conclusion: The results indicate that increased and decreased expression of hBDs in keratinocytes may alter their differentiation.