The Effects of PPAR-γ Activators on the Growth and Apoptosis of Melanocytes

Abstract

PURPOSE: Peroxisome proliferator-activated receptors (PPARs) play an important role in cellular responses including proliferation and differentiation. It was reported that 3 subtypes of PPAR are expressed in human melanocytes and activators for PPAR-α and PPAR-γ modulates melanocyte growth and melanogenesis. In this study, I investigated the effects of PPAR-γ activators on melanocyte growth and apoptosis. I further examined the regulation of extracellular signal regulated kinase (ERK) pathway under ciglitazone treatment. METHODS: The growth of melanocytes was measured using the Coulter counter and the effects on pigmentation were investigated with measurement of melanin contents of cultured melanocytes and skin. Apoptosis of cells were detected by TUNEL assay and flow cytometry. The expression of caspase-3 and extracellular signal regulated kinase in melanocytes was measured by Western blot analysis. RESULTS: All three PPAR-γ activators, ciglitazone, troglitazone, and 15d-PGJ₂, inhibited the melanocyte growth in a concentration-dependent fashion. At the highest concentration of 15d-PGJ₂, cytotoxicity was observed by trypan blue assay. Minimal, not statistically significant, increase of melanin content was observed in treatment with troglitazone and 15d-PGJ₂. Statistically significant differences were seen on ciglitazone-treated cells. Ciglitazone also induced pigmentation of cultured skin. Ciglitazone induced apoptosis and increased the expression of caspase 3. Ciglitazone also activated ERK, and mitogen-activated protein kinase kinase 1/2 inhibitor, PD 98059, decreased ciglitazone-induced ERK activation. Pretreatment with PD 98059 significantly prevented against the ciglitazone-induced cell growth inhibition, melanogenesis, and apoptosis. CONCLUSIONS: 1) PPAR-γ activators, ciglitazone, troglitazone, and 15-deoxy-PGJ₂, inhibited cell growth. 2) Ciglitazone increased melanin contents of melanocytes and cultured skin. 3) Ciglitazone inhibited growth of human melanocytes via the induction of apoptosis. 4) The effects of ciglitazone on melanocytes seems to be dependent on ERK signaling pathway.

연구목적: Peroxisome proliferator-activated receptors (PPARs)는 nuclear receptor superfamily 일종으로 다양한 세포의 성장 및 분화에 관여한다. 사람 멜라닌세포에 PPAR-α,β,γ 세가지 아형이 모두 존재하고 PPAR-α and PPAR-γ 활성제가 사람 멜라닌세포의 성장과 멜라닌생성 조절에 관여한다고 보고된 바 있다. 따라서 본 연구에서는 PPAR-γ 활성제가 멜라닌세포의 성장, 색소형성 및 세포고사에 미치는 영향을 알아보았다. 재료 및 방법: 사람 멜라닌세포를 배양하여 PPAR-γ 활성제가 멜라닌세포 증식에 미치는 영향을 Coulter counter를 이용하여 측정하였다. 또한 PPAR-γ 활성제가 멜라닌세포와 기관배양된 피부의 색소형성에 미치는 영향을 확인하였다. 멜라닌세포의 세포고사는 TUNEL 염색과 유식 세포분석기로 확인하고, Western blot법을 이용하여 caspase-3와 extracellular signal-regulated protein kinase (ERK)의 발현을 확인하였다. 결과: PPAR-γ 활성제 중 ciglitazone, troglitazone, 15-deoxy- Δ^(12,14)-prostaglandin J₂(15d-PGJ₂)는 멜라닌세포의 성장을 억제하였다. 15d-PGJ₂는 심한 세포독성을 보였다. 멜라닌세포의 멜라닌 양은 모든 PPAR- γ 활성제에 의해 증가하였으나, ciglitazone만 유의성을 보였고, 피부 기관배양에서 ciglitazone은 세포 수의 변화 없이 멜라닌 양을 증가시켰다. Ciglitazone 처리시 멜라닌세포에서 세포고사가 유도되었고, cleaved caspase-3가 발현되었다. Ciglitazone은 멜라닌세포에서 ERK를 활성화시키고, 그 억제제인 PD98059를 전처리 했을 때, ciglitazone에 의한 ERK 활성이 억제되었다. 또한 PD98059 전처리로 ciglitazone에 의한 멜라닌세포 성장 억제, 색소형성 및 세포고사의 유도가 유의하게 억제되었다. 결론: 1) PPAR-γ 활성제(ciglitazone, troglitazone, 15-deoxy-PGJ₂)는 멜라닌세포 성장을 억제한다. 2) Ciglitazone은 멜라닌세포와 피부 기관배양에서 멜라닌 양을 증가시킨다. 3) Ciglitazone은 세포고사를 통하여 멜라닌세포의 성장을 억제한다. 4)멜라닌세포에 대한 ciglitazone의 영향은 ERK 신호전달체계와 연관이 있을 것으로 생각한다.