Veli UĞURCU, Sümeyya AKYOL, Aynur ALTUNTAŞ, Rıdvan FIRAT, S. Fatih KURŞUNLU, Özlem ÇAKMAK, Kadir DEMİRCAN

The effects of insulin on the expression levels of ADAMTS6 & 19 in OUMS-27 cell

Amaç: A Disintegrin-like Metalloproteinase with Thrombospondin Motifs (ADAMTS) proteinleri ekstraselüler matrikste (ECM) bulunan bir çeşit matriks metalloproteinaz enzimidir. İnsulin birçok dokuda etki gösteren önemli bir anabolik hormondur. Bu çalışmanın amacı OUMS-27 insan kondrosarkom hücre kültüründe insülinin, fonksiyonları tam olarak bilinmeyen ADAMTS enzimlerinden ADAMTS6 ve 19 ekspresyonu üzerine zamana bağımlı olarak etkilerini değerlendirmek ve insülinin anabolik etkilerinden dolayı ADAMTS ekspresyonunu azaltma hipotezini test etmektir. Yöntemler: OUMS-27 hücrelerinin, 10 μg/mL insulin içeren ve içermeyen Dulbeccos modified Eagle besiyerinde (DMEM) kültürü yapıldı. 11. güne kadar iki günde bir besiyeri ile birlikte insülin takviyesi yapıldı. Hücreler 1, 3, 7 ve 11. günlerde harvest edilerek uygun zamanlarda RNA izolasyonu yapıldı. ADAMTS6 ve 19 RNA ekspresyonları uygun primerler kullanılarak qRT-PCR yöntemi ile ölçüldü. Bulgular: qRT-PCR cihazının verilerine göre, insülin uygulanmasından 1 gün sonra başlamak üzere deneyin son günü olan 11. güne kadar ADAMTS6nın mRNA düzeyinde ciddi bir azalma meydana geldi (p=0.008). İnsülin uygulanan gruplarda ADAMTS6 oranı kontrol grubuna kıyasla 1/2 ve 1/4 aralığında değişti. İnsülin verilen hücre grubunda ADAMTS19 mRNA düzeyindeki değişiklikler ise kontrol grubuna kıyasla istatistiksel olarak anlamsızdı. Sonuç: Sonuçlarımız, insülinin ADAMTS6 düzeylerini azaltarak ekstrasellüler matriks bileşenlerinin kaybının telafi edilmesinde potansiyel bir etkisinin olabileceğini gösterdi. Bu hipotez ve bulgunun anlaşılması için yetim ADAMTS proteinlerinin gerçek fonksiyonlarının inceleneceği çok sayıda araştırma yapılmasına ihtiyaç vardır.

İnsülinin OUMS-27 hücrelerinde ADAMTS6 ve 19 ekspresyon düzeylerine etkileri

Objective: A Disintegrin-like Metalloproteinase with Thrombospondin Motifs (ADAMTS) proteins are kind of matrix metalloproteinase enzymes that primarily founds in the extracellular matrix (ECM). Insulin is an important anabolic hormone, which acts on many tissues. The aim of this study is to evaluate the time-dependent effects of insulin on the two functionally unknown enzyme expressions (ADAMTS6 & 19) in OUMS-27 human chondrosarcoma cell line. Methods: OUMS-27 cells were cultured in Dulbeccos modified Eagle medium (DMEM) alone and DMEM containing 10 μg/mL insulin. The medium was changed every other day up to 11th day. Cells were harvested at 1, 3, 7, and 11th days and RNA isolation was performed at appropriate times according to study setup. The levels of RNA expression of ADAMTS6 and 19 were estimated by qRT-PCR using appropriate primers. Results: According to qRT-PCR analysis, ADAMTS6 mRNA expression was found to be decreased as early as one day after insulin application and continued up to day 11, the last day of insulin induction (p=0.008). The ratio of ADAMTS6 in insulin-applied groups was changed between 1/2 and 1/4 of control values. The changes in ADAMTS19 mRNA levels in insulin-applied cells were not statistically significant compared to control cell group. Conclusion: Our results demonstrated that insulin has a potential effect on alleviation of loss of extracellular matrix compounds by diminishing ADAMTS6 levels. To test this hypothesis and finding, more investigations are needed to recognize the real functions of orphan ADAMTS proteins.

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