ABSTRACT
Prostaglandin plays a significant role in the local control of bone metabolism associated with periodontal disease. delta12-PGJ2 is a natural PGD2 metabolite that is formed in vivo in the presence of plasma. It is known for delta12-PGJ2 to stimulate calcification in osteoblastic cells. Bone morphogenetic protein(BMP) stimulated osteoblastic differentiation in various types of cells and greatly enhanced healing of bony defects. The purpose of this study was to evaluate the effect of rhBMP-2 on delta12-PGJ2 induced osteoblastic differentiation and mineralization in vitro. A human osteosarcoma cells line Saos-2 were cultured. In the test groups, 10-7M of delta12-PGJ2 or mixture of 10-8M of delta12-PGJ2 and 100ng/ml of rhBMP-2 or 100ng/ml of rhBMP-2 were added to culture media. After 1 day, 2 days and 4 days of culture period, the cell number was measured. Alkaline phosphatase activity was measure at 3 days. Reverse transcription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein at 8 hours, 1 day and 7 days. The ability to produce mineralized nodules in rat osteoblasts(MC3T3-E1) was evaluated at 21 days. The results were as follows : 1. rhBMP-2 or mixture of rhBMP-2 and delta12-PGJ2 inhibited cell proliferation of human osteosarcoma cells. 2. rhBMP-2 or mixture of rhBMP-2 and delta12-PGJ2 stimulated alkaline phosphatase activity significantly higher than delta12-PGJ2 alone. 3. rhBMP-2 or mixture of rhBMP-2 and delta12-PGJ2 stimulated mineralization compared to delta12-PGJ2 alone. 4. mRNA of alkaline phosphatase, BMP-2, cbfa 1, Type I collagen were detected in the group treated with delta12-PGJ2/rhBMP-2, rhBMP-2 alone, delta12-PGJ2 alone. These results show that mixture of delta12-PGJ2 and rhBMP-2 causes more bone formation than delta12-PGJ2 alone while the bone formation effects of mixture of delta12-PGJ2 and rhBMP-2 are less than those of rhBMP-2 alone. Further researches would be necessary to clarify the interactions of these agents.
Subject(s)
Animals , Humans , Rats , Alkaline Phosphatase , Bone Matrix , Cell Count , Cell Proliferation , Collagen Type I , Culture Media , Metabolism , Osteoblasts , Osteogenesis , Osteosarcoma , Periodontal Diseases , Plasma , Prostaglandin D2 , Reverse Transcription , RNA, MessengerABSTRACT
delta12-Prostaglandin (PG) J2 is known to elicit an anti-neoplastic effects via apoptosis induction. Previous study showed delta12-PGJ2-induced apoptosis utilized caspase cascade through cytochrome c-dependent pathways in HeLa cells. In this study, the cellular mechanism of delta12-PGJ2- induced apoptosis in HeLa cells, specifically, the role of two mitochondrial factors; bcl-2 and apoptosis-inducing factor (AIF) was investigated. Bcl-2 attenuated delta12-PGJ2-induced caspase activation, loss of mitochondrial transmembrane potential (delta psi m), nuclear fragmentation, DNA laddering, and growth curve inhibition for approximately 24 h, but not for longer time. AIF was not released from mitochondria, even if the delta psi m was dissipated. One of the earliest events observed in delta12-PGJ2-induced apoptotic events was dissipation of delta psi m, the process known to be inhibited by bcl-2. Pre-treatment of z-VAD- fmk, the pan-caspase inhibitor, resulted in the attenuation of delta psi m depolarization in delta12-PGJ2- induced apoptosis. Up-regulation of Sox-4 protein by delta12-PGJ2 was observed in HeLa and bcl-2 overexpressing HeLa B4 cell lines. Bcl-2 overexpression did not attenuate the expression of Sox-4 and its expression coincided with other apoptotic events. These results suggest that delta12-PGJ2 induced Sox-4 expression may activate another upstream caspases excluding the caspase 9-caspase 3 cascade of mitochondrial pathway. These and previous findings together suggest that delta12-PGJ2-induced apoptosis in HeLa cells is caspase-dependent, AIF-independent events which may be affected by Sox-4 protein expression up-regulated by delta12-PGJ2.
Subject(s)
Female , Humans , Amino Acid Chloromethyl Ketones/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Caspases/physiology , Cytochromes c/physiology , Flavoproteins/metabolism , HeLa Cells , High Mobility Group Proteins/physiology , Membrane Proteins/metabolism , Mitochondria/metabolism , Prostaglandin D2/pharmacology , Protein Transport/physiology , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Transcriptional Activation , Trans-Activators/physiologyABSTRACT
We reported earlier that expression of Sox-4 was found to be elevated during prostaglandin (PG) A2 and delta(12)-PGJ(12) induced apoptosis in human hepatocarcinoma Hep3B cells. In this study, the role of Sox-4 was examined using human Hep3B and HepG2 cell lines. Sox-4 induction by several apoptotic inducer such as A23187 (Ca(2+) ionophore) and etoposide (topoisomerase II inhibitor) and Sox-4 transfection into the cells were able to induce apoptosis as observed by the cellular DNA fragmentation. Antisense oligonucleotide of Sox-4 inhibited the induction of Sox-4 expression and blocked the formation of DNA fragmentation by PGA(2) and delta(12)-PGJ(12) in Hep3B and HepG2 cells. Sox-4-induced apoptosis was accompanied with caspase-1 activation indicating that caspase cascade was involved in this apoptotic pathway. These results indicate that Sox-4 is involved in Hep3B and HepG2 cells apoptosis as an important apoptotic mediator.