ABSTRACT
AIMS: Joint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs). MAIN METHODS: In MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct. KEY FINDINGS: TNF-alpha (from 0.1 to 10 ng/ml) and IL-1beta (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-alpha-treated cultures did not reverse TNF-alpha effects, indicating that IL-1 was not involved in TNF-alpha-stimulated TNAP activity. Both TNF-alpha and IL-1beta decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures. SIGNIFICANCE: The different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.
Subject(s)
Alkaline Phosphatase/metabolism , Calcification, Physiologic/drug effects , Collagen/antagonists & inhibitors , Core Binding Factor Alpha 1 Subunit/antagonists & inhibitors , Interleukin-1beta/physiology , Mesenchymal Stem Cells/drug effects , Tumor Necrosis Factor-alpha/physiology , Adult , Cell Differentiation/drug effects , Cells, Cultured , Child , Child, Preschool , Collagen/biosynthesis , Core Binding Factor Alpha 1 Subunit/biosynthesis , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Female , Humans , Interleukin-1beta/immunology , Interleukin-1beta/pharmacology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/enzymology , Mesenchymal Stem Cells/immunology , Mesenchymal Stem Cells/metabolism , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/enzymology , Osteoblasts/immunology , Osteoblasts/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The effect of applying JHA ZR-515 at a concentration of 50 microliter/liter water in the larval rearing medium from the first larval instar on oxygen consumption, carbon dioxide out put and total carbohydrates was studied. The oxygen consumed by the pupae previously treated as larvae with ZR-515 was decreased and no adult could emerge. The carbon dioxide out put seems to follow a more or less the pattern of oxygen consumption. The concentration of total carbohydrate content in one day old pupae previously treated as larvae was significantly lower than normals. In three days old there was a marked significant increase and in five days old, the carbohydrate content was significantly higher than controls.