TNF-alpha and IL-1beta inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells.
OBJECTIVE
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).


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.


RESULTS
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.


CONCLUSIONS
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.
Ding
J
J
Laboratory of Cellular and Molecular Biology EA2603, IFR114, ULCO, Université Lille Nord de France, France.
Ghali
O
O
Lencel
P
P
Broux
O
O
Chauveau
C
C
Devedjian
J C
JC
Hardouin
P
P
Magne
D
D
D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism*; D002113:Calcification, Physiologic / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D003094:Collagen / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D050659:Core Binding Factor Alpha 1 Subunit / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D053583:Interleukin-1beta / Q000276:immunology / Q000494:pharmacology / Q000502:physiology*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000494:pharmacology / Q000502:physiology*
D050659:Core Binding Factor Alpha 1 Subunit; D053583:Interleukin-1beta; C493709:RUNX2 protein, human; D014409:Tumor Necrosis Factor-alpha; D003094:Collagen; D000469:Alkaline Phosphatase