NICD was overexpressed in primary chondrocytes from RosaNotch mice, where the Rosa26 promoter precedes a loxP-flanked STOP cassette followed by the NICD coding sequence. Cells were infected with adenoviral vectors expressing Cre to induce NICD or green fluorescent protein (GFP) as control. Gene expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Il6 protein concentration in the culture media was determined by enzyme-linked immunosorbent assay (ELISA). To test the mechanisms of Notch action on Il6 expression, cells were transfected with a fragment of the Il6 promoter or control vector pGL3, or transcriptionally arrested with 5,6-dichloro-1-¦Â-d-ribofuranosylbenzimidazole. Il6 was inhibited with a neutralizing antibody, whereas a normal immunoglobulin G (IgG) was used as control.
NICD induced Il6 mRNA and protein, and transactivated the Il6 promoter without affecting Il6 mRNA stability. Il6 neutralization had no impact on gene expression under basal conditions, and did not modify the effects of NICD on sex determining region-Y-related high mobility group-box gene (Sox)9, collagen type II ¦Á1 (Col2a1) and collagen type X ¦Á1 (Col10a1) expression. Conversely, Il6 neutralization opposed aggrecan (Acan) suppression and prevented matrix metalloprotease (Mmp)13 induction by NICD.
Il6 mediates suppression of Acan and induction of Mmp13 expression by Notch in chondrocytes.