Hes1, a new target for interleukin 1beta in chondrocytes.
OBJECTIVE
To investigate the effects of interleukin 1beta (IL1beta) treatment on the Notch1/Hes1 pathway in chondrocytes in vitro.


METHODS
Mouse articular chondrocytes in primary culture were challenged with IL1beta, alone or combined with Notch1 and IL1beta pathway inhibitors. Notch1 and Hes1 expressions were investigated by immunocytochemistry, western blot and real-time quantitative (q)PCR. IL1beta-responsive genes were assessed by real-time qPCR and a specific siRNA against Hes1 was used to identify Hes1 target genes.


RESULTS
Notch1 labelling remained nuclear and stable in intensity irrespective of treatment, suggesting a steady state activation of this pathway in our model. IL1beta transiently increased Hes1 mRNA (2.5-fold) and protein expression in treated versus naive chondrocytes. Hes1 mRNA level then decreased below control and its cyclic pattern of expression was lost. This was associated with nuclear translocation of the cytoplasmic Hes1 protein. IL1beta induced increase in Hes1 mRNA was transcriptional, occurred through nuclear factor (NF)kappaB activation and appeared to be associated with downregulation by its own protein. Hes1 induction was insensitive to the gamma-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), which suggested its independence from novel Notch1 activation. Hes1 expression was efficiently silenced by a specific siRNA. This experiment revealed that Hes1 did not mediate IL1beta-induced downregulation of Sox9, type II collagen and aggrecan transcription but mediated IL1beta induction of matrix metalloproteinase (MMP)13 and ADAM metallopeptidase with thrombospondin type 1 motif, 5 (ADAMTS5). The Hes1-related repressor Hey1 was expressed at a very low level and was not inducible by IL1beta.


CONCLUSIONS
Hes1 is a novel IL1beta target gene in chondrocytes which influences a discrete subset of genes linked to cartilage matrix remodelling and/or degradation.
Ottaviani
Sebastien
S
Universite Paris Descartes; INSERM UMRS-747, Paris, France.
Tahiri
Khadija
K
Frazier
Aline
A
Hassaine
Zohra Nabila
ZN
Dumontier
Marie-France
MF
Baschong
Werner
W
Rannou
François
F
Corvol
Marie-Therese
MT
Savouret
Jean-François
JF
Richette
Pascal
P
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D053583:Interleukin-1beta / Q000494:pharmacology*; D051379:Mice; D016328:NF-kappa B / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051881:Receptor, Notch1 / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction / Q000187:drug effects; D000072056:Transcription Factor HES-1
D051792:Basic Helix-Loop-Helix Proteins; D017931:DNA Primers; D018398:Homeodomain Proteins; D053583:Interleukin-1beta; D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051881:Receptor, Notch1; D000072056:Transcription Factor HES-1; C489734:Hes1 protein, mouse; C497762:Notch1 protein, mouse