Responses to the proinflammatory cytokines interleukin-1 and tumor necrosis factor alpha in cells derived from rheumatoid synovium and other joint tissues involve nuclear factor kappaB-mediated induction of the Ets transcription factor ESE-1.
OBJECTIVE
To investigate the expression of the novel Ets transcription factor ESE-1 in rheumatoid synovium and in cells derived from joint tissues, and to analyze the role of nuclear factor kappaB (NF-kappaB) as one of the central downstream targets in mediating the induction of ESE-1 by proinflammatory cytokines.


METHODS
ESE-1 protein expression was analyzed by immunohistochemistry using antibodies in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). ESE-1 messenger RNA (mRNA) levels were analyzed by reverse transcriptase-polymerase chain reaction or Northern blotting in human chondrocytes, synovial fibroblasts, osteoblasts, and macrophages, before and after exposure to interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), or lipopolysaccharide (LPS) with or without prior infection with an adenovirus encoding the inhibitor of nuclear factor kappaB (IkappaB). The wild-type ESE-1 promoter and the ESE-1 promoter mutated in the NF-kappaB site were cloned into a luciferase reporter vector and analyzed in transient transfections. Electrophoretic mobility shift assays (EMSAs) and supershift assays with antibodies against members of the NF-kappaB family were conducted using the NF-kappaB site from the ESE-1 promoter as a probe.


RESULTS
Immunohistochemical analysis showed specific expression of ESE-1 in cells of the synovial lining layer and in some mononuclear and endothelial cells in RA and OA synovial tissues. ESE-1 mRNA expression could be induced by IL-1beta and TNFalpha in cells such as synovial fibroblasts, chondrocytes, osteoblasts, and monocytes. Transient transfection experiments and EMSAs showed that induction of ESE-1 gene expression by IL-1beta requires activation of NF-kappaB and binding of p50 and p65 family members to the NF-kappaB site in the ESE-1 promoter. Overexpression of IkappaB using an adenoviral vector blocked IL-1beta-induced ESE-1 mRNA expression. Chromatin immunoprecipitation further confirmed that NF-kappaB binds to the ESE-1 promoter in vivo.


CONCLUSIONS
ESE-1 is expressed in synovial tissues in RA and, to a variable extent, in OA, and is specifically induced in synovial fibroblasts, chondrocytes, osteoblasts, and monocyte/macrophages by IL-1beta, TNFalpha, or LPS. This induction relies on the translocation of the NF-kappaB family members p50 and p65 to the nucleus and transactivation of the ESE-1 promoter via a high-affinity NF-kappaB binding site. ESE-1 may play a role in mediating some effects of proinflammatory stimuli in cells at sites of inflammation.
Grall
Franck
F
New England Baptist Bone and Joint Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, and Beth Israel Deaconess Medical Center Genomics Center, Boston, Massachusetts 02115, USA.
Gu
Xuesong
X
Tan
Lujian
L
Cho
Je-Yoel
JY
Inan
Mehmet Sait
MS
Pettit
Allison R
AR
Thamrongsak
Usanee
U
Choy
Bob K
BK
Manning
Cathy
C
Akbarali
Yasmin
Y
Zerbini
Luiz
L
Rudders
Susan
S
Goldring
Steven R
SR
Gravallese
Ellen M
EM
Oettgen
Peter
P
Goldring
Mary B
MB
Libermann
Towia A
TA
D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D015152:Blotting, Northern; D002460:Cell Line; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007375:Interleukin-1 / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics* / Q000276:immunology; D015345:Oligonucleotide Probes / Q000737:chemistry; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015534:Trans-Activators / Q000096:biosynthesis* / Q000235:genetics; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C105529:ELF3 protein, human; D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha