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.
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.
Franck
F
Grall
Xuesong
X
Gu
Lujian
L
Tan
Je-Yoel
JY
Cho
Mehmet Sait
MS
Inan
Allison R
AR
Pettit
Usanee
U
Thamrongsak
Bob K
BK
Choy
Cathy
C
Manning
Yasmin
Y
Akbarali
Luiz
L
Zerbini
Susan
S
Rudders
Steven R
SR
Goldring
Ellen M
EM
Gravallese
Peter
P
Oettgen
Mary B
MB
Goldring
Towia A
TA
Libermann
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