Regulation of Collagenase Gene Expression by IL-1 Beta Requires Transcriptional and Post-Transcriptional Mechanisms

Interleukin-1 beta is believed to contribute to the pathophysiology of rheumatoid arthritis by activating collagenase gene expression. We have used a cell culture model of rabbit synovial fibroblasts to examine the molecular mechanisms of IL-1 beta-mediated collagenase gene expression. Stimulation of rabbit synovial fibroblasts with 10 ng/ml recombinant human IL-1 beta resulted in a 20-fold increase in collagenase mRNA by 12 h. Transient transfection studies using collagenase promoter-CAT constructs demonstrated that proximal sequences responded poorly to IL-1 beta, possibly due to insufficient activation of AP-1 by this cytokine. More distal sequences were required for IL-1 beta responsiveness, with a 4700 bp construct showing approximately 5-fold induction above control. To examine post-transcriptional mechanisms, transcript from a human collagenase cDNA was constitutively produced by the simian virus 40 early promoter. IL-1 beta stabilized the constitutively expressed human transcript. Furthermore, mutation of the ATTTA motifs in the 3\u27 untranslated region of the human gene also stabilized the transcript. Finally, the rabbit collagenase 3\u27 untranslated region destabilized a constitutively transcribed chloramphenicol acetyltransferase transcript. These data indicate that in addition to activating transcription, IL-1 beta increases collagenase transcript stability by reversing the destabilizing effects of sequences in the 3\u27 untranslated region

Entwicklung muriner cDNA-Arrays zur Bestimmung der Proteinase-Expression arthritischer Fibroblasten im Modell der Antigen-Induzierten Arthritis

Das Ziel der Arbeit war die Entwicklung eines Custom-made-Array als kostengünstigere Alternative zu kommerziell erhältlichen high-density Arrays. Dieser Array sollte zur Untersuchung von Erkrankungen im tierexperimentellen Modell eingesetzt werden, bei welchem eine Veränderung des Proteinase-Expressionsprofils erwartet wird. Nach der Fertigstellung des cDNA-Arrays wurde er im Modell der Antigen-induzierten Arthritis auf seine Anwendbarkeit untersucht. Mit der Methode der Real Time PCR wurde die Gültigkeit der per cDNA-Arrays gewonnenen Daten überprüft

Expression and regulation of VCAM-1 and CD44 by cultured fibroblast-like synoviocytes.

The fibroblast-like synoviocyte (FLS) has been implicated in the destructive process associated with rheumatoid arthritis. In this thesis I demonstrate the expression and regulation of several adhesion molecules expressed on cultured FLSs obtained from inflamed synovium. Unlike fibroblasts from other areas of the body, FLSs constitutively express vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 on RA FLSs is constitutively expressed at high levels during the first 2 weeks of culture. At later time points (4 weeks in culture), VCAM-1 expression declined to low basal levels. A number of strategies were employed to determine the exogenous factors that determine the initially high levels of VCAM-1 in FLSs. Of the extracellular matrix components examined only collagen type I enhanced VCAM-1 expression but this had only limited success. The pro-inflammatory cytokines, IL-1β (10 ng/ml) and TNF-α (10 ng/ml), were also tested and found to induce only transient increases in cell surface VCAM-1 expression. However, the chronic administration of IL-4 or IL-13 in combination with TNF-α resulted in elevated levels of VCAM-1 with prolonged expression Prolonged VCAM-1 expression was found to result in part from the capacity of IL-4 and IL-13 to stabilize VCAM-1 mRNA transcripts. CD44 splice variants, isoforms of the CD44 receptor, that are implicated in the progression of a number of human tumours were also expressed by FLSs isolated from inflamed synovium. Immunohistochemistry, flow cytometry and RT-PCR analysis of CD44 splice variant expression revealed differential expression of a number of variant isoforms. Splice variant expression, at both the mRNA and cell surface protein level, was observed in a large percentage of RA FLSs, it is variable in those from OA synovium, and is absent in cells isolated from non-inflamed joints. However, RA FLSs showed a greater intensity of staining for variants v3, v5 and v7/8. VCAM-1-positive FLSs also demonstrated complex splice variant mRNA transcripts, comprising v3, v6, v7, v8, v9 and v10 in a variety of splicing combinations. These results indicate that the nature of CD44-splice variant expression is closely linked to the inflammatory state of the synovial joint. Moreover, expression of the CD44v7/8 epitope is associated with an increased cellular proliferation rate and could thus be functionally implicated in the hyperplasia observed in RA synovium