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THU0049 Protein kinase inhibition enhances steady state mrna levels encoding il -16 but reduces il-18 encoding message levels in fibroblasts
  1. H Schützle1,
  2. M Weis-Klemm1,
  3. RE Gay2,
  4. S Gay2,
  5. WK Aicher1
  1. 1Centre for Orthopedic Surgery, University of Tübingen Medical School, Tübingen, Germany
  2. 2WHO Rheumatology Research Centre, University Hospital, Zürich, Switzerland


Background In previous studies we have shown that synovial fibroblasts from rheumatoid arthritis (RA) patients transcribe elevated levels of IL-16 encoding mRNA. In addition, in RA synovial fluid high concentrations of IL-16 were detected. The chemotactic activity to CD4+ cells was associated with IL-16. In osteoarthritis synovial fibroblasts, IL-16 was not found.

Objectives Therefore we initiated experiments to study the activation pathway regulating IL-16 transcription in synovial versus skin fibroblasts.

Methods OA synovial fibroblasts (n = 3), skin fibroblasts (n = 6) and neonatal preputial fibroblasts (n = 3) were expanded (less than 4 passages). Cells were incubated in complete medium (10% FCS) supplemented with MAS-7, ocadaic acid (ODA), ionomycin, cAMP, forskolin, staurosporin, phorbolester, H7 and an unrelated peptide as mock control. Untreated cells served as controls. After 6 to 48 h of incubation, mRNA was extracted from experimental samples and controls. The mRNA yield was determined by spectrophotometry and, cDNA was generated from 1 μg RNA using the oligo (dT) priming procedure. Steady state transcript amounts encoding IL-16 or IL-18 were determined by real time quantitative RT/PCR (LightCycler, Roche) using the SYBR green method. The PCR products were visualised by EtBr fluorescence after gel electrophoresis. Quantitative GAPDH RT/PCR served as internal standard.

Results mRNA encoding IL-16 and IL-18 was detected in all fibroblasts analysed. Incubation of the fibroblasts for 24 to 48 h with staurosporin or forskolin enhanced IL-16 steady state mRNA levels two to three fold. Incubation with ODA or H7 reduced IL-16 mRNA three-fold and ten-fold, respectively. PMA reduced IL-16 signals to about 70%. Interestingly, IL-18 signals were not reduced in ODA treated fibroblasts whereas H7 reduced IL-18 signals about ten-fold. Staurosporin and forskolin did not enhance IL-18 encoding signals but reduced the IL-18 signals two to three fold. IL-18 encoding mRNA amounts were elevated in ionomycin treated cells. All other components did not show considerable activation or reduction of the IL-16 or IL-18 RT/PCR signals. In addition, there was no remarkable difference in IL-16 or IL-18 transcriptional responses to all agents analysed so far.

Conclusion Staurosporin is a broad protein kinase inhibitor and activated IL-16 transcript levels whereas phorbolester PMA activates protein kinase C and reduced IL-16 mRNA levels. We conclude that IL-16 transcription is regulated by kinase/phosphatase signal transduction pathways. In addition, a cAMP dependent enhancement of IL-16 endocing steady state mRNA levels was observed. By contrast, IL-18 RT/PCR signals are reduced upon kinase inhibition but not activated after PMA incubation. Raising intracellular calcium by ionomycin induced IL-18 encoding mRNA. Therefore the IL-16 and IL-18 gene activities are regulated differently. Since in RA synovial fibroblasts IL-16 mRNA is elevated when compared to OA fibroblasts and blocking kinase activity activated IL-16 mRNA levels in OA synovial or skin fibrobasts, we hypothesise that a reduced kinase activity may be associated with enhanced levels of IL-16 message in RA synovial fibroblasts.

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