| Summary: | The destruction of articular cartilage is a central feature of arthritis. The activity of collagenase enzymes, induced by pro-inflammatory cytokines, is a key step in this process. Collagenases have been targeted therapeutically but trials have not proven beneficial due to off-target effects. Understanding the signalling consequences which drive the expression of these proteolytic enzymes is therefore a major area of research. The aim of the present study was to elucidate the signalling events that regulate collagenase expression, focusing on the role of a small family of serine/threonine kinases termed protein kinase D (PKD). To understand the role of the individual PKD isoforms in the modulation of collagenase gene expression, each isoform was selectively silenced. Using a model of the pro-inflammatory milieu prevalent in arthritic disease, the consequences of isoform specific gene silencing on the expression of MMP-1 and MMP-13 was studied in primary chondrocytes stimulated with IL-1 in combination with OSM. Data suggest PKD1 to be ‘anabolic’, with gene silencing leading to increased collagenase gene expression. PKD2 was shown not to significantly modulate the collagenase expression, whereas, PKD3 silencing markedly reduced the collagenase gene expression. To understand the signalling consequences orchestrated by each isoform of PKD, the role each isoform in the regulation of signalling pathways known to modulate collagenase gene expression was examined. PKD1 and PKD3 silencing both abrogated the phosphorylation of the MAPK signalling pathways (ERK, JNK and p38). PKD3 silencing also led to decreased STAT-1 and STAT- 3 serine phosphorylation. This contrasted with the effects of PKD1 silencing, in which STAT-1 serine and tyrosine phosphorylation increased, as well as increased Akt and p65 phosphorylation being observed. These opposing roles may explain the differences in the regulation of collagenase gene expression between each isoform. To further understand this modulation the expression of the AP-1 components, Fos and Jun were examined, along with other recently identified post-AP-1 factors (ATF3, EGR2 NFATc1, and BMP-2). Data showed PKD3 silencing to reduce their expression, suggesting a potential mechanism by which PKD3 signalling can modulate MMP expression. In conclusion, this work identifies the distinct individual roles of PKD isoforms in the modulation of collagenase gene expression, illustrating the need to assess individual kinases within a family.
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