Human neutrophil lactoferrin trans-activates the matrix metalloproteinase 1 gene through stress-activated MAPK signaling modules

It has been proposed that human neutrophil lactoferrin (Lf) could be involved in gene expression as a DNA-binding protein after its translocation into the nucleus. However, the molecular basis of Lf action has not been defined, and Lf-regulated target genes have not been identified. We report here that overexpressed Lf functions as a specific trans-activator of matrix metalloproteinase 1 (MMP1) gene, and that induction of this AP-1-responsive gene is mediated via the stress-activated MAPK signaling modules. Transactivation of the MMP1 promoter by overexpressed Lf requires the presence of an AP-1 binding site. In gel shift experiments, Lf did not interact directly with AP-1-containing fragments of the MMP1 promoter. However, nuclear extracts from Lf-expressing cells contained increased levels of proteins that bound to AP-1 elements. This Lf-induced AP-1 DNA binding activity was reduced by a p38 MAPK inhibitor. Inhibitors of the MEK kinases had little effect on Lf-induced AP-1. However, expression of dominant-negative MKK4 or JNK1 inhibited Lf-induced gene expression. The JNK activity stimulated by Lf correlates with the enhanced AP-1 binding ability. These findings demonstrate that the Lf-induced activation of AP-1 is mediated via JNK and p38 MAPK pathways.     

Basic calcium phosphate crystals induce matrix metalloproteinase-1 through the Ras/mitogen-activated protein kinase/c-Fos/AP-1/metalloproteinase 1 pathway. Involvement of transcription factor binding sites AP-1 and PEA-3.

Synovial fluid basic calcium phosphate (BCP) crystals are common in osteoarthritis and are associated with severe degenerative arthropathy. Besides stimulating synovial fibroblast-like cells to proliferate, BCP crystals are a potent inducer of human matrix metalloproteinases (hMMPs), which can speed up the articular joint tissue degeneration of osteoarthritis patients. Here, we report that transfections with hMMP1 luciferase reporter plasmids in fibroblast-like synoviocytes revealed that the induction of hMMP1 promoter by BCP crystals was mainly mediated through the -72AP-1 element. Elimination of the -72AP-1 element either by mutation or deletion abolished the induction of hMMP1 promoter activity by BCP crystals almost completely. Interestingly, a mutation at the -88PEA-3 site also abolished the induction of hMMP1 promoter. Further mutation at the -181AP-1 site resumed the induction, indicating that the -181AP-1 element had an effect opposite to the -72AP-1 element. The effect of -181AP-1 could be inactivated either by a mutation at this -181AP-1 site or by the -88PEA-3 element. In addition, dominant negative Ras, Raf, and MEK1/2 could block the induction of hMMP1, and a MEK1/2-specific inhibitor (UO126) could block the induction of hMMP1 and c-Fos by BCP crystals. Taken together, these data indicate that multiple elements, including at least AP-1 and PEA-3, are involved in the induction of hMMP1 gene expression by BCP crystals and that the induction follows the Ras/MAPK/c-Fos/AP-1/MMP1 signaling pathway.