Specific pro-apoptotic fibronectin fragments modulate proteinase expression in periodontal ligament cells

Abstract

Background: During inflammation and periodontal disease, the extra-cellular matrix protein fibronectin (FN) is degraded into fragments whose activities differ from those of the intact molecule. In periodontal ligament cells in vitro, some fragments elevate the expression of matrix metalloproteinases and serine proteinases, while others induce apoptosis through a mechanism regulated by caspase proteases and specific signaling pathways. In this study, we investigated whether the pro-apoptotic V+H- fibronectin fragment modulates proteinase expression as part of the apoptotic mechanism in periodontal ligament cells. The importance of this study is that a structurally similar fibronectin fragment found in vivo (40-kDa) is associated with periodontal disease status. However, the recombinant V+H- FN protein which is structurally and functionally similar to the in vivo 40-kDa fragment was chosen as the focus of these studies over the in vivo fragment because of the ability to readily produce and purify large quantities of recombinant fragments for detailed studies. Thus, findings from this study relate to our understanding of how the extracellular matrix of the periodontal ligament in an inflamed environment may contribute to periodontal disease and its progression.

Methods: We used substrate zymography, reverse zymography, proteinase inhibitors, and partial sequencing to investigate whether the pro-apoptotic V+H- fibronectin fragment modulates proteinase expression as part of this apoptotic mechanism.

Results: Incubation with the V+H- fragment reduced the expression of all gelatinolytic proteinases and inhibitors commonly expressed by periodontal ligament cells. In the presence of caspase inhibitors, inhibitors known to suppress apoptosis, however, the reduced proteinase profile was rescued, showing that caspase inhibitors were able to reverse the reduced proteinase profile and indicating that caspase-mediated pathways are pertinent to fibronectin-fragment-mediated matrix metalloproteinase expression. In addition, the V+H- fragment also triggered the expression of a unique high molecular weight gelatinolytic proteinase of approximately 200 kDa. This proteinase was a serine proteinase, whose identity is not known.

Conclusion: These findings suggest that matrix-degrading proteinases may be involved in apoptosis as part of a unique mechanism of periodontal tissue breakdown, in which novel proteinases may help execute the dissolution of the extracellular matrix.