Date of Award
4-18-2008
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biology
First Advisor
Gene A. Homandberg
Abstract
It has been shown that fibronectin fragments (Fn-fs) greatly augment cartilage proteoglycan (PG) depletion by up-regulating matrix metalloproteinases (MMPs) and catabolic cytokines. The activities of three different Fn-fs have been studied: an amino terminal 29-kDa which is most potent with a weak affinity to the α5β1, integrin, the receptor for native fibronetin (Fn); a 50-kDa gelatin-binding Fn-f with no known interaction with Fn receptor; and a 140-kDa Fn-f which is the least potent and has a RGDS sequence which can bind to the α5β1 integrin. Confocal studies showed that Fn-fs caused diffusion of α5β 1 integrin clusters which were seen in untreated or Fn treated cells. Cortical actin and focal contacts were also affected by Fn-fs. These changes were associated with activation of proline rich tyrosine kinase 2 (Pyk2), the soluble form of focal adhesion kinase (FAK). Two lower MW Src-family kinases were also activated. However, the phosphorylation of FAK was not enhanced by Fn-fs. The mitogen activated protein kinases (MAP kinases)-p38, JNK1/2, and ERK1/2 were activated by Fn-fs while Fn only activated ERK1/2 and slightly enhanced phosphorylation Fn-fs also induced the activation of the inflammatory transcription factor-NF-κB. The activation of above signaling factors were demonstrated in kinetics and dose response studies by using phospho-specific antibodies. Confocal microscopy was also applied to visualize the location of activated kinases. The active roles of the kinases were confirmed by demonstration that specific inhibitors to Pyk2, MAP kinases and NF-κB blocked up-regulation of MMP-3 and -13 induced by Fn-fs and reversed the PG depletion caused by Fn-fs. The activation of Pyk2 but not of MAP kinases was required for the NF-κB activation. Investigation of the linkages of the kinases suggests the existence of multiple signaling arms. To summarize, Fn-fs, which can induce potent cartilage chondrolysis, alter normal Fn signaling to chondrocytes through changing the distribution of integrin receptors, the structure of focal contacts and actin and activation of integrin linked kinases, MAP kinases and NF-κB.
Recommended Citation
Ding, Lei, "Altered Signaling By Fibronectin Fragments Creates A Potent Chondrolytic State" (2008). Theses and Dissertations. 8008.
https://commons.und.edu/theses/8008