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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Investigation Of The Inflammatory Pathways In Spontaneously Differentiating Caco-2 Cells

Astarci, Erhan 01 July 2011 (has links) (PDF)
Intestinal epithelial differentiation entails the formation of highly specialized cells with specific absorptive, secretory, digestive and immune functions. Cell-cell and cell-microenvironment interactions appear to be crucial in determining the outcome of the differentiation process. Using the Caco-2 cell line that can undergo spontaneous differentiation when grown past confluency, we observed a loss of VCAM1 (vascular cell adhesion molecule-1) expression while ICAM1 (intercellular cell adhesion molecule-1) expression was seen to be stable in the course of differentiation. Protein kinase C theta (PKC&theta / ) acted downstream of PKC to inactivate Inhibitor of kappa B (IB) and activate NF-&kappa / B in the undifferentiated cells and this axis was inhibited in the differentiated cells. The increase in ICAM1 expression in the differentiated cells was due to a transcriptional upregulation by C/EBP. The protein expressions of both ICAM-1 and VCAM-1, however, were found to decrease in the course of differentiation, with both proteins getting post-translationally degraded in the lysosome. Functionally, a decrease in adhesion to HUVEC cells was observed in the differentiated Caco-2 cells. Thus, the regulation of ICAM-1 and VCAM-1, although both NF-B target genes, appear to be different in the course of epithelial differentiation. microRNAs are known to regulate many cellular pathways. miR-146a, which is known to target NF-&kappa / B, was shown to be highly upregulated in differentiated Caco-2 cells. As a predicted target of miR-146a, mRNA and protein expression of MMP16 was inversely correlated with miR-146a during differentiation of Caco-2 cells. miR-146a could bind to the 3&rsquo / UTR of MMP16 and ectopic expression of miR-146a resulted in a decreased mRNA and protein expression of MMP16 in the undifferentiated Caco-2 and HT-29 cells. Functionally, decreased gelatinase activity determined by gelatin zymography and reduced invasion and migration through Transwells was observed. In the final part of the thesis, the inhibition of NF-&kappa / B via PPAR&gamma / in 15-Lipoxygenase-1 (15LOX1) expressing cells was investigated. The expression of 15LOX1, a member of the inflammatory arachidonate cascade, could lower phosphorylation of I&kappa / B&alpha / and NF-&kappa / B DNA binding activity which was reversed with a 15LOX1 inhibitor. This inhibition was mediated by phospho-PPAR&gamma / , which in turn was phosphorylated by ERK1/2.

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