Meprin Metalloproteases Modulate Epithelial Barrier Integrity and Monocyte Migration

Open Access
- Author:
- Bao, Jialing
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 29, 2011
- Committee Members:
- Judith S Bond, Dissertation Advisor/Co-Advisor
Gail Lynn Matters, Committee Chair/Co-Chair
Sergei A Grigoryev, Committee Member
W. Brian Reeves, Committee Member
Harriet C Isom, Committee Member - Keywords:
- Meprin
Tight junctions
Occludin
Epithelial barrier
Monocyte - Abstract:
- Meprin metalloproteases have been implicated in a number of normal developmental and pathological processes. However, it has been difficult to establish the cellular and molecular basis for the biological role of meprin metalloproteases in health and disease, possibly because of the large number of metalloproteases, many having overlapping substrate specificities. A number of important biological molecules have been demonstrated to be meprin substrates in vitro, such as extracellular matrix and cytokines. With the development of congenic mice lacking meprin activity, it has been possible to relate in vitro results with in vivo data to examine cellular and molecular processes. The hypothesis of the thesis work is that meprins relax epithelial barriers by cleaving tight junction proteins, and facilitate monocyte migration. The study demonstrated that homomeric meprin A and meprin B cleaved the tight junction protein occludin, but not claudin-4, in membrane fractions from MDCK cells. Meprin A, but not meprin B, added exogenously to MDCK monolayers cleaved occludin. Meprin A, but not meprin B, cleaved recombinant occludin extracellular loops, and the cleavage site was determined in the first extracellular loop of occludin. Different cleavage site preferences at extracellular regions explain the different results between meprin A and meprin B to cleave occludin in intact cells and cell extacts. The biological relevance of the in vitro experiments was demonstrated by studies in cell culture, in vivo and ex vivo. Meprin A disrupted the immunostaining of tight junction proteins occludin and ZO-1 on MDCK monolayers. In addition, meprin A impaired the barrier function of MDCK monolayers, as shown by increased small molecule flux and decreased transepithelial electrical resistance. To elucidate the role of meprin A in acute urinary tract infections, meprin A was infused into the mouse bladder and the effects on bladder epithelium wall was investigated. The results showed that active meprin A increased the permeability of the epithelium as demonstrated by the influx of a fluorescene dye. The hypothesis that meprin A disrupts epithelial barriers and facilitates monocyte migration was further investigated by co-culturing monocytes with MDCK monolayers and measuring monocyte migration. Monocytes from mice lacking meprin A (meprin KO) were less able to migrate through MDCK monolayers than monocytes from wild-type mice. It is concluded that the capability of meprin A to disrupt epithelial barriers is one important factor by which meprin A modulates inflammation.