Date of Award

2013

Document Type

Thesis

First Advisor

Ongeri, Dr. Elimelda Moige

Abstract

Meprins, metalloproteases that are highly expressed in the brush-border membranes of kidney proximal tubules, have been associated with ischemia-reperfusion induced kidney injury. Osteosarcoma 9 (OS9), a peripheral membrane protein, has been shown to selectively interact with the carboxyl-terminal tail of meprin beta (β). Additionally, OS9 was shown to promote oxygen-dependent degradation of the hypoxia-inducible factor 1α (HIF1α), a protein involved in oxygen homeostasis. However, it is not known if OS9 is a meprin substrate. The objective of this study was to determine if OS9 is a meprin substrate, and whether there is a correlation between expression of meprins, OS9, and HIF1α under hypoxic conditions. To determine if meprins are capable of degrading OS9, purified human OS9 was co incubated with activated recombinant forms of meprin A and B. Our results show that meprin B cleaves/degradation OS9. This was not observed for meprin A, suggesting isoform-specific cleavage. Mardin-Darby canine kidney (MDCK) cells and human embryonic kidney 293 cells transfected with meprin α or β cDNA were depleted of oxygen by exposure to 125 µM cobalt chloride. Non-transfected cells served as controls. Western blot analysis was used to evaluate the nuclear fraction levels of OS9 and HIF1α. Our data showed that HIF-1α stabilized in the nucleus in a time-dependent manner and the levels of OS9 increased in non-transfected controls cells depleted of oxygen. To our knowledge, this is the first study to identify OS9 as a meprin B substrate. The degradation of OS9 by meprin B could impact the hypoxic response in kidney cells.

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