טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAvital Weiss
SubjectCharacterization of a 28kDa Form of Heme Oxygenase-1 in
Mouse Macrophages
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Dr. Meyron Holtz Esther
Full Thesis textFull thesis text - English Version


Abstract

I. A central step of iron recycling is the phagocytosis of senescent erythrocytes by cells of the reticuloendothelial system such as macrophages. In response to erythrophagocytosis, heme-oxygenase-1 (HO-1) levels are elevated, to catalyze the oxidative degradation of erythrocyte-heme to iron, CO and biliverdin. HO-1 is an inducible protein of 32kDa. Shorter forms of HO-1 were found in pathogenic bacteria and after trypsin treatment. In an in vivo model of hypertransfusion optimized in our lab two isoforms of HO-1 were detected after Western blot analysis, one of the expected 32kDa HO-1 and the other of about 28kDa.

The expression of HO-1 28kDa was further investigated in mouse macrophages. Opsonization or oxidation of red blood cells were used to induce erythrophagocytosis and subsequent elevation of HO-1 expression. HO-1 expression was tested using Western blot analysis. 28kDa HO-1 was found to be expressed as a function of time in an opposite pattern to the 32kDa HO-1, in mouse derived primary culture of macrophages but not in macrophage-cell lines. This trend was seen both in control and treated cells after erythrophagocytosis, but not in macrophages after phagocytosis of pathogenic bacteria.

The origin and processing pathways of the 28kDa HO-1 were analyzed. RNA processing was tested using Northern blot analysis and the technique of rapid amplification of cDNA ends. Protein processing was tested using pulse chase experiments and mass spectrometry analysis. It seems that HO-1 28kDa originates from the same gene as the 32kDa form and arises from RNA processing. The results suggest that splicing of HO-1 mRNA at the 3' end leads to several transcripts, possibly one with a shorter C-terminus. This result is reinforced by mass spectrometry.


II. A correlation between the cationic anti-microbial peptide (CAP), hepcidin, and the process of angiogenesis was tested. Hepcidin is known to attach to ferroportin, the sole known iron exporter and cause its internalization and degradation when systemic need for iron is low, thus, intra cellular iron levels are elevated. In the process of angiogenesis, there is massive growth of blood vessels. In cancer, angiogenesis enables blood and oxygen supply to the tumor. This process is mediated by the vascular endothelial growth factor (VEGF). VEGF is regulated both by hypoxia and iron levels. Preliminary result suggests that hepcidin can down regulate VEGF expression level, probably by the increase of intracellular iron. Many CAPs have been shown to inhibit angiogenesis and are considered as potential treatment for cancer.