(from the application): Lysyl oxidases are primordial enzymes which date back to the Cambrian explosion (550 million years ago), having, subserved the unique function of cross-linking collagen and elastin in the presence of molecular oxygen, producing the strong and flexible ligaments and tendons of large animal bodies. Lysyl oxidases are copper-dependent monoamine oxidase enzymes which even today are responsible for the lysine-derived cross-links in collagen and elastin of all animal species. Elastin and collagen are the major components of the extracellular matrix that impart elasticity and high tensile strength, respectively, to mammalian connective tissues. Cross-links are critical to the structural integrity of both collagen and elastin. In the human and now mouse, two lysyl oxidase genes have been identified: lysyl oxidase-l and lysyl oxidase-2. In the mouse, only lysyl oxidase-l (mLO-l) has been characterized but my preliminary work has identified the murine lysyl oxidase-2 gene (mLO-2). These two genes exhibit differential expression in human and mouse cell lines and tissues. In many adult mouse tissues, LO-2 mRNA expression is readily detectable but LO-1 mRNA expression is undetectable via Northern analysis. However in fetal skin and lung fibroblast cell lines, LO-1 mRNA expression is slightly greater than LO-2, but LO-2 expression is detectable. These data suggest differential mechanisms of gene regulation and potentially cell-specific or tissue-specific expression during normal matrix development and maintenance. Lysyl oxidases are intimately involved with normal extracellular matrix development and turnover, but if abnormally regulated, may contribute to a variety of developmental and fibrotic disorders. The long term goal of this proposal is to delineate the role of the lysyl oxidase-2 gene in the extracellular matrix of connective tissues during normal development and in the future, during pathological fibrosis. To accomplish this, the following specific aims are proposed: 1. Generate lines of mice deficient in mLO-2 (mLO-2 -/-) by targeted mutagenesis; 2. Characterize and determine the spectrum of mLO-2 versus mLO-l mRNA and protein expression in embryonic and normal (mice and human) cells and tissues to assess the phenotype of mLO-2 -/- mice; and 3. Examine the differential expression and regulation of LO-2 versus LO-1 by analysis of their specific promoters using deletion analysis, nuclease hypersensitivity, and in vivo footprinting. Our hypothesis is that mLO-2 deficient mice will have a specific phenotype due to the temporal and spatial differential expression of the mLO-2 gene. This phenotype will result from the specific and independent functions of mLO-2, and this phenotype will be defined in the mLO-2 -/- model. These specific functions of mLO-2 could not be identified with the use of non-specific inhibitors. In the unlikely event that no significant phenotype is observed or cannot be induced upon injury challenge, a double """"""""knockout"""""""" with both LOs would be constructed in the future.
