Katalin Csiszar

Functional analysis of the promoter and first intron of the human lysyl oxidase gene

Alterations in the synthesis and activity of lysyl oxidase occur concomitant with developmental changes in collagen and elastin deposition and with the pathogenesis of several acquired and heritable connective tissue disorders. To begin to unravel the mechanisms that control lysyloxidase gene expression, we have previously reported the complete exon-intron structure of the human lysyl oxidase gene. We have now sequenced this entire gene, including all six introns and 4 kb of DNA 5′ of exon 1. Analysis of over 13 kb of intervening sequence and 5′ flanking sequence revealed a concentration of conserved consensus sequence elements within the first intron and 1 kb immediately 5′ of exon 1. Analysis of intron 1 and the 5′ flanking domain, using recombinant plasmids containing the chloramphenicol acetyl transferase (CAT) reporter gene, identified functional DNA sequence elements within these non-coding domains responsible for inhibition and up-regulation of CAT activity in primary cultures of human skin fibroblasts, in smooth muscle cells, revertant cells derived from an osteosarcoma cell line and malignant c-Ha-ras-transformed osteosarcoma cells. DNA sequence elements within intron 1, in particular, resulted in a marked increase in CAT reporter activity in cultured fibroblasts, smooth muscle cells and osteosarcoma cells. In c-Ha-ras-transformed osteosarcoma cells, however, no such enhancer activity of intron 1 sequence was observed. Ras-transformed osteosarcoma cells exhibited reduced steady-state levels of lysyl oxidase mRNA that was primarily controlled through reduced transcription of the lysyl oxidase gene. The lack of any up-regulation of CAT activity in these ras-transformed cells by sequence elements within intron 1 suggests a complex interaction between cis-acting domains and trans-acting transcriptional factors in the 5′ promoter domain and the first intron of the lysyl oxidase gene.

The size heterogeneity of human lysyl oxidase mRNA is due to alternate polyadenylation site and not alternate exon usage

We have isolated the entire gene coding for human lysyl oxidase. Coding and untranslated domains of human lysyl oxidase mRNA were found in 7 exons, distributed throughout approximately 14kb of human genomic DNA. The appearance of exon sequences in lysyl oxidase mRNA in several human tissues was determined using a reverse transcriptase-PCR assay. In contrast to a previous report, this analysis has unambiguously shown that the size heterogeneity of lysyl oxidase mRNA was not due to alternate usage of any of the exons of the lysyl oxidase gene. Moreover, DNA sequence analysis of the entire 3.8 kb 3′-untranslated region (UTR) within exon 7 revealed multiple poly-adenylation sites which were shown to be differentially expressed in human skin fibroblasts. This differential usage of polyadenylation sites within the 3′-UTR explains the appearance of multiple lysyl oxidase mRNAs of different sizes.

Persistent Inflammatory Pathways Associated with Early Onset Myocardial Infarction in a Medicated Multiethnic Hawaiian Cohort

In spite of current standard therapies to target the major pathomechanisms in myocardial infarction (MI), inflammatory gene expression patterns have been consistently revealed in MI patients. In a multiethnic cohort, we aimed to identify MI-associated pathomechanisms that may be unresponsive to medical treatment to improve diagnosis and therapy. Gene expression profiles in whole blood were analyzed in medicated Asian, African American and Caucasian patients living in Hawaii with a history of early MI and age, ethnicity, risk factor and medication-matched controls. PANTHER ontological and Ingenuity Pathway analysis and functional evaluation of the consistently differentially expressed genes identified coordinated up-regulation of genes for inflammation (LGALS3, PTX3, ZBTB32, BCL2L1), T-cell activation (IL12RB1, VAV3, JAG1, CAMP), immune imbalance (IL-8, IL2RA, CCR7, AHNAK), and active atherosclerosis (NR1H4, BIN1, GSTT1, MARCO) that persist in MI patients in spite of concerted treatment efforts to control vascular pathology. Furthermore, significant ethnic differences appear to exist within the active disease mechanisms that need to be further investigated to identify key targets for effective medical intervention.