Bp

Fig. 2. Expression of LOX mRNA. RT-PCR was carried out with LOX-specific oligonucleotides using total RNA isolated from fibroblasts of MD patients and controls. The results suggest increase in LOX mRNA levels in the patient with the OHS phenotype (lane 1) as well as in the patients with the classical severe form (lanes 2 and 3) compared to controls (laned 4 and 5). M = molecular-weight marker.

5. EXPRESSION PROFILING IN MENKES DISEASE

5.1. Using Fibroblasts

Disturbance in copper homeostasis in MD is reflected in cultured skin fibroblasts, where a number of copper-dependent enzymes are expressed (see Section 3.2.). Fibroblasts thus provide a useful model for studying the effect of the disturbed copper metabolism on various copper-requiring enzymes and transport pathways through defining the differentially expressed genes.

Fibroblast is an excellent cell type, especially in studying connective tissue disturbance, which is a principal manifestation in Menkes disease, where lysyl oxidase deficiency plays a substantial role (Section 3.2.3.). However, neurodegeneration observed in MD is the result of a more complicated mechanism, where arterial abnormalities and disturbance of several copper enzymes add to the symptoms. The human brain is the most complex region of the body in terms of the diversity of gene expression and the brain copper metabolism is the result of a complex interplay of several copper enzymes in various cell types. Transcription profile obtained from the fibroblasts will, therefore, have obvious limitations in reflecting the gene expression profile of the brain. Still a simpler and easily accessible cell type like the fibroblast can provide useful information and may possibly direct the attention to new interactions.

5.2. LOX as a Differentially Expressed Gene in MD Fibroblasts

One of the differentially expressed genes in our displays was, indeed, lysyl oxidase and our initial studies with RT-PCR suggested an upregulation of LOX mRNA levels (Fig. 2). A likely explanation is that LOX levels are regulated by a positive feedback mechanism, where mRNA transcription is upregulated as a response to a defective posttranslational processing of LOX and/or elevated intrac-ellular copper levels. Supporting this hypothesis, the majority of protein-bound copper secreted from skin fibroblasts was found to be associated with LOX, suggesting that this enzyme is a vehicle for copper egress from connective tissue cells (85).

However, our results are not in line with the results of previous studies. In rat skin fibroblasts, copper deficiency influenced functional activity of lysyl oxidase but not the steady-state levels of mRNA (86). Similarly Yeowell and colleagues (87) did not observe a significant change in the steady-state level of LOX mRNA in MD fibroblasts. In contrast to these studies, levels of lysyl oxidase mRNA transcripts were decreased in Menkes and OHS fibroblasts (41,88). Further studies are necessary to understand the physiopathology of LOX and to reach a consensus in how LOX mRNA expression is reguated in MD.

6. OTHER STUDIES

A few studies have been published studying the influence of copper status on gene expression using the DD technique. One of the studies was carried out on the hepatocytes of copper-deficient

Fig. 2. Expression of LOX mRNA. RT-PCR was carried out with LOX-specific oligonucleotides using total RNA isolated from fibroblasts of MD patients and controls. The results suggest increase in LOX mRNA levels in the patient with the OHS phenotype (lane 1) as well as in the patients with the classical severe form (lanes 2 and 3) compared to controls (laned 4 and 5). M = molecular-weight marker.

rats (89). Among the 10 cDNAs confirmed to be differentially expressed were 2 mitochondrial rRNA genes, the intracellular gene-encoding ferritin, the iron storage protein, and fetuin which encodes tyrosine kinase inhibitor associated with rat insulin receptor. Elucidation of the influence of copper on the regulation of these genes requires further studies.

The other study was carried out to investigate the affect of Cu toxicity on cultured human hepatoma cells (HepG2) and they showed that copper regulated transcription of cytochrome-^ (58). Increased levels of copper led to substantial increase in cytochrome-b transcription, whereas the transcription was suppressed with low levels of copper. It is suggestive that cytochrome-b has a role in the mito-chondrial response to both deficiency and toxicity of copper. Mitochondrial damage has been shown previously under altered copper levels, including Menkes cells (90). Most interestingly in our initial studies, we have also observed differential expression of cytochrome-b in Menkes fibroblasts. Further studies are necessary to elucidate the role of cytochrome-^ in the mitochondrial dysfunction observed in Menkes disease.

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