Lysyl oxidase in development, aging and pathologies of the skin.

Lysyl oxidase (LOX) is a copper- and lysyl-tyrosyl cofactor containing amine oxidase that has been known to play a critical role in the catalysis of lysine-derived crosslinks in extracellular matrix (ECM) proteins in the dermis. Changes in the composition and crosslinked state of the ECM and alterations in LOX synthesis and activity are known to be associated with aging and a range of acquired and heritable skin disorders. It has been assumed until recently that the LOX-related changes in the skin are mediated through the catalytic activity of LOX. However, work by several laboratories over the last few years has shown that LOX is a multifunctional protein. In this review we discuss the regulation of expression, localization and activation of LOX in the normal developing and adult skin, and alterations in LOX expression and activity associated with skin aging and senescence, and in pathological conditions, including wound healing, fibrosis, hypertrophic scarring, keloids, scleroderma, and diabetic skin. We further evaluate the role of LOX in skin ECM changes associated with the normal aging process and with these pathological states. In addition to collagen and elastin cross-linkages, regulatory and activation mechanisms and cell type specific LOX interactions may contribute to a range of novel intra- and extracellular LOX functions that appear critical determinants of the cellular microenvironment in the normal skin and in these skin disorders.

The tissue distribution of murine Abcc6 (Mrp6) during embryogenesis indicates that the presence of Abcc6 in elastic tissues is not required for elastic fiber assembly.

Mutations in the gene coding for the ABC transporter, ABCC6, in humans cause Pseudoxanthoma elasticum, which is characterized by the deposition of aberrant elastic fibers. To investigate whether the presence of ABCC6 in tissues synthesizing elastin is required for elastin deposition and elastic fiber assembly, we have compared the steady-state levels and tissue distribution of Abcc6 and tropoelastin mRNAs during mouse embryogenesis. Whereas tropoelastin mRNA levels rose during embryogenesis and were the highest in neonatal mice, Abcc6 mRNA levels remained constantly low throughout embryogenesis. In some tissues, both Abcc6 and tropoelastin mRNA were detected. However, Abcc6 mRNA and protein were not detected in neonatal aorta and arteries, which produce large amounts of elastin indicating that the presence of Abcc6 in elastic tissues is not required for elastic fiber assembly.

Analysis of ABCC6 (MRP6) in normal human tissues.

To determine the tissue distribution of the ABC transporter ABCC6 in normal human tissues, we analyzed tissue arrays for the presence of ABCC6 mRNA by in situ hybridization and ABCC6 protein by immunohistochemistry using the polyclonal antibody HB-6. We detected ABCC6 mRNA and protein in various epithelial cells of exocrine and endocrine tissues, such as acinar cells in the pancreas, mucosal cells of the intestine and follicular epithelial cells of the thyroid. We obtained a very strong immunostaining for enteroendocrine G cells in the stomach. In addition, ABCC6 mRNA and protein were present in most neurons of the brain, in alveolar macrophages in the lungs and lymphocytes in the lymph node. Immunohistochemisty using the monoclonal antibody M6II-31 confirmed the widespread tissue distribution of ABCC6. The physiological substrate(s) of ABCC6 are yet unknown, but we suggest that ABCC6 fulfills multiple functions in different tissues. The strong immunostaining for ABCC6 in G cells suggests that it plays an important role in these endocrine cells.

Comparative immunocytochemical localization of lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) proteins: changes in the expression of LOXL during development and growth of mouse tissues.

Lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) are extracellular enzymes that deaminate peptidyl lysyl residues involved in the cross-linking of fibrillar collagens and elastin. While LOX is required for the survival of newborn mice, the role of LOXL during development remains unclear. Studies have shown that the same cell types express LOX and LOXL in the same tissues, but no functional differences have been established. We have compared the immunohistochemical localization of LOX and LOXL in various tissues from normal, young adult mice. LOX and LOXL were co-localized in the skin, aorta, heart, lung, liver and cartilage, but were localized to different areas in the kidney, stomach, small intestine, colon, retina, ovary, testis and brain. LOXL expression was further examined in tissues from different developmental stages. In embryonic mice (10.5-14.5 dpc), LOXL immunostaining was abundant in the heart, liver, intestine, and neural tube. LOXL was present in most major organs in late fetal (16.5 dpc) and newborn mice, but generally diminished as animals aged. Immunoreactivity was significantly reduced in the heart, lung, kidney and liver of 2 year-old mice, but remained prevalent in the skin and tongue. LOX and LOXL were also found in the nuclei of cells in a number of tissues. These results indicate that LOXL has a role during mouse development and in the maintenance of adult tissues.

The distribution of Abcc6 in normal mouse tissues suggests multiple functions for this ABC transporter.

We have studied the tissue distribution of Abcc6, a member of the ABC transmembrane transporter subfamily C, in normal C57BL/6 mice. RNase protection assays revealed that although almost all tissues studied contained detectable levels of the mRNA encoding Abcc6, the highest levels of Abcc6 mRNA were found in the liver. In situ hybridization (ISH) demonstrated abundant Abcc6 mRNA in epithelial cells from a variety of tissues, including hepatic parenchymal cells, bile duct epithelia, kidney proximal tubules, mucosa and gland cells of the stomach, intestine, and colon, squamous epithelium of the tongue, corneal epithelium of the eye, keratinocytes of the skin, and tracheal and bronchial epithelium. Furthermore, we detected Abcc6 mRNA in arterial endothelial cells, smooth muscle cells of the aorta and myocardium, in circulating leukocytes, lymphocytes in the thymus and lymph nodes, and in neurons of the brain, spinal cord, and the specialized neurons of the retina. Immunohistochemical analysis using a polyclonal Abcc6 rabbit antibody confirmed the tissue distribution of Abcc6 suggested by our ISH studies and revealed the cellular localization of Abcc6 in the basolateral plasma membrane in the epithelial cells of proximal convoluted tubules in the kidney. Although the function of Abcc6 is unknown, mutations in the human ABCC6 gene result in a heritable disorder of connective tissue called pseudoxanthoma elasticum (PXE). Our results demonstrating the presence of Abcc6 in epithelial and endothelial cells in a variety of tissues, including those tissues affected in PXE patients, suggest a possible role for Abcc6 in the normal assembly of extracellular matrix components. However, the presence of Abcc6 in neurons and leukocytes, two cell populations not associated with connective tissue, also suggests a more complex multifunctional role for Abcc6.

Evidence for a founder effect for pseudoxanthoma elasticum in the Afrikaner population of South Africa.

Pseudoxanthoma elasticum (PXE) is a heritable elastic tissue disorder recently shown to be attributable to mutations in the ABCC6 ( MRP6) gene. Whereas PXE has been identified in all ethnic groups studied to date, the prevalence of this disease in various populations is uncertain, although often assumed to be similar. A notable exception however is the prevalence of PXE among South African Afrikaners. A previous report has suggested that a founder effect may explain the higher prevalence of PXE in Afrikaners, a European-derived population that first settled in South Africa in the 17th century. To investigate this hypothesis, we performed haplotype and mutational analysis of DNA from 24 South African families of Afrikaner, British and Indian descent. Among the 17 Afrikaner families studied, three common haplotypes and six different disease-causing variants were identified. Three of these mutant alleles were missense variants, two were nonsense mutations and one was a single base-pair insertion. The most common variant accounted for 53% of the PXE alleles, whereas other mutant alleles appeared at lower frequencies ranging from 3% to 12%. Haplotype analysis of the Afrikaner families showed that the three most frequent mutations were identical-by-descent, indicating a founder origin of PXE in this population.

A serial analysis of gene expression in sun-damaged human skin.

To study the phenotypic changes in human skin associated with repeated sun exposure at the transcription level, we have undertaken a comparative serial analysis of gene expression of sun-damaged preauricular skin and sun-protected postauricular skin as well as sun-protected epidermis. Serial analyses of gene expression libraries, containing multiple mRNA-derived tag recombinants, were made to poly(A+)RNA isolated from human postauricular skin and preauricular skin, as well as epidermal nick biopsy samples. 5330 mRNA-derived cDNA tags from the postauricular serial analysis of gene expression library were sequenced and these tag sequences were compared to cDNA sequences identified from 5105 tags analyzed from a preauricular serial analysis of gene expression library. Of the total of 4742 different tags represented in both libraries we found 34 tags with at least a 4-fold difference of tag abundance between the libraries. Among the mRNAs with altered steady-state(1) levels in sun-damaged skin, we detected those encoding keratin 1, macrophage inhibitory factor, and calmodulin-like skin protein. In addition, a comparison of cDNA sequences identified in the serial analysis of gene expression libraries obtained from the epidermal biopsy samples (5257 cDNA tags) and from both full-thickness skin samples indicated that many genes with altered steady-state transcript levels upon sun exposure were expressed in epidermal keratinocytes. These results suggest a major role for the epidermis in the pathomechanism of largely dermal changes in chronically sun-exposed skin.

Connection between elastin haploinsufficiency and increased cell proliferation in patients with supravalvular aortic stenosis and Williams-Beuren syndrome.

To elucidate the pathomechanism leading to obstructive vascular disease in patients with elastin deficiency, we compared both elastogenesis and proliferation rate of cultured aortic smooth-muscle cells (SMCs) and skin fibroblasts from five healthy control subjects, four patients with isolated supravalvular aortic stenosis (SVAS), and five patients with Williams-Beuren syndrome (WBS). Mutations were determined in each patient with SVAS and in each patient with WBS. Three mutations found in patients with SVAS were shown to result in null alleles. RNA blot hybridization, immunostaining, and metabolic labeling experiments demonstrated that SVAS cells and WBS cells have reduced elastin mRNA levels and that they consequently deposit low amounts of insoluble elastin. Although SVAS cells laid down approximately 50% of the elastin made by normal cells, WBS cells deposited only 15% of the elastin made by normal cells. The observed difference in elastin-gene expression was not caused by a difference in the stability of elastin mRNA in SVAS cells compared with WBS cells, but it did indicate that gene-interaction effects may contribute to the complex phenotype observed in patients with WBS. Abnormally low levels of elastin deposition in SVAS cells and in WBS cells were found to coincide with an increase in proliferation rate, which could be reversed by addition of exogenous insoluble elastin. We conclude that insoluble elastin is an important regulator of cellular proliferation. Thus, the reduced net deposition of insoluble elastin in arterial walls of patients with either SVAS or WBS leads to the increased proliferation of arterial SMCs. This results in the formation of multilayer thickening of the tunica media of large arteries and, consequently, in the development of hyperplastic intimal lesions leading to segmental arterial occlusion.

Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6).

Mutations in the ABCC6 (MRP6) gene cause pseudoxanthoma elasticum (PXE), a rare heritable disorder resulting in the calcification of elastic fibers. In the present study a cDNA encoding a full-length normal variant of ABCC6 was amplified from a human kidney cDNA library, and the protein was expressed in Sf9 insect cells. In isolated membranes ATP binding as well as ATP-dependent active transport by ABCC6 was demonstrated. We found that glutathione conjugates, including leukotriene C(4) and N-ethylmaleimide S-glutathione (NEM-GS), were actively transported by human ABCC6. Organic anions (probenecid, benzbromarone, indomethacin), known to interfere with glutathione conjugate transport of human ABCC1 and ABCC2, inhibited the ABCC6-mediated NEM-GS transport in a specific manner, indicating that ABCC6 has a unique substrate specificity. We have also expressed three missense mutant forms of ABCC6, which have recently been shown to cause PXE. MgATP binding was normal in these proteins; ATP-dependent NEM-GS or leukotriene C(4) transport, however, was abolished. Our data indicate that human ABCC6 is a primary active transporter for organic anions. In the three ABCC6 mutant forms examined, the loss of transport activity suggests that these mutations result in a PXE phenotype through a direct influence on the transport activity of this ABC transporter.

Somatic mutations of the lysyl oxidase gene on chromosome 5q23.1 in colorectal tumors.

Lysyl oxidase (LOX), a copper-dependent amine oxidase, has been implicated in tumor suppression and cell growth regulation. The chromosomal locus of LOX, 5q23, is affected by loss of heterozygosity (LOH) in colon cancer, suggesting that the LOX gene could be affected by LOH and consequently, loss or reduction of LOX function contribute to the tumorigenic process. Identification of microsatellite markers within the LOX locus has allowed us to map the LOX gene within the 5q23.1 region. Analysis of this locus and flanking loci in matched tumor and blood DNA samples from a panel of colorectal cancer patients, demonstrated that 38% (16/42) of informative samples were affected by LOH or allelic imbalance. Furthermore, 75% (6/8) of these tumor samples were shown to have significantly reduced LOX mRNA levels. Similar reduction in LOX levels were detected in a panel of matched normal colon and colon tumor samples. Tumor samples demonstrating LOH by RFLP, were subject to mutational analysis, including RT-PCR, exonic deletion detection by PCR, cDNA and genomic DNA sequencing, and were found to have a spectrum of alterations and mutations affecting the LOX gene. These results confirm that loss or reduction of LOX function during tumor development is a direct consequence of somatic mutations and is associated with colon tumor pathogenesis.