Interaction of Gal-7 with HMGCS1 In Vitro May Facilitate Cholesterol Deposition in Cultured Keratinocytes.

Three-hydroxy-3-methylglutaryl coenzyme A synthase (HMGCS) 1 was identified to interact with Gal-7, a pro-apoptotic ß-galactoside‒binding protein, by yeast two-hybrid system. Their interaction was confirmed by in vitro ß-galactosidase, Biacore, and immunoprecipitation assays. A distinct interactive site of HMGCS1 was found to reside at phenylalanine 26. The expression of HMGCS1 in cultured keratinocytes was upregulated by exogenous Gal-7 and downregulated in LGALS7 small interfering RNA‒transfected cells. HMGCS1-overexpressing cells were found to induce Gal-7 expression, which suggests that Gal-7 and HMGCS1 expressions are both stimulated by positive feedback regulation. The amount of cholesterol, a final biosynthetic product of HMGCS1-involved pathway, was increased in Gal-7‒treated cells and was significantly reduced in LGALS7 small interfering RNA‒transfected cells. The increase of cholesterol level in Gal-7‒treated cells was inhibited by wild-type HMGCS1 peptide but not by phenylalanine 26‒mutated peptide, suggesting that the interaction of Gal-7/HMGCS1 is related to cellular cholesterol level. Foam cells in granulomatous tissues of the specimens from normolipidemic cutaneous xanthoma showed positive reactions with the antibodies for Gal-7 and HMGCS1 as well as for lipid markers. These results are likely to indicate that Gal-7 induction in epidermal keratinocytes causes both apoptotic cell death and HMGCS1-mediated cholesterol accumulation, which will be phagocytized by macrophages. This mechanism may explain the pathogenesis of normolipidemic cutaneous xanthoma.

Porphyrin accumulation in humans with common dysfunctional variants of ABCG2, a porphyrin transporter: potential association with acquired photosensitivity.

Photosensitivity is a skin reaction disorder mediated by phototoxic and/or photoallergic mechanisms. The accumulation of porphyrins is generally considered to induce phototoxicity. ATP-binding cassette subfamily G member 2 (ABCG2) has been identified as a transporter of porphyrins and its common variants-p.Gln126Ter (rs72552713) and p.Gln141Lys (rs2231142)-reportedly decrease the function of porphyrin transport in vitro; however, the physiological importance of ABCG2 as a porphyrin transporter remains to be fully elucidated. We herein investigated whether ABCG2 dysfunction could lead to porphyrin accumulation and photosensitivity in Japanese subjects, and found it to be significantly correlated with erythrocyte protoporphyrin levels (P = 0.012). This appears to be the first clinical finding of ABCG2 dysfunction-associated protoporphyrin accumulation in humans. We divided the patients into a chronic actinic dermatosis (CAD) group and a non-CAD group. CAD was diagnosed based on the criteria of reduced minimal erythema doses to ultraviolet B (UVB) and/or ultraviolet A (UVA). The non-CAD group was composed of patients who exhibited normal reactions to UVB and UVA on phototesting, but had histories of recurrent erythema/papules on sun-exposed areas. Estimated ABCG2 function according to ABCG2 genotypes in the non-CAD group was significantly lower than in the general Japanese population (P = 0.045). In contrast, no difference was found in ABCG2 function between the CAD group and the general population, suggesting that ABCG2 dysfunction might be a genetic factor in non-CAD patients with clinical photosensitivity. In this context, genetic dysfunction of ABCG2 might be an overlooked pathological etiology of "photosensitivity of unknown cause."

Accumulation of C-reactive protein in basal keratinocytes of normal skins.

BACKGROUND: C-reactive protein (CRP) is a prototypic acute phase protein which increases dramatically in the blood during the first 48h of tissue inflammation and has been recognized as a risk factor for atherosclerosis. CRP interacts with a variety of proteins. OBJECTIVE: To know the role of accumulated CRP in the skin. METHODS: Interaction of CRP with basal keratinocytes was studied using immunohistochemical method and keratinocyte culture system. RESULTS: We found an immunohistochemical deposition of CRP on the basal keratinocyte membrane in some normal human skins (23 out of 46 skins). When added to cultured keratinocytes, heat-denatured but not native CRP was found to adhere to keratinocyte cell membrane after 1h, then internalized into cytoplasm after 24h. The heat-denatured CRP recognized at least four keratinocyte polypeptides with the molecular weights of 56, 42, 32 and 24kDa. Ligand binding assays suggested that multiple populations of receptor-ligand interactions were involved in the binding between CRP and keratinocyte. Cultured dermal microvascular endothelial cells were found to express CRP of which expression was greatly induced by interleukin-1ß (IL-1ß) treatment, suggesting that the deposited CRP in the basal keratinocytes can be derived from local dermal microvasculatures as well as from systemic circulation (serum). Treatment of cultured keratinocytes with heat-denatured CRP induced interleukin-8 (IL-8) expression, a potent leukocyte chemotactic cytokine. CRP in the medium (liquid phase) and CRP-coated dishes (solid phase) both inhibited the adhesion of keratinocytes in culture. CONCLUSION: Accumulation of CRP may regulate the skin inflammation and keratinocyte proliferation by modulating keratinocyte cytokine expression and adhesion to substrate.

In vitro amyloidogenic peptides of galectin-7: possible mechanism of amyloidogenesis of primary localized cutaneous amyloidosis.

Pathogenesis of primary localized cutaneous amyloidosis (PLCA) is unclear, but pathogenic relationship to keratinocyte apoptosis has been implicated. We have previously identified galectin-7, actin, and cytokeratins as the major constituents of PLCA. Determination of the amyloidogenetic potential of these proteins by thioflavin T (ThT) method demonstrated that galectin-7 molecule incubated at pH 2.0 was capable of binding to the dye, but failed to form amyloid fibrils. When a series of galectin-7 fragments containing ß-strand peptides were prepared to compare their amyloidogenesis, Ser(31)-Gln(67) and Arg(120)-Phe(136) were aggregated to form amyloid fibrils at pH 2.0. The rates of aggregation of Ser(31)-Gln(67) and Arg(120)-Phe(136) were dose-dependent with maximal ThT levels after 3 and 48 h, respectively. Their synthetic analogs, Phe(33)-Lys(65) and Leu(121)-Arg(134), which are both putative tryptic peptides, showed comparable amyloidogenesis. The addition of sonicated fibrous form of Ser(31)-Gln(67) or Phe(33)-Lys(65) to monomeric Ser(31)-Gln(67) or Phe(33)-Lys(65) solution, respectively, resulted in an increased rate of aggregation and extension of amyloid fibrils. Amyloidogenic potentials of Ser(31)-Gln(67) and Phe(33)-Lys(65) were inhibited by actin and cytokeratin fragments, whereas those of Arg(120)-Phe(136) and Leu(121)-Arg(134) were enhanced in the presence of Gly(84)-Arg(113), a putative tryptic peptide of galectin-7. Degraded fragments of the galectin-7 molecule produced by limited trypsin digestion, formed amyloid fibrils after incubation at pH 2.0. These results suggest that the tryptic peptides of galectin-7 released at neutral pH, may lead to amyloid fibril formation of PLCA in the intracellular acidified conditions during keratinocyte apoptosis via regulation by the galectin-7 peptide as well as actin and cytokeratins.

Targeted ablation of the abcc6 gene results in ectopic mineralization of connective tissues.

Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6(-/-) mice but was not observed in Abcc6(+/-) or Abcc6(+/+) mice up to 2 years of age. A total body computerized tomography scan of Abcc6(-/-) mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease.

Expression of 67-kDa elastin receptor in annular elastolytic giant cell granuloma: elastin peptides induce monocyte-derived dendritic cells or macrophages to form granuloma in vitro.

Annular elastolytic giant cell granuloma (AEGCG) is characterized by non-palisading granuloma and elastophagocytic giant cells. Granulomas consist of structured masses of macrophages, dendritic cells, and T lymphocytes which play an essential role in granuloma formation. Two lineage systems of dendritic cells and macrophages originated from peripheral blood monocytes have been established in vitro. To know how elastin fragments are involved in the granuloma formation in AEGCG, we tested in vitro whether elastin fragments potentially induce monocyte-derived macrophages or dendritic cells to form granuloma and multinucleated giant cells. Immunohistochemical studies of the lesional skins of AEGCG (n = 5) revealed that the 67-kDa elastin receptor was specifically expressed in the epithelioid or multinucleated giant cells. Proliferation of factor XIIIa(+) cells and CD68(+) cells was also seen in the lesional skins of AEGCG. Factor XIIIa(+) dendritic cells or CD68(+) macrophages were established by the treatment of granulocyte/macrophage-colony stimulating factor (GM-CSF)/interleukin-4 or M-CSF, respectively. Further treatments of these dendritic cells or macrophages with elastin peptide resulted in the formation of granuloma or multinucleated giant cells which were immunoreactive with anti-67-kDa elastin receptor antibody. These findings suggest that elastic tissue induces factor XIIIa(+) cells and CD68(+) macrophages to form granuloma or multinucleated giant cells and plays an essential role in the formation of granuloma in AEGCG.

Eruptive vellus hair cyst in patients with chronic renal failure.

Two cases of eruptive vellus hair cysts associated with chronic renal failure are reported. Histologically the lesions of both cases showed cystic structures in the dermis lined by squamous epithelium which contained varying amounts of vellus hair shafts. Immunohistochemical studies using monoclonal anti-AGE (advanced glycation end product) antibody demonstrated that keratinous materials within the cystic structures were immunoreactive to the antibody, whereas those of cystic lesions (epidermal cyst, eruptive vellus hair cyst, steatocystoma multiplex, trichofolliculoma and trichilemmal cyst) seen in otherwise healthy individuals were negative. Because it has been reported that plasma and skin levels of AGE are elevated in renal failure patients, AGE-modified keratinous materials may be associated with the formation of cystic structures by stimulating the occlusion of the epithelium.

Amyloid deposition of systemic myeloma-associated amyloidosis excludes actinic elastotic material.

We report a 65-year-old Japanese man presenting with myeloma-associated systemic amyloidosis. A biopsy specimen taken from the purpuric papules on the periorbital area showed the presence of both amyloid and elastotic materials (actinic elastosis) in the upper dermis. Amyloid deposition and altered elastotic fibers were clearly bordered and never colocalized, and amyloid materials appeared to exclude elastotic materials. The mechanism of this phenomenon is discussed.

The presence of D-beta-aspartic acid-containing peptides in elastic fibers of sun-damaged skin: a potent marker for ultraviolet-induced skin aging.

Biologically uncommon d-aspartyl residues have been reported in proteins of various elderly tissues. We prepared a polyclonal antibody against d-beta-Asp-containing peptide and examined its immunoreactivity in the skin. The antibody recognized integrated or disintegrated elastic fibers in the sun-exposed skin but not in the sun-protected skin of the elderly donors. Western blot analysis of the proteins isolated from sun-damaged skin demonstrated that the 50 kDa protein was immunoreactive with both antibodies for d-beta-Asp-containing peptide and elastin. Ultraviolet (UV) irradiation on normal skin caused the appearance of d-beta-Asp-containing peptide-immunoreactive fibers in the dermis. These results suggest that UV irradiation is closely related to the formation of d-beta-Asp in the elastic fibers of skin. We propose that the antibody could be a useful indicator for sun damage of the skin.

Expression of 36-kDa microfibril-associated glycoprotein (MAGP-36) in human keratinocytes and its localization in skin.

Microfibril-associated glycoprotein-36 (MAGP-36) is a recently isolated elastin-binding protein and considered to be a member of microfibril-associated glycoproteins (MAGPs). We studied the expression of MAGP-36 in cultured normal human keratinocytes and its localization in the skin. MAGP-36 was found to be expressed in cultured human keratinocytes by Western blot and RT-PCR assays. The levels of MAGP-36 (polypeptide and mRNA) and the number of MAGP-36-producing keratinocytes were greatly increased during Ca(2+)-induced differentiation of keratinocytes. Immunohistochemical studies demonstrated that MAGP-36 colocalized with elastic fibers and formed candelabra like-fibers in the superficial dermis of normal skin. In the elderly skin of sun-exposed region, immunoreactivity of MAGP-36 in the superficial dermis disappeared. In the lesional skin of pseudoxanthoma elasticum which is an elastin-related disorder, immunoreactivity of MAGP-36 was found in the accumulation of disintegrated elastic fibers. The results show that MAGP-36 is a component of elastic fibers in the dermis and co-operates with elastin in normal and diseased conditions.

Advanced glycation end product-modified beta2-microglobulin is a component of amyloid fibrils of primary localized cutaneous nodular amyloidosis.

Primary localized cutaneous nodular amyloidosis is a rare form of cutaneous amyloidosis. Amyloid fibrils in primary localized cutaneous nodular amyloidosis have been reported to be originated from immunoglobulin light chains. Immunohistochemical studies on the lesional skins of four patients with primary localized cutaneous nodular amyloidosis demonstrated that amyloid deposits of all cases showed a positive reaction with the antibodies for beta2-microglobulin and advanced glycation end products as well as immunoglobulin light chain (kappa or lambda). No beta2-microglobulin and advanced glycation end product immunoreactivity was found in the amyloid deposits of other primary localized cutaneous amyloidosis (lichen amyloidosis and macular amyloidosis). Double immunofluorescence study of the lesional skin of primary localized cutaneous nodular amyloidosis showed that anti-kappa light chain, anti-beta2-microglobulin and anti-advanced glycation end product antibodies mostly reacted with the same area of amyloid deposit. Amyloid proteins were sequentially extracted with distilled water from one case of primary localized cutaneous nodular amyloidosis and recovered in the five water-soluble fractions (fractions I-V). Immunoblot assay of amyloid fibril proteins demonstrated that immunoreactive polypeptides with anti-kappa light chain antibody (29 kDa) and with anti-beta2-microglobulin antibody (12 kDa) were detected in fractions I-V, whereas immunoreactive polypeptide with anti-advanced glycation end product antibody (12 kDa) was detected exclusively in fractions III-V but not in fractions I and II. Two-dimensional polyacrylamide gel electrophoresis revealed that 12 kDa polypeptide in fractions I and II was electrophoretically identical with authentic beta2-microglobulin and that beta2-microglobulin in fractions III-V was advanced glycation end product-modified beta2-microglobulin with more acidic pI value. These results indicate that beta2-microglobulin is another major component of amyloid fibrils in primary localized cutaneous nodular amyloidosis and that beta2-microglobulin in primary localized cutaneous nodular amyloidosis is partly subjected to the modification of advanced glycation end product.