Atsushi Utani

Cloning and expression of laminin α2 chain (M-chain) in the mouse

Abstract Laminins are a family of heterotrimeric glycoporteins specific to basement membranes. Laminin-2, consisting of α2, β1 and γ1 chains, was originally identified in the basement membranes of skeletal muscle and peripheral nerve. We have isolated and sequenced the full-length cDNA for the mouse laminin α2 chain. Four overlapping clones spanning 9,330 bp encode a predicted polypeptide of 3,106 amino acids having a calculated molecular mass of 390 kDa including a 23-amino-acid signal peptide. The amino acid sequence of the α2 chain shares a 45.9%, identity with that of the α1 chain. Similar to the structure of the α1 chain, the α2 chain consists of several domains beginning at the N-terminus with three globular domains alternating with three epidermal growth factor-like domains followed by two α-helical domains and a C-terminal globular domain. The most N-terminal globular domain is highly conserved (77.3% identity) between the α2 and α1 chains, whereas the α-helical domains have low homology (30.3% identity). Northern blot and ribonuclease protection analysis revealed expression of mRNA for the α2 chain in heart, kidney, liver, skin, lung and skeletal muscle of newborn mice. Such a tissue distribution suggests a role for the α2 chain and, consequently, laminin-2 or -4 not only in the organization and the function of nerve and muscle tissue but possibly also in the mesenchymal components of certain tissues.

Near‐infrared irradiation stimulates cutaneous wound repair:laboratory experiments on possible mechanisms

Background/Aims: Several physical agents such as low‐energy lasers have been used in the treatment of chronic skin ulcers. This study was performed to investigate potential effects of a newly‐developed, specific near‐infrared light source on wound repair.

Fibulin-2 Binds to the Short Arms of Laminin-5 and Laminin-1 via Conserved Amino Acid Sequences

Epithelial cell-specific laminin-5, consisting of three chains, α3, β3, and γ2, is a component of the anchoring filament that traverses the lamina lucida beneath the hemidesmosomes of epidermal cells and functions to link these cells to the basement membrane. We have studied the molecular interaction between laminin-5 and extracellular matrix proteins using recombinant proteins and synthetic peptides. Affinity chromatography assays with recombinant fragments of the laminin γ2 short arm identified a 195-kDa binding protein in the conditioned media from the mouse epidermal cell line Pam 212 and from primary dermal fibroblasts. This molecule was identified by Western blotting as fibulin-2, a recently identified extracellular matrix protein. Using deletion mutants and various synthetic peptides in competition assays, the 9-amino acid sequence SADFSVHKI (residues 199-207) in domain IV of the γ2 chain was defined as a critical site for fibulin-2 binding. An anti-γ2 antibody co-immunoprecipitated fibulin-2 from the conditioned media, further confirming the interaction of fibulin-2 with laminin-5. Fibulin-2 was also found to interact with laminin-1 (α1β1γ1) through a region (residues 654-665) of the α1 chain short arm whose sequence is similar to that of the fibulin-2 binding site of the γ2 chain. Together these results suggest that fibulin-2 functions to bridge laminin-1 and laminin-5 with other extracellular matrix proteins, providing a linkage between the cell surface and the basement membrane.

Identification of Biologically Active Sequences in the Laminin α4 Chain G Domain

Laminins are a family of trimeric extracellular matrix proteins consisting of α, β, and γ chains. So far five different laminin α chains have been identified. The laminin α4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin α4 chain. Here we have focused on the biological functions of the laminin α4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-α4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-α4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-α4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-α4G. These results suggest that the four active sites are involved in the biological functions of the laminin α4 chain G domain. Furthermore, rec-α4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways.

The expression of nectin‐1α in normal human skin and various skin tumours

Summary Backgroundu2003A novel cell–cell adhesion system that consists of nectin and afadin has been identified at cadherin‐based cell–cell adherens junctions. Nectin is a Ca2+‐independent homophilic and heterophilic cell adhesion molecule that belongs to the immunoglobulin superfamily. Nectin has recently been shown to serve as an α‐herpesvirus entry and cell–cell spread mediator. In spite of the ubiquitous expression of nectin‐1α, its detailed localization in human skin has not been examined so far.

Primary Localized Cutaneous Nodular Amyloidosis in a Patient with Sjögren's Syndrome: A Review of the Literature

We report a 53‐year‐old Japanese woman with multiple, red, and elastic soft nodules on the left waist, left thigh, and right lower leg. She had had polyclonal hyperglobulinemia for one year, rheumatoid arthritis for 13 years, and Sjögrens syndrome (SjS) for 18 years. Histochemical examination of the nodule on the left thigh revealed a deposition of amyloid by Congo red staining. It was also positively stained with both anti‐κ and ‐λ light chain antibodies. Moreover, the cytoplasm of the infiltrating plasma cells also positively reacted to both antibodies. The major amyloid proteins of primary localized cutaneous nodular amyloidosis (PLCNA) generally consist of monoclonal immunoglobulin light chains. A review of literature demonstrates 13 cases of PLCNA with SjS, in which immunoglobulin light chains were demonstrated in the amyloid in 5 cases. Amyloid in the 3 cases was composed of a single class immunoglobulin light chain and that in the 2 cases was composed of both κ and λ light chains. Polyclonal immunoglobulin amyloid has been reported only in PLCNA with SjS, which may be related to the fact that a certain population of SjS develops polyclonal B cell proliferation and hyperglobulinemia.

Differential expression of laminin α chains during proliferative and differentiation stages in a model for skin morphogenesis

The purpose of this study was to determine the mRNA and protein expression of laminin alpha chains at various stages of in vitro skin morphogenesis. Fibroblasts in mono-cultures express low levels of the mRNA of laminin alpha1,alpha2, alpha3 and alpha4 chains. When co-cultured with keratinocytes for 28 days, they expressed the mRNA for all these chains. Keratinocytes in monolayer expressed the laminin alpha3 chain mRNA and very low levels of the mRNA of the alpha1 and alpha2 chains, although, when recombined with fibroblasts they also expressed laminin alpha1and alpha2 mRNA, but not the laminin alpha4 mRNA. Immunocytochemistry of cells in co-culture showed that laminin alpha1, alpha3 and alpha5 chains were expressed in the epidermis, while the laminin alpha2, beta1, and gamma1 chains were noted in the dermis and at the epidermo-dermal interface. The laminin alpha1chain was first expressed during the proliferative stage (14-21 days) and the laminin alpha2 and alpha5 chains appeared later, during the differentiation stage (28-42 days). The above results suggest that epithelial-mesenchymal interactions are involved in the expression of laminin alpha chain mRNA during in vitro skin morphogenesis. In addition, there is distinct temporal and spatial expression of these chains during proliferative and differentiation stages, possibly reflecting different functions.

Lamininα3 LG4 Module Induces Keratinocyte Migration: Involvement of Matrix Metalloproteinase-9

Abstract Laminin α 3 chain, a functionally key subunit of laminin-5, contains a large globular module (G module) which consists of a tandem repeat of five homologous LG modules (LG1∼ 5). We previously demonstrated that the LG4 module of laminin α 3 chain (α 3 LG4) induces a matrix metalloproteinase-1 (MMP-1) expression through the interaction with syndecans leading to MAPK activation/IL-1β expression signaling loop (Utani et al., J. Biol. Chem. 278, 34483–34490, 2003). Here, we show that a recombinant α 3 LG4 and synthetic peptides containing syndecan binding motif induced a cell motility and a MMP-9 expression in ketarinocytes. The synthetic peptide (A3G756)-induced cell migration and MMP-9 upregulation were inhibited by each application of a heparin and an IL-1 receptor antagonist (IL-1RA), suggesting the involvement of syndecans and IL-1β autocrine. Furthermore, the A3G756-induced cell motility was inhibited by an MMP-9 inhibitor and a neutralizing antibody of MMP-9, indicating induced cell motility was dependent on an MMP-9 activity. Taken these together, laminin-5 α 3 LG4 module may play an important role in re-epithelialization at tissue remodeling.

Deficiency of the decorin core protein in the variant form of Ehlers-Danlos syndrome with chronic skin ulcer

Decorin belongs to a family of small leucine-rich dermatan sulfate proteoglycans that are involved in the control of matrix organization and cell growth. Here, we described a patient whose skin glycosaminoglycans showed extremely decreased amount of dermatan sulfate compared with a normal control skin. This patient presented clinical features of Ehlers-Danlos syndrome with a chronic skin ulcer. Western blotting revealed that the deficiency of dermatan sulfate was due to the defect of decorin core protein. Beta-xyloside, an initiator of dermatan sulfate glycosaminoglycan chain elongation, enhanced the synthesis of dermatan sulfate in the fibroblasts of the patient to a similar extent to that of control. This result indicated that the enzymes for the elogation of dermatan sulfate side chains were normal. Northern blotting demonstrated remarkable reduction of decorin mRNA level, while biglycan mRNA level was concomitantly increased and procollagen alpha1(I) mRNA level was normal. cDNA and exons sequencing analysis showed there was no mutation in decorin gene of the patient. IL-1beta stimulated decorin expression to about 140% in control fibroblasts while about 110% in patient fibroblasts. On the other hand, TGF-beta1 resulted in 40% reductions of decorin expression in both control and patient fibroblasts. These data suggested that reduced decorin expression of fibroblasts from the patient of Ehlers-Danlos syndrome may be due to abnormalities in the regulatory regions, which is responsible for the IL-1beta stimulation.

Collagenoma in Down syndrome.

SIR, Retinoids, both naturally occurring and synthetic, are vitamin A derivatives. The widespread use of retinoids requires that not only dermatologists but also physicians must be aware of the wide spectrum of adverse reactions that may develop in patients receiving retinoids. Isotretinoin is a first-generation synthetic vitamin A derivative that is widely used orally. Adverse reactions involving skin and mucous membranes, cardiorespiratory, gastrointestinal, genitourinary, nervous and musculoskeletal systems, eyes and ears, have been the most commonly reported side-effects. Pregnant women must be also cautious of isotretinoin intake. Haematopoietic and lymphatic complications have also been described. We report two new adverse effects observed in an 18-yearold caucasian man who had been taking isotretinoin 1 mg kg daily for severe acne for 3 months. His medical history was noncontributory and he had not had any previous relevant illness. He was admitted to our hospital complaining of fever, headache, rigidity of the neck and limbs, masseter twitch and diffuse myalgias mainly localized at the buttocks and lumbar areas. At admission his acne severity was estimated as mild. Examination revealed fever (38 C), sinus tachycardia (140 beats min) that was not correlated with the fever level, and urine retention of 800 mL, which was collected by a bladder catheter. Acne lesions were observed on the face, neck and anterior chest. Lithiasis obstruction and ⁄ or urinary tract infection were excluded. Thus, urine retention was exclusively due to drug use. Hepatotoxicity with a fourfold elevation in liver enzymes was found. An electrocardiogram (ECG) showed sinus tachycardia with an accompanying right branch bundle block (RBBB). An electromyogram showed diffuse myopathy, while a muscle biopsy was not specific. A lung computed tomographic scan revealed characteristics of interstitial pneumonitis with a small amount of encysted pleural effusion. A whole-body bone scan detected dense radioactive areas at the upper wedge of the scapula and at the T4–T7 vertebrae. Hyperostosis was observed in the vertebrosternal joint areas. All other laboratory tests were normal or negative. Isotretinoin was discontinued on admission, and all the symptoms and signs disappeared after 3 weeks with the patient receiving only supportive therapy. Readministration 10 days later of isotretinoin 0Æ5 mg kg daily for 1 week induced myalgias and an RBBB again, but without sinus tachycardia. Clinical improvement and RBBB disappearance followed discontinuation of the drug, thus establishing by rechallenge the isotretinoin causality. It is well established that a wide spectrum of retinoid adverse effects may appear in a dose-dependent and ⁄ or idiosyncratic manner. To the best of our knowledge, all the adverse effects of isotretinoin noted in this patient have been reported in various frequencies except for the combination of RBBB with sinus tachycardia, and the urine retention. Sinus tachycardia alone has been reported once in a 26-year-old woman who developed tachycardia of 1 h duration after each dose of isotretinoin. Cardiac abnormalities or other arrhythmias besides tachycardia have not been reported until now. To the best of our knowledge, ECG changes have not been reported even in acne fulminans cases until now, except for the abovementioned case of Bigby and Stern. In summary, we report multisystem involvement with two new isotretinoin adverse effects: RBBB accompanied by sinus tachycardia, and urine retention. We propose that an ECG should be performed in patients with any systemic illness when on oral isotretinoin, as cardiac adverse effects may represent dangerous abnormalities of heart function. Furthermore, it may be wise to administer initial doses of the drug at less than 1 mg kg daily.