Epithelial cells maintained in culture medium containing low calcium proteolytically process laminin 5 (␣3␤3␥2) within the ␣3 and ␥2 chains (1). Experiments were designed to identify the enzyme(s) responsible for the laminin 5 processing and the sites of proteolytic cleavage. To characterize the nature of laminin 5 processing, we determined the N-terminal amino acid sequences of the proteolytic fragments produced by the processing events. The results indicate that the first ␣3 chain cleavage (200-l65 kDa ␣3) occurs within subdomain G4 of the G domain. The second cleavage (l65-l45 kDa ␣3) occurs within the lIla domain, 11 residues Nterminal to the start of domain II. The ␥ chain is cleaved within the second epidermal growth factor-like repeat of domain Ill. The sequence cleaved within the ␥2 chain matches the consensus sequence for the cleavage of type I, II, and III procollagens by bone morphogenetic protein-1 (BMP-1), also known as type I procollagen C-proteinase (2). Recombinant BMP-1 cleaves ␥2 in vitro, both within intact laminin 5 and at the predicted site of a recombinant ␥2 short arm. ␣3 is also cleaved by BMP-1 in vitro, but the cleavage site is yet to be determined. These results show the laminin ␣3 and ␥2 chains to be substrates for BMP-1 in vitro. We speculate that ␥2 cleavage is required for formation of the laminin 5-6 complex and that this complex is directly involved in assembly of the interhemidesmosomal basement membrane. This further suggests that BMP-1 activity facilitates basement membrane assembly, but not hemidesmosome assembly, in the laminin 5-rich dermalepidermal junction basement membrane in vivo.The occurrence of physiological, extracellular proteolytic processing of collagens is well documented, as is the important role that it plays in controlling the fibrillogenesis of banded collagen fibers (3). An enzyme responsible for removal of the C-terminal procollagen propeptides of the major fibrillar collagen types I-III has been identified as BMP-1 (2).1 BMP-1 was first identified in osteogenetic fractions of mammalian bone (4 -7) but was subsequently found to show substantial homology to proteins involved in morphogenetic patterning, such as the products of Drosophila genes tolloid (tld) and tlr-1 (12, 43) and of sea urchin gene products BP10 and SpAN (8, 9). Each contains an N-terminal astacin-like zinc-binding metalloendopeptidase domain (10) followed by varying numbers of epidermal growth factor-like (EGF-like) motifs and internal repeats termed CUB domains thought to be responsible for protein-protein interactions (44).There is abundant genetic and molecular evidence that Drosophila tld mediates dorsal-ventral patterning in the fly embryo (11-13), with null phenotypes of tld showing partial transformation of the dorsal ectoderm into ventral ectoderm (14). Genetic and developmental expression studies have also indicated that the tld gene product TLD participates within the same developmental pathway as the product of the decapentaplegic gene, DPP, the fly cognate of mammalian BMP-2 an...

Sulfur mustard (SM), a chemical weapon first employed during World War I, targets the skin, eyes, and lung. It remains a significant military and civilian threat. The characteristic response of human skin to SM involves erythema of delayed onset, followed by edema with inflammatory cell infiltration, the appearance of large blisters in the affected area, and a prolonged healing period. Several in vivo and in vitro models have been established to understand the pathology and investigate the mechanism of action of this vesicating agent in the skin. SM is a bifunctional alkylating agent which reacts with many targets including lipids, proteins, and DNA, forming both intra- and intermolecular cross-links. Despite the relatively nonselective chemical reactivity of this agent, basal keratinocytes are more sensitive, and blistering involves detachment of these cells from their basement membrane adherence zones. The sequence and manner in which these cells die and detach is still unresolved. Much has been discovered over the past two decades with respect to the mechanisms of SM-induced cytotoxicity and the intracellular and extracellular targets of this vesicant. In this review, the effects of SM exposure on the skin are described, as well as potential mechanisms mediating its actions. Successful therapy for SM poisoning will depend on following new mechanistic leads to develop drugs that target one or more of its sites of action.

Purpose: The goals of this study were (1) to compare the injury at the basement membrane zone (BMZ) of rabbit corneal organ cultures exposed to half mustard (2 chloroethyl ethyl sulfide, CEES) and nitrogen mustard with that of in vivo rabbit eyes exposed to sulfur mustard (SM); (2) to test the efficacy of 4 tetracycline derivatives in attenuating vesicant-induced BMZ disruption in the 24-h period postexposure; and (3) to use the most effective tetracycline derivative to compare the improvement of injury when the drug is delivered as drops or hydrogels to eyes exposed in vivo to SM. Methods: Histological analysis of hematoxylin and eosin-stained sections was performed; the ultrastructure of the corneal BMZ was evaluated by transmission electron microscopy; matrix metalloproteinase-9 was assessed by immunofluorescence; doxycycline as drops or a hydrogel was applied daily for 28 days to eyes exposed in vivo to SM. Corneal edema was assessed by pachymetry and the extent of neovascularization was graded by length of longest vessel in each quadrant. Results: Injury to the BMZ was highly similar with all vesicants, but varied in degree of severity. The effectiveness of the 4 drugs in retaining BMZ integrity did not correlate with their ability to attenuate matrix metalloproteinase-9 expression at the epithelial-stromal border. Doxycycline was most effective on organ cultures; therefore, it was applied as drops or a hydrogel to rabbit corneas exposed in vivo to SM. Eyes were examined at 1, 3, 7, and 28 days after exposure. At 7 and 28 days after SM exposure, eyes treated with doxycycline were greatly improved over those that received no therapy. Corneal thickness decreased somewhat faster using doxycycline drops, whereas the hydrogel formulation decreased the incidence of neovascularization. Conclusions: Corneal cultures exposed to 2-chloroethyl ethyl sulfide and nitrogen mustard were effective models to simulate in vivo SM exposures. Doxycycline as drops and hydrogels ameliorated vesicant injury. With in vivo exposed animals, the drops reduced edema faster than the hydrogels, but use of the hydrogels significantly reduced neovascularization. The data provide proof of principle that a hydrogel formulation of doxycycline as a daily therapy for ocular vesicant injury should be further investigated.

No abstract

Sulfur mustard (SM, bis(2-chloroethyl)sulfide) is a bifunctional alkylating agent that causes dermal inflammation, edema and blistering. To investigate the pathogenesis of SM-induced injury, we used a vapor cup model which provides an occlusive environment in which SM is in constant contact with the skin. The dorsal skin of SKH-1 hairless mice was exposed to saturated SM vapor or air control. Histopathological changes, inflammatory markers and DNA damage were analyzed 1–14 days later. After 1 day, SM caused epidermal thinning, stratum corneum shedding, basal cell karyolysis, hemorrhage and macrophage and neutrophil accumulation in the dermis. Cleaved caspase-3 and phosphorylated histone 2A.X (phospho-H2A.X), markers of apoptosis and DNA damage, respectively, were increased whereas proliferating cell nuclear antigen (PCNA) was down-regulated after SM exposure. By 3 days, epithelial cell hypertrophy, edema, parakeratosis and loss of epidermal structures were noted. Enzymes generating pro-inflammatory mediators including myeloperoxidase and cyclooxygenase-2 were upregulated. After 7 days, keratin-10, a differentiation marker, was evident in the stratum corneum. This was associated with an underlying eschar, as neoepidermis began to migrate at the wound edges. Trichrome staining revealed increased collagen deposition in the dermis. PCNA expression in the epidermis was correlated with hyperplasia, hyperkeratosis, and parakeratosis. By 14 days, there was epidermal regeneration with extensive hyperplasia, and reduced expression of cleaved caspase-3, cyclooxygenase-2 and phospho-H2A.X. These findings are consistent with the pathophysiology of SM-induced skin injury in humans suggesting that the hairless mouse can be used to investigate the dermatoxicity of vesicants and the potential efficacy of countermeasures.

No abstract

Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar ␣1(V), ␣1(XI), and ␣2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the ␣1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains ␣1(V) and ␣1(XI). However, a short imperfection in the triple helix makes ␣1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.Vertebrate collagens are a large family of extracellular proteins that provide mechanical stability to the connective tissue of virtually every organ system. There are at least 40 collagen chains that trimerize into 27 types (1-9), which, in turn, form a large variety of specialized macroaggregates. Structural considerations and the architecture of the resulting polymers have segregated individual collagen molecules into functionally distinct groups (10). Among them, the fibril-forming (fibrillar) collagens represent the most abundant product synthesized by connective tissue cells and include the highly expressed types I-III and the quantitatively minor collagen types V and XI. Types I, III, and V are distributed widely in non-cartilaginous tissues, and types II and XI collagen are found almost exclusively in cartilage and the eye. The pleiotropic manifestations of human fibrillar collagenopathies have dramatically underscored the importance of this ancient group of extracellular proteins in maintaining tissue integrity (1,(11)(12)(13)(14)(15).Fibrillar collagens display a common molecular structure that consists of a long collagenous domain made of ϳ330 Gly-X-Y triplets flanked at both ends by non-collagenous propeptides. Extracellular removal of propeptides initiates the process of maturation facilitating trimer self-assembly into fibrils (for review, see Ref 16). Ultimately, fibers are organized into specific spatial arrays that are responsible for the properties of individual tissues. It follows that the regulation of fibril diameter is an important determinant of connective tissue function. For instance, large diameter fibrils, spatially arranged in unidirectional bundles, are approp...

We have isolated the basement membrane component nicein and performed rotary-shadow analyses using electron microscopy that showed the presence of two forms (I and 11) of the protein. Molecular cloning of the cDNA that codes for the 100-kDa chain of the protein revealed that the sequence matches those independently identified for the 105 -155-kDa subunit of kalinin, a recently identified basement membrane component. These data demonstrate that nicein and kalinin contain an identical chain. The length of the open reading frame in the cDNA (~5 2 0 0 nucleotides) and amino acid sequences obtained from the N-terminus of the 105-kDa kalinin chain showed the occurrence of a precursor polypeptide. This immature polypeptide is probably related to form I, observed by rotary shadowing, while the mature form is related to form II. It is noteworthy that niceinkalinin subunits share discrete sequence similarities with the B2 chain of human laminin, but with a cleavage occurring within domain 111 that eliminates domains IV and V from the final product. The sequence of this subunit is nearly identical to that of B2t, a recently described polypeptide supposed to be related to a new laminin variant. Since niceinkalinin expression is specifically impaired in the severe genodermatosis Herlitz junctional epidermolysis bullosa, the role and structure of this tissue-restricted laminin variant is crucial for the understanding of epidermal-dermal adhesion.The basement membrane (BM) is a type of extracellular matrix which provides an attachment substrate for overlying cell populations and plays a crucial role in organizing tissue architecture, tissue compartmentalization, differentiation of adjacent cells and filtration of nutrients. Since mechanisms mediating these functions are poorly understood, a considerable amount of research has been devoted to the molecular characterization of BM components and elucidation of their biochemical properties. Some differences exist from one type of BM to another and no less than 14 shared macromolecules have already been found [l].We previously reported the isolation of a novel epidermal BM component, called nicein, which is found in various BM species that we investigated with the monoclonal antibody GB3 [2, 31. This glycosylated protein is composed of three major subunits of 100,125 and 150 kDa, as shown by immunoprecipitation assays performed on keratinocytes in culture [3]. Since nicein is secreted by these cells, we isolated the protein by immunoaffinity chromatography from conditioned