Eugene A. Bauer

Collagenase production by human skin fibroblasts.

Abstract Normal human skin fibroblasts, when cultured in serum free medium, produce collagenase in an inactive form. The enzyme in the crude medium can be activated by a brief preincubation with trypsin or by autoactivation. Once activated, the fibroblast collagenase is identical in its mechanism of action to human skin collagenase obtained from organ cultures. In addition, an inhibitor of collagenase is also present in the medium of fibroblast cultures. The inhibitor appears to be produced by the cells and its molecular weight is slightly higher than that of the enzyme. The presence of this inhibitor may account for previous inability to detect collagenase in human skin fibroblast cultures. It is also possible that some of the inactive enzyme exists in the medium in the form of a proenzyme.

Morphea and lichen sclerosus et atrophicus: Clinical and histopathologic studies in patients with combined features

Ten patients with skin lesions clinically consistent with morphea and lichen sclerosus et atrophicus were studied. In all cases, histopathologic changes of both morphea and lichen sclerosus et atrophicus were also present. The coexistence of morphea and lichen sclerosus et atrophicus in the same patient suggests that these lesions represent a spectrum which may reflect similar etiologic events or closely related pathologic processes in these two diseases.

Inhibition of collagen degradative enzymes by retinoic acid in vitro

The effects of a variety of retinoids on collagenase and gelatinase expression have been examined in skin fibroblast cultures derived from normal volunteers and from patients with the hereditary blistering disorder, recessive dystrophic epidermolysis bullosa. Both 13-cis- and all-trans-retinoic acid were effective inhibitors of collagenase production in both cell types. In the case of collagenase, the inhibition of collagenase activity was paralleled by a reduction in immunoreactive enzyme protein, suggesting that these retinoids act by inhibiting synthesis and/or secretion of the enzyme. Retinoic acid also inhibited production of the second enzyme in the collagen degradative pathway, gelatinase. In this case, the decrease in gelatinase activity was equal to or slightly greater than the achieved in collagenase expression. The observation that certain retinoids modulate the two crucial enzymes in the degradation of collagen in the skin suggests that they might be useful therapeutic agents in recessive dystrophic epidermolysis bullosa, a disease in which the pathogenesis of blistering is in part related to connective tissue destruction.

Immunologic relationship of purified human skin collagenase to other human and animal collagenases

Abstract Antibodies prepared to a number of animal and human collagenases have been used to investigate the relationships between these enzymes. The human skin collagenase used as an immunogen was one of two chromatographically separable peaks of collagenolytic activity obtained from culture media of normal human skin. On the basis of their electrophoretic mobility on polyacrylamide gel, they have been designated as fast-moving or slow-moving human skin collagenase. The fast moving enzyme was further purified and used to produce an anti-human skin collagenase antibody. Anti-fast-moving human skin collagenase antibody gives a reaction of complete identity on immunodiffusion with slow-moving skin collagenase as well as with preparations of human gingival and rheumatoid synovial collagenases. In addition, anti-fast-moving human skin collagenase antibody produces complete inhibition of these other human collagenases, suggesting that these enzymes are closely related proteins. Antibodies to tadpole and rat uterine collagenases are immunologically not identical when compared with one another or to the human skin enzyme. Crustacean hepatopancreas and bacterial collagenases also fail to react with anti-fast-moving human skin collagenase antibody, further indicating species specificity among collagenases.

Epidermolysis Bullosa: A Review

Abstract: Epidermolysis bullosa is a group of inherited disorders characterized by blistering of the skin as a result of minor trauma. The disease can be divided into three anatomical categories: Epidermolytic, where blister cleavage occurs within the epidermis; functional, which has blister cleavage within the lamina lucida; and Dermolytic, where blister cleavage occurs below the basal lamina in the upper papillary dermis. Each of these three categories can be divided into several distinct entities based on clinical and histologic criteria. Basic biochemical studies have increased our understanding of several of these diseases, most notably recessive dystrophic epidermolysis bullosa. Although therapy for patients with EB is largely supportive, increased knowledge of the biochemistry of these disorders is making direct therapeutic interventions possible.

Enzyme-Linked Immunosorbent Assay for Human Skin Collagenase

A specific and sensitive indirect inhibition enzyme-linked immunosorbent assay (ELISA) was develop for human skin collagenase. Using an antiserum dilution of 1: 8000, the ELISA could detect 0.2 ng of enzyme, which was approximately 10 times more sensitive than the previously described radioimmunoassay for human skin collagenase. The assay was also highly reproducible. In comparative studies, bacterial, tadpole and crab collagenases did not react in the ELISA. Rat uterine collagenase and collagenases from bovine gingival explants and fibroblasts displayed approximately 0.1% of the reactivity found with human skin collagenase. Human synovial and gingival collagenases and collagenase from skin fibroblasts from patients with recessive dystrophic epidermolysis bullosa showed almost complete identity with the normal human skin fibroblast enzyme.

Preparation of three vertebrate collagenases in pure form

Abstract Human skin, rheumatoid synovial and tadpole collagenases have been obtained in pure form using affinity chromatography with collagen coupled to Sepharose. This simple and reproducible method should have broad applicability in the purification of other animal and human collagenases in sufficient quantities to study in detail their physical and enzymologic properties.

Cell culture density as a modulator of collagenase expression in normal human fibroblast cultures

Abstract The effect of various stages of normal cell growth on human fibroblast collagenase found in the culture medium was studied, so that the regulatory mechanisms of synthesis, secretion and activity of the enzyme could be established. Specific activity of collagenase increased 6- to 10-fold shortly after confluence was reached when compared with low density levels and decreased in post-confluent cultures, suggesting that synthesis and/or release of the enzyme changes with culture density. To assess this possibility, culture medium was examined for immunoreactive collagenase protein by radioimmunoassay. After confluence was reached, immunoreactive collagenase had increased approx. 2-fold, indicating greater secretion, and probably synthesis, of the enzyme. However, the increase in specific activity of the enzyme observed shortly after confluence was greater than could be accounted for by an increase in immunoreactive enzyme protein. As a result of the disproportionate increase in collagenase activity, the collagenase activity per unit immunoreactive protein was also found to be greatest shortly after confluence and decreased in post-confluent cultures. This density-associated modulation of collagenase expression could be reproduced by initiating the cultures at high density after subculture. Expression of collagenase activity was dependent upon intact protein synthetic mechanisms, since cultures maintained in the presence of cycloheximide failed to secrete collagenase into the culture medium.

Studies on Purified Rheumatoid Synovial Collagenase In Vitro and In Vivo

Rheumatoid synovial collagenase obtained from culture medium can be separated by Sephadex gel filtration into two peaks of enzyme activity. These have been designated as fast-moving and slow-moving rheumatoid synovial collagenases on the basis of their electrophoretic mobility on polyacrylamide gels. The slow-moving rheumatoid synovial collagenase has been highly purified by affinity chromatography on collagen conjugated to Sepharose and used to prepare a monospecific anti-synovial collagenase antiserum. The antiserum against rheumatoid synovial collagenase has permitted the demonstration of immunoreactive collagenase in extracts of rheumatoid synovial tissue that have no detectable enzymatic activity. Collagenase has also been detected immunologically in enzymatically inactive culture medium from the first 24 hr of culture. Recovery of collagenase activity appears to be related to the chromatographic separation of the enzyme from serum antiproteases. The demonstration of collagenase in vivo in rheumatoid synovium adds further support for the concept that the enzyme is present in tissue at levels that are of significance in the pathogenesis of rheumatoid arthritis. In addition, rheumatoid synovial collagenase and human skin collagenase show complete immunologic identity when reacted with monospecific antiserum prepared against either of these purified enzymes, indicating that organ specificity between these two human collagenases is unlikely.

Hormonal interactions in mammalian collagenase regulation: Comparative studies in human skin and rat uterus

The production of collagenase by human skin explants in culture is prevented by 10(-8) M dexamethasone, 5 . 10(-4) M dibutyryl cyclic AMP, or 2.5 . 10(-3) M theophylline. Decreases in collagenase activity are paralleled by reductions in the degradation of explant collagen during the culture period. Progesterone, which effectively inhibits collagenase production in rat uterine explant cultures, has no effect on human skin explants. The inhibition by cyclic AMP is nucleotide specific. When partially inhibitory concentrations of dexamethasone and dibutyryl cyclic AMP, or dexamethasone and theophylline, are added to culture medium together, the resultant inhibition is that predicted by additivity. Synergistic inhibition, as observed in rat uterus between progesterone and dibutyryl cyclic AMP, fails to occur. Dexamethasone inhibits the production of collagenase by cultured explants of rat uterus, with complete inhibition occurring at 10(-7) M steroid. Synergism between glucocorticoids and dibutyryl cyclic AMP or between dexamethasone and progesterone could not be demonstrated in the uterine culture system. These results suggest the existence of three regulatory systems for the control of collagenase production in mammalian tissues, and that cooperativity between systems may occur on a tissue-specific basis.