Pedigree analysis in the Polish Red cattle population.

The objective of this study was to describe the population structure and inbreeding level of the population of Polish Red Cattle (PRC). The structure of the breed was analysed in the context of the existing genetic resources conservation programme. The level of genetic diversity and the effective population size were also determined. The analyses were carried out based on pedigree records of 9 170 animals. Data and pedigree information were collected during the time period of 1950-2014. Records were collected by the National Research Institute of Animal Production in Balice, Poland. The population structure was analysed using the CFC programme. All the animals were grouped into five classes according to their inbreeding coefficient: the first class included non-inbred animals; and the next classes included inbred animals 0% < F ≤ 5%, 5% < F ≤ 10%, 10% < F ≤ 20%, 20% < F ≤ 30% or F > 30%. The average inbreeding in PRC population was 4% and there were 2 182 (23.8%) inbred animals. The study also included the determination of ancestral paths for the PRC population. The longest ancestral path (LAP) consisted of 12 generations (three animals) while only 229 animals (2.53%) had an LAP comprising at least 10 generations. Therefore, a need exists, particularly in PRC as a small local breed, to manage selection and mating decisions to control future coancestry and inbreeding, which would lead to better handling of the effective population size. The study results showed the possibility of disrupting the balance of the structure of a small population like PRC. Hence, endangered populations need to be monitored on a continuous basis.

Recessive dystrophic epidermolysis bullosa. Evidence for increased collagenase as a genetic characteristic in cell culture.

Fibroblast cultures from patients with recessive dystrophic epidermolysis bullosa (RDEB) demonstrated an increased capacity to synthesize and secrete collagenase. This phenotypic trait appeared to distinguish RDEB from other genetically distinct forms of epidermolysis bullosa. The finding of increased collagenase may be a specific manifestation of these cells in that prototypic lysosomal and cytoplasmic enzymes were present in approximately normal concentrations. In addition, this trait persisted through many cell passages, suggesting that the property was genetically determined. The elevated concentrations of immunoreactive collagenase in fibroblast cultures of patients with RDEB reflected those previously observed in vivo (4) and support the concept of a pathogenetic role for the enzyme in the blistering phenomenon. In three of the cell lines, the increase in enzyme protein occurred in association with a structurally defective enzyme. The data suggest that this may be a characteristic of all RDEB cells.

Immunologic characterization of the membrane-bound collagen in normal human fibroblasts: identification of a distinct membrane collagen.

Collagen, the major extracellular matrix protein, is also a membrane protein. Two types of collagen are detected on the normal human fibroblast membrane in culture, type I collagen and a new immunologically and chemically distinct collagen, type M (membrane) collagen. Antibodies to type M collagen elicited complement-mediated cytotoxicity, which could be blocked by pretreatment of the cells with bacterial collagenase or the antibody with type M collagen. Pretreatment of the cells with other proteolytic enzymes or the antibody with type I collagen or type III collagen had no effect on this complement-mediated cytotoxicity. Although type I collagen is the major collagen synthesized by normal human fibroblasts type M collagen may be the major cell membrane collagen and may be a major cell membrane component.

Diminished response of Werner's syndrome fibroblasts to growth factors PDGF and FGF.

Patients with Werner's syndrome, an autosomal recessive disorder, undergo an accelerated aging process that leads to premature death. Fibroblasts from such patients typically grow poorly in culture. Here it is shown that fibroblasts from a patient with Werner's syndrome have a markedly attenuated mitogenic response to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). In contrast, they have a full mitogenic response to fetal bovine serum. Both PDGF binding and receptor numbers per cell are unaltered. The Werner's syndrome cells express high constitutive levels of collagenase in vitro. Although PDGF enhances collagenase expression through increased levels of hybridizable collagenase messenger RNA in normal skin fibroblasts, no induction of collagenase occurs in the Werner's syndrome fibroblasts. Moreover, the failure to respond to this agonist effect of PDGF is not restored by fetal bovine serum. The data suggest that failure of one or more PDGF-mediated pathways in Werner's syndrome cells may contribute to the phenotypic expression of the disorder.

Enhanced biosynthesis of human skin collagenase in fibroblast cultures from recessive dystrophic epidermolysis bullosa.

Using a sensitive, specific immunoprecipitation method, the biosynthesis of human skin collagenase was studied in fibroblast cultures from patients with recessive dystrophic epidermolysis bullosa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of solubilized immunoprecipitates showed two 3H-labeled procollagenase species that comigrated with those harvested from control cultures. Recessive dystrophic epidermolysis bullosa cultures accumulated increased amounts of collagenase. Both the initial rate of accumulation of intracellular enzyme and the rate of secretion were enhanced, suggesting that excessive accumulation is related to increased synthesis. Because the turnover of labeled collagenase was unaltered, the accumulation could not be attributed to diminishing enzyme degradation. No preferential incorporation of [3H]leucine into recessive dystrophic epidermolysis bullosa collagenase occurred. Furthermore, the mutant cultures displayed no alteration in total protein synthesis, the intracellular leucine pool, or the growth kinetics of the cells. Cells from a patient with dominant epidermolysis bullosa did not show enhanced accumulation of collagenase. The levels of collagenase synthesized in vitro correlated with those observed previously in vivo in recessive dystrophic epidermolysis bullosa patients, suggesting that this biochemical trait is pathogenetically significant in the disorder.

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.

Increased glycosaminoglycan accumulation as a genetic characteristic in cell cultures of one variety of dominant dystrophic epidermolysis bullosa.

Fibroblast cultures from patients with dominant dystrophic epidermolysis bullosa of the albopapuloid variety display deranged glycosaminoglycan metabolism. These cells accumulate increased amounts of sulfated glycosaminoglycans. The mechanism for the greater content of glycosaminoglycans appears to be related to increased synthesis. During the first 6-12 h, intracellular labeled glycosaminoglycans accumulated in the dominant dystrophic epidermolysis bullosa cells at about twice the rate as that of control fibroblasts. In addition, secretion of sulfated glycosaminoglycans was two- to threefold greater than in control cultures. In contrast, both pulse-chase and cross-correction experiments failed to show any evidence for defective degradation of the material. The biochemical trait is genetically specific for albopapuloid dominant dystrophic epidermolysis bullosa, since fibroblasts from patients with other varieties of epidermolysis bullosa did not accumulate increased glycosaminoglycans. The data suggest that in vitro abnormality in glycosaminoglycan metabolism could serve as an important marker for this variety of epidermolysis bullosa and be of genetic and prognostic value in the sporadic patient with epidermolysis bullosa. Although the precise relationship of the defect to the disease has not yet been defined, it is possible that excessive tissue accumulation of glycosaminoglycans may alter collagen fibril deposition, thus, impairing the structural integrity of the skin and leading to posttraumatic blisters and erosions that characterize the disease.

Scleroderma: increased biosynthesis of triple-helical type I and type III procollagens associated with unaltered expression of collagenase by skin fibroblasts in culture.

To assess potential abnormalities in collagen metabolism in systemic scleroderma, skin fibroblast lines from patients with this disease were established and compared to control cell lines derived from healthy subjects. For studies on the biosynthesis of procollagen, the cells were incubated with [(14)C]proline in a medium supplemented with ascorbic acid and beta-aminopropionitrile, and the synthesis of nondialyzable [(14)C]hydroxyproline, in relation to DNA or cell protein, was taken as an index of procollagen formation. Five of eight scleroderma fibroblast cell lines demonstrated procollagen biosynthesis rates significantly higher than the controls, and the mean rate of procollagen synthesis by scleroderma fibroblasts was about twice that of the control cells. Control experiments demonstrated that the specific activity of the intracellular free proline was not different in scleroderma and control fibroblasts, and the mean population doubling times of the scleroderma and the control fibroblast cell lines were the same. The relative synthesis of the genetically distinct procollagens was examined by isolating type I and type III procollagens from the cell culture medium using DEAE-cellulose chromatography. The ratios of type I/III procollagens in scleroderma cell lines did not differ from the controls. The helical stability of the collagenous portion of type I and type III procollagens, estimated by the resistance of (14)C-collagen to limited proteolytic digestion with pepsin under nondenaturing conditions, was the same in both scleroderma and control cultures. The capacity of the cells to synthesize enzymatically active and immunologically reacting collagenase was also studied; no marked differences in these parameters could be observed. The results suggest that cultured skin fibroblasts from patients with scleroderma demonstrate a metabolic abnormality expressed as increased synthesis of type I and type III procollagens in a normal ratio. This abnormality may play a role in the excessive accumulation of collagen in the skin and other organs affected in scleroderma.

Human skin collagenase in recessive dystrophic epidermolysis bullosa. Purification of a mutant enzyme from fibroblast cultures.

Recessive dystrophic epidermolysis bullosa, a genodermatosis characterized by dermolytic blister formation in response to minor trauma, is characterized by an incresaed collagenase synthesis by skin fibroblasts in culture. Since preliminary studies of partially purified recessive dystrophic epidermolysis bullosa collagenase suggested that the protein itself was aberrant, efforts were made to purify this enzyme to homogeneity, so that detailed biochemical and immunologic comparisons could be made with normal human skin fibroblast collagenase. Recessive dystrophic epidermolysis bullosa skin fibroblasts obtained from a patient documented to have increased synthesis of the enzyme were grown in large scale tissue culture and both serum-free and serum-containing medium collected as a source of collagenase. The recessive dystrophic epidermolysis bullosa collagenase was purified to electrophoretic homogeneity using a combination of salt precipitation, ion-exchange, and gel-filtration chromatography. In contrast to the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase bound to carboxymethyl-cellulose at Ca(2+) concentrations at least 10 times higher than those used with the normal enzyme. Additionally, this enzyme was significantly more labile to chromatographic manipulations, particularly when serum-free medium was used. However, rapid purification from serum-containing medium yielded a preparation enzymatically equivalent to normal human skin collagenase. Like the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase was secreted as a set of two closely related zymogens of approximately 60,000 and approximately 55,000 daltons that could be activated by trypsin to form enzymically active species of approximately 50,000 and approximately 45,000 daltons, respectively. Amino acid analysis suggested slight variations between the normal and recessive dystrophic epidermolysis bullosa collagenases. Cyanogen bromide digests demonstrated peptides unique to the enzyme from each source. The recessive dystrophic epidermolysis bullosa proenzyme was significantly more thermolabile at 60 degrees C than the normal, a finding that correlated with an approximate fourfold decrease in the affinity of the mutant enzyme for Ca(2+), a known activator and stabilizer of human skin collagenase. Aside from the altered affinity for this metal cofactor, kinetic analysis of the structurally altered recessive dystrophic epidermolysis bullosa collagenase revealed that its reaction rates and substrate specificity for human collagen types I-V were identical to those for the normal enzyme. Likewise, enzymes from both sources displayed identical energies of activation and deuterium isotope effects. Antisera were raised to the normal and putatively mutant procollagenases respectively, and, although they displayed a reaction of identity in double diffusion analysis, immunologic differences were present in enzyme inhibition and quantitative precipitation studies. These studies indicate that recessive dystrophic epidermolysis bullosa is characterized by the increased synthesis of an enzymically normal, but structurally aberrant, collagenase.

Enhanced collagenase production by fibroblasts derived from human basal cell carcinomas.

Fibroblast cultures derived from human basal cell carcinomas demonstrated an increased capacity to synthesize and secrete collagenase. Although the levels of collagenase were up to 8-fold greater than those of normal control cell lines, this phenotypic trait was not permanent and was expressed only for a few passages following primary explanation. The basal cell carcinoma fibroblast collagenase was secreted as a proenzyme. The kinetics of activation and the catalytic efficiency of the basal cell carcinoma fibroblast enzyme were equal to control collagenase, indicating that increased activity was due to increased synthesis of enzyme protein. Increased synthesis of collagenase was not due either to altered cell growth or to an overall increase in protein synthesis. Furthermore, synthesis of another major protein, of another major protein, collagen, was not enhanced. The data suggest that the tumors may have stimulated adjacent fibroblasts to produce more collagenase which is of importance in tumor invasion.

Environmental pH modulation of collagenase in normal human fibroblast cultures.

Normal human skin fibroblast cultures have been used to assess the effects of relatively minor changes in environmental pH on collagenase, a major extracellular gene product. Collagenase accumulation in the culture medium, measured both as enzyme activity and immunoreactive material, was 2- to 10-fold greater at pH 7.6--8.2 than at pH 6.8--7.2. The pH-associated increase in collagenase was paralleled by an increase in general protein synthesis. Nevertheless, prototypic lysosomal and cytoplasmic enzymes changed very little under identical culturing conditions. Although substantial intracellular protein degradation occurred at all pH values, the small differences either in general protein degradation or in specific collagenase degradation in the medium were of insufficient magnitude to account for the increased accumulation of collagenase.

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.

Glucocorticoid modulation of collagenase expression in human skin fibroblast cultures. Evidence for pre-translational inhibition.

Glucocorticoids inhibit collagenase accumulation in the medium of human skin explant cultures. To examine the mechanism for this process, skin fibroblasts were placed in serum-free medium containing various steroids. Dexamethasone produced a dose-dependent inhibition of trypsin-activatable collagenase in the culture medium with maximal inhibition of approx. 85% at 10(-6) M. Dexamethasone failed to inhibit collagenase activity directly. The decrease in activity in the medium was paralleled by a decrease in immunoreactive protein, suggesting inhibition of enzyme synthesis. The specificity of the effect was shown in two ways. At 10(-6) M steroid, only dexamethasone and hydrocortisone were inhibitory; estradiol, progesterone and testosterone produced less than 10% inhibition. In biosynthetic studies, exposure to 10(-7) M dexamethasone for 24 h produced approx. 50% inhibition of collagenase synthesis but caused no greater than 10% inhibition of total protein synthesis. The T1/2 for achieving the effect was approx. 16 h after initial exposure to dexamethasone. These kinetics were parallel to the inhibition caused by actinomycin D and cordycepin, two inhibitors of transcription, but were longer than that caused by cycloheximide (T 1/2 less than 3 h). To examine this process, cells were cultured in the presence or absence of 10(-6) M dexamethasone prior to harvesting mRNA for cell-free translation. In each case the inhibition or enzyme activity in the intact cells was paralleled by a reduction in translatable collagenase mRNA from the same cells. At the same time, there was no significant inhibition of total protein translation by the steroid. These data suggest that glucocorticoids regulate collagenase synthesis at a pre-translational level, possibly through inhibition of transcription.

Abnormalities in collagenase expression as in vitro markers for recessive dystrophic epidermolysis bullosa.

In order to correlate a characteristic clinical phenotype with biochemical abnormalities in recessive dystrophic epidermolysis bullosa, fibroblast cultures were established from 4 typical patients with the severe form of the disease. Collagenase, the enzyme implicated in the pathogenesis of blistering, was present in vitro in 2- to 4-fold greater concentrations than in control fibroblast cultures. Partially purified preparations of this enzyme displayed marked thermal lability and diminished affinity for Ca2+, a metal cofactor, suggesting the existence of a mutant enzyme. The data suggest that these 3 biochemical abnormalities, increased synthesis, decreased thermal stability and diminished affinity for Ca2+, should serve as reliable in vitro markers for genetic discrimination of recessive dystrophic epidermolysis bullosa.

Colchicine-induced modulation of collagenase in human skin fibroblast cultures. I. Stimulation of enzyme synthesis in normal cells.

Microtubule-active agents affect the secretion of a variety of proteins, including collagenase. To gain insight into the mechanisms involved in this process, we examined the effects of colchicine on the synthesis, secretion, and activity of human skin collagenase. When added to monolayer cultures of human skin fibroblasts, 10(-6) M colchicine produced a mean 3-fold increase in trypsin-activatable collagenase in the culture medium. Stimulation was not observed with lumicolchicine. The enhanced accumulation of collagenase was dose-dependent with 10(-9), 10(-8), 10(-7), and 10(-6) M colchicine giving collagenase activities/mg protein that were 100 +/- 6%, 165 +/- 20%, 186 +/- 34%, and 297 +/- 62% of control, respectively. Although the effect on collagenase was seen under conditions independent of cellular growth (i.e., in serum-free medium), maximum stimulation occurred in subconfluent cultures. The colchicine-induced increase in activity was paralleled by an increase in immunoreactive enzyme protein, suggesting stimulation of enzyme synthesis. The catalytic efficiency of the enzyme (activity per unit immunoreactive protein) was unchanged, however, indicating that a structurally normal enzyme was being synthesized. To examine the process in more detail, the biosynthesis of 3H-labeled collagenase was quantitated in these cultures by specific immunoprecipitation. Although 10(-6) M colchicine produced no increase in total protein synthesis, an increased rate of collagenase synthesis was seen after only 1.5 hr. These data suggest that colchicine has a specific effect on the synthesis of collagenase and may be a useful probe for studying its regulation.

Progressive nodular fibrosis of the skin: altered procollagen and collagenase expression by cultured fibroblasts.

A 33-year-old man presented with a spontaneous progressive cutaneous tumor-like fibrosis involving the right leg and buttock. Histologically the deep dermis was composed of numerous fibroblasts and dense bands of collagen, suggesting that the lesion might be related to an abnormality in collagen metabolism. Fibroblast cultures were established from the affected and normal-appearing skin. The growth rate of the lesional cells was essentially equal to that of control cells. The synthesis of procollagen was approximately 3.5-fold increased in the cells derived from the nodules when compared with control fibroblasts (p less than 0.001). The increase in procollagen synthesis was reflected by an approximate 6-fold increase in both type I and type III procollagen mRNA abundance in the lesional fibroblasts (p less than 0.001), thus suggesting an aberration in the pretranslational level of procollagen gene expression. In contrast, the synthesis of collagenase, the enzyme required for the initiation of collagen degradation, was decreased to approximately 25% of control values (p less than 0.0025), although the enzyme was catalytically normal. The data indicate that these cells are characterized by an increased synthesis of procollagen and decreased synthesis of collagenase, 2 phenotypic characteristics that could account pathophysiologically for the lesions. The unusual reciprocal nature of these biochemical parameters in 2 proteins important in connective tissue homeostasis suggests that this progressive tumor-like condition may have resulted from the expansion of a clonal population of cells.

Elevated levels of human collagenase inhibitor in blister fluids of diverse etiology.

Blister fluids from a variety of bullous disorders were examined for the presence of human collagenase inhibitor. A protein immunologically identical to the collagenase inhibitor produced by human skin fibroblasts was found in high concentrations within bullae of diverse etiologies. Levels of collagenase inhibitor in blister fluids ranged from 0.9-12.5 micrograms/ml, averaging 4.9 micrograms/ml. The mean values were 3- to 4-fold greater than those present in the sera of corresponding patients and exceeded plasma levels by 6- to 8-fold. The time course of collagenase inhibitor accumulation in blister fluid was studied using heat- and suction-induced bullae. The concentration in newly formed blisters was approximately 0.5 micrograms/ml, virtually identical to plasma inhibitor levels, and remained constant for approximately 4 h. Inhibitor concentrations then rose rapidly, reaching peak values of approximately 6 micrograms/ml after 48 h. We speculate that the role of this inhibitor in blister fluid involves the inhibitions of active proteinases within the bulla cavity and may occur to limit the extent of blister formation or to assist in wound repair.

Localization of type I human skin collagenase in developing embryonic and fetal skin.

Type I human skin collagenase (HSC-1) was localized in developing embryonic and fetal skin ranging from 6 to 20 weeks estimated gestational age using an antigen-specific, affinity-purified, polyclonal antiserum to HSC-1 and an avidin-biotin alkaline phosphatase procedure. Double immunolabeling with monoclonal antibodies for Factor VIII-related antigen, type IV collagen, and the 68-kilodalton neurofilament subunit was performed using a direct peroxidase procedure. By 8 weeks estimated gestational age, HSC-1 localized to the periderm, the basal cell epidermal keratinocytes, dermal fibroblasts, and surrounding extracellular matrix. At 12 weeks estimated gestational age, HSC-1 immunolabeling showed a continued association with the epidermis and dermis. Dermal and subcutaneous blood vessels and the surrounding extracellular matrix were positive for HSC-1 labeling. HSC-1 staining was also found around developing nerves and in association with dermal fibroblasts. In the developing hair follicle, HSC-1 was present in keratinocytes of the pre-germ, germ, hair peg, and bulbous hair peg. HSC-1 immunoreactivity was also found in association with the hair canal, the bulge, and the dermal papillae, but was absent from the fetal sebaceous gland. These data demonstrate the association of HSC-1 with the development of interfollicular epidermis, the dermal collagenous matrix, the process of angiogenesis, the development of nerves, and hair follicle morphogenesis.

Stimulation of skin fibroblast collagenase production by a cytokine derived from basal cell carcinomas.

Our previous studies of human basal cell carcinomas (BCC) revealed increased skin collagenase in vivo. Immunocytochemically the collagenase was localized to adjacent stroma, not to the tumor cells. When grown in culture, skin fibroblasts derived from tumor stroma showed a 3- to 4-fold increase in collagenase for the first 10-14 mean population doublings, after which collagenase expression reverted to control levels. These studies suggested that tumors stimulated adjacent fibroblasts to produce more collagenase. In the present study we sought direct evidence for epithelial-stromal interaction in this neoplasm. Under dissecting microscopy tumor islands were freed of stroma, homogenized, sonicated, and centrifuged to remove insoluble tissue. Tumor extracts were incubated with monolayer cultures of normal human skin fibroblasts to assess their effect on collagenase synthesis in these target cells. Culturing the fibroblasts for 24 h in the presence of individual BCC extracts resulted in a 1.6- to 3-fold increase in trypsin-activatable collagenase in the culture medium. This was paralleled by an equal increase in immunoreactive protein, suggesting enhanced enzyme synthesis. There was no change in the activity per immunoreactive protein, indicating a catalytically unaltered enzyme. Gel filtration of pooled BCC extracts showed that the stimulatory activity was contained in eluent fractions of Mr approximately 19Kd. These data suggest that BCCs elaborate a macromolecular cytokine that induces collagenase synthesis in skin fibroblasts and emphasize the importance of epithelial-stromal interactions in cutaneous tumor invasion.

Retinoic acid inhibition of collagenase and gelatinase expression in human skin fibroblast cultures. Evidence for a dual mechanism.

Human skin fibroblast cultures have been employed to study the effects of a variety of vitamin A analogues (retinoids) on the expression of two enzymes involved in collagen degradation in the skin, collagenase and a gelatinolytic protease. In normal and recessive dystrophic epidermolysis bullosa fibroblast cultures, retinoic acid compounds were effective inhibitors of the accumulation of both enzymes in the culture medium with half-maximal inhibitions occurring at 0.25-1 microM for collagenase and at 3-6 microM for the gelatinolytic protease. Various retinoids exhibited differing degrees of inhibitory actions, so that at a 1 microM concentration, relative inhibitions were: 13-cis-retinoic acid greater than all-trans-retinoic acid greater than aromatic retinoid (Ro 10-9359) much greater than retinol. The retinoic acid-mediated decrease in collagenase activity was accompanied by a parallel decrease in immunoreactive collagenase protein, suggesting that the retinoic acids were acting to inhibit synthesis of the enzyme. However, an additional effect of these agents was encountered. Although the retinoids themselves had no direct collagenase inhibitory action, medium derived from cultures maintained in these retinoids showed direct inhibitory capacity which was dependent both on the concentration of retinoic acid and on the length of time in culture. The results suggest that the retinoic acids modulate collagenase in vitro by two mechanisms: by decreasing the synthesis of enzyme protein and by modulating the expression of an inhibitory molecule.