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.

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.

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.

Colchicine-induced modulation of collagenase in human skin fibroblast cultures. II. A probe for defective regulation in epidermolysis bullosa.

The addition of colchicine to cultures of normal human skin fibroblasts produces a significant stimulation of collagenase. Because this finding implies a role for the microtubule system in the regulation of normal collagenase synthesis, we have used colchicine as a probe for aberrations in this enzyme in epidermolysis bullosa. In fibroblast cultures from the dominant simplex, dominant dystrophic, and recessive letalis forms of epidermolysis bullosa, 10(-6) M colchicine produced approximately a 2-fold increase in collagenase in the culture medium, a finding shown by biosynthetic studies to be attributable to enhanced synthesis of enzyme protein. In the case of typical recessive dystrophic epidermolysis bullosa, a disease characterized by excessive collagenase synthesis, the fibroblasts could also be stimulated to produce additional collagenase, despite having elevated baseline synthetic rates. In contrast, fibroblasts isolated from one recessive epidermolysis bullosa patient were resistant to the stimulatory effects of colchicine in concentrations up to 5 x 10(-6) M. In the absence of colchicine, collagenase synthesis in this patient's cells (termed REBc-) was 3-4 times that of normal controls, suggesting that the as yet undefined cellular function that is abrogated (or stimulated) by colchicine in normal cells may have been genetically impaired in these REBc- cells. Despite the resistance to colchicine, as manifested by the failure to stimulate collagenase, gross parameters of microtubular function, such as cell replication, were intact. Phenotypically, this patient had a form of epidermolysis bullosa intermediate between typical recessive dystrophic and recessive letalis forms of the disease. Although an experimentally induced blister was located in the lamina lucida, hypoplastic anchoring fibrils were also observed. These findings, in addition to the marked increase in collagenase synthesis, suggest the possibility that this patient may represent a compound heterozygote of two forms of epidermolysis bullosa and that colchicine may be useful in defining other such patients.

Enhanced cell-free translation of human skin collagenase in recessive dystrophic epidermolysis bullosa.

The regulatory mechanisms for collagenase synthesis in recessive dystrophic epidermolysis bullosa (RDEB) have been studied using messenger RNA (mRNA) harvested from normal and RDEB skin fibroblasts to direct protein synthesis in a rabbit reticulocyte lysate translation system. Fibroblast mRNA encoded the synthesis of approximately 60,000 and approximately 55,000 dalton forms of procollagenase in cell-free translation. In contrast to biosynthesis by intact cells, there was preferential translation of the approximately 60,000 dalton specie. For quantitative comparisons with mRNA from normal cells, mRNA was harvested from fibroblasts of 2 RDEB patents whose intact cells have been documented to have increased synthesis of collagenase. Although total translational activity was equal in normal and RDEB mRNA preparations, translatable collagenase mRNA was increased 3.5- to 10-fold, suggesting that the enhanced collagenase synthesis characteristic of RDEB is due to increased concentrations or preferential translation of collagenase mRNA.

The effect of nalidixic acid, rifampicin and chloramphenicol on the synthesis of phospholipase C in Bacillus cereus.

The effect of nalidixic acid, rifampicin and chloramphenicol on the synthesis of phospholipase C (EC 3.1.4.3) has been studied in washed Bacillus cereus cells resuspended in nutrient broth. In the absence of inhibitors, the synthesis showed a biphasic pattern. No synthesis of release of enzyme was found in the presence of chloramphenicol. When rifampicin was added, phospholipase C synthesis continued for 10-15 min. Nalidixic acid, at concentrations which inhibited DNA synthesis completely, permitted the synthesis of phospholipase C at the same rate and for a similar length of time as rifampicin.

Inhibition of the synthesis of phospholipase C in Bacillus cereus by a component of the growth medium.

A low molecular weight compound, probably of peptide nature, present in the Beef Heart Extract component of the growth medium, inhibits post-transcriptionally the biosynthesis of phospholipase C by a strain of Bacillus cereus. The compound also prevents the increase of proteolytic activity in the growth medium, thus suggesting that the synthesis of another enzyme, an extracellular protease, is also inhibited, and that the inhibitory compound may therefore have a more general effect on exoenzyme synthesis in this strain of Bacillus cereus.