Jo Louise Seltzer

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.

The function of Ca2+ in the action of mammalian collagenases

Abstract The removal of extrinsic Ca2+ from human skin, rat skin, and postpartum rat uterus collagenases results in a reversible loss of enzymatic activity, which becomes irreversible with increasing length of Ca2+-free incubation at physiological temperature and pH. Ca2+ is necessary for thermostabilization both in the presence and absence of the collagen substrate. Enzymes from all three sources display linear rates of reaction at Ca2+ concentrations from 0.5 to 20 m m and are half-maximally activated at 0.5 m m Ca2+. The increase in collagenase activity with increasing Ca2+ concentration is associated with an increase in thermostabilization. Ba2+ and Sr2+ are effective substitutes for Ca2+ in human skin collagenase but not in the collagenases from rat tissues. These studies also indicate that Ca2+ plays no role in the binding of collagenases to their substrate.

Evidence for mammalian collagenases as zinc ion metalloenzymes

Collagenases (EC 3.4.24.3) from human skin, rat skin and rat uterus were inhibited by the chelating agents EDTA, 1,10-phenanthroline and tetraethylene pentamine in the presence of excess Ca2+, suggesting that a second metal ion participates in the activity of the enzyme. Collagenase inhibition by 1,10-phenanthroline could be both prevented and reversed by a number of transition metal ions, specifically Zn2+, Co2+, Fe2+ and Cu2+. However, Zn2+ is effective in five-fold lower molar concentrations (1-10(-4) M) than the other ions. Furthermore, Zn2+ was the only ion tested able to prevent and reverse the inhibition of collagenase by EDTA in the presence of excess Ca2+. Atomic absorption analysis of purified collagenase for Zn2+ showed that Zn2+ was present in the enzyme preparations, and that the metal co-purifies with collagenase during column chromatography.

Protein tyrosine phosphorylation in signalling pathways leading to the activation of gelatinase A: activation of gelatinase A by treatment with the protein tyrosine phosphatase inhibitor sodium orthovanadate

Fibroblasts in monolayer culture secrete gelatinase A (MMP2; 72 kDa type IV collagenase) only in its proenzyme form. Unlike other secreted matrix metalloproteinases, progelatinase A is refractory to activation by serine proteinases. Disparate agents, including monensin, cytochalasin D, and concanavalin A, have been found to mediate the activation of gelatinase A zymogen secreted by fibroblast monolayers. Our finding that monensin-mediated activation can be reversed by the protein tyrosine kinase inhibitor genistein (Li et al., Experimental Cell Research 232 (1997) 332) prompted us to investigate the effect of the specific inhibitor of protein tyrosine phosphatases, sodium orthovanadate, on progelatinase A activation. Treatment of fibroblast monolayers with orthovanadate also results in the secretion of activated gelatinase A. This activation is dose- and time-dependent, requires protein synthesis, and is associated with cell membranes. Vanadate-mediated activation does not occur in the presence of herbimycin A, a protein tyrosine kinase inhibitor. As with progelatinase activation mediated by monensin, concanavalin A, and cytochalasin D, orthovanadate treatment results in increased synthesis of the membrane proteinase MT1-MMP, that can catalyze the activation of progelatinase A. Protein tyrosine kinase inhibitors are able to prevent the increase of MT1-MMP mRNA, as shown by Northern blot and RT-PCR. In addition, orthovanadate potentiates the effects of monensin and concanavalin A. While treatment with monensin or concanavalin A result only in an increase of the putative activator MT1-MMP, orthovanadate also reduces the production of the specific inhibitor TIMP-2. These experiments implicate protein tyrosine phosphorylation in the signal transduction pathways which lead to the activation of progelatinase A.

Enzyme levels in chick embryo heart and brain from 1 to 21 days of development

Abstract Enzyme activity levels were measured in chick embryo brain and heart during development, beginning with medullary plate and cardiogenic mesentoderm. To study heart and brain during the period of morphogenesis (1–4 days) a method for freezedrying whole chick embryos was developed. In three divisions of brain—diencephalon, telencephalon, and hindbrain-hexokinase, glyceraldehyde-3-P dehydrogenase, and 6-P-gluconic dehydrogenase maintained approximately constant levels of activity during this period. Brain glucose-6-P dehydrogenase levels fell somewhat, but contrary to earlier reports showed no wide fluctuations. In heart, glucose-6-P dehydrogenase activity fell to one-half between 1 and 4 days, 6-P-gluconic dehydrogenase activity remained constant, while hexokinase activity doubled in atrium from 1 to 2 days, and tripled in ventricle from 1 to 4 days. From 6 to 21 days of development, homogenates of hearts and brains were used. Hexokinase activity in brain increased four-fold during this period, while in heart the specific activity did not change. Glyceraldehyde-3-P dehydrogenase activity showed no change in either organ. NAD-dependent isocitric dehydrogenase increased in both heart and brain, fourfold in brain, nearly twofold in heart. α-Ketoglutaric dehydrogenase increased 50% in brain and 250% in heart. The increasing levels of citric acid cycle enzymes probably reflect an increasing energy demand in both organs during the last 2 weeks before hatching. Since adult brain depends primarily upon glucose for energy, it seems reasonable that the hexokinase activity continued to increase. Adult heart, however, obtains its energy from substrates other than glucose, which may account for the fact that during the last 2 weeks no change in heart hexokinase activity was seen.

The stoichiometric activation of human skin fibroblast pro-collagenase by factors present in human skin and rat uterus

Abstract Purified human skin fibroblast collagenase zymogen was used as a substrate for activators partially purified from the medium of cultured human skin and rat uterus. Analysis of the activated enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that these activators do not produce measurable changes in the molecular weight of the zymogen. Kinetic analysis indicated a noncatalytic mechanism of action for these activators, since zymogen activation was independent of incubation time, and dependent only upon the concentration of the activator fractions. These results are consistent with a Stoichiometric mechanism of procollagenase activation by these macromolecules.

Eriochrome black T inhibition of human skin collagenase, but not gelatinase, using both protein and synthetic substrates.

The intracellular degradation of interstitial collagen is accomplished by two neutral metalloproteases, collagenase and gelatinase. Both enzymes are inhibited by metal chelating agents, by certain sulfhydryl reagents, and by similar protein inhibitors. Here, we demonstrate that the dye eriochrome black T (EBT) appears to be unique in its capacity to inhibit collagenase but not gelatinase. Using native reconstituted helical collagen in gel form at 37 degrees C, half-maximal inhibition of collagenase activity by EBT occurs at approximately 45 microM. EBT more effectively inhibits the breakdown of native collagen in solution, with a KI of approximately 8 microM. Using a newly-developed spectrophotometric substrate, AcProLeuGly-S-LeuLeuGly-OC2H5, a KI of 1.4 microM was calculated for EBT on collagenase. Although this same thiopeptolide serves as a substrate for gelatinase with kinetics similar to those of collagenase, no inhibition by EBT was observed. EBT also did not inhibit the gelatinase-mediated breakdown of the natural substrate, gelatin. The data suggest that EBT may have significant potential for allowing the differentiation in biological fluids of two metalloproteases with similar cleavage site specificities.

A component of normal human serum which enhances the activity of vertebrate collagenases

Abstract The activity of vertebrate collagenase is increased by approximately 3-fold in the presence of saturating amounts of a macromolecule found in normal human serum. The activities of collagenases from human skin, rat skin, and tadpole tailfin are all markedly enhanced in the presence of this molecule, but activities of bacterial collagenase, trypsin, chymotrypsin, thermolysin, and a gelatin-specific neutral protease from human skin are unchanged. The enhancer itself has no proteolytic activity and does not change the normal cleavage products of human skin collagenase. The collagenase enhancer is an extremely stable molecule. It is resistant to heat, to extremes of pH at physiological temperature, and appears to be protein in nature. Of particular interest is the requirement that the collagen substrate be in fibrillar form in order for the enhancer to be effective.

Regulation of Gelatinase in Human Skin Organ Cultures by Glucocorticoids

Hydrocortisone and dexamethasone prevent the appearance of gelatinase in serum-free explant cultures of normal human skin. Hydrocortisone inhibits maximally at 10(-6) M and dexamethasone at 10(-8) M in culture medium. Glucocorticoids at these concentrations do not cause a generalized decrease in protein synthesis; thus the effect on gelatinase shows specificity. The reduction in gelatinase activity caused by dexamethasone can be overcome in the presence of dexamethasone 21-mesylate, a glucocorticoid antagonist that binds irreversibly to the cytoplasmic steroid receptor. These data suggest that the enzymes of collagen degradation, collagenase and gelatinase, may be coregulated.

The Effects of Strontium and Calcium on Mammalian Collagenases

Collagen is the major structural protein of skin, bone, cartilage, tendon, and teeth. It is, in fact, the most abundant protein in the body. The collagen molecule consists of three polypeptide chains, molecular weight approximately 95,000 each, which are coiled together in a triple helix. Chemically, the collagen molecule is unique in that every third amino acid residue is glycine, and in that it contains relatively large amounts of hydroxyproline and hydroxylysine.