Induction of 103-kDa gelatinase/type IV collagenase by acidic culture conditions in mouse metastatic melanoma cell lines.

Gelatinases/type IV collagenases have been shown to be involved in tumor invasion and metastasis. In this study, we examined the effect of culture medium pH on the secretion of the gelatinases from mouse B16 melanoma cell lines and human tumor cell lines using zymography analysis. The highly metastatic clone F10 of B16 melanoma did not secrete any gelatinase in neutral culture media (pH 7.1-7.3), whereas it secreted a high level of a 103-kDa gelatinase in an initial pH range of 5.4-6.1. The addition of an excess amount of glucose into a neutral culture medium also induced the gelatinase secretion from the cells by decreasing the medium pH during incubation. The extent of the acid-induced gelatinase secretion by the B16 melanoma cell lines was in the order of BL6 greater than F10 greater than F1 much greater than the parent B16 line, in good agreement with the order of their metastatic potentials. Two human cell lines (A549 and HT1080) secreted a higher level of a 90-kDa gelatinase at pH 6.8 compared with pH 7.3. The acid-induced gelatinase secretion from B16-F10 cells was blocked by cycloheximide, indicating that the enzyme induction was due to de novo synthesis. When in vitro tumor cell invasion was assayed in Boyden chambers, B16-F10 cells incubated in an acidic medium exerted a more active migration through type IV collagen gel than those in a neutral medium. These results suggest that the acidic environment formed around tumor tissues may be an important factor in invasion and metastasis of some types of tumors.     

Cytokines Regulate Gelatinase A and B (Matrix Metalloproteinase 2 and 9) Activity in Cultured Rat Astrocytes

Abstract: Under a tightly regulated expression mechanism, matrix metalloproteinases degrade extracellular matrix proteins and are though to play a role in injury repair and tumor metastasis in peripheral tissues. Little is known about the function of matrix metalloproteinases or agents that regulate their production in adult brain; however, it has been shown that the activity of a calcium-dependent metalloproteinase is elevated in Alzheimer's hippocampus. The goals of this study were to determine whether cultured rat astrocytes produce matrix metalloproteinases and to identify agents that regulate protease activity. Enriched astrocyte cultures were prepared from brains of 1-day-old rat pups, and experiments were performed 13 days later. Gelatinase activity in astrocyte conditioned medium was determined using zymography with gelatin copolymerized with acrylamide in the gel. Under basal conditions after a 24-h incubation, rat astrocytes produce gelatinases of 58 and 66 kDa. On stimulation of astrocytes with lipopolysaccharide, interleukin-1α or -β, or tumor necrosis factor-α for 24 h, a dose-dependent increase in the activity of the 58- and 66-kDa gelatinases and the induction of a 94-kDa gelatinase occurred. All three astrocyte-derived proteases showed maximal activity in the presence of millimolar levels of Ca²⁺, their activity was inhibited in the presence of 1,10-phenanthroline, and their proenzymes were cleaved and activated after incubation with p-aminophenylmercuric acetate. Using immunoblotting, immunopositive bands at the respective molecular sizes indicated that the 58-kDa gelatinase was gelatinase A (matrix metalloproteinase 2) and the 94-kDa activity was gelatinase B (matrix metalloproteinase 9). Induction of the 94-kDa gelatinase by lipopolysaccharide was not influenced when interleukin-1 receptor antagonist was included during the 24-h incubation period; however, the antagonist completely blocked interleukin-1β-induced 94-kDa activity and diminished the activity of the 58- and 66-kDa gelatinases. Dexamethasone inhibited both lipopolysaccharide and interleukin-1β stimulation of the 94-kDa gelatinase. These results indicate that cytokines regulate matrix metalloproteinase expression in cultured rat astrocytes. Because astrocytes become “activated” (are hypertrophic and express increased levels of glial fibrillary acidic protein) in the presence of several inflammatory cytokines, it is possible that these astrocyte-derived enzymes contribute to the activation process and may participate in tissue remodeling after brain injury.     

Three-dimensional collagen matrices induce delayed but sustained activation of gelatinase A in human endothelial cells via MT1-MMP

Gelatinase A, a member of the matrix metalloproteinase (MMP) family, plays an important role during angiogenesis. It is constitutively expressed by human endothelial cells as a latent enzyme and requires activation. Thrombin is the only described physiological inducer of gelatinase A in human endothelial cells. In this study, we investigated the mechanisms of gelatinase A activation by another physiological inducer, collagen. Endothelial cells were cultured on various ECM components for 24 h and the conditioned media were assessed for gelatinase A activity using gelatin zymography. The results demonstrated that type I collagen matrix specifically activates gelatinase A after 24 h in human umbilical vein and 48 h in neonatal foreskin endothelial cells. In contrast, thrombin activated gelatinase A after only 2 h. Activation by collagen was sustained over long periods of time in culture (96 h). Unlike thrombin-induced activation, collagen required active membrane type 1-MMP (MT1-MMP) on the endothelial cell surface to activate gelatinase A. In addition, collagen-induced activation of gelatinase A was inhibited by antibodies to the integrin receptor, alpha(2)beta(1), but not alpha(3)beta(1). Our findings, that collagen can provide long-term activation of gelatinase A are likely to be relevant to endothelial cell invasion during angiogenesis.     

UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes

While human dermal fibroblasts increase the expression and secretion of distinct matrix metalloproteinases (MMPs) in response to ultraviolet (UV) irradiation, much less is known about regulation of MMPs with regard to normal human epidermal keratinocytes (NHEK). In this in vitro study, the effect of ultraviolet A (UVA) irradiation on gelatinase expression and secretion by NHEK was investigated. Irradiation of NHEK with non-toxic doses of UVA resulted in a dose-dependent downregulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). A single dose of 30JUVA/cm(2) lowered MMP-2 activity to 26% and MMP-9 activity to 33% compared with mock-irradiated cells at 24h after irradiation. Downregulation of MMP-2 and MMP-9 steady-state mRNA levels was observed at 4h after UVA irradiation. The inhibitory effect of UVA on gelatinases was mediated by UVA-generated singlet oxygen (1O(2)). These findings suggest an inverse response to UVA irradiation in NHEK than in fibroblasts.     

Effect of human chorionic gonadotrophin supplementation during different culture periods on in vitro maturation of canine oocytes

The IVM of canine oocytes is characterized by low rates of metaphase II. The objective of this study was to evaluate the effects of hCG on meiotic development of canine oocytes for culture periods up to 96 h. Oocytes were collected after ovariohysterectomy. Only oocytes >110 microm in diameter, with a homogeneous dark cytoplasm and three or more layers of compact cumulus cells were used. For IVM, the COCs were cultured in TCM-199+10% fetal calf serum, without (medium A control) or supplement with 10 IU/mL of hCG (medium B), or with a combination of both media (treatment B/A). The COCs were randomly allocated into three groups. The first and second groups were cultured in either medium A or B, respectively for 24, 48, 72, and 96 h. Oocytes of the third group (treatment B/A) were incubated in medium with hCG (medium B) the first 48 h and then transferred to medium without hCG (medium A) for an additional 24 or 48 h. The proportion of COCs with cumulus cell expansion was also evaluated before fixation. Oocytes were stained with propidium iodide prior to nuclear assessment (with epifluorescence microscopy). COCs with cumulus expansion were evident after 48 h of culture. The proportion of COCs with cumulus expansion was higher (P<0.05) for media containing hCG (B or B/A) than for meda lacking hCG (A); this difference was maintained for 72 and 96 h in culture. In media A, B and B/A, 23.3, 31.7 and 29.5%, respectively, of oocytes were at metaphase II after 72 h, with 20.7, 33.1 and 43.4% at this stage after 96 h. The advancement of meiosis was directly proportional to the time of incubation; the highest percentage (P<0.05) of oocytes at metaphase II was observed after 96 h of culture when 10 IU/mL hCG was present for only the first 48 h of culture.     

Gelatinase B is involved in the in vitro wound repair of human respiratory epithelium

Following epithelial injury, extracellular matrix undergoes imposing remodelings. We examined the contribution of matrix metalloproteinases, gelatinases A and B, in an in vitro wound repair model of human respiratory epithelium. Confluent human surface respiratory epithelial (HSRE) cells cultured from dissociated surface cells of human nasal polyps were chemically injured. Over the next 3 to 5 days, cells migrated onto the injured area to repair the circular wound. Repair kinetics of these wounds was monitored until wound closure occurred. Gelatinolytic activities were analysed in culture supernates and in cell protein extracts derived from repairing migratory and non repairing stationary cells. Small amounts of gelatinase A were expressed by HSRE cells, and variations of this gelatinase remained very weak for the time of the wound repair. In contrast, gelatinase B was upregulated during the wound repair process, with a maximum peak observed at wound closure. A marked gelatinase B activation occurred only in cells involved in the repair process. Gelatinase B was localized in some migratory basal cells, recognized by an anti-cytokeratin 14 antibody and located around the wound. We could not detect any gelatinase A in repairing or in stationary HSRE cells. Addition of the 6-6B monoclonal antibody, known to inhibit gelatinase B activation, to the culture medium during the repair process resulted in a dose-dependent decrease of the wound repair speed. These results suggest that gelatinase B, produced by epithelial cells, actively contributes to the wound repair process of the respiratory epithelium. © 1996 Wiley-Liss, Inc.     

Collagenase and gelatinase messenger ribonucleic acid expression and activity during follicular development in the rat ovary.

Metalloproteinases are members of a family of proteinases that remodel the extracellular matrix throughout the body. To test the hypothesis that metalloproteinases are regulated by gonadotropin-induced changes during follicular growth, rats were injected with eCG (20 IU, s.c.), and ovaries and serum were collected at the time of eCG administration (0 h) and at 6, 12, 24, 36, or 48 h later for analysis of metalloproteinase mRNA expression, metalloproteinase activity, and steroidogenesis. Serum estradiol levels increased from 18.9 pg/ml at 0 h to 503.8 pg/ml at 48 h. Analysis of mRNA expression was performed for collagenase-3, 72-kDa gelatinase, and 92-kDa gelatinase (n = 3-4). For collagenase-3, eCG stimulated a 32-fold increase in collagenase-3 mRNA at 48 h after eCG injection as compared to that in ovaries collected at the time of eCG administration (i.e., 0-h control). The mRNA levels for 72-kDa gelatinase were 2.8-fold compared to 0 h at 36 h after eCG treatment and returned to control levels by 48 h after gonadotropin treatment. Levels of the 92-kDa mRNA expression peaked at 24 h (4. 2-fold compared to 0 h) and returned to control levels by 36 h. Gel zymography revealed 3 gelatinolytic bands corresponding to the gelatinases of approximately 72 kDa, 92 kDa, and 105 kDa. Analysis of metalloproteinase activity as the degradation of collagen or gelatin per ovary showed an increase in gelatinolytic and collagenolytic activity between 12 and 48 h after eCG treatment. In summary, these findings demonstrate that the gonadotropin induction of folliculogenesis results in changes in the metalloproteinases that may be responsible for extracellular matrix remodeling associated with follicular growth.     

The medium for determining the gelatinase activity of non-fermenting gram-negative microorganisms

To determine the gelatinase activity of Gram-negative nonfermenting microorganisms, a double-layer medium containing 2% of agar in the lower layer and 10-20% of gelatin in the upper layer has been developed. The medium ensures free access of atmospheric oxygen, possibility of rapid (24-42 h) determination of gelatinase activity at 22-25 degrees C, efficacy of the test. simultaneous determination of gelatin hydrolysis by 8-12 strains in one dish.     

Activation of progelatinase A by mammalian legumain, a recently discovered cysteine proteinase

The activation of progelatinase A to gelatinase A requires cleavage of an asparaginyl bond to form the N-terminus of the mature enzyme. We have asked whether the activation can be mediated by legumain, the recently discovered lysosomal cysteine proteinase that is specific for hydrolysis of asparaginyl bonds. Addition of purified legumain to the concentrated conditioned medium from HT1080 cell culture that contained both progelatinases A and B caused the conversion of the 72 kDa progelatinase A to the 62 kDa form. The progelatinase B in the medium was unaffected. Incubation of recombinant progelatinase A with legumain resulted in an almost instantaneous activation as judged by the fluorometric assay with a specific gelatinase A substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2. Legumain also activated progelatinase A when it was in complex with TIMP-2. Zymographic analysis and N-terminal sequencing revealed that legumain cleaved the 72 kDa progelatinase A at the bonds between Asn109-Tyr110 or Asn111-Phe112 to produce the 62 kDa mature enzyme, and that further cleavage at Asn430 also occurred to generate a 36 kDa active form. More 62 kDa gelatinase A was detected in cultures of C13 cells that over-expressed legumain than in those of the control HEK293 cells. We conclude that legumain is clearly capable of processing progelatinase A to the active enzyme in vitro and in cultured cells.     

Short-term and long-term alterations in neuronal excitability during injury-induced axonal regeneration in ganglia and cell culture

Helisoma neurons B5, regenerating axonal arbors following crush-axotomyin vivo, displayed a transient (<24 h) reduction in excitability followed by a sustained period of hyperexcitability (>13 d). Neurons isolated into outgrowth-permissive cell culture conditions expressed a similar pattern of hypo- and hyperexcitability; however, excitability of neurons B5 in culture was elevated for only 5 d and then declined to a lower, stable level. The expression of these alterations in excitability was neurite outgrowth-independent and required the presence of ganglia-derived conditioning factors in the culture medium. Excitability of neurons in medium lacking conditioning factors fell by day 3 to minimal levels. Conditioned medium was effective in rescuing excitability of neurons deprived of conditioning factors during their first 3 days of cell culture, but not following longer periods of deprivation. Exposure to the protein synthesis inhibitor, anisomycin, blocked the ability of conditioning factors to rescue B5 neuronal excitability. Together, results from cell culture suggest that mechanisms underlying neuronal excitability following nerve injury are independent of process outgrowth state, but require exposure to conditioning factors derived from injured neural tissue within several days of axonal insult. Although changes in B5 neurite outgrowth and neuronal excitability were expressed simultaneously following axonal injury, their independence suggests the existence of an underlying regenerative state that regulates both cellular modifications.     

Protease expression in the supernatant of Chinese Hamster Ovary cells grown in serum-free culture

The protease activity secreted by the Chinese Hamster Ovary (CHO-K1) cell line grown in serum-free medium was examined by substrate gel electrophoresis (zymography). The cell line expressed extracellular proteases that were active on gelatin zymograms but not on casein zymograms. The main protease band visible by gelatin zymography was approx. 92 kDa. Incubation of the conditioned medium with aminophenylmercuric acetate (APMA) resulted in the appearance of gelatinase activity at 82 kDa. Incubation of the conditioned media with EDTA significantly decreased the gelatinolytic activity of both the 92 kDa and 82 kDa forms, indicating the gelatinase responsible was a metalloprotease. Immunoblotting of the conditioned medium showed the gelatinase to be the pro- form of matrix metalloprotease-9 (pro-MMP-9), also known as gelatinase B.     

HepG2 cell LDL receptor activity and the accumulation of apolipoprotein B and E in response to docosahexaenoic acid and cholesterol.

In the present study, the accumulation of apolipoproteins (apo) A-I, B, and E in culture medium was measured after 0, 3, 6, 12, and 24 h of incubation with 150 microM docosahexaenoic acid complexed to 75 microM bovine serum albumin (BSA-22:6), either in the presence or absence of 50 micrograms/ml cholesterol and 4 micrograms/ml 25-hydroxycholesterol (C/25-OH). HepG2 cells incubated with BSA + C/25-OH for 24 h accumulated approximately 2.0-fold greater apoE and B as compared to BSA-treated cells. Moreover, HepG2 cell apoB accumulation after 24 h of BSA-22:6 treatment was approximately 2.0-fold greater than apoB accumulation from cells treated with BSA alone. When BSA-22:6 and C/25-OH were both included in the incubation, apoB accumulation was approximately 5.0-fold greater than BSA-treated cells. Comparative studies using BSA-18:1 were carried out for 24 h and showed similar levels of apoA-I, B, and E accumulation in culture medium as compared to BSA-22:6-treated cells. In addition, apoA-I, B, and E mRNA abundance were found to be unaffected by type of fatty acid treatment or length of incubation, averaging 48.2 +/- 7.5, 222 +/- 33.6, and 17.1 +/- 0.7 pg mRNA/micrograms RNA (mean +/- SEM), respectively. As the accumulation of apoB and apoE in culture medium may be modified by HepG2 cell LDL receptor expression, LDL receptor mRNA abundance and LDL receptor activity were quantified at various times over the course of the study. By 6 h of BSA + C/25-OH treatment, LDL receptor mRNA was reduced approximately 2.3-fold, while receptor activity was reduced approximately 1.5-fold, as compared to BSA controls. In an experiment designed to determine uptake of HepG2 cell lipoproteins, 3H-labeled apoB-containing lipoproteins derived from HepG2 cells were prepared. The 3H-labeled lipoproteins were 1.25-fold more likely to be removed from the media of HepG2 cells treated with BSA than from cells treated with BSA + C/25-OH. From these results, we postulate that HepG2 cell LDL receptor activity mediates the removal of apoB, E-containing lipoproteins from culture medium and contributes to the lower accumulation of apoB and E observed in culture medium from cells treated with BSA as compared to cells treated with C/25-OH.     

Effect of amylase accumulation on hydrogen production by Clostridium beijerinckii,strain AM21B

The maximal enzymatic activity of crude amylase produced in the batch culture of Clostridium beijerinckii strain AM21B grown in PY medium with starch was obtained at 55°C and in an acidic pH range of 4.6 to 5.4. Amylase was produced in the culture medium after 4 h (46.6 units) and reached a peak (405.5 units) after 12 h cultivation at 36°C, pH 6.0. Although the most efficient production of amylase, hydrogen and cells was achieved at 36°C and pH 6.0, the maximal hydrogen evolution rate was found at 41°C and pH 7.0.     

Proteoglycans synthesized by human glomerular mesangial cells in culture

Human fetal kidney mesangial cells were cultured for 24 h in the presence of 3H-amino acids and [35S] sulfate and chased for 24 h in nonradioactive medium. Incubation medium and cell layer proteoglycans were purified twice by high performance liquid chromatography-DEAE chromatography followed by gel filtration chromatography. The major medium 35S-macromolecules were chondroitin/dermatan-35SO4 proteoglycans. A small, Sepharose CL-6B Kav 0.14 dermatan-35SO4 proteoglycan was detected in the labeling medium and was released into both the early (time 0-0.5 h) and late (6-24 h) chase media. It contained 38 kDa 4-sulfated 35S-GAGs with a high content of iduronic acid and a 45-kDa protein core. A protein core of similar molecular weight was detected in the culture medium by Western analysis using antibodies to biglycan or proteoglycan-I (Fisher, L. W., Termine, J. D., and Young, M. F. (1989) J. Biol. Chem. 264, 4571-4576). This 35S-proteoglycan was not detected in the cell layer. However, a small dermatan-35SO4 with little or no protein core was present in the intracellular compartment. A large, Sepharose CL-6B excluded chondroitin-35SO4 proteoglycan was released into the culture medium and was detected between 6 and 24 h in chase medium. It eluted near the void volume of both associative and dissociative Sepharose CL-4B columns. It contained 30-kDa 4- and 6-sulfated 35S-GAGs and a 253-kDa protein core. A chondroitin-35SO4 proteoglycan with similar sized 35S-GAGs was detected in both the detergent-soluble and insoluble cell layer compartments. A Sepharose CL-6B Kav 0.11 heparin-35SO4 proteoglycan with a 220-kDa protein core and 38-kDa 35S-GAGs was rapidly released from the cell layer. This proteoglycan was larger than that previously described in isolated rat glomeruli or glomerular basement membranes, but had a core protein similar in size to one previously detected in these tissues. A larger heparan-35SO4 proteoglycan with larger 35S-GAGs was present in the detergent-insoluble cell layer compartment. The proteoglycans released by glomerular mesangial cells in culture resembled those synthesized by aortic smooth muscle cells in culture or extracted from aorta, supporting the notion that these cells are of vascular origin.