Hyaluronic Acid Synthetase Activity Research Articles

Synthesis and release of hyaluronic acid by Swiss 3T3 fibroblasts.

Hyaluronic acid (HA) and its synthesis were studied in intact Swiss 3T3 mouse fibroblasts and isolated membranes. HA chains in culture medium, attached to cells and in isolated membranes, were determined to possess average M(r) values of 5.2 x 10(6), 1.8 x 10(6) and 0.14 x 10(6) respectively. Log cells were determined to possess 680,000 HA molecules/cell, and to release 120,000 HA chains/h. The time required for intact cells to synthesize and release a complete HA chain was approximately 4 h, with elongation proceeding at a rate of 57 dimers/min. The amount of cell-associated HA of various cell populations correlated strongly with their rate of HA release into culture media and with the HA synthetase activity determined for their membranes. Prevention of protein synthesis with cycloheximide decreased the rate of HA synthesis of log cells and HA synthetase activity of isolated membranes by 50% within 2-3 h. Because of the similarity between the biological lifetime of HA synthetase and the time required to synthesize a HA chain, we propose a model where each synthetase makes only one HA chain; after synthesis of a complete HA chain, HA synthetase activity is terminated as its HA chain is released from the cell.

Hyaluronic acid synthesis is absent in normal human endothelial cells irrespective of hyaluronic acid synthetase inhibitor activity, but is significantly high in transformed cells

The characteristics of glycosaminoglycan (GAG) synthesis in normal and transformed human endothelial cells were analyzed by the incorporation of [ 3H]glucosamine and by the activities of GAG synthetases. The GAG synthesized by normal endothelial cells consisted of mainly heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate but little hyaluronic acid (HA) (less than 1%). The characteristics of GAG synthesis by normal cells reflected the synthetic enzyme activities for each individual GAG: the activity of HA synthetase was very low. In spite of this, the activity of HA synthetase inhibitor, induced in growth-retarded fibroblasts with low HA synthetase activity (Matuoka et al. (1987) J. Cell Biol., 104, 1105–1115), was very low in endothelial cells. In contrast to normal cells, transformed endothelial (ECV304) cells synthesized mainly HA (62% of total GAGs). These findings suggest that the regulatory system of GAG metabolism is cell type specific, and that transformation is accompanied by high levels of HA synthesis in endothelial cells.

Solubilization of hyaluronic acid synthetic activity from streptococci and its activation with phospholipids.

To date all hyaluronic acid synthetic systems have been of a particulate nature, and attempts at solubilization have been unsuccessful. This has hampered attempts to elucidate the mechanism by which hyaluronic acid is produced. In this paper we demonstrate that the hyaluronic acid synthetic activity from group C streptococcal membranes was solubilized using 2% digitonin and that the activity was optimized by reconstitution with cardiolipin at an optimum phospholipid/protein ratio (microgram/microgram) of 5:1. Furthermore, chromatography of the solubilized synthetase demonstrated that it eluted after the void volume of a Sepharose CL-6B column. CHAPSO, octyl glucopyranoside, sodium cholate, Triton X-100, and zwittergent 314 either inhibited or failed to solubilize the synthetic activity. Phospholipids other than cardiolipin also reconstituted the activity from the digitonin extract, particularly phosphatidylethanolamine and phosphatidylserine. In our system, the specific activity of hyaluronic acid synthetase was increased up to 63 times that of the system of the intact membrane. Furthermore, the total activity of the reconstituted system was 4.9 times greater than that of intact membranes. The soluble enzyme system showed similarities to the membrane-bound synthetase in the kinetics of production of trichloroacetic acid-soluble and -insoluble hyaluronic acid, and the hyaluronic acid produced was of comparable molecular weight.

The effect of the tumor promoter, phorbol myristate acetate (PMA), on hyaluronic acid (HA) synthesis by chicken embryo fibroblasts.

Treatment of chicken embryo fibroblasts (CEF) with the tumor promoter, phorbol myristate acetate (PMA), resulted in a rapid increase in their ability to synthesize the glycosaminoglycan, hyaluronic acid (HA), whereas the parent compound, phorbol, had no effect. CEF cultures incubated with PMA (100 ng/ml) for 6 h resulted in a 15-fold increase in HA synthetase activity compared with phorbol-treated control cultures. The incorporation of [3H]acetate into HA and chemical determination of this polymer also demonstrated increased synthesis of HA by cells treated with PMA. We have previously shown that CEF infected with a temperature-sensitive mutant of Rous sarcoma virus, LA24, exhibit increased synthesis of HA upon transformation. PMA treatment of cells transformed with RSV-LA24 results in a further 1.5-fold stimulation of HA synthesis. These data indicate that PMA causes an increased synthesis of HA in CEF which is analogous to the increased synthesis of HA found in virally transformed CEF.

A method for the hyaluronic acid synthetase assay in human skin.

A method for the determination of hyaluronic acid synthetase activity of human skin is described. Skin samples were crush-homogenized, and incubated with UDP-14C-glucuronic acid and UDP-N-acetylglucosamine in Tris-HCl-buffer containing MgCl2. After papain digestion of the samples, the 14C-labeled hyaluronic acid was separated in Sephadex G 50 chromatography. The radioactivity incorporated into hyaluronic acid was an expression of the activity of hyaluronic acid synthetase. This activity was found to be increased in skin biopsies obtained from psoriatic lesions and decreased after local treatment with potent corticoids.

Induction of hyaluronic acid synthetase by estrogen in the mouse skin

Hyaluronic acid synthetase activity was measured in male mouse skin following the topical application of estradiol in vivo. The enzyme activity increased in parallel with the hyaluronic acid content of the skin, and showed a similar response in the skin of ovariectomized female mice. The increase in enzyme activity was reduced by the anti-estrogen agents, tamoxifene citrate and clomiphene citrate, which block competitively the binding of estrogen to the estrogen receptor. The increase in hyaluronic acid synthetase activity was also reduced by topical application of cycloheximide or by subcutaneous injection of actinomycin D. The results suggest that the stimulation of hyaluronic acid synthesis in mouse skin in response to estrogen treatment is mediated through estrogen receptors and involves the induction of the enzyme hyaluronic acid synthetase.

Cell-free synthesis of hyaluronic acid in Marfan syndrome.

The cell-free synthesis of hyaluronic acid has been demonstrated in extracts of cultured human fibroblasts. Preparations from fibroblasts of normal individuals as well as those from patients with Marfan syndrome incorporate glucuronic acid and N-acetylglucosamine from their UDP derivatives into hyaluronic acid. Extracts from Marfan fibroblasts demonstrate 3 to 10 times more total and specific hyaluronic acid synthetase activity than do preparations from normal fibroblasts. All synthetic activity was found in particulate fractions with the bulk of activity localized in material sedimenting as large membrane fragments. Marfan and normal preparations exhibited similar properties with respect to substrate, cofactor, pH requirements, and heat stability. Neither the Marfan nor normal enzyme systems could be stimulated by exogenous acceptors, nor did either preparation contain a soluble factor which stimulated or inhibited the enzymic activity of the other. The genetic defect in Marfan syndrome appears to result in increased activity of hyaluronic acid synthetase without demonstrable changes in properties of the particulate enzymes involved.

Stimulatory effect of bleomycin on the hyaluronic acid synthetase in cultured fibroblasts

The mechanism of the stimulatory effect of bleomycin on the production of hexosamine-containing substances by cultured fibroblasts was studied by measuring changes in the activity of hyaluronic acid (HA) synthetase. HA synthetase activity was increased by 1 gmg bleomycin/ml, and its stimulatory effect could be detected 4 days after the start of bleomycin treatment and was enhanced by lenthening the time of treatment, i.e. by 96 per cent for an 8-day treatment. In cell-free systems, however, bleomycin showed no stimulatory effect on this enzyme activity. The enhancement of HA synthetase activity by bleomycin was inhibited by the addition of cycloheximide or actinomycin D. The synthesis of HA was much less influenced by cycloheximide than was that og glycoprotein and sulfated glycosaminoglycan. From our results it can be concluded that HA synthetase activity, induced when the old medium of stationary cultures was renewed with fresh medium containing 5% fetal bovine serum, was significantly enhanced by the addition of bleomycin. However, the detailed mechanism of enhancement of HA synthetase activity has not yet been elucidated.

Stimulatory effect of prostaglandins on the production of hexosamine-containing substance by cultured fibroblasts (3) induction of hyaluronic acid synthetase by prostaglandin F 2α

The mechanism of the stimulatory effect of prostaglandin(PG) F 2α on the production of hexosamine-containing substance by cultured fibroblasts was studied. Treatment of the cells with 1 μg/ml of PGF 2α resulted in a doubled net synthesis of acidic glycosaminoglycans during 20 hrs measured with uronic acid as index, and also resulted in 300 per cent increase of 3H-glucosamine incorporation into hexosamine-containing substances during the first 6 hrs. Fractionation of the PGF 2α-stimulated hexosamine-containing substances with double isotope technique revealed that hyaluronic acid was the most stimulated component. Prior to the increase of hyaluronic acid, hyaluronic acid synthetase activity was found to be augmented by PGF 2α as high as 4 times over the control. The augmentation of hyaluronic acid synthetase activity by PGF 2α did not take place if actinomycin D was simultaneously present in the culture medium, suggesting that PGF 2α induced the enzyme.

Effects of adenosine 3′:5′-cyclic monophosphate and serum on synthesis of hyaluronic acid in confluent rat fibroblasts

A small amount of hyaluronic acid is synthesized in confluent cultures of rat fibroblasts, which have a high content of cyclic AMP. Addition of calf serum caused a rapid decrease in the cellular cyclic AMP content and large increases in hyaluronic acid synthetase activity and hyaluronic acid production. Addition of cyclic AMP also caused a marked increase in hyaluronic acid synthetase activity within 2h and then increased hyaluronic acid production. The effects of cyclic AMP and serum on hyaluronic acid synthesis were additive. Prostaglandin E2, which increased the cyclic AMP by stimulating adenylate cyclase, was as effective as cyclic AMP in increasing hyaluronic acid synthetase activity, but AMP was far less effective than cyclic AMP. These results indicate that cyclic AMP itself stimulates the mucopolysaccharide synthesis and that the effect of serum is not due to a decrease in cyclic AMP in the cells.

The enhanced production of hyaluronic acid by cultured rat fibroblast cells treated with cyclic AMP and its dibutyryl derivative

Cells of a newly established rat fibroblast line (SEN) in culture synthesize mucopolysaccharides, which have been identified as hyaluronic acid, chondroitin-4-sulfate and heparan sulfate. Treatment of the cells with adenosine 3':5'-cyclic monophosphate resulted in a marked stimulation of production of hyaluronic acid, but not of the other mucopolysaccharides. Treated cells also showed increased activity of hyaluronic acid synthetase, a reduction in growth rate, and morphological alteration. In addition, 5-bromodeoxyuridine was found to counteract greatly the cyclic AMP effect.

Induction of hyaluronic acid synthetase activity in rat fibroblasts by medium change of confluent cultures.

Hyaluronic acid synthesis in cultured cells usually occurs during the growth phase. The relation between hyaluronic acid synthetase activity and cell proliferation is studied. The synthetase activity in rat fibroblasts is high during the growth phase, but low in the stationary phase. When the old medium of stationary cultures is renewed with fresh medium containing 20% calf serum, DNA synthesis occurs synchronously between 12 and 20 hours, followed by cell division. Under these conditions, the hyaluronic acid synthetase activity is significantly induced within two hours, reaching a maximum level at 5--8 hours, and then decreases gradually. This induction of the synthetase, which shows a high turnover rate, requires continued synthesis of both RNA and protein. Furthermore, the induction of both DNA and hyaluronic acid synthesis is found to be caused by calf serum added in the medium. However, dialysis and ultrafiltration of the serum permit us to concentrate an active fraction with a high molecular weight, which induces the synthetase activity, but not DNA synthesis.

Hyaluronic acid synthetase in cultured mammalian cells producing hyaluronic acid Oscillatory change during the growth phase and suppression by 5-bromodeoxyuridine

A hybrid line B-6 synthesizes and secretes a large amount of hyaluronic acid and this differentiated function is suppressed by growth in the presence of the thymidine analog, 5-bromodeoxyuridine. In order to elucidate the mechanism of this analog inhibition, the levels hyaluronic acid synthetase and UDPglucose dehydrogenase in the cells were investigated. The results obtained were as follows. 1.1.The addition of 5-bromodeoxyuridine at concentrations as low as 5 μg/ml to medium while having the effect on cell growth, caused a great drop in the activity of hyaluronic acid synthetase. By contrast, the specific activity of UDPglucose dehydrogenase was slightly increased.2.2. This suppression on the synthetase was detected 10 h after addition of the analog; when the culture treated for one generation (16 h) was transferred to the analog-free medium, the activity was recovered to control value during the next three to four generations. The analog effect was prevented by simultaneous addition of hydroxyurea, an inhibitor of DNA synthesis, with the analog. These results prove that incorporation of the analog into DNA is necessary for this inhibitory effect.3.3. In the presence of either actinomycin D or cycloheximide, cellular hyaluronic acid synthetase activity decayed with a half-life of 3.5 and 2 h, respectively, whereas UDPglucose dehydrogenase decayed with a half-life of 20 h.4.4. The activity of the synthetase, but not that of UDPglucose dehydrogenase, was correlated with high growth rate, falling sharply with decline of cell growth. Recovery from low activity in the cells transferred to fresh medium required new synthesis of both RNA and protein. 1.The addition of 5-bromodeoxyuridine at concentrations as low as 5 μg/ml to medium while having the effect on cell growth, caused a great drop in the activity of hyaluronic acid synthetase. By contrast, the specific activity of UDPglucose dehydrogenase was slightly increased. 2. This suppression on the synthetase was detected 10 h after addition of the analog; when the culture treated for one generation (16 h) was transferred to the analog-free medium, the activity was recovered to control value during the next three to four generations. The analog effect was prevented by simultaneous addition of hydroxyurea, an inhibitor of DNA synthesis, with the analog. These results prove that incorporation of the analog into DNA is necessary for this inhibitory effect. 3. In the presence of either actinomycin D or cycloheximide, cellular hyaluronic acid synthetase activity decayed with a half-life of 3.5 and 2 h, respectively, whereas UDPglucose dehydrogenase decayed with a half-life of 20 h. 4. The activity of the synthetase, but not that of UDPglucose dehydrogenase, was correlated with high growth rate, falling sharply with decline of cell growth. Recovery from low activity in the cells transferred to fresh medium required new synthesis of both RNA and protein.