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.     

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

Treatment of chicken embryo frbroblasts (CEF) with the tumor promoter, phorbol myristate accetate (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.     

Biosynthesis and secretion of collagen in normal and SV-40 virus-transformed human embryonal fibroblasts

The biosynthesis and secretion of collagen proteins was studied in cultures of normal human embryo fibroblasts at different passages and growth stages as well as in cultures of human embryo fibroblasts transformed by oncogenic virus SV-40. It was found that normal fibroblasts maintain at a constant level the collagen synthesis throughout 20 passages, which is typical of proliferating and resting cells. Virus-transformed cells produce 3-4 times less collagen proteins on a per cell count. Normal and transformed fibroblasts do not differ in terms of total protein synthesis. Secretion of collagen and non-collagen proteins in transformed cell cultures appeared to be much lower than in normal cell cultures. Study of synthesized proteins by polyacrylamide gel electrophoresis showed that both types of cells secrete collagen proteins predominantly as polymers containing interchain S-S bonds of 3-helix molecules. Study of the protein-synthesizing activity of two polysomal fractions, i.e. membrane bound and free polysomes, isolated from the cells of both types in a cell-free system showed that membrane-bound polysomes from transformed fibroblasts synthesize collagen much less actively in comparison with normal cells. However, in transformed cells free polysomes, in contrast with normal cells, are active participants of a cell-free collagen protein synthesis.     

Formation and utilization of methionine by rat liver cells in culture

The enzyme N5-methyltetrahydrofolate:homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells.     

Mononuclear cell-mediated modulation of synovial cell metabolism. II. Increased hyaluronic acid synthesis by a monocyte cell factor (MCF)

Treatment of cultured human synovial cells with a mononuclear cell factor (MCF) enhanced their ability to synthesize glycosaminoglycans (GAG), but GAG repartition between extracellular, pericellular and intracellular compartments was found to be the same as in control. Hyaluronic acid (HA) production, which represents 80-90% of all secreted GAG, was stimulated 2 1/2-3-fold, but the HA molecular weight was not modified. The MCF increased the hyaluronate synthetase activity of synovial cells in similar proportions. Actinomycin D inhibited the increase in hyaluronate synthetase activity produced by MCF, indicating that this increase involves new synthesis of mRNA. Stimulation of both HA synthesis and hyaluronate synthetase activity by MCF was suppressed by 10(-4)-10(-5) M indomethacin (an inhibitor of cyclo-oxygenase), suggesting that MCF effect is prostaglandin-dependent.     

Prostaglandin synthetase activity and hormone responsiveness in normal and SV40 transformed WI-38 fibroblasts.

Prostaglandin (PG) synthetase activity and selective hormone responsiveness were examined in normal and SV40 transformed WI-38 fibroblasts (VA13-2RA). The transformed VA13-2RA cells have significantly reduced rates of PGE1, PGE2, PGF1alpha and PGF2alpha synthesis as compared to the normal WI-38 fibroblast. The transformed cell in contrast to the normal cell hyperresponds to stimulation by L-epinephrine (10 muM) and PGE1 (8.5 muM) but is unresponsive to bradykinin (BK) as measured by the accumulation of intracellular cyclic AMP. Indomethacin treatment does not significantly alter the PGE1 and L-epinephrine (EPI) responsiveness of the normal WI-38 fibroblast, however it abolishes the BK response in these cells. These results provide further evidence for the dependency of cell activation by bradykinin on the PG synthetase system. No experimental data was found to support the role of PGs as negative regulators of PGE1 and EPI responsiveness in the WI-38 fibroblast. Using the VA13-2RA cells, limited attempts to recover PG synthetase activity comparable to that found in normal WI-38 cells were unsuccessful. The VA13-2RA cell and its normal counterpart represent models for investigating the role of PGs in cell function and the mechanism of BK activation and its effect on cell metabolism.     

Protein synthesis in 3T3 and SV40-transformed 3T3 cells. Activity of ribosomes and cytosol proteins in cell-free protein synthesis.

The activity of specific components involved in protein synthesis in 3T3 cells and its SV40-transformed derivative, SV3T3, were examined in a cell-free protein synthetic system, and the results correlated with previous studies, indicating that a decreasing rate of protein synthesis does not accompany the stationary phase of growth. We found that 3T3 and SV3T3 polysome preparations containing endogenous mRNA were equally efficient in supporting cell-free protein synthesis in this system. Further, the net protein synthesis observed was not altered by an increase in the population density of the cellular polysome source. The activity of the aminoacyl-tRNA synthetase enzymes from 3T3 and SV3T3 cells was examined in vitro after isolation by pH 5 precipitation and by ammonium sulfate fractionation. The activity of these preparations from stationary phase 3T3 and nonexponential phase SV3T3 cells was found to be approximately 3 times higher than the activity of fractions from the homologous exponential phase cell. However, at both growth stages, the SV3T3 preparations were 30 to 40 times more active than the 3T3 preparations. These findings may have implications for the different growth properties observed in the two cell types.     

Formation and utilization of methionine by rat liver cells in culture

Summary The enzymeN ⁵-methyltetrahydrofolate: homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells. Presented in part at the Conference on Differentiation and Carcinogenesis in Liver Cell Cultures sponsored by the New York Academy of Sciences, October 11, 1979 (see reference 1).     

Evidence for a defect of holocarboxylase synthetase activity in cultured lymphoblasts from a patient with biotin-responsive multiple carboxylase deficiency.

We report here the expression of biotin-responsive multiple carboxylase deficiency in cultured lymphoblasts of a patient whose fibroblasts belong to the bio genetic complementation group. Cultured lymphoblasts from the patient lost propionyl-CoA carboxylase (PCC) and beta-methylcrotonyl-CoA carboxylase (MCC) activities at a faster rate than normal cells when grown in biotin-deficient medium. Recovery of normal PCC and MCC activities, which was independent of protein synthesis, required a 2,500-fold higher biotin concentration than that required by normal lymphoblasts. Holocarboxylase synthetase activity was detected in cell-free extracts through the biotinylation of endogenous apo-PCC in the presence of ATP to form active holo-PCC. While the apo-PCC in extracts of normal biotin-starved lymphoblasts could be activated to 28% of maximal activity, extracts of patient lymphoblasts did not exhibit any ATP and biotin-dependent increase in PCC activity. A normal cell extract, cleared of apocarboxylases by immunoprecipitation, stimulated the PCC activity of a patient cell extract 20-fold. These results indicate that the apoenzyme in bio cells is normal and that the defect lies in the holocarboxylase synthetase.     

Hyaluronan synthesis is inhibited by adenosine monophosphate-activated protein kinase through the regulation of HAS2 activity in human aortic smooth muscle cells

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan (GAG) involved in cell motility, proliferation, tissue remodeling, development, differentiation, inflammation, tumor progression, and invasion and controls vessel thickening in cardiovascular diseases. Therefore, the control of HA synthesis could permit the fine-tuning of cell behavior, but the mechanisms that regulate HA synthesis are largely unknown. Recent studies suggest that the availability of the nucleotide-sugar precursors has a critical role. Because the formation of UDP-sugars is a highly energetically demanding process, we have analyzed whether the energy status of the cell could control GAG production. AMP-activated protein kinase (AMPK) is the main ATP/AMP sensor of mammalian cells, and we mimicked an energy stress by treating human aortic smooth muscle cells (AoSMCs) with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside and metformin. Under these conditions, HA synthesis, but not that of the other GAGs, was greatly reduced. We confirmed the inhibitory effect of AMPK using a specific inhibitor and knock-out cell lines. We found that AMPK phosphorylated Thr-110 of human HAS2, which inhibits its enzymatic activity. In contrast, the other two HAS isoenzymes (HAS1 and HAS3) were not modified by the kinase. The reduction of HA decreased the ability of AoSMCs to proliferate, migrate, and recruit immune cells, thereby reducing the pro-atherosclerotic AoSMC phenotype. Interestingly, such effects were not recovered by treatment with exogenous HA, suggesting that AMPK can block the pro-atherosclerotic signals driven by HA by interaction with its receptors.     

Hyaluronic acid induces survival and proliferation of human myeloma cells through an interleukin-6-mediated pathway involving the phosphorylation of retinoblastoma protein

Originating from a post-switch memory B cell or plasma cell compartment in peripheral lymphoid tissues, malignant myeloma cells accumulate in the bone marrow of patients with multiple myeloma. In this favorable microenvironment their growth and survival are dependent upon both soluble factors and physical cell-to-cell and cell-to-extracellular matrix contacts. In this report we show that hyaluronan (HA), a major nonprotein glycosaminoglycan component of the extracellular matrix in mammalian bone marrow, is a survival and proliferation factor for human myeloma cells. The effect of HA is mainly mediated through a gp 80-interleukin 6 (IL-6) receptor pathway by a CD44-independent mechanism, suggesting that HA retains and concentrates IL-6 close to its site of secretion, thus favoring its autocrine activity. In addition, we show that HA-mediated survival and proliferation of myeloma cells is associated with a down-regulation in the expression of p27(kip1) cyclin-dependent kinase inhibitor and a hyperphosphorylation of the retinoblastoma protein (pRb). These data suggest that HA could be an important component in the myeloma cell physiopathology in vivo by potentiating autocrine and/or paracrine IL-6 activities.     

Hyaluronic acid of high molecular weight inhibits proliferation and induces cell death in U937 macrophage cells

Hyaluronic acid (HA), a major glycosaminoglycan component of the extracellular matrix, has regulatory influences on cells and cellular activities. To explore the effects of a high concentration (1 mg/mL) of high molecular weight HA (500-730 kD) on U937 macrophage growth dynamics, three factors that influence overall cellular growth, namely proliferation, apoptosis, and cell death, were examined. Cells were cultured with HA and were analyzed by flow cytometry every 24 hours during a 168-hour period for proliferation and the presence of apoptotic and dead cells. These analyses demonstrated that HA inhibits U937 macrophage proliferation in a time-dependent manner. Through the first 72 hours, cells exhibited slowed proliferation. However, no evidence of cell division arrest or reduced cell viability was observed. Thereafter, HA continued to diminish proliferation, but induced apoptosis. This data is consistent with regulatory influences secondary to HA binding to CD44 and/or RHAMM cell surface receptors, both of which were shown to be expressed on U937 macrophages. This study demonstrates that a high concentration of high molecular weight HA greatly inhibits macrophage population growth by the dual actions of impeding cell proliferation and inducing apoptosis.     

Transformed cells have lost control of ribosome number through their growth cycle.

Previous studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus-transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in a post-mitotic pre-DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability. The rate of protein synthesis in the stationary phase of the transformed cells fell to 25-30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribisome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L-73 and LR-73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells. A model for growth control of normal cells and its derangement in transformed cells is presented.     

Distinct proteolytic mechanisms in serum-sufficient and serum-restricted fibroblasts. Transformed 3T3 cells fail to regulate proteolysis in relation to culture density only during serum-sufficiency.

Thymidine incorporation (reflecting cell division), degradation of long-half-life proteins and protein synthesis were compared in normal Swiss mouse 3T3 fibroblasts and their counterparts transformed by simian virus 40 at both high and low culture densities (no. of cells/cm2). Normal cells maintained faster proteolysis at high culture density than at low. Degradation was in all conditions enhanced by serum deprivation (1% serum). In serum-sufficient (10%) conditions, there was an inverse correlation between degradation and cell division, but in serum-restricted conditions proteolysis increased substantially as culture density was increased, without change in cell division. Protein synthesis generally changed in a converse sense to protein degradation. In serum-sufficient conditions, transformed 3T3 cells failed to regulate proteolysis in response to culture density. However, in serum-restricted conditions they can regulate proteolysis as do normal cells. Transformed 3T3 cells regulate protein synthesis and thymidine incorporation very poorly in response to culture density in both conditions studied. The failure of regulation of both protein synthesis and degradation may contribute to the exaggerated growth of transformed cells in serum-sufficient conditions. The retention by such cells of regulation of proteolysis during serum restriction may also aid their survival. Studies with several lysosomotropic agents indicated that lysosomes contribute to proteolysis in all conditions studied, but also that its regulation in serum restriction is distinct from that in serum sufficiency, and may involve primarily a non-lysosomal mechanism.     

Inhibition of hyaluronan degradation by dextran sulphate facilitates characterisation of hyaluronan synthesis: an in vitro and in vivo study

The concentration and molecular weight of hyaluronan often dictates its physiological function. Consequently full characterisation of the anabolic products and turnover rates of HA could facilitate understanding of the role that HA metabolism plays in disease processes. In order to achieve this it is necessary to interrupt the dynamic balance between concurrent HA synthesis and degradation, achievable through the inhibition of the hyaluronidases, a group of enzymes which degrade HA. The sulphated polysaccharide, dextran sulphate has been demonstrated to competitively inhibit testicular hyaluronidase in a non-biological system, but its application to in vitro biological systems had yet to be developed and evaluated. This study determined the inhibitory concentrations of dextran sulphate against both testicular and Streptomyces hyaluronidase in a cell-free and breast cancer model followed by characterisation of the effect that hyaluronidase inhibition exerted on HA synthesis and degradation. The IC(100) of dextran sulphate for both hyaluronidases in a cell-free and biological system was determined to be >or=400 microg/ml. At concentrations up to 10 mg/ml the dextran sulphate did not effect breast cancer cell proliferation or morphology, while at 400 microg/ml HA degradation was totally inhibited, enabling an accurate quantitation of HA production as well as characterisation of the cell-associated and liberated HA. FACS quantitation of the HA receptor CD44, HA synthase and the hyaluronidases HYAL 1 and HYAL 2 demonstrated that dextran sulphate down-regulated CD44 and HA synthase while upregulating the hyaluronidases. These results suggest dynamic feedback signalling and complex mechanisms occur in the net deposition of HA in vivo.     

Hyaluronan synthesis is required for IL-2-mediated T cell proliferation

Hyaluronan (HA) is a glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid subunits. Previous studies have suggested that CD44 expressed by T cells bind exogenous HA for their proliferation. However, HA endogenously synthesized by T cells may participate in their autocrine proliferation. In this study, we examined the role of endogenous HA in T cell proliferation using the highly specific HA synthase inhibitor, 4-methylumbelliferone (4-MU). We found that 4-MU inhibited the mitogen-induced synthesis of HA by T cells. Moreover, 4-MU inhibited T cell proliferation in a dose-dependent manner when cells were cultured with different stimuli, including Con A, PMA/ionomycin, and allogeneic spleen cells. Furthermore, 4-MU inhibited mitogen-stimulated IL-2 secretion, suggesting that HA may play a role in the production of this cytokine. Addition of IL-2 to T cells treated with 4-MU and Con A reversed the block in cell proliferation, showing that impaired IL-2 production is a likely mechanism for the inhibited division of T cells. Surprisingly, an anti-CD44 Ab antagonistic for HA binding did not reduce IL-2 secretion or T cell proliferation. Importantly, 4-MU did not alter the surface expression of CD44 or the ability of CD44 to bind to HA. Thus, HA-mediated IL-2 production and T cell proliferation are CD44 independent. Our results strongly suggest that HA synthesized by T cells themselves is critical for their IL-2-mediated proliferation and have revealed a previously unrecognized role for endogenous HA in T cell biology.     

Hyaluronan accumulation is elevated in cultures of low density lipoprotein receptor-deficient cells and is altered by manipulation of cell cholesterol content

The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2-4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2x10(6) Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains.     

Collagen synthesis in normal BHK cells and temperature-sensitive chemically transformed BHK cells.

Collagen synthesis in normal BHK 21/cl 13 and chemically transformed temperature sensitive BHK 21/cl 13 cells (Me2N4) was assessed by examination of hydroxyproline formation and collagenase-susceptible protein. The Me2N4 cells lost their ability to synthesize collagen at both permissive and nonpermissive temperatures for transformation. These conclusions were confirmed by polyacrylamide-gel eletrophoresis and CM-cellulose chromatography. Prolyl hydroxylase activity was present in both normal and transformed cells even when no collagen could be demonstrated. The production of noncollagen protein, although decreased in the transformed cell, did not change as drastically as the collagen synthesis.