Phospholipase C activity in cultured human skin fibroblasts

In this paper we report the detection of phospholipase C activity in cultured human skin fibroblasts by a rapid, sensitive method. Sonicates of fibroblasts were incubated with L-3-phosphatidyl-[U-14C]-inositol and the incubation mixture extracted with chloroform/methanol. The solvent components were then separated into 2 phases by the addition of 2 M KCl. Phospholipase C activity, determined from the amount of [14C] in the aqueous phase, agreed well with the enzyme activity assessed by other methods. The optimum pH for the enzyme was 7.0 and the enzyme was found to be dependent on Ca2+ and deoxycholate for optimal activity. The demonstration of phospholipase C activity by this method in cultured skin fibroblasts provides a useful means with which to study, in human tissues, the physiological control of this enzyme and its derangements in disease states in a controlled fashion.     

Absence of hyaluronidase in cultured human skin fibroblasts.

The in vitro degradation of [³⁵S]chondroitin sulfate was investigated in human fibroblasts and rat liver. In rat liver, preparations of chondroitin sulfate were shown to be degraded by the concerted action of endoglycosidase and exoglycosidases. However, with human skin fibroblast preparations, hyaluronidase activity was not detected and chondroitin sulfate was degraded by exoglycosidase action.     

Glucose transport and metabolism in cultured human skin fibroblasts

Human skin fibroblast cultures, seeded at $\text{10}{}^5\text{cells}\text{5}\text{cm}$ plate and allowed to grow to confluence at approx. $\text{10}{}^6\text{cells}\text{5}\text{cm}$ plate, utilized a glycolytic mode of metabolism where the ratio of glucose utilized to lactate produced wa 0.62±0.05 (Zielke, R.H., Ozand, P.T., Tyldon, J.I., Sevdalian, D.A. and Cornblath, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4110–4114) (mean±S.E.). When the glucose in the medium was exhausted, the lactate produced during the highly glycolytic phase was then reutilized. In monolayer cultures that had been washed with phosphate-buffered saline, rates of glucose utilization were measured at 0.25 and 2 mM glucose by monitoring the appearance of ³H₂O from [5-³H]glucose. Rate of utilization for each concentration of glucose decreased markedly as the cultures became more confluent. This decrease also correlated with a reduced ability to transport glucose as measured by 2-deoxy-[³H]glucose uptake in washed monolayer cultures. In washed confluent culture of fibroblasts, glucose utilization was markedly decreased by the presence of pyruvate and lactate but not by glutamine. The respiratory inhibitors, rotenone and antimycin, did not increase the rate of glucose utilization except when added in combination with pyruvate. We conclude that cultured skin fibroblasts posses a highly glycolytic mode of metabolism but that this mode can become more oxidative in the presence of sufficient quantities of pyruvate and lactate.     

Phytanic acid alpha-oxidation in human cultured skin fibroblasts.

We studied the oxidation of [1-14C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and 14CO2 in human skin fibroblasts. The specific activity for alpha-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid alpha-oxidation enzyme system in peroxisomes.     

Cell surface-associated lysosomal enzymes in cultured human skin fibroblasts

Trypsin released from the surface of intact human skin fibroblasts β-N-acetylglucosaminidase. The amount of trypsin removable β-N-acetylglucosaminidase in 4 control and 14 mucopolysaccharidosis cell lines was equivalent to 1.5% (range 0.5–4.3%) of the intracellular activity. Cell surface-associated β-N-acetylglucosaminidase was absent in mucolipidosis II and III fibroblasts that form lysosomal enzymes defective in binding to the cell surface receptors of fibroblasts and in β-N-acetylglucosaminidase deficient fibroblasts (Sandhoff's disease). Indirect immunofiuorescence with monospecific antisera allowed the demonstration of β-N-acetylglucosaminidase, α-N-acetylglucosaminidase, α-mannosidase and β-glucuronidase on the cell surface of fibroblasts, whereas these enzymes were absent on the cell surface of mucolipidosis II and III fibroblasts. Simultaneous staining for β-glucuronidase and β-N-acetylglucosaminidase showed presence of both enzymes in almost identical areas of the same cell. Cross-reacting material was present on the cell surface of fibroblasts with a deficiency of β-N-acetylglycosaminidase, α-N-acetylglucosaminidase (mucopolysaccharidosis III B), α-mannosidase (mannosidosis) and β-glucuronidase (mucopolysaccharidosis VII). The demonstration of lysosomal enzymes on the cell surface is in agreement with the hypothesis that in fibroblasts transport of lysosomal enzymes to the lysosomal apparatus involves cycling of lysosomal enzymes via the cell surface.     

Inactivation by chloroquine of alpha-galactosidase in cultured human skin fibroblasts

When human skin fibroblasts are cultured in the presence of chloroquine or NH₄Cl there is a decrease in the intracellular level of lysosomal hydrolases and a concomitant increase in the extracellular activity as compared with cells grown in the absence of a base (cf [18]). In a medium with 25 μM chloroquine or 5 mM NH₄Cl, the decrease in the intracellular activity of β-hexosaminidase, arylsulphatase and β-glucuronidase is 10–40% after 1 day. A similar decrease in α-galactosidase activity is observed in cells grown in the presence of 5 mM NH₄Cl. However, in the presence of 25 μM chloroquine, the intracellular activity of α-galactosidase decreases by 80–90% within 6 h. The inactivation is irreversible. After removal of the chloroquine and further culture of the cells in chloroquine-free medium, α-galactosidase activity gradually increases due to de novo synthesis. The turnover time of α-galactosidase was calculated to be 1.9 days. Inactivation of α-galactosidase also occurs when homogenates are incubated with chloroquine, but the concentration of the base required for maximum inactivation is at least three orders of magnitude higher than that which must be present in the medium of intact cells to obtain the same effect.     

Extracellular depolymerization of hyaluronic acid in cultured human skin fibroblasts

The chain length of [3H]hyaluronic acid synthesized by cultivating human skin fibroblasts in the presence of [3H]glucosamine was investigated. [3H]Hyaluronic acid obtained from the matrix fraction was excluded from a Sepharose CL-2B column irrespective of the incubation period, whereas that from the medium was depolymerized into a constant chain length (Mr = 40,000). The reducing and non-reducing terminals of the depolymerized hyaluronic acid were N-acetylglucosamine and glucuronic acid, respectively. Prolonged incubation produced no oligosaccharides as shown by examination of hyaluronidase digests, suggesting the presence of a novel endo-beta-N-acetylglucosaminidase in cultured human skin fibroblasts.     

Regulation of sugar transport in cultured diploid human skin fibroblasts

The regulation of hexose transport under glucose-starvation conditions was studied in cultured human skin fibroblasts. Glucose starvation enhanced the transport of 2-DG and 3-O-methyl-D-glucose (3-OMG) but not of L-glucose. Glucose-starvation enhanced transport was inhibited by cytochalasin B (10 μM). The starvation-induced change in 2-DG transport was due to an increase in the V_max of both the high and low affinity transport sites (2.8- and 2.4-fold, respectively) with no effect on their K_ms. The presence of 5.55 mM galactose, fructose, or L-glucose in the medium resulted in transport increases similar to those seen in glucose-starved cells, while the presence of 5.55 mM glucose, mannose, or 3-OMG repressed 2-DG transport. Glucose-starvation enhancement of 2-DG transport was blocked by cycloheximide (20 μg/ml) but not by actinomycin D (0.03 μg/ml) or α-amanitin (3.5 μM). Readdition of glucose (5.55 mM) for six hours to glucose-starved cells led to a rapid decrease in hexose transport that could be blocked by cycloheximide but not actinomycin D. Although readdition of 3-OMG to glucose-starved cells had little effect on reversing the transport increases, glucose plus 3-OMG were more effective than glucose alone. Serum containing cultures (10% v/v) of glucose-fed or glucose-starved cells exhibited rapid decreases in 2-DG transport when exposed to glucose-containing serum-free medium. These decreases were prevented by employing glucose-free, serum-free medium. The data indicate that hexose transport regulation in cultured human fibrob asts involves protein synthesis of hexose carriers balanced by interactions of glucose with a regulatory protein(s) and glucose metabolism as they affect the regulation and/or turnover of the carrier molecules.     

Receptor-dependent uptake of human chylomicron remnants by cultured skin fibroblasts

Human chylomicrons were isolated from plasma from a subject with familial hypertriglyceridemia and converted to chylomicron remnants by incubation with postheparin plasma. The interaction of these apolipoprotein E-containing, cholesterol-rich human chylomicron remnants with cultured skin fibroblasts was studied. Chylomicron remnants were internalized by skin fibroblasts as a unit, mainly via the low density lipoprotein (LDL)-receptor pathway, resulting in increased cell cholesterol content. After entering the fibroblast, chylomicron remnants stimulated cholesterol esterification, suppressed 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and down-regulated LDL receptor activity similar to the action of LDL. As a function of increasing lipolysis, remnant particles were progressively more effectively taken up by skin fibroblasts, despite a decrease in the apolipoprotein E content per lipoprotein particle. Remnant particles produced after hydrolysis of 70 to 80% of chylomicron triglyceride increased cell cholesterol content to an amount nearly identical to that observed with LDL when the two lipoproteins were incubated at an equal cholesterol concentration. However, when incubated on the basis of equal particle number, chylomicron remnants were 2 to 3 times more effective than LDL in delivering cholesterol to the cells. These results suggest that chylomicron remnants play a role in the regulation of postabsorptive cholesterol homeostasis in nonhepatic cells, and possibly in the pathogenesis of atherosclerosis.     

Monoamine oxidase and catechol-O-methyltransferase activities in cultured human skin fibroblasts

Human fibroblasts obtained from normal male children and children with the Lesch-Nyhan syndrome were found to contain both the A and B forms of monoamine oxidase, with the A form predominating. Both forms of monoamine oxidase showed decreased activities in Lesch-Nyhan, as compared to normal cells; while catechol-O-methyltrans-ferase activities were similar. This study demonstrates the usefulness of fibroblasts cultured from human skin biopsies in analyses of alterations in catecholamine catabolism associated with inherited neurologic diseases.     

Cell density-dependent changes of glycosphingolipid biosynthesis in cultured human skin fibroblasts

In this study, the glycosphingolipid biosynthesis was investigated in the sparse and the confluent cell populations of cultured human skin fibroblasts.The human skin fibroblast cell populations were metabolically pulse labeled with ¹⁴C-galactose (48 h). The amounts of ¹⁴C-radioactivity (cpm) incorporated into extracted and purified total cellular glycosphingolipid fractions were counted by -scintillation and the individual glycosphingolipid species were separated by high performance thin layer chromatography and visualized by autoradiography. The relative labeling (%) of individual newly synthesized glycosphingolipid species was detected by densitometric scanning of autoradiographic glycosphingolipid patterns.The incorporation of ¹⁴C-label into total glycosphingolipids per cell increased significantly as the cell-density increased, referring to five fold higher rate of glycosphingolipid biosynthesis de novo in cells at confluency vs. sparse populations. The total newly synthesized glycosphingolipid pattern (100%) of sparse cell populations showed a significant predominance of the gangliosides (70%) over the neutral glycosphingolipids (30%), with ganglioside GM2 as the major species followed by monohexosyl-ceramide. Oppositely, the newly synthesized neutral glycosphingolipids (67%) predominated over the gangliosides (33%) in cells at confluency (contact inhibition). Cells reaching confluency were characterized by: (a) a dramatic increase of absolute amount of all newly synthesized neutral glycosphingolipid species, particularly the most abundant monohexosyl-ceramide and trihexosyl-ceramide, but also of the ganglioside GM3; (b) a drastic decrease of absolute amount of newly synthesized ganglioside GM2. The specific shift in newly synthesized glycosphingolipid pattern in cells reaching confluency suggests a down-regulation of biosynthetic pathway primarily at the level of N-acetylgalactosaminyl-transferase. A possible involvement of glycosphingolipids in cell density-dependent regulation of cell growth through establishment of the direct intermolecular intermembrane interactions is discussed.     

Regulatory mechanisms of UDP-glucuronic acid biosynthesis in cultured human skin fibroblasts

Kinetic parameters and regulatory properties of UDPGDH extracted from cultured human skin fibroblasts were determined and compared with those of UDPGDH from cornea and epiphysial-plate cartilage. Fibroblast enzyme showed an affinity for UDPG 7 times higher than cartilage enzyme and 42 times higher than cornea enzyme. UDP-xylose acted as a co-operative allosteric inhibitor, but under the same experimental conditions fibroblast enzyme was significantly less inhibited. These results were in agreement with the different GAG production of the cells we studied. Fibroblast UDPGDH activity was regulated by the NAD/NADH ratio and it was also affected by modifications of extracellular matrix composition. A significant increase of UDPGDH affinity for UDPG was observed after the treatment of the monolayers with Chase ABC.     

Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts

Ascorbic acid (AA) is essential for collagen biosynthesis as a cofactor for prolyl and lysyl hydroxylase and as a stimulus for collagen gene expression. Many studies have evaluated the relationship between AA and collagen expression in short- and long-term effects on cells after a single administration of AA into the culture medium. However, no such study has monitored in detail the stability of AA in medium or the alterations of intracellular AA levels during a protracted interval. Therefore, we examined here intracellular AA levels and stability throughout its exposure to human skin fibroblasts in vitro. Moreover, we determined the effects on type 1 and type 4 collagen and sodium-dependent vitamin C transporter (SVCT) gene expression when medium containing 100 μM AA was replaced every 24 h for 5 days to avoid depletion of AA. Throughout this long-term culture, intracellular AA levels remained constant; the expression of type 1 and type 4 collagens and SVCT2 mRNA was enhanced, and type 1 procollagen synthesis increased. Thus, these results indicate that human skin fibroblasts exposed to AA over time had rising levels of type 1/type 4 collagens and SVCT2 mRNA expression and type 1 procollagen synthesis.     

Human breast milk stimulates prostaglandin synthesis in cultured human skin fibroblasts

Effect of human breast milk or its fractions on prostaglandin synthesis was investigated in cultured human skin fibroblasts. Prostaglandins released into the media were measured by radioimmunoassay. Incorporation of breast milk (2% level) into 10% fetal calf serum media (for 48 hours) stimulated the synthesis of 6-keto-PGF1 alpha (stable product of prostacyclin) by 800%. This stimulating effect of milk persisted after cold acetone extraction to remove phospholipids and potentiated further after dialysis. Stimulation by one of the commercial formulas (Similac) was less than 50% of the milk effect. Milk also stimulated PGE2 synthesis, although to a much lesser degree. These studies show for the first time that a) human breast milk contains potent factor(s) capable of influencing prostaglandin synthesis and suggest that b) these factors might have a role in the development of lipid synthetic pathways during early life.     

The effects of monensin on membrane lipids of cultured human skin fibroblasts

We have investigated the effects of monensin, a monovalent cationophore, on the metabolism of neutral lipids, fatty acids, ceramide and phospholipids in cultured human skin fibroblasts. Treatment with 1 microM monensin for 18 h reduced the cellular cholesterol ester content to less than one-third of untreated cells, and incorporation of [3H]acetate into cholesterol ester was also reduced, to less than one-fifth. Concomitantly, a greater conversion of [3H]acetate into free cholesterol occurred. There was a moderate increase in free fatty acids, but no change in triacylglycerol content, although the content of the latter appeared to increase in the presence of fetal calf serum in the culture medium. Phosphatidylcholine decreased in content and phosphatidylserine increased among the phosphatides, but ceramide remained unchanged after monensin treatment. These findings suggest that monensin influences the metabolic interrelationships of structural lipids in fibroblasts.     

The effect of hydrocortisone on cholesterol metabolism of cultured human skin fibroblasts

Hydrocortisone in physiologic concentrations resulted in a reduction in sterol synthesis by cultured normal human skin fibroblasts. These changes were observed when [14C]acetate, [14C]octanoic acid and 3H2O were used as precursors. However, the incorporation of [3H]mevalonic acid lactone into digitonin-precipitable sterols was not affected by hydrocortisone, suggesting that hydrocortisone inhibits sterol synthesis at a site prior to the formation of mevalonic acid. In contrast, the activity of hydroxymethylglutaryl-CoA reductase was stimulated several-fold by the hormone. Thus, the inhibitory effect of hydrocortisone on the cholesterol synthetic pathway may be on hydroxymethylglutaryl-CoA synthase.     

Effect of 2-deoxyglucose on lysosomal enzymes in cultured human skin fibroblasts

Addition of 2-deoxyglucose, an inhibitor of glycosylation of proteins, to the medium of confluent cultures of human skin fibroblasts prevents the increase in specific activity of lysosomal enzymes that normally occurs after confluence. Maximal inhibition is obtained at a concentration of about 1 mM 2-deoxyglucose. The inhibition by 2-deoxyglucose is reversible. The K_m, pH dependence and electrophoretic mobility of the acid hydrolases tested was the same in cells cultured with or without 2-deoxyglucose. In homogenates of cultured human skin fibroblasts, about 95% of the β-hexosaminidase and α-galactosidase activity and about 65 % of the acid phosphatase activity with β-glycerolphosphate as substrate binds to concanavalin A (ConA); 2-deoxyglucose affects only the activity able to bind to ConA. In cells cultured in the presence of 2-deoxyglucose, the specific activity of alkaline phosphodiesterase I, a plasma membrane glycoprotein is lowered. 2-Deoxyglucose has no effect on the specific activity of succinate dehydrogenase, lactate dehydrogenase or total cellular protein.