Mucopolysaccharide accumulation in cultured skin fibroblasts derived from patients with mucolipidosis IV.

Increased concentrations of total sulfated mucopolysaccharides (MPS), threefold, and hyaluronic acid (HA), 10-fold, were found in ML IV fibroblast extracts when compared to normal controls. Such accumulations altered the distribution of MPS:HA comprised 70% of total MPS in ML IV but only 30% in control cells. Intracellular sulfated MPS was observed accumulating almost linearly in ML IV fibroblasts. "Pulse-chase" experiments indicate that both HA and the sulfated MPS remain in the ML IV cells for long periods of time; in control cells, they are rapidly removed as low molecular weight, dialyzable fragments. These data suggest that the MPS accumulation in ML IV fibroblasts, is the consequence of a catabolic block, probably involving the lysosome.     

DHT formation and degradation in cultured human skin fibroblasts: DHT accumulation in the genital skin

The conversion of testosterone to dihydrotestosterone (DHT) by 5 alpha-reductase and the interconversion between DHT and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) by 3 alpha-hydroxy-steroid oxidoreductase (3 alpha-HSOR) were studied in fibroblasts derived from the genital skin of 22 males and 6 females, and from the nongenital skin of 19 males and 9 females with normal gonadal function. The formation of DHT from testosterone (5 alpha-reduction) was significantly greater in fibroblasts from genital skin than in those from nongenital skin in both males (2.15 +/- 1.43 vs 0.81 +/- 0.46 pmol/mg protein/h, mean +/- SD, P less than 0.001) and females (2.52 +/- 1.99 vs 0.69 +/- 0.18, P less than 0.01). Furthermore, DHT formation from 3 alpha-diol (3 alpha-HSOR oxidation) was also significantly greater in genital skin fibroblasts than in nongenital skin fibroblasts of males (5.47 +/- 3.37 vs 2.52 +/- 1.74 pmol/mg protein/h, P less than 0.01). However, the degradation of DHT to 3 alpha- and/or 3 beta-diol (3 alpha- and/or 3 beta-HSOR reductions) was not different between genital and nongenital skin fibroblasts of either males or females. Respective ratios of DHT formation to DHT degradation (5 alpha-reduction/3 alpha-HSOR reduction, 3 alpha-HSOR oxidation/3 alpha-HSOR reduction) were also significantly greater (P less than 0.002) in genital skin fibroblasts than in nongenital skin fibroblasts of males. On the other hand, both DHT formation and degradation were not different between male and female genital skin fibroblasts. These results suggest that the increased production of DHT in genital compared to nongenital skin results from increased 5 alpha-reduction and 3 alpha-HSOR oxidation.     

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.     

Altered glycosaminoglycan production in cultured osteogenesis-imperfecta skin fibroblasts.

Collagen and glycosaminoglycan syntheses were studied in skin fibroblasts cultured from patients with osteogenesis imperfecta (OI) and from age-matched controls. Collagen synthesis (measured as protein-bound [3H]hydroxyproline) was decreased in all four OI cell lines studied in the present experiments, comprising 16-24% of total protein synthesis (40% in normal cells). Hyaluronic acid production in OI skin fibroblasts per cell was higher than in age-matched controls, but the production of sulphated glycosaminoglycans was at the normal level. Thus the ratio of the hyaluronic acid and sulphated-glycosaminoglycan radioactivities was markedly higher in OI cultures than in control cultures, especially at the exponential phase of growth where the synthesis of hyaluronic acid was highest. Hyaluronic acid in OI had a normal molecular weight when determined by gel filtration on Sepharose 2B. The removal of high-molecular-weight hyaluronic acid from the medium by hyaluronidase had no effect on the rate of collagen secretion in OI cell line 1 (A.T.C.C. 1262), in which the rate of collagen secretion was lowest.     

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.     

Endocytosis of sulphated proteoglycans by cultured skin fibroblasts.

1. Human skin fibroblasts internalize homologous sulphated proteoglycans by adsorptive endocytosis. Endocytosis rate is half maximal when the concentration of the proteoglycans is 0.1 nM. At saturation, a single fibroblast may endocytose up to 8 X 10(6) proteoglycan molecules/h. 2. The kinetics of prote;glycan binding to the cell surface suggest the presence of 6 X 10(5) high-affinity binding sites per cell. The bulk of sulphated proteoglycans associates to low-affinity binding sites on the cell surface. 3. Glycosaminoglycans and other anionic macromolecules inhibit endocytosis of sulphated proteoglycans non-competitively. The lack of interaction of glycosaminoglycans with the cell-surface receptors for sulphated proteoglycans suggests that the protein core of proteoglycans is essential for binding to the cell surface. 4. The effects of trypsin, cell density, serum concentration and medium pH on endocytosis and degradation of endocytosed sulphated proteoglycans is described. 5. A comparison of the number of the high-affinity binding sites and the number of molecules endocytosed with respect to time suggests a recycling of the proteoglycan receptors between the cell surface and the endocytotic vesicles and/or the lysosomes.     

Abnormal ganglioside accumulation in cultured fibroblasts from patients with mucolipidosis IV.

Extracts of cultured skin fibroblasts derived from patients with mucolipidosis IV showed a marked increase and altered distribution of GM₃ and GD₃ gangliosides. GD₃ is elevated 1.5–2 times that of normal whereas GM₃ is elevated to a lesser extent. No abnormalities were found in the neutral glycolipids. These two gangliosides apparently comprise most of the accumulated lipid-like material observed on ultrastructural analysis in this disease.     

Glycoprotein metabolism in normal and beta-mannosidase-deficient cultured goat skin fibroblasts.

Cultured skin fibroblasts established from goats affected with beta-mannosidosis, an inherited neurovisceral storage disorder, showed an absence of lysosomal beta-mannosidase activity and the corresponding accumulation of a trisaccharide (TS) with the structure Man beta (1----4)GlcNAc beta (1----4)GlcNAc (0.4 mumol/g) and lesser amounts (0.15 mumol/g) of a Man beta (1----4)GlcNAc disaccharide (DS). By using purified storage TS isolated from fibroblasts metabolically labelled with [3H]GlcN, no conversion of TS into DS could be demonstrated in homogenates of affected cells at either lysosomal pH (4.4) or cytosolic pH (6.1), or in the culture medium (pH 7.0) of affected cells. Both TS and DS were secreted into the culture medium by affected fibroblasts. When affected fibroblasts were treated with tunicamycin before labelling with [3H]GlcN, the accumulation of both labelled TS and DS was completely inhibited. Treatment of both affected and normal goat fibroblasts with swainsonine resulted in the inhibition of lysosomal alpha-mannosidase activity and in the accumulation of the same labelled oligosaccharides in both. The major storage pentasaccharide from both normal and affected swainsonine-treated fibroblasts was sensitive to digestion with alpha-mannosidase and endo-beta-N-acetylhexosaminidase D, suggesting a branched mannose structure and a chitobiose core. In the absence of evidence for the existence of unusual N-linked glycoprotein-associated chitotriose oligosaccharide structures in affected goat fibroblasts, it must be concluded that degradative pathways for N-linked oligosaccharides are similar in both normal and affected goat fibroblasts, and that these pathways differ from catabolic pathways in human fibroblasts.     

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.     

Proteoglycan modifications in cultured osteogenesis imperfecta skin fibroblasts

The PGs produced in the growth medium by skin fibroblast cultures from two O.I. affected patients were investigated. After density gradient centrifugation, in the most dense fraction two main families of molecules appeared. The patient with the more severe clinical picture showed a lower content of the PGs with the highest molecular weight. The GAG composition of PGs was different in the two patients. The more severely affected one showed an increase of HS and a decrease of ChS content, in agreement with the lower value of galactosamine to glucosamine ratio in urinary GAGs.     

Lysosomal accumulation of phospholipids in mucolipidosis IV cultured fibroblasts

Pulse-chase studies were performed to investigate the metabolism of phosphatidylethanolamine (PEA) in cultured fibroblasts from patients with mucolipidosis type IV (MLIV) and normal controls. When cultured cells were incubated with 3H-ethanolamine, 80-90% of the intracellular radioactivity was associated with PEA. Compared to the metabolism of 3H-PEA in normal cells, the phospholipid was retained in greater amounts and degraded more slowly in the MLIV fibroblasts. The 3H-PEA concentration in lysosomal preparations isolated by Percoll gradients was more than 3-fold greater in MLIV than in normal cells after 10 days chase. These studies indicate that PEA catabolism is deranged in MLIV and suggest that the primary metabolic defect causes abnormal phospholipid catabolism in the lysosomes of affected individuals.     

Temperature effects on cyclic AMP accumulation in cultured fibroblasts

The kinetic parameters that determine the accumulation of cAMP in WI-38 cells stimulated with prostaglandin E1 have been determined at 37 degrees C and at lower temperatures. For desensitized cells, a reduction of temperatures from 37 degrees to 25 degrees C reduced both rate of synthesis and rate of elimination of cAMP by about 40%. The steady-state accumulation was, therefore, about the same at both temperatures. The extent of desensitization was also shown to be comparable at the two temperatures. It can be inferred that there was appreciable desensitization at 4 degrees C after a period of stimulation of less than one hour. This is contrasted with the behavior of C6-2B glioma cells at the same temperature. Escape of cAMP through the plasma membrane showed a greater temperature dependence than any of the other processes concerned with cAMP accumulation.     

Stimulation of hexose transport in cultured human skin fibroblasts by insulin.

The effect of insulin on hexose transport in cultured human skin fibroblasts. Studies were carried out on cultures of human skin fibroblasts to explore the effect of insulin on hexose transport in serum-starved monolayers. Insulin (100 mU/ml) stimulated 2-deoxy-D-glucose transport (30% above control values) after 30 minutes exposure time, the response being similar up to four hours exposure to insulin. In several experiments (n = 22) employing three cell strains, insulin (100 mU/ml) exposure led to variable stimulation of 2-deoxy-D-glucose transport (an average of 37% above control values, with a range of 0 = 120%). The insulin-induced stimulation of 2-deoxy-D-glucose transport showed a dose dependency with increasing amounts of insulin, the response being maximal at an insulin concentration of 100 mU/ml. Kinetic analysis of 2-deoxy-D-glucose transport showed that insulin addition resulted in a slight change in the transport K_m (3.13 to 4.06 mM) and a 1.8-fold increase in the transport V_max (17.6 nanomoles/mg protein/min to 32.1 nanomoles/mg protein/min). Insulin also stimulated the transport of 3-0-methyl-D-glucose while the hexokinase activity of the cells was not affected. Further, this insulin-induced stimulation of sugar transport was not blocked by cycloheximide. The results indicate that insulin stimulated the stereospecific carrier-mediated of hexose transport in cultured human skin fibroblasts.     

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.     

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.     

Sericin enhances attachment of cultured human skin fibroblasts

Human skin fibroblasts were cultured on sericin prepared from cocoon shells. The living cell number after 72 h was enhanced to 250% of the no-sericin control. The increase was due to the acceleration of the initial attachment of the cells. It was found that sericin M, the main component of about 400 kDa, and its serine-rich repetitive domain were the active principles.