The regulation of DNA synthesis by fibronectin and its proteolytic products in the skin fibroblasts of healthy donors and patients with systemic scleroderma and rheumatoid arthritis

To study the effect of fibronectin isolated from plasma and culture media and the effect of its tryptic hydrolyzates on DNA synthesis, cultured skin fibroblasts of healthy donors and these of patients with systemic scleroderma (SSD) and rheumatoid arthritis (RA) were employed. It was shown that both fibronectin and total products of its proteolysis markedly stimulated DNA synthesis only in skin fibroblasts of patients with SSD. Fibronectin fragments inhibited DNA synthesis in all fibroblast strains studied. The effect of fibronectin and all its Gel fragments on the DNA synthesis in skin fibroblasts of patients with SSD was dose-dependent. The activity of total fibronectin tryptate, Gel-fragment-free tryptate, and Gel fragments themselves depended on the duration of fibronectin proteolysis, i. e. on the size of the fragments obtained. Culture media collected after treatment of fibroblast monolayer with trypsin and subsequent removal of fibronectin Gel fragments had mitogenic effect on skin fibroblasts, especially on those of patients with SSD and RA. It is supposed that fibronectin Gel fragments are inhibitors of growth factors produced by fibroblasts. The results suggest that fibronectin and its fragments have an important regulatory role in fibroblast proliferation.     

Carnitine transport in cultured muscle cells and skin fibroblasts from patients with primary systemic carnitine deficiency

Summary l-Carnitine transport was studied in cultured muscle cells and skin fibroblasts of patients with primary systemic carnitine deficiency and control subjects. In both cell culture types, two systems for carnitine transport were identified. The kinetic parameters for carnitine transport were remarkably similar in cultured muscle cells and skin fibroblasts. Normal rates and kinetic properties of carnitine transport were observed for both cell lines from patients with systemic carnitine deficiency. These studies do not rule out a defect in carnitine transport in vivo. This study was supported by research grants AM27451 and NS06277 from the National Institutes of Health and by a Research Center Grant from the Muscular Dystrophy Association.     

Cholesterol synthesis in cultured skin fibroblasts from patients with Huntington's disease

In view of the proposed membrane defect in Huntington's disease, cultured skin fibroblasts from healthy volunteers and patients with Huntington's disease were compared with respect to their ability to carry out de novo synthesis of cholesterol. At confluency, values for incorporation of [14C]acetate and 3H2O into cholesterol, and activities of HMG-CoA reductase (the rate-limiting enzyme in the cholesterol biosynthetic pathway), did not differ significantly in the Huntington's disease cells compared to the controls. Determinations of total cellular cholesterol gave similar ratios of cholesterol/protein and cholesterol/phospholipid in the Huntington's disease and control fibroblasts. The data suggest that the proposed generalized cell membrane abnormality in Huntington's disease cannot be attributed to a defect in the cholesterol biosynthetic pathway.     

Sphingomyelinase activities in cultured skin fibroblasts from patients with Niemann-Pick disease

Summary Sphingomyelinase activity in cultured skin fibroblasts from a fetus affected with infantile-type Niemann-Pick disease was 0.5% of control activity; the activities in cells from two patients with adult-type disease (Cases 2 and 3) were 5.0% and 59.0%.Sphingomyelinase activity was separated into three peaks (I–III) by isoelectric focusing. The isoelectric points were 4.5, 4.9, and 5.2 for peaks I, II, and III, respectively. The three peaks in the Case 2 cells were drastically reduced; only a very small peak could be distinguished (pI of 4.7). On the other hand, three peaks were observed in the Case 3 cells. Peak I had a pI of 4.4, peak II a pI of 4.7, and peak III a pI of 5.2. Peak I was found at near normal level, but both peaks II and III were markedly reduced.Sphingomyelinase in the peak I fraction obtained from isoelectric focusing in Case 3 cells was found to have the same Km value as that in control cells.     

Small proteoglycan synthesis by skin fibroblasts cultured from elderly donors and patients with defined defects in types I and III collagen metabolism

The relative synthesis of two different types of small proteoglycans with potentially distinct roles in tissue function (PGI and PGII) was investigated in human skin fibroblast cultures initiated from donors of increasing age (fetal to 92 y) and from patients with defined defects in type I and type III collagen metabolism. Because these two small proteoglycans are not distinguished by the usual methods of ion-exchange and sieve chromatography, we have separated them using gel electrophoresis and confirmed this by specific immunoprecipitation. Small proteoglycans of the PGII type were the predominant species found in the medium of all cultures from normal donors, regardless of age. Most of the mutant cell lines showed a profile of small proteoglycan synthesis like that of the normal cells (i.e., predominantly PGII) although an increased ratio of PGI/PGII was seen for two cell strains from patients with Ehlers-Danlos syndrome type IV characterized by intracellular accumulation of type III procollagen. We conclude that mutations affecting collagen primary structure and secretion appear to have little effect on the cells' synthesis and secretion of small proteoglycans. These findings fail to support an hypothesis suggesting that the metabolism of normal cellular synthetic products (proteoglycans) is altered by abnormal cellular processing of a defective product (collagen).     

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.     

Impaired cholesterol esterification in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy

Cholesterol esterification was examined in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy by incubating cells pretreated without fetal calf serum for 48h, with (14C) cholesterol for 24h. Impaired cholesterol esterification was found in these cells and free cholesterol was accumulated in plasma membrane and Golgi fractions. This impairment was also induced in control cells by adding leupeptin (20 micrograms/ml) or monensin (2 micrograms/ml). These findings suggest the importance of the role of lysosomes for esterification of cholesterol and give a hint as to the basic defect in type C Niemann-Pick disease.     

Chemical and viral transformation of cultured skin fibroblasts from patients with familial polyposis coli

Treatment of skin fibroblasts from an FPC patient with 4NQO or MNNG followed by sequential passaging caused morphological changes of the cells, which showed characteristics of transformed cells such as a high frequency of colony formation in agarose, increased growth ability, and chromosomal abnormalities. This and other fibroblast lines from 5 of 12 FPC patients had an increased susceptibility to 4NQO cytotoxicity, which was caused by enhanced 4NQO-reductase activity rather than by reduced DNA repair. However, the susceptibility to cytotoxicity of MNNG and repair of MNNG-damaged DNA were normal in FPC cells. The tumor promoters TPA and DHTB enhanced the frequency of chemical transformation of the FPC fibroblasts, and protease inhibitors suppressed the promoter-enhanced transformation. The skin fibroblasts from many FPC patients exhibited increased susceptibility to transformation by murine sarcoma viruses. Analysis of the viral DNA and RNA after infection revealed that the increased susceptibility is determined at an early stage of transformation. Two out of 5 MNNG-transformed clones of FPC fibroblasts, isolated from agarose, had increased expression of c-Ki-ras or c-Ha-ras, and 4 of 4 MSV-transformed clones showed high expression of viral Ki-ras. These clones grew further after isolation from agarose, but were mortal and did not form tumors in nude mice. The present results suggest that additional changes in morphologically transformed FPC fibroblasts are required for malignant transformation.     

Properties of beta-glucosidase in cultured skin fibroblasts from controls and patients with Gaucher disease.

Membrane-bound beta-glucosidase from cultured skin fibroblasts can be solubilized in an active form by treatment of membrane preparations with a mixture of Triton X-100 and sodium taurocholate. Several properties of the solubilized enzyme have been studied in fibroblasts from normal, healthy individuals and from 14 patients with different clinical forms of Gaucher disease. The patients studied were classified as follows: group 1 consisted of 10 chronic patients, all (with one exception) of Ashkenazi Jewish origin; group 2 consisted of three black American patients with severe visceral symptoms, manifest from early childhood, but with no apparent neurological involvement; and group 3 consisted of a single white patient with the classical infantile form of the disease. Specific beta-glucosidase activity ranged from 6.6% to 16.5% mean control value in group 1 patients and from 4.1% to 5.8% in groups 2 and 3. When compared with the enzyme from control fibroblasts, the enzyme from chronic Gaucher patients (group 1) was more rapidly inactivated at 50 degrees C, had an altered pH curve, was less effectively inhibited by deoxycorticosterone-beta-glucoside, and was more effectively inhibited by deoxycorticosterone. The enzyme from patients in groups 2 and 3 was qualitatively indistinguishable from the control enzyme in terms of these parameters. No differences in Km (4-methylumbelliferyl-beta-glucoside) or sedimentation coefficient were found between the beta-glucosidases from control and Gaucher cells. The results demonstrate that cells from Ashkenazi Jewish patients with the chronic form of Gaucher disease contain a structurally altered form of beta-glucosidase. This enzyme differs both from normal beta-glucosidase and from the residual enzyme in patients of different ethnic origin and with clinically more severe forms of the disease.     

Hyaluronate synthesized by cultured skin fibroblasts derived from patients with Werner's syndrome.

Hyaluronate in cultured skin fibroblasts derived from patients with Werner's syndrome, who excrete large amounts of urinary hyaluronate, was investigated. The amount of hyaluronate secreted into the medium by Werner's fibroblasts was 2-3-times that of normal fibroblasts, whereas no difference in enzyme activities related to the degradation of hyaluronate was found. Werner's fibroblasts were then cultured in the presence of [3H]glucosamine, and the amount of [3H]hyaluronate and its chain lengths in the medium and matrix (trypsinate) fractions were compared with those of normal cells. No significant difference in the chain length of hyaluronate was observed between normal and Werner's fibroblasts. On the other hand, a significant increase of hyaluronate was found in the matrix fraction of Werner's fibroblasts when the cells reached confluency. In addition, a hyaluronate of small chain length was found in the matrix fraction of Werner's fibroblasts, although this was absent from that of normal cells. It was concluded that the constituents of the extracellular matrix of Werner's fibroblasts differed from those of normal cells, characterized by the presence of a large amount of hyaluronate and a relatively small hyaluronate chain.     

Protein, RNA and DNA synthesis in skin fibroblast cultures from healthy donors and patients with rheumatic diseases

Synthesis of protein, RNA and DNA was studied in skin fibroblast cultures of healthy donors and patients with systemic scleroderma (SSD) and in those with rheumatoid arthritis (RA) with the use of 14C-protein hydrolyzate, 14C-uridine and 14C-thymidine, respectively. A study was also made of the stimulation of 14C-proline incorporation in protein fibroblasts upon addition to serum-free media of 5% bovine embryonic serum. The stability of RNA in fibroblasts was tested. It was shown that the rate of protein synthesis was 11 times higher in fibroblasts of RA patients and 6 times higher in those of SSD patients as compared to the rate of protein synthesis in fibroblasts of normal subjects. The rate of DNA synthesis in skin fibroblasts of RA patients was 15 times higher and in those of SSD patients 4 times higher than normal. In both RA and SSD patients, the synthesis of short-labeled RNA was 2-3 times higher than normal. The addition of embryonic serum increased 2-3 times the incorporation of 14C-proline in protein skin fibroblasts of SSD patients. It was found that all RNA in skin fibroblasts was represented by long-living molecules and that 30-40% of short-labeled RNA in skin fibroblasts of healthy donors and SSD patients underwent degradation within 1-2 hours. The data obtained indicate that fibroblasts of the two pathologies under study are characterized by considerable differences in the synthesis of DNA and the activity of the protein-synthesizing system.     

3H]biotin-labeled proteins in cultured human skin fibroblasts from patients with pyruvate carboxylase deficiency

Biotin containing carboxylases in cultured human skin fibroblasts were radioactively labeled by addition of [8,9-3H]biotin to biotin-depleted cell cultures. Three major bands were visualized by fluorography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fibroblast proteins. These bands corresponded to pyruvate carboxylase (Mr = 125,000), the biotin-containing subunit of methyl crotonyl-CoA carboxylase (Mr = 75,000) and the biotin-containing subunit of propionyl-CoA carboxylase (Mr = 73,000) as judged by molecular weight markers, purified carboxylase protein standards, and interaction with monospecific antisera. Four out of 5 cell lines from patients with classical pyruvate carboxylase deficiency (less than 5% of normal activity) labeled with this technique displayed a normal band in the position of pyruvate carboxylase while one cell line showed complete absence of any labeled protein in this area. These results demonstrate heterogeneity in the etiology of pyruvate carboxylase deficiency.     

Accumulation of phosphorus compounds in tissues and cultured skin fibroblasts in patients with hypophosphatasia

Patients with hypophosphatasia caused by a deficiency of alkaline phosphatase first showed marked accumulation of phosphoethanolamine and other phosphorus compounds in kidney and liver, while in placenta and intestine contents of these compounds were within a normal range. Furthermore, 32P-incorporation in cultured skin fibroblasts of patients with hypophosphatasia was increased about two to three times of control. FPLC chromatographic analysis also indicates that the accumulated phosphorus compounds in hypophosphatasia was smaller molecular phosphorus containing compounds. These data provide new pathophysiological aspect of hypophosphatasia.     

Increased retraction of collagen lattices by fibroblasts from patients with scleroderma

Skin fibroblasts from eight scleroderma patients were seeded in collagen lattices, and their capacity of retraction was compared to that of fibroblasts from normal volunteers. In all cases, pathological fibroblasts retracted collagen lattices earlier and more intensively than controls. This in vitro feature may be related to the cutaneous retraction which characterizes scleroderma lesions in vivo.     

Collagen synthesis by cultured skin fibroblasts from siblings with hydroxylysine-deficient collagen

It has been previously shown that dermis from subjects with hydroxylysine-deficient collagen contains approximately 5% of normal levels of hydroxylysine and sonicates of skin fibroblasts contain less than 15% of normal levels of collagen lysyl hydroxylase activity. However, cultures of dermal fibroblasts from two siblings with hydroxylysine-deficient collagen (Ehlers-Danlos Syndrome Type VI) compared to fibroblasts from normal subjects synthesize collagen containing approximately 50% of normal amounts of hydroxylysine. The lysyl hydroxylase deficient cultures synthesize both Type I and Type III collagen in the same proportion as control cultures. Both α1(I) and α2 chains are similarly reduced in hydroxylysine content. Collagen prolyl hydroxylation by normal and mutant cells is severely depressed without ascorbate but in all cultures collagen lysyl hydroxylation is the same with or without ascorbate supplementation. In mutant cells the rate of prolyl hydroxylation measured after release of inhibition by α,α′-dipyridyl is the same as in control cells. The rate of lysyl hydroxylation is reduced in mutant cells but only to approximately 50% of normal.     

Levels of gamma-glutamyltranspeptidase in cultured skin fibroblasts from cystinotics and normals

The activity of gamma-glutamyltranspeptidase (GGTP) was determined in cultured skin fibroblasts initiated from normals and cystinotics. For the most part, fibroblasts cell lines from patients with nephropathic cystinosis had elevated levels of gamma-glutamyltranspeptidase averaging 37.01 ± 5.88 (16 determinations) as compared with levels in cells from normals of 14.53 ± 1.43 (17 determinations) nanomoles p-nitroaniline released/hr/mg protein with glycylglycine as acceptor substrate. Enzymatic activities also were elevated in affected cells when cystine was the receptor substrate. Increased GGTP is not secondary to the abnormal amounts of intracellular free cystine since the depletion of the cystine pool did not affect the elevated transpeptidase levels. Whether the increased transpeptidase is closely related to the genetic basis for cystinosis, however, remains to be elucidated.