Glycosyl acceptors in intact and permeabilized normal and cystic fibrosis fibroblasts

Using cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 m?g/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP-xylose, UDP-galactose, and UDP-glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP-galactose and UDP-glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the normal cells.     

Functional effects of enhancing or silencing adenosine A2b receptors in cardiac fibroblasts

Cardiac fibroblasts (CF) express adenosine (ADO) receptors, and pharmacological evidence suggests the possible involvement of the A2 (A2a and A2b) receptor (A2aR and A2bR) subtypes in inhibiting cell functions involved in fibrosis. The main objective of this study was to define the contributions of A2a and/or A2b receptors in modulating ADO-induced decreases in CF functions. For this purpose, CF were either treated pharmacologically or had the A2aR or A2bR levels modified through the use of recombinant adenovirus or siRNA. The assessment of mRNA expression in adult rat CF yielded evidence for A1R, A2bR, A2a), and A3R. Endogenously or exogenously enhanced ADO significantly inhibits CF proliferation, collagen, and protein synthesis. A2R and A2aR agonists, although capable of inhibiting CF protein and collagen synthesis, were unable to define the contributions derived from A2aR or A2bR. Overexpression of A2bR in CF yielded significant decreases in basal levels of collagen and protein synthesis and correlated with increases in cAMP levels. However, at higher doses of ADO receptor agonists, significant increases in protein and collagen synthesis were observed. CF with underexpression of A2bR yielded increases in protein and collagen synthesis. In contrast, A2aR underexpression did not modify ADO-induced decreases in CF protein or collagen synthesis. In conclusion, results derived from the molecular manipulation of receptor levels indicate that A2bR are critically involved in ADO-mediated inhibition of CF functions.     

Coordination of growth and differentiation in the fetal lung

The male fetal lung begins to synthesize surfactant later in gestation than the female. This delay appears to be caused by androgens. We hypothesized that male fetal lung differentiation is delayed as a consequence of an extended phase of growth which is elicited by androgens. We observed that in vivo fetal lung protein synthesis relative to DNA synthesis peaked earlier in gestation in the female fetal lung and that this event was synchronous with the onset of differentiation. Pregnant rats were treated with dihydrotestosterone (DHT) during pregnancy, and fetal lung growth parameters were measured. Lung wet weight, dry weight, and DNA and protein concentrations were significantly elevated by DHT treatment. Type II cells and fibroblasts were isolated from lungs of DHT-treated fetuses. The number of total cells recovered was increased by 30%; the number of type II cells recovered was increased by 87%; and the number of fibroblasts recovered was increased by 42%. The type II cells which were recovered exhibited increased incorporation of [3H]thymidine into DNA and a reduced ratio of radiolabeled protein to radiolabeled DNA compared to that of cells from control lungs. Further studies were done in vitro with fibroblasts and type II cells isolated from untreated fetal rat lungs. Treatment of the fibroblasts with DHT during culture caused an increase in thymidine incorporation into DNA. This effect was not blocked by simultaneous treatment with cortisol, which normally causes reduced DNA synthesis and induces fibroblast differentiation. Treatment of the type II cells with DHT in culture caused a dose-dependent increase in cell number but a decrease in synthesis of disaturated phosphatidylcholine. These studies provide more direct evidence of the interrelationships between the control of growth and the control of differentiation in the fetal lung. DHT, a signal which delays the onset of expression of differentiation, also induces growth. We conclude that the controls of growth and of differentiation of the fetal lung are reciprocally linked.     

Profibrotic influence of high glucose concentration on cardiac fibroblast functions: effects of losartan and vitamin E

Long-standing diabetes can result in the development of cardiomyopathy, which can be accompanied by myocardial fibrosis. Although exposure of cultured kidney and skin fibroblasts to high glucose (HG) concentration is known to increase collagen synthesis, little is known about cardiac fibroblasts (CFs). Therefore, we determined the influence of HG conditions on CF functions and the effects of losartan and vitamin E in these responses. We cultured rat CFs in either normal glucose (NG; 5.5 mM) or HG (25 mM) media and assessed changes in protein and collagen synthesis, matrix metalloproteinase (MMP) activity, and levels of mRNA for ANG II type 1 (AT(1)) receptors. Results indicate that HG-level CFs synthesized more protein and collagen, and these effects were not due to changes in osmotic pressure. The addition of ANG II stimulated protein and collagen synthesis in NG-concentration but not HG-concentration CFs. Interestingly, losartan pretreatment blocked the HG- or ANG II-induced increases in both protein and collagen synthesis. HG or ANG II decreased total MMP activity. Decreases in MMP activity were blocked by losartan. AT(1) mRNA levels were upregulated with HG concentration. Vitamin E pretreatment blocked the effects of HG on total protein synthesis and stimulated MMP activity. Results suggest that HG levels may promote fibrosis by increasing CF protein and collagen synthesis and decreasing MMP activity. HG levels may cause these effects via the upregulation of AT(1) receptors, which can be blocked by losartan. However, vitamin E can alter HG concentration-induced changes in CF functions independently of AT(1) mRNA levels.     

Relationship of macromolecular synthesis to competence induction in a group H streptococcus.

Group H streptococcus strain Wicky, which was induced to competence for genetic transformation with competence factor (CF) derived from a related strain, displayed reduced rates of ribonucleic acid (RNA) and peptidoglycan synthesis. Pulse-labeling studies revealed that the inhibition of both RNA and peptidoglycan synthesis was maximal at the peak of competence and decreased as competence declined. These studies indicated that competence induction had only a slight effect on the rate of protein synthesis. Trypsin inactivation of CF prevented the reductions in synthesis normally elicited by CF preparations. If the addition of trypsin was delayed until 5 min after the addition of CF, competence induction and decreased synthesis of RNA and peptidoglycan were again apparent. Thus, the alterations in the synthesis of these macromolecules appeared to be related to the induction of competence. Further studies indicated that the apparent reductions in biosynthesis were not caused by decreased uptake of the labeled precursors by intact Wicky cells. In addition, these effects were probably not the result of turnover of macromolecules induced by CF. The lack of turnover of labeled peptidoglycan suggested that competence induction may not involve an autolysin.     

Differential glucocorticoid regulation of collagen mRNAs in human dermal fibroblasts. Keloid-derived and fetal fibroblasts are refractory to down-regulation

Abnormal regulation of collagen synthesis has been observed in fibroblasts from keloids, benign collagenous tumors that develop as a result of an inherited defect in dermal wound healing. Hydrocortisone reduces the rate of collagen synthesis in fibroblasts from normal adult dermis and scars, but fails to down regulate collagen synthesis in keloid-derived fibroblasts. We show here that loss of glucocorticoid control of collagen synthesis in keloid cells is due to an inability of hydrocortisone to reduce the levels of types I, III, and V collagen mRNA, whereas it coordinately lowers these RNAs in normal adult cells. The defective regulatory mechanism is expressed only in fibroblasts from the lesion. Fibroblasts from uninvolved dermis respond normally to hydrocortisone. Not all glucocorticoid-modulated matrix proteins are abnormally regulated in this disorder; fibronectin mRNA is induced to a similar extent in normal and keloid cells. The failure of hydrocortisone to reduce collagen gene expression is also seen in fibroblasts from fetal dermis. We have reported similarities between keloid and fetal cells with regard to growth factor requirements and growth response to hydrocortisone. Thus, keloids may be due to the inappropriate expression of a pattern of growth and matrix production that is developmentally regulated.     

The energy-corrective and antioxidative effect of cytoflavin in the postischemical period of human dermal fibroblasts in vitro

The effect of the metabolic drug Cytoflavin (CF) with antihypoxic and antioxidative properties on human dermal fibroblasts in the model of ischemia/reoxygenation in vitro was studied. It was revealed that, during the reoxygenation period after ischemia, the cell death rate in culture with CF appeared to be 2–2.7 times lower than in control cultures. Recovery of ATP synthesis after ischemia was 2.1 times faster in culture with CF. The drug effectively reduced the level of reactive oxygen species (ROS) in fibroblasts after H₂O₂ treatment, which allowed their survival to be maintained at the level of control cells. Pretreatment of cells with CF for one day ensured the maintenance of a normal level of ROS during the investigated time period in fibroblasts subjected to H₂O₂ treatment and reduced H₂O₂-induced cell death by almost one-third as compared to non-pretreatment control cells. Addition of CF into the culture medium in the postischemic period showed no effect on Hsp70 synthesis, but led to a decrease in GRP78 synthesis, raised after ischemia, to the control level, indicating a resolution of unfolded protein response in these cells and normalization of the functioning of their endoplasmic reticulum.     

G protein-coupled receptor kinase-2 is a novel regulator of collagen synthesis in adult human cardiac fibroblasts

Cardiac fibroblasts (CF) make up 60-70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction and the transition to heart failure. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. We investigated the potential role of G protein-coupled receptor kinase-2 (GRK2) in modulating collagen synthesis by adult human CF isolated from normal and failing left ventricles. Baseline collagen synthesis was elevated in failing CF and was not inhibited by β-agonist stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in the failing CF, and expression and activity of GRK2 were increased 3-fold. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following β-agonist stimulation. In contrast, knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity in failing CF by expression of the GRK2 inhibitor, GRK2ct, or siRNA-mediated knockdown restored β-agonist-stimulated inhibition of collagen synthesis and decreased collagen synthesis in response to TGFβ stimulation. GRK2 appears to play a significant role in regulating collagen synthesis in adult human CF, and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.     

Synthesis of biologically active transforming growth factor alpha

A 50-amino acid residue transforming growth factor, type alpha (TGF alpha), secreted in culture by feline-sarcoma-virus-transformed rat embryo fibroblasts, was synthesized by an improved stepwise solid-phase method with an overall yield of 31%. A deprotection strategy based on the SN2 mechanism using either a low concentration of HF or CF3SO3H-CF3CO2H in dimethylsulfide was employed to remove most of the benzyl-derived protecting groups. The more acid resistant protecting groups of Cys and Arg were removed by the SN2 condition using a high concentration of HF. Synthetic TGF alpha was purified to homogeneity in three steps. Synthetic and natural TGF alpha were indistinguishable from each other in HPLC and in different assays, including the assay for anchorage-independent growth of normal rat kidney fibroblasts in soft agar, binding, and stimulating to epidermal growth factor (EGF)-receptor protein kinase. Furthermore, synthetic TGF alpha showed similar biological activities when compared with EGF in these assays. Thus, the chemical synthesis of TGF alpha provided convincing evidence that TGF alpha is functionally related to EGF and is one of the active principles required for cellular transformation.     

Keloid fibroblasts are refractory to inhibition of DNA synthesis by phorbol esters. Altered response is accompanied by reduced sensitivity to prostaglandin E2 and altered down-regulation of phorbol ester binding sites.

To investigate abnormal growth regulation in keloid fibroblasts, responses to phorbol esters were examined. Treatment of quiescent cultures with phorbol 12-myristate 13-acetate (PMA) blocked a normally occurring (20-24 h) peak of serum-stimulated thymidine incorporation in normal and keloid cells. In keloid fibroblasts PMA induced a delayed peak of DNA synthesis. When indomethacin was added with PMA the delayed peak appeared in normal fibroblasts. The ED50 for inhibition of the 20-24-h peak was 1 nM, whereas the delayed peak required a 50-fold-higher PMA concentration. In both cell types PMA induced prostaglandin E2 (PGE2) synthesis, and exogenous PGE2 caused 50% inhibition of the 20-24-h peak. When PMA and indomethacin were added with PGE2 the delayed peak was inhibited 90% in normal fibroblasts, whereas inhibition of keloid cells was the same as with PGE2 alone. Normal and keloid fibroblasts had the same number of phorbol ester binding sites. However, in normal cells, phorbol 12,13-dibutyrate bound with greater affinity, and down-regulation of phorbol ester binding occurred to a greater extent. These findings suggest that altered expression of protein kinase C isozymes or another molecule that binds phorbol esters may play a role in abnormal growth regulation of keloid cells.     

Can glutamine and growth hormone promote protein anabolism in children with cystic fibrosis?

To determine whether recombinant human growth hormone (rhGH), glutamine (GLN) or a combination of both agents can enhance protein synthesis in cystic fibrosis (CF) patients, six 9.6 +/- 0.5-year-old prepubertal children (4 M, 2 F) with CF and stable lung disease with undernutrition (weight/height <50th percentile) or delayed growth (height <5th percentile) received stable isotope infusions, in the postabsorptive state and on 4 separate study days: (a) at baseline, and after a 4-week treatment with either, (b) oral GLN (0.7 g/kg/day), (c) rhGH (0.3 mg/kg/week, SC), or (d) both GLN and rhGH. Four-hour infusions of (13)C-leucine were used to assess leucine appearance rate (Ra, an index of protein breakdown), oxidation (Ox), and non-oxidative leucine disposal (NOLD, an index of protein synthesis). Results are expressed as changes (%) from baseline:We conclude that in children with CF: (1) due to high inter-subject variability, oral glutamine does not significantly enhance protein gain; (2) rhGH has significant anabolic effects which are mediated by stimulation of protein synthesis, and (3) glutamine does not enhance the effect of rhGH.     

Modulation of uterine morphology and growth by estradiol-17beta and an estrogen antagonist

The estrogen antagonist C1628 maintains sustained hypertrophy of the uterine epithelium and the synthesis of many proteins including peroxidase. C1628 is a progestogen, inducing secretion of the protein by surface epithelial and glandular cells. C1628 is a connective tissue mitogen, inducing DNA synthesis in fibroblasts and the endothelium. C1628 and estrogen share these properties mentioned above. Estrogen, however, induced moderate growth of the mucosa within a 24-h period and massive hyperplasia of the mucosa within a 24-h period thereafter. C1628 alone, or in combination with estradiol, does not have mitogenic effect on the mucosa, and in fact blocks the mitotic response normally induced by estrogen alone.     

Effect of Competence Induction on Macromolecular Synthesis in a Group H Streptococcus

The effects of competence induction by competence factor (CF) on macromolecular synthesis in group H streptococcus strain Wicky were investigated. CF preparations (culture filtrates from competent group H streptococcus strain Challis) were either heated or partially purified to remove a bacteriocin. These preparations did not inhibit growth, although they induced high levels of competence in strain Wicky. The action of the CF preparations did not affect the overall rates of deoxyribonucleic acid and protein synthesis, but caused a reduction in the rates of ribonucleic acid (RNA) and peptidoglycan synthesis. When competence induction by CF was prevented, no alterations in RNA or peptidoglycan synthesis were observed, indicating that these changes are in fact related to the development of competence.     

Pleiotropic drug resistance in cystic fibrosis fibroblasts: increased resistance to cyclic AMP.

In previous studies, cystic fibrosis (CF) fibroblasts were demonstrated to be resistant to the cytotoxic effects of ouabain, dexamethasone, and the sex hormones, dihydrotestosterone, 17beta-estradiol, and progesterone. We now show that CF fibroblasts also exhibit greatly increased resistance to the cytotoxic effects of exogenous dibutyryl cyclic AMP (cAMP), as well as to isoproterenol and theophylline, drugs which are known to increase endogenous levels of cAMP. CF cells were also shown to have normal amounts of (3H)cAMP binding to protein kinase as well as normal amounts of cAMP-stimulated protein kinase activity. Phosphodiesterase in CF cells was also found to be stimulated by cAMP to the same degree as in normal cells. These findings suggest that there is no detectable protein kinase deficiency in CF cells. cf cells thus appear to be unlike some cAMP-resistant mutants described by others which are defective in protein kinase activity and cAMP regulation of phosphodiesterase levels. The cross-resistance of CF fibroblasts to ouabain, steroid hormones, and cAMP may provide a unique opportunity to study the biochemical events involved in the metabolism of these drugs as well as the basic biochemical defect in a common human genetic disease.     

Synthesis of collagen, collagenase and collagenase inhibitors by cloned human gingival fibroblasts and the effect of concanavalin A

Individual clones of human gingival fibroblasts that differ in morphology and growth characteristics have been found to synthesize collagen (types I, III and V), collagenase and collagenase inhibitors, and to be capable of degrading native collagen mats. Although collagenase activity was normally low, synthesis of the enzyme could be stimulated ten-fold by Concanavalin A. These results demonstrate that individual fibroblasts have the ability to both synthesize and degrade collagen.     

Effect of lymphokines on the production of cytostatic protein factors from human monocytes

The effect of activating human monocytes in vitro with lymphokines on the production of cytostatic protein factor(s) (CF) was investigated. Upon exposing the monocytes to either lymphokines or lipopolysaccharide (LPS) the amount of CF released was increased approximately fivefold compared to the amount released from unexposed monocytes. With sequential lymphokine and LPS treatment CF release increased nearly 10-fold. Even 10 min lymphokine activation before LPS exposure enhanced CF production significantly. The enhanced CF production was detected between 5 and 10 hr after lymphokine activation. The RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide reduced the lymphokine-induced CF production in a dose-dependent manner, indicating that lymphokines augment both CF mRNA and CF protein synthesis. When monocytes were exposed to LPS on both Day 2 and Day 4 of culture, the amount of CF obtained on Day 4 was reduced compared to that obtained on Day 2. A significant increase in CF production, however, was observed when the monocytes were activated with lymphokines before the second exposure to LPS on Day 4, supporting the view that lymphokines initiate synthesis of CF in monocytes. Upon ion exchange chromatography, chromatofocusing, and gel filtration the same elution profiles of CF were obtained irrespectively of whether the monocytes had been activated with lymphokines or not. This indicates that lymphokines induce an increased production of the same factor(s) which was obtained in the absence of lymphokines.