CRP,immunologically cross-reacting protein of prolyl hydroxylase. Its role in assembly of active prolyl hydroxylase and cellular localization in L-929 fibroblasts

There are two forms of prolyl hydroxylase in L-929 flbroblasts. One is the enzymatically active tetramer having two α- and two β-subunits. The other is monomeric cross-reacting protein which is enzymatically inactive but is structurally related to β-subunit of the enzyme. Cultured L-929 fibroblasts at mid-log phase were labeled by ³H-labeled amino acid mixture and the radioactivity was chased for 24 h while cells were harvested and plated at higher cell densities in cultures. The results indicated that both α-subunit of the tetrameric prolyl hydroxylase and cross-reacting protein were labeled, but the β-subunit of the tetrameric active prolyl hydroxylase was not labeled until the cells were crowded for 24 h. Using immunofluorescent techniques with antibodies directed against pure tetrameric prolyl hydroxylase, capping or patching was observed when the cells were incubated at 37 °C. Also, it was found that phagosomes prepared from L-929 flbroblasts contained about 30% of total enzyme protein as determined immunologically but contained no significant prolyl hydroxylase activities. Labeling cells with ¹²⁵I by lactoperoxidase, cross-reacting protein was labeled but both α- and β-subunits of tetrameric active prolyl hydroxylase were not labeled. The results indicate that cross-reacting protein can be utilized as the precursor of β-subunit by the cells to form tetrameric active prolyl hydroxylase and that cross-reacting protein is found associated with cytoplasmic membranes.     

Anti-steroid action in cultured L-929 mouse fibroblasts

L-929 mouse fibroblast growth is arrested by the glucocorticosteroid dexamethasone (dex), which also decreases adhesiveness to culture plates. Both dex effects were abolished when RU 486, a new synthetic anti-hormonal steroid, was added to the culture medium. Using [3H]RU 486, binding studies revealed that RU 486 bound approximately 25,000 sites/cell of the glucocorticosteroid receptor (GR) with affinity higher than that of dex and translocated GR to the nucleus. However, the distribution of steroid-receptor complexes between cytosol and nuclei was different for the agonist and the antagonist, with more nuclear accumulation in the case of dex. Estradiol increases L-929 cell growth and adhesiveness to culture plates, but not if the anti-estrogen tamoxifen (tam) was added. These observations initially made in serum containing medium, were confirmed in serum-free, chemically defined culture medium (SF). In SF medium, tam (1 microM) provoked death of most L-929 cells after 10 days of culture, leading to the selection of a variant clone LB of tam-resistant cells. Tam binds to the estrogen receptor (ER), but with less affinity than estradiol. ER concentration, estimated by the binding of 4-hydroxytamoxifen (OH-tam) and of estradiol was lower in LB cells than in original tam-sensitive L-929 cells. The concentration of specific anti-estrogen binding sites in the particulate fraction of the cells, measured by OH-tam binding, non competed by estradiol, was also significantly diminished in tam-resistant LB cells.     

The stimulatory effect of platelet homogenate on prolyl hydroxylase activity in L-929 cells

We have found that the addition of platelet homogenate to confluent cultures of L-929 cells increases 2-3 times the activity of prolyl hydroxylase in these cells. Furthermore, it was found that the platelet homogenate potentiates the effect of ferrous ions and ascorbic acid, which are known activators of prolyl hydroxylase. The effect of the platelet homogenate is diminished by cycloheximide. It seems probable that some products present in the platelet homogenate may promote biosynthesis of the enzyme or they stimulate glycolysis and accumulation of lactic acid, an activator of the hydroxylase.     

Characterization of activatable form of prolyl hydroxylase in L929 fibroblasts

Prolyl hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate : oxygen oxidoreductase, EC 1.14.11.2) activity in a sonicated preparation of early log-phase L929 cells could be increased 3-4-times by preincubation of the sonicate with all cofactors of proline hydroxylation, such as ascorbate, Fe2+ and alpha-ketoglutarate. An "activatable" form of the enzyme is produced in these cells due to a deficiency of one of the cofactors in these cultures. The activatable form is found to be different from the active enzyme with respect to its stability to heat and dithiothreitol denaturation. The activatable form has different ionic properties and could be separated from the active enzyme by DEAE-Sephadex chromatography. The available evidence suggests that the activatable form is a tight complex produced by the enzyme with underhydroxylated collagen, the latter being produced by a cofactor deficiency in the cells. Activation of this complex follows the hydroxylation of the substrate and its subsequent release from the bound enzyme.     

Cell density-dependent increase in prolyl hydroxylase activity in cultured L-929 cells requires de novo protein synthesis.

Prolyl hydroxylase activity in cultured L-929 cells was found to increase when cells grew from log phase to stationary phase and when cells were harvested at the mid-log phase and replated at higher cell densities. Cycloheximide and actinomycin D inhibited the cell density-dependent increase in prolyl hydroxylase activity indicating that the increase in prolyl hydroxylase activity required de novo synthesis of protein and RNA. Prolyl hydroxylase was purified from cultured L-929 cells and antibodies against the protein were raised in rabbits. The antibodies were used to demonstrate that L-929 cells contained two forms of prolyl hydroxylase: an enzymatically active, tetrameric form consisting of two alpha and two beta polypeptide chains and an enzymatically inactive form containing immunologically cross-reacting protein. The polypeptide chains alpha, beta and cross-reacting protein were obtained by immunoadsorption. Peptide map analysis indicated that cross-reacting protein was similar if not identical to beta in primary structure, and alpha was different from both beta and cross-reacting protein. The results suggested that the prolyl hydroxylase levels in cells or tissues may be regulated by new protein and/or RNA synthesis.     

Prolyl hydroxylase activity in L-929 fibroblasts incubated with and without ascorbate

An improved procedure was used to assay prolyl hydroxylase activity in both early-log and late-log L-929 fibroblasts grown on plastic surfaces. When 40 μg/ml of ascorbate was added to early-log phase cultures, the rate of hydroxy-[¹⁴C] proline synthesis increased 2-fold within 4 h, but there was no change in prolyl hydroxylase activity per cell. The results indicated therefore that ascorbate did not “activate” prolyl hydroxylase in the sense of converting inactive enzyme protein to active enzyme protein. Instead ascorbate appeared to increase hydroxyproline synthesis in early-log L-929 fibroblasts because the prolyl hydroxylase reaction in such cells was limited by the availability of ascorbate or a similar cofactor. When 40 μg/ml of ascorbate was added to late-log phase cultures, there was essentially no effect on the rate of hydroxyl[¹⁴C]-proline synthesis or prolyl hydroxylase activity. The late-log phase cells, however, contained three times more enzyme activity and about two times more immuno-reactive enzyme protein than early-log phase cells. In addition, the rate of protein synthesis per cell in late-log phase cells was only one-tenth the rate in early-log phase cells. The results suggested that as the cells grew to confluency, collagen polypeptides were more completely hydroxylated in part because the rate of polypeptide synthesis decreased and at the same time prolyl hydroxylase activity per cell increased. The results appear to provide an alternate explanation for previous observations on the effects of ascorbate and “crowding” on hydroxy[roline synthesis in cultures of L-929 fibroblasts.     

Regulation of prolyl and lysyl hydroxylase activities in cultured human skin fibroblasts by ascorbic acid

Studies with confluent human skin fibroblasts maintained in 0.5% serum supplemented medium have given new insight into the regulatory influences of ascorbate. These include a reduction of prolyl hydroxylase activity, a stimulation of lysyl hydroxylase activity, and an acceleration of collagen production. The lack of parallel between prolyl hydroxylase activity and collagen production indicates that the rate of collagen synthesis is not controlled by the level of prolyl hydroxylase.     

A paradoxical effect of hydralazine on prolyl and lysyl hydroxylase activities in cultured human skin fibroblasts

The effect of hydralazine on several parameters of collagen biosynthesis has been studied in cultured human skin fibroblasts. Cells treated with hydralazine synthesized procollagen which was severely deficient in hydroxyproline and hydroxylysine, indicating inhibition of prolyl and lysyl hydroxylase reactions in the cell. Assays of prolyl and lysyl hydroxylase activities, however, revealed markedly increased levels in hydralazine-treated cells. The stimulatory effect of hydralazine could not be simulated in cell extracts, demonstrating its requirement for intact cells. The effect occurred slowly over a period of 96 h and was dependent on hydralazine concentration between 10 and 100 microM. This phenomenon was also observed in lysyl hydroxylase-deficient mutants. In both normal and mutant cells the relative magnitude of the hydralazine effect could be modified by ascorbic acid in the culture medium. Ascorbic acid increased the response of prolyl hydroxylase to hydralazine from 1.5- to 2-fold to 3- to 7-fold, whereas it decreased the response of lysyl hydroxylase to hydralazine from 4- to 8-fold to 2- to 3-fold. Total collagen synthesis was substantially reduced in hydralazine-treated cells; the time course and the dose-response relationship were similar to those observed for the hydroxylases. alpha, alpha'-Dipyridyl, an iron chelator, mimicked these effects of hydralazine. The studies suggest the existence in cultured cells of a compensatory mechanism for overproduction of these crucial enzymes in collagen biosynthesis, a mechanism which remains functional in cells derived from patients afflicted with hydroxylysine-deficient collagen disease.     

Assay of prolyl hydroxylase in cultured fibroblast monolayers

Prolyl hydroxylase may be assayed in cultured cell monolayers after a cold acetone treatment or air-drying. Enzyme activity of dried cells is stable when cell monolayers are stored frozen and desiccated, thus permitting simultaneous assay of cells harvested at different times, e.g., on different days of a culture cycle. Uniformity of cell monolayers permits use of separate dishes for assay of protein or DNA to determine specific enzyme activity. This procedure greatly facilitates harvesting and assay of cells for a variety of biochemical measurements.     

An intramembranous reductant which participates in the proline hydroxylation reaction with intracisternal prolyl hydroxylase and unhydroxylated procollagen in isolated microsomes from L-929 cells

We previously have described a substance present in crude sonicates of L-929 cells which replaced ascorbate in vitro as a reductant for prolyl hydroxylase (B. Peterkofsky, D. Kalwinksy and R. Assad, 1980, Arch. Biochem. Biophys.199, 362–373). In the present study we found that almost 90% of the substance was particulate after differential centrifugation of stationary phase L-929 cell homogenates. The substance was not localized in nuclei or mitochondria and was found in the same fractions as microsomes, but these fractions also contained lysosomes and cell membranes. The reductant could not be solubilized from particles by Brij-35, indicating that it is an intrinsic component of a membrane rather than intracisternally located. The intramembranous cofactor, in the absence of ascorbate, participated in the in vitro hydroxylation of [4-³H]proline in radio-actively labeled, intracisternal unhydroxylated procollagen in isolated microsomes which also contained prolyl hydroxylase. Hydroxylation was determined by measuring tritiated water formed from release of the 4-trans tritium atom. Since it is unlikely that such participation could occur if the cofactor were located within the membrane of another subcellular organelle, we have concluded that it is in the same particle as prolyl hydroxylase and unhydroxylated procollagen, that is, the microsome. With the endogenous reductant the reaction was slower than with saturating ascorbate and was increased by NADH. Maximum hydroxylation with the endogenous reductant was close to that which could be achieved with ascorbate. These results provide strong evidence that the endogenous reductant alone can account for the phenomenon of ascorbate-independent proline hydroxylation in L-929 cells. As in the case of ascorbate, the microsomal reductant functioned only in the presence of α-ketoglutarate and Fe²⁺ and served as reductant for lysyl hydroxylase. It also was detected in the particulate fraction of virally transformed BALB 3T3 cells and in purified microsomes from bones of intact chick embryos. Since ascorbate could be taken up and concentrated in bone microsomes, it is unlikely that the endogenous reductant serves as an intermediary between ascorbate and intracisternal prolyl hydroxylase.     

Effect of L-3,4-dehydroproline on collagen synthesis and prolyl hydroxylase activity in mammalian cell cultures.

The incorporation of DL-3,4-dehydro[14C]proline into collagen and total protein of 3T3 cells occurred at approximately one-fifth the rate observed for L-[14C]proline. Addition of L-3,4-dehydroproline to the culture medium inhibited markedly the incorporation of [14C]glycine and L-[3H]lysine into the collagen of 3T3 cells, but there was only slight inhibition of the incorporation of the radiolabeled amino acids into total cellular proteins, indicating that the action of L-3,4-dehydroproline is specific for collagen. When 1 mM L-3,4-dehydroproline was added to the culture medium the [14C]hydroxyproline content was reduced 40% in the cell layer and 70% in the medium. The D isomer of 3,4-dehydroproline did not inhibit [14C]hydroxyproline formation. These findings indicate that L-3,4-dehydroline reduced the hydroxylation of the susceptible prolyl residues in the collagen molecule and the secretion of collagen from the cell. The reduction in the hydroxyproline content is probably related in part to a reduction in the activity of prolyl hydroxylase; when various mammalian cell cultures were exposed to 0.2 mM L-3,4-dehydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroxylase was not affected. Under these conditions, cell growth and lactic dehydrogenase required protein synthesis. Removal of L-3,4-dehydroproline from the growth medium resulted in a time-dependent increase in the specific activity of prolyl hydroxylase.     

A substance in L-929 cell extracts which replaces the ascorbate requirement for prolyl hydroxylase in a tritium release assay for reducing cofactor; correlation of its concentration with the extent of ascorbate-independent proline hydroxylation and the level of prolyl hydroxylase activity in these cells

Reductant used as cofactor for the prolyl hydroxylase reaction, was measured by a tritium release assay modified from an enzyme assay by making all components of the assay system saturating except for the reductant, but including prolyl hydroxylase. Reduced glutathione (6 mm), which had little activity as a cofactor, and thymol (0.1 mm), an antioxidant which exhibited no cofactor activity at all, were required for optimal proline hydroxylation dependent on reducing cofactor, with thymol fulfilling the previously described requirement for catalase. Ascorbate, cysteine and 6,7-dimethyltetrahydropterin were active as cofactors, in descending order of activity at equimolar concentrations, and activity was concentration dependent for all of these compounds. Sonicates of stationary phase L-929 cells which exhibit ascorbate-independent proline hydroxylation in culture contained reducing cofactor which could replace ascorbate in the cofactor assay, while sonicates of log phase cells which exhibit an ascorbate requirement in culture contained about one-third or less of that amount. NADH and NADPH, which themselves have little or no activity as cofactor, increased the cofactor activity of log phase cell sonicates but had relatively little effect on the activity of stationary cell sonicates suggesting that the cofactor is in a more reduced state in stationary phase. Within 24 h after replating dense, stationary phase cell cultures at low density, conditions where cells return to ascorbate dependence, prolyl hydroxylase activity had decreased to one-fifth the original activity while the concentration of functional reducing cofactor had decreased to less than 1% of its original concentration, largely as a result of oxidation. Ascorbate was not present in L-929 cells sonicates and the levels of tetrahydropterin and cysteine in sonicates could not account for the amount of cofactor activity exhibited by the sonicates in the assay system. Treatment of L-929 cultures with aminopterin did not decrease ascorbate independence, suggesting that tetrahydrofolate did not contribute significantly to cellular proline hydroxylation. These results suggest that an unidentified reductant present in L-929 cells can account for ascorbate-independent proline hydroxylation and also regulate prolyl hydroxylase activity in these cells and that cellular levels of reduced pyridine nucleotides may regulate the reduction state of this substance.     

Characterization of the lysosomal cystine transport system in mouse L-929 fibroblasts

We characterize here a lysosomal cystine transporter in mouse L-929 fibroblasts. Granular fractions from cells preloaded with cystine demonstrated countertransport that showed no dependence on Na+ or K+. The Michaelis constant for infinite-trans influx was 0.27 +/- 0.06 mM (n = 3), and a nonsaturable component of cystine entry was observed with Kd = 0.8-1.8 nmol of cystine.min-1.unit of hexosaminidase-1.mM-1. We found no evidence that cystine was also carried on any of the other known lysosomal amino acid transporters. Over 50 analogs were tested for their ability to inhibit countertransport. The inhibition constants are reported for selenocystine, cystathionine, selenomethionine, and leucine. Significant requirements for recognition by the transporter were the presence of amino groups, L configuration, and a chain length not greater than eight atoms. A net positive or negative charge was not required. Some di- as well as tetrapolar amino acids were recognized. We have surmised that the binding site has polar and apolar domains, the latter being large enough to accommodate branching on C-3 and the substitution of selenium or carbon in place of sulfur.     

Mycobacterial growth and ultrastructure in mouse L-929 fibroblasts and bone marrow-derived macrophages: Evidence that infected fibroblasts secrete mediators capable of modulating bacterial growth in macrophages

The intracellular growth kinetics ofMycobacterium avium and H37Rv (virulent) and H37Ra (avirulent) strains ofMycobacterium tuberculosis were compared by use of both the professional (mouse bone marrow-derived macrophages, BMMØ) and nonprofessional (mouse L-929 fibroblast cell line) phagocytes. The results obtained showed that all the mycobacterial strains grew more actively in fibroblasts than in BMMØ. This difference was paralleled by lesser acid phosphatase (AcP) labeling of noninfected fibroblasts and the observation that upon infection both the proportion of AcP-positive cells and AcP content were higher in BMMØ than in L-cells during the 7 days of infection. In parallel experiments, intracellular growth ofM. tuberculosis H37Rv andM. avium was compared inside BMMØ from both theBcg ^s (C57BL/6) andBcg ^r (DBA-2) mice, which were matured and differentiated with either an L-cell-conditioned medium (LCM) obtained from control, noninfected L-929 cells, or a LCM obtained withM. tuberculosis-orM. avium-infected L-cells. Upon mycobacterial infection, fibroblasts were able to secrete mediators that stimulated the BMMØ to better control the infection by pathogenic mycobacteria. These results are discussed in terms of the mycobacteria-fibroblast interactions and their eventual role in the immune modulation of the host's response to invading mycobacteria.