Lipopolysaccharide and serum synergistically stimulate ornithine decarboxylase in Chinese hamster ovary cells

Summary Lipopolysaccharide (LPS), the active component of bacterial endotoxin, caused no significant increase in ornithine decarboxylase (ODC) activity in serum-starved, Chinese hamster ovary fibroblasts. However, concurrent addition of LPS with 10% fetal bovine serum caused a synergistic 30 to 40-fold increase in enzyme activity as compared to the 10 to 20-fold increase seen after addition of serum alone. This synergism was not due to an alteration in the time course of enzyme induction after serum addition. The LPS-induced synergy of ODC induction by serum was inhibited by the concurrent addition of the specific LPS-antagonist, Polymyxin B. This investigation was supported by PHS Grant CA32444, awarded by the National Cancer Institute. A. R. L. G. is a recipient of a USPHS fellowship, GM09226-01, and S. M. T. was supported by NIH training Grant AMO 7282.     

Bacterial lipopolysaccharide induction of ornithine decarboxylase in the macrophage-like cell line RAW264: requirement of an inducible soluble factor

Ornithine decarboxylase (ODC, EC 4.1.1.17) activity is induced in the RAW264 macrophage-like cell line by bacterial lipopolysaccharide (LPS). As little as 0.1 ng/ml LPS promoted an increase in ODC activity, while maximal ODC activity (30-fold above control) was induced with 1.0 microgram/ml LPS. An increase in ODC activity was detectable within 90 min of LPS addition. The LPS-induced increase in ODC activity was prevented by inhibitors of protein and RNA synthesis. The induction of the enzyme by LPS was not dependent on prostaglandin production. However, PGE2 (1 microgram/ml) and 8-bromo-cyclic AMP (1 mM), neither of which had an effect on ODC activity when added alone, each acted synergistically to enhance the LPS induction of ODC activity. Enzyme induction was not associated with an alteration in Km for ornithine, which remained constant at 0.04 mM. The extent of the increase in ODC in response to LPS increased with increasing cellular density. This relationship was dependent not on absolute cell density of the monolayer but on the cell number in relation to medium volume, and this dependence could be extrapolated to the origin. Addition of conditioned media from LPS-stimulated but not unstimulated cultures enhanced the ODC increase in sparsely plated cultures in response to a maximal concentration of LPS. The addition of polymyxin B, a reagent that blocks the effects of LPS, including the increase in ODC activity, did not totally inhibit the conditioned medium stimulation. This data indicates that two signals, LPS and a LPS-induced mediator, are involved in the induction of ODC activity in RAW264 cells.     

The induction of ornithine decarboxylase by tumor promoting phorbol ester analogues in Reuber H35 hepatoma cells

The induction of ornithine decarboxylase activity was studied in a rat hepatoma cell line (Reuber H35) incubated with a group of structurally-related phorbol ester analogues. A single application of 1.6 μM of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to H35 cells caused a dramatic increase in the activity of ornithine decarboxylase. The stimulation of the enzyme activity was rapid but transient, peaking at 4 to 5 hr with a value which was 116-fold greater than control and then declining to the basal level after 8 hr. In addition, the increase in ODC activity was dependent upon the concentration of TPA added to the culture medium and the EC₅₀ was estimated to be about 2.63 × 10^?7 M. Our studies of the effect of various phorbol ester analogues on the H35 ODC activity indicated an apparent correlation between the ability of phorbol ester derivatives to induce ODC activity in the H35 cells and their activity to promote papilloma formation in the mouse skin in that the various derivatives possessed the following relative abilities to increase ODC activity: TPA > PDB > PDA > 4 α-P > 4 α-PDD. Concurrent addition of either actinomycin D or cycloheximide abolished the increase in ODC activity after TPA treatment. Changes of intracellular concentrations of polyamines, particularly putrescine, were in good agreement with the increase in ODC activity in response to TPA: a 10-fold increase in putrescine over the control level was observed at 6 hr. Our data suggest that cultured Reuber H35 hepatoma cells exhibit a marked and specific response to the phorbol ester tumor promoters and may be of great value in studying the biochemical mechanism of ODC induction by these agents.     

Effect of sodium arsenite on the induction and turnover of ornithine decarboxylase activity in erythroleukemia cells

Sodium arsenite proved effective in preventing the induction of ornithine decarboxylase (ODC) activity elicited by dilution of Friend erythroleukemia cells in fresh medium. A 50 per cent inhibition was produced at approximately 1 microM arsenite and complete inhibition was obtained at concentrations above 10 microM. However, addition of arsenite 5 h after cell dilution, i.e. when ODC was already induced, appeared to stabilize the enzyme. The half-life of ODC activity, measured after cycloheximide treatment, increased almost six-fold after addition of sodium arsenite. Agents known to provoke oxidative alteration of the thiol-redox status in cells, also caused a similar effect on the induction and stability of ODC.     

Effects of putrescine on synthesis and degradation of ornithine decarboxylase in primary cultured hepatocytes

Changes in both synthesis rate and degradation rate of ornithine decarboxylase (ODC) were pursued in primary cultures of adult rat hepatocytes during the process of ODC induction caused by asparagine and glucagon and also during the process of rapid ODC decay caused by putrescine. The synthesis rate of ODC was determined by [35S]methionine incorporation into the enzyme, which was separated afterwards by immunoprecipitation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The degradation rate of ODC was determined by following the decay of prelabeled ODC. The enzyme induction caused by asparagine (10 mM) and glucagon (1 microM) was due both to an increase in the synthesis rate and to a decrease in the degradation rate. Addition of 10 mM putrescine caused a rapid decay of ODC activity, which was faster than ODC decay in the presence of cycloheximide. This rapid decay in ODC activity was accompanied by slightly slower decay in ODC protein, which was due both to partial suppression of ODC synthesis and to several fold acceleration of ODC degradation.     

A stable, inducible, dose-responsive ODC overexpression system in human cell lines

ODC is a labile protein subject to rapid turnover, and a conditional expression system providing long-term overexpression may be helpful in further understanding the biochemical properties of this enzyme and elucidating aspects of the polyamine biosynthetic pathway that have otherwise been difficult to study. HEK293 and LNCaP cell lines were engineered to stably and inducibly overexpress ODC using a Tet-on inducible construct. Clones from both cell lines were characterized by evaluating ODC mRNA expression, ODC activity, intracellular and extracellular polyamine levels, SSAT activity and growth kinetics. The ODC-inducible cell lines were time- and dose-responsive providing a mechanism to increase ODC and putrescine accumulation to a desired level in a flexible and controllable manner. The findings demonstrate that LNCaP ODC overexpressing cells maintained over a 100-fold increase in ODC activity and over a 10-fold increase in intracellular putrescine after 6 h. ODC induction at the highest levels was accompanied by a slight decline in intracellular spermidine and spermine levels and this observation was supported by the finding that SSAT activity was induced over 40-fold under these conditions. Growth rate remained unaffected following at least 12 h of ODC overexpression. Similar results were observed in the HEK293 ODC overexpressing cells.     

Stimulation of ornithine decarboxylase activity in chick fibroblasts by non-suppressible insulin-like activity (NSILA), insulin and serum.

A factor isolated from human serum (nonsuppressible insulin-like activity, NSILA) stimulates multiplication of serum-starved chick embryo fibroblasts and stimulates activity of ornithine decarboxylase (ODC). Physiological doses of NSILA (200 muU/ml) and pharmacological doses of insulin (200 mU/ml) stimulate ODC 4-5-fold, 10% fetal calf serum about 18-fold. Combined addition of NSILA and insulin does not result in higher activities, suggesting a common mechanism of action. The increase in cell number obtained with NSILA, insulin or serum parallels the degree of ODC stimulation. Treatment of cells with pronase also stimulates ODC activity. A sharp increase in ODC activity occurs between 2, 5 and 5.0 hours after addition of the growth factors with a peak at 4.0-4.5 hours ("activation period"). As cells leave G1 phase, ODC activity decreases rapidly. To achieve maximal activity of ODC, the growth factors have to be present during the entire "activation period." The potential to reactivate ODC decreases as cells pass through S phase. Results obtained using cycloheximide suggest that ODC is translated only in the second half of the "activation period." Data on effects of dbcAMP and dbcGMP on ODC activation by serum are discussed.     

Involvement of the ornithine decarboxylase pathway in macrophage collagenase production

Definition of the cellular events involved in the production of collagenase by macrophages following activation has revealed prostaglandin E2 (PGE2)- and cAMP-dependent steps. Since ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is regulated by cAMP and is associated with certain aspects of protein synthesis, the potential role of this enzyme and its polyamine product, putrescine, in collagenase synthesis was examined. Lipopolysaccharide (LPS) activation of macrophages resulted in a maximal ODC response after 6 to 9 h with a 10- to 12-fold elevation in enzyme activity. This elevation in ODC appeared to be regulated by PGE2 since indomethacin inhibited LPS-induced macrophage ODC levels by 70%. Associated with the indomethacin-mediated inhibition of ODC was a loss of collagenase synthesis. Furthermore, partial restoration of collagenase production in indomethacin-inhibited cultures could be achieved by the addition of putrescine. In additional studies alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, also inhibited collagenase production when added to LPS-treated macrophages. This inhibition by DFMO could be reversed by the exogenous addition of putrescine. These findings demonstrate that the ODC pathway is an important intracellular component in the sequence of events that lead to macrophage collagenase synthesis.     

Cyclic AMP-dependent regulation of ornithine decarboxylase activity in Chinese hamster ovary cells maintained with a salts/glucose medium.

Ornithine decarboxylase activity (ODC) increased about 7 fold 6--8 h following 10mM asparagine (ASN) addition to confluent cultures that had been previously serum deprived and then placed in a salts/glucose medium. Optimal concentrations of dibutyryl cAMP (dB cAMP) when incubated with the ASN caused up to a 50 fold increase in the activity of this enzyme after 7--8 h. The enhancement of ODC activity by ASN and dB cAMP was not sensitive to continuous (0--7 h) treatment with actinomycin D but similar treatment with cycloheximide depressed enzyme activity 40--60%. The synergistic stimulation of ODC activity by dB cAMP added with ASN was dose dependent and the dB cAMP stimulation of ODC activity displayed an absolute requirement for ASN when cells were maintained in the salts/glucose medium. The addition of dB cAMP always further enhanced ODC activity above the levels produced by addition of various levels of ASN (1 to 40mM) to the salts/glucose medium. Other agents which elevated cAMP levels such as 1-methyl-3-isobutylxanthine (IBMX) also enhanced ODC activity when administered with ASN. Additionally, treatment with sodium butyrate at concentrations ranging from 0.001mM to 5.0mM did not elevate ODC activity above the activity obtained with ASN alone. Addition of dB cAMP at various times after placing cells in salts/glucose medium with ASN further stimulated ODC activity only when added during the first 3-4 h. These results demonstrate the involvement of cAMP in the ASN mediated stimulation of ODC activity using cells maintained in a salts/glucose medium.     

Modulation of macrophage Ia-expression by lipopolysaccharide. I. Induction of Ia expression in vivo

Experiments were performed to analyze the modulation of macrophage Ia expression and biosynthesis by Salmonella minnesota-derived lipopolysaccharide (LPS) in vivo. The i.p. injection of LPS into LPS-responder mice caused a dramatic increase in the Ia expression of the peritoneal macrophage population harvested 1 wk after injection. As little as 1 ng of lipid-rich Re595 LPS per mouse caused a significant I-Ak increase, and 1 microgram was optimal; wild-type S. minnesota LPS was less active. No I-Ak induction by LPS was observed in the LPS-nonresponder strain C3H/HeJ. LPS-induced macrophages showed a 6- to 16-fold increase in I-Ak expression by radioimmunoassay (RIA), a 3- to 10-fold increase in the proportion of I-Ak-positive cells, and a 10- to 15-fold increase in I-Ak biosynthetic capacity. The magnitude of this induction by LPS was comparable to increases observed after injection of live Listeria monocytogenes. The kinetics of I-Ak induction by LPS and by L. monocytogenes were different: LPS caused an initial decrease in I-Ak expression 1 day after injection, and I-Ak induction by LPS occurred more slowly and maintained heightened expression longer. Several H-2 gene products (H-2Kk, I-Ak, and I-Ek) were augmented in LPS-induced macrophages. In keeping with increased I-A and I-E expression, LPS-induced macrophages were more effective than normal macrophages in presenting antigen to T lymphocytes. We suggest that the modulation of macrophage Ia expression is one important mechanism contributing to the immunoregulatory activity of LPS.     

Early alterations of polyamine metabolism induced after acute administration of clenbuterol in mouse heart.

An acute treatment of mice with clenbuterol, a beta-adrenergic agonist, produced a marked increase of polyamines levels in heart, particularly during the early phase of administration of the drug. A single dose of 1.5 mg/kg caused as much as a 10 fold induction in activity of ornithine decarboxylase (ODC) and 3 to 4 fold increase in levels of putrescine, spermidine and spermine in mouse heart. Maximum changes were observed 3 to 4 hours post-administration of clenbuterol. This treatment did not produce any change in S-adenosylmethionine decarboxylase activity. The induction of cardiac ODC by clenbuterol was also dose dependent with a peak at about 5 micromol/kg. Co-administration of difluoromethylornithine, an irreversible inhibitor of ODC, or propranolol, a nonspecific beta-antagonist, with clenbuterol completely prevented the induction of ODC activity as well as the increase in polyamine levels in heart. However, pretreatment with alprenolol or metoprolol, the specific beta1 and beta2-antagonists, respectively, produced only partial prevention. The cardiac ODC from controls as well as clenbuterol treated mice exhibited similar affinity (Km) for its substrate, ornithine, while maximum enzyme activity (Vmax) was about 14 fold higher in clenbuterol treated mouse heart than in the control. Clenbuterol produced no change in the level of specific ODC mRNA or the protein, but the enzyme from the drug-treated mouse heart was considerably more stable than the control. Pretreatment of mice with either cycloheximide or actinomycin D followed by administration of clenbuterol could not prevent the induction in ODC activity suggesting that de novo biosynthesis of the enzyme protein or ODC mRNA was not responsible for induction of ODC activity. Post-translational changes in ODC may be responsible for an early increase of ODC activity due to clenbuterol treatment.     

Cultured hepatoma cells for the study of enzyme regulation: induction of ornithine decarboxylase by insulin and asparagine

The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combination of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10(-5) M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present.     

Induction of ornithine decarboxylase in macrophages by bacterial lipopolysaccharides (LPS) and mycobacterial cell wall material

Lipopolysaccharide from $\text{E. Coli}$ (LPS) and BCG cell walls (BCGcw) are recognized immunoadjuvants that directly stimulate some macrophage functions. The macrophage cell line J774.1 and peritoneal exudate cells (PEC) from mice can be stimulated by LPS or other adjuvants $\text{in vitro}$ to synthesize and release protein factor(s) that activate thymus-derived lymphocytes. We have utilized J774.1 cells and PEC to demonstrate that an increase in ornithine decarboxylase (ODC) activity is a marker of early biochemical changes in adjuvant-stimulated macrophages. BCGcw and LPS increased ODC within 2 hours in J774.1 cells as well as murine peritoneal exudate macrophages. Maximal increases in ODC were detected 4 hours after the addition of adjuvants to J774.1 cells. The marked increases (12–23 fold) in ODC observed with BCGcw (20 μg/ml) did not appear to involve an effect on cell proliferation which was suppressed by this adjuvant. Cycloheximide inhibited the induction of ODC by LPS and BCGcw in the macrophage cell line. Evidence that the induction of ODC may be promoted by an increase in cyclic AMP was provided by experiments demonstrating that prostaglandin E₁ (PGE₁) and 8-bromo-adenosine-3′:5′-monophosphate (8Br-cyclic AMP) can mimic the effects of LPS and BCGcw in J774.1 cells. These observations indicate that one of the early biochemical changes in macrophages promoted by adjuvants is an induction of ODC.