Chronic inflammation and lung fibrosis: pleotropic syndromes but limited distinct phenotypes.

Experimental models of lung fibrosis have been disappointing in predicting therapeutic responses to a wide variety of interventions in clinical fibrosing lung diseases. There are multiple potential reasons, but this fundamentally calls into question the validity of the models and their fidelity to clinical syndromes. We propose that the clinical diseases associated with pulmonary fibrosis, although manifesting a broad array of widely different clinical presentations and features, result in essentially two distinct phenotypes of fibrosis that we will describe. The most common and problematic of these are not effectively modeled experimentally. In this review, we present several clinical entities as examples of the phenotypic distinctions. The first two represent the extremes: postinflammatory fibrosis observed in hypersensitivity pneumonitis (HP) and dysregulated matrix deposition as observed in idiopathic pulmonary fibrosis (IPF). We also present a third clinical entity, that of lung disease associated with rheumatoid arthritis (rheumatoid lung), representing a condition that can manifest as either phenotype, and offering a potential opportunity to explore the mechanisms underlying the pathogenesis of the two distinct fibrotic phenotypes.

Brain-derived neurotrophic factor mediates the anti-apoptotic effect of NMDA in cerebellar granule neurons: signal transduction cascades and site of ethanol action.

Cerebellar granule neurons cultured in medium containing a physiological concentration of KCl (5 mM) undergo apoptosis. The cells can be rescued by the in vitro addition of NMDA. The protective effect of NMDA is thought to reflect the in vivo innervation of developing cerebellar granule neurons by glutamatergic afferents. In the current work, we investigated the mechanism of the anti-apoptotic (protective) effect of NMDA. NMDA treatment reduced caspase-3-like activity in cerebellar granule neurons, and the time course and concentration dependence of the protective effect of NMDA mirrored the ability of NMDA to induce brain-derived neurotrophic factor (BDNF) expression. Furthermore, a Trk receptor antagonist, K252a, as well as a blocking antibody to BDNF, attenuated the protective effects of both NMDA and BDNF. These results suggest that NMDA-induced BDNF expression mediates the anti-apoptotic effect of NMDA. The protective effects of NMDA and BDNF were reduced by inhibitors of the phosphatidylinositol 3'-OH kinase (PI 3-kinase) signal transduction cascade (wortmannin and LY29004) but not by a MAP kinase kinase (MEK) inhibitor (PD98059) or a protein kinase A inhibitor (Rp-cAMPS). BDNF increased phosphorylation of Akt, a target of PI 3-kinase, and NMDA also induced Akt phosphorylation, but only after an exposure that was long enough to induce BDNF expression. Furthermore, ethanol, which interferes with NMDA receptor function, inhibited the NMDA-induced increase in BDNF levels but did not block the protective effect of BDNF. These findings further support the role of BDNF in the anti-apoptotic effect of NMDA in cerebellar granule neurons and suggest that the NMDA-BDNF interaction may play a key role in in vivo cerebellar granule neuron development, as well as in the deleterious effects of ethanol on the developing cerebellum.

The 90-kDa ribosomal S6 kinase (pp90rsk) phosphorylates the N-terminal regulatory domain of IkappaBalpha and stimulates its degradation in vitro.

Nuclear factor kappaB (NF-kappaB) is a eukaryotic member of the Rel family of transcription factors whose biological activity is post-translationally regulated by its assembly with various ankyrin-rich cytoplasmic inhibitors, including IkappaBalpha. Expression of NF-kappaB in the nucleus occurs after signal-induced phosphorylation, ubiquitination, and proteasome-mediated degradation of IkappaBalpha. The induced proteolysis of IkappaBalpha unmasks the nuclear localization signal within NF-kappaB, allowing its rapid migration into the nucleus, where it activates the transcription of many target genes. At present, the identity of the IkappaBalpha kinase(s) that triggers the first step in IkappaBalpha degradation remains unknown. We have investigated the potential function of the 90-kDa ribosomal S6 kinase, or pp90(rsk), as a signal-inducible IkappaBalpha kinase. pp90(rsk) lies downstream of mitogen-activated protein (MAP) kinase in the well characterized Ras-Raf-MEK-MAP kinase pathway that is induced by various growth factors and phorbol ester. We now show that pp90(rsk), but not pp70(S6K) or MAP kinase, phosphorylates the regulatory N terminus of IkappaBalpha principally on serine 32 and triggers effective IkappaBalpha degradation in vitro. When co-expressed in vivo in COS cells, IkappaBalpha and pp90(rsk) readily assemble into a complex that is immunoprecipitated with antibodies specific for either partner. While phorbol 12-myristate 13-acetate produced rapid activation of pp90(rsk), in vivo, other potent NF-kappaB inducers, including tumor necrosis factor alpha and the Tax transactivator of human T-cell lymphotrophic virus, type I, failed to activate pp90(rsk). These data suggest that more than a single IkappaBalpha kinase exists within the cell and that these IkappaBalpha kinases are differentially activated by different NF-kappaB inducers.

Role of ornithine decarboxylase suppression and polyamine depletion in the antiproliferative activity of polyamine analogs.

Two transfected cell lines, one carrying a mammalian ornithine decarboxylase (ODC) that is suppressed by polyamines and one carrying a trypanosomal ODC that is not, were used to ask whether ODC suppression is necessary for the antiproliferative activities of two polyamine analogs, N1,N8-bis(ethyl)spermidine (BES) and N1,N14-bis(ethyl)homospermine (BE444). Both analogs accumulated within cells and suppressed S-adenosylmethionine decarboxylase, as well as polyamine-sensitive mouse ODC activity. Neither drug was able to suppress the activity of the polyamine-refractory trypanosome ODC. But, whereas BE444 was able to inhibit growth of both cell lines, BES could inhibit only growth of cells carrying the polyamine-sensitive ODC, under conditions that cause prolonged depletion of endogenous polyamines. We conclude from these studies that the antiproliferative activity of BES, a less potent drug, requires the suppression of ODC. The efficacy of BE444 is enhanced by its ability to suppress ODC. However, it can function without ODC suppression, whereas BES cannot.

Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation.

Mammalian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is rapidly degraded in cells, an attribute important to the regulation of its activity. Mutant and chimeric ODCs were created to determine the structural requirements for two modes of proteolysis. Constitutive degradation requires the carboxy terminus and is independent of intracellular polyamines. Truncation of five or more carboxy-terminal amino acids prevents this mode of degradation, as do several internal deletions within the 37 carboxy-most amino acids that spare the last five residues. Polyamine-dependent degradation of ODC requires a distinct region outside the carboxy terminus. The ODC of a parasite, Trypanosoma brucei, is structurally very similar to mouse ODC but lacks the carboxy-terminal domain; it is not a substrate for either pathway. The regulatory properties of enzymatically active chimeric proteins incorporating regions of the two ODCs support the conclusion that distinct domains of mouse ODC confer constitutive degradation and polyamine-mediated regulation. Mouse ODC contains two PEST regions. The first was not required for either form of degradation; major deletions within the second ablated constitutive degradation. When mouse and T. brucei ODC RNAs were translated in vitro in a reticulocyte lysate system, the effects of polyamine concentration on ODC protein production and activity were similar for the two mRNAs, which contradicts claims that this system accurately reflects the in vivo effects of polyamines on responsive ODCs.

Expression and post-transcriptional regulation of ornithine decarboxylase during early Xenopus development.

In this paper we show that large changes in ornithine decarboxylase (ODC) activity occurred during early Xenopus development. Following fertilization, this enzyme activity rises with a quantitatively correlated accumulation of putrescine and spermidine. This increase in ODC activity was associated with an increased translation of the maternal ODC mRNA, which was stable in the embryo and whose polyadenylation increased slightly between fertilization and the mid-blastula transition (MBT). ODC activity was stable in cycloheximide-treated embryos, indicating that before the MBT this enzyme was not degraded. After the MBT, ODC activity fell, but no decrease in this mRNA was observed. In gastrulae, ODC mRNA was both increased in amount and polyadenylated. The reduced ODC activity at this stage of development was not associated with a fall in ribosome loading of the mRNA. Treatment of post-MBT embryos with cycloheximide lead to an accentuation of the normally observed decrease in ODC activity. Expression of Xenopus ODC in mutant ODC-deficient Chinese hamster ovary cells (C 55.7 cells) showed that the Xenopus enzyme was rapidly degraded and can be regulated post-translationally by polyamines, indicating that the post-MBT fall in ODC activity could be caused by a change in protein turnover or by polyamine-mediated regulation.

Trypanosome ornithine decarboxylase is stable because it lacks sequences found in the carboxyl terminus of the mouse enzyme which target the latter for intracellular degradation.

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Mouse ODC is rapidly degraded in mouse cells, whereas ODC within Trypanosoma brucei, a protozoan parasite infesting cattle, is stable. We have expressed cloned ODC genes of both T. brucei and mouse in ODC-deficient Chinese hamster ovary (CHO) cells. The T. brucei enzyme is stable, whereas the mouse ODC similarly expressed in CHO cells is unstable. This shows that the observed difference in intracellular stability is a property of the ODC protein itself, rather than the cellular environment in which it is expressed. A chimeric ODC composed of the amino terminus of trypanosome and the carboxyl terminus of mouse ODC is rapidly degraded in CHO cells, suggesting that peptide sequences in the mouse ODC carboxyl terminus determine its stability.

Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation.

Ornithine decarboxylase (ODC) was converted from a protein with a short intracellular half-life in mammalian cells to a stable protein by truncating 37 residues at its carboxyl terminus. Cells expressing wild-type protein lost ODC activity with a half-life of approximately 1 hour. Cells expressing the truncated protein, however, retained full activity for at least 4 hours. Pulse-chase experiments in which immunoprecipitation and gel electrophoresis were used confirmed the stabilizing effect of the truncation. Thus, a carboxyl-terminal domain is responsible for the rapid intracellular degradation of murine ODC.

Substrate specificities of 5'-deoxy-5'-methylthioadenosine phosphorylase from Trypanosoma brucei brucei and mammalian cells.

The separation by chromatofocusing of two distinct purine nucleoside cleaving activities from crude extracts of Trypanosoma brucei brucei is described. One catalyzes the reversible phosphorolysis of 5'-deoxy-5'-methylthioadenosine (MeSAdo) and adenosine (Ado) and was designated an MeSAdo/Ado phosphorylase, while the other catalyzes the hydrolysis of adenosine, inosine, and guanosine but not MeSAdo. The substrate specificity of trypanosomal MeSAdo/Ado phosphorylase differed from that of a mammalian MeSAdo phosphorylase (derived from murine Sarcoma 180 cells) in that it was able to phosphorolyze 2'-deoxyadenosine, 3'-deoxyadenosine and 2',3'-dideoxyadenosine. In addition, the trypanosomal phosphorylase was able to utilize the nucleoside analog, 6-methylpurine 2'-deoxyribonucleoside, as an alternative substrate, whereas the mammalian enzyme could not. Because of these differences, cytotoxic analogs of MeSAdo may be designed that are selectively activated by the trypanosomal MeSAdo/Ado phosphorylase.

Characterization of a defect in the pathway for converting 5'-deoxy-5'-methylthioadenosine to methionine in a subline of a cultured heterogeneous human colon carcinoma.

5'-Deoxy-5'-methylthioadenosine (methylthioadenosine) is cleaved to adenine and 5-methylthioribose-1-phosphate (methylthioribose-1-P). Methylthioribose-1-P is converted to 2-keto-4-methylthiobutyrate ( ketomethylthiobutyrate ) which is transaminated to methionine. We report that one subline of a heterogeneous human colon carcinoma, DLD-1 Clone D, only forms methylthioribose-1-P from methylthioadenosine or 5'-deoxy-5'-methylthioinosine (methylthioinosine), a deaminated derivative of methylthioadenosine, whereas Clone A converts methylthioadenosine and methylthioinosine to methionine, as shown by growth studies in culture of Clone A and Clone D cells and radioactive studies utilizing [methyl-14C]methylthioadenosine or [methyl-14C]methylthioinosine in the presence of extracts of these cells lines. To characterize this defect, we utilized three protein fractions isolated from rat liver which together convert methylthioribose-1-P to ketomethylthiobutyrate . Addition of only Fraction A to Clone D sonicates restores its ability to convert methylthioadenosine to methionine. This fraction is responsible for converting methylthioribose-1-P to 5- methylthioribulose -1-phosphate; radioactive studies confirm this observation. Thus, Clone D is deficient in an enzyme contained in Fraction A; this represents a qualitative biochemical difference between the two clones derived from a single human tumor.

Conversion of 5'-deoxy-5'-methylthioadenosine and 5'-deoxy-5'-methylthioinosine to methionine in cultured human leukemic cells.

5'-Deoxy-5'-methylthioadenosine and 5'-deoxy-5'-methylthioinosine, which are metabolized to the methionine precursor, 5-methylthioribose-1-phosphate, by 5'-deoxy-5'-methylthioadenosine phosphorylase and purine nucleoside phosphorylase, respectively, can serve as sources of methionine for cultured HL-60 promyelocytic leukemia cells. CCRF-CEM T-cell leukemia cells, which lack 5'-deoxy-5'-methylthioadenosine phosphorylase, convert 5'-deoxy-5'-methylthioinosine (but not 5'-deoxy-5'-methylthioadenosine) to methionine; this conversion is blocked by purine nucleoside phosphorylase inhibitors. Therefore, the pathway for the conversion of 5-methylthioribose-1-phosphate to methionine is present in both human leukemic lines.

Biochemical pharmacology and toxicology of formycin alone and in combination with 2'-deoxycoformycin (pentostatin).

The toxicology and pharmacology of formycin both as a single agent and combined with the adenosine deaminase inhibitor 2'-deoxycoformycin (dCF) were examined in outbred Swiss mice heterozygous for the nude gene (nu/+). The LD10 for formycin alone given on a daily x 5 schedule was 21 mg/kg. When the animals were pretreated with 1 mg/kg of dCF 1 hour prior to each dose of formycin, toxicity was approximately doubled, ie, LD10 was reduced to 10 mg/kg. Death was associated with hepatic toxicity in both treatment regimens; suppression of leukocyte counts was mild except at doses greater than the LD10. Formycin nucleotides were detected by high-performance liquid chromatography in the livers of mice treated with formycin either alone or combined with dCF. When isolated rat hepatocytes were incubated for 2 hours with either formycin or dCF plus formycin, analog nucleotides accumulated in the cells. Cellular ATP decreased to below the limits of detection, whereas a large peak corresponding to formycin-5'-triphosphate was present. This replacement of cellular ATP by formycin-5'-triphosphate may help explain the hepatic toxicity observed.

Biochemical pharmacology and toxicology of 8-azaadenosine alone and in combination with 2'-deoxycoformycin (pentostatin).

The toxicology and metabolism of 8-azaadenosine (8-azaAdo) were examined both as a single agent and in combination with the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF). The LD10 (mice) for 8-azaAdo alone on a once daily for 5 days (q.d. x 5) schedule was 30 mg . kg-1 . day-1. When the animals were pretreated with 0.1 mg . kg-1 . day-1 of dCF, the LD10 dose was reduced to 10 mg . kg-1 . day-1 x 5. The major organ toxicity seen was hepatic. Bone marrow cellularity was only slightly altered at the LD10 dose. 8-AzaAdo nucleotides were detected in the livers of treated mice as determined by high performance liquid chromatography. Further, after 2 hr of incubation, isolated rat hepatocytes accumulated 8-azaATP to levels of 2.2 mumoles/g of cells with 8-azaAdo (1 mM) alone and to 4.3 mumoles/g of cells when 8-azaAdo was used in combination with dCF (1 microgram/ml). ATP levels decreased to below the limits of detection after 2 hr in cells treated with the combination. The replacement of cellular ATP by 8-azaATP may provide an explanation for the hepatotoxicity observed in the murine toxicology studies.