Ornithine Decarboxylase Protein Expression Research Articles

Regulatory role of l-proline in fetal pig growth and intestinal epithelial cell proliferation.

l-proline (Pro) is a precursor of ornithine, which is converted into polyamines via ornithine decarboxylase (ODC). Polyamines plays a key role in the proliferation of intestinal epithelial cells. The study investigated the effect of Pro on polyamine metabolism and cell proliferation on porcine enterocytes in vivo and in vitro. Twenty-four Huanjiang mini-pigs were randomly assigned into 1 of 3 groups and fed a basal diet that contained 0.77% alanine (Ala, iso-nitrogenous control), 1% Pro or 1% Pro + 0.0167% α-difluoromethylornithine (DFMO) from d 15 to 70 of gestation. The fetal body weight and number of fetuses per litter were determined, and the small and large intestines were obtained on d 70 ± 1.78 of gestation. The in vitro study was performed in intestinal porcine epithelial (IPEC-J2) cells cultured in Dulbecco's modified Eagle medium-high glucose (DMEM-H) containing 0 μmol/L Pro, 400 μmol/L Pro, or 400 μmol/L Pro + 10 mmol/L DFMO for 4 d. The results showed that maternal dietary supplementation with 1% Pro increased fetal weight; the protein and DNA concentrations of the fetal small intestine; and mRNA levels for potassium voltage-gated channel, shaker-related subfamily, member 1 (Kv1.1) in the fetal small and large intestines (P < 0.05). Supplementing Pro to either gilts or IPEC-J2 cells increased ODC protein abundances and polyamine concentrations in the fetal intestines and IPEC-J2 cells (P < 0.05). In comparison with the Pro group, the combined administration of Pro and DFMO reduced the expression of ODC protein and spermine concentration in the fetal intestine, as well as the concentrations of putrescine, spermidine and spermine in IPEC-J2 cells (P < 0.05). Meanwhile, the percentage of cells in the S-phase and the mRNA levels of proto-oncogenes c-fos and c-myc were increased in response to Pro supplementation, whereas depletion of cellular polyamines with DFMO increased tumor protein p53 (p53) mRNA levels (P < 0.05). Taken together, dietary supplementation with Pro improved fetal pig growth and intestinal epithelial cell proliferation via enhancing polyamine synthesis.

Aβ stimulates microglial activation through antizyme-dependent downregulation of ornithine decarboxylase.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative disorders. Its pathology is associated with the deposition of amyloid β (Aβ), an abnormal extracellular peptide. Moreover, its pathological progression is closely accompanied by neuroinflammation. Specifically, Aβ-associated microglial overactivation may have the central role in AD pathogenesis. Interestingly, arginine metabolism may contribute to the equilibrium between M1 and M2 microglia. However, little is known about the involvement of arginine metabolism in Aβ-induced microglial neuroinflammation and neurotoxicity. Moreover, the underlying mechanism by which Aβ induces the transition of microglia to the M1 phenotype remains unclear. In this study, we investigated the role of Aβ in mediating microglial activation and polarization both in vitro and in vivo. Our results demonstrated that under the Aβ treatment, ornithine decarboxylase (ODC), a rate-limiting enzyme in the regulation of arginine catabolism, regulates microglial activation by altering the antizyme (AZ) + 1 ribosomal frameshift. Furthermore, the restoration of ODC protein expression levels has profound effects on inhibition of Aβ-induced M1 markers and thus attenuates microglial-mediated cytotoxicity. Altogether, our findings suggested that Aβ may contribute to M1-like activation by disrupting the balance between ODC and AZ in microglia.

Mitogen-Activated Protein Kinase and Intracellular Polyamine Signaling Is Involved in TRPV1 Activation-Induced Cardiac Hypertrophy.

BackgroundThe transient receptor potential vanilloid type 1 (TRPV1) is expressed in the cardiovascular system, and increased TRPV1 expression has been associated with cardiac hypertrophy. Nevertheless, the role of TRPV1 in the pathogenesis of cardiac hypertrophy and the underlying molecular mechanisms remain unclear.Methods and ResultsIn cultured cardiomyocytes, activation of TRPV1 increased cell size and elevated expression of atrial natriuretic peptide mRNA and intracellular calcium level, which was reversed by TRPV1 antagonist capsazepine. Increased expression of phosphorylated calmodulin‐dependent protein kinase IIδ and mitogen‐activated protein kinases were found in TRPV1 agonist capsaicin‐treated cardiomyocytes. Selective inhibitor of calmodulin‐dependent protein kinase IIδ decreased phosphorylation of extracellular signal–regulated kinases and p38. Capsaicin induced an increase in expression of ornithine decarboxylase protein, which is the key enzyme in polyamine biosynthesis in cardiomyocytes. Nevertheless, there was no obvious change of ornithine decarboxylase expression in TRPV1 knockdown cells after capsaicin treatment, and specific inhibitors of calmodulin‐dependent protein kinase IIδ or p38 downregulated the capsaicin‐induced expression of ornithine decarboxylase. Capsazepine alleviated the increase in cross‐sectional area of cardiomyocytes and the ratio of heart weight to body weight and improved cardiac function, including left ventricular internal end‐diastolic and ‐systolic dimensions and ejection fraction and fractional shortening percentages, in mice treated with transverse aorta constriction. Capsazepine also reduced expression of ornithine decarboxylase and cardiac polyamine levels. Transverse aorta constriction induced increases in phosphorylated calmodulin‐dependent protein kinase IIδ and extracellular signal–regulated kinases, and p38 and Serca2a were attenuated by capsazepine treatment.ConclusionsThis study revealed that the mitogen‐activated protein kinase signaling pathway and intracellular polyamines are essential for TRPV1 activation–induced cardiac hypertrophy.

Abstract 681: Heat shock-mediated suppression of ODC protein expression in colorectal cancer cells

Abstract Polyamines are small, positively charged metabolites that have important roles in several cancer hallmarks, including cell proliferation, invasion, and angiogenesis. Difluoromethylornithine (DFMO) is an FDA-approved drug that is in clinical trials for treatment of colorectal cancer. DFMO functions by inhibiting ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in polyamine biosynthesis. By reducing polyamine levels, DFMO has shown much promise as an anti-cancer chemotherapeutic. One potential limitation to DFMO is that it causes a profound increase in ODC expression, which we suspect may counteract the intended use of the drug. However, strategies to suppress ODC expression would theoretically increase DFMO efficacy. We have discovered that transient hyperthermia (heat shock, 42°C) causes a profound and rapid suppression in ODC protein levels in RKO colorectal cancer cells. To investigate the responsible mechanism, we began by examining ODC mRNA expression and stability. Hyperthermia caused only a moderate reduction in ODC transcript levels. Also, mRNA stability, which was assessed using actinomycin D, was not reduced by heat shock. We next examined the effect of hyperthermia on the control of ODC protein turnover, which is known to be mediated by ODC antizyme (OAZ1). We performed real-time PCR to assess the effect of heat shock on OAZ1 expression. Data reveal a significant increase in the level of OAZ1 transcript. Since heat shock is known to activate the transcription factor HSF1, we next investigated the role of HSF1 in OAZ1 induction. Using siRNA to silence HSF1, we found that the induction of OAZ1 by heat shock is fully dependent on HSF1 expression. In addition to heat shock, HSF1 is also activated by various chemotherapeutics, but most notably by Hsp90 inhibitors. We therefore tested if Hsp90 inhibitors, geldanamycin and 17-AAG could be used to induce OAZ1 and promote ODC turnover. These results show that both Hsp90 inhibitors caused a significant activation of HSF1, an induction in OAZ1 expression, and decrease in ODC protein levels. We therefore propose that Hsp90 inhibitors may be effective in attenuating ODC expression and polyamine metabolism. We suggest that they may be used in combination with DFMO to prevent the compensatory increase in ODC levels and increase anti-cancer effects. Citation Format: Christina T.K. Wales, Aaron T. Jacobs. Heat shock-mediated suppression of ODC protein expression in colorectal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 681. doi:10.1158/1538-7445.AM2014-681

Akt and Erk1/2 activate the ornithine decarboxylase/polyamine system in cardioprotective ischemic preconditioning in rats: the role of mitochondrial permeability transition pores

Ornithine decarboxylase (ODC) is the first rate-limiting enzyme in polyamine biosynthesis, which is essential for cell survival. We hypothesized that the ODC/polyamine system is involved in ischemic preconditioning (IPC)-mediated cardioprotection through the activation of Erk1/2 and Akt and through the inhibition of the mitochondrial permeability transition (mPT). Isolated rat hearts were subjected to 40 min of ischemia either with or without IPC (3 cycles of 5-min global ischemia), and ODC protein expression, polyamine content, and Akt and Erk1/2 phosphorylation were evaluated after 30 min of reperfusion. IPC significantly upregulated the ODC/polyamine pathway, promoted Erk1/2 and Akt phosphorylation, and reduced the infarct size and heart dysfunction after reperfusion. An inhibitor of ODC, α-difluoromethylornithine (DFMO), abolished the IPC-induced cardioprotection. Moreover, the inhibition of the IPC-induced activation of Erk1/2 and Akt using PD98059 or wortmannin downregulated the ODC/polyamine system. In separate studies, the Ca(2+) load required to open the mPT pore was significantly lower in DFMO-treated cardiac mitochondria than in mitochondria from IPC hearts. Furthermore, spermine or spermidine significantly inhibited the mPT induced by CaCl2. These results suggest that IPC upregulates the ODC/polyamine system and mediates preconditioning cardioprotection, which may depend on the phosphorylation/activation of Erk1/2 and Akt and on the inhibition of the mPT during reperfusion.

Hypoxia-Mediated Induction of the Polyamine System Provides Opportunities for Tumor Growth Inhibition by Combined Targeting of Vascular Endothelial Growth Factor and Ornithine Decarboxylase

Hypoxia is a hallmark of solid tumors, which may offer opportunities for targeted therapies of cancer; however, the mechanisms that link hypoxia to malignant transformation and tumor progression are not fully understood. Here, we show that up-regulation of the polyamine system promotes cancer cell survival during hypoxic stress. Hypoxia was found to induce polyamine transport and the key enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), in a variety of cancer cell lines. Increased ODC protein expression was shown in hypoxic, GLUT-1-expressing regions of tumor spheroids and experimental tumors, as well as in clinical tumor specimens. Hypoxic induction of the polyamine system was dependent on antizyme inhibitor (i.e., a key positive regulator of ODC and polyamine transport), as shown by RNA interference experiments. Interestingly, depletion of the polyamines during hypoxia resulted in increased apoptosis, which indicates an essential role of the polyamines in cancer cell adaptation to hypoxic stress. These results were supported by experiments in an in vivo glioma tumor model, showing significantly enhanced antitumor effects of the antiangiogenic, humanized anti-vascular endothelial growth factor (VEGF) antibody bevacizumab when used in combination with the well-established, irreversible inhibitor of ODC, alpha-difluoromethylornithine. Our results provide important insights into the hypoxic stress response in malignant cells and implicate combined targeting of VEGF and ODC as an alternative strategy to treat cancer disease.

Inhibition of Inducible Nitric Oxide Synthase Alters Thy-1 Glomeruonephritis in Rats

Background/Aims: Inducible nitric oxide (NO) synthase (iNOS) generated NO increases in the early phase of Thy-1 glomerulonephritis concurrently with mesangiolysis and reduction in glomerular filtration rate (GFR). Activation of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, is upregulated to allow mesangial cell proliferation which constitutes the repair phase in this model. Antiproliferative high-output NO generation inhibits proproliferative ODC activity, thereby temporally separating the early ‘bactericidal’ phase from the later ‘growth’ repair phase. Methods: Renal function, ODC protein expression, arginine, ornithine, and polyamines by high-performance liquid chromatography, and histological changes were assessed in rats after induction of Thy-1 nephritis with and without NOS inhibition. Results: Thy-1 significantly reduced the GFR relative to untreated controls. Treatment with a nonspecific NOS inhibitor, but not a selective iNOS inhibitor, further decreased the GFR at day 1. This implys a protective role for constitutive NOS in the early phase of this inflammatory model. Selective iNOS inhibition abrogated increased plasma NO₂/NO₃ levels in Thy-1 glomerulonephritis, but did not significantly reduce mesangiolysis. However, inhibition of iNOS did result in significantly more nuclei/glomerulus during the proliferative phase, increasing the hypercellularity component of this disease model. This correlates with increased levels of polyamines, ornithine, and arginine beyond those observed with Thy-1 administration alone. Conclusions: These studies provide evidence that NO generation from different NOS isoforms can be protective in the temporal course of Thy-1 glomerulonephritis. The finding that iNOS attenuates hypercellularity in the repair phase of this inflammatory model adds cautionary insight in the therapeutic use of selective iNOS inhibition in vivo.

Spermine Causes Loss of Innate Immune Response to Helicobacter pylori by Inhibition of Inducible Nitric-oxide Synthase Translation

Helicobacter pylori infection of the stomach elicits a vigorous but ineffective host immune and inflammatory response, resulting in persistence of the bacterium for the life of the host. We have reported that in macrophages, H. pylori up-regulates inducible NO synthase (iNOS) and antimicrobial NO production, but in parallel there is induction of arginase II, generating ornithine, and of ornithine decarboxylase (ODC), generating polyamines. Spermine, in particular, has been shown to restrain immune response in activated macrophages by inhibiting proinflammatory gene expression. We hypothesized that spermine could prevent the antimicrobial effects of NO by inhibiting iNOS in macrophages activated by H. pylori. Spermine did not affect the up-regulation of iNOS mRNA levels but in a concentration-dependent manner significantly attenuated iNOS protein levels and NO production. Reduction in iNOS protein was due to inhibition of iNOS translation and not due to iNOS degradation. ODC knockdown with small interfering (si) RNA resulted in increased H. pylori-stimulated iNOS protein expression and NO production without altering iNOS mRNA levels. When macrophages were cocultured with H. pylori, killing of bacteria was enhanced by transfection of ODC siRNA and prevented by addition of spermine. These results identify a mechanism of immune dysregulation induced by H. pylori in which stimulated spermine synthesis by the arginase-ODC pathway inhibits iNOS translation and NO production, leading to persistence of the bacterium and risk for peptic ulcer disease and gastric cancer.

Increased expression of ornithine decarboxylase in distal tubules of early diabetic rat kidneys: are polyamines paracrine hypertrophic factors?

Polyamines are small biogenic molecules that are essential for cell cycle entry and progression and proliferation. They can also contribute to hypertrophy. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, increases in the early diabetic kidney to enable renal hypertrophy. Inhibition of ODC in early diabetes attenuates diabetic renal hypertrophy and glomerular hyperfiltration. The current studies examine the temporal profile of renal ODC protein expression and localization, intrarenal polyamine levels, and sites of proliferation in kidneys of rats during the first 7 days of streptozotocin diabetes. ODC mRNA and protein content were increased in diabetic kidneys. High-performance liquid chromatography analysis showed increased intrarenal polyamine concentrations peaking after 24 h of diabetes. A subsequent increase in the number of proliferating proximal tubular cells was detected by in vivo 5-bromodeoxyuridine (BrdU) incorporation on day 3. Surprisingly, immunohistochemical studies revealed that increased ODC protein was apparent only in distal nephrons, whereas the main site of diabetic kidney hypertrophy is the proximal tubule. These findings raise the possibility that polyamines produced in the distal nephron may mediate the early diabetic kidney growth of the proximal tubules via a paracrine mechanism.

Flavanones structure-related inhibition on TPA-induced tumor promotion through suppression of extracellular signal-regulated protein kinases: involvement of prostaglandin E2 in anti-promotive process.

Biological functions of flavanones have been studied extensively, however, the structure-related activities of flavanones on 12-o-tetradecanoylphorbol 13-acetate (TPA)-induced promotive effects are still unclear. In this study, flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone showed the most significant dose-dependent inhibition on TPA-induced proliferative effects among eight tested flavanones in NIH3T3 cells. TPA-induced mitogen activated protein kinases (MAPK) phosphorylation, ornithine decarboxylase (ODC), c-Jun, and cyclooxygenase 2 (COX-2) protein expressions in a time-dependent manner, and the maximal inductive time point is at 1 h for MAPK phosphorylation and 6 h for others. Flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone showed the dose-dependent inhibition on TPA-stimulated MAPK phosphorylation, COX-2, ODC, c-Jun protein expressions. Induction of, prostaglandin E(2) (PGE(2)) production was detected in TPA-treated NIH3T3 cells, and flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone inhibited significantly PGE(2) production induced by TPA. Addition of PGE(2) reverses the inhibitory activities of flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone on TPA-induced proliferation. And, PD98059, a specific inhibitor of ERKs, inhibited TPA-induced MAPK phosphorylation, accompanied by decreasing COX-2, c-Jun, and ODC protein expression, and showed dose-dependent inhibition on TPA-induced proliferation in cells. These results demonstrated that PGE(2) is an important mediator in TPA-induced proliferation, and MAPK phosphorylation was located at the upstream of COX-2, c-Jun, and ODC gene expressions in TPA-induced responses. Furthermore, flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone (100 microM) suppressed TPA-induced colony formation associated with blocking MAPK phosphorylation, ODC, c-Jun, and COX-2 proteins expression. And, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay showed that flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone did not perform potent anti-radical activities among these eight tested compounds. In conclusion, this study provided molecular evidences to demonstrate that flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone were potent inhibitors on TPA-induced responses without notable cytotoxicity through suppression of PGE(2) production; and anti-radical activity of flavanones was not correlated with preventing the occurrence of tumor promotion. We proposed that blocking TPA-induced intracellular signaling responses might be involved in the anti-promotive mechanism of flavanones.

Decreased ornithine decarboxylase activity in the kidneys of Dahl salt-sensitive rats.

To assess the roles of polyamines (putrescine, spermidine, and spermine) and ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, in the development of salt-sensitive hypertension, we evaluated activity and expression of ODC, urinary polyamine excretion, and antizyme (endogenous ODC inhibitor protein) expression in Dahl salt-sensitive (SS) and salt-resistant (SR) rats after they were fed on a low (0.3%) or high (4%) salt diet for 4 weeks. We also examined the effects of spermidine and difluoromethylornithine (DFMO: a specific inhibitor of ODC) on the systolic blood pressure and ODC protein expression in SS rats fed a high salt diet. Renal ODC activity and urinary polyamine excretion in SS rats were lower than those in SR rats after 4 weeks treatment with a low or high salt diet. The renal ODC protein expression of SS rats was paradoxically increased as compared to the SR group. A high salt diet did not alter ODC activity but increased ODC protein only in SS rats. ODC mRNA and antizyme protein expressions were not significantly different among the four groups. Spermidine supplementation attenuated and DFMO exaggerated hypertension in SS rats fed a high salt diet. Spermidine down-regulated and DFMO up-regulated renal ODC protein in SS rats on a high salt diet. ODC activity was decreased but protein was paradoxically increased in kidneys of SS rats. ODC protein was suggested to increase in compensation for the inhibition of its activity. Impaired ODC activity and polyamine production in the kidney may exaggerate salt-sensitive hypertension in SS rats.

A mechanistic study of proliferation induced by Angelica sinensis in a normal gastric epithelial cell line

It has been reported that an extract from Angelica sinensis mainly consisting of polysaccharides (95%) prevented ethanol- or indomethacin-induced gastric mucosal damage (Cho CH et al. Planta Med 2000;66:348–51). However, it is not known whether Angelica sinensis has a direct stimulatory effect on the healing of gastric mucosal lesions. To study the hypothesis that Angelica sinensis has a direct mucosal healing effect in rats and in isolated gastric epithelial cells, we assessed the wound repair in both animals and normal cell culture (RGM-1), as well as [ 3H]thymidine incorporation, ornithine decarboxylase (ODC) activity, and ODC protein and c-Myc protein expression after different treatments in RGM-1 cells. We found that Angelica sinensis crude extract (ASCE) dose-dependently enhanced gastric ulcer healing in rats and promoted wound repair in RGM-1 cells. It also significantly stimulated [ 3H]thymidine incorporation and ODC activity in RGM-1 cells in a concentration-dependent manner. ODC and c-Myc protein expression was also increased as a result of this process. dl-α-difluoromethyl-ornithine repressed the [ 3H]thymidine incorporation and ODC activity induced by ASCE. Pretreatment with c-Myc antisense oligodeoxynucleotides blocked the stimulatory action of ASCE on [ 3H]thymidine incorporation and ODC protein expression. These data suggest that ASCE has a direct mucosal healing effect on gastric epithelial cells, while ODC and c-Myc are closely associated with this effect.

Increased expression of ornithine decarboxylase by gamma-ray in mouse epidermal cells: relationship with protein kinase C signaling pathway.

The correlation between ornithine decarboxylase (ODC) protein induction and specific protein kinase C (PKC) isozyme expression by gamma-ray in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated normal and v-rasHa transformed mouse keratinocytes was examined. TPA at 100 nM was treated in primary mouse keratinocytes immediately after 4 Gy, 8 Gy and 16 Gy gamma-ray irradiation. After 4 hrs, cells were harvested and the protein expression levels of PKC isozymes (PKC alpha, -delta, -epsilon, -eta and -zeta) and ODC were examined. For v-rasHa infection, primary keratinocytes were infected with a defected retrovirus containing the v-rasHa gene. After 3 hrs of irradiation, each PKC isozyme and ODC protein expression were tested. Gamma-ray increases ODC protein expression in both TPA-treated normal and v-rasHa transformed mouse keratinocytes and this phenomenon correlated to the increased induction of PKC alpha without altering other PKC isozymes. Tyrosine phosphorylation of epidermal growth factor receptor protein was also stimulated during gamma-ray induced cellular changes in TPA-treated normal mouse keratinocytes. These results indicate that PKC alpha as an important regulator of mouse epidermal changes by gamma-radiation, contributes to the ODC expression occurring during exposure to tumor promoter, such as TPA, and epidermal neoplasia induced by ras activation.

Localization of ornithine decarboxylase (ODC) in the cochlea of the immature rat

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is important in cochlear development. Whereas tissue specific differences in cochlear ODC activity have been demonstrated, cellular localization of ODC protein in the inner ear of the immature rat has not. ODC was localized in inner ear structures using an ODC polyclonal antibody and the effects of cycloheximide on ODC immunoreactivity and enzymatic activity were determined. Tissues demonstrating elevated enzymatic activity contained cells with the strong immunoreactivity. ODC activity was highest in the organ of Corti and lateral wall followed by the cochlear nerve. Immunoreactivity was demonstrated throughout the cochlea with intense staining of the hair cells, pillar cells, Deiter's cells, inner sulcus cells, basilar membrane, stria vascularis, spiral ganglion cell bodies and cochlear nerve fibers. Cycloheximide rapidly diminished cochlear ODC activity and expression of ODC protein. The half-life of cochlear ODC was 30 min. Localization of cellular sites of ODC is important in understanding the role of the ODC-polyamine pathway in cochlear development and will be a valuable marker for tissue damage from ototoxic agents.