ROLES OF CELLULAR POLYAMINES IN MUCOSAL HEALING IN THE GASTROINTESTINAL TRACT

Natural polyamines (putrescine, spermidine and spermine) are low molecular weight and highly charged aliphatic polycations which intimately involved in many distinct cellular functions. For example, polyamines interact with polyanionic DNA molecules, thereby modulating chromatin structure, gene transcription, translation, and DNA stabilization (7). Several studies showed that polyamines are also required for signal transduction, receptor-ligand interactions, cell proliferation, migration, RNA stabilization and function of ion channels (8). An increasing body of evidence has advanced our understanding of the cellular and molecular functions of polyamines, therefore, this review will briefly highlight the roles and mechanisms of cellular polyamines in GI mucosal healing and also point out their potential clinical applications in patients with mucosal injury-associated disorders.

POLYAMINE METABOLISM IN THE GASTROINTESTINAL TRACT

Polyamine levels and subcellular distribution in the gastrointestinal tract

Polyamines are mainly located in the nucleus and cytoplasm (9). The concentration of polyamines is higher in rapidly proliferating tissues and increases rapidly when growth or proliferation is induced (10). In the GI tract, there is a polyamine gradient in the villi-crypt cell axis and along the digestive tube. The greatest luminal polyamines content is observed in the jejunum and colon (11).

Although the intracellular polyamines are in the millimolar range, free polyamines are considerably less abundant (only 7–10% of the total cellular polyamines pool), as polyamines are mostly ionically bound to different polyanionic in the cells (mainly DNA, RNA, proteins and phospholipids) (8). Only the free intracellular polyamines are available for immediate cellular needs. The major sources of exogenous polyamines come from diet and luminal bacteria in GI tract. A rat consumes daily about 160 µmoles/kg body weight (b.w.) polyamines of dietary origin, 30–40 µmoles/kg b.w. come from gut bacteria and 140 µmoles/kg b.w. from endogenous biosynthesis (12). The average daily polyamines intake of adolescents was 316±170 mmol/day (13). The concentration of intracellular polyamines is actively maintained and regulated by dynamic balance among exogenous transport, endogenous biosynthesis, interconversion, and degradation (14).

Transport of polyamines in the gastrointestinal tract

Transiently after meal, the concentration of polyamines in the duodenal and jejunal lumen reaches its maximum level. However, as early as 2 hours after the meal, the concentration of polyamines in the luminal side gradually reduces and returns to the fasting level, which indicates that the polyamines are rapidly absorbed by epithelial cells (15). Five models are currently applied to explain mammalian polyamines transport from lumen of the intestine. Firstly, transepithelial transport of polyamines occurs by passive diffusion (16). Secondly, mammalian polyamine transport proceeds in two tightly connected steps. Polyamines are first transported into the cell by a membrane transporter/carrier which is powered by electronegative membrane potential, then polyamines immediately accumulate into polyamines-sequestering vesicles which involve proton exchange and are driven by a vacuolar-ATPase pH gradient (17). Thirdly, polyamine uptake is a process involving the cell surface-associated heparan sulfate proteoglycans (HSPGs) and glypican 1 (Gpc1) (18, 19). Fourthly, absorption of polyamines is mediated by caveolin-1 dependent endocytosis and NOS2-dependent mechanism (20, 21). In addition, putrescine and acetylated polyamines are exported by an amino acid transporter SLC3A2 via a diamine/arginine exchange activity (21, 22), and other membrane transporters, such as SLC22A1, SLC22A2, SLC22A3, SLC47A1, SLC7A1, SLC12A8A, and SLC22A16, might also be involved in polyamine transport (23).

Biosynthesis and degradation of polyamines in the gastrointestinal tract

Polyamines are synthesized from methionine and ornithine. The first rate-limiting step in polyamine biosynthesis is the production of putrescine from ornithine. Ornithine is decarboxylated to putrescine by ornithine decarboxylase (ODC), which is a key enzyme of polyamine biosynthesis (Fig. 1). Putrescine is subsequently converted into spermidine through the catalysis of S-adenosyl-methionine decarboxylase (AdoMetDC) and spermidine synthase (SPDS). Spermidine is then converted into spermine by AdoMetDC and spermine synthase (SPMS) (Fig. 1). Methionine is the precursor for S-adenosyl-methionine (AdoMet) (8, 24). The decarboxylation product of AdoMet (DCAdoMet) is the precursor of the aminopropyl moieties of spermidine and spermine. Spermine, spermidine and putrescine are interconverted, depending on the physiological needs (14). Polyamines are degraded by spermine oxidases (SPMO), spermidine oxidases (SPDO) and spermine/spermidine-N-acetyltransferase (SSAT) (Fig. 1) (3, 25). In regard to regulating ODC, the ODC-antizyme binds ODC and facilitates proteasomal ODC degradation, thus inhibiting the endogenous biosynthesis of polyamines from ornithine (14, 26). The activity of ODC could also be specifically inhibited by the chemical compound DL-α-difluoromethylornithine (DFMO).

Fig. 1. Polyamine metabolism pathway. Abbreviations: AdoMet, S-adenosyl-methionine; AdoMetDC, S-adenosyl-methionine decarboxylase; DCAdoMet, decarboxylation product of AdoMet; DFMO, DL-α-difluoromethylornithine; ODC, ornithine decarboxylase; SPDO, spermidine oxidases; SPDS, spermidine synthase; SPMO, spermine oxidases; SPMS, spermine synthase; SSAT, spermine/spermidine-N-acetyltransferase.

THE ROLE OF POLYAMINES IN MUCOSAL HEALING AFTER INJURY

Restitution

Minor injuries in GI epithelia are routinely caused by mechanical strain. After injury, rapid repair of the epithelial barrier is accomplished by a complex process termed epithelial restitution. Epithelial restitution refers to resealing of superficial wounds occurring as a consequence of epithelial cell migration into the defect rather than cell proliferation (4, 27). The process of epithelial restitution is that polarized epithelial cells adjacent to the defect, flatten, migrate, and ultimately repolarize over the exposed basement membrane (27). Restitution is stimulated by a wide range of highly divergent factors including prostaglandins, trefoil proteins, mucins, growth factors and polyamines. In contrast, the blockage of these factors significantly delays restitution (28-31).

Esophageal epithelia

Esophageal mucosa consists of keratinized stratified squamous epithelium. Mucosal injuries in the oesophagus could be induced by exposure to gastric acid. In vitro, the restitution and proliferation of esophageal epithelial cell are affected by several growth factors, by which hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1) and epidermal growth factor (EGF) stimulate the restitution, whereas, transforming growth factor beta (TGFb) inhibits restitution (32, 33). However, epithelial restitution may not involve in the healing of acid-induced injury, because restitution of esophageal epithelial cells are gradually inhibited at pH from 3.0 to 6.5 (34). Alternatively, cell proliferation is stimulated by acid in the normal esophageal tissues (35, 36). Thus, it could be estimated that cell proliferation but not restitution appears to be the major reparative defense for acid-induced esophageal epithelium injury.

Gastric epithelia

Restitution is an important process in the repair of gastric mucosal injury. Growth factors such as HGF and EGF seem to be involved in gastric restitution (37-39). EGF facilitated gastric restitution via basolateral EGF receptors by stimulation of basolateral Na+/H+ exchangers and phosphatidylinositol 3-kinases (PI3K) activation (37, 40). In addition, HGF-mediated epithelial restitution involves cyclooxygenase-2 and polyamines (41, 42). Polyamines facilitated the distribution, diffusion and assembly of microtubules and actin cytoskeleton in normal and damaged cells in vivo, which demonstrate that polyamines are important for gastric mucosal restitution (43-46). Additionally, the reduction of mucous secreting layer thickness is accompanied with suppression of gastric mucosal ODC activity in rats with gastric ulcers and spermidine supplementation reversed the delay in healing (47).

Intestinal epithelia

Restitution is a precisely regulated process involving epithelial cells to polarize and migrate to reseal wounded area. Polyamines play an important role in intestinal epithelia cell (IEC) restitution. Several studies manifested that migration of IEC was inhibited by DFMO and the decreased migration was reversed by exogenous addition of polyamines (48-50). However, the exact molecular mechanisms remain unclear. It has been demonstrated that K+ channel activity, cytoplasmic free Ca2+ ([Ca2+]cyt) and membrane potential (Em) were involved in polyamine-depended IEC migration (49-51). Polyamines could increase [Ca2+]cyt not only by enhancing K+ channel expression which cause membrane hyperpolarization and [Ca2+] influx, but also by canonical transient receptor potential 1 (TRPC1)-mediated [Ca2+] release from internal stores though altering the ratio of stromal interaction molecule 1 (STIM1) to STIM2 (Fig. 2) (51-55). The increased [Ca2+]cyt stimulates IEC migration by regulation of specific proteins during restitution, including up-regulation of phospholipase C-γ1 (PLC-γ1), small GTP binding proteins such as RhoA, Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) (Fig. 2) (49, 50, 56, 57). Additionally, activation of RhoA by cellular polyamines results in increasing polymerization of myosin regulatory light chain (MRLC) which leading to the enhanced formation of myosin stress fiber and lamellipodia during cell migration after wounding (Fig. 2) (58-60).

Fig. 2. The Integrated polyamine signaling pathway related to restitution, proliferation, apoptosis, angiogenesis and GI barrier function.
Abbreviations: ATF2, activating transcription factor 2; AUF, AU-binding factor 1; Bax, Bcl-2-associated X protein; Bcl2, B cell lymphoma gene 2; [Ca2+]cyt, cytoplasmic free Ca2+; cdc42, cell division control protein 42; CDK4, cyclin-depen-dent kinase 4; Chk2, checkpoint kinase 2; CREB, cAMP response element-binding protein; CUGBP1, CUG-binding protein 1; HuR, human antigen R; H2O2, hydrogen peroxide; IκBα, inhibitor of NF-κB α; MCL1, myeloid cell leukemia sequence 1; MEK1, MAPK mitogen-activated protein kinase kinase-1; MLC, myosin light chain; MRLC, myosin regulatory light chain; NDRG1, N-Myc downregulated gene 1; NFκB, nuclear factor-κB; NPM, nucleophosmin; PDK1, 3-phosphoinositide-dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinases; PLC-γ1, phospholipase C-γ1; Rac1, Ras-related C3 botulinum toxin substrate 1; RhoA, Ras homolog gene family, member A; Rock1, Rho-associated coiled-coil containing protein kinase 1; SGs, stress granules; SPMO, spermine oxidases; STIM, stromal interaction molecule; TGFβ, transforming growth factor beta; TGFβR, TGFβ receptor; TIA1, T-cell intracellular antigen 1; TIAR, TIA1-related protein; TRPC1, canonical transient receptor potential 1; XIAP, X chromosome-linked inhibitor of apoptosis protein; ZO-1, zona occludens-1.

PROLIFERATION

Esophageal epithelia

Epithelial cell proliferation is subsequent to restitution in order to restore normal mucosal architecture if the defect is large. Several studies indicated that polyamines and ODC levels were increased after esophageal epithelial injury (Barrett’s esophagus (BE), BE-associated dysplasia and carcinoma) (61). However, DFMO inhibits the growth of Barrett’s epithelium in vitro and in vivo probably by down-regulation of Kruppel like factor 5 (KLF5) and suppression of replication factor C subunit 5 (RFC5) (62).

Gastric epithelia

Several studies have shown that the cellular polyamines are necessary for cell proliferation in gastric epithelium. Polyamines are associated with increased 3H-thymidine incorporation in gastric ulcer model of rats (63). Reduction of polyamines by inhibiting ODC activity significantly suppressed gastric epithelial cell proliferation (64). Furthermore, lipopolysaccharides (LPS) stimulate gastric enterochromaffin-like cell (ECL) DNA synthesis by activation of the intracellular polyamine pathway and ODC activity via a cluster of differentiation 14 (CD14) receptor on the ECL cells (65). Additionally, polyamines and ODC regulate cell proliferation by altering cytoskeletal reorganization via RhoA (66). In the restraint-stress-induced gastric lesions, pretreatment with DFMO resulted into a remarkable decrease in mucosal DNA synthesis and thereby significantly delayed the mucosal healing. In contrast, treatment with polyamines remarkably accelerated ulcer healing (67, 68).

Intestinal epithelia

IEC proliferation is stimulated by polyamines and repressed by polyamine depletion in vivo and in vitro, which manifested that polyamines are absolutely essential for IEC growth and division (69, 70). Although the precise molecular mechanisms have not yet fully elucidated, increasing evidence reveals that polyamines enhances IEC proliferation by regulating large number of growth related genes. Recent studies revealed that polyamines up-regulate the transcription of growth-promoting genes such as c-Fos, c-Jun, and c-Myc (71, 72) and down-regulate growth-inhibiting genes such as p53, N-Myc downregulated gene 1 (NDRG1), nucleophosmin (NPM), JunD, and TGFβ/TGFβ receptor at the posttranscriptional level (Fig. 2) (69, 73-78). Additionally, polyamines promote translation of cyclin-dependent kinase 4 (CDK4) through CUG-binding protein 1 (CUGBP1), JunD and microRNA-222, thereby induction of IEC cell proliferation (Fig. 2) (79, 80). Polyamines regulate gene expression by binding to negatively charged macromolecules, such as DNA, RNA and proteins. In transcriptional level, polyamines could condense and aggregate DNA and induce B-Z and B-A conformational transitions in vitro (81, 82). In posttranscriptional level, polyamines could regulate the subcellular localization of the RNA-binding proteins human antigen R (HuR) and CUGBP1 which stabilizes its target transcripts such as JunD, CDK4, c-Myc, p53 and NPM mRNA (Fig. 2) (70, 79, 83-85).

APOPTOSIS

Esophageal and gastric epithelia

The GI epithelial homeostasis depends on a balance between cell proliferation and apoptosis. Studies showed that polyamine depletion by DFMO induces esophageal epithelial apoptosis by altering both Bcl-2 and Bax expression (61). Helicobacter pylori (H. pylori) induced oxidative stress could induce apoptosis in host gastric epithelial cells and macrophages by a mitochondrial-dependent cell death pathway (86). The oxidative stress mediated by hydrogen peroxide (H2O2) is generated by the polyamines via the inducible SPMO in H. pylori infected cells (86, 87). Increased expression of polyamines, ODC and SPMO were observed in both H. pylori infected human and mouse gastritis tissues (86, 88, 89). Inhibition or knockdown of SPMO or ODC remarkably attenuates apoptosis and DNA damage in gastric epithelial cells (86). Macrophage apoptosis was stimulated by the polyamines and was abolished by inhibition of ODC or SPMO activity (87, 90). H. pylori infected mice treated with DFMO significantly reduced both H. pylori colonization levels and gastritis severity (88). The phosphorylated extracellular regulated protein kinases (p-ERK) › phosphorylated c-Fos/c-Jun › c-Myc › ODC › SPMO › H2O2 › cytochrome C › caspase-3 pathway might involve in the polyamine-mediated apoptosis in H. pylori induced gastric diseases (Fig. 2) (87, 89, 91).

Intestinal epithelia

Apoptosis occurs in the crypt area or the position of epithelial stem cells (92). It has been shown that polyamine depletion promotes the resistance of IECs to apoptosis by stabilization of activating transcription factor 2 (ATF-2), X chromosome-linked inhibitor of apoptosis protein (XIAP) and MAPK mitogen-activated protein kinase kinase-1 (MEK1) mRNA and increases translation though enhancing HuR levels (Fig. 2). Elevation of cytoplasmic HuR levels by inhibition of cellular polyamines increased ATF-2, XIAP and MEK1 mRNA and protein levels, whereas HuR silencing make these mRNAs unstable and suppressed ATF-2, XIAP and MEK1 expression (92-94). Additionally, depletion of cellular polyamines by DFMO protects normal IEC-6 cells from tumor necrosis factor-α (TNF-α) induced apoptosis. Elevation of nuclear factor-κB (NF-κB) transcriptional activity by reduction of inhibitor of NF-κB a (IκBα) and decreased TRPC1-mediated [Ca2+]cyt were observed after polyamine depletion, indicating that these pathways might also involve in the regulation of polyamine-dependent IEC apoptosis (Fig. 2) (95, 96). Furthermore, polyamine depletion could increase phosphorylation of Akt by enhancing 3-phosphoinositide-dependent protein kinase-1 (PDK1) activity, thereby protecting IECs against TNF-α/cycloheximide (CHX) induced apoptosis (Fig. 2) (97). The stress granules (SGs) also implicate the protection of IECs from apoptosis (98). Following polyamine depletion, SGs formation was activated by silencing SGs resident proteins Sort1 and T-cell intracellular antigen 1 (TIA1), which in turn increase resistance to TNF-α/CHX induced IEC-6 apoptosis (Fig. 2) (99).

Gastrointestinal stem cells

In adult, stem cells are characterized by their self-renewal capacity to generate multiple differentiated cell types (100). The GI epithelium represents one of the most rapidly self-renewing tissues in adult mammals, which continuously self-renews for every 2–5 days (101). It has been suggested that GI stem cells are important for the maintenance of GI mucosal homeostasis (102).

Esophageal and gastric stem cells

Recent studies reveal that esophageal epithelium is maintained and repaired by stem cells (103, 104). Esophageal stem cells are thought to reside within the basal layer of the stratified squamous epithelium (102). Esophageal stem cells are a reversible switch between homeostatic and regenerative in response to wound healing (104).

Each gastric unit of mammalian adult stomach self-renews continually by a tiny population of stem cells (105). Gastric stem cells are important for gastric epithelial repair after injury (106). Although gastric stem cells have been investigated for more than two decades, it has not yet been identified for lack of specific markers (102). Putative gastric stem cells are supposed to locate to the isthmus, the middle portion of the tubule (107). Recently, a subpopulation of gastric progenitors with multiple potential has been identified (108). By using genetic and chemical inhibition approaches, Notch signal pathway is demonstrated to be required for the maintenance of gastric stem cell homeostasis (109).

Intestinal stem cells

Two types of stem cells have been currently defined in the intestine: leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) positive crypt base columnar (CBC) cells and Bmi1 positive quiescent ‘+4’ cells. CBC cells are predominantly located at the crypt base and cycle rapidly, while the ‘+4’ cells are situated above the crypt base and are largely reside (110-112). Additionally, these two stem cell populations could dynamically interconvert in distinct niches (101). The population of intestinal stem cells are descending gradient from duodenum to colon (113). In a recent study, Lgr5+ stem cells were grown in vitro to produce organoids of epithelial tissue, and the damaged epithelium was rescued by which the organoids were engraft onto the damaged colon within the dextran sulfate sodium (DSS)-induced colitis in mice (114). This study provides proof of principle that stem-cell-based therapies can be applied for repairing damaged epithelium.

The exact role of polyamines in the differentiation of GI stem cells has not been characterized. The polyamine pathway was recently identified to be essential for self-renewal and differentiation of embryonic stem cells, adipose-derived stem cells and mesenchymal stem cell (115-118), which indicated that polyamines might be important for self-renewal and differentiation of GI stem cells.

ANGIOGENESIS

Restitution and subsequent proliferation are effective processes to repair superficial lesions but not deeper lesions in the GI tract. Angiogenesis and proliferation of fibroblasts are involved in the healing of mucosal lesions in deeper damages into muscularis mucosae and lamina propria (119).

Angiogenesis, the development of new blood vessels from the existing vasculature, brings new capillaries and larger blood vessels which are essential for clearance of necrotic tissue and stimulates fibroblasts proliferation as it delivers oxygen and nutrients to injury sites (5, 120). Thus, angiogenesis might be a fundamental process in the wound healing of the GI tract. Angiogenesis in the damaged GI tract is stimulated by various factors, including vascular endothelial growth factor (VEGF), play a pivotal role in the GI mucosa (121).

Angiogenesis in healing of esophagus and stomach

In an eosinophilic esophagitis mice model, mice challenged with egg ovalbumin developed a significant increase in the number of small vessels within the esophageal and remarkably elevated the number of VEGF-positive cells (122), which reveals that angiogenesis might involve in the healing of esophageal injuries.

Previous studies have demonstrated that angiogenesis is commonly associated with gastritis, peptic ulcer, and gastric carcinoma (123). The angiogenesis which is stimulated to repair the gastric mucosa is subsequently increased in patients with H. pylori infection (124). Additionally, there are an increased concentration of angiogenic factors and the formation of new blood vessels in H. pylori-associated diseases. Compared with H. pylori negative patients, the expression of VEGF mRNA and protein was increased in the H. pylori positive dyspeptic patients (125). In a water-immersion and restraint stress induced gastric ulcer rat model and thermal cauterization induced gastric ulcer mice model, VEGF expression and angiogenesis were remarkably increased compared with normal controls (126, 127). The increased VEGF and angiogenesis is important for protection and healing of gastric ulcers (128-130). In contrast, inhibition of VEGF delayed ulcer healing (131). Thus, angiogenesis might facilitate the healing of gastric lesions.

Angiogenesis in healing of intestine

Angiogenesis is accompanied with chronic inflammation in many diseases (132). Stimulation of angiogenesis by treatment with genes or peptides of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) or VEGF are sufficient to accelerate duodenal ulcer healing in the GI tract respectively, while blockade of these angiogenic factors resulted in delaying ulcer healing (119, 133, 134). Furthermore, it has been illustrated that angiogenesis might be implicated in the pathogenesis of ulcerative colitis (UC) and Crohn’s disease (CD), the two major forms of inflammatory bowel diseases (IBD) (135, 136). Increased mucosal vascular density and pro-angiogenic molecules content (e.g. VEGF, placental growth factor (PIGF), PDGF and bFGF) were associated with progression of human IBD and animal models of UC (137, 138). Promoting angiogenesis with these pro-angiogenic molecules in experimental models of colitis increased mucosal angiogenesis (139, 140). Blockade of angiogenesis by specific antibodies to neutralize VEGF, integrin avb3 and bFGF is effective to ameliorate experimental colitis (135, 139, 141). Thus, pharmacological inhibition of angiogenesis may represent a new and promising therapeutic target in experimental models of IBD.

However, given the beneficial effect of anti-angiogenesis treatment in IBD, additional studies indicated that stimulation of angiogenesis by PIGF, PDGF or bFGF remarkably accelerated healing of experimental models of UC (137, 138, 142). Blockade of angiogenesis by specific inhibition of PIGF leads an increased disease activity during DSS-induced colitis, which was subsequently prevented by exogenous administration of recombinant PIGF (143). In the early stages of IBD, increased angiogenesis could accelerate the influx of inflammatory cells. However, reduced vessel diameter, decreased vascular density and diminished blood flow were observed in advanced IBD lesions which resulting in sustained tissue hypo-perfusion and ischemia in the colon (132, 144). Thus, anti-angiogenesis therapy might be beneficial in the early stage of IBD but harmful in the advanced stage of IBD.

The effect of polyamines on angiogenesis is mainly explored in cancer cells. The angiogenesis in tumor was completely abrogated with DFMO, and the anti-angiogenesis effect of DFMO could be reversed by the exogenous addition of polyamines (145). Although polyamines are widely applied in the wound healing of the GI tract, mechanisms of polyamines on angiogenesis are not fully understood. Polyamines significantly increased gastric blood flow and accelerated ulcer healing. Inhibition of ODC activity with DFMO remarkably reduced gastric blood flow and delayed ulcer healing (67). Thus, the beneficial effect of polyamines on the wound healing is also facilitated by angiogenesis.

GASTROINTESTINAL BARRIER FUNCTION

Most GI mucosa are covered by mucus gel barrier which is secreted by specialized epithelial cells and created the first barrier to protect large particles from directly contacting epithelial cells (146). Additionally, the surface of GI mucosa is lined by epithelial cells which establish the second barrier between hostile external environments and the underlying tissue. The effectiveness and stability of these epithelial barriers are maintained by tight junctions, adherents, junctions, desmosomes, and gap junctions (2, 146). Defective of epithelium barriers have been demonstrated to be crucial in the pathogenesis of many GI disorders, such as gastroesophageal reflux, H. pylori related gastritis and IBD (147-150).

Esophageal barrier function

Mucus gel barrier is absent in the normal esophagus (151), thus, the stratified squamous epithelium lined within esophagus establish the first barrier to protect the underlying tissue from mechanical and chemical damage. Reflux of acid and bile acids is the most important agent damaging esophageal epithelial barrier which could lead to gastroesophageal reflux diseases (148). Although the influence of refluxed agents on esophageal epithelial barrier function remains unclear, decreased expression of tight junction proteins, e.g. claudin and occludin, might contribute to refluxed agents induced esophageal epithelial barrier impairments (152).

Gastric barrier function

Mucus-bicarbonate-phospholipid barrier and epithelial barrier of the stomach maintain an effective defense against toxins, H. pylori, hydrochloric acid, pepsin, bile acids and other noxious factors. A large number of studies have manifested that H. pylori impairs the gastric barriers. In a chronic H. pylori infection mice model, the firmly adherent mucus layer was significantly reduced in infected mice compared within control mice. And the ability of the gastric mucosa to maintain a neutral pH at the epithelial cell surface was also altered after chronic H. pylori infection (153). Furthermore, several studies have demonstrated that H. pylori could disrupt localization of occludin, claudin and zona occludens-1 (ZO-1) at the tight junction and reduce expression of E-cadherin at adherens junctions, which ultimately resulting alteration of epithelial barrier (154-156). Spermidine, but not putrescine or spermine, not only reversed reduction of mucus synthesis but also significantly promoted ulcer healing in gastric ulcer bearing rats (47).

Intestinal barrier function

Epithelial barrier (forming by monolayer columnar epithelium) and pre-epithelial barrier (containing a thick mucus layer, trefoil peptides and immunoglobulin A) are physical and functional barriers to separate million of bacteria from intestinal lumen to the underlying host tissues (157). Disruption of these barriers is associated with a variety of intestinal diseases, including diarrhea, IBD and cancer (157-159). Increasing evidences have demonstrated that polyamines are required for maintenance of intestinal barrier function. Decreased polyamine level was associated with dysfunction of the epithelial barrier, which was ameliorated by exogenous polyamines. The epithelial barrier function was regulated by polyamines though modulating the expression of ZO-1, occludin and E-cadherin (Fig. 2). Occludin, claudins, ZO-1 and ZO-2 are the major transmembrane and cytosolic tight junction proteins. Suppression of polyamines result in increased JunD expression which regulate ZO-1 expression through both cAMP response element-binding protein-binding (CREB) site within ZO-1 proximal promoter region and enhancing the interaction of the ZO-1 3’-untranslated region (3’UTR) with RNA-binding protein TIA1-related protein (TIAR) (Fig. 2) (160). Reduction of checkpoint kinase 2 (Chk2) by polyamine depletion reduces HuR phosphorylation and substantially inhibits occludin translation (161). Alternatively, the translation of occludin might also modulate through competitive binding of CUGBP1 and HuR to occludin 3’UTR (Fig. 2) (162). E-cadherin is mainly located at the adherens junctions and plays a critical role in maintenance of GI epithelial barrier function. Depletion of cellular polyamine decrease E-cadherin expression though c-Myc mediated reduction of E-cadherin promoter activity and its mRNA expression (Fig. 2) (163, 164).

ROLE OF POLYAMINES IN COMMON GASTROINTESTINAL DISEASES

Polyamine metabolism is involved with a wide variety of GI diseases. Numerous factors may influence polyamine homeostasis, however the changes seem to be tissue-specific. Chronic biliopancreatic reflux which causes severe esophagitis increases ODC activity and polyamine levels (165). The altered polyamine metabolism contributes to epithelial cell proliferation and esophageal carcinogenesis in experimental animals (165, 166). ODC activity is upregulated in BE with a premalignant lesion of adenocarcinoma and correlated with the degree of dysplasia (167). DFMO, an irreversible inhibitor of ODC, is considered as a promising chemoprevention agent for esophageal adenocarcinoma by suppressing polyamine content in BE mucosa (166).

Polyamine metabolism is also involved in gastric mucosal injury. H. pylori, a major pathological factor of gastric tissue, induces immune dysregulation by increasing ODC activity and polyamine content in macrophages (88, 89, 168). Interestingly, H. pylori can not induce ODC in gastric epithelium (89), but up-regulates polyamine catabolism and aggravates polyamine-mediated oxidative stress (89). On the other hand, polyamines seem to provide a protective role against other detrimental factors of gastric tissue such as aspirin, smoking and stress (47, 68, 169). During stress ulcers, both ODC activity and polyamine level are up-regulated and ultimately improved mucosal healing (170). In an animal experiment, oral administration of polyamines, putrescine, cadaverine, spermidine or spermine, immediately after stress accelerate the normal rate of healing (68). DFMO inhibits the early phase of ulcer healing, however, it does not influence ulcer recovery eventually, which may indicate an alternate source of polyamines besides ODC pathway are required in these experimental models (171).

The relationship between polyamines and IBD is extensively studied. Conflicting results are reported in different studies regarding the ODC activity and polyamine content in IBD patients. It has been found that ODC activity was decreased in IBD patients both in involved and uninvolved mucosal tissues (172). Furthermore, the decrease of ODC was related to the severity of disease (172). In contrast, ODC was found to be elevated both in human and animal studies (173-175). The discrepancy may be due to the content of tissue sample as previously indicated in gastric tissue (171, 176). A few studies indicate that increased levels of spermidine and polyamine catabolism in IBD patients and experimental models which may relate to the accelerated proliferation of injured tissues (175, 177). Spermine exerts an inhibitory role on the inflammatory reaction and is down-regulated in severe ulcerative colitis patients and in chronic colitis experimental models (176, 178). It has been noticed that the decrease of spermine content may further aggravate the disease (176). The role of polyamine metabolism in IBD is further supported by the fact that L-arginine improves colitis by enhancing the formation of polyamines in animal models while DFMO worsen the disease (175).

Pathological conditions outside the GI tract also closely relate to polyamine metabolism and interfere with internal polyamine pool. In experimental pancreatitis, polyamine catabolism correlates with the degree of pancreatic necrosis with depletion of blood spermidine and spermine levels and elevation of putrescine level (179). Furthermore, increase of putrescine level is also found in remote organs such as liver, lung and kidney which may relate to remote organ injury during pancreatitis (180). However, in humans only the increase of blood putrescine level is noticed in necrotic pancreatitis (181). During cirrhosis and primary hepatic tumor, erythrocyte polyamines (spermine and spermidine) are elevated which may be due to hepatic cell regeneration (182). Other factors such as surgery, trauma and radiation also influence blood polyamine levels (183, 184). However, the exact mechanisms on the role of internal polyamine pool affecting GI mucosal homeostasis are not yet clearly understood.

MALNUTRITION AND POLYAMINES

Polyamine metabolism is closely related to the whole-body energy and protein metabolism (185). In animal experiments, ODC activity and polyamine content in liver drop significantly after starvation for three days, and re-feeding can rapidly increase these parameters (186). Protein-deprivation diet, on the other hand, induces ODC activity and polyamine content in the early phases in liver and colon tissues (187, 188). It has been reported that the ODC activity and polyamine content were not decreased in liver tissue until the fifth week of protein restriction (188). Feeding with polyamine-related amino acids like methionine, arginine or ornithine does not increase muscle or liver concentrations of polyamines in rats (189). Also, in humans, correction of nutritional deficits does not contribute significantly to the red blood cells (RBC) polyamine pool in patients with nonmalignant diseases (190). Studies indicate that the dietary polyamines are essential for intestinal polyamine pool and significantly contribute to the integrity of GI mucosa (176, 191). Recent studies reveal the relationship between dietary polyamine levels and risk of colorectal adenoma, and low polyamine diet may contribute to the control of certain tumors (192-194). Future studies are warranted to clarify polyamine metabolism in the GI tract under critical situation like fasting and malnutrition, and explore the possible therapeutic use of dietary polyamines.

CONCLUSIONS

An increasing body of evidence indicating that polyamines are necessary for normal GI mucosal growth and that decreasing cellular polyamines inhibits cell proliferation and disrupts epithelial mucosal integrity. Polyamines are shown to regulate intestinal epithelial cell renewal by virtue of their ability to modulate expression of various growth promoting genes. Increasing the levels of cellular polyamines accelerate GI growth and cell proliferation by enhancement of several gene transcriptional and translational abilities, whereas growth inhibition following polyamine depletion results primarily from the activation of growth-inhibiting genes. Moreover, polyamines also regulate apoptosis and barrier function of intestinal epithelial cells through multiple signaling pathways. However, there are still many critical issues that remain to be addressed regarding the roles of polyamines in maintenance of gut epithelial integrity.

Acknowledgements: This study are supported by National Natural Science Fund of China (Grant NO. 81170413, to Cheng-Wei Tang).

Conflict of interests: None declared.

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