RESUMO
The growth factor-like lipid mediator, lysophosphatidic acid (LPA), is a potent signaling molecule that influences numerous physiologic and pathologic processes. Manipulation of LPA signaling is of growing pharmacotherapeutic interest, especially because LPA resembles compounds with drug-like features. The action of LPA is mediated through activation of multiple types of molecular targets, including six G protein-coupled receptors that are clear targets for drug development. However, the LPA signaling has been linked to pathological responses that include promotion of fibrosis, atherogenesis, tumorigenesis, and metastasis. Thus, a question arises: Can we harness, in an LPA-like drug, the many beneficial activities of this lipid without eliciting its dreadful actions? We developed octadecyl thiophosphate (OTP; subsequently licensed as Rx100), an LPA mimic with higher stability in vivo than LPA. This article highlights progress made toward developing analogs like OTP and exploring prosurvival and regenerative LPA signaling. We determined that LPA prevents cell death triggered by various cellular stresses, including genotoxic stressors, and rescues cells condemned to apoptosis. LPA2 agonists provide a new treatment option for secretory diarrhea and reduce gastric erosion caused by nonsteroidal anti-inflammatory drugs. The potential uses of LPA2 agonists like OTP and sulfamoyl benzoic acid-based radioprotectins must be further explored for therapeutic uses.
Assuntos
Descoberta de Drogas/métodos , Receptores de Ácidos Lisofosfatídicos/agonistas , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Humanos , Receptores de Ácidos Lisofosfatídicos/química , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco-2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco-2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF-α/CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner.
Assuntos
Apoptose/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Células Epiteliais/enzimologia , Intestinos/citologia , Animais , Apoptose/efeitos dos fármacos , Células CACO-2 , Camptotecina/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Células Epiteliais/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Intestinos/enzimologia , Purinas/farmacologia , Pirimidinas/farmacologia , RatosRESUMO
Polyamine-depletion inhibited apoptosis by activating ERK1/2, while, preventing JNK1/2 activation. MKP-1 knockdown by SiRNA increased ERK1/2, JNK1/2, and p38 phosphorylation and apoptosis. Therefore, we predicted that polyamines might regulate MKP1 via MEK/ERK and thereby apoptosis. We examined the role of MEK/ERK in the regulation of MKP1 and JNK, and p38 activities and apoptosis. Inhibition of MKP-1 activity with a pharmacological inhibitor, sanguinarine (SA), increased JNK1/2, p38, and ERK1/2 activities without causing apoptosis. However, pre-activation of these kinases by SA significantly increased camptothecin (CPT)-induced apoptosis suggesting different roles for MAPKs during survival and apoptosis. Inhibition of MEK1 activity prevented the expression of MKP-1 protein and augmented CPT-induced apoptosis, which correlated with increased activities of JNK1/2, caspases, and DNA fragmentation. Polyamine depleted cells had higher levels of MKP-1 protein and decreased JNK1/2 activity and apoptosis. Inhibition of MEK1 prevented MKP-1 expression and increased JNK1/2 and apoptosis. Phospho-JNK1/2, phospho-ERK2, MKP-1, and the catalytic subunit of PP2Ac formed a complex in response to TNF/CPT. Inactivation of PP2Ac had no effect on the association of MKP-1 and JNK1. However, inhibition of MKP-1 activity decreased the formation of the MKP-1, PP2Ac and JNK complex. Following inhibition by SA, MKP-1 localized in the cytoplasm, while basal and CPT-induced MKP-1 remained in the nuclear fraction. These results suggest that nuclear MKP-1 translocates to the cytoplasm, binds phosphorylated JNK and p38 resulting in dephosphorylation and decreased activity. Thus, MEK/ERK activity controls the levels of MKP-1 and, thereby, regulates JNK activity in polyamine-depleted cells.
Assuntos
Apoptose , Intestinos/citologia , MAP Quinase Quinase 4/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Poliaminas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Camptotecina/farmacologia , Linhagem Celular , Fosfatase 1 de Especificidade Dupla/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Intestinos/enzimologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação , Proteína Fosfatase 2/metabolismo , RatosRESUMO
Amino acids, especially glutamine (GLN) have been known for many years to stimulate the growth of small intestinal mucosa. Polyamines are also required for optimal mucosal growth, and the inhibition of ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, blocks growth. Certain amino acids, primarily asparagine (ASN) and GLN stimulate ODC activity in a solution of physiological salts. More importantly, their presence is also required before growth factors and hormones such as epidermal growth factor and insulin are able to increase ODC activity. ODC activity is inhibited by antizyme-1 (AZ) whose synthesis is stimulated by polyamines, thus, providing a negative feedback regulation of the enzyme. In the absence of amino acids mammalian target of rapamycin complex 1 (mTORC1) is inhibited, whereas, mTORC2 is stimulated leading to the inhibition of global protein synthesis but increasing the synthesis of AZ via a cap-independent mechanism. These data, therefore, explain why ASN or GLN is essential for the activation of ODC. Interestingly, in a number of papers, AZ has been shown to inhibit cell proliferation, stimulate apoptosis, or increase autophagy. Each of these activities results in decreased cellular growth. AZ binds to and accelerates the degradation of ODC and other proteins shown to regulate proliferation and cell death, such as Aurora-A, Cyclin D1, and Smad1. The correlation between the stimulation of ODC activity and the absence of AZ as influenced by amino acids is high. Not only do amino acids such as ASN and GLN stimulate ODC while inhibiting AZ synthesis, but also amino acids such as lysine, valine, and ornithine, which inhibit ODC activity, increase the synthesis of AZ. The question remaining to be answered is whether AZ inhibits growth directly or whether it acts by decreasing the availability of polyamines to the dividing cells. In either case, evidence strongly suggests that the regulation of AZ synthesis is the mechanism through which amino acids influence the growth of intestinal mucosa. This brief article reviews the experiments leading to the information presented above. We also present evidence from the literature that AZ acts directly to inhibit cell proliferation and increase the rate of apoptosis. Finally, we discuss future experiments that will determine the role of AZ in the regulation of intestinal mucosal growth by amino acids.
Assuntos
Aminoácidos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Animais , Apoptose , Proliferação de Células , Mucosa Intestinal/citologia , Mucosa Intestinal/crescimento & desenvolvimento , Ornitina Descarboxilase/metabolismo , Inibidores da Ornitina Descarboxilase , Poliaminas/metabolismo , Proteínas/metabolismoRESUMO
Although intracellular polyamine levels are highly regulated, it is unclear whether intracellular putrescine (PUT), spermidine (SPD), or spermine (SPM) levels act as a sensor to regulate their synthesis or uptake. Polyamines have been shown to induce AZ1 expression through a unique +1 frameshifting mechanism. However, under physiological conditions which particular polyamine induces AZ1, and thereby ODC activity, is unknown due to their inter-conversion. In this study we demonstrate that SPD regulates AZ1 expression under physiological conditions in IEC-6 cells. PUT and SPD showed potent induction of AZ1 within 4 h in serum-starved confluent cells grown in DMEM (control) medium. Unlike control cells, PUT failed to induce AZ1 in cells grown in DFMO containing medium; however, SPD caused a robust AZ1 induction in these cells. SPM showed very little effect on AZ1 expression in both the control and polyamine-depleted cells. Only SPD induced AZ1 when S-adenosylmethionine decarboxylase (SAMDC) and/or ODC were inhibited. Surprisingly, addition of DENSpm along with DFMO restored AZ1 induction by putrescine in polyamine-depleted cells suggesting that the increased SSAT activity in response to DENSpm converted SPM to SPD, leading to the expression of AZ1. This study shows that intracellular SPD levels controls AZ1 synthesis.
Assuntos
Proteínas/metabolismo , Putrescina/farmacologia , Espermidina/farmacologia , Espermina/farmacologia , Adenosilmetionina Descarboxilase/antagonistas & inibidores , Animais , Linhagem Celular , Eflornitina/farmacologia , Homeostase , Ornitina Descarboxilase/metabolismo , Inibidores da Ornitina Descarboxilase/farmacologia , Ratos , Espermina/análogos & derivadosRESUMO
Since antizyme (AZ) is known to inhibit cell proliferation and to increase apoptosis, the question arises as to whether these effects occur independently of polyamines. Intestinal epithelial cells (IEC-6) were grown in control medium and medium containing 5 mM difluoromethylornithine (DFMO) to inhibit ODC, DFMO + 5 µM spermidine (SPD), DFMO + 5 µM spermine (SPM), or DFMO + 10 µM putrescine (PUT) for 4 days and various parameters of growth were measured along with AZ levels. Cell counts were significantly decreased and mean doubling times were significantly increased by DFMO. Putrescine restored growth in the presence of DFMO. However, both SPD and SPM when added with DFMO caused a much greater inhibition of growth than did DFMO alone, and both of these polyamines caused a dramatic increase in AZ. The addition of SPD or SPM to media containing DFMO + PUT significantly inhibited growth and caused a significant increase in AZ. IEC-6 cells transfected with AZ-siRNA grew more than twice as rapidly as either control cells or those incubated with DFMO, indicating that removal of AZ increases growth in cells in which polyamine synthesis is inhibited as well as in control cells. In a separate experiment, the addition of SPD increased AZ levels and inhibited growth of cells incubated with DFMO by 50%. The addition of 10 mM asparagine (ASN) prevented the increase in AZ and restored growth to control levels. These results show that cell growth in the presence or absence of ODC activity and in the presence or absence of polyamines depends only on the levels of AZ. Therefore, the effects of AZ on cell growth are independent of polyamines.
Assuntos
Poliaminas Biogênicas/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Proteínas/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/citologia , Humanos , Mucosa Intestinal/citologia , Proteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
In a glucose-salt solution (Earle's balanced salt solution), asparagine (Asn) stimulates ornithine decarboxylase (ODC) activity in a dose-dependent manner, and the addition of epidermal growth factor (EGF) potentiates the effect of Asn. However, EGF alone fails to activate ODC. Thus, the mechanism by which Asn activates ODC is important for understanding the regulation of ODC activity. Asn reduced antizyme-1 (AZ1) mRNA and protein. Among the amino acids tested, Asn and glutamine (Gln) effectively inhibited AZ1 expression, suggesting a differential role for amino acids in the regulation of ODC activity. Asn decreased the putrescine-induced AZ1 translation. The absence of amino acids increased the binding of eukaryotic initiation factor 4E-binding protein (4EBP1) to 5'-mRNA cap and thereby inhibited global protein synthesis. Asn failed to prevent the binding of 4EBP1 to mRNA, and the bound 4EBP1 was unphosphorylated, suggesting the involvement of the mammalian target of rapamycin (mTOR) in the regulation of AZ1 synthesis. Rapamycin treatment (4 h) failed to alter the expression of AZ1. However, extending the treatment (24 h) allowed expression in the presence of amino acids, indicating that AZ1 is expressed when TORC1 signaling is decreased. This suggests the involvement of cap-independent translation. However, transient inhibition of mTORC2 by PP242 completely abolished the phosphorylation of 4EBP1 and decreased basal as well as putrescine-induced AZ1 expression. Asn decreased the phosphorylation of mTOR-Ser(2448) and AKT-Ser(473), suggesting the inhibition of mTORC2. In the absence of amino acids, mTORC1 is inhibited, whereas mTORC2 is activated, leading to the inhibition of global protein synthesis and increased AZ1 synthesis via a cap-independent mechanism.
Assuntos
Asparagina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Capuzes de RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Ornitina Descarboxilase/biossíntese , Ornitina Descarboxilase/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Biossíntese de Proteínas/fisiologia , Proteínas/genética , Capuzes de RNA/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cell migration is important to the integrity of the gastrointestinal tract for the normal movement of cells from crypt to villi and the healing of wounds. Polyamines are essential to cell migration, mucosal restitution, and, hence, healing. Polyamine depletion by α-difluoromethyl ornithine (DFMO) inhibited migration by decreasing lamellipodia and stress fiber formation and preventing the activation of Rho-GTPases. Polyamine depletion increased the association of the thick F-actin cortex with phosphorylated myosin regulatory light chain (pMRLC). In this study, we determined why MRLC is constitutively phosphorylated as part of the actin cortex. Inhibition of myosin light chain kinase (MLCK) decreased RhoA and Rac1 activities and significantly inhibited migration. Polyamine depletion increased phosphorylation of MRLC (Thr18/Ser19) and stabilized the actin cortex and focal adhesions. The Rho-kinase inhibitor Y27632 increased spreading and migration by decreasing the phosphorylation of MRLC, remodeling focal adhesions, and by activating Rho-GTPases. Thus phosphorylation of MRLC appears to be the rate-limiting step during the migration of IEC-6 cells. In addition, increased localization of RhoA with the actin cortex in polyamine-depleted cells appears to activate Rho-kinase. In the absence of polyamines, activated Rho-kinase phosphorylates myosin phosphatase targeting subunit 1 (MYPT1) at serine-668 leading to its inactivation and preventing the recruitment of phosphatase (protein phosphastase, PP1cδ) to the actomyosin cortex. In this condition, MRLC is constitutively phosphorylated and cycling does not occur. Thus activated myosin binds F-actin stress fibers and prevents focal adhesion turnover, Rho-GTPase activation, and the remodeling of the cytoskeleton required for migration.
Assuntos
Movimento Celular/fisiologia , Células Epiteliais/metabolismo , Mucosa Intestinal/citologia , Cadeias Leves de Miosina/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular , Linhagem Celular , Citoesqueleto/fisiologia , Adesões Focais/fisiologia , Regulação da Expressão Gênica/fisiologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Transporte Proteico , Ratos , Quinases Associadas a rho/metabolismoRESUMO
Camptothecin (CPT) and Nutlin-3 caused apoptosis by increasing p53 protein and its activation in intestinal epithelial cells (IEC-6). We studied the effectiveness of these inducers on apoptosis in human colon cancer cells (Caco2) lacking p53 expression. CPT failed to activate caspase-3 and cause apoptosis in these cells. The absence of p53 expression, higher basal Bcl-xL and lower Bax proteins prevented CPT-induced apoptosis. However, the Mdm2 antagonist Nutlin-3 induced apoptosis in a dose dependent manner by activating caspases-9 and -3. Nutlin-3 prevented the activation of AKT via PTEN-mediated inhibition of the PI3K pathway. Nutlin-3 increased the phosphorylation of retinoblastoma protein causing E2F1 release leading to induction of Siva-1. Nutlin-3-mediated degradation of Mdm2 caused the accumulation of p73, which induced the expression of p53 up-regulated modulator of apoptosis (PUMA). E2F1 and p73 knockdown decreased the expression of Siva and PUMA, respectively and abolished Nutlin-3-induced caspase-3 activation. Cycloheximide (CHX) inhibited Nutlin-3-induced Siva, Noxa, and PUMA expression and inhibited apoptosis in IEC-6 and Caco2 cells. These results indicate that translation of mRNAs induced by Nutlin-3 is critical for apoptosis. In summary, apoptosis in Caco2 cells lacking functional p53 occurred following the disruption of Mdm2 binding with p73 and Rb leading to the expression of pro-apoptotic proteins, PUMA, Noxa, and Siva-1.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias do Colo/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Western Blotting , Células CACO-2 , Camptotecina/farmacologia , Caspases/genética , Caspases/metabolismo , Linhagem Celular Transformada , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Fragmentação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Expressão Gênica , Humanos , Imidazóis/farmacologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Interferente Pequeno/farmacologia , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
We have studied apoptosis of gastrointestinal epithelial cells by examining the receptor-mediated and DNA damage-induced pathways using TNF-α and camptothecin (CPT), respectively. TNF-α requires inhibition of antiapoptotic protein synthesis by cycloheximide (CHX). CHX also results in high levels of active JNK, which are necessary for TNF-induced apoptosis. While CPT induces apoptosis, the increase in JNK activity was not proportional to the degree of apoptosis. Thus the mechanism of activation of JNK and its role in apoptosis are unclear. We examined the course of JNK activation in response to a combination of TNF-α and CPT (TNF + CPT), which resulted in a three- to fourfold increase in apoptosis compared with CPT alone, indicating an amplification of apoptotic signaling pathways. TNF + CPT caused apoptosis by activating JNK, p38, and caspases-8, -9, and -3. TNF-α stimulated a transient phosphorylation of JNK1/2 and ERK1/2 at 15 min, which returned to basal by 60 min and remained low for 4 h. CPT increased JNK1/2 activity between 3 and 4 h. TNF + CPT caused a sustained and robust JNK1/2 and ERK1/2 phosphorylation by 2 h, which remained high at 4 h, suggesting involvement of MEKK4/7 and MEK1, respectively. When administered with TNF + CPT, SP-600125, a specific inhibitor of MEKK4/7, completely inhibited JNK1/2 and decreased apoptosis. However, administration of SP-600125 at 1 h after TNF + CPT failed to prevent JNK1/2 phosphorylation, and the protective effect of SP-600125 on apoptosis was abolished. These results indicate that the persistent activation of JNK might be due to inhibition of JNK-specific MAPK phosphatase 1 (MKP1). Small interfering RNA-mediated knockdown of MKP1 enhanced TNF + CPT-induced activity of JNK1/2 and caspases-9 and -3. Taken together, these results suggest that MKP1 activity determines the duration of JNK1/2 and p38 activation and, thereby, apoptosis in response to TNF + CPT.
Assuntos
Apoptose/fisiologia , Células Epiteliais/fisiologia , Mucosa Intestinal/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Antracenos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Western Blotting , Camptotecina/farmacologia , Caspases/metabolismo , Linhagem Celular , Fragmentação do DNA , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Mucosa Intestinal/enzimologia , MAP Quinase Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase Quinase 4/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , RNA Interferente Pequeno , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Integrin binding to the extracellular matrix (ECM) activated Rho GTPases, Src, and focal adhesion kinase in intestinal epithelial cells (IEC)-6. Polyamine depletion inhibited activities of Rac1, RhoA, and Cdc42 and thereby migration. However, constitutively active (CA) Rac1 expression abolished the inhibitory effect of polyamine depletion, indicating that polyamines are involved in a process upstream of Rac1. In the present study, we examined the role of polyamines in the regulation of the guanine nucleotide exchange factor, diffuse B-cell lymphoma (Dbl), for Rho GTPases. Polyamine depletion decreased the level as well as the activation of Dbl protein. Dbl knockdown by siRNA altered cytoskeletal structure and decreased Rac1 activity and migration. Cells expressing CA-Dbl increased migration, Rac1 activity, and proliferation. CA-Dbl restored migration in polyamine-depleted cells by activating RhoA, Rac1, and Cdc42. CA-Dbl caused extensive reorganization of the F-actin cortex into stress fibers. Inhibition of Rac1 by NSC23766 significantly decreased migration of vector-transfected cells and CA-Dbl-transfected cells. However, the inhibition of migration was significantly higher in the vector-transfected cells compared with that seen in the CA-Dbl-transfected cells. Dbl localized in the perinuclear region in polyamine-depleted cells, whereas it localized with the stress fibers in control cells. CA-Dbl localized with stress fibers in both the control and polyamine-depleted cells. These results suggest that polyamines regulate the activation of Dbl, a membrane-proximal process upstream of Rac1.
Assuntos
Movimento Celular , Células Epiteliais/enzimologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mucosa Intestinal/enzimologia , Poliaminas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Aminoquinolinas/farmacologia , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Fatores de Troca do Nucleotídeo Guanina/genética , Pirimidinas/farmacologia , Interferência de RNA , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Fibras de Estresse/metabolismo , Transfecção , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Neointimal lesions are characterized by accumulation of cells within the arterial wall and are a prelude to atherosclerotic disease. Here we report that a brief exposure to either alkyl ether analogs of the growth factor-like phospholipid lysophosphatidic acid (LPA), products generated during the oxidative modification of low density lipoprotein, or to unsaturated acyl forms of LPA induce progressive formation of neointima in vivo in a rat carotid artery model. This effect is completely inhibited by the peroxisome proliferator-activated receptor (PPAR)gamma antagonist GW9662 and mimicked by PPARgamma agonists Rosiglitazone and 1-O-hexadecyl-2-azeleoyl-phosphatidylcholine. In contrast, stearoyl-oxovaleryl phosphatidylcholine, a PPARalpha agonist and polypeptide epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor failed to elicit neointima. The structure-activity relationship for neointima induction by LPA analogs in vivo is identical to that of PPARgamma activation in vitro and disparate from that of LPA G protein-coupled receptor activation. Neointima-inducing LPA analogs up-regulated the CD36 scavenger receptor in vitro and in vivo and elicited dedifferentiation of cultured vascular smooth muscle cells that was prevented by GW9662. These results suggest that selected LPA analogs are important novel endogenous PPARgamma ligands capable of mediating vascular remodeling and that activation of the nuclear transcription factor PPARgamma is both necessary and sufficient for neointima formation by components of oxidized low density lipoprotein.
Assuntos
Anilidas/farmacologia , Arteriosclerose/induzido quimicamente , Doenças das Artérias Carótidas/induzido quimicamente , Lisofosfolipídeos/toxicidade , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Análise de Variância , Animais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Células Cultivadas , Primers do DNA , Modelos Animais de Doenças , Substâncias de Crescimento/metabolismo , Ligantes , Lipoproteínas LDL/metabolismo , Masculino , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rosiglitazona , Relação Estrutura-Atividade , Tiazolidinedionas/toxicidade , Fatores de Tempo , Fatores de Transcrição/agonistasRESUMO
Intracellular polyamine synthesis is regulated by the enzyme ornithine decarboxylase (ODC), and its inhibition by alpha-difluromethylornithine (DFMO), confers resistance to apoptosis. We have previously shown that DFMO leads to the inhibition of de novo polyamine synthesis, which in turn rapidly activates Src, STAT3 and NF-kappaB via integrin beta3 in intestinal epithelial cells. One mechanism to explain these effects involves the activation of upstream growth factor receptors, such as the epidermal growth factor receptor (EGFR). We therefore hypothesized that EGFR phosphorylation regulates the early response to polyamine depletion. DFMO increased EGFR phosphorylation on tyrosine residues 1173 (pY1173) and 845 (pY845) within 5 min. Phosphorylation declined after 10 min and was prevented by the addition of exogenous putrescine to DFMO containing medium. Phosphorylation of EGFR was concomitant with the activation of ERK1/2. Pretreatment with either DFMO or EGF for 1 h protected cells from TNF-alpha/CHX-induced apoptosis. Exogenous addition of polyamines prevented the protective effect of DFMO. In addition, inhibition of integrin beta3 activity (with RGDS), Src activity (with PP2), or EGFR kinase activity (with AG1478), increased basal apoptosis and prevented protection conferred by either DFMO or EGF. Polyamine depletion failed to protect B82L fibroblasts lacking the EGFR (PRN) and PRN cells expressing either a kinase dead EGFR (K721A) or an EGFR (Y845F) mutant lacking the Src phosphorylation site. Conversely, expression of WT-EGFR (WT) restored the protective effect of polyamine depletion. Fibronectin activated the EGFR, Src, ERKs and protected cells from apoptosis. Taken together, our data indicate an essential role of EGFR kinase activity in MEK/ERK-mediated protection, which synergizes with integrin beta3 leading to Src-mediated protective responses in polyamine depleted cells.
Assuntos
Apoptose/efeitos dos fármacos , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Receptores ErbB/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Inibidores da Ornitina Descarboxilase , Poliaminas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibronectinas/metabolismo , Integrina beta3/metabolismo , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Mutação , Oligopeptídeos/farmacologia , Ornitina Descarboxilase/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Putrescina/metabolismo , Pirimidinas/farmacologia , Quinazolinas , Ratos , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo , Tirosina/química , Tirfostinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Inhibition of ornithine decarboxylase by DFMO (alpha-difluromethylornithine) and subsequent polyamine depletion increases p21Cip1 protein, induces cell cycle arrest and confers resistance to apoptosis on intestinal epithelial cells. However, the mechanism by which polyamines regulate p21Cip1 expression and apoptosis is unknown. On the basis of the involvement of p21Cip1 as an anti-apoptotic protein, we tested the role of p21Cip1 in providing protection from apoptosis. Simultaneously, we investigated the role of E47, a basic helix-loop-helix protein, in the regulation of p21Cip1 gene transcription. Gene-specific siRNA (small interfering RNA) decreased E47 protein levels, increased p21Cip1 promoter activity and protein levels and protected cells from TNFalpha (tumour necrosis factor alpha)-induced apoptosis. Knockdown of p21Cip1 protein by siRNA resulted in cells becoming more susceptible to apoptosis. In contrast, incubation with EGF (epidermal growth factor) stimulated p21Cip1 mRNA and protein levels and rescued cells from apoptosis. During apoptosis, the level of E47 mRNA increased, causing a concomitant decrease in p21Cip1 mRNA and protein levels. Polyamine depletion decreased E47 mRNA levels and cell survival. Caspase 3-mediated cleavage of p130Cas has been implicated in p21Cip1 transcription. The progression of apoptosis led to a caspase 3-dependent cleavage of p130Cas and generated a 31 kDa fragment, which translocated to the nucleus, associated with nuclear E47 and inhibited p21Cip1 transcription. Polyamine depletion inhibited all these effects. Transient expression of the 31 kDa fragment prevented the expression of p21Cip1 protein and increased apoptosis. These results implicate p21Cip1 as an anti-apoptotic protein and suggest a role for polyamines in the regulation of p21Cip1 via the transcription repressor E47. Caspase-mediated cleavage of p130Cas generates a 31 kDa fragment, inhibits p21Cip1 transcription and acts as an amplifier of apoptotic signalling.
Assuntos
Apoptose , Proteína Substrato Associada a Crk/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Regulação da Expressão Gênica/fisiologia , Mucosa Intestinal/citologia , Fatores de Transcrição TCF/fisiologia , Animais , Linhagem Celular , Proteína Substrato Associada a Crk/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Poliaminas , Ratos , Proteína 1 Semelhante ao Fator 7 de Transcrição , Transcrição GênicaRESUMO
IMPACT STATEMENT: A critical barrier in treating diarrheal disease is easy-to-use effective treatments. Rx100 is a first in class, novel small molecule that has shown efficacy after both subcutaneous and oral administration in a mouse cholera-toxin- and Citrobacter rodentium infection-induced diarrhea models. Our findings indicate that Rx100 a metabolically stable analog of the lipid mediator lysophosphatidic acid blocks activation of CFTR-mediated secretion responsible for fluid discharge in secretory diarrhea. Rx100 represents a new treatment modality which does not directly block CFTR but attenuates its activation by bacterial toxins. Our results provide proof-of-principle that Rx100 can be developed for use as an effective oral or injectable easy-to-use drug for secretory diarrhea which could significantly improve care by eliminating the need for severely ill patients to regularly consume large quantities of oral rehydration therapies and offering options for pediatric patients.
Assuntos
Toxinas Bacterianas/toxicidade , Toxina da Cólera/toxicidade , Diarreia/tratamento farmacológico , Diarreia/prevenção & controle , Lisofosfolipídeos/farmacologia , Animais , Diarreia/induzido quimicamente , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , CamundongosRESUMO
Intestinal epithelial (IEC-6) cells are resistant to apoptosis following the inhibition of ODC (ornithine decarboxylase) and subsequent polyamine depletion. The depletion of polyamines rapidly activates NF-kappaB (nuclear factor kappaB) and STAT3 (signal transducer and activator of transcription 3), which is responsible for the observed decrease in apoptosis. Since both NF-kappaB and STAT3 signalling pathways can be activated by Src kinase, we examined its role in the antiapoptotic response. Inhibition of ODC by DFMO (alpha-difluoromethylornithine) increased the activity of Src and ERK1/2 (extracellular-signal-regulated kinase 1/2) within 30 min, which was prevented by exogenous polyamines added to the DFMO-containing medium. Conversely, epidermal growth factor-mediated Src and ERK1/2 activation was not prevented by the addition of polyamines. Inhibition of Src with PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine} and a DN-Src (dominant-negative Src) construct prevented the activation of Akt, JAK (Janus kinase) and STAT3. Spontaneous apoptosis was increased in DN-Src-expressing cells and the protective effect of polyamine depletion was lost. Polyamine depletion by DFMO increased integrin beta3 Tyr785 phosphorylation. Cells plated on fibronectin had significantly higher beta3 phosphorylation and Src activation compared with plastic. Exogenous polyamines added to the fibronectin matrix prevented Src activation. Arg-Gly-Asp-Ser inhibited beta3, Src and Akt phosphorylation and sensitized polyamine-depleted cells to tumour necrosis factor alpha/cycloheximide-mediated apoptosis. Fibronectin activated Src and subsequently protected cells from apoptosis. Together, these results suggest that the inhibition of ODC rapidly removes a small pool of available polyamines triggering the activation of beta3 integrin, which in turn activates Src. The subsequent Akt and JAK activation is accompanied by translocation of NF-kappaB and STAT3 to the nucleus and the synthesis of antiapoptotic proteins.
Assuntos
Apoptose , Integrina beta3/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Fator de Transcrição STAT3/fisiologia , Quinases da Família src/metabolismo , Animais , Linhagem Celular , Cicloeximida/farmacologia , Eflornitina/farmacologia , Ativação Enzimática , Fibronectinas/metabolismo , Mucosa Intestinal/citologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Inibidores da Ornitina Descarboxilase , Fosforilação , Poliaminas/metabolismo , Poliaminas/farmacologia , Pirimidinas/farmacologia , Ratos , Fator de Necrose Tumoral alfa/metabolismo , Tirosina/metabolismo , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
Activation of STAT3 (signal transducer and activator of transcription 3) plays a crucial role in cell survival and proliferation. The aim of the present study was to clarify the role of STAT3 signalling in the protection of polyamine-depleted intestinal epithelial cells against TNF-alpha (tumour necrosis factor-alpha)-induced apoptosis. Polyamine depletion by DFMO (alpha-difluoromethylornithine) caused phosphorylation of STAT3 at Tyr-705 and Ser-727. Phospho-Tyr-705 STAT3 was immunolocalized at the cell periphery and nucleus, whereas phospho-Ser-727 STAT3 was predominantly detected in the nucleus of polyamine-depleted cells. Sustained phosphorylation of STAT3 at tyrosine residues was observed in polyamine-depleted cells after exposure to TNF-alpha. Inhibition of STAT3 activation by AG490 or cell-membrane-permeant inhibitory peptide (PpYLKTK; where pY represents phospho-Tyr) increased the sensitivity of polyamine-depleted cells to apoptosis. Expression of DN-STAT3 (dominant negative-STAT3) completely eliminated the protective effect of DFMO against TNF-alpha-induced apoptosis. Polyamine depletion increased mRNA and protein levels for Bcl-2, Mcl-1 (myeloid cell leukaemia-1) and c-IAP2 (inhibitor of apoptosis protein-2). Significantly higher levels of Bcl-2 and c-IAP2 proteins were observed in polyamine-depleted cells before and after 9 h of TNF-alpha treatment. Inhibition of STAT3 by AG490 and DN-STAT3 decreased Bcl-2 promoter activity. DN-STAT3 decreased mRNA and protein levels for Bcl-2, Mcl-1 and c-IAP2 in polyamine-depleted cells. siRNA (small interfering RNA)-mediated inhibition of Bcl-2, Mcl-1 and c-IAP2 protein levels increased TNF-alpha-induced apoptosis. DN-STAT3 induced the activation of caspase-3 and PARP [poly(ADP-ribose) polymerase] cleavage in polyamine-depleted cells. These results suggest that activation of STAT3 in response to polyamine depletion increases the transcription and subsequent expression of anti-apoptotic Bcl-2 and IAP family proteins and thereby promotes survival of cells against TNF-alpha-induced apoptosis.
Assuntos
Apoptose , Proteínas Inibidoras de Apoptose/genética , Proteínas de Neoplasias/genética , Poliaminas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fator de Transcrição STAT3/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Eflornitina/farmacologia , Ativação Enzimática , Proteínas Inibidoras de Apoptose/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transcrição Gênica , Fator de Necrose Tumoral alfa/farmacologia , Regulação para CimaRESUMO
Tissue polyamine levels are largely determined by the activity of ornithine decarboxylase (ODC, EC 4.1.17), which catalyzes the conversion of ornithine to the diamine putrescine. The activity of the enzyme is primarily regulated by a negative feedback mechanism involving ODC antizyme (AZ). Our previous studies demonstrated that AZ synthesis is stimulated by the absence of amino acids, the levels of which are sensed by the mTOR complex containing TORC1, which is stimulated by amino acids and inhibited by their absence, and TORC2 the function of which is not well defined. Polyamines, which cause a +1 ribosomal frameshift during the translation of AZ mRNA are required to increase AZ synthesis in both the presence and absence of amino acids. Amino acid starvation increases TORC2 activity. We have demonstrated that mTORC2 activity is necessary for AZ synthesis in the absence of amino acids. Tuberous sclerosis protein (TSC), a negative regulator of mTOR function regulates the activities of both the TORC1 and TORC2. TSC2 knockdown increased mTORC1 activity with concomitant inhibition of mTORC2 activity eliminating AZ induction in the absence of amino acids as well as that induced by spermidine. Thus, these results clearly demonstrate that in addition to polyamines, mTORC2 activity is necessary for AZ synthesis. Moreover, our results support a role for mTORC2 in the synthesis of a specific protein, AZ, which regulates growth of intestinal epithelial cells.
Assuntos
Poliaminas Biogênicas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Mutantes , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas/genética , Proteínas/genética , Serina-Treonina Quinases TOR/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
We have previously demonstrated that the small molecule octadecenyl thiophosphate (OTP), a synthetic mimic of the growth factor-like mediator lysophosphatidic acid (LPA), showed radioprotective activity in a mouse model of total-body irradiation (TBI) when given orally or intraperitoneally 30 min before exposure to 9 Gy γ radiation. In the current study, we evaluated the effects of OTP, delivered subcutaneously, for radioprotection or radiomitigation from -24 h before to up to +72 h postirradiation using a mouse TBI model with therapeutic doses at around 1 mg/kg. OTP was injected at 10 mg/kg without observable toxic side effects in mice, providing a comfortable safety margin. Treatment of C57BL/6 mice with a single dose of OTP over the time period from -12 h before to +26 h after a lethal dose of TBI reduced mortality by 50%. When administered at +48 h to +72 h postirradiation (LD50/30 to LD100/30), OTP reduced mortality by ≥34%. OTP administered at +24 h postirradiation significantly elevated peripheral white blood cell and platelet counts, increased crypt survival in the jejunum, enhanced intestinal glucose absorption and reduced endotoxin seepage into the blood. In the 6.4-8.6 Gy TBI range using LD50/10 as the end point, OTP yielded a dose modification factor of 1.2. The current data indicate that OTP is a potent radioprotector and radiomitigator ameliorating the mortality and tissue injury of acute hematopoietic as well as acute gastrointestinal radiation syndrome.
Assuntos
Síndrome Aguda da Radiação/prevenção & controle , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/efeitos da radiação , Hematopoese/efeitos dos fármacos , Hematopoese/efeitos da radiação , Lisofosfolipídeos/metabolismo , Compostos Organofosforados/farmacologia , ATPases Associadas a Diversas Atividades Celulares , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos CD34/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/efeitos da radiação , Materiais Biomiméticos/efeitos adversos , Materiais Biomiméticos/farmacocinética , Materiais Biomiméticos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/efeitos da radiação , Relação Dose-Resposta a Droga , Feminino , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Glucose/metabolismo , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Proteínas com Domínio LIM/metabolismo , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos C57BL , Compostos Organofosforados/efeitos adversos , Compostos Organofosforados/farmacocinética , Fosfoproteínas/metabolismo , Contagem de Plaquetas , Complexo de Endopeptidases do Proteassoma , Protetores contra Radiação/efeitos adversos , Protetores contra Radiação/farmacocinética , Protetores contra Radiação/farmacologia , Trocadores de Sódio-Hidrogênio/metabolismo , Fatores de Transcrição/metabolismo , Irradiação Corporal Total/efeitos adversosRESUMO
PURPOSE: To determine whether polyamines are present in corneal cells, whether corneal cell polyamines can be depleted by blocking the first rate-limiting enzyme in the polyamine synthesis pathway, ornithine decarboxylase (ODC), and whether polyamines are required for proliferation in all three corneal cell types. METHODS: Cultured corneal epithelial cells, keratocytes, and endothelial cells were exposed to the specific ODC blocker difluoromethylornithine (DFMO), and ODC activity, intracellular polyamine concentrations, and cell proliferation were measured. RESULTS: DFMO blocked ODC activity in a dose- and time-dependent manner in all three cell types. DFMO treatment completely depleted putrescine and spermidine by 2 days and also significantly depleted spermine. DFMO treatment also inhibited cell growth in all three cell types and this inhibition could be completely reversed by adding exogenous putrescine to the culture medium. CONCLUSIONS: Polyamines are present in all cell types of the cornea, their formation is catalyzed at least in part by ODC, and they are an important component of corneal cell proliferation.