RESUMO
Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.
Assuntos
Lisofosfolipídeos , Osteogênese , Receptores de Lisofosfolipídeos , Perda de Dente , Animais , Camundongos , Diferenciação Celular/fisiologia , Lisofosfolipídeos/metabolismo , Osteoblastos/metabolismo , Ligamento Periodontal/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Receptores de Lisofosfolipídeos/metabolismoRESUMO
While immune checkpoint inhibitors (ICIs) are promising in the treatment of metastatic melanoma, about half of patients do not respond well to them. Low levels of human leukocyte antigen-DR (HLA-DR) in tumors have been shown to negatively influence prognosis and response to ICIs. Lysophosphatidic acid (LPA) is produced in large amounts by melanoma and is abundantly present in the tumor microenvironment. LPA induces the release of various cytokines and chemokines from tumor cells, which affect cancer development, metastasis, and tumor immunity. In the present study, we investigated the role of LPA-induced IL-10 release in regulating HLA-DR expression and the underlying mechanisms in human melanoma cells. We showed that LPA (0.001-10 µM) dose-dependently increased DR6 transcript levels through activating LPAR1 in HEK293T cells. Knockdown of NF-κB1 abrogated the LPA-increased DR6 expression without affecting basal DR6 expression in both A2058 and A375 melanoma cell lines. LPA (10 µM) significantly increased IL-10 transcripts in A2058 and A375 melanoma cells, the effect was abolished by pharmacological inhibition of LPAR1 or knockdown of DR6. We found a statistically significant correlation between the expression of LPAR1, DR6 and IL-10 in human melanoma tissue and an association between increased expression of LPAR1 and reduced effectiveness of ICI therapy. We demonstrated that LPA (10 µM) markedly suppressed HLA-DR expression in both A375 and A2058 melanoma cells via activating the LPAR1-DR6-IL-10 pathway. These data suggest that the LPAR1-DR6-IL-10 autocrine loop could constitute a novel mechanism used by tumor cells to evade immunosurveillance by decreasing HLA-DR expression.
RESUMO
The ATX-LPA-LPAR1 signaling pathway plays a universal role in stimulating diverse cellular responses, including cell proliferation, migration, survival, and invasion in almost every cell type. The ATX-LPAR1 axis is linked to several metabolic and inflammatory diseases including cancer, fibrosis, and rheumatoid arthritis. Numerous selective ATX or LPAR1 inhibitors have been developed and so far, their clinical efficacy has only been evaluated in idiopathic pulmonary fibrosis. None of the ATX and LPAR1 inhibitors have advanced to clinical trials for cancer and rheumatoid arthritis. Nonetheless, several research groups, including ours, have shown considerable benefit of simultaneous ATX and LPAR1 inhibition through combination therapy. Recent research suggests that dual-targeting therapies are superior to combination therapies that use two selective inhibitors. However, limited reports are available on ATX-LPAR1 dual inhibitors, potentially due to co-expression of multiple different LPARs with close structural similarities at the same target. In this review, we discuss rational design and future directions of dual ATX-LPAR1 inhibitors.
Assuntos
Artrite Reumatoide , Fibrose Pulmonar Idiopática , Neoplasias , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Lisofosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) plays important roles as a signaling lipid in a variety of physiological and pathophysiological processes. S1P signals via a family of G-protein-coupled receptors (GPCRs) (S1P1-5) and intracellular targets. Here, we report on photoswitchable analogs of S1P and its precursor sphingosine, respectively termed PhotoS1P and PhotoSph. PhotoS1P enables optical control of S1P1-3, shown through electrophysiology and Ca2+ mobilization assays. We evaluated PhotoS1P in vivo, where it reversibly controlled S1P3-dependent pain hypersensitivity in mice. The hypersensitivity induced by PhotoS1P is comparable to that induced by S1P. PhotoS1P is uniquely suited for the study of S1P biology in cultured cells and in vivo because it exhibits prolonged metabolic stability compared to the rapidly metabolized S1P. Using lipid mass spectrometry analysis, we constructed a metabolic map of PhotoS1P and PhotoSph. The formation of these photoswitchable lipids was found to be light dependent, providing a novel approach to optically probe sphingolipid biology.
Assuntos
Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Animais , Lisofosfolipídeos/química , Camundongos , Modelos Moleculares , Estrutura Molecular , Imagem Óptica , Processos Fotoquímicos , Esfingosina/química , Esfingosina/metabolismoRESUMO
Lysophosphatidic acid (LPA) is a phospholipid that acts as an extracellular signaling molecule and activates the family of lysophosphatidic acid receptors (LPA1-6). These G protein-coupled receptors (GPCRs) are broadly expressed and are particularly important in development as well as in the nervous, cardiovascular, reproductive, gastrointestinal, and pulmonary systems. Here, we report on a photoswitchable analogue of LPA, termed AzoLPA, which contains an azobenzene photoswitch embedded in the acyl chain. AzoLPA enables optical control of LPA receptor activation, shown through its ability to rapidly control LPA-evoked increases in intracellular Ca2+ levels. AzoLPA shows greater activation of LPA receptors in its light-induced cis-form than its dark-adapted (or 460 nm light-induced) trans-form. AzoLPA enabled the optical control of neurite retraction through its activation of the LPA2 receptor.
Assuntos
Lisofosfolipídeos/metabolismo , Humanos , Lisofosfolipídeos/química , Processos Fotoquímicos , Receptores de Ácidos Lisofosfatídicos/química , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de SinaisRESUMO
The lysophospholipase D autotaxin (ATX) generates lysophosphatidic acid (LPA) that activates six cognate G-protein coupled receptors (GPCR) in cancerous cells, promoting their motility and invasion. Four novel compounds were generated aided by molecular docking guided design and synthesis techniques to obtain new dual inhibitors of ATX and the lysophosphatidic acid receptor subtype 1 (LPAR1). Biological evaluation of these compounds revealed two compounds, 10 and 11, as new ATX enzyme inhibitors with potencies in the range of 218-220 nM and water solubility (>100 µg/mL), but with no LPAR1 inhibitory activity. A QSAR model was generated that included four newly designed compounds and twenty-one additional compounds that we have reported previously. The QSAR model provided excellent predictability of the pharmacological activity and potency among structurally related drug candidates. This model will be highly useful in guiding the synthesis of new ATX inhibitors in the future.
Assuntos
Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Piranos/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/metabolismo , Ligação Proteica , Piranos/síntese química , Piranos/metabolismo , Relação Quantitativa Estrutura-Atividade , Ratos , Receptores de Ácidos Lisofosfatídicos/metabolismoRESUMO
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
Stem cells are a rare subpopulation defined by the potential to self-renew and differentiate into specific cell types. A population of stem-like cells has been reported to possess the ability of self-renewal, invasion, metastasis, and engraftment of distant tissues. This unique cell subpopulation has been designated as cancer stem cells (CSC). CSC were first identified in leukemia, and the contributions of CSC to cancer progression have been reported in many different types of cancers. The cancer stem cell hypothesis attempts to explain tumor cell heterogeneity based on the existence of stem cell-like cells within solid tumors. The elimination of CSC is challenging for most human cancer types due to their heightened genetic instability and increased drug resistance. To combat these inherent abilities of CSC, multi-pronged strategies aimed at multiple aspects of CSC biology are increasingly being recognized as essential for a cure. One of the most challenging aspects of cancer biology is overcoming the chemotherapeutic resistance in CSC. Here, we provide an overview of autotaxin (ATX), lysophosphatidic acid (LPA), and their signaling pathways in CSC. Increasing evidence supports the role of ATX and LPA in cancer progression, metastasis, and therapeutic resistance. Several studies have demonstrated the ATX-LPA axis signaling in different cancers. This lipid mediator regulatory system is a novel potential therapeutic target in CSC. In this review, we summarize the evidence linking ATX-LPA signaling to CSC and its impact on cancer progression and metastasis. We also provide evidence for the efficacy of cancer therapy involving the pharmacological inhibition of this signaling pathway.
Assuntos
Neoplasias/enzimologia , Neoplasias/patologia , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Humanos , Lisofosfolipídeos/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) has been implicated recently in the physiology and pathology of the cardiovascular system including regulation of vascular tone. Pilot experiments showed that the vasoconstrictor effect of S1P was enhanced markedly in the presence of phenylephrine (PE). Based on this observation, we hypothesized that S1P might modulate α1-adrenergic vasoactivity. In murine aortas, a 20-minute exposure to S1P but not to its vehicle increased the Emax and decreased the EC50 of PE-induced contractions indicating a hyperreactivity to α1-adrenergic stimulation. The potentiating effect of S1P disappeared in S1P2 but not in S1P3 receptor-deficient vessels. In addition, smooth muscle specific conditional deletion of G12/13 proteins or pharmacological inhibition of the Rho-associated protein kinase (ROCK) by Y-27632 or fasudil abolished the effect of S1P on α1-adrenergic vasoconstriction. Unexpectedly, PE-induced contractions remained enhanced markedly as late as three hours after S1P-exposure in wild-type (WT) and S1P3 KO but not in S1P2 KO vessels. In conclusion, the S1P-S1P2-G12/13-ROCK signaling pathway appears to have a major influence on α1-adrenergic vasoactivity. This cooperativity might lead to sustained vasoconstriction when increased sympathetic tone is accompanied by increased S1P production as it occurs during acute coronary syndrome and stroke.
Assuntos
Lisofosfolipídeos/farmacologia , Receptores Adrenérgicos alfa 1/fisiologia , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Vasoconstrição/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Animais , Sinergismo Farmacológico , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenilefrina/farmacologia , Piridinas/farmacologia , Esfingosina/farmacologia , Receptores de Esfingosina-1-Fosfato/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Autotaxin (ATX), through its lysophospholipase D activity controls physiological levels of lysophosphatidic acid (LPA) in blood. ATX is overexpressed in multiple types of cancers, and together with LPA generated during platelet activation promotes skeletal metastasis of breast cancer. However, the pathophysiological sequelae of regulated interactions between circulating LPA, ATX, and platelets remain undefined in cancer. In this study, we show that ATX is stored in α-granules of resting human platelets and released upon tumor cell-induced platelet aggregation, leading to the production of LPA. Our in vitro and in vivo experiments using human breast cancer cells that do not express ATX (MDA-MB-231 and MDA-B02) demonstrate that nontumoral ATX controls the early stage of bone colonization by tumor cells. Moreover, expression of a dominant negative integrin αvß3-Δ744 or treatment with the anti-human αvß3 monoclonal antibody LM609, completely abolished binding of ATX to tumor cells, demonstrating the requirement of a fully active integrin αvß3 in this process. The present results establish a new mechanism for platelet contribution to LPA-dependent metastasis of breast cancer cells, and demonstrate the therapeutic potential of disrupting the binding of nontumor-derived ATX with the tumor cells for the prevention of metastasis.
Assuntos
Plaquetas/imunologia , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Integrina alfaVbeta3/imunologia , Diester Fosfórico Hidrolases/imunologia , Animais , Plaquetas/patologia , Neoplasias Ósseas/sangue , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/patologia , Osso e Ossos/imunologia , Osso e Ossos/patologia , Mama/imunologia , Mama/patologia , Neoplasias da Mama/sangue , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Lisofosfolipídeos/imunologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/imunologia , Invasividade Neoplásica/patologia , Ativação PlaquetáriaRESUMO
High expression of autotaxin in cancers is often associated with increased tumor progression, angiogenesis and metastasis. This is explained mainly since autotaxin produces the lipid growth factor, lysophosphatidate (LPA), which stimulates cell division, survival and migration. It has recently become evident that these signaling effects of LPA also produce resistance to chemotherapy and radiation-induced cell death. This results especially from the stimulation of LPA(2) receptors, which depletes the cell of Siva-1, a pro-apoptotic signaling protein and stimulates prosurvival kinase pathways through a mechanism mediated via TRIP-6. LPA signaling also increases the formation of sphingosine 1-phosphate, a pro-survival lipid. At the same time, LPA decreases the accumulation of ceramides, which are used in radiation therapy and by many chemotherapeutic agents to stimulate apoptosis. The signaling actions of extracellular LPA are terminated by its dephosphorylation by a family of lipid phosphate phosphatases (LPP) that act as ecto-enzymes. In addition, lipid phosphate phoshatase-1 attenuates signaling downstream of the activation of both LPA receptors and receptor tyrosine kinases. This makes many cancer cells hypersensitive to the action of various growth factors since they often express low LPP1/3 activity. Increasing our understanding of the complicated signaling pathways that are used by LPA to stimulate cell survival should identify new therapeutic targets that can be exploited to increase the efficacy of chemo- and radio-therapy. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Lisofosfolipídeos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Diester Fosfórico Hidrolases/metabolismo , Tolerância a Radiação , Animais , Humanos , Neoplasias/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de SinaisRESUMO
Mesenchymal stem cells in the dental tissue indicate a disposition for differentiation into diverse dental lineages and contain enormous potential as the important means for regenerative medicine in dentistry. Among various dental tissues, the dental pulp contains stem cells, progenitor cells and odontoblasts for maintaining dentin homeostasis. The conventional culture of stem cells holds a limit as the living tissue constitutes the three-dimensional (3D) structure. Recent development in the organoid cultures have successfully recapitulated 3D structure and advanced to the assembling of different types. In the current study, the protocol for 3D explant culture of the human dental pulp tissue has been established by adopting the organoid culture. After isolating dental pulp from human tooth, the intact tissue was placed between two layers for Matrigel with addition of the culture medium. The reticular outgrowth of pre-odontoblast layer continued for a month and the random accumulation of dentin was observed near the end. Electron microscopy showed the cellular organization and in situ development of dentin, and immunohistochemistry exhibited the expression of odontoblast and stem cell markers in the outgrowth area. Three-dimensional explant culture of human dental pulp will provide a novel platform for understanding stem cell biology inside the tooth and developing the regenerative medicine.
RESUMO
Lysophosphatidic acid (LPA) is a bioactive lipid mediator that regulates a variety of cellular functions such as cell proliferation, migration, survival, calcium mobilization, cytoskeletal rearrangements, and neurite retraction. The biological actions of LPA are mediated by at least six G protein-coupled receptors known as LPAR1-6. Given that LPAR1-3 were among the first LPARs identified, the majority of research efforts have focused on understanding their biology. This review provides an in-depth discussion of LPAR5, which has recently emerged as a key player in regulating normal intestinal homeostasis and modulating pathological conditions such as pain, itch, inflammatory diseases, and cancer. We also present a chronological overview of the efforts made to develop compounds that target LPAR5 for use as tool compounds to probe or validate LPAR5 biology and therapeutic agents for the treatment of inflammatory diseases and cancer.
Assuntos
Neoplasias , Receptores de Ácidos Lisofosfatídicos , Humanos , Neoplasias/tratamento farmacológico , Transdução de Sinais/fisiologia , Lisofosfolipídeos/metabolismo , Proliferação de Células , DorRESUMO
Background/purpose: Human dental pulp stem cells (hDPSCs) possess excellent proliferative and osteogenic differentiation potentials. This study aimed to elucidate the role of lysophosphatidic acid (LPA) signaling in the proliferation and osteogenic differentiation of hDPSCs. Materials and methods: hDPSCs were treated with LPA and proliferation was measured using the cell counting kit-8 assay. Following the osteogenic differentiation of hDPSCs using osteogenic medium in the presence or absence of LPA, alkaline phosphatase (ALP) staining, ALP activity measurements, and RT-qPCR were performed to analyze the osteoblast differentiation. Small interfering RNA (siRNA)-mediated LPAR3 silencing and extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase inhibitors were used to elucidate the molecular mechanisms underlying LPA-induced proliferation and differentiation of hDPSCs. Results: LPA treatment significantly induced proliferation and osteogenic differentiation of hDPSCs. The depletion of LPAR3 expression by LPAR3-speicifc siRNA in hDPSCs diminished LPA-induced proliferation and osteogenic differentiation. The LPAR3-mediated proliferation and osteogenic differentiation of hDPSCs in response to LPA were significantly suppressed by U0126, a selective inhibitor of ERK. Conclusion: These findings suggest that LPA induces the proliferation and osteogenic differentiation of hDPSCs via LPAR3-ERK-dependent pathways.
RESUMO
LPA (lysophosphatidic acid, 1-acyl-2-hydroxy-sn-glycero-3-phosphate), is a growth factor-like lipid mediator that regulates many cellular functions, many of which are unique to malignantly transformed cells. The simple chemical structure of LPA and its profound effects in cancer cells has attracted the attention of the cancer therapeutics field and drives the development of therapeutics based on the LPA scaffold. In biological fluids, LPA is generated by ATX (autotaxin), a lysophospholipase D that cleaves the choline/serine headgroup from lysophosphatidylcholine and lysophosphatidylserine to generate LPA. In the present article, we review some of the key findings that make the ATX-LPA signalling axis an emerging target for cancer therapy.
Assuntos
Neoplasias/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Humanos , Lisofosfolipídeos/metabolismo , Terapia de Alvo Molecular , Invasividade Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Organofosfonatos/farmacologia , Organofosfonatos/uso terapêutico , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/uso terapêutico , Diester Fosfórico Hidrolases/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vascular endothelial growth factor (VEGF) appears to be an important mediator of pathologic retinal angiogenesis. In understanding the mechanisms of pathologic retinal neovascularization, we found that VEGF activates PLD1 in human retinal microvascular endothelial cells, and this event is dependent on Src. In addition, VEGF activates protein kinase C-gamma (PKCgamma) via Src-dependent PLD1 stimulation. Inhibition of Src, PLD1, or PKCgamma via pharmacologic, dominant negative mutant, or siRNA approaches significantly attenuated VEGF-induced human retinal microvascular endothelial cell migration, proliferation, and tube formation. Hypoxia also induced Src-PLD1-PKCgamma signaling in retina, leading to retinal neovascularization. Furthermore, siRNA-mediated down-regulation of VEGF inhibited hypoxia-induced Src-PLD1-PKCgamma activation and neovascularization. Blockade of Src-PLD1-PKCgamma signaling via the siRNA approach also suppressed hypoxia-induced retinal neovascularization. Thus, these observations demonstrate, for the first time, that Src-dependent PLD1-PKCgamma activation plays an important role in pathologic retinal angiogenesis.
Assuntos
Endotélio Vascular/metabolismo , Fosfolipase D/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Retina/metabolismo , Neovascularização Retiniana , Western Blotting , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Imunofluorescência , Humanos , Fosforilação , Retina/patologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Although metastases are the principal cause of cancer-related deaths, the molecular aspects of the role of stromal cells in the establishment of the metastatic niche remain poorly understood. One of the most prevalent sites for cancer metastasis is the lungs. According to recent research, lung stromal cells such as bronchial epithelial cells and resident macrophages secrete autotaxin (ATX), an enzyme with lysophospholipase D activity that promotes cancer progression. In fact, several studies have shown that many cell types in the lung stroma could provide a rich source of ATX in diseases. In the present study, we sought to determine whether ATX derived from alveolar type II epithelial (ATII) pneumocytes could modulate the progression of lung metastasis, which has not been evaluated previously. To accomplish this, we used the B16-F10 syngeneic melanoma model, which readily metastasizes to the lungs when injected intravenously. Because B16-F10 cells express high levels of ATX, we used the CRISPR-Cas9 technology to knock out the ATX gene in B16-F10 cells, eliminating the contribution of tumor-derived ATX in lung metastasis. Next, we used the inducible Cre/loxP system (Sftpc-CreERT2/Enpp2fl/fl) to generate conditional knockout (KO) mice in which ATX is specifically deleted in ATII cells (i.e., Sftpc-KO). Injection of ATX-KO B16-F10 cells into Sftpc-KO or Sftpc-WT control littermates allowed us to investigate the specific contribution of ATII-derived ATX in lung metastasis. We found that targeted KO of ATX in ATII cells significantly reduced the metastatic burden of ATX-KO B16-F10 cells by 30% (unpaired t-test, p = 0.028) compared to Sftpc-WT control mice, suggesting that ATX derived from ATII cells could affect the metastatic progression. We detected upregulated levels of cytokines such as IFNγ (unpaired t-test, p < 0.0001) and TNFα (unpaired t-test, p = 0.0003), which could favor the increase in infiltrating CD8+ T cells observed in the tumor regions of Sftpc-KO mice. Taken together, our results highlight the contribution of host ATII cells as a stromal source of ATX in the progression of melanoma lung metastasis.
RESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor-2 and CFTR through a PSD95/Dlg/ZO-1-based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.
Assuntos
Toxina da Cólera/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia/tratamento farmacológico , Lisofosfolipídeos/farmacologia , Análise de Variância , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Toxina da Cólera/toxicidade , Cricetinae , AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/metabolismo , Diarreia/induzido quimicamente , Proteína 4 Homóloga a Disks-Large , Células Epiteliais/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio , Proteína da Zônula de Oclusão-1RESUMO
Recent studies have evaluated alternatives to the use of live animals in colony health monitoring. Currently, an alternative method that is suitable for all rack types and that has been verified to detect the infectious agents most commonly excluded from mouse colonies is unavailable. We compared the use of filter paper placed on the inside floor of mouse cages to the traditional use of sentinel mice in the detection of several prevalent murine pathogens including mouse hepatitis virus (MHV), murine norovirus (MNV), minute virus of mice (MVM), mouse parvovirus (MPV), Theiler murine encephalomyelitis virus (TMEV), Helicobacter spp., Syphacia obvelata, and Aspiculuris tetraptera. Experimental groups comprised 7 cages containing either 2 pieces of filter paper on the cage floor or 2 ICR sentinel mice. Soiled bedding from pet-store mice was transferred to the experimental cages weekly for 8 wk. At 1 and 2 mo after bedding transfer, the filter papers were evaluated by PCR and sentinel mice were tested by serology and fecal PCR. Filter papers detected all pathogens as effectively (MHV, MNV, MPV, MVM, TMEV S. obvelata, and A. tetraptera) or more effectively (Helicobacter spp.) than sentinel mice at both time points. Filter papers more readily detected pathogens with a high copy number per RT-PCR analysis than a low copy number. Helicobacter spp. were not detected by sentinel mice at either time point. These results indicate that the use of filter paper placed on the interior floor of empty mouse cages and exposed to soiled bedding is efficient in detecting bacteria, endoparasites, and most of the common mouse viruses included in an animal health monitoring program.
Assuntos
Abrigo para Animais , Papel , Infecções por Parvoviridae/veterinária , Doenças dos Roedores/transmissão , Vírus , Animais , Infecções Bacterianas/microbiologia , Infecções Bacterianas/prevenção & controle , Infecções Bacterianas/transmissão , Infecções Bacterianas/veterinária , Camundongos , Camundongos Endogâmicos ICR , Infecções por Parvoviridae/transmissão , Doenças dos Roedores/microbiologia , Doenças dos Roedores/parasitologia , Doenças dos Roedores/prevenção & controle , Vigilância de Evento Sentinela , Viroses/prevenção & controle , Viroses/transmissão , Viroses/veterinária , Viroses/virologiaRESUMO
OBJECTIVES: SSc (scleroderma) is an often fatal disease characterized by widespread tissue fibrosis. Fibroblasts play a key role in SSc-associated fibrosis. This study was designed to determine: (i) whether fibroblasts isolated from skin of patients with SSc have increased lysophosphatidic acid-activated Cl- current (IClLPA) activity vs healthy controls; (ii) whether myofibroblast differentiation is involved in SSc skin fibrosis; and (iii) whether SSc fibroblasts have different proliferation rates vs controls. METHODS: Skin biopsies were taken from involved and uninvolved skin of SSc patients and controls. Whole-cell perforated patch-clamping was used to measure IClLPA activity in fibroblasts isolated and cultured from these biopsies. Western blotting was used to measure α-smooth muscle actin (α-SMA). Proliferation was measured using a colorimetric assay. RESULTS: Fibroblasts cultured from SSc skin show significantly increased IClLPA activity following LPA exposure compared with control skin fibroblasts. α-SMA protein was significantly increased in cultured SSc skin fibroblasts vs controls. No significant differences in proliferation rates were found. CONCLUSIONS: Elevated IClLPA activity is a hallmark of SSc skin fibroblasts. Blocking IClLPA activation may be a new therapeutic approach for treating SSc-associated fibrosis.