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Head and neck squamous cell carcinomas (HNSCCs) are a heterogenous group of tumors and among the top 10 most common cancers and they arise from the epithelial tissues of the mucosal surfaces of the oral cavity, oropharynx, and larynx. Aberrant purinergic signaling has been associated with various cancer types. Here, we studied the role of the P2Y2 purinergic receptor (P2Y2R) in the context of oral cancer. We utilized bioinformatics analysis of deposited datasets to examine purinome gene expression in HNSCC tumors and cells lines and functionally characterized nucleotide-induced P2 receptor signaling in human FaDu and Cal27 and murine MOC2 oral cancer cell lines. Utilizing tumorigenesis assays with wild-type or P2ry2 knockout MOC2 cells we evaluated the role of P2Y2Rs in tumor growth and the host anti-tumor immune responses. Our data demonstrate that human and murine oral cancer cell lines express numerous P2 receptors, with the P2Y2R being highly expressed. Using syngeneic tumor grafts in wild-type mice, we observed that MOC2 tumors expressing P2Y2R were larger than P2Y2R-/- tumors. Wild-type MOC2 tumors contained a lower population of tumor-infiltrating CD11b+F4/80+ macrophages and CD3+ cells, which were revealed to be CD3+CD4+IFNγ+ T cells, compared to P2Y2R-/- tumors. These results were mirrored when utilizing P2Y2R-/- mice, indicating that the changes in MOC2 tumor growth and to the host anti-tumor immune response were independent of host derived P2Y2Rs. Results suggest that targeted suppression of the P2Y2R in HNSCC cells in vivo, rather than systemic P2Y2R antagonism, may be a more effective treatment strategy for HNSCCs.
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Purpose: To develop a mouse model of human dry eye disease (DED) for investigation of sex differences in autoimmune-associated dry eye pathology. Methods: Ocular surface disease was assessed by quantifying corneal epithelial damage with lissamine green stain in the NOD.H-2h4,IFNγ-/-,CD28-/- (NOD.H-2h4 DKO) mouse model of Sjögren's syndrome (SS). Lacrimal gland function was assessed by tear volume quantification with phenol red thread and lacrimal gland inflammation (i.e., dacryoadenitis) was assessed by quantification of immune cell foci, flow cytometric analysis of immune cell composition, and expression of proinflammatory markers. Results: The NOD.H-2h4 DKO mouse model of SS exhibits greater age-dependent increases in corneal damage than in NOD.H-2h4 parental mice and demonstrates an earlier disease onset in females compared to males. The severity of ocular surface disease correlates with loss of goblet cell density, increased conjunctivitis, and dacryoadenitis that is more pronounced in NOD.H-2h4 DKO than NOD.H-2h4 mice. B cells dominate lacrimal infiltrates in 16-week-old NOD.H-2h4 and NOD.H-2h4 DKO mice, but T helper cells and macrophages are also present. Lacrimal gland expression of proinflammatory genes, including the P2X7 and P2Y2 purinergic receptors, is greater in NOD.H-2h4 DKO than NOD.H-2h4 mice and correlates with dacryoadenitis. Conclusions: Our results demonstrate for the first time that autoimmune dry eye disease occurs in both sexes of NOD.H-2h4 DKO and NOD.H-2h4 mice, with earlier onset in female NOD.H-2h4 DKO mice when compared to males of the same strain. This study demonstrates that both NOD.H-2h4 and NOD.H-2h4 DKO mice are novel models that closely resemble SS-related and sex-dependent DED.
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Dacriocistitis , Síndromes de Ojo Seco , Aparato Lagrimal , Síndrome de Sjögren , Animales , Dacriocistitis/patología , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/metabolismo , Femenino , Aparato Lagrimal/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Síndrome de Sjögren/genética , Síndrome de Sjögren/metabolismoRESUMEN
Annually, >600,000 new cases of head and neck cancer (HNC) are diagnosed worldwide with primary treatment being surgery and radiotherapy. During ionizing radiation (IR) treatment of HNC, healthy salivary glands are collaterally damaged, leading to loss of function that severely diminishes the quality of life for patients due to increased health complications, including oral infections and sores, cavities, and malnutrition, among others. Therapies for salivary hypofunction are ineffective and largely palliative, indicating a need for further research to uncover effective approaches to prevent or restore loss of salivary gland function following radiotherapy. Previous work in our lab implicated prostaglandin E2 (PGE2) as an inflammatory mediator whose release from radiation-exposed cells promotes salivary gland damage and loss of function. Deletion of the P2X7 purinergic receptor for extracellular ATP reduces PGE2 secretion in irradiated primary parotid gland cells, and salivary gland function is enhanced in irradiated P2X7R-/- mice compared to wild-type mice. However, the role of PGE2 signaling in irradiated salivary glands is unclear and understanding the mechanism of PGE2 action is a goal of this study. Results show that treatment of irradiated mice with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, which reduces PGE2 production via inhibition of cyclooxygenase-1 (COX-1), improves salivary gland function compared to irradiated vehicle-treated mice. To define the signaling pathway whereby PGE2 induces salivary gland dysfunction, primary parotid gland cells treated with PGE2 have increased c-Jun N-terminal Kinase (JNK) activation and cell proliferation and reduced amylase levels and store-operated calcium entry (SOCE). The in vivo effects of blocking PGE2 production were also examined and irradiated mice receiving indomethacin injections have reduced JNK activity at 8 days post-irradiation and reduced proliferation and increased amylase levels at day 30, as compared to irradiated mice without indomethacin. Combined, these data suggest a mechanism whereby irradiation-induced PGE2 signaling to JNK blocks critical steps in saliva secretion manifested by a decrease in the quality (diminished amylase) and quantity (loss of calcium channel activity) of saliva, that can be restored with indomethacin. These findings encourage further attempts evaluating indomethacin as a viable therapeutic option to prevent damage to salivary glands caused by irradiation of HNC in humans.
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OBJECTIVE: Sjögren's syndrome (SS) is a chronic autoimmune exocrinopathy characterized by lymphocytic infiltration of the salivary and lacrimal glands and decreased saliva and tear production. Previous studies indicate that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is upregulated in numerous models of salivary gland inflammation (i.e., sialadenitis), where it has been implicated as a key mediator of chronic inflammation. Here, we evaluate both systemic and localized P2Y2R antagonism as a means to resolve sialadenitis in the NOD.H-2h4,IFNγ-/-,CD28-/- (NOD.H-2h4 DKO) mouse model of SS. DESIGN: Female 4.5 month old NOD.H-2h4 DKO mice received daily intraperitoneal injections for 10 days of the selective P2Y2R antagonist, AR-C118925, or vehicle-only control. Single-dose localized intraglandular antagonist delivery into the Wharton's duct was also evaluated. Carbachol-induced saliva was measured and then submandibular glands (SMGs) were isolated and either fixed and paraffin-embedded for H&E staining, homogenized for RNA isolation or dissociated for flow cytometry analysis. RESULTS: Intraperitoneal injection, but not localized intraglandular administration, of AR-C118925 significantly enhanced carbachol-induced salivation and reduced lymphocytic foci and immune cell markers in SMGs of 5 month old NOD.H-2h4 DKO mice, compared to vehicle-injected control mice. We found that B cells represent the primary immune cell population in inflamed SMGs of NOD.H-2h4 DKO mice that express elevated levels of P2Y2R compared to C57BL/6 control mice. We further demonstrate a role for P2Y2Rs in mediating B cell migration and the release of IgM. CONCLUSION: Our findings suggest that the P2Y2R represents a novel therapeutic target for the treatment of Sjögren's syndrome.
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Sialadenitis , Síndrome de Sjögren , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Sialadenitis/tratamiento farmacológico , Síndrome de Sjögren/tratamiento farmacológico , Glándula SubmandibularRESUMEN
Purinergic receptors for extracellular nucleotides and nucleosides contribute to a vast array of cellular and tissue functions, including cell proliferation, intracellular and transmembrane ion flux, immunomodulation and thrombosis. In mammals, the purinergic receptor system is composed of G protein-coupled P1 receptors A1, A2A, A2B and A3 for extracellular adenosine, P2X1-7 receptors that are ATP-gated ion channels and G protein-coupled P2Y1,2,4,6,11,12,13 and 14 receptors for extracellular ATP, ADP, UTP, UDP and/or UDP-glucose. Recent studies have implicated specific P2Y receptor subtypes in numerous oncogenic processes, including cancer tumorigenesis, metastasis and chemotherapeutic drug resistance, where G protein-mediated signaling cascades modulate intracellular ion concentrations and activate downstream protein kinases, Src family kinases as well as numerous mitogen-activated protein kinases. We are honored to contribute to this special issue dedicated to the founder of the field of purinergic signaling, Dr. Geoffrey Burnstock, by reviewing the diverse roles of P2Y receptors in the initiation, progression and metastasis of specific cancers with an emphasis on pharmacological and genetic strategies employed to delineate cell-specific and P2Y receptor subtype-specific responses that have been investigated using in vitro and in vivo cancer models. We further highlight bioinformatic and empirical evidence on P2Y receptor expression in human clinical specimens and cover clinical perspectives where P2Y receptor-targeting interventions may have therapeutic relevance to cancer treatment.
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Antineoplásicos/administración & dosificación , Progresión de la Enfermedad , Líquido Extracelular/metabolismo , Neoplasias/metabolismo , Nucleótidos/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Líquido Extracelular/efectos de los fármacos , Humanos , Neoplasias/tratamiento farmacológico , Agonistas del Receptor Purinérgico P2Y/administración & dosificación , Antagonistas del Receptor Purinérgico P2Y/administración & dosificación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Thermoresponsive cell culture plates release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining functional extracellular matrix proteins and tight junctions, both of which indicate formation of intact and functional tissue. Our recent studies demonstrated that cell sheets are highly effective in promoting mouse submandibular gland (SMG) cell differentiation and recovering tissue integrity. However, these studies were performed only at early time points and extension of the observation period is needed to investigate duration of the cell sheets. Thus, the goal of this study was to demonstrate that treatment of wounded mouse SMG with cell sheets is capable of increasing salivary epithelial integrity over extended time periods. The results indicate that cell sheets promote tissue organization as early as eight days after transplantation and that these effects endure through Day 20. Furthermore, cell sheet transplantation in wounded SMG induces a significant time-dependent enhancement of cell polarization, differentiation and ion transporter expression. Finally, this treatment restored saliva quantity to pre-wounding levels at both eight and twenty days post-surgery and significantly improved saliva quality at twenty days post-surgery. These data indicate that cell sheets engineered with thermoresponsive cell culture plates are useful for salivary gland regeneration and provide evidence for the long-term stability of cell sheets, thereby offering a potential new therapeutic strategy for treating hyposalivation.
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Saliva/fisiología , Glándula Submandibular/metabolismo , Animales , Anoctamina-1/metabolismo , Acuaporina 5/metabolismo , Diferenciación Celular , Femenino , Ratones , Ratones Endogámicos C57BL , Saliva/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Glándula Submandibular/citología , Glándula Submandibular/patología , Cicatrización de Heridas , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
OBJECTIVES: To assess functional expression of the P2Y2 nucleotide receptor (P2Y2R) in head and neck squamous cell carcinoma (HNSCC) cell lines and define its role in nucleotide-induced epidermal growth factor receptor (EGFR) transactivation. The use of anti-EGFR therapeutics to treat HNSCC is hindered by intrinsic and acquired drug resistance. Defining novel pathways that modulate EGFR signaling could identify additional targets to treat HNSCC. MATERIALS AND METHODS: In human HNSCC cell lines CAL27 and FaDu and the mouse oral cancer cell line MOC2, P2Y2R contributions to extracellular nucleotide-induced changes in intracellular free Ca2+ concentration and EGFR and extracellular signal-regulated kinase (ERK1/2) phosphorylation were determined using the ratiometric Ca2+ indicator fura-2 and immunoblot analysis, respectively. Genetic knockout of P2Y2Rs using CRISPR technology or pharmacological inhibition with P2Y2R-selective antagonist AR-C118925 defined P2Y2R contributions to in vivo tumor growth. RESULTS: P2Y2R agonists UTP and ATP increased intracellular Ca2+ levels and ERK1/2 and EGFR phosphorylation in CAL27 and FaDu cells, responses that were inhibited by AR-C118925 or P2Y2R knockout. P2Y2R-mediated EGFR phosphorylation was also attenuated by inhibition of the adamalysin family of metalloproteases or Src family kinases. P2Y2R knockout reduced UTP-induced CAL27 cell proliferation in vitro and significantly reduced CAL27 and FaDu tumor xenograft volume in vivo. In a syngeneic mouse model of oral cancer, AR-C118925 administration reduced MOC2 tumor volume. CONCLUSION: P2Y2Rs mediate HNSCC cell responses to extracellular nucleotides and genetic or pharmacological blockade of P2Y2R signaling attenuates tumor cell proliferation and tumorigenesis, suggesting that the P2Y2R represents a novel therapeutic target in HNSCC.
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Although often overlooked in our daily lives, saliva performs a host of necessary physiological functions, including lubricating and protecting the oral cavity, facilitating taste sensation and digestion and maintaining tooth enamel. Therefore, salivary gland dysfunction and hyposalivation, often resulting from pathogenesis of the autoimmune disease Sjögren's syndrome or from radiotherapy of the head and neck region during cancer treatment, severely reduce the quality of life of afflicted patients and can lead to dental caries, periodontitis, digestive disorders, loss of taste and difficulty speaking. Since their initial discovery in the 1970s, P2 purinergic receptors for extracellular nucleotides, including ATP-gated ion channel P2X and G protein-coupled P2Y receptors, have been shown to mediate physiological processes in numerous tissues, including the salivary glands where P2 receptors represent a link between canonical and non-canonical saliva secretion. Additionally, extracellular nucleotides released during periods of cellular stress and inflammation act as a tissue alarmin to coordinate immunological and tissue repair responses through P2 receptor activation. Accordingly, P2 receptors have gained widespread clinical interest with agonists and antagonists either currently undergoing clinical trials or already approved for human use. Here, we review the contributions of P2 receptors to salivary gland function and describe their role in salivary gland dysfunction. We further consider their potential as therapeutic targets to promote physiological saliva flow, prevent salivary gland inflammation and enhance tissue regeneration.
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Head and neck cancer treatments typically involve a combination of surgery and radiotherapy, often leading to collateral damage to nearby tissues causing unwanted side effects. Radiation damage to salivary glands frequently leads to irreversible dysfunction by poorly understood mechanisms. The P2X7 receptor (P2X7R) is a ligand-gated ion channel activated by extracellular ATP released from damaged cells as "danger signals." P2X7R activation initiates apoptosis and is involved in numerous inflammatory disorders. In this study, we utilized P2X7R knockout (P2X7R-/-) mice to determine the role of the receptor in radiation-induced salivary gland damage. Results indicate a dose-dependent increase in γ-radiation-induced ATP release from primary parotid gland cells of wild-type but not P2X7R-/- mice. Despite these differences, apoptosis levels are similar in parotid glands of wild-type and P2X7R-/- mice 24-72 h after radiation. However, γ-radiation caused elevated prostaglandin E2 (PGE2) release from primary parotid cells of wild-type but not P2X7R-/- mice. To attempt to uncover the mechanism underlying differential PGE2 release, we evaluated the expression and activities of cyclooxygenase and PGE synthase isoforms. There were no consistent trends in these mediators following radiation that could explain the reduction in PGE2 release in P2X7R-/- mice. Irradiated P2X7R-/- mice have stimulated salivary flow rates similar to unirradiated controls, whereas irradiated wild-type mice have significantly decreased salivary flow rates compared with unirradiated controls. Notably, treatment with the P2X7R antagonist A438079 preserves stimulated salivary flow rates in wild-type mice following γ-radiation. These data suggest that P2X7R antagonism is a promising approach for preventing γ-radiation-induced hyposalivation.
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Rayos gamma , Glándula Parótida/metabolismo , Traumatismos por Radiación/prevención & control , Receptores Purinérgicos P2X7/deficiencia , Salivación , Xerostomía/prevención & control , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Ratones Endogámicos C57BL , Ratones Noqueados , Glándula Parótida/efectos de los fármacos , Glándula Parótida/fisiopatología , Prostaglandina-E Sintasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Traumatismos por Radiación/genética , Traumatismos por Radiación/metabolismo , Traumatismos por Radiación/fisiopatología , Receptores Purinérgicos P2X7/efectos de los fármacos , Receptores Purinérgicos P2X7/genética , Salivación/efectos de los fármacos , Xerostomía/genética , Xerostomía/metabolismo , Xerostomía/fisiopatologíaRESUMEN
Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1ß and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1ß, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R-/-). P2X7R-mediated IL-1ß release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1ß release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28-/-, IFNγ-/-, NOD.H-2h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.
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Células Epiteliales/metabolismo , Interleucina-1beta/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Piridinas/farmacología , Receptores Purinérgicos P2X7/metabolismo , Síndrome de Sjögren/metabolismo , Glándula Submandibular/metabolismo , Tetrazoles/farmacología , Animales , Antígenos CD28/genética , Antígenos CD28/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/patología , Inflamasomas , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Transporte Iónico/efectos de los fármacos , Transporte Iónico/genética , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Potasio/metabolismo , Receptores Purinérgicos P2X7/genética , Síndrome de Sjögren/genética , Síndrome de Sjögren/patología , Sodio/metabolismo , Glándula Submandibular/patologíaRESUMEN
Sixty to seventy percent of IFN-γ(-/-) NOD.H-2h4 mice given sodium iodide (NaI)-supplemented water develop a slow onset autoimmune thyroid disease, characterized by thyrocyte epithelial cell (TEC) hyperplasia and proliferation (H/P). TEC H/P develops much earlier in CD28(-/-) mice and nearly 100% (both sexes) have severe TEC H/P at 4 mo of age. Without NaI supplementation, 50% of 5- to 6-mo-old CD28(-/-)IFN-γ(-/-) mice develop severe TEC H/P, and 2-3 wk of NaI is sufficient for optimal development of severe TEC H/P. Mice with severe TEC H/P are hypothyroid, and normalization of serum thyroxine levels does not reduce TEC H/P. Activated CD4(+) T cells are sufficient to transfer TEC H/P to SCID recipients. Thyroids of mice with TEC H/P have infiltrating T cells and expanded numbers of proliferating thyrocytes that highly express CD40. CD40 facilitates, but is not required for, development of severe TEC H/P, as CD40(-/-)IFN-γ(-/-)CD28(-/-) mice develop severe TEC H/P. Accelerated development of TEC H/P in IFN-γ(-/-)CD28(-/-) mice is a result of reduced regulatory T cell (Treg) numbers, as CD28(-/-) mice have significantly fewer Tregs, and transfer of CD28(+) Tregs inhibits TEC H/P. Essentially all female IFN-γ(-/-)CD28(-/-) NOD.H-2h4 mice have substantial lymphocytic infiltration of salivary glands and reduced salivary flow by 6 mo of age, thereby providing an excellent new model of autoimmune exocrinopathy of the salivary gland. This is one of very few models where autoimmune thyroid disease and hypothyroidism develop in most mice by 4 mo of age. This model will be useful for studying the effects of hypothyroidism on multiple organ systems.
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Enfermedades Autoinmunes/etiología , Modelos Animales de Enfermedad , Hipotiroidismo/etiología , Enfermedades de las Glándulas Salivales/etiología , Enfermedades de la Tiroides/etiología , Animales , Antígenos CD28/fisiología , Antígenos CD40/fisiología , Células Cultivadas , Células Epiteliales/patología , Hiperplasia , Interferón gamma/fisiología , Yodo/farmacología , Ratones , Ratones Endogámicos C57BL , Linfocitos T/fisiología , Glándula Tiroides/patología , Tiroxina/sangreRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of memory and cognitive ability and is a serious cause of mortality. Many of the pathological characteristics associated with AD are revealed post-mortem, including amyloid-ß plaque deposition, neurofibrillary tangles containing hyperphosphorylated tau proteins and neuronal loss in the hippocampus and cortex. Although several genetic mutations and risk factors have been associated with the disease, the causes remain poorly understood. Study of disease-initiating mechanisms and AD progression in humans is inherently difficult as most available tissue specimens are from late-stages of disease. Therefore, AD researchers rely on in vitro studies and the use of AD animal models where neuroinflammation has been shown to be a major characteristic of AD. Purinergic receptors are a diverse family of proteins consisting of P1 adenosine receptors and P2 nucleotide receptors for ATP, UTP and their metabolites. This family of receptors has been shown to regulate a wide range of physiological and pathophysiological processes, including neuroinflammation, and may contribute to the pathogenesis of neurodegenerative diseases like Parkinson's disease, multiple sclerosis and AD. Experimental evidence from human AD tissue has suggested that purinergic receptors may play a role in AD progression and studies using selective purinergic receptor agonists and antagonists in vitro and in AD animal models have demonstrated that purinergic receptors represent novel therapeutic targets for the treatment of AD. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.
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Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Encefalitis/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Astrocitos/metabolismo , Encéfalo/metabolismo , Humanos , Microglía/metabolismo , Placa Amiloide/metabolismoRESUMEN
Transforming growth factor-ß (TGF-ß) is a multi-functional cytokine with a well-described role in the regulation of tissue fibrosis and regeneration in the liver, kidney and lung. Submandibular gland (SMG) duct ligation and subsequent deligation in rodents is a classical model for studying salivary gland damage and regeneration. While previous studies suggest that TGF-ß may contribute to salivary gland fibrosis, the expression of TGF-ß signaling components has not been investigated in relation to mouse SMG duct ligation-induced fibrosis and regeneration following ductal deligation. Following a 7 day SMG duct ligation, TGF-ß1 and TGF-ß3 were significantly upregulated in the SMG, as were TGF-ß receptor 1 and downstream Smad family transcription factors in salivary acinar cells, but not in ductal cells. In acinar cells, duct ligation also led to upregulation of snail, a Smad-activated E-cadherin repressor and regulator of epithelial-mesenchymal transition, whereas in ductal cells upregulation of E-cadherin was observed while snail expression was unchanged. Upregulation of these TGF-ß signaling components correlated with upregulation of fibrosis markers collagen 1 and fibronectin, responses that were inhibited by administration of the TGF-ß receptor 1 inhibitors SB431542 or GW788388. After SMG regeneration following a 28 day duct deligation, TGF-ß signaling components and epithelial-mesenchymal transition markers returned to levels similar to non-ligated controls. The results from this study indicate that increased TGF-ß signaling contributes to duct ligation-induced changes in salivary epithelium that correlate with glandular fibrosis. Furthermore, the reversibility of enhanced TGF-ß signaling in acinar cells of duct-ligated mouse SMG after deligation indicates that this is an ideal model for studying TGF-ß signaling mechanisms in salivary epithelium as well as mechanisms of fibrosis initiation and their resolution.
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Transducción de Señal/genética , Glándula Submandibular/metabolismo , Glándula Submandibular/patología , Factor de Crecimiento Transformador beta/genética , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Células Acinares/patología , Animales , Benzamidas/farmacología , Biomarcadores/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Ligadura , Masculino , Ratones Endogámicos C57BL , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/farmacología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Regeneración/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factores de Transcripción de la Familia Snail , Glándula Submandibular/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their structure-activity relationship, encouraged us to develop new NPP1 inhibitors. Specifically, we synthesized ATP-α-thio-ß,γ-CH2 (1), ATP-α-thio-ß,γ-CCl2 (2), ATP-α-CH2-γ-thio (3), and 8-SH-ATP (4) and established their resistance to hydrolysis by NPP1,3 and NTPDase1,2,3,8 (<5% hydrolysis) (NTPDase = ectonucleoside triphosphate diphosphohydrolase). Analogues 1-3 at 100 µM inhibited thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis by NPP1 and NPP3 by >90% and 23-43%, respectively, and only slightly affected (0-40%) hydrolysis of ATP by NTPDase1,2,3,8. Analogue 3 is the most potent NPP1 inhibitor currently known, Ki = 20 nM and IC50 = 0.39 µM. Analogue 2a is a selective NPP1 inhibitor with Ki = 685 nM and IC50 = 0.57 µM. Analogues 1-3 were found mostly to be nonagonists of P2Y1/P2Y2/P2Y11 receptors. Docking analogues 1-3 into the NPP1 model suggested that activity correlates with the number of H-bonds with binding site residues. In conclusion, we propose analogues 2a and 3 as highly promising NPP1 inhibitors.
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Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/síntesis química , Organofosfatos/síntesis química , Organofosfonatos/síntesis química , Organotiofosfatos/síntesis química , Pirofosfatasas/antagonistas & inhibidores , Adenosina Trifosfato/farmacología , Animales , Sitios de Unión , Células COS , Dominio Catalítico , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Enlace de Hidrógeno , Hidrólisis , Simulación del Acoplamiento Molecular , Organofosfatos/farmacología , Organofosfonatos/farmacología , Organotiofosfatos/farmacología , Hidrolasas Diéster Fosfóricas , Agonistas del Receptor Purinérgico P2Y/síntesis química , Agonistas del Receptor Purinérgico P2Y/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Hyposalivation resulting from salivary gland dysfunction leads to poor oral health and greatly reduces the quality of life of patients. Current treatments for hyposalivation are limited. However, regenerative medicine to replace dysfunctional salivary glands represents a revolutionary approach. The ability of dispersed salivary epithelial cells or salivary gland-derived progenitor cells to self-organize into acinar-like spheres or branching structures that mimic the native tissue holds promise for cell-based reconstitution of a functional salivary gland. However, the mechanisms involved in salivary epithelial cell aggregation and tissue reconstitution are not fully understood. This study investigated the role of the P2Y2 nucleotide receptor (P2Y2R), a G protein-coupled receptor that is upregulated following salivary gland damage and disease, in salivary gland reconstitution. In vitro results with the rat parotid acinar Par-C10 cell line indicate that P2Y2R activation with the selective agonist UTP enhances the self-organization of dispersed salivary epithelial cells into acinar-like spheres. Other results indicate that the P2Y2R-mediated response is dependent on epidermal growth factor receptor activation via the metalloproteases ADAM10/ADAM17 or the α5ß1 integrin/Cdc42 signaling pathway, which leads to activation of the MAPKs JNK and ERK1/2. Ex vivo data using primary submandibular gland cells from wild-type and P2Y2R(-/-) mice confirmed that UTP-induced migratory responses required for acinar cell self-organization are mediated by the P2Y2R. Overall, this study suggests that the P2Y2R is a promising target for salivary gland reconstitution and identifies the involvement of two novel components of the P2Y2R signaling cascade in salivary epithelial cells, the α5ß1 integrin and the Rho GTPase Cdc42.
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Agregación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Glándula Parótida/efectos de los fármacos , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/efectos de los fármacos , Glándula Submandibular/efectos de los fármacos , Uridina Trifosfato/farmacología , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Animales , Línea Celular , Células Epiteliales/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfa5beta1/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glándula Parótida/citología , Glándula Parótida/metabolismo , Fosforilación , Inhibidores de Proteasas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Receptores Purinérgicos P2Y2/deficiencia , Receptores Purinérgicos P2Y2/genética , Glándula Submandibular/citología , Glándula Submandibular/metabolismo , Transfección , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Neuroinflammation is a prominent feature in Alzheimer's disease (AD) and activation of the brain's innate immune system, particularly microglia, has been postulated to both retard and accelerate AD progression. Recent studies indicate that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is an important regulator of innate immunity by assisting in the recruitment of monocytes to injured tissue, neutrophils to bacterial infections and eosinophils to allergen-infected lungs. In this study, we investigated the role of the P2Y2R in progression of an AD-like phenotype in the TgCRND8 mouse model that expresses Swedish and Indiana mutations in amyloid precursor protein (APP). Our results indicate that P2Y 2 R expression is upregulated in TgCRND8 mouse brain within 10 weeks of age and then decreases after 25 weeks of age, as compared to littermate controls expressing low levels of the P2Y 2 R. TgCRND8 mice with homozygous P2Y 2 R deletion survive less than 5 weeks, whereas mice with heterozygous P2Y 2 R deletion survive for 12 weeks, a time point when TgCRND8 mice are fully viable. Heterozygous P2Y 2 R deletion in TgCRND8 mice increased ß-amyloid (Aß) plaque load and soluble Aß1-42 levels in the cerebral cortex and hippocampus, decreased the expression of the microglial marker CD11b in these brain regions and caused neurological deficits within 10 weeks of age, as compared to age-matched TgCRND8 mice. These findings suggest that the P2Y2R is important for the recruitment and activation of microglial cells in the TgCRND8 mouse brain and that the P2Y2R may regulate neuroprotective mechanisms through microglia-mediated clearance of Aß that when lost can accelerate the onset of an AD-like phenotype in the TgCRND8 mouse.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Receptores Purinérgicos P2Y2/deficiencia , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/patología , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patología , Receptores Purinérgicos P2Y2/genéticaRESUMEN
The pro-inflammatory cytokine interleukin-1ß (IL-1ß), whose levels are elevated in the brain in Alzheimer's and other neurodegenerative diseases, has been shown to have both detrimental and beneficial effects on disease progression. In this article, we demonstrate that incubation of mouse primary cortical neurons (mPCNs) with IL-1ß increases the expression of the P2Y2 nucleotide receptor (P2Y2R) and that activation of the up-regulated receptor with UTP, a relatively selective agonist of the P2Y2R, increases neurite outgrowth. Consistent with the accepted role of cofilin in the regulation of neurite extension, results indicate that incubation of IL-1ß-treated mPCNs with UTP increases the phosphorylation of cofilin, a response absent in PCNs isolated from P2Y2R(-/-) mice. Other findings indicate that function-blocking anti-αv ß3/5 integrin antibodies prevent UTP-induced cofilin activation in IL-1ß-treated mPCNs, suggesting that established P2Y2R/αv ß3/5 interactions that promote G12 -dependent Rho activation lead to cofilin phosphorylation involved in neurite extension. Cofilin phosphorylation induced by UTP in IL-1ß-treated mPCNs is also decreased by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), suggesting a role for P2Y2R-mediated and Gq-dependent calcium mobilization in neurite outgrowth. Taken together, these studies indicate that up-regulation of P2Y2Rs in mPCNs under pro-inflammatory conditions can promote cofilin-dependent neurite outgrowth, a neuroprotective response that may be a novel pharmacological target in the treatment of neurodegenerative diseases.
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Corteza Cerebral/citología , Interleucina-1beta/farmacología , Neuronas/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Integrina alfaVbeta3/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuritas/ultraestructura , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Fosforilación , Cultivo Primario de Células , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/genética , Receptores de Vitronectina/metabolismo , Regulación hacia Arriba , Uridina Trifosfato/farmacologíaRESUMEN
Inflammation of the salivary gland is a well-documented aspect of salivary gland dysfunction that occurs in Sjogren's syndrome (SS), an autoimmune disease, and in γ-radiation-induced injury during treatment of head and neck cancers. Extracellular nucleotides have gained recognition as key modulators of inflammation through activation of cell surface ionotropic and metabotropic receptors, although the contribution of extracellular nucleotides to salivary gland inflammation is not well understood. In vitro studies using submandibular gland (SMG) cell aggregates isolated from wild-type C57BL/6 mice indicate that treatment with ATP or the high affinity P2X7R agonist 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) induces membrane blebbing and enhances caspase activity, responses that were absent in SMG cell aggregates isolated from mice lacking the P2X7R (P2X7R(-/-)). Additional studies with SMG cell aggregates indicate that activation of the P2X7R with ATP or BzATP stimulates the cleavage and release of α-fodrin, a cytoskeletal protein thought to act as an autoantigen in the development of SS. In vivo administration of BzATP to ligated SMG excretory ducts enhances immune cell infiltration into the gland and initiates apoptosis of salivary epithelial cells in wild-type, but not P2X7R(-/-), mice. These findings indicate that activation of the P2X7R contributes to salivary gland inflammation in vivo, suggesting that the P2X7R may represent a novel target for the treatment of salivary gland dysfunction.
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Mediadores de Inflamación/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Glándula Submandibular/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/fisiología , Animales , Epitelio/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glándulas Salivales/metabolismoRESUMEN
The activation by extracellular nucleotides of pancreatic P2Y receptors, particularly, the P2Y(1)R subtype, increases insulin secretion. Therefore, we developed analogues of the P2Y(1)R receptor agonist 2-MeS-ADP, as potential antidiabetic drugs. Analogue 3A was found to be a potent P2Y(1)R agonist (EC(50) = 0.038 µM vs 0.0025 µM for 2-MeS-ADP) showing no activity at P2Y(2/4/6)Rs. Analogue 3A was stable at pH 1.4 (t(1/2) = 7.3 h) and resistant to hydrolysis vs 2-MeS-ADP by alkaline phosphatase (t(1/2) = 6 vs 4.5 h), human e-NPP1 (4% vs 16% hydrolysis after 20 min), and human blood serum (30% vs 50% hydrolysis after 24 h). Intravenous administration of 3A in naive rats decreased blood glucose from 155 mg/dL to normal values, ca. 87 mg/dL, unlike glibenclamide, leading to subnormal values (i.e., 63 mg/dL). Similar observations were made for streptozotocin (STZ)-treated and db(+)/db(-) mouse models. Furthermore, 3A inhibits platelet aggregation in vitro and elongates bleeding time in mice (iv administration of 30 mg of 3A/kg), increasing bleeding time to 16 vs 9 min for Prasugrel. Oral administration of 30 mg/kg 3A to rats increased tail bleeding volume, similar to aspirin. These findings suggest that 3A may be an effective treatment for type 2 diabetes by reducing both blood glucose levels and platelet aggregation.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Agonistas del Receptor Purinérgico P2Y/química , Agonistas del Receptor Purinérgico P2Y/uso terapéutico , Animales , Glucemia/análisis , Línea Celular Tumoral , Diabetes Mellitus Experimental/tratamiento farmacológico , Prueba de Tolerancia a la Glucosa , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos ICR , Estructura Molecular , Agonistas del Receptor Purinérgico P2Y/farmacología , Ratas , Ratas WistarRESUMEN
Extracellular nucleotides induce cellular responses in the central nervous system (CNS) through the activation of ionotropic P2X and metabotropic P2Y nucleotide receptors. Activation of these receptors regulates a wide range of physiological and pathological processes. In this review, we present an overview of the current literature regarding P2X and P2Y receptors in the CNS with a focus on the contribution of P2X7 and P2Y(2) receptor-mediated responses to neuroinflammatory and neuroprotective mechanisms.