Cell type-specific transforming growth factor-ß (TGF-ß) signaling in the regulation of salivary gland fibrosis and regeneration.

Salivary gland damage and hypofunction result from various disorders, including autoimmune Sjögren's disease (SjD) and IgG4-related disease (IgG4-RD), as well as a side effect of radiotherapy for treating head and neck cancers. There are no therapeutic strategies to prevent the loss of salivary gland function in these disorders nor facilitate functional salivary gland regeneration. However, ongoing aquaporin-1 gene therapy trials to restore saliva flow show promise. To identify and develop novel therapeutic targets, we must better understand the cell-specific signaling processes involved in salivary gland regeneration. Transforming growth factor-ß (TGF-ß) signaling is essential to tissue fibrosis, a major endpoint in salivary gland degeneration, which develops in the salivary glands of patients with SjD, IgG4-RD, and radiation-induced damage. Though the deposition and remodeling of extracellular matrix proteins are essential to repair salivary gland damage, pathological fibrosis results in tissue hardening and chronic salivary gland dysfunction orchestrated by multiple cell types, including fibroblasts, myofibroblasts, endothelial cells, stromal cells, and lymphocytes, macrophages, and other immune cell populations. This review is focused on the role of TGF-ß signaling in the development of salivary gland fibrosis and the potential for targeting TGF-ß as a novel therapeutic approach to regenerate functional salivary glands. The studies presented highlight the divergent roles of TGF-ß signaling in salivary gland development and dysfunction and illuminate specific cell populations in damaged or diseased salivary glands that mediate the effects of TGF-ß. Overall, these studies strongly support the premise that blocking TGF-ß signaling holds promise for the regeneration of functional salivary glands.

The minimally important difference for the Xerostomia Inventory among Sjögren's disease patients.

OBJECTIVE: Until now, the clinically relevant improvement for the Xerostomia Inventory (XI) has not been defined. Therefore, our aim was to determine the Minimally Important Difference (MID) of the XI for improvement in dry-mouth symptoms in SjD patients. METHOD: The study recruited 34 SjD patients who underwent sialendoscopy of major salivary glands and 15 SjD patients in a nonintervention control group. XI scores were assessed at several time points. The MID was determined from the mean difference in XI scores between the groups with and without improvement. RESULTS: In the control group, no significant XI score changes were seen. In the sialendoscopy group, a clinically relevant XI score change of four scale points was identified after 1 week. For a prolonged duration (≥16 weeks), a minimum reduction of seven scale points in the XI score was required to indicate clinically relevant improvement. CONCLUSION: In SjD patients, a minimum change of four points in the XI score indicates a clinically relevant improvement for evaluating short-term effects. For prolonged effects, a clinically relevant improvement requires a MID of seven points. The determination of the MID in XI could assist in future studies that evaluate changes in xerostomia.

A combination treatment of low-dose dexamethasone and aspirin-triggered resolvin D1 reduces Sjögren syndrome-like features in a mouse model.

Background: Sjögren syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration and diminished secretory function of the salivary glands. Dexamethasone (DEX) resolves dry mouth and lymphocytic infiltration; however, this treatment is difficult to maintain because of multiple adverse effects (eg, osteoporosis and skin thinning); likewise, aspirin-triggered resolvin D1 (AT-RvD1) increases saliva secretion but cannot eliminate lymphocytic infiltration. Previous studies showed that a combination of low-dose DEX with AT-RvD1 before disease onset prevents SS-like features in a mouse model; however, this is not clinically practical because there are no reliable indicators of SS before disease onset. Therefore, the authors applied the combined treatment at disease onset to show its efficacy and comparative lack of adverse effects, so that it may reasonably be maintained over a patient's lifetime. Methods: NOD/ShiLtJ mice were treated with ethanol (vehicle control), high-dose DEX alone, AT-RvD1 alone, or a combination of low-dose DEX with AT-RvD1 at disease onset for 8 weeks. Then saliva flow rates were measured, and submandibular glands were harvested for histologic analyses. Results: A combined treatment of low-dose DEX with AT-RvD1 significantly decreased mast cell degranulation and lymphocytic infiltration, increased saliva secretion, and restored apical aquaporin-5 expression in submandibular glands of NOD/ShiLtJ mice. Conclusions: Low-dose DEX combined with AT-RvD1 reduces the severity of SS-like manifestation and prevents the development of advanced and potentially irreversible damage, all in a form that can reasonably be administered indefinitely without the need to cease treatment because of secondary effects.

Tumoral P2Y2 receptor modulates tumor growth and host anti-tumor immune responses in a syngeneic murine model of oral cancer.

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.

Intraoperative Visualization and Treatment of Salivary Gland Dysfunction in Sjögren's Syndrome Patients Using Contrast-Enhanced Ultrasound Sialendoscopy (CEUSS).

In sialendoscopy, ducts are dilated and the salivary glands are irrigated with saline. Contrast-enhanced ultrasound sialendoscopy (CEUSS), using microbubbles, may facilitate the monitoring of irrigation solution penetration in the ductal system and parenchyma. It is imperative to test CEUSS for its safety and feasibility in Sjögren's syndrome (SS) patients. CEUSS was performed on 10 SS patients. The primary outcomes were safety, determined by the occurrence of (serious) adverse events ((S)AEs), and feasibility. The secondary outcomes were unstimulated and stimulated whole saliva (UWS and SWS) flow rates, xerostomia inventory (XI), clinical oral dryness score, pain, EULAR Sjögren's syndrome patient reported index (ESSPRI), and gland topographical alterations. CEUSS was technically feasible in all patients. Neither SAEs nor systemic reactions related to the procedure were observed. The main AEs were postoperative pain (two patients) and swelling (two patients). Eight weeks after CEUSS, the median UWS and SWS flow had increased significantly from 0.10 to 0.22 mL/min (p = 0.028) and 0.41 to 0.61 mL/min (p = 0.047), respectively. Sixteen weeks after CEUSS, the mean XI was reduced from 45.2 to 34.2 (p = 0.02). We conclude that CEUSS is a safe and feasible treatment for SS patients. It has the potential to increase salivary secretion and reduce xerostomia, but this needs further investigation.

Synthesis, structure-activity relationships and biological evaluation of benzimidazole derived sulfonylurea analogues as a new class of antagonists of P2Y1 receptor.

The P2Y receptors are responsible for the regulation of various physiological processes including neurotransmission and inflammatory responses. These receptors are also considered as novel potential therapeutic targets for prevention and treatment of thrombosis, neurological disorders, pain, cardiac diseases and cancer. Previously, number of P2Y receptor antagonists has been investigated but they are less potent and non-selective with poor solubility profile. Herein, we present the synthesis of new class of benzimidazole derived sulfonylureas (1a-y) as potent antagonists of P2Y receptors, with the specific aim to explore selective antagonists of P2Y1 receptors. The efficacy and selectivity of the synthesized derivatives 1) against four P2Y receptors i.e., t-P2Y1, h-P2Y2, h-P2Y4, and r-P2Y6Rs was carried out by calcium mobilization assay. The results revealed that except 1b, 1d, 1l, 1m, 1o, 1u, 1v, 1w, and 1y, rest of the synthesized derivatives exhibited moderate to excellent inhibitory potential against P2Y1 receptors. Among the potent antagonists, derivative 1h depicted the maximum inhibition of P2Y1 receptor in calcium signalling assay, with an IC50 value of 0.19 ± 0.04 µM. The potential of inhibition was validated by computational investigations where bonding and non-bonding interactions between ligand and targeted receptor further strengthen the study. The best identified derivative 1h revealed the same binding mechanism as that of already reported selective antagonist of P2Y1 receptor i.e (1-(2- (2-tert-butyl-phenoxy) pyridin-3-yl)-3-4-(trifluoromethoxy) phenylurea but the newly synthesized derivative exhibited better solubility profile. Hence, this derivative can be used as lead candidate for the synthesis of more potential antagonist with much better solubility profile and medicinal importance.

Therapeutic potential for P2Y2 receptor antagonism.

G protein-coupled receptors are the target of more than 30% of all FDA-approved drug therapies. Though the purinergic P2 receptors have been an attractive target for therapeutic intervention with successes such as the P2Y12 receptor antagonist, clopidogrel, P2Y2 receptor (P2Y2R) antagonism remains relatively unexplored as a therapeutic strategy. Due to a lack of selective antagonists to modify P2Y2R activity, studies using primarily genetic manipulation have revealed roles for P2Y2R in a multitude of diseases. These include inflammatory and autoimmune diseases, fibrotic diseases, renal diseases, cancer, and pathogenic infections. With the advent of AR-C118925, a selective and potent P2Y2R antagonist that became commercially available only a few years ago, new opportunities exist to gain a more robust understanding of P2Y2R function and assess therapeutic effects of P2Y2R antagonism. This review discusses the characteristics of P2Y2R that make it unique among P2 receptors, namely its involvement in five distinct signaling pathways including canonical Gαq protein signaling. We also discuss the effects of other P2Y2R antagonists and the pivotal development of AR-C118925. The remainder of this review concerns the mounting evidence implicating P2Y2Rs in disease pathogenesis, focusing on those studies that have evaluated AR-C118925 in pre-clinical disease models.

Tuft Cells Are Present in Submandibular Glands Across Species.

Tuft cells are bottle-shaped, microvilli-projecting chemosensory cells located in the lining of a variety of epithelial tissues and, following their identification approximately 60 years ago, have been linked to immune system function in a variety of epithelia. Until recently, Tuft cells had not been convincingly demonstrated to be present in salivary glands with their detection by transmission electron microscopy only shown in a handful of earlier studies using rat salivary glands, and no follow-up work has been conducted to verify their presence in salivary glands of other species. Here, we demonstrate that Tuft cells are present in the submandibular glands of various species (i.e., mouse, pig and human) using transmission electron microscopy and confocal immunofluorescent analysis for the POU class 2 homeobox 3 (POU2F3), which is considered to be a master regulator of Tuft cell identity.

Early Dry Eye Disease Onset in a NOD.H-2h4 Mouse Model of Sjögren's Syndrome.

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.

Indomethacin Treatment Post-irradiation Improves Mouse Parotid Salivary Gland Function via Modulation of Prostaglandin E2 Signaling.

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.

Evolution, correlation, structural impact and dynamics of emerging SARS-CoV-2 variants.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections remain unmanageable in some parts of the world. As with other RNA viruses, mutations in the SARS-CoV-2 gene have been continuously evolving. Recently, four variants have been identified, B.1.1.7, B.1.351, P.1 and CAL.20C. These variants appear to be more infectious and transmissible than the original Wuhan-Hu-1 virus. Using a combination of bioinformatics and structural analyses, we show that the new SARS-CoV-2 variants emerged in the background of an already known Spike protein mutation D614G together with another mutation P323L in the RNA polymerase of SARS-CoV-2. The phylogenetic analysis showed that the CAL.20C and B.1.351 shared one common ancestor, whereas the B.1.1.7 and P.1 shared a different ancestor. Structural comparisons did not show any significant difference between the wild-type and mutant ACE2/Spike complexes. Structural analysis indicated that the N501Y mutation may increase hydrophobic interactions at the ACE2/Spike interface. However, reported greater binding affinity of N501Y Spike with ACE2 does not seem to be entirely due to increased hydrophobic interactions, given that Spike mutation R417T in P.1 or K417N in B.1.351 results in the loss of a salt-bridge interaction between ACE2 and S-RBD. The calculated change in free energy did not provide a clear trend of S protein stability of mutations in the variants. As expected, we show that the CAL.20C generally migrated from the west coast to the east coast of the USA. Taken together, the analyses suggest that the evolution of variants and their infectivity is complex and may depend upon many factors.

Factors Associated with Emerging and Re-emerging of SARS-CoV-2 Variants.

Global spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has triggered unprecedented scientific efforts, as well as containment and treatment measures. Despite these efforts, SARS-CoV-2 infections remain unmanageable in some parts of the world. Due to inherent mutability of RNA viruses, it is not surprising that the SARS-CoV-2 genome has been continuously evolving since its emergence. Recently, four functionally distinct variants, B.1.1.7, B.1.351, P.1 and CAL.20C, have been identified, and they appear to more infectious and transmissible than the original (Wuhan-Hu-1) virus. Here we provide evidence based upon a combination of bioinformatics and structural approaches that can explain the higher infectivity of the new variants. Our results show that the greater infectivity of SARS-CoV-2 than SARS-CoV can be attributed to a combination of several factors, including alternate receptors. Additionally, we show that new SARS-CoV-2 variants emerged in the background of D614G in Spike protein and P323L in RNA polymerase. The correlation analyses showed that all mutations in specific variants did not evolve simultaneously. Instead, some mutations evolved most likely to compensate for the viral fitness.

P2Y2 receptor antagonism resolves sialadenitis and improves salivary flow in a Sjögren's syndrome mouse model.

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.

P2Y receptors for extracellular nucleotides: Contributions to cancer progression and therapeutic implications.

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.

Radiation-Induced Salivary Gland Dysfunction: Mechanisms, Therapeutics and Future Directions.

Salivary glands sustain collateral damage following radiotherapy (RT) to treat cancers of the head and neck, leading to complications, including mucositis, xerostomia and hyposalivation. Despite salivary gland-sparing techniques and modified dosing strategies, long-term hypofunction remains a significant problem. Current therapeutic interventions provide temporary symptom relief, but do not address irreversible glandular damage. In this review, we summarize the current understanding of mechanisms involved in RT-induced hyposalivation and provide a framework for future mechanistic studies. One glaring gap in published studies investigating RT-induced mechanisms of salivary gland dysfunction concerns the effect of irradiation on adjacent non-irradiated tissue via paracrine, autocrine and direct cell-cell interactions, coined the bystander effect in other models of RT-induced damage. We hypothesize that purinergic receptor signaling involving P2 nucleotide receptors may play a key role in mediating the bystander effect. We also discuss promising new therapeutic approaches to prevent salivary gland damage due to RT.

Cell Sheets Restore Secretory Function in Wounded Mouse Submandibular Glands.

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.

P2Y2 receptors mediate nucleotide-induced EGFR phosphorylation and stimulate proliferation and tumorigenesis of head and neck squamous cell carcinoma cell lines.

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.

Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess.

Copper (Cu) is an essential, yet potentially toxic nutrient, as illustrated by inherited diseases of copper deficiency and excess. Elevated expression of the ATP7A Cu exporter is known to confer copper tolerance, however, the contribution of metal-binding metallothioneins is less clear. In this study, we investigated the relative contributions of ATP7A and the metallothioneins MT-I and MT-II to cell viability under conditions of Cu excess or deficiency. Although the loss of ATP7A increased sensitivity to low Cu concentrations, the absence of MTs did not significantly affect Cu tolerance. However, the absence of all three proteins caused a synthetic lethal phenotype due to extreme Cu sensitivity, indicating that MTs are critical for Cu tolerance only in the absence of ATP7A. A lack of MTs resulted in the trafficking of ATP7A from the trans-Golgi complex in a Cu-dependent manner, suggesting that MTs regulate the delivery of Cu to ATP7A. Under Cu deficiency conditions, the absence of MTs and / or ATP7A enhanced cell proliferation compared to wild type cells, suggesting that these proteins compete with essential Cu-dependent pathways when Cu is scarce. These studies reveal new roles for ATP7A and metallothioneins under both Cu deficiency and excess.

P2 Receptors as Therapeutic Targets in the Salivary Gland: From Physiology to Dysfunction.

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.

ATP7A delivers copper to the lysyl oxidase family of enzymes and promotes tumorigenesis and metastasis.

Lysyl oxidase (LOX) and LOX-like (LOXL) proteins are copper-dependent metalloenzymes with well-documented roles in tumor metastasis and fibrotic diseases. The mechanism by which copper is delivered to these enzymes is poorly understood. In this study, we demonstrate that the copper transporter ATP7A is necessary for the activity of LOX and LOXL enzymes. Silencing of ATP7A inhibited LOX activity in the 4T1 mammary carcinoma cell line, resulting in a loss of LOX-dependent mechanisms of metastasis, including the phosphorylation of focal adhesion kinase and myeloid cell recruitment to the lungs, in an orthotopic mouse model of breast cancer. ATP7A silencing was also found to attenuate LOX activity and metastasis of Lewis lung carcinoma cells in mice. Meta-analysis of breast cancer patients found that high ATP7A expression was significantly correlated with reduced survival. Taken together, these results identify ATP7A as a therapeutic target for blocking LOX- and LOXL-dependent malignancies.