TRPC1 expression and function inhibit ER stress and cell death in salivary gland cells.
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ABSTRACT:
SUBMITTER: Sukumaran P
PROVIDER: S-EPMC6524637 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Calcium-activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca(2+) influx that activates ion channels such as CaCC to initiate Cl(-) efflux. However direct evidence as well as the molecular identity of the Ca(2+) channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl(-) current was activated by increasing [Ca(2+)]i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh-A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store-depletion and activates TRPC1-mediated Ca(2+) entry, potentiated the Cl(-) current, which was inhibited by the addition of a non-specific TRPC channel blocker SKF96365 or removal of external Ca(2+). Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca(2+) entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl(-) currents upon increasing [Ca(2+)]i. These Cl(-) currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh-A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl(-) currents without decreasing TMEM16a expression. Together the data suggests that Ca(2+) entry via the TRPC1 channels is essential for the activation of CaCC.
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Project description:Agonist-induced Ca(2+) entry via store-operated Ca(2+) (SOC) channels is suggested to regulate a wide variety of cellular functions, including salivary gland fluid secretion. However, the molecular components of these channels and their physiological function(s) are largely unknown. Here we report that attenuation of SOC current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). Neurotransmitter-regulated salivary gland fluid secretion in TRPC1-deficient TRPC1(-/-) mice was severely decreased (by 70%). Further, agonist- and thapsigargin-stimulated SOC channel activity was significantly reduced in salivary gland acinar cells isolated from TRPC1(-/-) mice. Deletion of TRPC1 also eliminated sustained Ca(2+)-dependent potassium channel activity, which depends on Ca(2+) entry and is required for fluid secretion. Expression of key proteins involved in fluid secretion and Ca(2+) signaling, including STIM1 and other TRPC channels, was not altered. Together, these data demonstrate that reduced SOC entry accounts for the severe loss of salivary gland fluid secretion in TRPC1(-/-) mice. Thus, TRPC1 is a critical component of the SOC channel in salivary gland acinar cells and is essential for neurotransmitter-regulation of fluid secretion.
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Project description:ObjectivesThis study aims to determine a role of interleukin-17A (IL-17) in salivary gland (SG) dysfunction and therapeutic effects of targeting IL-17 in SG for treating autoimmune sialadenitis in primary Sjögren's syndrome (pSS).MethodsSalivary IL-17 levels and IL-17-secreting cells in labial glands of pSS patients were examined. Kinetic changes of IL-17-producing cells in SG from mice with experimental Sjögren's syndrome (ESS) were analysed. To determine a role of IL-17 in salivary secretion, IL-17-deficient mice and constructed chimeric mice with IL-17 receptor C (IL-17RC) deficiency in non-hematopoietic and hematopoietic cells were examined for saliva flow rates during ESS development. Both human and murine primary SG epithelial cells were treated with IL-17 for measuring cholinergic activation-induced calcium movement. Moreover, SG functions were assessed in ESS mice with salivary retrograde cannulation of IL-17 neutralisation antibodies.ResultsIncreased salivary IL-17 levels were negatively correlated with saliva flow rates in pSS patients. Both IL-17-deficient mice and chimeric mice with non-hematopoietic cell-restricted IL-17RC deficiency exhibited no obvious salivary reduction while chimeric mice with hematopoietic cell-restricted IL-17RC deficiency showed significantly decreased saliva secretion during ESS development. In SG epithelial cells, IL-17 inhibited acetylcholine-induced calcium movement and downregulated the expression of transient receptor potential canonical 1 via promoting Nfkbiz mRNA stabilisation. Moreover, local IL-17 neutralisation in SG markedly attenuated hyposalivation and ameliorated tissue inflammation in ESS mice.ConclusionThese findings identify a novel function of IL-17 in driving salivary dysfunction during pSS development and may provide a new therapeutic strategy for targeting SG dysfunction in pSS patients.
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Project description:Salivary glands produce and secrete saliva, which is essential for maintaining oral health and overall health. Understanding both the unique structure and physiological function of salivary glands, as well as how they are affected by disease and injury, will direct the development of therapy to repair and regenerate them. Significant recent advances, particularly in the OMICS field, increase our understanding of how salivary glands develop at the cellular, molecular, and genetic levels: the signaling pathways involved, the dynamics of progenitor cell lineages in development, homeostasis, and regeneration, and the role of the extracellular matrix microenvironment. These provide a template for cell and gene therapies as well as bioengineering approaches to repair or regenerate salivary function.
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Project description:Salivary gland function is severely disrupted by radiation therapy used to treat patients diagnosed with head and neck cancer and by Sjögren's syndrome. The resulting condition, which results in xerostomia or dry mouth, is due to irreversible loss of the secretory acinar cells within the major salivary glands. There are presently no treatments for the resolution of xerostomia. Cell-based approaches could be employed to repopulate acinar cells in the salivary gland but investigations into potential therapeutic strategies are limited by the challenges of maintaining and expanding acinar cells in vitro. We investigate the encapsulation of salivary gland cell aggregates within PEG hydrogels as a means of culturing secretory acinar cells. Lineage tracing was used to monitor the fate of acinar cells isolated from murine submandibular gland (SMG). Upon initial formation in vitro, SMG aggregates comprise both acinar and duct cells, with the majority cells of acinar origin. With longer culture times, acinar cells significantly decreased the expression of specific markers and activated the expression of keratins normally found in duct cells. A similar acinar-to-duct cell transition was also observed in vivo, following duct ligation injury. These results indicate that under conditions of stress (mechanical and enzymatic isolation from glands) or injury (duct ligation), salivary gland acinar cells exhibit plasticity to adopt a duct cell phenotype.
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Project description:TH17 cells and their associated signature cytokines, IL-17 and IL-22, are highly elevated in primary Sjögren's syndrome (pSjS). The levels of IL-22 present in sera showed significant correlations with many disease parameters, specifically hyposalivation, anti-SSB, anti-SSA/SSB, hypergammaglobulinemia and rheumatoid factor. The present study aims to examine the biological function of IL-22 on human salivary glands. To accomplish the goal, microarray analysis using the HumanHT-12 v4 Expression BeadChip was utilized to determine the biological function of IL-22. Differential expression analyses were conducted using the LIMMA package from the Bioconductor project. MTT assay, flow cytometry and Western blotting were used to identify the function of IL-22 on human salivary gland cells. Results indicate an extensive effect of IL-22 on many major molecular functions including activation of antimicrobial genes and downregulation of immune-associated pathways. Functional studies performed in-vitro using human salivary gland cells treated with IL-22 indicated a direct effect of IL-22 on cell cycling, specifically reducing cellular proliferation at the G2-M phase by activation of STAT3. These results suggest the important role of IL-22 in the salivary gland function. The present study suggests that IL-22 might be involved in regulating inflammation and controlling the cell proliferation in SjS.
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Project description:Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.
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Project description:New strategies for tissue engineering have great potential for restoring and revitalizing impaired tissues and organs, including the use of smart hydrogels that can be modified to enhance organization and functionality of the salivary glands. For instance, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel (L1pM-FH) promote cell cluster formation in vitro and salivary gland regeneration in vivo when compared with fibrin hydrogel (FH) alone; however, L1pM-FH produce only weak expression of acinar differentiation markers in vivo (e.g., aquaporin-5 and transmembrane protein 16). Since previous studies demonstrated that a greater impact can be achieved when trimeric forms were used as compared with monomeric or dimeric forms, we investigated the extent to which trimers of laminin-111 chemically conjugated to FH (L1pT-FH) can increase the expression of acinar differentiation markers and elevate saliva secretion. In vitro studies using Par-C10 acinar cells demonstrated that when compared with L1pM-FH, L1pT-FH induced similar levels of acinar-like cell clustering, polarization, lumen formation, and calcium signaling. To assess the performance of the trimeric complex in vivo, we compared the ability of L1pM-FH and L1pT-FH to increase acinar differentiation markers and restore saliva flow rate in a salivary gland wound model of C57BL/6 mice. Our results show that L1pT-FH applied to wounded mice significantly improved the expression of the acinar differentiation markers and saliva secretion when compared with the monomeric form. Together, these positive effects of L1pT-FH warrant its future testing in additional models of hyposalivation with the ultimate goal of applying this technology in humans.
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