Human Submandibular Gland Cells Research Articles

ZnR/GPR39 mediated human salivary secretion

Zn2+ is divalent cation that is essential for many biological events by affecting various ion channels. Zn2+ can serve G‐protein coupled receptor signaling via activation of metabotropic zinc receptors, ZnR/GPR39. In spite of several evidences of the existence of ZnR/GPR39 in salivary gland cells, there has been no evidence of ZnR/GPR39‐mediated modulation of the salivary gland functions. Here we have characterized the role of ZnR/GPR39 in human submandibular gland cells. Mouth wash with Zn‐containing solution evoked the secretion of unstimulated and stimulated whole saliva in human. We found that ZnR/GPR39 is expressed in the human submandibular glands and HSG cells. Zn2+ increased cytosolic Ca2+ concentration ([Ca2+]i) in a concentration‐dependent manner. Both muscarinic antagonist and histaminergic antagonists did not have any effect on Zn2+‐induced increase [Ca2+]i, which is completely blocked by the inhibitor of phospholipase C‐beta. Like as muscarinic and histaminergic stimulation, Zn2+ induced the translocation of aquaporin‐5 (AQP5) into the plasma membrane. These data suggest that metabotropic zinc receptors can modulate salivary secretion in human submandibular gland cells independently from the muscarinic receptor signaling.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (2017R1A2B4002176 to H.K.P.; 2018R1A5A2024418 to S.Y.C.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential regulation of vacuolar H+‐ATPase subunits by Transforming Growth Factor beta 1 (TGF‐β1) in salivary ducts

Bicarbonate concentration in saliva is controlled by the action of acid‐base transporters in salivary duct cells. We have previously shown expression of ATP6V1E1 and ATP6V1A in human and rodent salivary ducts and adaptive redistribution to the apical plasma membrane following acute and chronic metabolic acidosis. However, whether such redistribution has any physiological significance has not been elucidated. Using the human submandibular gland cell line HSG, we show for the first time functional expression of V‐ATPase in submandibular duct cells and introduce Transforming Growth Factor beta (TGF‐β) as a novel regulator of V‐ATPase subunits. Using QRT‐PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, and intracellular H(+) recording with H(+) ‐sensitive dye 2′,7′‐bis‐(carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, we show that activation of TGF‐β signalling upregulates ATP6V1E1 and ATP6V1B2, and has no effect on ATP6V1A. Following acidosis, neutralization of TGF‐βs or blocking of Smad2/3 phosphorylation decreased membrane expression of ATP6V1A, and prevented the acidosis‐induced increased V‐ATPase activity. The results suggest multiple modes of action of TGF‐β1 on V‐ATPase subunits in HSG cells: TGF‐β1 may regulate transcription or protein synthesis of certain subunits and trafficking of other subunits in a context‐dependent manner. Moreover, V‐ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Estradiol Induced Estrogen Receptor-mediated Transcription and Expression of Aquaporin5

In this study, we constructed the recombinant plasmid of pGL2/Aquaporin5(AQP5) promoter (pGL2/AQP5p) luciferase reporter, then found estradiol(E2) induced AQP5 promoter activation in a dose-dependent manner. Further, we identified endogenous estrogen receptors(ER), including ERα and ERβ, expressed in human submandibular gland(HSG) cells, which responded to E2. Then demonstrated by the stimulation of E2, AQP5 was upregulated in protein level, meanwhile AQP5 located in cytomembrane was elevated in immunofluorescence. Furthermore, we revealed the roles of ERα and ERβ in AQP5 upregulation by E2. When ERα and ERβ were overexpressed, the AQP5 transcription level and protein expression were augmented obviously. While when knockdown ER by ERα-shRNA or ERβ-shRNA, AQP5 transcription and expression attenuated. Moreover, we detected the effect of E2 in Sjogren’s syndrome(SS) mice model in vivo. SS mice models were constructed by injecting submandibular gland antigen immune induction combined with estrogen deprivation, which were administrated with saline and E2. The salivary secretion was decreased, and the AQP5 expression downregulated in the submandibular gland in the SS model group. When SS mice were administrated with E2, the salivary secretion was significantly increased, and the AQP5 expression upregulated in the submandibular gland. These results suggest E2 activates AQP5p transcription and upregulates AQP5 protein expression, and E2 promotes salivary secretion in SS model in vivo. Taken together, we provided the evidence that E2 increased salivary secretion by activating AQP5 transcription and expression.

Differential regulation of vacuolar H+ -ATPase subunits by transforming growth factor-β1 in salivary ducts.

Bicarbonate concentration in saliva is controlled by the action of acid-base transporters in salivary duct cells. We show for the first time expression of ATP6V1B1 in submandibular gland and introduce transforming growth factor-beta (TGF-β) as a novel regulator of V-ATPase subunits. Using QRT-PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, chromatin immunoprecipitation, and intracellular H(+ ) recording with H(+ )-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein we show that in the human submandibular gland (HSG) cell line, activation of TGF-β signaling upregulates ATP6V1E1 and ATP6V1B2, downregulates ATP6V1B1, and has no effect on ATP6V1A. TGF-β1 effects on ATP6V1B1 are mediated through the canonical, the soluble adenylate cyclase, and ERK signaling. A CREB binding sequence was identified in the ATP6V1B1 promoter and CREB binding decreased after TGF-β1 treatment. Following acidosis, a bafilomycin-sensitive and Na+ -independent cell pH recovery was observed in HSG cells, an effect that was not influenced after disruption of acidic lysosomes. Moreover, neutralization of TGF-βs, inhibition of TGF-β receptor, or inhibition of the canonical pathway decreased membrane expression of ATP6V1A and prevented the acidosis-induced increased V-ATPase activity. The results suggest multiple modes of action of TGF-β1 on V-ATPase subunits in HSG cells: TGF-β1 may regulate transcription or protein synthesis of certain subunits and trafficking of other subunits in a context-dependent manner. Moreover, surface V-ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.

The synergistic effect of NOD2 and TLR4 on the activation of autophagy in human submandibular gland inflammation.

Sialadenitis is a nonneoplastic disease that causes salivary dysfunction. Autophagy may be involved in helping protect salivary function when the salivary gland is impaired; this process is primarily activated by sensors of innate immunity, such as Toll-like receptors and nucleotide-binding oligomerization domain (NOD)-like receptors. The role of these pattern recognition receptors (PRRs) in the regulation of salivary gland tissue defense and homeostasis has been underappreciated. This study hypothesized that NOD2 and TLR4 have a synergistic effect on the activation of autophagy in human submandibular gland (HSG) inflammation. Submandibular gland inflammation was modeled by treating HSG cell lines invitro with muramyl dipeptide (MDP) and lipopolysaccharide (LPS) for 24hours. The mRNA and protein expression of NOD2, TLR4 and autophagy-related proteins (ATG5, LC3, Beclin1) were evaluated by real-time PCR and Western blot. Immunohistochemistry and double immunofluorescence were used to analyze the presence, distribution and colocalization of the aforementioned indicators in HSG tissues. The mRNA and protein expression of autophagy-related proteins were significantly increased in HSG cells costimulated with LPS and MDP for 24hours. NOD2, TLR4 and the autophagy-related proteins were also highly expressed in residual acini and dilated ducts of chronic submandibular sialadenitis tissues. In addition, PRRs and autophagy markers were obviously colocalized in chronic submandibular sialadenitis tissues and HSG cells. TLR4 and NOD2 have unique expression sites in salivary glands, and they may synergistically activate autophagy in salivary glands under conditions of inflammation.

The activation of Akt/mTOR pathway by bleomycin in Epithelial-to-mesenchymal transition of human submandibular gland cells: A treatment mechanism of bleomycin for mucoceles of the salivary glands

ObjectiveBleomycin (BLM) has been found safe and highly effective in the treatment of the mucoceles by intralesional injection in our previous study. The present research was designed to investigate whether epithelial-to-mesenchymal transition (EMT) contributes to the therapeutic effects of BLM for mucoceles of the salivary glands. Material and methodsThe cell proliferation and apoptosis of human submandibular gland cells (HSG cells) were examined by Cell Counting Kit-8 assay and Annexin V binding assay respectively. Epithelial and mesenchymal markers of HSG cells were measured by real-time quantitative PCR and Western blot analysis. Acinar differentiation and cell migration assays were performed to evaluate HSG cells function. ResultsHigh-dose BLM (≥0.5μg/mL) significantly inhibited the cell proliferation and induced the cell apoptosis, while the treatment with low-dose BLM (0.05 and 0.1μg/mL) for 48h induced EMT in HSG cells. Furthermore, Akt/mTOR pathway, rather than MAPK pathway, was activated through treated with 0.05 and 0.1μg/mL BLM, as well as activation of the transcription factor Slug and Zeb 1. The migration of HSG cells was also enhanced through 0.05 and 0.1μg/mL BLM, but the ability of acinar differentiation was diminished. ConclusionOur results indicated that an EMT process was involved in the BLM-induced therapeutic effects on the HSG cells through the Akt/mTOR pathway. Importantly, the results indicated the potential role of this process in the BLM sclerotherapy of mucoceles of the salivary glands.

Human salivary gland cells express bradykinin receptors that modulate the expression of proinflammatory cytokines.

Bradykinin is an important peptide modulator that affects the function of neurons and immune cells. However, there is no evidence of the bradykinin receptors and their functions in human salivary glands. Here we have identified and characterized bradykinin receptors on human submandibular gland cells. Both bradykinin B1 and B2 receptors are expressed on human submandibular gland cells, A253 cells, and HSG cells. Bradykinin increased the intracellular Ca2+ concentration ([Ca2+ ]i ) in a concentration-dependent manner. Interestingly, a specific agonist of the B1 receptor did not have any effect on [Ca2+ ]i in HSG cells, whereas specific agonists of the B2 receptor had a Ca2+ mobilizing effect. Furthermore, application of the B1 receptor antagonist, R715, did not alter the bradykinin-mediated increase in cytosolic Ca2+ , whereas the B2 receptor antagonist, HOE140, showed a strong inhibitory effect, which implies that bradykinin B2 receptors are functional in modulating the concentration of cytosolic Ca2+ . Bradykinin did not affect a carbachol-induced rise of [Ca2+ ]i and did not modulate translocation of aquaporin-5. However, bradykinin did promote the expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), implying the role of bradykinin in salivary gland inflammation. These data suggest that bradykinin receptors are involved in Ca2+ signaling in human submandibular gland cells and serve a unique role, which is separate from that of other salivary gland G protein-coupled receptors.

Study of the viability of cultured human cells in suspensions

The success of cell therapy is directly related to the viability of cells used for transplantation. The cells used for transplantation are in some cases injected in suspension. However, the optimal conditions for the preservation of cell viability upon the preparation and storage of cell suspensions for transplantation have not been defined yet. The aim of the present work consisted in the selection of optimal conditions for the storage of suspensions of human submandibular salivary gland cells, differentiated cells of the submandibular salivary gland, and dermal fibroblasts in biocompatible solutions. Standard procedures of cell isolation and cultivation were used in the study. An automatic cell counter from BioRad was used to count the cells, and viability of the cells was assessed using staining with 4% Trypan Blue. The biocompatible solutions tested included phosphate-buffered saline, physiological saline for injections, and a 2% solution of human albumin in phosphate-buffered saline. The study showed that the human cells under investigation remained viable in suspension at both +4°С and +25°С for at least 24 hours, regardless of the carrier solution used. The highest content of viable cells of the salivary gland (more than 50%) at both temperatures examined was observed when cells were suspended in phosphate-buffered saline. However, the adhesive and proliferative properties of the salivary gland cells were better preserved at +4°С in case of 24 hours of incubation under the conditions described above. Fibroblasts maintained in physiological saline formed a homogeneous single-cell suspension that remained stable for 30 hours at +4°С; virtually no loss of cell viability was observed. The addition of 2% albumin resulted in a decrease of the viability of fibroblasts. Thus, storage and transportation in phosphate- buffered saline at +4°С can be recommended for suspensions of cells of the human submandibular salivary gland, whereas human fibroblast suspensions should be maintained at +4°С in physiological saline.

SU‐F‐T‐129: Impact of Radial Fluctuations in RBE for Therapeutic Proton Beams

Purpose:To evaluate the off axis relative biological effectiveness (RBE) for actively scanned proton beams and determine if a constant radial RBE can be assumed.Methods:The PHITS Monte Carlo code paired with a microscopic analytical function was used to determine probability distribution functions of the lineal energy in 0.3µm diameter spheres throughout a water phantom. Twenty million primary protons were simulated for a 0.6cm diameter pencil beam. Beam energies corresponding to Bragg Peak depths of 50, 100, 150, 200, 250, and 300mm were used and evaluated transversely every millimeter and radially for annuli of 1.0, 2.0, 3.0, 3.2, 3.4, 3.6, 4.0, 5.0, 10.0, 15.0, 20.0 and 25.0mm outer radius. The acquired probability distributions were reduced to dose‐mean lineal energies and applied to the modified microdosimetric kinetic model, for human submandibular gland (HSG) cells, to calculate relative biological effectiveness (RBE) compared to 60Co beams at the 10% survival threshold.Results:RBE was generally seen to increase as distance from the central axis (CAX) increased. However, this increase was only seen in low dose regions and its overall effects on the transverse biological dose remains low. In the entrance region of the phantom (10mm depth), minimum and maximum calculated RBEs varied between 15.22 and 18.88% for different energies. At the Bragg peak, this difference ranged from 3.15 to 26.77%. Despite these rather large variations the dose‐weighted RBE and the CAX RBE varied by less than 0.14% at 10mm depth and less than 0.16% at the Bragg peak. Similarly small variations were found at all depths proximal of the Bragg peak.Conclusion:Although proton RBE does vary radially, its overall effect on biological dose is minimal and the use of a radially constant RBE in treatment planning for scanned proton beams would not produce large errors.

Prevention of irradiation-induced salivary hypofunction by rapamycin in swine parotid glands.

Radiotherapy is commonly used in patients with oral cavity and pharyngeal cancers, usually resulting in irreversible salivary hypofunction. Currently management of radiation damage to salivary glands still remains a great challenge. Recent studies show that activation of mammalian target of rapamycin (mTOR) occurs in salivary gland lesions, making it possible to apply mTOR inhibitor for treatment. Our results indicate inhibition of mTOR by rapamycin significantly alleviated irradiation-induced salivary hypofunction by restoring 46% salivary flow rate and protecting histological structures in swine. Furthermore, rapamycin protected human submandibular gland cell line (HSG) from irradiation-induced cell depletion and loss of cell proliferation capacity. These findings lay the foundation for a new clinical application of rapamycin to prevent irradiation-induced salivary hypofunction.

Transcription factors related to chondrogenesis in pleomorphic adenoma of the salivary gland: a mechanism of mesenchymal tissue formation

In salivary gland pleomorphic adenoma, expression of extracellular matrix (ECM) substances indicates that tumor epithelial cells are becoming chondrogenic and will produce cartilage-like mesenchymal tissues. Sox9, the master transcription factor of chondrogenesis, is expressed in mouse salivary gland cells. To clarify the mechanism behind chondrogenesis in tumor epithelial cells, we examined the expression of transcription factors related to chondrogenesis in tumors and salivary glands. Reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, and immunostaining were performed on pleomorphic adenoma tissues, salivary gland tissues, and human submandibular gland (HSG) cells. The mRNAs of essential transcription factors for chondrogenesis—Sox9, Sox6, and Sox5—were detected in both tumor and salivary gland tissues. The mRNAs of aggrecan and type II collagen—cartilage-specific ECM substances—were detected only in tumors. Sox9 and Sox6 proteins were colocalized in many epithelial cells in tumors and salivary glands. Tumor epithelial cells also possessed aggrecan protein and occasionally type II collagen protein. Moreover, mRNAs for transcription repressors of chondrogenesis δEF1 and AP-2α were detected in both tumors and salivary glands, whereas Twist1 mRNA was detected only in salivary glands and was at significantly low-to-undetectable levels in tumors. Twist1 protein was localized in the Sox9-expressing salivary gland cells. HSG cells expressed Sox9, Sox6, and Twist1, but not aggrecan or type II collagen, and thus were similar to salivary gland cells. Twist1 depletion by Twist1 siRNA led to the upregulation of aggrecan and type II collagen mRNA expression in HSG cells. In contrast, forced expression of Twist1, using Twist1 cDNA, resulted in the downregulation of both these genes. Taken together, these results indicate that salivary gland cells have a potential for chondrogenesis, and Twist1 depletion concomitant with neoplastic transformation, which would permit tumor epithelial cells to produce cartilage-like mesenchymal tissues in salivary gland pleomorphic adenoma.

Salivary mucins induce a Toll-like receptor 4-mediated pro-inflammatory response in human submandibular salivary cells: are mucins involved in Sjögren's syndrome?

A hallmark characteristic of SS patients is the ectopic presence of the mucins MUC5B and MUC7 in the extracellular matrix of salivary glands that have lost apical-basolateral acinar-cell polarity. This study aims to determine whether exogenous salivary mucins induce gene expression of pro-inflammatory cytokines, as well as to evaluate whether the Toll-like receptor-4 (TLR4) pathway is involved in this response. Differentiated human submandibular gland (HSG) cells were stimulated with mucins or oligosaccharide residues at different concentrations and for different periods of time. The expression of pro-inflammatory cytokines and their receptors was determined by semi-quantitative real time PCR (sqPCR). TLR4-mediated responses induced by mucin were evaluated with the Toll-IL-1 receptor domain containing adaptor protein (TIRAP) inhibitory peptide or using anti-hTLR4 blocking antibody. TLR4-receptor expression was also determined in SS patients, controls and HSG cells. Mucins induced a significant increase in CXCL8, TNF-α, IFN-α, IFN-β, IL-6 and IL-1β, but not B cell activating factor (BAFF). Cytokine induction was mediated by TLR4, as shown using TIRAP or using anti-hTLR4 antibody. Sugar residues present in MUC5B, such as sulpho-Lewis (SO3-3Galβ1-3GlcNAc), also induced cytokines. Unexpectedly, mucins induced MUC5B, but not MUC7 expression. Salivary mucins were recognized by TLR4 in epithelial cells initiating a pro-inflammatory response that could attract inflammatory cells to amplify and perpetuate inflammation and thereby contribute to the development of a chronic state characteristic of SS. The ectopic localization of MUC5B and MUC7 in the salivary gland extracellular matrix from SS patients and the current results reveal the importance of salivary epithelial cells in innate immunity, as well as in SS pathogenesis.

Effect of Antimuscarinic Autoantibodies in Primary Sjögren’s Syndrome

The presence of functional autoantibodies against the muscarinic type 3 receptor (M3R) has been reported in primary Sjögren’s syndrome (pSS). However, the pathogenic role of these autoantibodies in pSS development remains to be elucidated. In this experiment, we investigated a pathologic role of pSS autoantibodies (pSS IgG) associated with downregulation of the major histocompatibility complex I (MHC I) molecule with M3R through internalization. Anti-M3R autoantibodies in purified control and pSS IgG were detected using 4 synthesized cyclic M3R peptides by enzyme-linked immunosorbent assay. The binding reactivity of pSS IgG to M3R in situ was analyzed by a dual immunostaining method. Surface expression, interaction, and internalization of M3R with MHC I were analyzed by immunofluorescence confocal microscopy and biochemical assays. Synthetic cyclic peptides M3RP205-221 and M3RP520-527 showed significantly high reactivity with pSS IgG compared to the control IgG or the other 3 peptides (P < 0.05). Significantly high reactivity of pSS IgG to M3R in situ was observed. PSS IgG increased the interaction of membrane M3R with MHC I and induced their internalization in primary human submandibular gland cells. The pSS IgG-induced internalization of M3R with MHC I was significantly inhibited by the cholesterol-sequestering drug filipin. Our novel finding—namely, strong downregulation of the membrane MHC I with M3R through internalization of the cholesterol-rich microdomain associating with anti-M3R autoantibodies—could be an important mechanism contributing to the impaired salivation seen in pSS and linking secretory hypofunction to autoimmune pathogenesis.

Anti-oxidative and anti-inflammatory effects of astaxanthin on H2O2-induced oxidative stress in human submandibular gland cells

Anti-oxidative and anti-inflammatory effects of astaxanthin on H₂O₂-induced oxidative stress in human submandibular gland cells

TRPC1 contributes to the Ca2+-dependent regulation of adenylate cyclases.

SOCE (store-operated Ca2+ entry) is mediated via specific plasma membrane channels in response to ER (endoplasmic reticulum) Ca2+ store depletion. This route of Ca2+ entry is central to the dynamic interplay between Ca2+ and cAMP signalling in regulating the activity of Ca2+-sensitive adenylate cyclase isoforms (AC1, AC5, AC6 and AC8). Two proteins have been identified as key components of SOCE: STIM1 (stromal interaction molecule 1), which senses ER Ca2+ store content and translocates to the plasma membrane upon store depletion, where it then activates Orai1, the pore-forming component of the CRAC (Ca2+ release-activated Ca2+) channel. Previous studies reported that co-expression of STIM1 and Orai1 in HEK-293 (human embryonic kidney 293) cells enhances Ca2+-stimulated AC8 activity and that AC8 and Orai1 directly interact to enhance this regulation. Nonetheless, the additional involvement of TRPC (transient receptor potential canonical) channels in SOCE has also been proposed. In the present study, we evaluate the contribution of TRPC1 to SOCE-mediated regulation of Ca2+-sensitive ACs in HEK-293 cells stably expressing AC8 (HEK-AC8) and HSG (human submandibular gland) cells expressing an endogenous Ca2+-inhibited AC6. We demonstrate a role for TRPC1 as an integral component of SOCE, alongside STIM1 and Orai1, in regulating Ca2+ fluxes within AC microdomains and influencing cAMP production.

Preparation and antitumor effect of a toxin-linked conjugate targeting vascular endothelial growth factor receptor and urokinase plasminogen activator.

The aberrant signaling activation of vascular endothelial growth factor receptor (VEGFR) and urokinase plasminogen activator (uPA) is a common characteristic of many tumors, including lung cancer. Accordingly, VEGFR and uPA have emerged as attractive targets for tumor. KDR (Flk-1/VEGFR-2), a member of the VEGFR family, has been recognized as an important target for antiangiogenesis in tumor. In this study, a recombinant immunotoxin was produced to specifically target KDR-expressing tumor vascular endothelial cells and uPA-expressing tumor cells and mediate antitumor angiogenesis and antitumor effect. Based on its potent inhibitory effect on protein synthesis, Luffin-beta (Lβ) ribosome-inactivating protein was selected as part of a recombinant fusion protein, a single-chain variable fragment against KDR (KDRscFv)-uPA cleavage site (uPAcs)-Lβ-KDEL (named as KPLK). The KDRscFv-uPAcs-Lβ-KDEL (KPLK) contained a single-chain variable fragment (scFv) against KDR, uPAcs, Lβ, and the retention signal for endoplasmic reticulum proteins KDEL (Lys-Asp-Glu-Leu). The KPLK-expressing vector was expressed in Escherichia coli, and the KPLK protein was isolated with nickel affinity chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis test demonstrated KPLK was effectively expressed. Result of invitro cell viability assay on non-small cell lung cancer (NSCLC) H460 cell line (uPA-positive cell) revealed that KPLK significantly inhibited cell proliferation, induced apoptosis, and accumulated cells in S and G2/M phases, but the normal cell line (human submandibular gland cell) was unaffected. These effects were enhanced when uPA was added to digest KPLK to release Lβ. For invivo assay of KPLK, subcutaneous xenograft tumor model of nude mice were established with H460 cells. Growth of solid tumors was significantly inhibited in animals treated with KPLK up to 21 days, tumor weights were decreased, and the expression of angiogenesis marker CD31 was downregulated; meanwhile, the apoptosis-related protein casspase-3 was upregulated. These results suggested that the recombinant KPLK may have therapeutic applications on tumors, especially uPA-overexpressing ones.

Caffeine increases the expression of cystatin SN in human submandibular acinar-like HSG cells

ObjectiveThe study aimed at evaluating in vitro the effect of caffeine on expression of cystatin SN, a potential marker of sensitivity to bitterness in humans. MethodsDifferentiation of human submandibular gland (HSG) cells was induced by culturing cells on Matrigel. Caffeine cytotoxicity was assessed over 3 days by the Resazurin test. Finally, effects of 5, 50 and 100μM caffeine exposure on cystatin SN expression were explored over 3 days by ELISA. ResultsAt concentrations relevant to human adult plasma levels (5, 50 and 100μM), caffeine did not affect cell viability whether cells were differentiated or not. Cystatin SN levels were overall higher in differentiated cells and increased with time in both conditions. There was a significant (p<0.001) effect of caffeine on cystatin SN expression specifically in differentiated cells. ConclusionsThe HSG cell line proved to be a relevant tool to study in vitro the effect of caffeine at concentrations consistent with dietary intake in human subjects. The results suggest that salivary cystatin SN abundance may depend on caffeine intake, with possible consequences on taste sensitivity.

Chaperone Stress 70 Protein (STCH) Binds and Regulates Two Acid/Base Transporters NBCe1-B and NHE1 *

Regulation of intracellular pH is critical for the maintenance of cell homeostasis in response to stress. We used yeast two-hybrid screening to identify novel interacting partners of the pH-regulating transporter NBCe1-B. We identified Hsp70-like stress 70 protein chaperone (STCH) as interacting with NBCe1-B at the N-terminal (amino acids 96-440) region. Co-injection of STCH and NBCe1-B cRNA into Xenopus oocytes significantly increased surface expression of NBCe1-B and enhanced bicarbonate conductance compared with NBCe1-B cRNA alone. STCH siRNA decreased the rate of Na(+)-dependent pHi recovery from NH4(+) pulse-induced acidification in an HSG (human submandibular gland ductal) cell line. We observed that in addition to NBCe1-B, Na(+)/H(+) exchanger (NHE)-dependent pHi recovery was also impaired by STCH siRNA and further confirmed the interaction of STCH with NHE1 but not plasma membrane Ca(2+) ATPase. Both NBCe1-B and NHE1 interactions were dependent on a specific 45-amino acid region of STCH. In conclusion, we identify a novel role of STCH in the regulation of pHi through site-specific interactions with NBCe1-B and NHE1 and subsequent modulation of membrane transporter expression. We propose STCH may play a role in pHi regulation at times of cellular stress by enhancing the recovery from intracellular acidification.

Distinct Contributions of Orai1 and TRPC1 to Agonist-Induced [Ca2+]i Signals Determine Specificity of Ca2+-Dependent Gene Expression

Regulation of critical cellular functions, including Ca2+-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca2+ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca2+]i due to intracellular Ca2+ release and Ca2+ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca2+]i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca2+]i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca2+ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca2+ store depletion in these cells, it is not clear how the cells decode individual Ca2+ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca2+]i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca2+ entry generates [Ca2+]i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca2+ entry generates sustained [Ca2+]i elevation at high [CCh] and contributes to frequency of [Ca2+]i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca2+ dependent gene expression pathways, consistent with the different patterns of [Ca2+]i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca2+]i generated by Orai1, independent of global [Ca2+]i changes or TRPC1-mediated Ca2+ entry. In contrast, Ca2+ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca2+ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca2+-dependent gene expression pathways.

Acidosis-induced V-ATPase trafficking in salivary ducts is initiated by cAMP/PKA/CREB pathway via regulation of Rab11b expression

Changes in systemic acid–base homeostasis cause a series of organ-specific cellular responses, among them changes of acid–base transporter activities, and recruitment or retrieval of these transporters from intracellular pools to the plasma membrane and vice versa. The purpose of this study was to investigate the impact of protein phosphorylation in the acidosis-induced translocation of vacuolar-type H+-ATPase (V-ATPase) in salivary ducts and to identify molecular targets. Therefore, the human submandibular gland cell line HSG was exposed to acidosis and V-ATPase trafficking was investigated in the presence or absence of inhibitors and activators of sAC/PKA and Src/ERK signaling pathways. Putative target genes have been identified by RT-PCR and immunoblotting, and validated by loss-of-function experiments. Acidosis caused activation of cAMP/PKA and Src signaling and inhibition of either pathway significantly impaired acidosis-induced V-ATPase redistribution and incorporation into the plasma membrane. Activation of ERK1/2 was Src-independent, whereas activation of PKA caused phosphorylation of cAMP response element-binding (CREB) and activation to regulate Rab11b transcription. Loss-of-function of CREB down-regulated Rab11b transcript and protein and significantly impaired acidosis-induced V-ATPase translocation in HSG cells. These data demonstrate that the cAMP/PKA/CREB signaling pathway initiates acidosis-induced V-ATPase trafficking in salivary ducts via regulation of Rab11b expression and provide first evidence for a molecular mechanism underlying cAMP/PKA-dependent transporter trafficking that could account for accumulation and activity of transporters in other cellular systems as well.