Mouse Salivary Gland Cells Research Articles

Sappanone A Aggrandises Ionising Radiation-induced Damage in Oral Epithelial Cells.

To analyse the role played by Sappanone A, a bioactive ingredient isolated from the heartwood of Caesalpinia sappan, in the regulation of oral epithelial cell viability under radiation. Cell viability of human oral keratinocytes (HOKs) and mouse salivary gland cells under ionising radiation was analysed. Expression of Ki67 was measured by immunohistochemical staining. Fragmentation of deoxyribonucleic acid (DNA) was measured by comet assay. Cell death was analysed using trypan blue exclusion assay. Cell viability was measured using a Cell Counting Kit 8 (CCK8; Abcam, Cambridge, UK) assay. Sappanone A decreased cell viability of HOK cells and mouse salivary gland cells under ionising radiation. In addition, Sappanone A enhanced radiation-induced genomic DNA fragmentation, accompanied by impaired homologous recombination and non-homologous end joining DNA repair. Mechanistic evaluation revealed that Sappanone A counteracted radiation-induced inosine monophosphate dehydrogenase 2 (IMPDH2) activation, and that this effect could be abolished by reconstituted expression of a Sappanone A-binding defective IMPDH2 mutant. The present study highlights a novel role played by Sappanone A in the modulation of radiosensitivity of oral epithelial cells.

Reversely immortalized mouse salivary gland cells presented a promising metabolic and fibrotic response upon BMP9/Gdf2 stimulation

The submandibular gland (SMG) and the sublingual gland (SLG) are two of the three major salivary glands in mammals. In mice, they are adjacent to each other and open into the oral cavity, producing saliva to lubricate the mouth and aid in food digestion. Though salivary gland dysfunction accompanied with fibrosis and metabolic disturbance is common in clinic, in-depth mechanistic research is lacking. Currently, research on how to rescue salivary function is challenging, as it must resort to using terminally differentiated acinar cells or precursor acinar cells with unknown differentiation. In this study, we established reversely immortalized mouse primary SMG cells (iSMGCs) and SLG cells (iSLGCs) on the first postnatal day (P0). The iSMGCs and iSLGCs grew well, exhibited many salivary gland characteristics, and retained the metabolism-related genes derived from the original tissue as demonstrated using transcriptome sequencing (RNA-seq) analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of these two cell lines, which overlapped with those of the SMG and SLG, were enriched in cysteine and methionine metabolism. Furthermore, we investigated the role of bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2(Gdf2), on metabolic and fibrotic functions in the SMG and SLG. We demonstrated that iSMGCs and iSLGCs presented promising adipogenic and fibrotic responses upon BMP9/Gdf2 stimulation. Thus, our findings indicate that iSMGCs and iSLGCs faithfully reproduce characteristics of SMG and SLG cells and present a promising prospect for use in future study of salivary gland metabolism and fibrosis upon BMP9/Gdf2 stimulation.

Slc4a11 disruption causes duct cell loss and impairs NaCl reabsorption in female mouse submandibular glands.

Slc4a11, a member of the Slc4 HCO3 − transporter family, has a wide tissue distribution. In mouse salivary glands, the expression of Slc4a11 mRNA was more than eightfold greater than the other nine members of the Slc4 gene family. The Slc4a11 protein displayed a diffuse subcellular distribution in both the acinar and duct cells of mouse submandibular glands (SMG). Slc4a11 disruption induced a significant increase in the Na+ and Cl− concentrations of stimulated SMG saliva, whereas it did not affect the fluid secretion rate in response to either β‐adrenergic or cholinergic receptor stimulation. Heterologous expressed mouse Slc4a11 acted as a H+/OH− transporter that was uncoupled of Na+ or Cl− movement, and this activity was blocked by ethyl‐isopropyl amiloride (EIPA) but not 4,4′‐Diisothiocyanato‐2,2′‐stilbenedisulfonic acid (DIDS). Slc4a11 disruption revealed that Slc4a11 does not play a major role in intracellular pH regulation in mouse salivary gland cells. In contrast, NaCl reabsorption was impaired in the SMG saliva of female compared to male Slc4a11 null mice, which correlated with the loss of duct cells and a decrease in expression of the duct‐cell‐specific transcription factor Ascl3. Together, our results suggest that Slc4a11 expression regulates the number of ducts cells in the mouse SMG and consequently NaCl reabsorption.

Enhancer hijacking activates oncogenic transcription factor NR4A3 in acinic cell carcinomas of the salivary glands

The molecular pathogenesis of salivary gland acinic cell carcinoma (AciCC) is poorly understood. The secretory Ca-binding phosphoprotein (SCPP) gene cluster at 4q13 encodes structurally related phosphoproteins of which some are specifically expressed at high levels in the salivary glands and constitute major components of saliva. Here we report on recurrent rearrangements [t(4;9)(q13;q31)] in AciCC that translocate active enhancer regions from the SCPP gene cluster to the region upstream of Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3) at 9q31. We show that NR4A3 is specifically upregulated in AciCCs, and that active chromatin regions and gene expression signatures in AciCCs are highly correlated with the NR4A3 transcription factor binding motif. Overexpression of NR4A3 in mouse salivary gland cells increases expression of known NR4A3 target genes and has a stimulatory functional effect on cell proliferation. We conclude that NR4A3 is upregulated through enhancer hijacking and has important oncogenic functions in AciCC.

Rho-associated kinase inhibitor enhances the culture condition of isolated mouse salivary gland cells in vitro.

Hyposalivation because of curative radiation therapy in patients with head and neck cancer is a major concern. At present, there is no effective treatment for hyposalivation, highlighting the importance of cell therapy as a new therapeutic approach. To provide functional cells for cell replacement therapy, it is important to overcome the limitations of current in vitro culture methods for isolated salivary gland cells. Here, we suggest an improved culture condition method for the cultivation of isolated salivary gland cells. The dissociated submandibular salivary gland cells of mice were seeded and treated with Rho-associated kinase (ROCK) inhibitor (Y-27632), which resulted in an increase in their cell adhesion, viability, migration, and proliferation. In particular, ROCK inhibitor treatment maintained the expression of α-amylase in the primary cultured salivary gland cells for a long time as compared with untreated cells. The expression of C-Met, a ductal cell marker, was increased in cells treated with ROCK inhibitor. This modified culture condition may serve as an easy and convenient tool for culturing primary salivary gland cells for their application in hyposalivation therapy.

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.

DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca2+ signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca2+ signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca2+-activated Cl- channels (CaCCs) and increased intracellular calcium concentrations ([Ca2+]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca2+]i in mouse salivary gland cells and human corneal epithelial cells. [Ca2+]i increase of DA-6034 was dependent on the Ca2+ entry from extracellular and Ca2+ release from internal Ca2+ stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca2+ stores. These results suggest that DA-6034 induces Ca2+ signaling via extracellular Ca2+ entry and RyRs-sensitive Ca2+ release from internal Ca2+ stores in epithelial cells.

Regeneration of irradiated salivary glands with stem cell marker expressing cells

BackgroundStem cell therapy could be a potential way for reducing radiation-induced hyposalivation and improving the patient’s quality of life. However, the identification and purification of salivary gland stem cells have not been accomplished. This study aims to better characterize the stem/progenitor cell population with regenerative potential residing in the mouse salivary gland. MethodsMouse submandibular gland tissue, isolated cells and cultured 3day old salispheres were tested for their expression of stem cell markers c-Kit, CD133, CD49f, and CD24 using immunohistochemistry for tissue and flow cytometry for cells. Mice were locally irradiated with a single dose of 15Gy and transplanted with cells expressing defined markers. ResultsCells expressing known stem cell markers are localized in the larger ducts of the mouse salivary gland. Isolated cells and cells from day 3 salispheres also express these markers: c-Kit (0.058% vs. 0.65%), CD133 (6% vs. 5%), CD49f (78% vs. 51%), and CD24 (60% vs. 60%, respectively). Intraglandular transplantation of these cells into irradiated salivary glands of mice resulted in stem cell marker-specific recovery of salivary gland function. ConclusionsDifferent stem cell-associated markers are expressed in mouse salivary gland cells, which upon transplantation are able to regenerate the irradiation damaged salivary gland.

Dispersed donor salivary gland cells are widely distributed in the recipient gland when infused up the ductal tree

The salivary glands often are severely and permanently damaged by therapeutic irradiation for cancer of the head and neck. The markedly reduced quantity and quality of saliva results in greatly increased susceptibility to dental caries and infection of the oral mucosa and alveolar bone. Recently, subcapsular injection of cultured mouse salivary gland cells has achieved a significant degree of regeneration in a previously irradiated mouse salivary gland; however, the recovery was limited to one lobule. We describe here a method for delivering donor rat salivary gland cells via the main duct that distributes several thousand cells throughout the recipient rat's salivary gland. The donated cells exhibited the cytodifferentiation of the structures in which they lodged, i.e., acini, granular convoluted tubules, and the several types of ducts. This method may facilitate the simultaneous functional recovery of almost all of the lobules of irradiated rat salivary glands.

Development of Autoimmune Exocrinopathy Resembling Sjögren's Syndrome in Estrogen-Deficient Mice of Healthy Background

Although a number of autoimmune diseases are known to develop in postmenopausal women, the mechanisms by which estrogen deficiency influences autoimmune lesions remain unclear. We speculate that antiestrogenic actions might be a potent factor in the formation of pathogenic autoantigens. Previously, we have identified 120-kd alpha-fodrin as an important autoantigen in Sjögren's syndrome (SS). When healthy C57BL/6 (B6) mice were treated with an ovariectomy (Ovx), we found a significant increase in TUNEL(+)-apoptotic epithelial cells in the salivary gland cells associated with alpha-fodrin cleavage during 2 and 3 weeks after Ovx. By contrast, no apoptotic cells were found in estrogen receptor-alpha knockout mice. In in vitro studies using primary cultured mouse salivary gland cells and human salivary gland cells, we found a cleavage product of 120-kd alpha-fodrin in cells that had undergone tamoxifen (Tam)-induced apoptosis through caspase activation, especially caspase-1. Adoptive transfer of alpha-fodrin-reactive T cells into Ovx-B6 and -SCID mice resulted in the development of autoimmune exocrinopathy quite similar to SS. These results suggest that estrogen deficiency exerts a crucial influence on autoantigen cleavage, and may cause, in part, autoimmune exocrinopathy in postmenopausal women.

Cathepsin S inhibitor prevents autoantigen presentation and autoimmunity

The cysteine endoprotease cathepsin S mediates degradation of the MHC class II invariant chain Ii in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on autoantigen presentation and organ-specific autoimmune diseases in a murine model for Sjögren syndrome. Specific inhibitor of cathepsin S (Clik60) in vitro markedly impaired presentation of an organ-specific autoantigen, 120-kDa α-fodrin, by interfering with MHC class II–peptide binding. Autoantigen-specific T cell responses were significantly and dose-dependently inhibited by incubation with Clik60, but not with inhibitor s of cathepsin B or L. Clik60 treatment of mouse salivary gland cells selectively inhibited autopeptide-bound class II molecules. Moreover, the treatment with Clik60 in vivo profoundly blocked lymphocytic infiltration into the salivary and lacrimal glands, abrogated a rise in serum autoantibody production, and led to recovery from autoimmune manifestations. Thus, inhibition of cathepsin S in vivo alters autoantigen presentation and development of organ-specific autoimmunity. These data identify selective inhibition of cysteine protease cathepsin S as a potential therapeutic strategy for autoimmune disease processes.

Cathepsin S inhibitor prevents autoantigen presentation and autoimmunity

The cysteine endoprotease cathepsin S mediates degradation of the MHC class II invariant chain Ii in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on autoantigen presentation and organ-specific autoimmune diseases in a murine model for Sjögren syndrome. Specific inhibitor of cathepsin S (Clik60) in vitro markedly impaired presentation of an organ-specific autoantigen, 120-kDa α-fodrin, by interfering with MHC class II–peptide binding. Autoantigen-specific T cell responses were significantly and dose-dependently inhibited by incubation with Clik60, but not with inhibitor s of cathepsin B or L. Clik60 treatment of mouse salivary gland cells selectively inhibited autopeptide-bound class II molecules. Moreover, the treatment with Clik60 in vivo profoundly blocked lymphocytic infiltration into the salivary and lacrimal glands, abrogated a rise in serum autoantibody production, and led to recovery from autoimmune manifestations. Thus, inhibition of cathepsin S in vivo alters autoantigen presentation and development of organ-specific autoimmunity. These data identify selective inhibition of cysteine protease cathepsin S as a potential therapeutic strategy for autoimmune disease processes.

Cathepsin S inhibitor prevents autoantigen presentation and autoimmunity.

The cysteine endoprotease cathepsin S mediates degradation of the MHC class II invariant chain Ii in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on autoantigen presentation and organ-specific autoimmune diseases in a murine model for Sjögren syndrome. Specific inhibitor of cathepsin S (Clik60) in vitro markedly impaired presentation of an organ-specific autoantigen, 120-kDa alpha-fodrin, by interfering with MHC class II-peptide binding. Autoantigen-specific T cell responses were significantly and dose-dependently inhibited by incubation with Clik60, but not with inhibitor s of cathepsin B or L. Clik60 treatment of mouse salivary gland cells selectively inhibited autopeptide-bound class II molecules. Moreover, the treatment with Clik60 in vivo profoundly blocked lymphocytic infiltration into the salivary and lacrimal glands, abrogated a rise in serum autoantibody production, and led to recovery from autoimmune manifestations. Thus, inhibition of cathepsin S in vivo alters autoantigen presentation and development of organ-specific autoimmunity. These data identify selective inhibition of cysteine protease cathepsin S as a potential therapeutic strategy for autoimmune disease processes.

Prevention and induction of autoimmune exocrinopathy is dependent on pathogenic autoantigen cleavage in murine Sjögren's syndrome.

The in vivo role of autoantigen cleavage during apoptosis in autoimmune diseases remains unclear. Previously, we found a cleavage product of 120-kDa alpha-fodrin as an important autoantigen in the pathogenesis of primary Sjögren's syndrome (SS). In the murine primary SS model, tissue-infiltrating CD4(+) T cells purified from the salivary glands bear a large proportion of Fas ligand, and the salivary gland duct cells constitutively possess Fas. Infiltrating CD4(+) T cells, but not CD8(+) T cells, identified significant (51)Cr release against mouse salivary gland cells. In vitro studies demonstrated that apoptotic mouse salivary gland cells result in a specific alpha-fodrin cleavage into 120 kDa and that preincubation with caspase inhibitor peptides blocked alpha-fodrin cleavage. In vivo treatment with caspase inhibitors N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone and N-acetyl-Asp-Glu-Val-Asp-al-CHO into the murine model results in dramatic inhibitory effects on the development of autoimmune lesions and in restoration of sicca syndrome. Furthermore, we found that immunization with recombinant alpha-fodrin protein identical with an autoantigen into normal recipients induced autoimmune lesions similar to SS. These data indicate that prevention and induction of autoimmune exocrinopathy is dependent on autoantigen cleavage via caspase cascade and that caspase inhibitors might provide a new therapeutic option directed at reducing tissue damage in the murine model for SS.

Severe Destructive Autoimmune Lesions with Aging in Murine Sjögren's Syndrome through Fas-Mediated Apoptosis

When we evaluated the age-associated changes in autoimmune exocrinopathy in a NFS/sld murine model for primary Sjögren's syndrome (SS), severe destructive autoimmune lesions developed in the salivary and lacrimal glands in the aged mice, compared with those observed in the younger model. We detected a decreased secretion of saliva and tear flow in the aged group. A significant increase of TUNEL(+)-apoptotic epithelial duct cells in the salivary glands was detected in the aged SS animal model. A higher proportion of mouse salivary gland cells bearing Fas was found in the aged group, whereas no significant changes were seen on tissue-infiltrating CD4(+) T cells bearing FasL in the salivary glands from young and aged mice. We detected an increased cleavage product of organ-specific autoantigen, 120-kd alpha-fodrin, in the aged salivary gland tissues on immunoblotting, and an increase in serum autoantibody production against 120-kd alpha-fodrin by enzyme-linked immunosorbent assay. An increase in the proliferative response of splenic T cells against organ-specific autoantigen was observed, whereas nonspecific concanavalin A responsiveness was decreased in the aged mice. In addition, a decrease in Fas expression was found on splenic CD4(+) T cells in the aged mice, and anti-Fas mAb-stimulated apoptosis was down-regulated on CD4(+) T cells. These results indicate that age-associated dysregulation of CD4(+) T cells may play a crucial role on acceleration of organ-specific autoimmune lesions in a murine model for primary SS through Fas-mediated apoptosis.

Effect of T lymphocytes on proliferation of rat and mouse salivary gland cells induced by isoproterenol

This paper describes a study of the ability of lymphocytes to take part in the regulation of isoproterenol-induced proliferation of submaxillary salivary gland cells of rats and mice. Experiments were carried out on female August rats and female mice. To judge activation of the lymphocytes in the course of induction of salivary gland proliferation by isoproterenol, incorporation of /sup 3/H-thymidine into spleen cells of the experimental animals was used. An increase in /sup 3/H-thymidine incorporation into fragments of rat or mouse salivary glands in culture was observed 16 h after injection of isoproterenol into the animals. An increase in incorporation of /sup 3/H-thymidine into the spleen cells was also observed.

Latent and Persistent Lethal Injury in Mouse Salivary Gland Cells Following Gamma Irradiation

Newly synthesized DNA in previously irradiated and isoproterenol-stimulated mouse salivary gland cells was found to be quickly degraded when the stimulation for DNA synthesis was given 10 days after a dose of 1000 rad ..gamma.. radiation. The degradation of the DNA was due to degeneration of acinar cells prior to mitosis. When the stimulation with isoproterenol was given 1 or 3 months after irradiation, DNA degradation in parotids was not detectable. An autoradiographic analysis revealed, however, that about half of the acinar cells labeled with tritiated thymidine were eliminated from irradiated parotids in a few days, even when the stimulation with isoproterenol was given 3 months after irradiation. This indicates that irradiation of mouse salivary gland cells produced latent lethal damage and that this damage is unmasked by the stimulation for DNA synthesis and cell division.

Membrane potential measurement in mouse salivary gland cells.

Acetylcholin erzeugt auf das Membranpotential von Azinuszellen der Glandula submandibularis der Maus eine diphasische Veranderung des Membranpotentials (rasche Hyperpolarisation und langsame, verzogerte Depolarisation oder rasche Depolarisation und langsame Hyperpolarisation). Hinweise sprechen dafur, dass Acetylcholin die Permeabilitat fur Na+-und K+-Ionen erhoht.