Salivary Gland Tissue Recombination Can Modify Cell Fate.

Although mesenchyme is essential for inducing the epithelium of ectodermal organs, its precise role in organ-specific epithelial fate determination remains poorly understood. To elucidate the roles of tissue interactions in cellular differentiation, we performed single-cell RNA sequencing and imaging analyses on recombined tissues, where mesenchyme and epithelium were switched ex vivo between two types of embryonic mouse salivary glands: the parotid gland (a serous gland) and the submandibular gland (a predominantly mucous gland). We found partial induction of molecules that define gland-specific acinar and myoepithelial cells in recombined salivary epithelium. The parotid epithelium recombined with submandibular mesenchyme began to express mucous acinar genes not intrinsic to the parotid gland. While myoepithelial cells do not normally line parotid acini, newly induced myoepithelial cells densely populated recombined parotid acini. However, mucous acinar and myoepithelial markers continued to be expressed in submandibular epithelial cells recombined with parotid mesenchyme. Consequently, some epithelial cells appeared to be plastic, such that their fate could still be modified in response to mesenchymal signaling, whereas other epithelial cells appeared to be already committed to a specific fate. We also discovered evidence for bidirectional induction: transcriptional changes were observed not only in the epithelium but also in the mesenchyme after heterotypic tissue recombination. For example, parotid epithelium induced the expression of muscle-related genes in submandibular fibroblasts that began to mimic parotid fibroblast gene expression. These studies provide the first comprehensive unbiased molecular characterization of tissue recombination approaches exploring the regulation of cell fate.

SPM Receptor Expression and Localization in Irradiated Salivary Glands.

Radiation therapy-mediated salivary gland destruction is characterized by increased inflammatory cell infiltration and fibrosis, both of which ultimately lead to salivary gland hypofunction. However, current treatments (e.g., artificial saliva and sialagogues) only promote temporary relief of symptoms. As such, developing alternative measures against radiation damage is critical for restoring salivary gland structure and function. One promising option for managing radiation therapy-mediated damage in salivary glands is by activation of specialized proresolving lipid mediator receptors due to their demonstrated role in resolution of inflammation and fibrosis in many tissues. Nonetheless, little is known about the presence and function of these receptors in healthy and/or irradiated salivary glands. Therefore, the goal of this study was to detect whether these specialized proresolving lipid mediator receptors are expressed in healthy salivary glands and, if so, if they are maintained after radiation therapy-mediated damage. Our results indicate that specialized proresolving lipid mediator receptors are heterogeneously expressed in inflammatory as well as in acinar and ductal cells within human submandibular glands and that their expression persists after radiation therapy. These findings suggest that epithelial cells as well as resident immune cells represent potential targets for modulation of resolution of inflammation and fibrosis in irradiated salivary glands.

Parasympathetic neurons in the human submandibular ganglion.

The submandibular ganglion (SMG) contains parasympathetic neurons which innervate the submandibular gland. In this study, immunohistochemistry for vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), choline acetyltransferase (ChAT), dopamine ß-hydroxylase (DBH), tyrosine hydroxylase (TH), and the transient receptor potential cation channel subfamily V members 1 (TRPV1) and 2 (TRPV2) was performed on the human SMG. In the SMG, 17.5 % and 8.9 % of parasympathetic neurons were immunoreactive for VIP and TRPV2, respectively. SMG neurons mostly contained ChAT- and DBH-immunoreactivity. In addition, subpopulations of SMG neurons were surrounded by VIP (69.6 %)-, TRPV2 (54.4 %)- and DBH (9.5 %)-immunoreactive (-ir) nerve fibers. SMG neurons with pericellular VIP- and TRPV2-ir nerve fibers were significantly larger than VIP- and TRPV2-ir SMG neurons, respectively. Other neurochemical substances were rare in the SMG. In the human submandibular gland, TRPV1- and TRPV2-ir nerve fiber profiles were seen around blood vessels. Double fluorescence method also demonstrated that TRPV2-ir nerve fiber profiles were located around myoepithelial and acinar cells in the submandibular gland. VIP and TRPV2 are probably expressed by both pre- and post-ganglionic neurons innervating the submandibular and sublingual glands. VIP, DBH and TRPV2 may have functions about regulation of salivary components in the salivary glands and neuronal activity in the SMG.

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.

Diverse epithelial cell populations contribute to the regeneration of secretory units in injured salivary glands.

Salivary glands exert exocrine secretory function to provide saliva for lubrication and protection of the oral cavity. Its epithelium consists of several differentiated cell types, including acinar, ductal and myoepithelial cells, that are maintained in a lineage-restricted manner during homeostasis or after mild injuries. Glandular regeneration following a near complete loss of secretory cells, however, may involve cellular plasticity, although the mechanism and extent of such plasticity remain unclear. Here, by combining lineage-tracing experiments with a model of severe glandular injury in the mouse submandibular gland, we show that de novo formation of acini involves induction of cellular plasticity in multiple non-acinar cell populations. Fate-mapping analysis revealed that, although ductal stem cells marked by cytokeratin K14 and Axin2 undergo a multipotency switch, they do not make a significant contribution to acinar regeneration. Intriguingly, more than 80% of regenerated acini derive from differentiated cells, including myoepithelial and ductal cells, that appear to dedifferentiate to a progenitor-like state before re-differentiation into acinar cells. The potential of diverse cell populations serving as a reserve source for acini widens the therapeutic options for hyposalivation.

Sex-dependent Regeneration Patterns in Mouse Submandibular Glands.

Our previous studies indicated that YIGSR-A99 peptides chemically conjugated to fibrin hydrogel (FH) and applied to wounded submandibular gland (SMG) in vivo, formed new organized salivary tissue, whereas wounded SMG treated with FH alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. While these studies indicated that damaged SMG grow and differentiate when treated with FH containing L1 peptide, they were performed only in female mice. However, there is a well-established sexual dimorphism present in mouse SMG (e.g., males develop well-differentiated granular convoluted tubules, but these structures are poorly developed in females) and little is known about how these sex differences influence wound healing events. Therefore, the goal of this study was to conduct comparative analyses of regeneration patterns in male and female mice using L1p-FH in a wounded SMG mouse model. Particularly, we focused on sex-dependent wound healing events such as macrophage polarization, vascularization, tissue organization, and collagen deposition, and how these events affect salivary gland functioning.

Stress or injury induces cellular plasticity in salivary gland acinar cells.

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.

Aberrant MUC1 accumulation in salivary glands of Sjögren's syndrome patients is reversed by TUDCA in vitro.

OBJECTIVES: Xerostomia in SS patients has been associated with low quality and quantity of salivary mucins, which are fundamental for the hydration and protection of the oral mucosa. The aim of this study was to evaluate if cytokines induce aberrant mucin expression and whether tauroursodeoxycholic acid (TUDCA) is able to counteract such an anomaly. METHODS: Labial salivary glands from 16 SS patients and 15 control subjects, as well as 3D acini or human submandibular gland cells stimulated with TNF-α or IFN-γ and co-incubated with TUDCA, were analysed. mRNA and protein levels of Mucin 1 (MUC1) and MUC7 were determined by RT-qPCR and western blot, respectively. Co-immunoprecipitation and immunofluorescence assays for mucins and GRP78 [an endoplasmic reticulum (ER)-resident protein] were also performed. mRNA levels of RelA/p65 (nuclear factor-κB subunit), TNF-α, IL-1ß, IL-6, SEL1L and EDEM1 were determined by RT-qPCR, and RelA/p65 localization was evaluated by immunofluorescence. RESULTS: MUC1 is overexpressed and accumulated in the ER of labial salivary gland from SS patients, while MUC7 accumulates throughout the cytoplasm of acinar cells; however, MUC1, but not MUC7, co-precipitated with GRP78. TUDCA diminished the overexpression and aberrant accumulation of MUC1 induced by TNF-α and IFN-γ, as well as the nuclear translocation of RelA/p65, together with the expression of inflammatory and ER stress markers in 3D acini. CONCLUSION: Chronic inflammation alters the secretory process of MUC1, inducing ER stress and affecting the quality of saliva in SS patients. TUDCA showed anti-inflammatory properties decreasing aberrant MUC1 accumulation. Further studies are necessary to evaluate the potential therapeutic effect of TUDCA in restoring glandular homeostasis in SS patients.

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 ß-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.

Physical exercise stimulates salivary secretion of cystatins.

Physical exercise is known to activate the sympathetic nervous system, which influences the production of saliva from salivary glands. Our examination of saliva collected from highly trained athletes before and after a number of physical competititions showed an increase in the secretion of S-type cystatins and cystatin C as a subacute response to aerobic and anaerobic exercise. The elevation in salivary cystatins was transient and the recovery time course differed from that of amylase and other salivary proteins. An in vitro assay was developed based on a cell line from a human submandibular gland (HSG) that differentiated into acinus-like structures. Treatments with the ß-adrenergic agonist isoproterenol caused a shift in the intracellular distribution of S-type cystatins and cystatin C, promoting their accumulation at the outer regions of the acinus prior to release and suggesting the activation of a directional transport involving co-migration of both molecules. In another treatment using non-differentiated HSG cells, it was evident that both expression and secretion of cystatin C increased upon addition of the ß-adrenergic agonist, and these effects were essentially eliminated by the antagonist propranolol. The HSG cell line appears to have potential as a model for exploring the mechanism of cystatin secretion, particularly the S-type cystatins that originate primarily in the submandibular glands.

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (αSMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sjögren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

Establishment of a Murine Pro-acinar Cell Line to Characterize Roles for FGF2 and α3ß1 Integrins in Regulating Pro-acinar Characteristics.

Radiation therapy for head and neck cancers results in permanent damage to the saliva producing acinar compartment of the salivary gland. To date, a pure pro-acinar cell line to study underlying mechanisms of acinar cell differentiation in culture has not been described. Here, we report the establishment of a pro-acinar (mSG-PAC1) and ductal (mSG-DUC1) cell line, from the murine submandibular salivary gland (SMG), which recapitulate developmental milestones in differentiation. mSG-DUC1 cells express the ductal markers, keratin-7 and keratin-19, and form lumenized spheroids. mSG-PAC1 cells express the pro-acinar markers SOX10 and aquaporin-5. Using the mSG-PAC1 cell line, we demonstrate that FGF2 regulates specific steps during acinar cell maturation. FGF2 up-regulates aquaporin-5 and the expression of the α3 and α6 subunits of the α3ß1 and α6ß1 integrins that are known to promote SMG morphogenesis and differentiation. mSG-DUC1 and mSG-PAC1 cells were derived from genetically modified mice, homozygous for floxed alleles of the integrin α3 subunit. Similar to SMGs from α3-null mice, deletion of α3 alleles in mSG-PAC1 cells results in the up-regulation of E-cadherin and the down-regulation of CDC42. Our data indicate that mSG-DUC1 and mSG-PAC1 cells will serve as important tools to gain mechanistic insight into salivary gland morphogenesis and differentiation.

Sox9 regulates the luminal stem/progenitor cell properties of salivary glands.

Exocrine glands share a common morphology consisting of ductal, acinar, and basal/myoepithelial cells, but their functions and mechanisms of homeostasis differ among tissues. Salivary glands are an example of exocrine glands, and they have been reported to contain multipotent stem cells that differentiate into other tissues. In this study, we purified the salivary gland stem/progenitor cells of adult mouse salivary glands using the cell surface marker CD133 by flow cytometry. CD133+ cells possessed stem cell capacity, and the transplantation of CD133+ cells into the submandibular gland reconstituted gland structures, including functional acinar. CD133+ cells were sparsely distributed in the intercalated and exocrine ducts and expressed Sox9 at higher levels than CD133- cells. Moreover, we demonstrated that Sox9 was required for the stem cell properties CD133+ cells, including colony and sphere formation. Thus, the Sox9-related signaling may control the regeneration salivary glands.

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α, ß, γ in the three major salivary glands in situ of mice and their response to ß-adrenoceptor stimulation.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which is composed of three isozymes (α, ß and γ), catalyzes the production of phosphatidylinositol bisphosphate (PIP2). This phospholipid functions in membrane trafficking, as an anchor for actin cytoskeletons and as a regulator of intramembranous channels/transporters. It is also a precursor of such second messengers as diacylglycerol, inositol triphosphate and phosphatidylinositol (3,4,5)-triphosphate. In the present study, the expression and localization of endogenous PIP5Ks were examined in the three major salivary glands of young adult mice in situ. In western blotting of normal control glands, immunoreactive bands for individual PIP5Ks were detectable, with the highest density in the parotid gland and the weakest density in the submandibular gland. In immuno-light microscopy under non-stimulated condition, weak immunoreactivity for PIP5Kα was confined to the apical plasmalemma in parotid, but not sublingual or submandibular, acinar cells. Immunoreactivity for PIP5Kß was weak to moderate and confined to ductal cells but not acinar cells, whereas that for PIP5Kγ was selectively and intensely detected in myoepithelial cells but not acinar cells, and it was weak in ductal cells in the three glands. In western blot of the parotid gland stimulated by isoproterenol, a ß-adrenoceptor agonist, no changes were seen in the intensity of immunoreactive bands for any of the PIP5Ks. In contrast, in immuno-light microscopy, the apical immunoreactivity for PIP5Kα in parotid acinar cells was transiently and distinctly increased after the stimulation. The increased immunoreactivity was ultrastructurally localized on most apical microvilli and along contiguous plasma membrane, where membranous invaginations of various shapes and small vesicles were frequently found. It was thus suggested that PIP5Kα is involved in post-exocytotic membrane dynamics via microvillous membranes. The present finding further suggests that each of the three isoforms of PIP5K functions through its product PIP2 discretely in different cells of the glands to regulate saliva secretion.

Potential therapeutic effects of induced pluripotent stem cells on induced salivary gland cancer in experimental rats.

Salivary gland neoplasms exhibit complex histopathology in a variety of tumor types and treatment options depend largely on the stage of the cancer. Induced pluripotent stem cells (iPS) have been investigated for treating induced salivary gland cancer and for restoring salivary gland function. We investigated iPS treatment for salivary gland cancer both in vitro and in vivo. For our study in vitro, we re-programmed human skin fibroblasts to form iPS cells using a plasmid containing Oct4, Sox2, L-MYC and LIN28. For our study in vivo, we used 30 white male albino rats divided into the following groups of 10: group 1 (control): rats were injected with phosphate-buffered saline (PBS), group 2 induced squamous cell carcinoma (SCC): rat submandibular glands were injected with squamous carcinoma cells (SCC), group 3 (induced SCC/iPS): SCC treated rats treated with 5 × 106 iPS cells. Submandibular glands from rats of all groups were examined histologically and real time PCR was performed for amylase, and COX I and COX II gene expression. We confirmed that submandibular gland specimens included tumor tissue before starting treatment with iPS. iPS treated cases exhibited regeneration of salivary glands, although minor degenerative and vascularization changes remained. The acinar cells regained their proper organization, but continued to exhibit abnormal activity including hyperchromatism. iPS cells may be useful for treating salivary gland carcinomas.

Genetic and scRNA-seq Analysis Reveals Distinct Cell Populations that Contribute to Salivary Gland Development and Maintenance.

Stem and progenitor cells of the submandibular salivary gland (SMG) give rise to, maintain, and regenerate the multiple lineages of mature epithelial cells including those belonging to the ductal, acinar, basal and myoepithelial subtypes. Here we have exploited single cell RNA-sequencing and in vivo genetic lineage tracing technologies to generate a detailed map of the cell fate trajectories and branch points of the basal and myoepithelial cell populations of the mouse SMG during embryonic development and in adults. Our studies show that the transcription factor p63 and alpha-smooth muscle actin (SMA) serve as faithful markers of the basal and myoepithelial cell lineages, respectively and that both cell types are endowed with progenitor cell properties. However, p63+ basal and SMA+ myoepithelial cells exhibit distinct cell fates by virtue of maintaining different cellular lineages during morphogenesis and in adults. Collectively, our results reveal the dynamic and complex nature of the diverse SMG cell populations and highlight the distinct differentiation potential of the p63 and SMA expressing subtypes in the stem and progenitor cell hierarchy. Long term these findings have profound implications towards a better understanding of the molecular mechanisms that dictate lineage commitment and differentiation programs during development and adult gland maintenance.

The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells.

The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3- and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing-based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl- for oxalate and HCO3-, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl-/oxalate exchange. Of note, both Cl-/oxalate exchange and Cl-/HCO3- exchange were significantly reduced in acinar cells isolated from the submandibular glands of Slc26a6-/- mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6-/- mice, but HCO3- secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinar cells, where it mediates Cl-/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.

Expression and localization of phosphodiesterase 2A in the submandibular gland of mice.

OBJECTIVES: Phosphodiesterases comprise a superfamily of enzymes that hydrolyze and inactivate cyclic AMP (cAMP) and/or cyclic GMP (cGMP), thereby regulating cellular signaling mechanisms. We herein investigated the production of phosphodiesterase 2A (PDE2A) in the mouse submandibular gland. DESIGN: The expression and localization of the mRNA and protein of PDE2A were examined in the submandibular gland of male and female mice using the reverse transcription-polymerase chain reaction, in situ hybridization, Western blotting, and immunohistochemistry. RESULTS: Among the different species of phosphodiesterases examined in the mouse submandibular gland, PDE2A, which hydrolyzes cAMP and cGMP, exhibited a marked sexual difference; it was more abundantly expressed in females. The mRNA and protein signals for PDE2A were intense in all acinar and duct portions, including the striated duct, in females, whereas in males, these signals were markedly weaker in the granular convoluted duct, the counterpart of the female striated duct, than in acini and other duct portions. Furthermore, the signals for protein kinases A and G1, which are intracellular effectors of cAMP and cGMP, respectively, were markedly weaker in the male granular convoluted duct. CONCLUSIONS: These results suggest that cyclic nucleotide-dependent signaling mechanisms function poorly in granular convoluted duct cells in the mouse submandibular gland.

Functional characterization and genomic studies of a novel murine submandibular gland epithelial cell line.

A better understanding of the normal and diseased biology of salivary glands (SG) has been hampered, in part, due to difficulties in cultivating and maintaining salivary epithelial cells. Towards this end, we have generated a mouse salivary gland epithelial cell (mSGc) culture system that is well-suited for the molecular characterization of SG cells and their differentiation program. We demonstrate that mSGc can be maintained for multiple passages without a loss of proliferation potential, readily form 3D-spheroids and importantly express a panel of well-established salivary gland epithelial cell markers. Moreover, mSGc 3D-spheroids also exhibit functional maturation as evident by robust agonist-induced intracellular calcium signaling. Finally, transcriptomic characterization of mSGc by RNA-seq and hierarchical clustering analysis with adult organ RNA-seq datasets reveal that mSGc retain most of the molecular attributes of adult mouse salivary gland. This well-characterized mouse salivary gland cell line will fill a critical void in the field by offering a valuable resource to examine various mechanistic aspects of mouse salivary gland biology.