Granular Convoluted Tubule Research Articles

Abstract 7446: Transcriptomic and histological analysis of radiation-induced damage in the murine submandibular gland

Abstract Many head and neck cancer patients develop radiation-induced xerostomia (RIX). There are few clinical solutions for this condition and there is a critical need for novel therapeutics for this condition. To better understand the molecular alterations induced by radiation to the salivary glands, we used transcriptional profiling to assess acute and long-term alterations. Based on evidence that immunologic changes are induced by radiation, we used immune-depletion experiments to investigate the role of B-cells and macrophages in RIX. The submandibular gland (SMG) of male-C57/BL6-mice was irradiated (15Gy) mice were followed for 90 days. Saliva was collected throughout the time-course. SMGs were collected 3-, 7-, 60-, and 90-days following radiation. Tissues were analyzed by Nanostring Digital-Spatial-Profiling (DSP) utilizing the murine whole-transcriptome panel. Regions of interest corresponding to acini, ducts, and granular convoluted tubules (GCTs) were delineated and gene expression was assessed. Immunohistochemical staining was performed on SMG’s for fibrosis (masons trichrome), α-amylase, F4/80, Lgr5, Sox2, and MIST1 to evaluate glandular structure, salivary proteins, immune-microenvironment and pro-acinar salivary-stem-cell presence. Specific immune populations were depleted using anti-CD20 and clodronate-liposome prior to radiation and salivary function and histology was assessed at 3-, 7-, and 60-days following radiation. Radiation resulted in a significant decrease in salivary production (p-value = 0.03) in immune competent models. Transcriptional pathway analysis identified early alterations in signal-transduction and innate immune-response. Late timepoints identified alterations in B-cell activity and Wnt signaling pathways. Histological evaluation of the tissues showed increased disorganization of the gland, with decreases in size and distribution of the acinar compartments (mean area stained: No RT=25.81% vs 90d RT=19.98%, p-value=0.005). Acutely, there was an increase in expression of MIST1 and macrophages (mean area stained: No RT=14.8% vs 7d RT=18.76%, p-value=0.0004) within the SMG that resolved by the 60- and 90-day time-points. Results of immune depletion studies will be presented.Results implicate alterations in macrophages and B-cells in radiation-induced damage. These data along with the data from our immune-depletion experiments will provide valuable mechanistic insight guiding modulation of cell-therapies for treatment of RIX which can be further tested in preclinical models. Citation Format: Cristina Paz, Annemarie Glassey, Abigail Frick, Lauryn Melzer, Jasmine M. Robinson, Rebecca M. Baus, Randall J. Kimple. Transcriptomic and histological analysis of radiation-induced damage in the murine submandibular gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7446.

Putative effect of chronic renal failure on granular convoluted tubule of submandibular gland in male rats: Immunohistochemical and ultrastructural study

ObjectiveThis study aimed to investigate the ultrastructural and immunohistochemical changes in the granular convoluted tubule (GCT) of rodents' submandibular gland (SMG) upon theinduction of chronic renal failure. Material and methodsThirty young adult Sprague-Dawley rats were randomized into three groups: the Control group, rats received no intervention; the Sham group, rats underwent surgical incision without nephrectomy; the Experimental group, rats underwent surgical procedures to induce chronic renal failure. Afterward, SMG was examined for histological and ultrastructural changes and immunohistochemical staining for Renin. ResultsHistologically, the experimental group demonstrated cytoplasmic vacuolization within the seromucous acini and ducts. Several GCTs were proliferating, whereas others exhibited degenerative changes in the form of disturbed cytoplasmic architecture. On the ultrastructural level, both acini and ductal segments showed degenerative changes Interestingly, immunohistochemical examination of the lining cells of GCT and intralobular ducts of the experimental group revealed the presence of Renin. ConclusionRenal failure induced histological, immunohistochemical, and ultrastructural variations within GCTs of SMG.

Pathway Analysis of Transcriptional Alterations in the Salivary Gland Following Radiation Exposure

Radiation-induced xerostomia (RIX) is the subjective condition of dry mouth caused by radiation therapy to the head and neck and a result of hyposalivation and altered sialochemistry. Our long-term goal is to develop novel approaches to improve salivary function after radiation therapy. To better understand the molecular alterations induced by radiation in the salivary glands, we performed transcriptional profiling aimed at describing acute and long-term alterations in the murine salivary glands induced by radiation to better design effective regenerative therapies. The submandibular gland (SMG) of male C57Bl/6 mice received 15 Gy in a single fraction using a small animal image-guided irradiator. The mice were followed for 90 days. Saliva was collected at baseline and 7, 60 and 90 days after radiation. Salivary glands were collected for histology 3, 7, 60, and 90 days after radiation. The tissues were analyzed using a multiplex analysis platform processed with the murine whole transcriptome panel to investigate changes in gene expression over time. Using the multiplex analysis platform software, 96 regions of interest corresponding to acini, ducts, and granular convoluted tubules (GCTs) were delineated and gene expression was assessed separately in each area. To confirm the identified alterations, SMG tissue was histologically evaluated for fibrosis (masons tri-chrome), mucin (alcian blue), and amylase (α-amylase). Alterations in the immune microenvironment (CD19, CD3, and F4/80) and salivary gland stem cells (MIST1, SCA, Sox2, and c-Kit) were assessed by immunohistochemistry. Radiation resulted in a significant decrease in salivary production compared to nonirradiated controls (p-value = 0.03). The analysis of the transcriptional pathway identified early alterations in cell communication, DNA damage, and the immune response. Protein metabolism and extracellular matrix remodeling were up-regulated at later time points. Histological evaluation of tissues showed an increase in glandular structure disorganization over time, with a decrease in the size and distribution of the acinar compartments throughout the gland (mean area stained: No RT = 25.81% vs 90d RT = 19.98%, p-value = 0.005). There was an increase in periductal fibrosis within the tissue after 60 days compared to controls. Acutely, there was an increase in the expression of MIST1 and macrophages (mean area stained: No RT = 14.8% vs 7d RT = 18.76%, p-value = 0.0004) within the SMG that had resolved by the 60- and 90-day time points. The results implicate macrophage alterations in radiation-induced salivary gland damage and provide valuable mechanistic insight guiding potential approaches to intervene in RIX. We are pursuing cell therapy-based approaches to prevent and repair damage caused by radiation in order to improve the lives of our patients.

P130Cas is required for androgen-dependent postnatal development regulation of submandibular glands

Salivary glands develop through epithelial-mesenchymal interactions and are formed through repeated branching. The Crk-associated substrate protein (p130Cas) serves as an adapter that forms a complex with various proteins via integrin and growth factor signaling, with important regulatory roles in several essential cellular processes. We found that p130Cas is expressed in ductal epithelial cells of the submandibular gland (SMG). We generated epithelial tissue-specific p130Cas-deficient (p130CasΔepi–) mice and aimed to investigate the physiological role of p130Cas in the postnatal development of salivary glands. Histological analysis showed immature development of granular convoluted tubules (GCT) of the SMG in male p130CasΔepi– mice. Immunofluorescence staining showed that nuclear-localized androgen receptors (AR) were specifically decreased in GCT cells in p130CasΔepi– mice. Furthermore, epidermal growth factor-positive secretory granules contained in GCT cells were significantly reduced in p130CasΔepi– mice with downregulated AR signaling. GCTs lacking p130Cas showed reduced numbers and size of secretory granules, disrupted subcellular localization of the cis-Golgi matrix protein GM130, and sparse endoplasmic reticulum membranes in GCT cells. These results suggest that p130Cas plays a crucial role in androgen-dependent GCT development accompanied with ER-Golgi network formation in SMG by regulating the AR signaling.

Cimetidine-induced androgenic failure causes cell death and changes in actin, EGF and V-ATPase immunoexpression in rat submandibular glands.

Submandibular gland (SMG) is responsive to androgens via androgen receptor (AR). We verified whether cimetidine induces androgenic dysfunction in SMG, and evaluated the structural integrity, cell death and immunoexpression of actin, EGF and V-ATPase in androgen-deficient SMG. Male rats received cimetidine (CMTG) and control animals (CG) received saline. Granular convoluted tubules (GCTs) diameter and number of acinar cell nuclei were evaluated. TUNEL and immunofluorescence reactions for detection of AR, testosterone, actin, EGF and V-ATPase were quantitatively analysed. In CG, testosterone immunolabelling was detected in acinar and ductal cells cytoplasm. AR-immunolabelled nuclei were observed in acinar cells whereas ductal cells showed AR-immunostained cytoplasm, indicating a non-genomic AR action. In CMTG, the weak testosterone and AR immunoexpression confirmed cimetidine-induced androgenic failure. A high cell death index was correlated with decreased number of acinar cells, GCTs diameter and EGF immunoexpression under androgenic dysfunction. Actin immunofluorescence decreased in the SMG cells, but an increased and diffuse cytoplasmic V-ATPase immunolabelling was observed in striated ducts, suggesting a disruption in the actin-dependent V-ATPase recycling due to androgenic failure. Our findings reinforce the androgenic role in the maintenance of SMG histophysiology, and point to a potential clinical use of cimetidine against androgen-dependent glandular tumour cells.

Reversible Conversion among Subtypes of Salivary Gland Duct Cells as Identified by Production of a Variety of Bioactive Polypeptides.

Four major kallikreins (mK1, mK22, mK9, and mK13) were identified in the mouse submandibular gland (SMG). mK1, a true tissue kallikrein, was used as a protein marker to identify different types of SMG granular convoluted tubule (GCT) cells along with epidermal growth factor (EGF), nerve growth factor (NGF), and renin. Kallikrein mK1 was localized in a very small number (~5%) of GCT cells, which were scattered throughout the GCT, indicating that the majority of GCT cells are mK1-negative. Among mK1-positive cells, particularly strong signals were observed in a small number of narrow cells, recognized as slender granular cells (SG cells, Type IV), in the GCT. After postnatal development of the SMG, GCT cells are no longer uniform based on the bioactive substances (mK1, EGF, NGF, and renin) that they produce and secrete. GCT cells were classified into four subtypes, Types I–IV, and it became clear that these subtypes are complicatedly and reversibly converted by the endocrine hormones 5α-dihydrotestosterone (DHT) and triiodothyronine (T3). Duct segments with similar morphology or hormone dependency were recognized in the sublingual and parotid glands. The presence of duct cells with such characteristics is therefore a common feature of the three major salivary glands of rodents.

Expression of endogenous phospholipase D1, localized in mouse submandibular gland, is greater in females and is suppressed by testosterone.

To clarify the signal transduction mechanism in the differentiation and secretion of salivary glandular cells, the present study was attempted to examine in the submandibular gland (SMG) of mice, the expression and localization of phospholipase D1 (PLD1), one of the important effector molecules working in response to the activation of intramembranous receptors by first messengers. In immunoblotting analysis, the expression of PLD1 was high at postnatal 4weeks (P4W) and decreased at P8W, and it was at negligible levels at newborn stage (P0W) and postnatal 2weeks (P2W). The expression of PLD1 was greater in females, and it was suppressed by administration of testosterone to female mice. In immuno-light microscopy, immunoreactivity for PLD1 at P4W was moderate to intense, in the forms of dots and globules mainly in the apical domains of immature granular convoluted tubule (GCT)-cells localized largely in the proximal portion of the female GCT. By P8W, it decreased in intensity and remained weak to moderate along the apical plasmalemma of cells throughout the course of the female GCT, whereas it was faint throughout the GCT of the male SMG at P4W and negligible at P8W. In immuno-electron microscopy, immature GCT-cells characterized by electron-lucent granules were immunoreactive and the immunoreactive materials were deposited close to, but not within, those granules. Typical GCT cells, characterized by electron-dense granules, were immunonegative. No significant immunoreaction for PLD1 was seen in acini of SMGs of either sex at any time point examined. It is suggested that PLD1 is involved in the signaling for secretion of immature GCT cells and influences differentiation of these cells, probably through their own secretory substances.

Loss of Fam20c causes defects in the acinar and duct structure of salivary glands in mice.

Family with sequence similarity 20-member C (FAM20C), a recently characterized Golgi kinase, performs numerous biological functions by phosphorylating more than 100 secreted proteins. However, the role of FAM20C in the salivary glands remains undefined. The present study demonstrated that FAM20C is mainly located in the cytoplasm of duct epithelial cells in the salivary glands. Fam20cf/f; Mmtv-Cre mice were created in which Fam20c was inactivated in the salivary gland cells and observed that the number of ducts and the ductal cross-sectional area increased significantly, while the number of acinar cells was reduced. The granular convoluted tubules (GCTs) exhibited an accumulation of aberrant secretory granules, along with a reduced expression and altered distribution patterns of β nerve growth factor, α-amylase and bone morphogenetic protein (BMP) 4. This abnormality suggested that the GCT cells were immature and exhibited defects in developmental and secretory functions. In accordance with the morphological alterations and the reduced number of acinar cells, FAM20C deficiency in the salivary glands significantly decreased the salivary flow rate. The Na+, Cl− and K+ concentrations in the saliva were all significantly increased due to dysfunction of the ducts. Furthermore, Fam20c deficiency significantly increased BMP2 and BMP7 expression, decreased BMP4 expression, and attenuated the canonical and noncanonical BMP signaling pathways in the salivary glands. Collectively, the results of the present study demonstrate that FAM20C is a key regulator of acinar and duct structure and duct maturation and provide a novel avenue for investigating novel therapeutic targets for oral diseases including xerostomia.

Cholesterol metabolism plays a crucial role in the regulation of autophagy for cell differentiation of granular convoluted tubules in male mouse submandibular glands

It has been long appreciated that sex hormone receptors are expressed in various non-gonadal organs. However, it remains unclear how sex hormones regulate the morphogenesis of these non-gonadal organs. To address this issue, we used a male mouse model of androgen-dependent salivary gland morphogenesis. Mice with excessive cholesterol synthesis in the salivary glands exhibited defects in the maturation of granular convoluted tubules (GCTs), which is regulated through sex hormone-dependent cascades. We found that excessive cholesterol synthesis resulted in autophagy failure specifically in the duct cells of salivary glands, followed by the accumulation of NRF2, a transcription factor known as one of the specific substrates for autophagy. The accumulated NRF2 suppressed the expression of Foxa1, which forms a transcriptional complex with the androgen receptor to regulate target genes. Taken together, our results indicate that cholesterol metabolism plays a crucial role in GCT differentiation through autophagy.

Distinct hormonal regulation of two types of sexual dimorphism in submandibular gland of mice.

The submandibular gland (SMG) of mice exhibits prominent sexual dimorphism in two aspects: the preferential development of granular convoluted tubule (GCT) cells and the earlier disappearance of granular intercalated duct (GID) cells in males after puberty. The former is dependent on androgens and thyroid hormones, whereas the hormonal dependence of the latter remains obscure. In the present study, we examined the effects of the postnatal administration of androgens and thyroid hormones to wild-type (WT) and androgen-receptor-knockout (ARKO) mice on these two types of sexual dimorphism by counting the numbers of GCT and GID cells labeled with nerve growth factor and submandibular gland protein C, respectively, as immunohistochemical markers. WT females and ARKO males and females exhibited a lower number of GCT cells and higher number of GID cells at 5 and 11weeks postpartum than WT males. The administration of dihydrotestosterone for 1-2weeks prior to these ages caused an increase in GCT cells and decrease in GID cells in WT females to similar levels as those in WT males, whereas it had no effects in ARKO, indicating that both types of sexual dimorphism are androgen-dependent. In contrast, the administration of thyroxine caused an increase in GCT cells but did not cause a decrease in GID cells in WT females or ARKO, indicating that the former is dependent on thyroid hormones, whereas the latter is not. The present results suggest that the two types of sexual dimorphism in the mouse SMG undergo distinct forms of hormonal regulation and, therefore, have different mechanisms.

Mechanistic effect of human umbilical cord blood derived mesenchymal stem cells on the submandibular salivary gland in ovariectomized rats.

We performed this study to understand the effect of human umbilical cord blood derived mesenchymal stem cells (hUCB-MSCs) on the submandibular gland after bilateral ovariectomy. For this, 21 adult female rats were distributed equally among 3 groups: the sham-operated group (SHAM); the ovariectomized group (OVX); and the OVX group that received repeated intravenous injections of the hUCB-MSCs (OVX + hUCB-MSCs). We used reverse transcription - PCR to analyze for the gene expression of AQPs 3, 4, 5, and BMP-6. The cellular localization and expression of human CD105, human CD34, proliferating nuclear antigen (PCNA), single-stranded DNA (ss-DNA), caspase 3, AQP1, and α smooth muscle actin (α-SMA) were determined immunohistochemically. In the OVX group, a significant decrease in the gene expression of AQP3, AQP4, and BMP6, as well as the acinar area % was detected, while area % of granular convoluted tubules (GCTs) showed a significant increase. A significant decrease in area % staining positively for AQP1 and α-SMA was noted. An obvious improvement in the structure of the submandibular gland was demonstrated in the group injected with hUCB-MSCs, as well as a significant increase in the gene expression of AQP3, AQP4, and BMP6. The acinar and GCT area %, as well as the different measured markers, were relatively normal. This demonstrates that E2-deficiency induces structural changes to the submandibular gland. Moreover, a definite amelioration of the structure and function of the submandibular gland was detected after the administration of hUCB-MSCs.

Effects of ovariectomy, estrogen and soy isoflavones in rats submandibular glands

Objective: A decrease in granular convoluted tubule (GCT) cells and acini occurs in the submandibular glands of castrated female rats, while in rats submitted to hormone replacement and phytotherapy with soy isoflavones, this effect is reversed. This study aimed to elucidate the mechanisms through which these changes occur. Material and Methods: Rats (n=84) were ovariectomized and 21 were sham-operated. Ovariectomized rats were randomly subdivided and orally administered the following: 17 ?-estradiol (OVX-E; n=21), 15 mg/kg/day of soy isoflavone extract (OVX-I; n=21), 17 ?-estradiol + soy isoflavone extract (OVX-A; n=21); and water as placebo (OVX; n=21). The rats were euthanized three, five and eight weeks after ovariectomy. The submandibular salivary glands were submitted to histological processing with HE stain and immunohistochemistry was performed using the streptavidin-biotin-peroxidase complex. The cell area and the expression of proliferating cell nuclear antigen and estrogen receptor ? were evaluated. Results: The results were statistically analyzed by ANOVA and Tukey test. A decrease in the area of GCT cells in the OVX, was observed, in contrast with an increase in the OVX-E. PCNA in the acinar cells and estrogen receptors were elevated in the OVX-I group. Conclusion: Castration exerts an immediate reductive effect on the volume of GCT cells. Estrogens, soy isoflavones and their combination have different mechanisms of action on the homeostasis of the gland. Estrogens cause an increase in GCT cells area, while isoflavones enhance cell proliferation and the expression of estrogen receptor-?. Their association showed no additional increase in the effect studied.KeywordsEstrogen; Morphometry; Salivary glands; Soy isoflavones.

Runx1 mediates the development of the granular convoluted tubules in the submandibular glands.

The mouse granular convoluted tubules (GCTs), which are only located in the submandibular gland (SMG) are known to develop and maintain their structure in an androgen-dependent manner. We previously demonstrated that the GCTs are involuted by the epithelial deletion of core binding factor β (CBFβ), a transcription factor that physically interacts with any of the Runt-related transcription factor (RUNX) proteins (RUNX1, 2 and 3). This result clearly demonstrates that the Runx /Cbfb signaling pathway is indispensable in the development of the GCTs. However, it is not clear which of the RUNX proteins plays useful role in the development of the GCTs by activating the Runx /Cbfb signaling pathway. Past studies have revealed that the Runx /Cbfb signaling pathway plays important roles in various aspects of development and homeostatic events. Moreover, the Runx genes have different temporospatial requirements depending on the biological situation. In the present study, the GCTs of the SMG showed a remarkable phenotype of, which phenocopied the epithelial deletion of Cbfb, in epithelial-specific Runx1 conditional knock-out (cKO) mice. The results indicate that Runx1 works as a partner of Cbfb during the development of the GCTs. We also discovered that the depletion of Runx1 resulted in the reduced secretion of saliva in male mice. Consistent with this finding, one of the water channels, Aquaporin-5 (AQP5) was mislocalized in the cytoplasm of the Runx1 mutants, suggesting a novel role of Runx1 in the membrane trafficking of AQP5. In summary, the present findings demonstrated that RUNX1 is essential for the development of the GCTs. Furthermore, RUNX1 could also be involved in the membrane trafficking of the AQP5 protein of the acinar cells in the SMG in order to allow for the proper secretion of saliva.

Synthesis, localization and possible function of serine (or cysteine) peptidase inhibitor, clade B, member 6a (Serpinb6a) in mouse submandibular gland.

The granular convoluted tubule (GCT) in the duct system of the submandibular gland (SMG) develops preferentially in male mice and produces a number of bioactive peptides including proteases such as renin and kallikrein. We examine the synthesis and localization of the serine (or cysteine) peptidase inhibitor, clade B, member 6a (Serpinb6a), the mouse ortholog of the human intracellular serine protease inhibitor SERPINB6, in the mouse SMG by using reverse transcription plus the polymerase chain reaction, in situ hybridization, immunoblotting and immunohistochemistry. Serpinb6a mRNA expression was more abundant in the male than in the female SMG and in the GCT than in other duct portions or acini. Within GCT cells, immunoreactivity for Serpinb6a was localized in the nucleus and cytosol but was absent in the secretory granules. The binding target of Serpinb6a in the SMG was investigated by using a mass spectrometric analysis of immunoprecipitation products and kallikrein-1-related peptidase b26 (Klk1b26), a serine protease, was identified. These results raise the possibility that Serpinb6a functions in the protection of GCT cells from intracellular kallikreins that may leak from secretory granules.

Expression and localization of calpain 3 in the submandibular gland of mice

ObjectivesCalpains comprise a family of intracellular Ca2+-dependent cysteine proteases and are considered to play roles in various physiological phenomena with limited proteolytic activities against specific substrates. We herein revealed the expression and localization of calpain 3, the muscle-type calpain, in the submandibular gland (SMG) of mice. DesignThe expression of the mRNA for conventional, ubiquitous calpains 1 and 2 and skeletal muscle-specific calpain 3 was examined in the major salivary glands of mice using RT-PCR, and the expression and localization of calpain 3 protein was examined in the SMG of mice using immunohistochemistry and Western blotting. ResultsThe large catalytic subunits of calpains 1 and 2 and the small regulatory subunit common to calpains 1 and 2 were weakly expressed in the parotid gland, sublingual gland, and SMG at similar levels in males and females. In contrast, the single large catalytic subunit of calpain 3 was expressed predominantly in the SMG at markedly higher levels in males than in females and in a manner dependent on androgens. Immunoreactivity for calpain 3 was mainly localized in cells of the granular convoluted tubules (GCT) that developed preferentially in the male SMG. In GCT cells, calpain 3 immunoreactivity was localized predominantly in the cytosolic region and was absent in the secretory granules. ConclusionsThese results revealed that the GCT is the primary site of production of calpain 3 in the mouse SMG.

Runx/Cbfb signaling regulates postnatal development of granular convoluted tubule in the mouse submandibular gland.

The rodent salivary gland is not fully developed at birth and the cellular definitive differentiation takes place postnatally. However, little is known about its molecular mechanism. Here we provide the loss-of-function genetic evidence that Runx signaling affects postnatal development of the submandibular gland (SMG). Core binding factor β (Cbfb) is a cotranscription factor which forms a heterodimer with Runx proteins. Cbfb was specifically expressed in the duct epithelium, specifically in the SMG. Epithelial Cbfb deficiency resulted in decrease in the size of the SMG and in the saliva secretion on postnatal day 35. The Cbfb mutant SMG specifically exhibited involution of the granular convoluted tubules (GCT), with a down-regulated expression of its marker genes, such as Klk1, Ngf, and Egf. The induction of GCT is under the control of androgens, and the Cbfb mutant SMG demonstrated down-regulated expression of Crisp3, an androgen-dependent transcript. Because the circulating testosterone or tissue dihydrotestosterone levels were not affected in the Cbfb mutants, it appears that Runx/Cbfb signaling regulate androgen receptor pathway, but does not affect the circulating testosterone levels or the enzymatic conversion to DHT. Runx signaling is important in the postnatal development of androgen-dependent GCT in the SMG.

Morphology and gene expression profile of the submandibular gland of androgen-receptor-deficient mice

ObjectivesIn the submandibular gland (SMG) of mice, the granular convoluted tubule (GCT) develops preferentially in males dependent on androgens. To clarify the molecular mechanism of androgen action in SMG, we examined the SMG of mice deficient for the androgen receptor (ARKO). DesignThe morphological features and gene expression in the SMG of control and ARKO mice with or without hormone treatments were analysed by immunohistochemistry, DNA microarray, and RT-PCR. ResultsThe development of GCT and expression of GCT-specific products such as NGF were even lower in ARKO male SMG than in control female SMG. The administration of androgens to ARKO males had no effect on SMG, whereas the administration of thyroid hormone (T4) caused the extensive conversion of striated duct cells to GCT cells with the increase of NGF mRNA. Gene expression profiles in control and ARKO male SMG were analysed by DNA microarrays, and genes with higher or lower expression in ARKO male SMG were determined. They were then classified into groups according to their responsiveness to the administration of dihydotestosterone (DHT) or T4 to ARKO males. RT-PCR revealed that, while no gene was responsive to DHT, expression of many genes was up- or down-regulated by T4. ConclusionsThese results confirmed that GCT cell differentiation induced by androgens is dependent on the classical androgen receptor (AR), whereas that by T4 is independent of AR. Differential reactivity of genes to androgens and thyroid hormone in ARKO mice may shed light on the mechanism of androgen action in the SMG.

Why do male mice spit soluble enzymes that hydrolyze extracellular nucleotides? Focus on “Prostatic acid phosphatase is the main acid phosphatase with 5′-ectonucleotidase activity in the male mouse saliva and regulates salivation”

the 5′-ectonucleotidases found on the surface of most mammalian cells and in blood serum serve as a source of adenosine derived from extracellular nucleotide degradation (11). The 5′-ectonucleotidases are phosphatases whose substrate is extracellular adenosine 5′-monophosphate (AMP) generated from adenosine 5′-tri- and diphosphates (ATP and ADP) released from injured tissues to stimulate cellular responses via P2 purinergic receptors (7). A recent study by Quintero et al. (10) investigated prostatic acid phosphatase (PAP), a 5′-ectonucleotidase that plays a role in the development of adenocarcinoma of the prostate gland. These authors showed that PAP exists as two alternative splice variants, a transmembrane prostatic acid phosphatase (TMPAP) and secreted PAP (sPAP). TMPAP and sPAP are distinguished functionally from other 5′-nucleotidases through inhibition by l−(+)−tartrate, ability to cleave phosphomonoesters besides AMP, and activity over a wide pH range (i.e., 3–8). TMPAP activation inhibits endocytosis/exocytosis through interaction with snapin (10), a soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE)-associated protein (5) that negatively regulates secretory vesicle docking and fusion with plasma membranes (Fig. 1). In addition, adenosine generated by PAP is an agonist for G protein-coupled A1 adenosine receptors (A1R) that counteract the nociceptive (i.e., pain-producing) effects of ATP (4) occurring through activation of P2X receptor ATP-gated ion channels (2; Fig. 1). However, little information is available concerning physiological roles of sPAP in biological fluids. Fig. 1. Prostatic acid phosphatase (PAP) signaling in salivary glands and saliva. In this issue of American Journal of Physiology-Cell Physiology, Araujo et al. (1) attempt to understand the role of TMPAP and sPAP in a secretory organ, the submandibular gland (SMG), which secretes fluid, proteins, and other factors into saliva, thereby bathing the oral cavity with antimicrobial and digestive agents. Using PAP−/− mice, the authors evaluate effects of deletion of this 5′-ectonucleotidase on SMG function. Comparisons between PAP−/− and wild-type SMG indicate that TMPAP is primarily expressed in granular convoluted tubule (GCT) cells of the duct but not acinar cells. Remarkably, deletion of PAP increases α-adrenergic and muscarinic cholinergic receptor-mediated saliva secretion and enhances expression of genes promoting inflammation and proliferation, particularly interferon-dependent proinflammatory genes. The authors suggest that this increase in saliva secretion relates to loss of negative control by TMPAP of exocytosis (Fig. 1). PAP deletion also increases B and T cell infiltration of SMG and levels of miR-146a that have been associated with prevention of tissue damage. Together with observations of proliferating acinar cells after PAP deletion, these results strongly suggest that SMG homeostasis in PAP−/− mice is maintained by an immune response without salivary gland degeneration seen with immune cell infiltration of SMG in the autoimmune disease Sjogren's syndrome (SS; 3, 8). Another important finding is that sPAP is only present in male mouse saliva, which correlates with reduced levels of GCT cells in female versus male SMG and is consistent with sexual dimorphism in rodent salivary glands (6). The authors suggest that mouse sPAP plays a male-specific function in which licking of wounds (perhaps caused by fighting) deposits sPAP-containing saliva that induces an anti-nociceptive response in wounded tissue by participating in the generation of adenosine from released nucleotides to activate A1 receptors (Fig. 1). Since SS has a female predominance in rodents and humans (3, 8), it would be interesting to determine whether PAP in males plays a protective role in SS by promoting gland homeostasis and delay in the onset of salivary gland dysfunction and degenerative immune cell infiltration. Another question remaining is whether extracellular nucleotides are released into the oral cavity due to tissue injury of oral epithelium caused by inflammation, exposure to toxins, SS, and radiation-induced damage occurring as a side effect of cancer therapy. Once released, degradation of extracellular nucleotides by ectonucleotide diphosphohydrolases (eNTPDases), ectoATPases, and ectoADPases would generate AMP whereupon 5′-ectonucleotidases including PAP would yield adenosine that could either be taken up by nucleoside transporters in cells and salvaged or activate P1 adenosine receptors (7, 9, 11). Further study is needed to investigate whether sPAP in saliva plays a role in the regulation of purinergic signaling in the oral cavity under physiological and pathological conditions.

Expression and localization of trefoil factor family genes in rat submandibular glands

The trefoil factor (TFF) family, which comprises TFF1, TFF2 and TFF3, plays an essential role in epithelial regeneration within the gastrointestinal tract. All three TFFs are present in human saliva; TFF3 is the predominant trefoil peptide. Little is known about the expression and tissue distribution of TFFs in rats, which are commonly used as a model system for human studies. We investigated the localization of the TFF genes that encode secretory peptides in rat submandibular glands (SMG). All three TFFs were expressed in rat SMG, although their location varied. Substantial amounts of TFF1 were detected only in the cytoplasm of epithelial cells in the SMG granular convoluted tubules (GCT), while TFF2 and TFF3 were widely distributed in the cytoplasm of epithelial cells of intercalated ducts (ID), striated ducts (SD) and interlobular ducts (ILD). The three TFFs also were detected especially in the lumens of the SD and ILD. Semi-quantitative RT-PCR and in situ hybridization experiments confirmed TFF1, TFF2 and TFF3 mRNA expressions in the SMG. Greater expression of TFF peptides and mRNA was observed in male rats than in females. The broad expression of TFFs in rat SMG cells and lumens suggests that TFFs function in this organ by their secretion into the duct lumens. We also found differences in TFF expression profiles between rat and human SMG; therefore, caution should be exercised when using rats as a model for human TFF studies.

Expression and Localization of α-amylase in the Submandibular and Sublingual Glands of Mice.

In the major salivary glands of mice, acinar cells in the parotid gland (PG) are known to be the main site for the production of the digestive enzyme α-amylase, whereas α-amylase production in the submandibular gland (SMG) and sublingual gland (SLG), as well as the cell types responsible for α-amylase production, has been less firmly established. To clarify this issue, we examined the expression and localization of both the mRNA and protein of α-amylase in the major salivary glands of male and female mice by quantitative and histochemical methods. α-amylase mRNA levels were higher in the order of PG, SMG, and SLG. No sexual difference was observed in α-amylase mRNA levels in the PG and SLG, whereas α-amylase mRNA levels in the female SMG were approximately 30% those in the male SMG. Using in situ hybridization and immunohistochemistry, signals for α-amylase mRNA and protein were found to be strongly positive in acinar cells of the PG, serous demilune cells of the SLG, and granular convoluted tubule (GCT) cells of the male SMG, weakly positive in seromucous acinar cells of the male and female SMG, and negative in mucous acinar cells of the SLG. These results clarified that α-amylase is produced mainly by GCT cells and partly by acinar cells in the SMG, whereas it is produced exclusively by serous demilune cells in the SLG of mice.