TAZ inhibits acinar cell differentiation but promotes immature ductal cell proliferation in adult mouse salivary glands

There are currently no treatments for salivary gland diseases, making it vital to understand signaling mechanisms operating in acinar and ductal cells so as to develop regenerative therapies. To date, little work has focused on elucidating the signaling cascades controlling the differentiation of these cell types in adult mammals. To analyze the function of the Hippo-TAZ/YAP1 pathway in adult mouse salivary glands, we generated adMOB1DKO mice in which both MOB1A and MOB1B were TAM-inducibly deleted when the animals were adults. Three weeks after TAM treatment, adMOB1DKO mice exhibited smaller submandibular glands (SMGs) than controls with a decreased number of acinar cells and an increased number of immature dysplastic ductal cells. The mutants suffered from reduced saliva production accompanied by mild inflammatory cell infiltration and fibrosis in SMGs, similar to the Sjogren's syndrome. MOB1-deficient acinar cells showed normal proliferation and apoptosis but decreased differentiation, leading to an increase in acinar/ductal bilineage progenitor cells. These changes were TAZ-dependent but YAP1-independent. Biochemically, MOB1-deficient salivary epithelial cells showed activation of the TAZ/YAP1 and β-catenin in ductal cells, but reduced SOX2 and SOX10 expression in acinar cells. Thus, Hippo-TAZ signaling is critical for proper ductal and acinar cell differentiation and function i n adult mice.     

小鼠下颌下腺颗粒曲管细胞的免疫细胞化学观察

本采用免疫细胞化学ABC法，观察到小鼠下颌下腺颗粒曲管细胞呈现降钙素基因相关肽和生长抑素免疫反应阳性，但两在颗粒曲管细胞内分布特点不尽相同，腺泡细胞呈免疫反应阴性，本就下颌下腺中降钙素基因相关肽和生长抑素存在的意义进行了讨论。     

涎腺腺泡细胞癌临床病理分析并文献复习

目的：探讨涎腺腺泡细胞癌的临床病理学特点及诊断要点。方法：对1例涎腺腺泡细胞癌进行临床资料、病理形态学及免疫组织化学观察,并结合文献对其诊断及鉴别诊断进行探讨。结果：镜下瘤细胞胞体宽大,胞浆嗜碱性呈细颗粒状,核圆形瘤细胞生长呈腺泡状或实性片状生长,部分区域呈乳头状改变,免疫组化显示AE1/AE3(+)、CK8/18(+)、CK7(+)、AAT(+)、S-100(+)。结论：涎腺腺泡细胞癌发病率低,但根据其常见的发病部位及特征性的组织形态,结合免疫组织化学方法,有助于其诊断及鉴别诊断。     

Distribution of dendritic cells in normal human salivary glands

Dendritic cells (DC) are believed to contribute to development of autoimmune sialadenitis, but little is known about their distribution in normal salivary glands. In this study, DC were identified and their distribution was determined in normal human parotid and submandibular glands. For light microscopy, salivary gland sections were stained with H&E or immunocytochemically using antibodies to DC markers. Transmission electron microscopy (TEM) was used to evaluate the ultrastructural characteristics of DC. In H&E sections, elongated, irregularly shaped nuclei were occasionally seen in the striated and excretory duct epithelium. Immunolabeling with anti-HLA-DR, anti-CD11c and anti-S100 revealed DC with numerous processes extending between ductal epithelial cells, often close to the lumen. Morphometric analyses indicated that HLA-DR-positive DC occupied approximately 4-11% of the duct wall volume. Similar reactive cells were present in acini, intercalated ducts and interstitial tissues. TEM observations revealed cells with indented nuclei containing dense chromatin, pale cytoplasm with few organelles, and lacking junctional attachments to adjacent cells. These results indicate that DC are abundant constituents of normal human salivary glands. Their location within ductal and acinar epithelium suggests a role in responding to foreign antigens and/or maintaining immunological tolerance to salivary proteins.     

MUC-1 mucin in normal human salivary glands detected by HMFG-1 and HMFG-2 monoclonal antibodies

The aim of this study was to determine the immunoexpression of normal salivary gland cells using two human milk fat globule membrane protein antibodies (HMFG-1 and HMFG-2). Paraffin-embedded, 5 microm-thick slides from parotid and submandibular gland tissues were cut and immunostained with monoclonal antibodies HMFG-1 and HMFG-2 against MUC1 protein. HMFG antigens were found in secretory epithelial cells and ductal cells lining the striated and intercalated ducts in the intraductal secretion material, and sometimes in the myoepithelial cells. Our results suggest that HMFG-1 and HMFG-2 proteins may play a role in normal salivary gland function, mainly in the manufacturing and secretion of saliva.     

Disruption of tight junctions contributes to hyposalivation of salivary glands in a mouse model of type 2 diabetes

Tight junction (TJ) plays an important role in regulating paracellular fluid transport in salivary glands; however, little is known about the involvement of TJs in diabetes salivary glands. This study aimed to investigate the alterations of TJs and their possible contribution in diabetes-induced hyposalivation. Here, we observed that the morphologies of submandibular glands (SMGs) were impaired, characterized by enlarged acini accumulation with giant secretory granules, which were significantly reduced in atrophic ducts in SMGs of db/db mice, a spontaneous model of type-2 diabetes. However, the secretory granules were increased and scattered in the acini of diabetes parotid glands (PGs). Other ultrastructural damages including swollen mitochondria, expansive endoplasmic reticulum, and autophagosomes were observed in the diabetes group. The levels of TJ proteins including claudin-1 (Cldn1) and claudin-3 (Cldn3) were increased, whereas those of claudin-4 (Cldn4), occludin (Ocln), and zonula occludens-1 (ZO-1) were decreased in SMGs of db/db mice. Higher Cldn1 and Cldn3 and lower claudin-10 (Cldn10) and Ocln levels were observed in PGs of diabetes mice. Taken together, the structures of SMGs and PGs were impaired in diabetes mice, and the disruption of TJ integrity in both SMGs and PGs may contribute to diabetes-induced hyposalivation.     

Diverse roles of E-cadherin in the morphogenesis of the submandibular gland: insights into the formation of acinar and ductal structures

The formation of acinar and ductal structures during epithelial tissue branching morphogenesis is not well understood. We report that in the mouse submandibular gland (SMG), acinar and ductal cell fates are determined early in embryonic morphogenesis with E-cadherin playing pivotal roles in development. We identified two morphologically distinct cell populations at the single bud stage, destined for different functions. The outer layer of columnar cells with organized E-cadherin junctions expressed the neonatal acinar marker B1 by E13.5, demonstrating their acinar fate. The interior cells initially lacked distinct E-cadherin junctions, but with morphogenesis formed cytokeratin 7 (K7) -positive ductal structures with organized E-cadherin junctions and F-actin filaments. Inhibition of E-cadherin function with either siRNA or function blocking antibody caused extensive apoptosis of ductal cells and aberrantly dilated lumens, providing the first evidence that E-cadherin regulates ductal lumen formation during branching morphogenesis of the salivary gland.     

Amiloride-sensitive Na+ current in the granular duct cells of mouse mandibular glands

Whole-cell patch clamp studies on dispersed cells from the intralobular ducts of mouse mandibular glands reveal the presence of an amiloride-sensitive Na⁺- selective conductance that is not found in the cells of the secretory endpieces. Since studies on ripped-off outsideout patches from the basolateral membranes of isolated intralobular ducts show that the membrane conductance is not altered by amiloride, it can be inferred that the Na⁺ conductance seen in whole-cell experiments is located in the luminal membrane. These studies thus provide evidence for the presence of a luminal Na⁺ conductance in salivary ducts and indicate that intralobular and extralobular ducts may handle Na⁺ in a similar manner.     

Polymorphous low-grade adenocarcinoma of the major salivary glands: report of three cases in an unusual location

AIMS: Polymorphous low-grade adenocarcinoma (PLGA) is the second most common type of malignant neoplasm in minor salivary glands. Its origin in major salivary glands is considered exceedingly rare. Herein, we present three cases of de novo PLGA arising in major salivary glands. METHODS AND RESULTS: Three cases of PLGA were identified in a large series of primary tumours of major salivary glands. We investigated their clinicopathological profiles, including immunohistochemical features. The three patients (two men and one woman) were 51, 65, and 79 years old. The tumours were 20-30 mm large; two were in the parotid gland and one in the submandibular gland. Histologically, all the tumours had a polymorphous architectural pattern showing predominantly solid, tubular, and cribriform features and invasive growth. Papillary areas were observed focally in two tumours and an 'Indian-file' array in one. The tumour cells had a bland cytological appearance and low mitotic count. Two tumours showed perineural invasion. No preexisting pleomorphic adenoma component was identified. In all cases, tumour cells were positive for epithelial markers, S100 protein, and vimentin but negative for alpha-smooth muscle actin, muscle-specific actin, and glial fibrillary acidic protein. Proliferative activities assessed with the Ki67 labelling index were 4.3%, 7.1%, and 7.6%; no p53 overexpression was observed. Two patients had local recurrence, but none had metastasis or died of tumour. CONCLUSIONS: PLGAs arising in major salivary glands and those in minor salivary glands have similar clinicopathological and immunohistochemical characteristics. It is important to recognize that PLGA can occur ab initio in the major salivary glands, although it is extremely rare.     

Long‐term dexamethasone treatment alters the histomorphology of acinar cells in rat parotid and submandibular glands

Glucocorticoids (GCs) induce insulin resistance (IR), a condition known to alter oral homeostasis. This study investigated the effects of long‐term dexamethasone administration on morphofunctional aspects of salivary glands. Male Wistar rats received daily injections of dexamethasone [0.1 mg/kg body weight (b.w.), intraperitoneally] for 10 days (DEX), whereas control rats received saline. Subsequently, glycaemia, insulinaemia, insulin secretion and salivary flow were analysed. The parotid and submandibular glands were collected for histomorphometric evaluation and Western blot experiments. The DEX rats were found to be normoglycaemic, hyperinsulinaemic, insulin resistant and glucose intolerant (P < 0.05). DEX rat islets secreted more insulin in response to glucose (P < 0.05). DEX rats had significant reductions in the masses of the parotid (29%) and submandibular (16%) glands (P < 0.05) that was associated with reduced salivary flux rate. The hypotrophy in both glands observed in the DEX group was associated with marked reduction in the volume of the acinar cells in these glands of 50% and 26% respectively (P < 0.05). The total number of acinar cells was increased in the submandibular glands of the DEX rats (P < 0.05) but not in the parotid glands. The levels of proteins related to insulin and survival signalling in both glands did not differ between the groups. In conclusion, the long‐term administration of dexamethasone caused IR, which was associated with significant reductions in both mass and flux rate of the salivary glands. The parotid and submandibular glands exhibited reduced acinar cell volume; however, the submandibular glands displayed acinar hyperplasia, indicating a gland‐specific response to GCs. Our data emphasize that GC‐based therapies and insulin‐resistant states have a negative impact on salivary gland homeostasis.     

CD109, a new marker for myoepithelial cells of mammary, salivary, and lacrimal glands and prostate basal cells

The CD109 gene encodes a glycosylphosphatidylinositol (GPI)-anchored cell surface protein. Herein it is shown that CD109 is highly expressed in myoepithelial cells of mammary, salivary, and lacrimal glands; and in prostate basal cells. The anti-CD109 antibody generated by the authors was available for formalin-fixed paraffin section, and it strongly stained myoepithelial cells and basal cells but not ductal, acinar, and secretory cells in these glands. CD109 expression was negative in examined breast ductal carcinomas and prostate adenocarcinomas. These findings indicate that CD109 is a useful marker for the diagnosis of invasive breast and prostate carcinomas.     

Diagnostic difficulties in lesions of the minor salivary glands

A wide range of lesions arise from the intra-oral salivary glands, and often present a diagnostic challenge to specialists and generalists alike. Of the salivary neoplasms, pleomorphic adenoma is the most common, but its morphological diversity may bring several other entities to mind, notably polymorphous adenocarcinoma, particularly in a small incisional biopsy. Polymorphous adenocarcinoma in turn shares features with adenoid cystic carcinoma. Immunohistochemistry and molecular cytogenetic studies can assist diagnosis in the face of overlapping morphology. The other salivary neoplasms most likely to be encountered in the oral cavity are canalicular adenoma, mucoepidermoid carcinoma, secretory carcinoma and acinic cell carcinoma. Of the non-neoplastic conditions, necrotising sialometaplasia is most likely to be misdiagnosed as neoplastic on both clinical and histological grounds. However, careful consideration of the clinicopathological features of an adequate tissue specimen will enable correct diagnosis.     

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

Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K), which is composed of three isozymes (α, β 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 PIP5Kβ 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 β‐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.     

Expression of Ca2+-dependent synaptotagmin isoforms in mouse and rat parotid acinar cells

Synaptotagmin is a Ca2+ sensing protein, which triggers a fusion of synaptic vesicles in neuronal transmission. Little is known regarding the expression of Ca2+-dependent synaptotagmin isoforms and their contribution to the release of secretory vesicles in mouse and rat parotid acinar cells. We investigated a type of Ca2+-dependent synaptotagmin and Ca2+ signaling in both rat and mouse parotid acinar cells using RT-PCR, microfluorometry, and amylase assay. Mouse parotid acinar cells exhibited much more sensitive amylase release in response to muscarinic stimulation than did rat parotid acinar cells. However, transient [Ca2+]i increases and Ca2+ influx in response to muscarinic stimulation in both cells were identical, suggesting that the expression or activity of the Ca2+ sensing proteins is different. Seven Ca2+-dependent synaptotagmins, from 1 to 7, were expressed in the mouse parotid acinar cells. However, in the rat parotid acinar cells, only synaptotagmins 1, 3, 4 and 7 were expressed. These results indicate that the expression of Ca2+-dependent synaptotagmins may contribute to the release of secretory vesicles in parotid acinar cells.     

Epidermal growth factor and insulin-like growth factor I are localized in different compartments of salivary gland duct cells. lmmunohistochemical evidence

Immunohistochemical methods were used to map EGF (epidermal growth factor) and IGF-I (insulin-like growth factor I; somatomedin C) immunoreactivities in salivary glands of adult rodents. Epidermal growth factor is, as is NGF (nerve growth factor), limited in distribution to the granules in granular duct cells in the submandibular gland. Insulin-like growth factor I is, in contrast, cytoplasmic and has a much more widespread distribution. It is seen in intercalated, striated and granulated duct cells as well as in apical parts of excretory duct cells. The parotid and the palatine salivary glands, lacking EGF immunoreactivity, have their IGF-I immunoreactivity similarly distributed as the submandibular gland. Isoproterenol treatment of adult male rats results in rapid and extensive growth of the submandibular and the parotid glands, which double their weights in just a few days. Isoproterenol causes release of granules from the submandibular granular duct cells and decrease in frequency of EGF immunoreactive cells. However, there is no or only minor concomitant changes in the distribution and intensity of the IGF-I immunoreactivity in these duct cells. Our results indicate that the trophic peptides EGF (and NGF) and IGF-I are localized in different compartments in salivary gland duct cells and that divergent pathways control their release.     

Intercalated duct hyperplasia: possible relationship to epithelial-myoepithelial carcinoma and hybrid tumours of salivary gland

AIMS: The aims of this study were to ascertain the incidence of intercalated duct hyperplasia in association with cases of epithelial-myoepithelial carcinoma (EMC), and to explore a possible relationship between them and hybrid carcinomas of salivary glands. METHODS AND RESULTS: Seven cases of EMC with sufficient surrounding non-tumour parotid were examined. Three cases contained foci of intercalated duct hyperplasia adjacent to the tumour. One of the cases was a hybrid tumour composed of EMC and mucoepidermoid carcinoma. The hyperplastic intercalated ducts formed multiple foci within the salivary parenchyma and were composed of bland, uniform ducts. Cytological atypia was not identified. CONCLUSIONS: Intercalated duct hyperplasia may be a precursor lesion to EMC. Furthermore, it may also explain why EMC is frequently associated with other salivary gland carcinomas, so-called hybrid tumours, as well as sharing histological features with adenoid cystic carcinoma. Recognition of the latter is of particular importance because adenoid cystic carcinoma carries a poor prognosis.     

小鼠下颌下腺发育中转化生长因子β受体的表达

背景：小鼠的下颌下腺是研究唾液腺的发育的良好模型,转化生长因子β是器官发育和疾病中重要的生长因子,但是在下颌下腺中转化生长因子β受体的表达以及作用机制至今并不明确。 目的：观察胚胎小鼠下颌下腺发育过程中转化生长因子βⅠ型受体和Ⅱ型受体以及p-ERK1/2的表达,揭示转化生长因子β在小鼠涎腺发育中的作用。 方法：取C57BL/6J小鼠胚胎期第14.5天的标本,使用转化生长因子βⅠ型受体和Ⅱ型受体以及p-ERK1/2抗体,对小鼠的下颌下腺进行免疫组化染色。取新生小鼠标本,大体观察下颌下腺,并且使用苏木精-伊红染色观察其形态。 结果与结论：①小鼠出生时,下颌下腺位于下颌骨下方;苏木精-伊红染色发现小鼠下颌下腺的腺泡、导管和闰管细胞也已经分化完成。②在胚胎期第14.5天,转化生长因子βⅠ型和Ⅱ型受体在腺泡上皮和导管上皮内高表达,而腺体上皮细胞外的间充质没有表达。③p-ERK1/2主要也是表达在下颌下腺的上皮细胞中,与转化生长因子βⅠ型受体和Ⅱ型受体在下颌下腺中的表达基本一致。说明在小鼠下颌下腺的发育过程中,转化生长因子β蛋白可能通过与上皮细胞表面的受体结合,激活ERK信号通路来调节涎腺腺泡和导管的发育。