Fibronectin-induced ductal formation in salivary gland self-organization model.

BACKGROUND: Recent advances in tissue regeneration approaches including 3D organoids, were based on various 3D organogenesis models. However, 3D models are generally technique-sensitive and time-consuming. Thus, we utilized an existing model of submandibular salivary gland (SMG) to modify a simple and highly reproducible in vitro 3D culture model of primary SMG cells self-organization into a well-developed cell spheroid inside Matrigel substrate. We used this model to observe the collective multicellular behavior during spheroid formation. Further, we applied various quantitative approaches including real-time live imaging and immune histochemical image analysis to dissect the cellular dynamics during tissue patterning. RESULTS: On a time-scale of hours, we observed marked size and shape transformations in the developed 3D spheroid which resulted in a spatially-controlled growth differential from the canter to the periphery of the formed aggregates. Moreover, we investigated the effect of fibronectin (FN) on SMG cells self-organization using our simplified culture model. Interestingly, we discovered a novel role of FN in inducing duct-like elongation during initial stages of SMG bud formation. CONCLUSION: This in vitro model provides an excellent tool for analyzing the intercellular dynamics during early SMG tissue development as well as revealing a novel role of FN in SMG ductal expansion.

Activation of TLR9-dependent p38MAPK pathway in the pathogenesis of primary Sjögren's syndrome in NOD/Ltj mouse.

OBJECTIVE: The objective of this study was to investigate the potential role of Toll-like receptor 9-dependent p38 MAPK signaling pathway in the pathogenesis of primary Sjögren's syndrome (pSS) in NOD/Ltj mouse, aiming to identify an ideal target therapy model for human pSS. METHODS: NOD/Ltj mice were chosen as a model of pSS. The Toll-like receptor 9 and p-p38 MAPK double-positive peripheral blood mononuclear cells (PBMCs) of 4-, 5-, 8-, 10-, and 15-week-old NOD/Ltj mouse were analyzed by flow cytometry. The expressions of Toll-like receptor 9 and p-p38 MAPK in the submandibular gland (SMG) were also examined by immunohistochemistry. The change of stimulated salivary flow rate was dynamically measured, and the histopathology of SMG was evaluated by hematoxylin and eosin stain. RESULTS: The stimulated salivary flow rate in NOD/Ltj was reduced to 50-60% of the flow rate of control mice since the fifth week onwards. The Toll-like receptor 9 and p-p38 MAPK double-positive PBMCs in both groups increased gradually from 5 weeks, peaked at 8 weeks and then gradually decreased at 10 weeks, yet the percentage of Toll-like receptor 9 and p-p38MAPK double-positive PBMCs in 5-, 8-, and 10-week-old NOD/Ltj mouse was significantly increased compared with those in control subjects. After the 10th week onwards, there were no significant differences in the Toll-like receptor 9 and p-p38 MAPK double-positive PBMCs between NOD/Ltj mice and controls. Immunohistochemical staining showed that Toll-like receptor 9 was positive in the acinar epithelium cells and infiltrating lymphocytes in NOD/Ltj mice. p-p38 MAPK was detected in infiltrating lymphocytes and few ductal or acinar epithelium cells adjacent to infiltrating lymphocytes in NOD/Ltj mice. CONCLUSIONS: From the fifth week till the tenth week, Toll-like receptor 9 and p-p38 MAPK double-positive PBMCs were significantly increased in NOD/Ltj mice, accompanied with reduced stimulated salivary flow rate and Toll-like receptor 9 or p-p38 MAPK positive infiltrating lymphocytes observed in the SMG of NOD/Ltj mouse. Our results indicated that activation of Toll-like receptor 9-depended p38 MAPK signal pathway in PBMCs was an early event in pSS which made NOD/Ltj as an ideal therapy model to test the treatment effects of p38 MAPK or Toll-like receptor 9 inhibitors on pSS.

Rab11b and its effector Rip11 regulate the acidosis-induced traffic of V-ATPase in salivary ducts.

Redistribution of acid-base transporters is a crucial regulatory mechanism for many types of cells to cope with extracellular pH changes. In epithelial cells, however, translocation of acid-base transporters ultimately leads to changes in vectorial transport of H+ and HCO3-. We have previously shown that the bicarbonate-secreting epithelium of salivary ducts responds to changes of systemic acid-base balance by adaptive redistribution of H+ and HCO3- transporters, thereby influencing the ionic composition and buffering capacity of saliva. However, the specific proteins involved in regulated vesicular traffic of acid-base transporters are largely unknown. In the present study we have investigated the impact of Rab11 family members on the acidosis-induced trafficking of the vacuolar-type H+-ATPase (V-ATPase) in salivary duct cells in vitro using the human submandibular cell line of ductal origin HSG as an experimental model. The results show that Rab11b is expressed in salivary ducts and exhibits a significantly higher co-localization with V-ATPase than Rab11a and Rab25. We also show that Rab11 but not Rab25 interacts with the ε subunit of V-ATPase. Extracellular acidosis up-regulates Rab11b expression and protein abundance in HSG cells and causes translocation of the V-ATPase from intracellular pools toward the plasma membrane. Loss-of-function experiments using specific siRNA either against Rab11b or against its effector Rip11 prevent acidosis-induced V-ATPase translocation. These data introduce Rab11b as a crucial regulator and Rip11 as mediator of acidosis-induced V-ATPase traffic in duct cells of submandibular gland.

Organic secretion in ductal cells of the sublingual salivary gland of ferret: Histochemical observations.

The presence of organic secretion in ductal cells of the sublingual salivary gland of ferret has been questioned, which prompted the present investigation. Paraffin or cryostat sections from aldehyde fixed or quenched sublingual glands of this species were tested for some amino acid residues, mucosubstances, oxidative and hydrolytic enzymes, and lectins. The glands showed inconspicuous ducts of simple appearances on routine histology. The histochemical procedures, however, revealed a granulated substance in the apical (periluminal) region of ductal cells, which contained tryptophan, disulphides, neutral mucosubstances, αFuc and GalNAc, and showed chloroacetate esterase activity. Occurrence of the substance varied between different ducts of the same gland and/or cells of the same duct. The ductal cells also showed diffuse peroxidase and acid phosphatase, and Golgi-like thiamine pyrophosphatase activities. Acetylcholinesterase-positive nerve fibres embraced the ducts. The results support a particular localisation of protein-bound amino acid residues and enzymatic catalytic activities indicative of organic secretion, possibly tissue kallikrein, in sublingual ductal cells of ferret.

Expression of two drug-metabolizing cytochrome P450-enzymes in human salivary glands.

OBJECTIVE: The oral cavity is constantly lubricated by saliva and even small amounts of xenobiotics and / or their metabolites in the saliva may affect the oral mucosa. Our aim was therefore to clarify if xenobiotic metabolizing enzymes CYP1A2 and CYP3A4 are expressed in salivary glands. METHODS: Formalin-fixed paraffin-embedded specimens from parotid (10), submandibular (7) and labial (10) salivary glands were examined immunohistochemically and by in situ hybridization for expression of CYP1A2 and CYP3A4 protein and mRNA. RESULTS: CYP1A2 and CYP3A4 protein and mRNA were detected in ductal and seromucous / serous acinar cells in all gland types although to a varying degree and intensity. Mucous acinar cells were positive to a lesser extent. CONCLUSION: The results indicate a xenobiotic metabolizing capability of salivary glands. This may have implications for development of oral mucosal disease as a result of mucosal exposure to metabolites originating from internal sources (blood) as well as from saliva.

Interlobular excretory ducts of mammalian salivary glands: structural and histochemical review.

In the major salivary glands of mammals, excretory ducts (EDs) succeed striated ducts. They are for the most part interlobular in position, although their proximal portions sometimes are on the periphery of a lobule, where they occasionally retain some of the structural features of striated ducts. Based on a survey of a broad range of mammalian species and glands, the predominant tissue type that composes EDs is pseudostratified epithelium. In some species, there is a progression of epithelial types: the proximal EDs are composed of simple cuboidal or columnar epithelium that, in the excurrent direction, usually gives way to the pseudostratified variety. Secretory granules are visible in the apical cytoplasm of the principal cells of the EDs of only a few species, but histochemistry has shown the presence of a variety of glycoproteins in these cells in a spectrum of species. Moreover, the latter methodology has revealed the presence of a variety of oxidative, acid hydrolytic, and transport enzymes in the EDs, showing that, rather than simply acting as a conduit for saliva, these ducts play a metabolically active role in gland function. It is difficult to describe a "typical" mammalian ED because it can vary along its length and interspecific variation does not follow a phylogenetic pattern. Moreover, in contrast to intercalated and striated ducts, ED cellular features do not exhibit a relationship to diet.

Cyclooxygenase-2 expression in Warthin's tumour.

OBJECTIVES: To determine whether cyclooxygenase-2 (COX-2) is overexpressed in Warthin's tumours, and to characterize its pattern of expression. METHODS: Twenty-one paraffin-embedded Warthin's tumour specimens were analysed by immunohistochemical staining for expression of human COX-2. Semi-quantitative analysis of the staining was performed. RESULTS: In all of the specimens, we found that there was overexpression of COX-2 within the epithelial component of the tumours, with no expression in the lymphoid components. There was also overexpression of COX-2 in the salivary duct system of normal parotid tissue. CONCLUSIONS: Our results suggest that COX-2 is up-regulated in the epithelial component of Warthin's tumours. Our findings support the hypothesis that Warthin's tumours originate from heterotopic ductal epithelial cells of the parotid gland. The role of COX-2 expression in the pathogenesis of Warthin's tumours remains to be determined.

Immunolocalization of vacuolar-type H+-ATPase in rat submandibular gland and adaptive changes induced by acid-base disturbances.

Using antibodies against the 31-kD and 70-kD subunits of vacuolar type H+-ATPase (V-ATPase) and light microscopic immunocytochemistry, we have demonstrated the presence of this V-ATPase in rat submandibular gland. We have also investigated the adaptive changes of this transporter during acid-base disturbances such as acute and chronic metabolic acidosis or alkalosis. Our results show intracellularly distributed V-ATPase in striated, granular, and main excretory duct cells in controls, but no V-ATPase immunoreaction in acinar cells. Both acute and chronic metabolic acidosis caused a shift in V-ATPase away from diffuse distribution towards apical localization in striated and granular duct cells, suggesting that a V-ATPase could be involved in the regulation of acid-base homeostasis. In contrast, during acidosis the main excretory duct cells showed no changes in the V-ATPase distribution compared to controls. With acute and chronic metabolic alkalosis, no changes in the V-ATPase distribution occurred. (J Histochem Cytochem 46:91-100, 1998)

Immunocytochemical evidence for the coexistence of catecholestrogen and catechol-O-methyltransferase in the rat parotid gland.

Catechol-O-methyltransferase (COMT) (EC 2.1.1.6) and catecholestrogen were localized in the parotid gland of the rat by immunocytochemical methods. Specific immunoreactive deposits for COMT and catecholestrogen were found in the cytoplasm of duct cells, but only those for COMT in myo-epithelial cells. The pattern of localization of COMT and catecholestrogen in the parotid gland suggests a functional relationship between COMT and catecholestrogen.

Expression of SIBLINGs and their partner MMPs in salivary glands.

Three members of the SIBLING family of integrin-binding phosphoglycoproteins (bone sialoprotein, BSP; osteopontin, OPN; and dentin matrix protein-1, DMP1) were recently shown to bind with high affinity (nM) and to activate 3 different matrix metalloproteinases (MMP-2, MMP-3, and MMP-9, respectively) in vitro. The current study was designed to document the possible biological relevance of the SIBLING-MMP activation pathway in vivo by showing that these 3 SIBLINGs and their known MMP partners are co-expressed in normal adult tissue. BSP, OPN, and DMP1 were invariably co-expressed with their partner MMPs in salivary glands of humans and mice. The 2 SIBLING proteins without known MMP partners, dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE), were also expressed in salivary glands. Expression of all SIBLINGs in this normal, non-mineralizing epithelial tissue suggests that they serve at least one function in vivo other than directly promoting matrix mineralization--a function we hypothesize involves local activation of MMPs.

Immunohistochemical localization of kallikrein in salivary glands of the Japanese monkey, Macaca fuscata.

The immunofluorescence staining using anti-human urinary kallikrein polyclonal antiserum was intense in the luminal rim of the striated duct cells, but decreased in the excretory duct cells and was weak in the intercalated duct cells. Western blot analysis demonstrated the monospecificity of the antiserum to glandular kallikrein in Macaca submandibular gland homogenate. The staining was observed to a similar degree as that in salivary glands with long and numerous striated ducts in the following order: the submandibular, the parotid and the sublingual gland. These findings suggest that kallikrein is secreted by exocytosis from the duct cells, mainly from the striated duct cells.

An enzyme histochemical and biochemical study of the activity of three oxidative enzymes in the developing rat parotid gland.

Information on ductal differentiation in the developing rat parotid gland is sparse. One of the main functions of the striated and excretory ducts in this gland is the selective exchange of electrolytes from the primary fluid secreted by the acini. These ducts are rich in a number of enzymes involved in this task, suggesting that they might be useful as markers of ductal differentiation. The objective of this investigation was to delineate the developmental changes in activity of three of these, cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), nicotinamide adenine phosphate dinucleotide (reduced form)-dehydrogenase (NADPH-DH). Histochemical localization of all three enzymes in fresh frozen sections was complemented by biochemical assays of CCO and SDH and cytochemical localization of CCO. Biochemically, CCO- and SDH-specific activity in gland homogenates increased progressively after birth, reaching adult levels at 21-28 days. Histochemically, deposits of reaction products of all three enzymes increased more in the striated and excretory ducts, especially in their basal cytoplasm, than in other glandular structures between 19 days in utero and 28 days after birth. During the same age span, the mitochondria in the striated and excretory ducts increased markedly in both number and size, migrated to a mostly basal location, and increased from many to virtually all showing strong cytochemical CCO reactions. These histochemical and cytochemical patterns of changes in enzyme activity at the cellular level accounted for the overall increases in CCO and SDH seen in the biochemical assays. Only the SDH histochemical reaction was consistently weak in the acini and intercalated ducts, and thus provided the most contrast with the progressively stronger reactions in the larger ducts. We conclude that of the three enzymes evaluated in these experiments, SDH is the best marker of the functional differentiation of the striated and excretory ducts in the developing rat parotid gland.

Distribution of V-ATPase in rat salivary glands.

Using immunohistochemistry we have investigated the presence and cellular distribution of the 31-kDa subunit of vacuolar-type H+-ATPase (V-ATPase) in secretory endpieces and the duct system of rat major salivary glands. In all three salivary glands studied the 31-kDa subunit of V-ATPase was not expressed in secretory endpieces. In rat parotid gland V-ATPase was luminally located in main excretory and striated duct cells. In contrast, both rat submandibular and sublingual glands showed a diffuse intracellular V-ATPase distribution. The differences in V-ATPase immunolocalization in rat salivary glands probably reflect the structural heterogeneity of the different glands. The data also suggest that the duct systems of major salivary glands may modify the H+ and HCO3- concentration of the final saliva in different ways.

Expression of nitric oxide synthase in pleomorphic adenomas of the parotid.

The actions of nitric oxide (NO) in the pathology of solid tumours are complicated and many are poorly understood because NO has both inhibitory and tumour-promoting activities. In the current study we aimed to find out immunohistochemically whether the expression of both the inducible (iNOS) and endothelial (eNOS) forms of the enzyme nitric oxide synthase (NOS) were changed in pleomorphic adenomas of the parotid compared with normal salivary tissue. There was a significant difference in staining for iNOS between the tumour and normal salivary tissue, with tumour epithelial cells being stained in 29 cases of the 30 cases studied (P< 0.0001). The luminal cells of the salivary ducts also stained, but not the normal salivary tissue. Immunohistochemistry for the eNOS isoenzyme showed moderate staining of the tumour epithelium in only three specimens. There was also mild staining in the salivary duct cells of the normal glandular tissue and in endothelium of blood vessels in both tumour and normal glandular tissue in the same 29 cases.

PIK3CA mutations and PTEN loss in salivary duct carcinomas.

Salivary duct carcinoma (SDC) is an aggressive malignancy that frequently presents at an advanced stage. Mutations/amplification of the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PIK3CA) and/or loss of the phosphatase and tensin homolog (PTEN) are known to activate the phosphoinositide 3-kinase (PI3K) pathway and may represent a therapeutic target. In 7 of 34 SDCs (20.5%) a SNaPshot polymerase chain reaction detected PIK3CA exon 9 [p.E545K (n=3) and p.E542K (n=2)] or exon 20 [p.H1047R (n=2)] mutations. PIK3CA p.E545K mutation was identified in 3 de novo SDCs with conventional morphology. The only case of SDC with anaplastic transformation showed PIK3CA p.H1047R mutation, whereas 1 of 2 PIK3CA p.E542K mutations was identified in SDC arising in a pleomorphic adenoma. None of the 16 tested SDCs showed PIK3CA amplification by fluorescence in situ hybridization. Fluorescence in situ hybridization identified PTEN loss in 8 of 16 tested SDCs (50%) [homozygous deletion (n=3), chromosome 10 monosomy (n=3), hemizygous deletion (n=2)]. Two cases showed both PIK3CA mutation and PTEN loss, suggesting that these events are not mutually exclusive. These findings offer a molecular rationale for therapeutic targeting of the PI3K pathway in patients with SDC.

A vacuolar-type H+-ATPase and a Na+/H+ exchanger contribute to intracellular pH regulation in cockroach salivary ducts.

Cells of the dopaminergically innervated salivary ducts in the cockroach Periplaneta americana have a vacuolar-type H(+)-ATPase (V-ATPase) of unknown function in their apical membrane. We have studied whether dopamine affects intracellular pH (pH(i)) in duct cells and whether and to what extent the apical V-ATPase contributes to pH(i) regulation. pH(i) measurements with double-barrelled pH-sensitive microelectrodes and the fluorescent dye BCECF have revealed: (1) the steady-state pH(i) is 7.3+/-0.1; (2) dopamine induces a dose-dependent acidification up to pH 6.9+/-0.1 at 1 micromol l(-1) dopamine, EC(50) at 30 nmol l(-1) dopamine; (3) V-ATPase inhibition with concanamycin A or Na(+)-free physiological saline (PS) does not affect the steady-state pH(i); (4) concanamycin A, Na(+) -free PS and Na(+)/H(+) exchange inhibition with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) each reduce the rate of pH(i) recovery from a dopamine-induced acidification or an acidification induced by an NH(4)Cl pulse; (5) pH(i) recovery after NH(4)Cl-induced acidification is almost completely blocked by concanamycin A in Na(+)-free PS or by concanamycin A applied together with EIPA; (6) pH(i) recovery after dopamine-induced acidification is also completely blocked by concanamycin A in Na(+)-free PS but only partially blocked by concanamycin A applied together with EIPA. We therefore conclude that the apical V-ATPase and a basolateral Na(+)/H(+) exchange play a minor role in steady-state pH(i) regulation but contribute both to H(+) extrusion after an acute dopamine- or NH(4)Cl-induced acid load.

Morphological and functional differentiation of HSG cells: role of extracellular matrix and trpc 1.

A human salivary intercalated duct cell line (HSG) is capable of morphological change to acinar-type cells, and of salivary amylase (AMY1) expression, by culturing on basement membrane extracts (BME). The aim of this study was to determine the critical conditions for functional and morphological differentiation of HSG cells and to establish if the processes are related. Cells were grown on BMEs that had different protein concentrations and growth factor content, and then examined with respect to morphology and AMY1 expression. To investigate the role of intracellular calcium in amylase expression, a pcDNA3.1-TRPC1alpha construct was used to overexpress htrp1alpha, which mediates the store-operated calcium entry in HSG cells. Expression of the AMY1, TRPC1alpha and beta genes was quantified by means of real time RT-PCR. Growth factor-reduced BME (12.8 mg/ml) induced multicellular acinar structures with lumen formation but without stimulation of either AMY1 or TRPC1. HSG cells cultured on higher concentration BME (17.5 or 16.4 mg/ml) formed reticular networks. AMY1 expression increased both on growth factor-reduced BME (17.5 mg/ml: 3.0-fold, P < 0.001) and on regular BME (16.4 mg/ml: 3.7-fold, P < 0.001) accompanied by a slight increase in expression of TRPC1alpha and TRPC1beta. Overexpression of htrp1alpha did not cause any significant changes in AMY expression, though it attenuated the BME (17.5 mg/ml)-induced AMY1 upregulation. Overall, the higher protein concentration BME favors amylase expression in HSG cells, whereas the lower concentration causes marked morphological changes.

Temporal reduction in size of salivary acinus in rats induced by theophylline.

Repeated administration of theophylline, a phosphodiesterase inhibitor, induces the enlargement of the salivary glands in rats. Time-course changes after a single administration of theophylline were examined in the salivary glands, including phosphodiesterase enzyme activity, and the expression of aquaporin 5 (AQP5), a water channel. We also examined the contribution of beta-adrenergic receptors to theophylline-induced salivary changes. Male F344 rats were given 50 mg/kg of theophylline intraperitoneally either alone or concurrently with a 10 mg/kg subcutaneous injection of propranolol. After treatment with theophylline alone, the weight and histology of the submaxillary and parotid glands were examined. Phosphodiesterase activity and AQP5 were detected by enzyme- and immuno-histochemistry, respectively. At 4 hours, 8 hours, or both, organ weights were decreased with depletion of secretory vesicles in the acinar cells. In the submaxillary glands, reduced activity of phosphodiesterase and increased expression of AQP5 in the intercalated ducts were observed at 4 hours. When co-administered, propranolol partially abolished theophylline-induced glandular reduction. These results suggest that the theophylline-induced transient reduction in size of the salivary glands is attributable not only to phosphodiesterase inhibition but also to beta-adrenergic receptor activation and that the intercalated ducts in submaxillary glands play a role in the production of saliva.

Endothelial constitutive nitric oxide synthase (ecNOS) localization in normal and neoplastic salivary tissue.

BACKGROUND: Nitric oxide (NO.) has been implicated in the process of carcinogenesis in various organs. This study was designed to investigate the expression of endothelial constitutive nitric oxide synthase (ecNOS) in normal and neoplastic salivary tissues. METHODS: Paraffin-embedded tissue from 48 salivary tumors and adjacent non-neoplastic tissue was immunohistochemically evaluated for both frequency (percentage) and intensity (1-4+) of staining using a commercially available anti-ecNOS monoclonal antibody. RESULTS: Expression of ecNOS was predominantly localized to vascular endothelium, skeletal muscle, and to salivary duct luminal epithelium in normal salivary tissue (n = 37). All salivary tumors demonstrated at least 1 + cytoplasmic staining for ecNOS without apparent correlation to most clinical parameters. A tendency toward increased frequency and intensity of ecNOS expression in oncocytic cells, relative to cells with myoepithelial or acinar differentiation, was noted. CONCLUSIONS: Expression of ecNOS is localized to the luminal cells of normal salivary ducts. Limited expression of ecNOS was found in all the salivary gland tumors examined. This suggests a common histogenesis for this diverse group of tumors, which may reflect different degrees of differentiation toward luminal duct epithelium. The possible role of ecNOS and NO. in salivary gland carcinogenesis is intriguing and warrants further study.

Significant correlation between matrix metalloproteinase activity and tumor necrosis factor-alpha in salivary extravasation mucoceles.

We examined the matrix metalloproteinase (MMP) activity and tumor necrosis factor (TNF)-alpha in luminal fluid of 18 extravasation mucoceles and in saliva from Wharton's duct of five patients by means of gelatin zymography and enzyme immunoassay, respectively. The luminal fluid showed a high level of MMP activity compared with the saliva. Quantitative determination by enzyme immunoassay revealed that the luminal fluid contained higher levels of TNF-alpha than the saliva. In addition, the amount of TNF-alpha in luminal fluid exhibited a direct correlation with MMP activity estimated by densitometric analysis of gelatin zymograms. Since TNF-alpha stimulates the production of MMPs from cells such as fibroblasts, these results suggest that TNF-alpha is one of the causal molecules that enhance the accumulation of proteolytic enzymes in luminal fluid of mucoceles.