Comment on "Reciprocity between p63 and p21 via TNFα/NF-κB signaling in p63-positive salivary duct adenocarcinoma cell".

Reciprocity between p63 and p21 via TNFα/NF-κB signaling in p63-positive salivary duct adenocarcinoma cell.

Currently, all salivary ducts (intercalated, striated and collecting) are assumed to function broadly in a similar manner, reclaiming ions that were secreted by the secretory acinar cells while preserving fluid volume and delivering saliva to the oral cavity. Nevertheless, there has been minimal investigation into the structural and functional differences between distinct types of salivary duct cells. Therefore, in this study, the expression profile of proteins involved in stimulus-secretion coupling, as well as the function of the intercalated duct (ID) and striated duct cells, was examined. Particular focus was placed on defining differences between distinct duct cell populations. To accomplish this, immunohistochemistry and in situ hybridization were utilized to examine the localization and expression of proteins involved in reabsorption and secretion of ions and fluid. Further, in vivo calcium imaging was employed to investigate cellular function. Based on the protein expression profile and functional data, marked differences between the IDs and striated ducts were observed. Specifically, the ID cells express proteins native to the secretory acinar cells while lacking proteins specifically expressed in the striated ducts. Further, the ID and striated duct cells display different calcium signalling characteristics, with the IDs responding to a neural stimulus in a manner similar to the acinar cells. Overall, our data suggest that the IDs have a distinct role in the secretory process, separate from the reabsorptive striated ducts. Instead, based on our evidence, the IDs express proteins found in secretory cells, generate calcium signals in a manner similar to acinar cells, and, therefore, are likely secretory cells. KEY POINTS: Current studies examining salivary intercalated duct cells are limited, with minimal documentation of the ion transport machinery and the overall role of the cells in fluid generation. Salivary intercalated duct cells are presumed to function in the same manner as other duct cells, reclaiming ions, maintaining fluid volume and delivering the final saliva to the oral cavity. Here we systematically examine the structure and function of the salivary intercalated duct cells using immunohistochemistry, in situ hybridization and by monitoring in vivo Ca2+ dynamics. Structural data revealed that the intercalated duct cells lack proteins vital for reabsorption and express proteins necessary for secretion. Ca2+ dynamics in the intercalated duct cells were consistent with those observed in secretory cells and resulted from GPCR-mediated IP3 production.

Mammalian salivary glands are mainly composed of two epithelial cell types, the acinar cells that secrete the salivary fluid as well as most of the salivary proteins, and the ductal cells that secrete some protein and modify the ionic composition of the saliva as they convey it to the mouth. For a variety of reasons, including their physiological interest, their relative abundance and ease of preparation, and their robust stimulation-secretion responses, salivary acinar cells been intensively studied and many of their properties are well, albeit certainly not completely, understood. Although salivary ducts have also received considerable experimental attention, particularly over the past 10 years, less is known about their behavior. Because of this and because of the central role of the acinar cells in salivary fluid and protein secretion we will concentrate almost exclusively on them in this article. For more information on salivary ducts the interested reader is referred to several recent reviews and the references therein (7; 62; 6). It is also worth mentioning at this point that most of the information presented below has been obtained from the salivary glands of experimental animals (mainly rats and rabbits). Except for morphologic studies and several non-invasive procedures, such as the collection of saliva after the application of various stimuli, experiments using human salivary tissues are rare. However, in those cases where the functional properties of human salivary acini have been investigated they appear to conform well to the results and conclusions derived from animal studies. The easiest way to understand the proposed mechanisms of salivary fluid secretion is to consider a specific model. Figure 1 shows a schematic representation of a salivary acinar cell. The cell contains four ion transporters, the Na+/K+ adenosine triphosphatase (ATPase), a Na+–K+–2Cl− cotransporter and a Ca2+-activated K+ channel, all located in the basolateral membrane, and a Ca2+ activated Cl− channel located in the apical membrane. Fluid secretion is thought to arise from the concerted actions of these four transporters as follows. The Na+/K+ ATPase maintains intracellular Na+ concentration low and intracellular K+ concentration high relative to the interstitium by exchanging 3Na+ for 2K+ at the expense of cellular adenosine triphosphate (ATP). The Na+–K+–2 Cl− cotransporter (also known as NKCC1) is a secondary active transport system that transports 1Na+, 1K+ and 2Cl− into the cell in a tightly coupled fashion. Because of the extracellular to intracellular Na+ gradient maintained by the Na+/K+ ATPase, Cl− is likewise concentrated in the acinar cytoplasm above electrochemical equilibrium by NKCC1. In the resting (unstimulated) state intracellular Ca2+ concentration is low and the Ca2+ activated K+ and Cl− channels are therefore closed. But when the cell is stimulated by secretagogues (in situ, typically the muscarinic agonist acetylcholine) intracellular Ca2+ concentration rises and the K+ and Cl− channels open. These Ca2+ associated changes in K+ and Cl− conductance allow KCl to flow out of the cell resulting in the accumulation of Cl− ions and their associated negative electrical charge in the acinar lumen. Na+ is then thought to follow Cl− by leaking from the interstitium through the tight junctions between the cells to preserve electroneutrality, and the resulting osmotic gradient for NaCl causes a transepithelial movement of water from interstitium to lumen. In the continued presence of the secretagogue a net transepithelial Cl− flux and a concomitant secretion of fluid is sustained as a result of Cl− entry via NKCC1 and exit via the apical Cl− channel. When the stimulus is removed the intracellular Ca2+ concentration falls to resting levels, the K+ and Cl− channels close, and the cell returns to its resting state. Model for salivary fluid secretion based on active transepithelial Cl– transport and the osmotic coupling of salt and water fluxes (see text for details) As discussed in more detail below, considerable experimental evidence indicates that the mechanism presented in Figure 1 can account for most of the salivary secretion from rat, rabbit and presumably human, major salivary glands. However, there is also evidence that two alternate mechanisms based on the same osmotic coupling principle outlined in Figure 1 (viz., transepithelial anion transport driving the secretion of salt followed by osmotically obliged water) can also make significant contributions to salivary fluid secretion. The first of these differs in the Cl− entry step. Here, NKCC1 is replaced by a Cl−/HCO3− exchanger and a Na+/H+ exchanger (Figure 2a). In this model the decrease in intracellular Cl− concentration resulting from secretagogue-induced KCl loss leads to increased Cl− entry via the Cl−/HCO3− exchanger. The HCO3− is then replaced by the diffusion of CO2 into the cell and its conversion into HCO3− plus H+ by carbonic anhydrase (CA). Finally the H+ is pumped out of the cell by the Na+/H+ exchanger using the extracellular to intracellular Na+ gradient generated by Na+/K+ ATPase. The net result is the movement of NaCl into the cell in exchange for CO2 that simply recycles across the basolateral membrane. The other alternate mechanism (Figure 2b) involves the secretion of HCO3− rather than Cl−. Here CO2 enters the acinar cell across the basolateral membrane and is converted to HCO3− plus H+ by intracellular CA. HCO3− is secreted across the apical membrane via an anion channel, possibly the same channel involved in Cl− secretion, and the H+ is extruded by the basolateral Na+/H+ exchanger. Recent data suggest that a basolateral Na+–HCO3− cotransporter may also be responsible for some of the HCO3− entry (38) although this is still rather speculative. Two additional models for salivary fluid secretion based on active transepithelial anion transport (see text for details) Space does not permit us to review all of the experimental data related to the mechanism of salivary fluid secretion. Instead we will briefly discuss some of the early evidence that supports the involvement of the three mechanisms of fluid secretion introduced above and then mention some studies involving more recent experimental approaches. For more comprehensive treatments we suggest several review articles (29; 52; 7; 62) as well as more recent original reports referenced below. Briefly stated, the existing data from whole animal studies, perfused glands, isolated acini, and acinar plasma membranes demonstrate that all of the membrane transport systems illustrated in Figures 1 and 2 are present in salivary acinar cells and function in a way consistent with the proposed mechanisms for fluid secretion. In addition they provide convincing evidence that these mechanisms can account for the fluid secretion observed. As already alluded to above, salivary fluid secretion appears to be a two-stage process as first proposed by 51); that is, saliva is initially formed as a near isotonic plasma-like primary secretion in the acinar lumen (the first stage), then the salivary ducts modify this primary fluid by removing sodium and chloride and adding potassium and bicarbonate to produce a final hypotonic fluid that enters the mouth. The plasma-like ionic composition of the primary saliva has been confirmed by analysing the electrolyte content of fluid collected from the acinar lumen using micropuncture techniques (62; 27). This property of the primary saliva is in general agreement with the operation of some combination of the secretory mechanisms discussed above and with the prevailing view that osmotically driven fluid transport is typically near isotonic owing to the relatively high water permeability of fluid transporting epithelia (43). The impermeability of the salivary ducts to water and their ability to modify the ionic composition of the primary fluid as required have also been confirmed (27; 62). A series of experiments with perfused rat and rabbit submandibular glands carried out in the 1980s were particularly important in focusing experimental attention on the mechanisms discussed above (see 52 for a more detailed review and specific references). These studies showed that acetylcholine-induced salivary secretion is markedly reduced (˜70%) when Cl− is replaced in a Cl−/HCO3−-replete perfusate by a physiologically inert anion, or when inhibitors of NKCC1 were present in the perfusate. The effect of NKCC1 inhibitors was even more dramatic in HCO3−-free media where the inhibition of fluid secretion was >95%. These results, taken together with other supporting data, strongly suggested that the mechanism illustrated in Figure 1 was responsible for most of the fluid secretion. In addition, the residual secretion observed in Cl− free but HCO3− replete media was HCO3− rich and blocked by inhibitors of CA or the Na+/H+ exchanger. This component of fluid secretion was therefore consistent with the presence of the mechanism illustrated in 2Figure 2b. Other less direct observations indicated the operation of the mechanism illustrated in 2Figure 2a as well. In the perfused rat submandibular gland 33) estimated that the mechanisms illustrated in Figures 1, 22a and b contributed to anion secretion and thus fluid secretion in the ratio 16:3:2. Although there is convincing evidence that all three of the mechanisms discussed above make significant contributions to the fluid secreted by the rabbit submandibular, rat submandibular and rat parotid glands, this is not the case for all salivary glands. Thus, for example, the mechanisms shown in 2Figure 2a and b appear to play little if any role in fluid secretion from human labial glands (32), while only the mechanism shown in 2Figure 2b is operative in the bovine parotid (24). This latter observation is consistent with the observation that ruminant saliva contains very high levels of bicarbonate (≈100 mM), which is required to buffer the acid produced by microbial fermentation in the rumen. The reason for the presence of three fluid secretory mechanisms in the same gland is still not yet clear. Moreover, somewhat surprisingly the available evidence indicates that when multiple mechanisms are present they coexist in the same acinar cells (53; 23). It is possible that the cell is able to modulate the contributions of the various mechanisms according to certain physiological circumstances, for example brief versus sustained periods of salivary flow. But at present there is no convincing evidence that anything like this occurs. In a recent paper 13) have studied salivary secretion in mice in which the gene for NKCC1 has been disrupted and no expression of this protein is detectable. Stimulated salivary flow rates in these NKCC1 knockout mice are only 40% of those observed in normal littermates. Interestingly, these authors also found that the activity of the Cl−/HCO3− exchanger was increased in parotid acinar cells from NKCC1 knockout mice suggesting that fluid secretion via the mechanism shown in 2Figure 2a is increased in these animals to compensate for the loss of NKCC1. In addition to reduced salivary flow, NKCC1 knockout mice also exhibited a number of other abnormalities, the most dramatic of which were profound deafness caused by a collapse of the membranous labyrinth of the inner ear (10; 16) and male infertility because of defective spermatogenesis (30). Both of these problems are thought to result from fluid secretory defects in the respective tissues. The mechanisms illustrated in Figures 1 and 2 do not address the question of whether the water that follows salt secretion flows between the cells (via the tight junctions) or through the cell body (via the cytoplasm). In fact this issue has been a source of considerable interest and experimentation in both secretory and absorptive epithelia for many years (43). In salivary acini this question is made more complicated by the pyramidal shape of the acinar cells that results in a rather small area for both the luminal membrane and the tight junctional complex. Thus whichever route the water takes would necessarily require a relatively high water permeability. A major milestone in the resolution of these problems was the discovery and cloning of the aquaporins, a family of plasma membrane water channel proteins (3). It had been known for some time that the membranes of many cells had very high water permeabilities while those of others were relatively water impermeant (e.g. the luminal membranes of salivary ducts, see above) and still others could regulate their water permeability according to need (3). It is now clear that cell membranes have very low intrinsic water permeabilities and that the large water permeabilities observed in the membranes of many tissues are because of the presence of aquaporins. Surprisingly, early studies failed to demonstrate the presence of any aquaporins in salivary acinar cell membranes, however, it was subsequently found that a new aquaporin isoform, AQP5, was localized to the apical membranes of many secretory epithelia, including salivary acinar cells (37). More recent experiments have shown that stimulated salivary flows are reduced >60% in AQP5 knockout mice relative to normal controls (26). Thus it would appear that most, but probably not all of the secreted water flows through the acinar cells. Consistent with its central role in salivary fluid secretion, the activity of NKCC1 has been shown to be dramatically up-regulated by secretagogues. 14) have demonstrated a 20-fold increase in the NKCC1 transport activity of rat parotid acinar cells following the application of muscarinic and other Ca2+ mobilizing stimuli. This effect appears to be mediated by a metabolite of arachadonic acid (14) but the exact mechanism is still uncertain. This observation is consistent with the idea that NKCC1 activity is down-regulated while the cell is at rest to prevent futile cycling of the transporter, then up-regulated during stimulation when its activity is required. Interestingly, in the rat parotid NKCC1 is also activated, although to a lesser degree, by β-adrenergic stimulation (31) which increases intracellular cAMP levels without affecting intracellular Ca2+ concentration. This is because of a phosphorylation of NKCC1 that involves, but does not appear to be directly caused by, cAMP-dependent protein kinase A (PKA) (50; 22). At first glance, it seems odd that NKCC1 would be up-regulated by such a stimulus because, as discussed in detail below, increased intracellular cAMP concentration results in robust acinar protein secretion but typically produces little salivary fluid. However, there is good evidence that sympathetic (adrenergic) stimulation, arising for example from mastication, when superimposed on parasympathetic (muscarinic) stimulation has a synergistic effect on salivary flow (20). This synergetic effect may arise from the cAMP-dependent up regulation of NKCC1. It is also interesting to speculate that the unexplained symptoms of dry mouth (xerostomia) and dry eyes that accompany the use of many commonly prescribed medications (44) may be related to interference with the secretagogue-induced up regulation of NKCC1. Indeed, among these drugs are a number of β-blockers (44). Finally, we should mention that, although the osmotic coupling hypothesis (that water follows salt secretion osmotically) underlying the models in Figures 1 and 2 is widely accepted among physiologists, there are some dissenting views (57). One of the central problems here is that it has not been possible, to date, to experimentally demonstrate the existence of the osmotic gradient between the interstitium and lumen that is required for the functioning of the models. In terms of the osmotic coupling model, the explanation for this problem is that, because of the high water permeability of the epithelium, only a very small gradient is required to account for the water fluxes observed (43). The problem for the dissenting views, a major one in our opinion, is to account for the large body of existing evidence consistent with the osmotic gradient hypothesis not only in salivary glands but also in numerous other secretory and absorptive epithelia. Saliva contains a wide variety of secreted proteins, including: α-amylase, an enzyme involved in the digestion of starch; lysozyme, peroxidase, immunoglobulins (IgA) and many additional proteins that have antibacterial and/or antiviral properties; and mucins, which are multifunctional glycoproteins involved in mechanical protection and prevention of dehydration of the oral epithelia, as well as in lubrication for solid food and trapping of microorganisms (62; Amerongen & Veerman in press). As already indicated, most of the proteins in saliva are secreted by the acinar cells. Salivary proteins exhibit vectorial transport from the rough endoplasmic reticulum, where they are synthesized, through a succession of membrane-bounded compartments including the Golgi complex, condensing vacuoles, and secretory granules (54). The secretory granules migrate to particular locations within the cell close to the apical membrane prior to the release of their contents into the acinar lumen. Exocytosis is the process by which cells release the contents of their secretory granules. This involves the fusion of the granule membrane with the luminal plasma membrane of the secretory cell followed by the rupture of the fused membranes. This process is continuous in most cells (`constitutive' exocytosis), but it can be greatly accelerated following an appropriate cellular signal such as neural stimulation (`regulatory' exocytosis). In the three major salivary glands, parotid, submandibular and sublingual, exocytotic protein secretion is primarily controlled by the autonomic nervous system; sympathetic stimulation elicits protein release from parotid and submandibular gland acini, and parasympathetic stimulation elicits protein release from sublingual gland acini as well as some release from parotid acini (35; 42). We will focus here on amylase secretion from rat parotid acinar cells as recent studies on this system are promoting a better understanding of the cellular events involved in salivary gland exocytosis. Salivary protein secretion, like fluid secretion, is evoked when neurotransmitters bind to specific receptors on the basolateral membrane of the secretory cells and generate intracellular second messengers that, in turn, activate the cellular mechanisms responsible for secretion. cAMP is the primary second messenger for amylase secretion from rat parotid acinar cells (4). Noradrenaline, released from sympathetic nerves, binds to and activates β-adrenergic receptors leading to increased intracellular cAMP levels. cAMP is thought to mediate most of its effects through the activation of a cAMP-dependent protein kinase, also known as PKA. In parotid acinar cells, PKA activation is essential for cAMP-dependent exocytotic secretion (34; 49). However, the target proteins phosphorylated by PKA have not yet been identified. In contrast to rat parotid acini, in many other secretory cells Ca2+ has been found to be the primary intracellular second messenger for exocytosis. But it has been shown that cAMP mediates parotid amylase secretion without the elevation of cytosolic Ca2+ (48). In addition, in permeabilized acinar cells significantly enhanced amylase release was observed at all concentrations of free Ca2+ tested (1). Stimulation of muscarinic, substance P peptidergic or α-adrenergic receptors also elicits significant amylase release from the rat parotid, albeit at levels that are significantly lower than those observed from a β-adrenergic receptor-mediated response. These receptors are activated by acetylcholine and substance P released from parasympathetic nerves, and by noradrenaline released from sympathetic nerves, respectively. The stimulation of these receptors activates phosphatidylinositide metabolism and induces an increase in intracellular Ca2+ concentration without affecting intracellular cAMP levels (4; 46). Yoshimura et al (61; 59, 60) have developed a perfusion system for isolated rat parotid acinar cells that allows one to obtain a detailed time course for amylase release. They have demonstrated that the activation of the cAMP by β-adrenergic results in a increase in the of amylase secretion. the other the activation of Ca2+ mobilizing receptors changes in amylase release of an large but followed by a lower sustained In the of extracellular only the is These time of amylase release the in intracellular Ca2+ that Ca2+ can play a significant role in the regulation of amylase secretion. the stimulation of both cAMP and Ca2+ mobilizing results in that are than the of the by stimulus More detailed discussed below suggest that this synergistic effect is caused by a of the and of the process by It has been shown that the of neurotransmitters can be into three of to the plasma membrane a by and by the elevation of intracellular Ca2+ concentrations that Ca2+ the final fusion process for parotid amylase release and that cAMP the of secretory granules thus in the effect of Ca2+ as the for fusion (Figure confirmed that the experimental data on amylase release from the rat parotid could be for by such a model. More amylase release as two first the first and the second fusion of secretory granules. of the experimental data of with this model indicated that the number of granules is small in cells. Thus considerable amylase release could be by a stimulus even at resting intracellular Ca2+ levels, for the observation that amylase release appears to be mainly It is also worth that in stimulation is to noradrenaline which also results in the stimulation of α-adrenergic that acinar cells a in intracellular Ca2+ concentration with that of A model for the mechanism of and Ca2+ amylase secretion in rat parotid acinar cells. cAMP the of secretory granules and the effect of Ca2+ as the for (see text for details) In cells, proposed the that to a target membrane through the of and target membrane proteins referred to as receptors is a and 1 and are target membrane an protein localized to the secretory granule membrane of rat parotid acinar cells as by that it with an and was by a specific for In rat parotid acinar cells permeabilized with these authors also showed that amylase but not Ca2+ suggesting that is involved in amylase as a In parotid acinar cells, such as 1 and which are found in cells, were not In cells, and are for Both of these proteins were in rat parotid acinar cells, but they were not with suggesting that their with may be studied the of using a rabbit a to of This of the involved in the of This was able to from rat parotid secretory granule membranes from cells in the presence of cAMP and acinar but not in their and this was by an of PKA. These observations suggest that the on to which the was is in resting cells and that PKA is able to this via the phosphorylation of some cytosolic (Figure Two and proteins found to be with are for these proteins, but additional studies are required to this as well as their A possible model for the mechanism of between and by the activation of the cAMP-dependent may be by an as yet protein is removed from as a result of the phosphorylation of a cytosolic protein by cAMP-dependent PKA. of from then allows it to with Two of proteins are known to be involved in signal the proteins, which have well in coupling and the or proteins, which we discuss The small proteins have in the In contrast to the proteins, they of a and have specific proteins that their intrinsic The small proteins are into and These are involved in cell secretion, membrane between the endoplasmic and the Golgi of and protein transport into the respectively. it has been suggested that small proteins are involved in exocytotic in secretory cells, including parotid acinar cells proteins are of the proteins are into two and has been in the secretory granule membrane, plasma membrane and of rat parotid acinar cells and has been observed to to the following β-adrenergic activation proteins have been shown to be involved in the and fusion of transport with membranes in various tissues and have been has been in the secretory granule membrane, plasma membrane and of rat parotid acinar cells like β-adrenergic activation has been shown to the of from the to the membrane has been shown to be localized to secretory granules in rat parotid acinar cells and of from acinar cells following β-adrenergic stimulation was as a for of have been and as multifunctional involved in a number of events such as membrane and have investigated a possible involvement of in from rat parotid acinar cells. They have demonstrated that is mainly in the cytosolic of these cells and is to the secretory granule membrane in a a from the of amylase secretion when it was to permeabilized rat parotid cells The above observations strongly suggest the involvement of small proteins in the of amylase from rat parotid acinar cells. for these proteins based on observations from other tissues are indicated proteins between a cytosolic state and a state with the of many cycling between membranes and can be generated through In cells, proteins are thought to be of is an important in exocytosis. have proposed a role for the in in salivary glands. In human the of protein with has been suggested to be involved in via fusion may be by the of membrane acid may be a In cells, associated with secretory granules to the plasma membrane cell stimulation, resulting in the activation of and its of generated at exocytotic appear to be involved in exocytosis. is also thought to be a of exocytosis. In cells, has been shown to to the activation of following the receptors by the of via an as yet mechanism proteins could have in addition to as yet to those above in protein secretion from salivary acinar cells. The of proteins such as and small proteins in from salivary gland cells to be A number of new and techniques are available to these and other which bind to cytosolic and the of specific proteins, and expression systems for and of specific proteins, and specific should us to understand the of various proteins in salivary protein and as a us to their involvement in mechanisms of oral We for and This was in by for the of and of

Expression of integrin subunits in normal and malignant human salivary gland cell clones and its regulation by transforming growth factor-beta 1.

The regenerating gene, Reg, was originally isolated from a rat regenerating islet complementary DNA (cDNA) library, and its human homologue was named REG Iα. Recently, we reported that REG Iα messenger RNA (mRNA), as well as its product, was overexpressed in ductal epithelial cells in the salivary glands of Sjögren's syndrome patients. Furthermore, autoantibodies against REG Iα were found in the sera of Sjögren's syndrome patients, and the patients who were positive for the anti-REG Iα antibody showed significantly lower saliva secretion than antibody-negative patients. We found the mechanism of REG Iα induction in salivary ductal epithelial cells. Reporter plasmid containing REG Iα promoter (-1190/+26) upstream of a luciferase gene was introduced into human NS-SV-DC and rat A5 salivary ductal cells. The cells were treated with several cytokines (interleukin (IL)-6, IL-8, etc.), upregulated in Sjögren's syndrome salivary ducts, and the transcriptional activity was measured. IL-6 stimulation significantly enhanced the REG Iα promoter activity in both cells. Deletion analysis revealed that the -141∼-117 region of the REG Iα gene was responsible for the promoter activation by IL-6, which contains a consensus sequence for signal transducer and activator of transcription (STAT) binding. The introduction of small interfering RNA for human STAT3 abolished IL-6-induced REG Iα transcription. These results indicated that IL-6 stimulation induced REG Iα transcription through STAT3 activation and binding to the REG Iα promoter in salivary ductal cells. This dependence of REG Iα induction upon IL-6/STAT in salivary duct epithelial cells may play an important role in the pathogenesis/progression of Sjögren's syndrome.

[Ca2+]i and the Cl- current were measured in isolated submandibular gland acinar and duct cells to characterize and localize the purinergic receptors expressed in these cells. In both cell types 2'-3'-benzoylbenzoyl (Bz)-ATP and ATP increased [Ca2+]i mainly by activation of Ca2+ influx. UTP had only minimal effect on [Ca2+]i at concentrations between 0.1 and 1 mM. However, a whole cell current recording showed that all nucleotides effectively activated Cl- currents. Inhibition of signal transduction through G proteins by guanyl-5'-beta-thiophosphate revealed that the effect of ATP on Cl- current was mediated in part by activation of a G protein-coupled and in part by a G protein-independent receptor. BzATP activated exclusively the G protein-independent portion, whereas UTP activated only the G protein-dependent portion of the Cl- current. Measurement of [Ca2+]i in the microperfused duct showed that ATP stimulated a [Ca2+]i increase when applied to the luminal or the basolateral sides. BzATP increased [Ca2+]i only when applied to the luminal side, whereas UTP at 100 microM increased -Ca2+-i only when applied to the basolateral side. The combined results suggest that duct and possibly acinar cells express P2z receptors in the luminal and P2u receptors in the basolateral membrane.

Molecular and Functional Expression of the Best2 Ca2±activated Cl- Channel in Mouse Submandibular Salivary Gland

To understand the molecular mechanisms whereby normal human salivary gland cells become malignant and escape growth-inhibitory control by transforming growth factor (TGF)-betaI, we examined the effect of TGF-betaI on the proliferation and expression of TGF-beta receptors in cells and the expression of TGF-beta type II receptor (TbetaR-II) mRNA. An SV40-immortalized normal human salivary gland duct cell clone (NS-SV-DC) with no tumorigenic ability, originally obtained via s.c. implantation into nude mice, was partially resistant to the growth-inhibitory effect of TGF-betaI, while a neoplastic human salivary gland duct cell clone (HSGc) with tumorigenic, but not metastatic, potential in nude mice was more resistant to the growth-suppressive effect of TGF-betaI than NS-SV-DC. Metastatic cell clones derived from carcinogen-treated HSGc were completely refractory to the anti-proliferative effect of TGF-betaI. Affinity cross-linking revealed that NS-SV-DC possesses the types I, II (TbetaR-II) and III receptors. However, HSGc and metastatic cell clones lacked expression of detectable levels of the TbetaR-II protein. Moreover, we evaluated TbetaR-II mRNA expression in these cell clones by Northern blot analysis and observed that, although NS-SV-DC expressed a large amount of TbetaR-II mRNA, a small amount of TbetaR-II mRNA was detectable in HSGc. In contrast, no significant bands were detected in metastatic cell clones. Our results, therefore, suggest that one of the possible mechanisms of escape from autocrine or paracrine growth inhibition by TGF-betaI during human salivary gland carcinogenesis involves reduced expression or lack of TbetaR-II.

The effects of ATP on salivary glands have been recognized since 1982. Functional and pharmacological studies of the P2 nucleotide receptors that mediate the effects of ATP and other extracellular nucleotides have been supported by the cloning of receptor cDNAs, by the expression of the receptor proteins, and by the identification in salivary gland cells of multiple P2 receptor subtypes. Currently, there is evidence obtained from pharmacological and molecular biology approaches for the expression in salivary gland of two P2X ligand-gated ion channels, P2Z/P2X7 and P2X4, and two P2Y G protein-coupled receptors, P2Y1 and P2Y2. Activation of each of these receptor subtypes increases intracellular Ca2+, a second messenger with a key role in the regulation of salivary gland secretion. Through Ca2+ regulation and other mechanisms, P2 receptors appear to regulate salivary cell volume, ion and protein secretion, and increased permeability to small molecules that may be involved in cytotoxicity. Some localization of the various salivary P2 receptor subtypes to specific cells and membrane subdomains has been reported, along with evidence for the co-expression of multiple P2 receptor subtypes within specific salivary acinar or duct cells. However, additional studies in vivo and with intact organ preparations are required to define clearly the roles the various P2 receptor subtypes play in salivary gland physiology and pathology. Opportunities for eventual utilization of these receptors as pharmacotherapeutic targets in diseases involving salivary gland dysfunction appear promising.

IRF6 regulates adherens junction proteins and inflammatory cytokines in salivary acinar cells and its expression is elevated in Sjӧgren's syndrome.

Mouse mandibular salivary duct cells contain an amiloride-sensitive Na+ current and express all three subunits of the epithelial Na+ channel, ENaC. This amiloride-sensitive Na+ current is subject to feedback regulation by intracellular Na+ and we have previously demonstrated that this regulation is mediated by an ubiquitin-protein ligase, which we identified as Nedd4. The evidence supporting this identification is as follows: (1) antibodies raised against murine Nedd4 block Na+ feedback inhibition; (2) a mutant of murine Nedd4 containing the WW domains but no HECT domain (ubiquitin-protein ligase) blocks Na+ feedback inhibition; and (3) Nedd4 is expressed in mouse mandibular salivary duct cells. In the present studies, we have used whole-cell patch-clamp methods to further investigate the mechanisms by which ubiquitin-protein ligases regulate the amiloride-sensitive Na+ conductance in mouse salivary duct cells. In particular, we have examined the possibility that the ubiquitin-protein ligase, KIAA0439, which is closely related to Nedd4, may mediate Na+ feedback control of amiloride-sensitive Na+ channels. Furthermore, we have attempted to define the mechanism by which ubiquitin-protein ligases inhibit Na+ channels. We have found that KIAA0439 is expressed in mouse mandibular ducts and interacts with the PY motifs of the alpha-, beta-, and gamma-subunits of ENaC in vitro. Furthermore, in whole-cell patch-clamp studies, a glutathione-S-transferase (GST)-fusion protein containing the WW motifs of human KIAA0439 was able to inhibit feedback regulation of the amiloride-sensitive Na+ current by intracellular Na+. We also examined whether GST-fusion proteins containing the C-termini of the alpha-, beta-, and gamma-subunits of ENaC are able to interrupt Na+ feedback regulation of the amiloride-sensitive Na+ current. We found that the C-termini of the beta- and gamma-subunits were able to do so, whereas the C-terminus of the alpha-subunit was not. We conclude that KIAA0439 is, together with Nedd4, a potential mediator of the control of epithelial Na+ channels in salivary duct cells by intracellular Na+. We further conclude that ubiquitin-protein ligases interact with the Na+ channels through the C-termini of the beta- and gamma-subunits of the Na+ channels.

BackgroundThe regenerating gene, Reg, was originally isolated from a rat regenerating islet cDNA library, and its human homologue was named REG Iα. Reg gene product acts as a growth factor...

BackgroundThe regenerating gene, Reg, was originally isolated from a rat regenerating islet cDNA library, and its human homologue was named REG Iα. Reg gene product acts as a growth factor...

Bicarbonate concentration in saliva is controlled by the action of acid‐base transporters in salivary duct cells. We have previously shown expression of ATP6V1E1 and ATP6V1A in human and rodent salivary ducts and adaptive redistribution to the apical plasma membrane following acute and chronic metabolic acidosis. However, whether such redistribution has any physiological significance has not been elucidated. Using the human submandibular gland cell line HSG, we show for the first time functional expression of V‐ATPase in submandibular duct cells and introduce Transforming Growth Factor beta (TGF‐β) as a novel regulator of V‐ATPase subunits. Using QRT‐PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, and intracellular H(+) recording with H(+) ‐sensitive dye 2′,7′‐bis‐(carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, we show that activation of TGF‐β signalling upregulates ATP6V1E1 and ATP6V1B2, and has no effect on ATP6V1A. Following acidosis, neutralization of TGF‐βs or blocking of Smad2/3 phosphorylation decreased membrane expression of ATP6V1A, and prevented the acidosis‐induced increased V‐ATPase activity. The results suggest multiple modes of action of TGF‐β1 on V‐ATPase subunits in HSG cells: TGF‐β1 may regulate transcription or protein synthesis of certain subunits and trafficking of other subunits in a context‐dependent manner. Moreover, V‐ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Salivary gland homeostasis is maintained through acinar cell self-duplication.