Role of aquaporins in saliva secretion

Introduction Main function of salivary gland is saliva secretion. This function is essential for proper oral homeosta-sis. Aquaporins are water-permeable trans-membrane proteins involved in trans-cellular water flow. Aim of this review is to give an overview of the expression of aquaporins in the salivary gland; their role in saliva secretion and in pathophysiological conditions. Discussion Several aquaporins are expressed in salivary glands, amongst which aquaporin-5, plays an essential physiological role. Consequently, modulation of aquaporin expression and/ or trafficking may contribute to the pathogenesis of diseases affecting salivary glands such as xerostomia conditions. Indeed, therapeutic tools increasing aquaporins expression might be valuable for the treatment of xerostomia conditions. Conclusion Aquaporins are involved in salivary gland physiological and pathophysi-ological processes. Therefore, they could represent novel therapeutic targets for the treatment of diseases affecting the salivary glands.


Introduction
The existence of water channels was anticipated based on the existence of biological membranes presenting high-membrane water permeability that could not be explained solely by passive water diffusion though a lipid bilayer 1 .The existence of water channels, also called aquaporins, was discovered following the isolation of a 28 kDa membrane protein from red blood cells 2 termed channel-forming integral protein of 28 kDa (CHIP28), conferring increased water permeability to Xenopus laevis oocytes following microinjection with an in vitro-transcribed CHIP28 RNA 3 , and to proteoliposomes containing pure CHIP28 protein 4 .Later, related mammalian and plant proteins were sequenced and shown to allow water transport.The term aquaporin (AQP) was proposed for the family of water channel and the name CHIP28 was changed to AQP1 5 .CHIP28 was predicted to have a homotetrameric structure, each monomer containing six trans-membrane domains and the N-and C-termini of the proteins located intra-cellularly 6 .As two halves of the AQP1 protein are symmetric forming a channel pore in the membrane bilayer, with two highly conserved asparagineproline-alanine (NPA) motifs juxtaposed within the channel, serve as a water selective gate.AQP1's threedimensional structure resembles a hourglass 7 .Some AQPs transport only water, while other transport other solutes such as glycerol, urea, anions, ammonia, hydrogen peroxide, arsenite, antimonite, carbon dioxide and nitric oxide 8 .According to their permeability and structure, the 13 mammalian AQPs have been subdivided into classical AQPs, primarily permeable to water but also to gasses and ions (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, AQP8) 9 ; aquaglyceroporins are also permeable to glycerol and other small solutes (AQP3, AQP7, AQP9, AQP10) 9 ; and non-classical AQPs with permeability specificity that has not been clearly established 10 .The review discusses the expression and roles of AQPs in salivary secretion.

Discussion
The author has referenced some of its own studies in this review.These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.All human subjects, in these referenced studies, gave informed consent to participate in these studies.Animal care was also in accordance with the institution guidelines.

Salivary glands morphology
Salivary glands (SG) consist of acinar (serous, mucous or seromucous), ductal and myoepithelial cells 11 .The acinar cells secrete a primary fluid, composed of proteins and fluid, into ducts.The ductal cells then modify the primary fluid secreted by the acinar cells.Contraction of myoepithelial cells surrounding the basal area of acinar and ductal cells forces fluid out of the ducts 11 .
Mammals possess three major pairs of SG: parotid (PG), submandibular (SMG) and sub-lingual glands (SLG), as well as minor labial SG (LSG) scattered throughout the oral cavity 11 .Rodent and human PGs are exclusively composed of serous acinar cells, whereas SMG and LSG contain both serous and mucous acinar cells.Human SMG contain more serous acinar cells than mucous acinar cells, while it is opposite in LSG.Still, both human SMG and LSG contain some seromucous acinar cells 12 .expression of AQP4 in SMG acinar cells remains controversial, as for the expression of AQP5 in ductal cells 13 .In PG acinar cells, AQP5 is expressed at the apical membrane, while AQP6 is expressed at the plasma membrane near tight junctions and the secretory granule membranes 13 .AQP8 is located in myoepithelial cells from SG acinar cells 13 .
In a mouse, AQP1, AQP3, AQP4 and AQP5 mRNA have been detected 14 .Mouse PG expresses AQP5 at the apical membrane and basolateral membrane of acinar cells 15 .In a human, AQP1 is localised on capillaries and myoepithelial cells 13 of all SG.AQP3 is expressed at the basolateral membrane of both serous and mucous acinar cells, and not on ductal cells of all SG 13 .Despite the presence of AQP4 mRNA in all SG, AQP4 protein expression has not been confirmed 13 .AQP5 is expressed and localised to the APM of acinar cells, but not on the ductal cells of all SG 13 .Furthermore, AQP5 expression is confined to the serous acinar cells and absent from the mucous acinar cells 13 .AQP6 and AQP7 mRNAs are present in SMG 13 .

Physiology of saliva secretion
In humans, SGs secrete daily 750 to 1,000 ml of saliva.Mainly, SMG and LSG secrete saliva under resting conditions, while PG secrete saliva upon stimulation.Endocrine, paracrine and neuronal inputs control saliva secretion 16 .Neuronal regulation of saliva secretion is controlled by parasympathetic and sympathetic nerve endings.In response to acetylcholine, activating M3 and M1 (to a lesser extent) receptors in both acinar and ductal cells, intra-cellular calcium release leads mainly to fluid secretion 17 .On the other hand, noradrenalin, activating ß-adrenergic receptors, induces intra-cellular cAMP increase leading mainly to enzyme secretion from acinar cells and fluid and electrolyte transport in ductal cells 16 .
Several important physiological functions, such as protection and hydratation of mucosal structures, initiation of digestion and antimicrobial defences, are ensured by saliva.Therefore, multiple clinical manifestations are encountered in response to dysfunction of saliva secretion 18 .
Saliva secretion results from a two step process (Figure 1).During the first step of the saliva secretion process, an isotonic-like fluid rich in NaCl is secreted by acinar cells 19 .Accumulation of ions in the acinar lumen generates a trans-epithelial osmotic gradient driving large water efflux to the acinar lumen via apical AQP5 channels and possible paracellular pathways.This resulting primary fluid then flows into the ductal lumen.During the second step of the saliva secretion process, the ductal cells, relatively impermeable to water, modify the composition of the fluid by reabsorbing most of the Na + and Cl − , while secreting HCO 3 − and K +19 .Upon reaching the mouth, the final saliva is hypotonic 19 .

Role of AQPs in saliva secretion
The involvement of AQP5 in transcellular water movement during saliva secretion has been exposed using AQP5 knockout mice 20,21 .Indeed, the knockout mice displayed a 60% decrease in pilocarpinestimulated saliva production and a more viscous and hypertonic saliva than their wild-type littermates 20,21 .Furthermore, PG and SLG acinar cells, prepared from AQP5-knockout mice, presented, respectively, a 65 and a 77% decrease in water permeability in response to osmotic challenge, compared to acinar cells prepared from wild-type littermates 21 .AQP1, AQP4 and AQP8 seemingly do not participate to saliva secretion as knocking out their expression had not impact on mice pilocarpine-induced salivation, compared to wild-type littermates 20,22 .The implication of other AQPs in salivation has not been demonstrated either.In light of the still debated expression of AQP5 in ductal cells, further studies will be required to establish its possible functional role and regulation in that cell type.
In addition to the well-documented trans-cellular water movement, via AQP5, occurring during saliva secretion, several experiments have provided evidences for the existence of a concurrent paracellular water flow, via tight junctions 23 .The data generated from such experiments suggested an osmosensor feedback model in which osmosensor, likely AQP5 in SGs, controls the tonicity of the transported fluid by mixing trans-cellular and paracellular flows 23,24 .However, another secretory model based a trans-cellular-only osmotic mechanism is sufficient to predict the results obtained from AQP5-knockout mice studies, despite the fact that AQP5-knockout mice studies do not give enough information to definitively rule out the existence of an additional paracellular water pathway 25 .Indeed, whether paracellular water flow also participates remains an ongoing debate.Therefore, further studies will be required to ultimately assess which of the two proposed secretory models accounts best for saliva secretion.

Role of AQP5 traf icking in saliva secretion
Sub-cellular localisation of plasma membrane proteins is dynamically regulated by post-translational modifications such as phosphorylation and ubiquitination occurring on sorting signal located into the cytosolic domains of the transported proteins.
Phosphorylation-dependent trafficking of AQP5 in SGs remains poorly understood as compared to that of its most closely sequencerelated family member AQP2 in the kidney 26 .AQP5 shares homologous structural and amino acid features with AQP2, namely in the carboxyterminus domain, the latter being involved in AQP2 cellular trafficking and apical membrane targeting in the kidney 26 .Targeting of AQP5 to the apical membrane of epithelial cells seemingly result from the presence of a specific targeting signal present in the C-terminal part of the protein 27 .AQP5 trafficking, resulting from the fusion of intra-cellular vesicles expressing AQP5 to the apical membrane, can be induced by cAMP and acetylcholine 28,29 (Figure 2).AQP5 amino acids Ser156 30 and Thr259 31 have been shown to be phosphorylated by a cAMP-dependent mechanism but are not involved in AQP5 trafficking.So far, in vivo trafficking of AQP5 could not be detected in SGs 32 .Additional studies will be necessary to identify the phosphorylated amino acids involved in stimuli-induced AQP5 trafficking and concomitant saliva secretion.

Implication of AQPs in xerostomia conditions
With senescence, declined salivation occurs gradually as age increases in humans and mice.In senescent rats, decreased AQP5 translocation results from a reduction in PG nitric oxide synthase 33 .Civimeline could be used as a therapeutic agent for the treatment of age-related xerostomia as it increases AQP5 expression at the acinar apical membrane 34 .DNA demethylation agents increased AQP5 expression and restored salivation in a murine aging model 35 .Therefore, increase of AQP5 expression by analogs of acetylcholine, activators of nitric oxide synthase and DNA demethylation agents could be useful for xerostomia treatment in elderly individuals.
Radiation therapy is often used as a therapeutic component for patients diagnosed with head and neck cancer.However, this therapy damages the SG acinar cells lying in the radiation field and induces loss of salivation 36 .This loss of salivation could result from decreased AQP5 expression [37][38][39][40][41] and AQP5 trafficking 39 .Based on the physiological mechanisms of saliva secretion, it was hypothesised that the introduction of a facilitated water pathway in irradiated ductal cells could generate an osmotic gradient (extra-cellular lumen > intracellular) allowing fluid secretion 37 .The introduction of such facilitated water pathway was performed using a recombinant adenoviral vector coding for human AQP1 (AdhAQP1) that drove both AQP1 expression and osmotically driven fluid secretion in epithelial cells 37,42,43 , and restored saliva secretion in irradiated SMG from rats 37 , non-human primates 44 and miniature pigs 45 .Furthermore, a clinical trial showed that AdhAQP1 delivery was safe in subjects with irradiated PG, induced increased parotid flow rate and relieved symptoms in a subset of subjects 46 .This clinical trial demonstrated that AQP could be used as a therapeutic agent in a particular pathological condition affecting SG.
Sjögren's syndrome (SS) is an autoimmune disease inducing secretory gland dysfunction, including SG dysfunction resulting in saliva loss 47 .Patients may present primary or secondary SS 47 .Worldwide prevalence of primary SS is about 0.5% with a 9:1 female to male ratio.Several animal models for SS have been generated to study the progression and the multiple aspects of the pathogenesis of SS 48 .The involvement of AQPs in the pathogenesis of SS has been examined.Treatment of SS patient, presenting altered AQP1 LSG expression, with rituximab (an anti-CD20 monoclonal antibody) increased both AQP1 expression in myoepithelial cells and saliva flow 49 .However, the participation of AQP1 to saliva flow has not been demonstrated using AQP1 knockout mice 21,22 .Therefore, it remains to be investigated if rituximab could restore saliva secretion in SS patients by a mechanism independent of the increased in AQP1 expression.Salivary AQP5 levels, shown to be proportional to saliva flow 50 , could be of diagnostic value in xerostomic conditions, such as SS.Besides, abnormal AQP5 localisation (mostly basolateral instead of being apical), has been detected in SG 51 from SS patients, and from animal models for SS [52][53][54][55][56] .In some animal models for SS, altered SG AQP5 distribution appears to be concomitant with the presence of inflammatory infiltrates and acinar destruction 54 .Toll-like receptor activation and tumour necrosis factor α decreased AQP5 expression in SG 57,58 , while interferon α (improving xerostomia in SS patients) increased AQP5 expression 59 .Besides, auto-antibodies against M3 receptors, present in SS patients, inhibited AQP5 trafficking in SG cells 60 .Therefore, altered AQP5 localisation could participate to SS pathogenesis, even though it could not directly account for saliva impairment 53 .Finally, rituximab improved xerostomia and glandular manifestations of the disease while it increased AQP5 expression at the apical membrane of SG acinar cells 46 .In the future, it remains to clearly evaluate the benefits of increasing AQP expression, by the use of various therapeutic tools, in SG dysfunction occurring in SS.
Diabetes, with a prevalence estimated to be 4.4% in 2030, represents a common cause of xerostomia 61 .Contradictory data exist concerning the modulation of AQP5 expression in animals with streptozotocin-induced type-1 diabetes 62,63 .Further studies will be required to fully assess the role of AQP5 in diabetic xerostomia.

Conclusion
Several AQPs have been shown to be expressed in SG.Direct participation of AQP5 to saliva secretion has been established.Therapeutic tools driving increased AQP expression in SG could be useful for the treatment of several xerostomia conditions.Additional studies are required to fully understand the involvement of AQPs in pathophysiological conditions of SG

FirstFigure 1 :
Figure 1: Molecular mechanisms of saliva secretion.Acinar cells secrete a large volume of isotonic-like fluid rich in NaCl.The generation of a trans-epithelial osmotic gradient drives water flow through apical AQP5 and possibly paracellular pathways.Ductal cells, relatively impermeable to water, re-absorb most of the NaCl and secrete K + and HCO 3 − .

Figure 2 :
Figure 2: AQP5 intra-cellular trafficking.Acetylcholine (Ach) and noradrenalin (NA) bind to their receptors at the basolateral membrane of acinar cells, resulting in intra-cellular calcium and cAMP[OAPL2]: [AQ2: Please define cAMP.]increases and subsequent activation of protein kinase C, calcium-calmodulindependent protein kinase (Ca 2+ -CaMK) and protein kinase A. These kinases are likely to induce the phosphorylation (P) of AQP5 present in intra-cellular vesicles, leading to their fusion with the apical membrane and the subsequent insertion of AQP5 in the cell plasma membrane.Once inserted into the apical membranes, AQP5 then allow water to flow to the gland lumen.