Developmental role of hyaluronic acid and its application in salivary gland tissue engineering.

Dry mouth, or xerostomia, caused by salivary gland dysfunction significantly impacts oral/systemic health and quality of life. Although in vitro-generated artificial salivary glands have been considered as the fundamental solution, its structural complexity is difficult to reproduce using current biomaterials. Therefore, understanding and recapitulating the roles of biomacromolecules in salivary gland organogenesis is needed to solve these problems. Hyaluronic acid (HA) is a macromolecule abundant during salivary gland organogenesis, but its role remains unknown. Here, we verify the effects of HA on salivary gland organogenesis and artificial organ germ formation in solubilized and substrate-immobilized forms. In embryonic submandibular glands (eSMG), we found dense HA layers encapsulating proliferative c-Kit+ progenitor cells that were expressing CD44, an HA receptor. The blockage of HA synthesis, or degradation of HA, impaired eSMG growth by ablating the c-Kit+ progenitor cell population. We also found that high-molecular-weight (HMW) HA has a significant role in eSMG growth. Based on these findings, we discovered that HA is also crucial for in vitro formation of salivary gland organ germs, one of the most promising candidates for salivary gland tissue regeneration. We significantly enhanced salivary gland organ germ formation by supplementing HMW HA in solution; this effect was further increased when the HMW HA was immobilized on the substrate by polydopamine/HA co-immobilization. Our study suggests that the current use of HA in salivary gland tissue engineering can be further optimized.

Epitaxially Strained CeO2 /Mn3 O4 Nanocrystals as an Enhanced Antioxidant for Radioprotection.

Nanomaterials with antioxidant properties are promising for treating reactive oxygen species (ROS)-related diseases. However, maintaining efficacy at low doses to minimize toxicity is a critical for clinical applications. Tuning the surface strain of metallic nanoparticles can enhance catalytic reactivity, which has rarely been demonstrated in metal oxide nanomaterials. Here, it is shown that inducing surface strains of CeO2 /Mn3 O4 nanocrystals produces highly catalytic antioxidants that can protect tissue-resident stem cells from irradiation-induced ROS damage. Manganese ions deposited on the surface of cerium oxide (CeO2 ) nanocrystals form strained layers of manganese oxide (Mn3 O4 ) islands, increasing the number of oxygen vacancies. CeO2 /Mn3 O4 nanocrystals show better catalytic activity than CeO2 or Mn3 O4 alone and can protect the regenerative capabilities of intestinal stem cells in an organoid model after a lethal dose of irradiation. A small amount of the nanocrystals prevents acute radiation syndrome and increases the survival rate of mice treated with a lethal dose of total body irradiation.

NiCHE Platform: Nature-Inspired Catechol-Conjugated Hyaluronic Acid Environment Platform for Salivary Gland Tissue Engineering.

Recently, there has been growing interest in replacing severely damaged salivary glands with artificial salivary gland functional units created in vitro by tissue engineering approaches. Although various materials such as poly(lactic-co-glycolic acid), polylactic acid, poly(glycolic acid), and polyethylene glycol hydrogels have been used as scaffolds for salivary gland tissue engineering, none of them is effective enough to closely recapitulate the branched structural complexity and heterogeneous cell population of native salivary glands. Instead of discovering new biomaterial candidates, we synthesized hyaluronic acid-catechol (HACA) conjugates to establish a versatile hyaluronic acid coating platform named "NiCHE (nature-inspired catechol-conjugated hyaluronic acid environment)" for boosting the salivary gland tissue engineering efficacy of the previously reported biomaterials. By mimicking hyaluronic acid-rich niche in the mesenchyme of embryonic submandibular glands (eSMGs) with NiCHE coating on substrates including polycarbonate membrane, stiff agarose hydrogel, and polycaprolactone scaffold, we observed significantly enhanced cell adhesion, vascular endothelial and progenitor cell proliferation, and branching of in vitro-cultured eSMGs. High mechanical stiffness of the substrate is known to inhibit eSMG growth, but the NiCHE coating significantly reduced such stiffness-induced negative effects, leading to successful differentiation of progenitor cells to functional acinar and myoepithelial cells. These enhancement effects of the NiCHE coating were due to the increased proliferation of vascular endothelial cells via interaction between CD44 and surface-immobilized HAs. As such, our NiCHE coating platform renders any kind of material highly effective for salivary gland tissue culture by mimicking in vivo embryonic mesenchymal HA. Based on our results, we expect the NiCHE coating to expand the range of biomaterial candidates for salivary glands and other branching epithelial organs.

Epigenetic Modification as a Regulatory Mechanism for Spatiotemporal Dynamics of ANO1 Expression in Salivary Glands.

Anoctamin1 (ANO1), a calcium activated chloride channel, is known to play a critical role in salivary secretion. In the salivary gland, ANO1 is expressed exclusively in the acinar cells, with no expression in the ductal cells. However, the mechanisms that determine this distinctive cell type-dependent expression pattern of ANO1 remain unknown. In this study, we discovered that the cell-dependent expression of ANO1 during salivary gland organogenesis is regulated by DNA methylation of ANO1 CpG islands. ANO1 CpG islands in e12 embryonic submandibular glands (eSMG) are highly methylated, but those in e14 eSMG or adult SMG are significantly unmethylated. The differential expression pattern of ANO1 in duct and acini is defined at e14. Artificial demethylation by treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR), induced the expression of ANO1 in both the ductal cell line Human Submandibular Gland (HSG) and in the duct cells of adult mouse SMG. During the trans-differentiation in Matrigel of duct-origin HSG cells into acinar-like phenotype, significant demethylation of ANO1 CpG islands is observed. This may be due to the reduced expression of DNA methyltransferase (DNMT) 3a and 3b. These results suggest that the differential expression of ANO1 in salivary glands during organogenesis and differentiation is mainly regulated by epigenetic demethylation of the ANO1 gene.

Differential regulation of vacuolar H+ -ATPase subunits by transforming growth factor-ß1 in salivary ducts.

Bicarbonate concentration in saliva is controlled by the action of acid-base transporters in salivary duct cells. We show for the first time expression of ATP6V1B1 in submandibular gland and introduce transforming growth factor-beta (TGF-ß) as a novel regulator of V-ATPase subunits. Using QRT-PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, chromatin immunoprecipitation, and intracellular H(+ ) recording with H(+ )-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein we show that in the human submandibular gland (HSG) cell line, activation of TGF-ß signaling upregulates ATP6V1E1 and ATP6V1B2, downregulates ATP6V1B1, and has no effect on ATP6V1A. TGF-ß1 effects on ATP6V1B1 are mediated through the canonical, the soluble adenylate cyclase, and ERK signaling. A CREB binding sequence was identified in the ATP6V1B1 promoter and CREB binding decreased after TGF-ß1 treatment. Following acidosis, a bafilomycin-sensitive and Na+ -independent cell pH recovery was observed in HSG cells, an effect that was not influenced after disruption of acidic lysosomes. Moreover, neutralization of TGF-ßs, inhibition of TGF-ß receptor, or inhibition of the canonical pathway decreased membrane expression of ATP6V1A and prevented the acidosis-induced increased V-ATPase activity. The results suggest multiple modes of action of TGF-ß1 on V-ATPase subunits in HSG cells: TGF-ß1 may regulate transcription or protein synthesis of certain subunits and trafficking of other subunits in a context-dependent manner. Moreover, surface V-ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.

Effect of anti-muscarinic autoantibodies on leukocyte function in Sjögren's syndrome.

Patients with primary Sjögren's syndrome, a systemic autoimmune disease, have been shown to have serum autoantibodies that react with the muscarinic acetylcholine type 3 receptor (M3R).Primary Sjögren's syndrome is a systemic autoimmune disease. Patients with primary Sjögren's syndrome have been shown to have serum autoantibodies that react with the muscarinic acetylcholine type 3 receptor (M3R). Leukopenia has been reported to be significantly more common in primary Sjögren's syndrome patients who have anti-M3R-autoantibodies in their sera. In this study, we investigated whether these anti-M3R autoantibodies have effects on M3R and MHCI expression in Jurkat T cells. Purified IgG antibodies were isolated from the serum of healthy individuals and primary Sjögren's syndrome patients. Jurkat cell line was used to represent T lymphocytes. In situ immunofluorescence confocal microscopy was used to confirm the binding reactivity of primary Sjögren's syndrome IgG antibodies to M3R. Co-immunoprecipitation and immunofluorescence results suggested a direct interaction between M3R and MHC I. Co-internalization of M3R and MHC I was observed when Jurkat cells were exposed to the primary Sjögren's syndrome IgG, but this primary Sjögren's syndrome IgG-induced co-internalization of M3R and MHC I was prevented by the presence of exogenous IFN-γ. Primary Sjögren's syndrome IgG itself did not affect the viability of Jurkat cells, but Jurkat cells exposed to primary Sjögren's syndrome IgG were observed to undergo significant cell death when co-cultured with primary Natural Killer cells. Our results suggest that anti-M3R autoantibodies in primary Sjögren's syndrome induce downregulation of plasma membrane-resident M3R and MHC class I molecules in leukocytes followed by NK cell-mediated cell death. This mechanism may explain the frequency of leukopenia occurrence in patients with primary Sjögren's syndrome.

Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands.

Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 µM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.

Epigenetic alteration of the purinergic type 7 receptor in salivary epithelial cells.

Purinergic receptors, particularly type 7 (P2RX7), are involved in apoptotic cell death. However, the expression and function of P2RX7 are suppressed in HSG cells. In the present study, we explored whether P2RX7 function is regulated by epigenetic alteration of the receptors in two different cell lines, HSG cells derived from human submandibular ducts, and A253 cells, originated from human submandibular carcinoma. We discovered that HSG cells expressed all subtypes of purinergic receptors, excluding P2RX7, at the mRNA level. However, treatment of the cells with 5-Aza-CdR, a DNA demethylating agent, increased the mRNA expression levels of P2RX7 in a time-dependent manner. Furthermore, 5-Aza-CdR completely rescued the calcium response induced by P2RX7 agonist BzATP, a response that was absent in untreated HSG cells. In contrast, A253 cells showed a moderate methylation pattern in the P2RX7 CpG island. Most CG pairs from the first to the 21st were methylated in untreated HSG cells, but 5-Aza-CdR-treatment partially demethylated the methylated CG pairs. We obtained similar results when investigated human tissues; the CG pairs in the P2RX7 CpG islands showed hypermethylation and hypomethylation patterns in human normal and cancer tissues, respectively. Our results suggest that the expression level and function of P2RX7 are regulated by DNA methylation in epithelial cells.

Epigenetic modulation of the muscarinic type 3 receptor in salivary epithelial cells.

Muscarinic receptors, particularly the type 3 subtype (M3R), have an important role in exocrine secretion. M3R normally function in HSG cells originated from human submandibular gland ducts, but not in A253 and SGT cells, derived from human submandibular carcinoma and salivary gland adenocarcinoma. However, the underlying mechanism of this suppression has remained elusive. In this study, we examined whether M3R function is suppressed by epigenetic modulation of the receptor. To this end, we investigated the mRNA transcript and protein levels of the M3R using reverse transcriptase-PCR, western blot, and confocal microscopy analyses. Global DNA methylation assays, methylation-specific PCR, and bisulfite sequencing were also performed to understand the epigenetic status of the M3R CpG island. We found that A253 cells expressed all subtypes of muscarinic receptors, except M3R, on the mRNA level. However, treatment of cells with 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA-demethylating agent, increased the expression levels of both M3R mRNA transcript and protein in proportion to the incubation period. 5-Aza-CdR completely restored the carbachol-induced calcium response, which was not observed in untreated A253 cells. In untreated A253 cells, all CG pairs from the 1st to 14th were methylated and 5-Aza-CdR treatment demethylated one of the methylated CG pairs. We also examined the methylation pattern of M3R CpG island in human cancer tissue. Interestingly, the result was very similar to those of A253 cells. All CG pairs in M3R CpG island were also methylated. Another salivary gland tumor cell line, SGT, also showed the similar methylation pattern, heavy methylation in M3R CpG island. It is concluded that CpG island in M3R is hypermethylated in cancer cell lines and human cancer. Our results further suggest that 5-Aza-CdR could potentially be used to restore the function of M3R, suppressed in some cancer cell types.

Role of LPA and the Hippo pathway on apoptosis in salivary gland epithelial cells.

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid involved in numerous physiological responses. However, the expression of LPA receptors and the role of the Hippo signaling pathway in epithelial cells have remained elusive. In this experiment, we studied the functional expression of LPA receptors and the associated signaling pathway using reverse transcriptase-PCR, microspectrofluorimetry, western blotting and immunocytochemistry in salivary gland epithelial cells. We found that LPA receptors are functionally expressed and involved in activating the Hippo pathway mediated by YAP/TAZ through Lats/Mob1 and RhoA/ROCK. Upregulation of YAP/TAZ-dependent target genes, including CTGF, ANKRD1 and CYR61, has also been observed in LPA-treated cells. In addition, based on data suggesting that tumor necrosis factor (TNF)-α induces cell apoptosis, LPA upregulates TNF-induced caspase-3 and cleaved Poly(ADP-ribose)polymerase (PARP). However, small interfering RNA treatment to Yes-associated protein (YAP) or transcriptional co-activator with a PDZ-binding motif (TAZ) significantly decreased TNF-α- and LPA-induced apoptosis, suggesting that YAP and TAZ modulate the apoptotic pathway in salivary epithelial cells.

TRPV1 in Salivary Gland Epithelial Cells Is Not Involved in Salivary Secretion via Transcellular Pathway.

Transient receptor potential vanilloid subtype 1 (TRPV1) was originally found in sensory neurons. Recently, it has been reported that TRPV1 is expressed in salivary gland epithelial cells (SGEC). However, the physiological role of TRPV1 in salivary secretion remains to be elucidated. We found that TRPV1 is expressed in mouse and human submandibular glands (SMG) and HSG cells, originated from human submandibular gland ducts at both mRNA and protein levels. However, capsaicin (CAP), TRPV1 agonist, had little effect on intracellular free calcium concentration ([Ca(2+)]i) in these cells, although carbachol consistently increased [Ca(2+)]i. Exposure of cells to high temperature (>43℃) or acidic bath solution (pH5.4) did not increase [Ca(2+)]i, either. We further examined the role of TRPV1 in salivary secretion using TRPV1 knock-out mice. There was no significant difference in the pilocarpine (PILO)-induced salivary flow rate between wild-type and TRPV1 knock-out mice. Saliva flow rate also showed insignificant change in the mice treated with PILO plus CAP compared with that in mice treated with PILO alone. Taken together, our results suggest that although TRPV1 is expressed in SGEC, it appears not to play any direct roles in saliva secretion via transcellular pathway.

Chaperone stress 70 protein (STCH) binds and regulates two acid/base transporters NBCe1-B and NHE1.

Regulation of intracellular pH is critical for the maintenance of cell homeostasis in response to stress. We used yeast two-hybrid screening to identify novel interacting partners of the pH-regulating transporter NBCe1-B. We identified Hsp70-like stress 70 protein chaperone (STCH) as interacting with NBCe1-B at the N-terminal (amino acids 96-440) region. Co-injection of STCH and NBCe1-B cRNA into Xenopus oocytes significantly increased surface expression of NBCe1-B and enhanced bicarbonate conductance compared with NBCe1-B cRNA alone. STCH siRNA decreased the rate of Na(+)-dependent pHi recovery from NH4(+) pulse-induced acidification in an HSG (human submandibular gland ductal) cell line. We observed that in addition to NBCe1-B, Na(+)/H(+) exchanger (NHE)-dependent pHi recovery was also impaired by STCH siRNA and further confirmed the interaction of STCH with NHE1 but not plasma membrane Ca(2+) ATPase. Both NBCe1-B and NHE1 interactions were dependent on a specific 45-amino acid region of STCH. In conclusion, we identify a novel role of STCH in the regulation of pHi through site-specific interactions with NBCe1-B and NHE1 and subsequent modulation of membrane transporter expression. We propose STCH may play a role in pHi regulation at times of cellular stress by enhancing the recovery from intracellular acidification.

Effect of probenecid on the biliary excretion of belotecan.

The purpose of this study was to investigate the effect of probenecid, an inhibitor of the MRP2/ ABCC transporter, on the pharmacokinetics and transport of belotecan (7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin). The effect of probenecid on the pharmacokinetics of belotecan was studied in rats. When belotecan was injected as a bolus dose of 5 mg/kg after probenecid was infused at a rate of 42.8 mg/2 mL/h/kg, the cumulative biliary excretion amounts and biliary clearance (CL(b)) of belotecan decreased (28.29 +/- 2.83 versus 19.96 +/- 1.45% of dose and 161.01 +/- 26.95 versus 92.66 +/- 1.45 mL/min/kg), whereas the systemic pharmacokinetics did not change. This indicates that the MRP2 transporter is involved in the biliary excretion of belotecan. The involvement of MRP2 in the secretory transport was further characterized using Caco-2 cell monolayers expressing MRP2. The apparent permeability across Caco-2 cell monolayers from basolateral to apical was 2.3 times greater than that from the apical to the basolateral side at the 50 microM belotecan. In addition, probenecid significantly decreased the basolateral-to-apical transport of belotecan (52.9%). These results indicate that MRP2 is involved in the secretory transport of belotecan in biliary excretion.