Salivary gland development : its mediation by a subtilisinlike proprotein convertase, PACE4

The submandibular gland (SMG) develops under the epithelial-mesenchymal interaction. Its process is regulated by various growth/differentiation factors, which are synthesized as inactive precursors and activated via the limited proteolysis at their multi basic amino acid site(s) such as Arg-X-Lys/Arg-Arg. Although many of these processing steps are elucidated to be catalyzed by subtilisin-like proprotein convertases (SPCs), little is known about the role of SPCs in the SMG development. Here, we focused upon the physiological role of PACE4 (SPC4), a member of SPC family, in the SMG development. In the organ culture system of rat embryonic SMG (E15), Dec-RVKR-CMK, a potent inhibitor for SPCs, inhibited the salivary branching and the expression of an exocrine gland type water channel, AQP5. However, other peptidyl-CMKs and inhibitors for trypsin-like serine proteases including leupeptin did not affect the salivary branching and AQP5 expression. Dec-RVKR-CMK also suppressed the expression of PACE4, but not furin, another member of the family. The specific antibody for the catalytic domain of PACE4 suppressed the salivary branching and AQP5 expression similarly. These inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2 whose precursor is a candidate for the physiological substrates of PACE4. Further, the transcriptional silencing of PACE4 by its specific siRNAs caused the suppression of both the salivary branching and AQP5 expression in the present organ culture system. These observations strongly support the idea that PACE4 mediates the SMG development

Down-regulation of submandibular gland AQP5 following parasympathetic denervation in rats

Following chorda tympani denervation (CTD, parasympathetomy), the protein levels of aquaporin5 (AQP5) as well as AQP1 and Na+K+ATPase α-subunit in the rat submandibular gland (SMG) were found to be decreased significantly. However, the level of another membrane protein, dipeptidyl peptidase IV was not affected by CTD, suggesting a selective reduction of AQP5, AQP1, and Na+K+ATPase1α-subunit proteins by CTD. However, the AQP5 mRNA level was scarcely affected by CTD, which suggested that transcription process of AQP5 was unaffected by this operation. AQP5 protein was shown to be degraded in vitro by the extract of the SMG obtained from normal rat ; inhibitor experiments in vitro suggested cathepsin B was a responsible enzyme. Co-localization of AQP5 and LAMP-2, a lysosomal marker, implicated AQP5 is degraded in lysosomes. A significant increase in the protein levels of LC3-II, an autophagy marker, at day 1 after CTD, and co-localization of the LC3 protein and AQP5, suggested that CTD activated autophagy of SMG, leading to AQP5 degradation

Trafficking of GFP-AQP5 chimeric proteins conferred with unphosphorylated amino acids at their PKA-target motif (152SRRTS) in MDCK-II cells

Three constructs having mutated PKA-target motif at 152SRRTS of AQP5, an exocrine type water channel, were prepared and fused to C-terminus of green fluorescence protein cDNA to examine the effects of blocking of phosphorylation at 152SRRTS (a consensus PKA-target motif of AQP5) on translocation or trafficking of the chimeric proteins expressed in the Madin-Darby canine kidney-II (MDCK-II) cells. H-89 treatment increased translocation of wild-type GFP-AQP5 to the apical membrane. All 3 mutant molecules translocated 1.5 to 2 times more than the control wild-type GFP-AQP5. Colchicine but not cytochalasin B inhibited the translocation of wild-type GFP-AQP5. Present results suggest dephosphorylation of this consensus sequence increase GFP-AQP5 translocation, and that microtubules but not microfilaments are involved in this event

Induction of calprotectin mRNAs by lipopolysaccharide in the salivary gland of mice

Calprotectin is a major cytosolic calcium-binding protein of leukocytes which belongs to the S100 protein family. S100A8 and S100A9, major types of calprotectin are heterodimeric complexes being composed of light- and heavy-chain subunits. The calprotectin levels in the plasma, feces, synovial fluid, gingival crevicular fluid, dental calculus and saliva change when the host animal suffers from several inflammatory diseases. Members of Toll-like receptor (TLR) family are pattern-recognition receptors for lipopolysaccharide (LPS) and other pathogens. Here we examined if the biological role of TLR receptor is reflected to the calprotectin expression in the salivary gland. Time course study by using real-time RT-PCR detected higher levels of S100A8 and S100A9 mRNA at 1.5-3 h after injection of LPS in both the submandibular gland (SMG) and parotid gland (PG) of C3H/HeN mice but not in the same tissues of C3H/HeJ, a TLR-4 mutant strain, indicating that this induction is mediated via the TLR-4. These results indicate that, an inflammatory marker, calprotectin, is expressed in the mouse salivary gland and that LPS stimulated its synthesis. Calprotectin (S100A8/A9) showed minimum expression in all cellular segments in the SMG except excretory duct cells, which showed strong signal at the cytoplasm. LPS induced their expressions in the granular convoluted tubular cells and striated duct cells. In the PG, these proteins were expressed very weakly in both duct and acinar cells with a little stronger staining for the former cells. LPS injection induced calprotectin (S100A8/A9) in both duct and acinar cells especially in the former cells

Effects of natural point mutation of rat aquaporin 5 expressed in vitro on its capacity of water permeability and membrane trafficking

In the colony of Sprague-Dawley (SD) strain, we found that there were rats expressing a mutant AQP5, which has a point mutation at nt 308 (G308A), leading to a replacement of 103Gly with 103Asp in the 3rd transmembrane domain. The mutant molecule scarcely expressed in the acinar cells, probably because of ineffective trafficking. The mutant molecule, however, showed normal water permeability when assessed by the oocyte system

THE INCIDENCE OF PARTIAL EDENTULISM AND DENTURE STATUS AMONG MONGOLIANS

<p><strong>Abstract</strong></p><p><strong>Introduction: </strong>Since 2013, the prevalence of tooth loss among adults in rural areas has not been studied in Mongolia. Therefore, our goal of the survey was to determine the level of tooth loss and denture needs among the population of Mongolia's. <strong>Materials and Methods: </strong>According to the geographical region of Mongolia, 1638 subjects over 18 years old were examined from target provinces and their partial edentulous status and denture status were determined. <strong>Results: </strong>The prevalence of partial edentulous level in total subjects was 1478 (90.2%), and partial edentulous level was high (93.5%) in over < 45 year-olds. The incidence of Kennedy's class in both arches was 880 (36.0%), Class III was 853 (34.8%), and Class II was 675 (27.6%), respectively. Class IV was lower at 40 (1.6%) in both dental arches (P< 0.01). Evaluating denture status in people: 435 (67.8%) have acrylic dentures and 171 (26.6%) have flexible dentures. The number of subjects who needed prosthetic treatment was 861 (57.3%).<strong> Conclusion: </strong>The level and pattern of partial edentulism among adults Mongolia were high, and the level and pattern of tooth loss were significantly associated with age groups. Prosthetic needs among rural people are high, and all these findings are also being considered in oral health promotion strategies designed to reduce tooth loss in Mongolia's adults. </p&gt

Potential Down-Regulation of Salivary Gland AQP5 by LPS via Cross-Coupling of NF-κB and p-c-Jun/c-Fos

The mRNA and protein levels of aquaporin (AQP)5 in the parotid gland were found to be potentially decreased by lipopolysaccharide (LPS) in vivo in C3H/HeN mice, but only weakly in C3H/HeJ, a TLR4 mutant mouse strain. In the LPS-injected mice, pilocarpine-stimulated saliva production was reduced by more than 50%. In a tissue culture system, the LPS-induced decrease in the AQP5 mRNA level was blocked completely by pyrrolidine dithiocarbamate, MG132, tyrphostin AG126, SP600125, and partially by SB203580, which are inhibitors for IκB kinase, 26S proteasome, ERK1/2, JNK, and p38 MAPK, respectively. In contrast, the expression of AQP1 mRNA was down-regulated by LPS and such down-regulation was blocked only by SP600125. The transcription factors NF-κB (p65 subunit), p-c-Jun, and c-Fos were increased by LPS given in vivo, whereas the protein-binding activities of the parotid gland extract toward the sequences for NF-κB but not AP-1–responsive elements present at the promoter region of the AQP5 gene were increased by LPS injection. Co-immunoprecipitation by using antibody columns suggested the physical association of the three transcription factors. These results suggest that LPS-induced potential down-regulation of expression of AQP5 mRNA in the parotid gland is mediated via a complex(es) of these two classes of transcription factors, NF-κB and p-c-Jun/c-Fos