ABSTRACT
Klotho is an antiaging protein that has multiple functions. The purpose of this study is to investigate whether soluble klotho plays a role in cellular stress response pathways. We found that klotho deficiency (kl-/-) largely decreased HSF1 levels and impaired heat shock protein expression. Interestingly, recombinant soluble klotho-induced HSF1 and HSPs such as HSP90, HSP70, and HSP27 in kl-/- mouse embryonic fibroblasts (MEFs). Soluble Klotho treatment also induced cell proliferation and HSF1 promoter activity in MEF kl-/- cells in a concentration-dependent manner. Furthermore, using point mutagenesis, we identified regulatory/binding sites of transcription factors EGR1 regulated by soluble klotho in the HSF1 promoter. Taken together, our findings unravel the molecular basis of klotho and provide molecular evidence supporting a direct interaction between soluble klotho and HSF1-mediated stress response pathway.
ABSTRACT
Aging affects salivary gland function and alters saliva production and excretion. This study aimed to investigate whether ascorbic acid can be used to treat salivary gland dysfunction in an extensive aging mouse model of SAMP1/Klotho-/- mice. In our previous study, we found that ascorbic acid biosynthesis was disrupted in the salivary glands of SAMP1/Klotho (-/-) mice subjected to metabolomic profiling analysis. In SAMP1/Klotho -/- mice, daily supplementation with ascorbic acid (100 mg/kg for 18 days) significantly increased saliva secretion compared with the control. The expression of salivary gland functional markers (α-amylase, ZO-1, and Aqua5) is upregulated. Additionally, acetylcholine and/or beta-adrenergic receptors (M1AchR, M3AchR, and Adrb1) were increased by ascorbic acid in the salivary glands of aging mice, and treatment with ascorbic acid upregulated the expression of acetylcholine receptors through the DNA demethylation protein TET2. These results suggest that ascorbic acid could overcome the lack caused by dysfunction of ascorbic acid biosynthesis and induce the recovery of salivary gland function.
Subject(s)
Acetylcholine , Dioxygenases , Aging/physiology , Animals , Ascorbic Acid/metabolism , Ascorbic Acid/pharmacology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dioxygenases/metabolism , Klotho Proteins/metabolism , Membrane Proteins/metabolism , Mice , Nuclear Proteins/metabolism , Receptors, Cholinergic/metabolism , Salivary Glands/physiologyABSTRACT
Aging-related salivary dysfunction commonly induces the poor oral health, including decreased saliva flow and dental caries. Although the clinical significance of the salivary glands is well-known, the complex metabolic pathways contributing to the aging-dysfunction process are only beginning to be uncovered. Here, we provide a comprehensive overview of the metabolic changes in aging-mediated salivary gland dysfunction as a key aspect of oral physiology. Several metabolic neuropeptides or hormones are involved in causing or contributing to salivary gland dysfunction, including hyposalivation and age-related diseases. Thus, aging-related metabolism holds promise for early diagnosis, increased choice of therapy and the identification of new metabolic pathways that could potentially be targeted in salivary gland dysfunction.
Subject(s)
Aging/metabolism , Energy Metabolism , Salivary Glands/metabolism , Animals , Biomarkers , Disease Management , Disease Susceptibility , Hormones/metabolism , Humans , Metabolomics/methods , Saliva/metabolism , Salivary Gland Diseases/diagnosis , Salivary Gland Diseases/etiology , Salivary Gland Diseases/metabolism , Salivary Gland Diseases/therapy , Salivary Glands/pathologyABSTRACT
Salivary gland dysfunction induces salivary flow reduction and a dry mouth, and commonly involves oral dysfunction, tooth structure deterioration, and infection through reduced salivation. This study aimed to investigate the impact of aging on the salivary gland by a metabolomics approach in an extensive aging mouse model, SAMP1/Klotho -/- mice. We found that the salivary secretion of SAMP1/Klotho -/- mice was dramatically decreased compared with that of SAMP1/Klotho WT (+/+) mice. Metabolomics profiling analysis showed that the level of acetylcholine was significantly decreased in SAMP1/Klotho -/- mice, although the corresponding levels of acetylcholine precursors, acetyl-CoA and choline, increased. Interestingly, the mRNA and protein expression of choline acetyltransferase (ChAT), which is responsible for catalyzing acetylcholine synthesis, was significantly decreased in SAMP1/Klotho -/- mice. The overexpression of ChAT induced the expression of salivary gland functional markers (α-amylase, ZO-1, and Aqua5) in primary cultured salivary gland cells from SAMP1/Klotho +/+ and -/- mice. In an in vivo study, adeno-associated virus (AAV)-ChAT transduction significantly increased saliva secretion compared with the control in SAMP1/Klotho -/- mice. These results suggest that the dysfunction in acetylcholine biosynthesis induced by ChAT reduction may cause impaired salivary gland function.
Subject(s)
Acetylcholine/metabolism , Aging/metabolism , Choline O-Acetyltransferase/metabolism , Glucuronidase/metabolism , Membrane Proteins/metabolism , Nuclear Proteins/metabolism , Salivary Glands/metabolism , Acetyl Coenzyme A/metabolism , Acetylcholine/genetics , Aging/genetics , Animals , Cell Line , Choline/metabolism , Choline O-Acetyltransferase/genetics , Down-Regulation , Gene Expression Regulation/genetics , Gene Knockdown Techniques , Glucuronidase/genetics , Humans , Klotho Proteins , Membrane Proteins/genetics , Metabolomics , Mice , Mice, Knockout , Nuclear Proteins/genetics , Salivary Glands/enzymology , Up-Regulation , Zonula Occludens-1 Protein/genetics , Zonula Occludens-1 Protein/metabolism , alpha-Amylases/genetics , alpha-Amylases/metabolismABSTRACT
Klotho is a transmembrane protein known to regulate aging and lifespan. Soluble Klotho (sKL), a truncated form of Klotho, regulates various cell signaling pathways, including bone development. Here, we investigated the relationship between sKL and the zinc finger transcription factor early growth response protein 1 (EGR-1) on bone formation. We find that sKL induces the expression of EGR-1 mRNA and protein. Through mutational analysis, we identify the 130 bp region on the EGR-1 promoter that is responsive to sKL overexpression. Additionally, sKL induces the expression of markers of bone differentiation (BMP2, RUNX2, ALP, COL1A, and osteocalcin) in osteoblast MC3T3 cells. EGR-1 siRNA decreases the bone mineralization induced by sKL or ascorbic acid/glycerol 2-phosphate in MC3T3 cells. Our results suggest that sKL may regulate bone development through EGR-1 expression.
