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.

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

The dysfunction of salivary glands commonly induces dry mouth, infections, and dental caries caused by a lack of saliva. This study was performed to determine the genetic and functional changes in salivary glands using a klotho (-/-) mouse model. Here, we confirmed the attenuation of KLF4 expression in the salivary glands of klotho (-/-) mice. Soluble klotho overexpression induced KLF4 transcription and KLF4-mediated signaling pathways, including mTOR, AMPK, and SOD1/2. Silencing klotho via siRNA significantly down-regulated KLF4 expression. Additionally, we monitored the function of salivary glands and soluble klotho and/or KLF4 responses and demonstrated that soluble klotho increased the expression of KLF4 and markers of salivary gland function (α-amylase, ZO-1, and Aqua5) in primary cultured salivary gland cells from wild type and klotho (-/-) mice. In a 3D culture system, cell sphere aggregates were observed in soluble klotho- or KLF4-expressing cells and exhibited higher expression levels of salivary gland function-related proteins than those in nontransfected cells. These results suggest that activation of the klotho-mediated KLF4 signaling pathway contributes to potentiating the function of salivary glands.