Transient receptor potential melastatin 6 (TRPM6) channel is involved in the reabsorption of magnesium in the kidney. We recently found that TRPM6 expression is up-regulated by EGF, but the regulatory mechanism has not been clear. TRPM6 mRNA was endogenously expressed in HEK293 cells. TRPM6 mRNA expression was increased by EGF, which was inhibited by U0126, an MEK inhibitor. Promoter activity of human TRPM6 was observed in the TRPM6 5'-flanking region from -1,214 to -718. This promoter activity was enhanced by EGF and inhibited by U0126. Three putative AP-1 binding sites were identified within the region of -1,214/-718. The mutation of the putative AP-1 binding site (-741/-736) completely inhibited the EGF-induced promoter activity. EGF increased p-ERK1/2, c-Fos, c-Jun, and p-c-Jun levels, which were inhibited by U0126. The introduction of c-Fos or c-Jun siRNA inhibited the EGF-induced promoter activity. A chromatin immunoprecipitation assay revealed that c-Fos and c-Jun bind to the AP-1 binding site within the region of -1,214/-718. These results suggest that EGF up-regulates TRPM6 mRNA expression mediate via the activation of ERK/AP-1-dependent pathway.

Background: Disease-associated claudin-16 mutants cause mislocalization and decrease reabsorption of Mg 2ϩ in the kidney. Results: Knockdown of syntaxin 8 in kidney cells decreased the tight junctional localization of claudin-16 in parallel with a decrease in Mg 2ϩ permeability. Conclusion: Syntaxin 8 controls the localization of claudin-16. Significance: Our findings provide a new insight into the trafficking mechanism of tight junctional proteins.

The dermis is a tissue rich in collagenous and elastic fibres and supports the epidermal tissue. The network structure of extracellular matrix in the dermis is maintained by molecules such as collagen and elastin produced by fibroblasts. When DNA in fibroblasts is damaged by ageing, UV ray, oxidative stress or other reasons, the production of such fibre molecules is decreased, resulting in dermal dysfunction. DNA damage is able to be sensed and repaired by ATM and ATR kinases and downstream DNA damage response signalling pathway. Fibroblasts that have irreparable damage, however, become senescent or are subject to apoptosis. [1-3] Cell senescence is well known to be associated with reduced function of the cell itself, and recently, it has also been found that the accumulation of senescent cells damages the surrounding tissue. Age-related accumulation of senescent cells has been observed in the dermis, [4,5] and impairment of the dermis by senescent cells needs to be recovered to maintain dermal homeostasis.

Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), is used in treatments for transplantation and cancer. Rapamycin causes hypomagnesemia, although precisely how has not been examined. Here, we investigated the effect of rapamycin on the expression of transient receptor potential melastatin 6 (TRPM6), a Mg2+ channel. Rapamycin and LY-294002, an inhibitor of phosphatidilinositol-3 kinase (PI3K) located upstream of mTOR, inhibited epidermal growth factor (EGF)-induced expression of the TRPM6 protein without affecting TRPM7 expression in rat renal NRK-52E epithelial cells. Both rapamycin and LY-294002 decreased EGF-induced Mg2+ influx. U0126, a MEK inhibitor, inhibited EGF-induced increases in c-Fos, p-ERK, and TRPM6 levels. In contrast, neither rapamycin nor LY-294002 inhibited EGF-induced increases in p-ERK and c-Fos levels. EGF increased p-Akt level, an effect inhibited by LY-294002 and 1L-6-hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (Akt inhibitor). Akt inhibitor decreased TRPM6 level similar to rapamycin and LY-294002. These results suggest that a PI3K/Akt/mTOR pathway is involved in the regulation of TRPM6 expression. Rapamycin inhibited the EGF-induced increase in TRPM6 mRNA but did not inhibit human TRPM6 promoter activity. In the presence of actinomycin D, a transcriptional inhibitor, rapamycin accelerated the decrease in TRPM6 mRNA. Rapamycin decreased the expression and activity of a luciferase linked with the 3'-untranslated region of human TRPM6 mRNA. These results suggest that TRPM6 expression is up-regulated by a PI3K/Akt/mTOR pathway and rapamycin reduces TRPM6 mRNA stability, resulting in a decrease in the reabsorption of Mg2+.

Periodontitis is an inflammatory disease associated with severe alveolar bone loss and is dominantly induced by lipopolysaccharide from Gram-negative bacteria; however, the role of Gram-positive bacteria in periodontal bone resorption remains unclear. In this study, we examined the effects of lipoteichoic acid (LTA), a major cell-wall factor of Gram-positive bacteria, on the progression of inflammatory alveolar bone loss in a model of periodontitis. In coculture of mouse primary osteoblasts and bone marrow cells, LTA induced osteoclast differentiation in a dose-dependent manner. LTA enhanced the production of PGE2 accompanying the upregulation of the mRNA expression of mPGES-1, COX-2 and RANKL in osteoblasts. The addition of indomethacin effectively blocked the LTA-induced osteoclast differentiation by suppressing the production of PGE2. Using ex vivo organ cultures of mouse alveolar bone, we found that LTA induced alveolar bone resorption and that this was suppressed by indomethacin. In an experimental model of periodontitis, LTA was locally injected into the mouse lower gingiva, and we clearly detected alveolar bone destruction using 3D-μCT. We herein demonstrate a new concept indicating that Gram-positive bacteria in addition to Gram-negative bacteria are associated with the progression of periodontal bone loss.