Leucine reduces the proliferation of MC3T3-E1 cells through DNA damage and cell senescence.

Leucine (Leu) is an essential branched-chain amino acid, present in dairy products, which has been investigated for its important role in cell signaling. The effects of Leu on several kinds of cells have been studied, altough little is known on its action upon bone cells and cell proliferation. Thus, the aim of this study is to investigate the effects of Leu supplementation on the proliferation of pre-osteoblasts from MC3T3-E1 lineage. MC3T3-E1 cells were kept in Alpha medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimitotic. Cells were treated during 48h by adding 50µM of Leu, which corresponds to a 12.5% increase of the amino acid in the culture medium. The evaluation of viability and proliferation of cultured cells was performed using Trypan Blue dye. In order to identify the mechanisms related to the decreased cellular proliferation, assays were performed to assess cytotoxicity, apotosis, oxidative stress, inflammation, autophagy, senescence and DNA damage. Results showed that Leu supplementation decreased cell proliferation by 40% through mechanisms not related to cell necrosis, apoptosis, oxidative stress, autophagy or inhibition of the mTORC1 pathway. On the other hand, Leu supplementation caused DNA damage. In conclusion, Leu caused a negative impact on bone cell proliferation by inducing cell senescence through DNA damage.

Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration.

BACKGROUND: Osteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC) recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca2+-containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO4) on MSC migration. In addition, to evaluate the influence of CaSO4 on MSC differentiation and the potential molecular mechanisms involved. METHODS: A circular calvarial bone defect (5 mm diameter) was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO4 concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO4 effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO4 treatment was also evaluated by qPCR. RESULTS: CaSO4 increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO4 significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO4-containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO4 and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO4 effects on MSC migration. CONCLUSIONS: Specific CaSO4 concentrations induce bone regeneration of calvarial defects in part by acting on the host's undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO4 regulates BMP-2-induced MSC migration by differentially activating the PI3K/AKT pathway. Altogether, these results suggest that CaSO4 scaffolds could have potential applications for bone regeneration.

The transcriptional activation of the cyclooxygenase-2 gene in zymosan-activated macrophages is dependent on NF-kappa B, C/EBP, AP-1, and CRE sites.

Zymosan is a yeast cell wall particle that activates several cell lines, especially macrophages, resulting in the stimulated secretion of inflammatory products including tumor necrosis factor alpha (TNF-α) and arachidonic acid. Cyclooxygenase-2 (COX-2) is an immediate early gene induced by several stimuli in macrophages. The following research aimed to investigate the regions of COX-2 promoter gene that mediate the inductive effects of zymosan. Transient transfections with a series of COX-2 promoter-mutation constructs were performed to further elucidate the effects of zymosan on COX-2 transcription. Exposure to zymosan (50 µg/mL for 24 h) markedly enhanced the relative luciferase activity in RAW 264.7 macrophages (mouse leukemic monocyte macrophage cell line) transfected with COX-2 luciferase promoter constructs. Deletion on the CCAAT-enhancer binding protein (C/EBP) and nuclear factor kappa B (NF-kappa B) binding site resulted in an important decrease in reporter gene activity and a deletion of NF-kappa B and C/EBP with mutation of the cyclic adenosine monophosphate response element (CRE) and/or activator protein-1 totally abolished the reporter gene activity induced by zymosan. These findings provide further insight into the signal transduction pathways involved in COX-2 gene activated by zymosan in macrophages.