To define the regulatory role of Kruppel-like factor 2 ( KLF2 ) during osteoblast (OB) differentiation of dental pulp-derived stem cell (DPSC)s, herein, we show that the levels of KLF2 and autophagy-related molecules were significantly increased in differentiated cells. Gain-of-function and loss-of-function approaches of KLF2 confirmed that KLF2 modulated autophagic and OB differentiation-related molecules. In addition, knockdown of the autophagic molecule ( ATG7 or BECN1 ) in DPSCs resulted in reduced levels of KLF2 and OB differentiation-related molecules. Conversely, the induction of autophagy increased levels of KLF2 and OB differentiation-related molecules. Moreover, OB differentiation induced mitophagy and mitochondrial membrane potential-related molecules. In addition, OB differentiation reduced the generation of total and mitochondrial ROS productions and induced intracellular Ca 2+ production. Measurements of glycolysis and oxidative phosphorylation simultaneously in live cells revealed that OB differentiation decreased the oxygen consumption rate, which is an indicator of mitochondrial respiration and reduced the level of ATP production. Furthermore, flux analysis also revealed that OB differentiation increased the extracellular acidification rate (ECAR) in the non-glycolytic acidification, and the glycolytic capacity conditions, increasing the lactate production and reducing the metabolic activity of the cells. Thus, a metabolic shift from mitochondrial respiration to the glycolytic pathway was observed during OB differentiation. Finally, chromatin immunoprecipitation (ChIP) analysis confirmed that the KLF2 and active epigenetic marks (H3K27Ac and H3K4me3) were upregulated in the promoter region of ATG7 during OB differentiation. These results provide evidence that the mitophagy process is important during OB differentiation, and KLF2 critically regulates it.

Osteoporosis is a silent systemic disease that causes bone deterioration, and affects over 10 million people in the US alone. This study was undertaken to develop a potential stem cell therapy for osteoporosis. We have isolated and expanded human dental pulp-derived stem cells (DPSCs), characterized them, and confirmed their multipotential differentiation abilities. Stem cells often remain quiescent and require activation to differentiate and function. Herein, we show that ferutinin activates DPSCs by modulating the Wnt/β-catenin signaling pathway and key osteoblastsecreted proteins osteocalcin and collagen 1A1 both mRNA and protein levels. To confirm that ferutinin modulates the Wnt pathway, we inhibited glycogen synthase kinase 3 (GSK3) and found that protein expression patterns were similar to those found in ferutinin-treated DPSCs. To evaluate the role of ferutinin in epigenetic regulation of canonical Wnt signaling, the pathway molecules Wnt3a and Dvl3 were analyzed using chromatin immunoprecipitation (ChIP)quantitative PCR approaches. We confirmed that active marks of both H3K9 acetylation and H3K4 trimethylation were significantly enhanced in the promoter sites of the WNT3A and DVL3 genes

Osteoclasts (OCs) differentiate from the monocyte/macrophage lineage, critically regulate bone resorption and remodelling in both homeostasis and pathology. Various immune and non‐immune cells help initiating activation of myeloid cells for differentiation, whereas hyper‐activation leads to pathogenesis, and mechanisms are yet to be completely understood. Herein, we show the efficacy of dental pulp–derived stem cells (DPSCs) in limiting RAW 264.7 cell differentiation and underlying molecular mechanism, which has the potential for future therapeutic application in bone‐related disorders. We found that DPSCs inhibit induced OC differentiation of RAW 264.7 cells when co‐cultured in a contact‐free system. DPSCs reduced expression of key OC markers, such as NFATc1, cathepsin K, TRAP, RANK and MMP‐9 assessed by quantitative RT‐PCR, Western blotting and immunofluorescence detection methods. Furthermore, quantitative RT‐PCR analysis revealed that DPSCs mediated M2 polarization of RAW 264.7 cells. To define molecular mechanisms, we found that osteoprotegerin (OPG), an OC inhibitory factor, was up‐regulated in RAW 264.7 cells in the presence of DPSCs. Moreover, DPSCs also constitutively secrete OPG that contributed in limiting OC differentiation. Finally, the addition of recombinant OPG inhibited OC differentiation in a dose‐dependent manner by reducing the expression of OC differentiation markers, NFATc1, cathepsin K, TRAP, RANK and MMP9 in RAW 264.7 cells. RNAKL and M‐CSF phosphorylate AKT and activate PI3K‐AKT signalling pathway during osteoclast differentiation. We further confirmed that OPG‐mediated inhibition of the downstream activation of PI3K‐AKT signalling pathway was similar to the DPSC co‐culture–mediated inhibition of OC differentiation. This study provides novel evidence of DPSC‐mediated inhibition of osteoclastogenesis mechanisms.