Ferutinin induces osteoblast differentiation of DPSCs via induction of KLF2 and autophagy/mitophagy

Abstract

Osteoblast differentiation is critically reduced in various bone-related pathogenesis, including arthritis and osteoporosis. For future development of effective regenerative therapeutics, herein, we reveal the involved molecular mechanisms of a phytoestrogen, ferutinin-induced initiation of osteoblast differentiation from dental pulp-derived stem cell (DPSC). We demonstrate the significantly increased expression level of a transcription factor, Kruppel-like factor 2 (KLF2) along with autophagy-related molecules in DPSCs after induction with ferutinin. The loss-of-function and the gain-of-function approaches of KLF2 confirmed that the ferutinin-induced KLF2 modulated autophagic and OB differentiation-related molecules. Further, knockdown of the autophagic molecule (ATG7 or BECN1) from DPSC resulted not only in a decreased level of KLF2 but also in the reduced levels of OB differentiation-related molecules. Moreover, mitochondrial membrane potential-related molecules were increased and induction of mitophagy was observed in DPSCs after the addition of ferutinin. The reduction of mitochondrial as well as total ROS generations; and induction of intracellular Ca2+ production were also observed in ferutinin-treated DPSCs. To test the mitochondrial respiration in DPSCs, we found that the cells treated with ferutinin showed a reduced extracellular acidification rate (ECAR) than that of their vehicle-treated counterparts. Furthermore, mechanistically, chromatin immunoprecipitation (ChIP) analysis revealed that the addition of ferutinin in DPSCs not only induced the level of KLF2, but also induced the transcriptionally active epigenetic marks (H3K27Ac and H3K4me3) on the promoter region of the autophagic molecule ATG7. These results provide strong evidence that ferutinin stimulates OB differentiation via induction of KLF2-mediated autophagy/mitophagy.

Epigallocatechin-3-gallate inhibits osteoclastic differentiation by modulating mitophagy and mitochondrial functions

“…To assess the mitochondrial function during OC differentiation in the presence or absence of EGCG, we performed the cell MitoStress and glycolytic properties according to our previously described protocol by using an Agilent XFe 24 flux analyzer [ 50 , 51 ]. In brief, RAW264.7 (1 × 10 4 cells/well) were cultured on a seahorse cell culture plate for 6 days in OC differentiation media with or without EGCG.…”

Mitophagy—A New Target of Bone Disease

“…Accumulating evidence suggests that aberrant mitophagy plays a key role in stem cell differentiation and in bone metabolism disorders [ 12 , 118 ]. Recent studies have found that mitophagy is involved in the regulation of OP [ 119 , 120 ], which may be related to signals such as phosphoinositide 3-kinase (PI3K) [ 115 ] and Pink1 [ 121 ] (as shown in Figure 2 ).…”

Autophagy: An important target for natural products in the treatment of bone metabolic diseases

“…An increase in mitochondrial mass implies an accumulation of damaged mitochondria and requires a corresponding mitochondrial autophagic activity to maintain a good mitochondrial mass. The induction of mitochondrial autophagy eliminates damaged and unnecessary mitochondria from dental pulp stem cells and preserves healthy mitochondria, which promotes their differentiation into osteoblasts (Maity et al, 2022). During TNF-α-induced osteoblast senescence, the impaired bone anabolic activity can be ameliorated by restoring mitochondrial dysfunction and promoting mitochondrial autophagy (Lu et al, 2022).…”