Ferulic acid improves self‐renewal and differentiation of human tendon‐derived stem cells by upregulating early growth response 1 through hypoxia

Abstract

We aimed to investigate the potential beneficial effect of ferulic acid (FA) on stemness of human tendon-derived stem cells (hTSCs) in vitro and to elucidate the underlying molecular mechanism.The self-renewal ability of hTSCs was evaluated by colony formation and cell proliferation was determined by CCK-8 kit. Adipogenesis, osteogenesis, and chondrogenesis were determined by Oil Red O, Alizarin Red, and Alcian Blue stainings, respectively. Relative mRNA levels of PPARγ, Col2A1, Acan, Runx2, HIF1α, and EGR1 were measured with real-time PCR. Protein levels of HIF1α and EGR1 were detected by western blot. Direct binding of HIF1α with EGR1 promoter was analyzed by ChIP assay. Hypoxia-induced expression of EGR1 was interrogated by luciferase reporter assay. We demonstrated that FA treatment improved both self-renewal ability and multi-differentiation potential of hTSCs. FA induced hypoxia which in turn upregulated EGR1 expression via direct association with its hypoxia response element consensus sequence.Furthermore, we showed that both HIF1α and EGR1 were required for the enhancing effects of FA on hTSC self-renewal and differentiation. We hereby characterize the beneficial effect of FA on the stemness of hTSCs and highlight the critical role of HIF1α-EGR1 axis in this process. K E Y W O R D SEGR1, ferulic acid, human tendon-derived stem cells, hypoxia, multi-differentiation

EGR1 Transcription Factor is a Multifaceted Regulator of Matrix Production in Tendons and Other Connective Tissues

“…Moreover, EGR1 induces tenogenic differentiation in rabbit tendon stem cells [97]. EGR1 mediates the promoting effect of the anti-miR124 on collagen production in human tendon-derived stem cells [98] and the promoting effect of ferulic acid on self-renewal ability of human tendon-derived stem cells [99]. Consistent with the positive regulation of Col1a gene transcription by EGR1 observed both in vivo and in vitro, chromatin immunoprecipitation (ChIP) experiments show the recruitment of EGR1 to the tendon regulatory regions of the Col1a1 promoter in E18.5 limbs [73] and to Col1a1 and Col1a2 regulatory regions in adult Achilles tendons [76].…”

Insight into Hypoxia Stemness Control

“…Qiu et colleagues investigated the beneficial effect of FA on stemness of human tendonderived stem cells (hTSCs) and demonstrated that FA treatment promoted proliferation, self-renewal, and multi-differentiation potential (adipogenesis, chondrogenesis, and osteogenesis) in hTSCs cultures, in a dose-dependent manner. However, the authors suggest that, in in vitro experiments, high FA concentration might present slightly adverse effects on cells suggesting that FA beneficial action falls within an optimal range [94]. Then, the critical involvement of HIF-1α in mediating the FA-elicited pro-stemness effect on hTSCs was further disclosed by a specific knockdown assay, which readily abolished this beneficial influence of FA [94].…”

“…However, the authors suggest that, in in vitro experiments, high FA concentration might present slightly adverse effects on cells suggesting that FA beneficial action falls within an optimal range [94]. Then, the critical involvement of HIF-1α in mediating the FA-elicited pro-stemness effect on hTSCs was further disclosed by a specific knockdown assay, which readily abolished this beneficial influence of FA [94].…”