RUNX2 co‐operates with EGR1 to regulate osteogenic differentiation through Htra1 enhancers

Abstract

Runt‐related transcription factor 2 (Runx2) has been shown to regulate osteoblast differentiation by directly or indirectly regulating numerous osteoblast‐related genes. However, our understanding of the transcriptional mechanisms of RUNX2 is mainly restricted to its transactivation, while the mechanism underlying its inhibitory effect during osteoblast differentiation remains largely unknown. Here, we incorporated the anti‐RUNX2 chromatin immunoprecipitation (ChIP) sequencing in MC3T3‐E1 cells and RNA‐sequencing of parietal bone from Runx2 heterozygous mutant mice, to identify the putative genes negatively regulated by RUNX2. We identified HtrA serine peptidase 1 (Htra1) as a target gene and found ten candidate Htra1 enhancers potentially regulated by RUNX2, among which seven were verified by dual‐luciferase assays. Furthermore, we investigated the motifs in the vicinity of RUNX2‐binding sites and identified early growth response 1 (EGR1) as a potential partner transcription factor (TF) potentially regulating Htra1 expression, which was subsequently confirmed by Re‐ChIP assays. RUNX2 and EGR1 co‐repressed Htra1 and increased the expression levels of other osteoblast marker genes, such as osterix, osteocalcin, and osteoprotegerin at the messenger RNA and protein level. Moreover, Alizarin red staining combined with alkaline phosphatase (ALP) staining showed decreased calcified nodules and ALP activity in the siRUNX2+siEGR1 group compared with siRUNX2 group. Our findings revealed the detailed mechanism of the inhibitory function of RUNX2 towards its downstream genes, along with its partner TFs, to promote osteoblast differentiation.

Analysis of ceRNA networks during mechanical tension‐induced osteogenic differentiation of periodontal ligament stem cells

“…Three hub genes ([EGR1], receptor interacting serine/threonine kinase 4 [RIPK4], and activating transcription factor 3 [ATF3] of differentially expressed mRNAs) were found to be in ceRNA networks. EGR1 is a transcriptional regulator controlling osteogenic differentiation by cooperating with RUNX2 and BMP2 [60,61]. RIPK4 has been shown to regulate NF-κB, PKC, Wnt, and MAPK signaling pathways in the development and maintenance of the epidermis [62]; however, there has been no study on the role of RIPK4 in stem cells and bone metabolism, which stimulated us to further investigate.…”

Pterostilbene attenuates the proliferation and differentiation of TNF‑α‑treated human periodontal ligament stem cells

“…The downregulation of HDACs reduces the removal of acetylated groups on the lysine residues of histones, destabilizing the binding between histones and DNA (43). This detachment promotes the combination of the DNA promoter regions with transcription factors (44), which increases the transcription of genes related to proliferation and differentiation (45,46). This may be the mechanism through which PTE can regulate proliferation and differentiation via HDACs.…”

EGR1 plays an important role in BMP9‐mediated osteoblast differentiation by promoting SMAD1/5 phosphorylation

“…EGR1 regulates the chondrocyte extracellular matrix through PPAR/RUNX2 signalling pathways during skeletal development [18] and promotes dental stem cell mineralization through DLX3 and BMP2 expression [22]. Through cooperation with RUNX2, EGR1 inhibits HtrA serine peptidase 1 (Htra1) transcription and is apparently important for osteoblast differentiation induced by ascorbic acid [37]. Moreover, Egr1‐deficient mice are smaller, have decreased bone volume, and a relatively limited degree of mineralization [18], but how EGR1 regulates skeletal development and osteoblast differentiation remain unclear.…”