Runx1 is a central regulator of osteogenesis for bone homeostasis by orchestrating BMP and WNT signaling pathways

Abstract

Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific (Runx1f/fTwist2-Cre) and osteoblast-specific (Runx1f/fCol1α1-Cre) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/β-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7, Alk3, and Atf4, and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1-deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1-deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2-/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebpα and Pparγ and largely increased adipogenesis. Co-culture of Runx1-deficient and wild-type cells demonstrated that Runx1 regulates osteoblast−adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/β-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis.

Long Non-coding RNA 332443 Inhibits Preadipocyte Differentiation by Targeting Runx1 and p38-MAPK and ERK1/2-MAPK Signaling Pathways

“…The top downregulated gene clusters were associated with fatty acid biosynthetic process , fatty acid metabolic process , lipid metabolic process and so on ( Figure 2A ), which were all closely associated with adipocyte differentiation. To find out the target genes of lnc-FR332443 in adipocyte differentiation, we analyzed the potential transcription factors of lnc-FR332443 and found that Runx1 which had been previously shown in the negative regulation of adipogenesis ( Tang et al, 2021 ) is among them ( Supplementary Material 2 ). Further we analyzed mRNA expression in the F72h mouse and control mouse mWAT using microarrays and measured 1,353 downregulated mRNAs and 494 upregulated mRNAs (fold change > 2, P < 0.05) ( Figure 2B ) among the total 33,420 mRNAs ( Supplementary Material 3 ).…”

“…Considering the essential functions of Runx1 in many biological programs related to development ( Ichikawa et al, 2004 ) and our previous research results that Runx1 could cell autonomously and cell non-autonomously regulate adipocyte differentiation ( Tang C.Y. et al, 2020 ; Tang et al, 2021 ), we hypothesized that Runx1 might be involved in the process of fat formation in the WAT of the AAV-Lnc-FR332443 mice.…”

“…Subsequently, we further explored the potential molecular mechanism underlying the function of Lnc-FR332443 in regulating adipogenesis. Our previous RNA-seq analysis results showed that MAPK signaling pathway was involved in Runx1 deficient osteoblasts within upregulated adipogenesis ( Tang et al, 2021 ). Another research also showed that the MAPK signaling pathway involving p38 and ERK1/2 plays crucial positive roles in adipogenesis ( Xu et al, 2014 ).…”

“…However, to date, the specific regulatory mechanism of lncRNAs in adipogenic differentiation was still unclear, and there were few lncRNAs that could be used as targets of antiobesity drugs. In this study, in order to identify lncRNAs/mRNAs selectively involved in adipocyte differentiation, we analyzed mWAT using microarray, and the data demonstrated that Runx1, which had been shown to negatively regulate adipogenesis in our previous research (Tang et al, 2021), was among the downregulated mRNAs in the F72h mice mWAT compared to the control mice. Further, we found Lnc-FR332443, which is the antisense LncRNA of Runx1 through UCSC and Ensembl database search, was among the top 40 downregulated lncRNAs in the microarray analysis.…”

“…In our previous research, we found that the specific deletion of Runx1 in osteoblasts and chondrocytes promotes adipogenic differentiation, suggesting that Runx1 can cell autonomously and cell non-autonomously regulate adipocyte differentiation (Tang C.Y. et al, 2020;Tang et al, 2021). However, the pathway upstream or downstream of Runx1 that causes inhibition of adipocyte differentiation remains unclear.…”