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Reactive oxygen species derived from Nox4 mediate BMP2 gene transcription and osteoblast differentiation.

ABSTRACT: BMP-2 (bone morphogenetic protein-2) promotes differentiation of osteoblast precursor cells to mature osteoblasts that form healthy bone. In the present study, we demonstrate a novel mechanism of BMP-2-induced osteoblast differentiation. The antioxidant NAC (N-acetyl-L-cysteine) and the flavoprotein enzyme NAD(P)H oxidase inhibitor DPI (diphenyleneiodonium) prevented BMP-2-stimulated alkaline phosphatase expression and mineralized bone nodule formation in mouse 2T3 pre-osteoblasts. BMP-2 elicited a rapid generation of ROS (reactive oxygen species) concomitant with increased activation of NAD(P)H oxidase. NAC and DPI inhibited BMP-2-induced ROS production and NAD(P)H oxidase activity respectively. NAD(P)H oxidases display structurally similar catalytic subunits (Nox1-5) with differential expression in various cells. We demonstrate that 2T3 pre-osteoblasts predominantly express the Nox4 isotype of NAD(P)H oxidase. To extend this finding, we tested the functional effects of Nox4. Adenovirus-mediated expression of dominant-negative Nox4 inhibited BMP-2-induced alkaline phosphatase expression. BMP-2 promotes expression of BMP-2 for maintenance of the osteoblast phenotype. NAC and DPI significantly blocked BMP-2-stimulated expression of BMP2 mRNA and protein due to a decrease in BMP2 gene transcription. Dominant-negative Nox4 also mimicked this effect of NAC and DPI. Our results provide the first evidence for a new signalling pathway linking BMP-2-stimulated Nox4-derived physiological ROS to BMP-2 expression and osteoblast differentiation.

SUBMITTER: Mandal CC

PROVIDER: S-EPMC4539275 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Reactive oxygen species derived from Nox4 mediate BMP2 gene transcription and osteoblast differentiation.

Mandal Chandi C CC Ganapathy Suthakar S Gorin Yves Y Mahadev Kalyankar K Block Karen K Abboud Hanna E HE Harris Stephen E SE Ghosh-Choudhury Goutam G Ghosh-Choudhury Nandini N

The Biochemical journal 20110101 2

BMP-2 (bone morphogenetic protein-2) promotes differentiation of osteoblast precursor cells to mature osteoblasts that form healthy bone. In the present study, we demonstrate a novel mechanism of BMP-2-induced osteoblast differentiation. The antioxidant NAC (N-acetyl-L-cysteine) and the flavoprotein enzyme NAD(P)H oxidase inhibitor DPI (diphenyleneiodonium) prevented BMP-2-stimulated alkaline phosphatase expression and mineralized bone nodule formation in mouse 2T3 pre-osteoblasts. BMP-2 elicite ...[more]

PMID: 21029048

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Project description:Transcription factors (TFs) regulate the expression of target genes, inducing changes in cell morphology or activities needed for cell fate determination and differentiation. The BMP signaling pathway is widely regarded as one of the most important pathways in vertebrate skeletal biology, of which BMP2 is a potent inducer, governing the osteoblast differentiation of bone marrow stromal cells (BMSCs). However, the mechanism by which BMP2 initiates its downstream transcription factor cascade and determines the direction of differentiation remains largely unknown. In this study, we used RNA-seq, ATAC-seq, and animal models to characterize the BMP2-dependent gene regulatory network governing osteoblast lineage commitment. Sp7-Cre; Bmp2fx/fx mice (BMP2-cKO) were generated and exhibited decreased bone density and lower osteoblast number (n > 6). In vitro experiments showed that BMP2-cKO mouse bone marrow stromal cells (mBMSCs) had an impact on osteoblast differentiation and deficient cell proliferation. Osteogenic medium induced mBMSCs from BMP2-cKO mice and control were subjected to RNA-seq and ATAC-seq analysis to reveal differentially expressed TFs, along with their target open chromatin regions. Combined with H3K27Ac CUT&Tag during osteoblast differentiation, we identified 2338 BMP2-dependent osteoblast-specific active enhancers. Motif enrichment assay revealed that over 80% of these elements were directly targeted by RUNX2, DLX5, MEF2C, OASIS, and KLF4. We deactivated Klf4 in the Sp7 + lineage to validate the role of KLF4 in osteoblast differentiation of mBMSCs. Compared to the wild-type, Sp7-Cre; Klf4fx/+ mice (KLF4-Het) were smaller in size and had abnormal incisors resembling BMP2-cKO mice. Additionally, KLF4-Het mice had fewer osteoblasts and decreased osteogenic ability. RNA-seq and ATAC-seq revealed that KLF4 mainly "co-bound" with RUNX2 to regulate downstream genes. Given the significant overlap between KLF4- and BMP2-dependent NFRs and enriched motifs, our findings outline a comprehensive BMP2-dependent gene regulatory network specifically governing osteoblast differentiation of the Sp7 + lineage, in which Klf4 is a novel transcription factor.

Dataset Information

Reactive oxygen species derived from Nox4 mediate BMP2 gene transcription and osteoblast differentiation.

Publications

Reactive oxygen species derived from Nox4 mediate BMP2 gene transcription and osteoblast differentiation.

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