Involvement of PU.1 in NFATc1 promoter function in osteoclast development
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Background: The transcription factors NFATc1 and PU.1 play important roles in osteoclast development. NFATc1 and PU.1 transactivate osteoclast-speciﬁc gene expression and a deﬁciency in NFATc1 or PU.1 genes causes osteopetrosis due to an insufﬁcient development of osteoclasts. However, the existence of cross-regulation between NFATc1 and PU.1 is largely unknown. In the present study, the role of PU.1 in NFATc1 expression was investigated.Methods: Osteoclasts were generated from mouse bone marrow cells. PU.1 knockdown was performed with siRNA introduction. The mRNA levels in siRNA-introduced cells were determined by quantitative RT-PCR. The involvement of PU.1 in the NFATc1 promoter was analyzed by using a chromatin immu- noprecipitation (ChIP) assay and a reporter assay. Retrovirus vector was used for enforced expression of PU.1.Results: Introduction of PU.1 siRNA into bone marrow-derived osteoclasts resulted in a decrease in NFATc1 mRNA level. A ChIP assay showed that PU.1 bound to the NFATc1 promoter in osteoclasts. NFATc1 promoter activity was reduced in PU.1 knockdown cells as assessed by a reporter assay. PU.1 siRNA introduction also downregulated the expression of osteoclast-speciﬁc genes and tartrate resistant acid phosphatase (TRAP) activity. Enforced expression of PU.1 using a retrovirus vector increased NFATc1 expression and TRAP activity. When NFATc1 expression was knocked down by using siRNA, the induction of osteoclast-speciﬁc genes and TRAP-positive cells was suppressed without affecting the expression level of PU.1.Conclusions: These results indicate that PU.1 is involved in osteoclast development by transactivating NFATc1 expression via direct binding to the NFATc1 promoter.
- Allergology International
Allergology International 64(3), 241-247, 2015
Japanese Society of Allergology