Abstract
The bone morphogenic proteins (BMPs) play a key role in skeletal development and patterning. Using the technique of differential display polymerase chain reaction (ddPCR), we have identified a novel gene whose expression is increased during BMP-2-induced differentiation of the prechondroblastic cell line, MLB13MYC clone 17, to an osteoblastic phenotype. The 6.5-kilobase mRNA recognized by this ddPCR product is increased 10-fold by BMP-2 treatment of the MLB13MYC clone 17 cells. The mRNA recognized by this ddPCR product is also increased as MC3T3-E1 cells recapitulate the program of osteoblast differentiation during prolonged culture. The full-length transcript corresponding to this ddPCR product was cloned from a MLB13MYC clone 17 cell cDNA library. Analysis of the deduced amino acid sequence demonstrated that this gene encodes a novel 126-kDa putative serine/threonine protein kinase containing a nuclear localization signal. The kinase domain, expressed in Escherichia coli, is capable of autophosphorylation as well as phosphorylation of myelin basic protein. The gene was, therefore, named BIKe (BMP-2-Inducible Kinase). The BIKe nuclear localization signal is able to direct green fluorescent protein to the nucleus in transfected COS-7 cells. When stably expressed in MC3T3-E1 cells, BIKe significantly decreases alkaline phosphatase activity and osteocalcin mRNA levels and retards mineral deposition relative to vector control. This novel kinase, therefore, is likely to play an important regulatory role in attenuating the program of osteoblast differentiation.
Highlights
Numerous investigations have been directed at elucidating factors that regulate osteoblast differentiation [1]
Using the technique of differential display polymerase chain reaction, we have identified a novel gene whose expression is increased during bone morphogenic proteins (BMPs)-2-induced differentiation of the prechondroblastic cell line, MLB13MYC clone 17, to an osteoblastic phenotype
RNA isolated from cells, treated with rhBMP-2 for 72 h, or left untreated was used for differential display PCR
Summary
The induction of Cbfa by BMPs has been shown to be a pivotal event in endochondral bone formation, the identification of genes induced prior to and after Cbfa will be essential to elucidate other factors, the interaction of which is required for normal skeletal differentiation. These factors are likely to play an important role in skeletal homeostasis postnatally as well. We have identified a novel protein kinase containing a nuclear localization signal and a glutamine-rich region characteristic of many transcription factors When stably expressed, this kinase markedly attenuates the program of osteoblast differentiation recapitulated during prolonged culture of MC3T3-E1 cells
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