Influence of bone morphogenetic protein type IA receptor conditional knockout in lens on expression of bone morphogenetic protein 4 in lens

AIM: To investigate the influence of bone morphogenetic protein type IA receptor [BMPR-IA(ALK3)] conditional knockout in lens on expression of bone morphogenetic protein 4(BMP4) in lens during the development of the vertebrate eye.METHODS: Cre-positive mice were mated with Crenegative mice to generate 50% Cre-positive(conditional knockout, CKO) 4 embryos, 8 eyes and 50% Cre-negative offspring(wild type, WT) 4 embryos, 8 eyes. The embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm.Removal of paraffin wax and dehydrating for sections,and then the procedure of in situ hybridization was processed, BMP4 MK1784-m(BOSTER) was used, and observed the expression of BMP4 in the lens in experimental group and control group. We selected SPSS11.0 software for statistical analysis, P【0.05 showed that the difference was statistically significant.· RESULTS: Four embryos of each genotype were examined, totally we had 8 embryos, 16 eyes. We got the uniform outcomes in all the embryos. We found ALK3 was required during lens growing, but was not essential for the formation of lens. We observed that the expression of BMP4 in the lens was significantly reduced in all 8 ALK3 CKO lens, BMP4 expression was normal in all the 8 WT lens, P 【0.01. This phenomenon became increasingly visible in accordance with embryo development. The most apparent alteration was present at stage E15.5.CONCLUSION: ALK3 is essential for lens growth. The influence of ALK3 on the expression of BMP4 is present during the development of mice lens.

CLONING AND SEQUENCING OF MATURE FRAGMENT OF HUMAN BMP4 GENE

Objective To study the cloning and sequencing of mature fragment of human bone morphogenetic protein 4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT PCR method, the cDNA coding for the mature fragment of BMP 4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99%. Sequence analysis showed that there were two differences, one was at base 1154(201): G→C, which had no influence on the corresponding amino acids(Val). Another was at base1222(269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.

Activation of p38 mitogen-activated protein kinase contribute to BMP4-induced alkaline phosphatase expression in MC3T3-E1 preosteoblast

Bone morphogenetic proteins （BMPs） induce ectopic bone formation and promote osteoblast differentiation. It has been documented that Smad transcriptional factors function as primary mediators of BMPs activity. Receptor-regulated Smad （Smad1, 5, 8） could be phosphorylated by activated BMPR-I and form complex with Smad4. The Smad complex translocates to the nucleus and regulate target gene transcription. Recently, several reports suggested that Mitogen-Activated Protein Kinase （MAPK） signaling pathways could be initiated downstream of the BMP receptor complex. Alkaline phosphatase （ALP） is an early marker of osteoblast differentiation Both ALP activity and its mRNA expression level could be increased by BMP4 treatment. Previously, we demonstrated that mutation of ERK1/2 phosphorylation sites in Smad5 partially rescued Smad transcriptional activity. However, fibroblast growth factor2-suppressed ALP activity could not be rescued similarly by introduction of Smad5 mutant in MC3T3-E1. These results prompted us to further evaluate the effect of BMP4-stimulated Smad transcriptional activity on ALP expression in this study.