Cloning and characterization of a novel WD-40 repeat protein that dramatically accelerates osteoblastic differentiation.

The bone morphogenetic proteins (BMPs) play a pivotal role in endochondral bone formation. Using differential display polymerase chain reaction, we ha ve identified a novel gene, named BIG-3 (BMP-2-induced gene 3 kb), that is induced as a murine prechondroblastic cell line, MLB13MYC clone 17, acquire s osteoblastic features in response to BMP-2 treatment. The 3-kilobase mRNA encodes a 34-kDa protein containing seven WD-40 repeats. Northern and West ern analyses demonstrated that BIG-3 mRNA and protein were induced after 24 h of BMP-2 treatment. BIG-3 mRNA was expressed in conditionally immortaliz ed murine bone marrow stromal cells, osteoblasts, osteocytes, and growth pl ate chondrocytes, as well as in primary calvarial osteoblasts. Immunohistoc hemistry demonstrated that BIG-3 was expressed in the osteoblasts of calvar iae isolated from mouse embryos. To identify a role for BIG-3 in osteoblast differentiation, MC3T3-E1 cells were stably transfected with the full-leng th coding region of BIG-3 (MC3T3E1-BIG-3) cloned downstream of a cytomegalo virus promoter in pcDNA3.1. Pooled MC3T3E1-BlG-3 clones expressed alkaline phosphatase activity earlier and achieved a peak level of activity 10-fold higher than cells transfected with the empty vector (MC3T3E1-EV) at 14 days . Cyclic AMP production in response to parathyroid hormone was increased 10 - and 14-fold at 7 and 14 days, respectively, in MC3T3E1-BlG-3 clones, rela tive to MC3T3E1-EV clones. This increase in cAMP production was associated with an increase in PTH binding. Expression of BIG-3 increased mRNA levels encoding Cbfa1, type I collagen, and osteocalcin and accelerated formation of mineralized nodules. In conclusion, we have identified a novel WD-40 pro tein, induced by BMP-2 treatment, that dramatically accelerates the program of osteoblastic differentiation in stably transfected MC3T3E1 cells.