title: Cloning and characterization of a novel WD-40 repeat protein that dramatically accelerates osteoblastic differentiation.
abstract: The bone morphogenetic proteins (BMPs) play a pivotal role in endochondral bone formation. Using differential display polymerase chain reaction, we have 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, acquires osteoblastic features in response to BMP-2 treatment. The 3-kilobase mRNA encodes a 34-kDa protein containing seven WD-40 repeats. Northern and Western analyses demonstrated that BIG-3 mRNA and protein were induced after 24 h of BMP-2 treatment. BIG-3 mRNA was expressed in conditionally immortalized murine bone marrow stromal cells, osteoblasts, osteocytes, and growth plate chondrocytes, as well as in primary calvarial osteoblasts. Immunohistochemistry demonstrated that BIG-3 was expressed in the osteoblasts of calvariae isolated from mouse embryos. To identify a role for BIG-3 in osteoblast differentiation, MC3T3-E1 cells were stably transfected with the full-length coding region of BIG-3 (MC3T3E1-BIG-3) cloned downstream of a cytomegalovirus promoter in pcDNA3.1. Pooled MC3T3E1-BIG-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-BIG-3 clones, relative 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 protein, induced by BMP-2 treatment, that dramatically accelerates the program of osteoblastic differentiation in stably transfected MC3T3E1 cells.
authors.affiliation: Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: F
authors.initials: F
authors.lastname: Gori
authors.forename: P
authors.initials: P
authors.lastname: Divieti
authors.forename: M B
authors.initials: MB
authors.lastname: Demay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000166:cytology*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D012333:RNA, Messenger; C443118:Wdr5 protein, mouse