Important Role In Osteoblast Differentiation Research Articles

Overexpression of Dlx5 in chicken calvarial cells accelerates osteoblastic differentiation.

Our laboratory and others have shown that a homeodomain protein binding site plays an important role in transcription of the Collal gene in osteoblasts. This suggests that homeodomain proteins have an important role in osteoblast differentiation. We have investigated the role of Dlx5 in osteoblastic differentiation. In situ hybridization studies indicated that Dlx5 is expressed in chick calvarial osteoblasts (cCOB) in vivo. Northern blot analysis indicated that Dlx5 expression in cultured cCOBs is induced concurrently with osteoblastic markers. To study the effect of overexpression of Dlx5 on osteoblast differentiation, we infected primary osteoblast cultures from 15-day-old embryonal chicken calvaria with replication competent retroviral vectors [RCASBP(A)] expressing Dlx5 or control replication competent avian splice acceptor brianhightiter polymerase subtype A [RCASBP(A)]. Expression of Collal, osteopontin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) occurred sooner and at higher levels in cultures infected with RCASBP(A)DLX5 than in RCASBP(A)-infected cultures. Mineralization of Dlx5-expressing cultures was evident by days 12-14, and RCAS-infected control osteoblasts did not begin to mineralize until day 17. Dlx5 also stimulated osteoblastic differentiation of calvarial cells that do not normally undergo osteoblastic differentiation in vitro. Our results suggest that Dlx5 plays an important role in inducing calvarial osteoblast differentiation.

Discovery of osteoblast-associated genes using cDNA microarrays

Osteoblast maturation is a complex process and involves distinct genotypic changes that are accompanied by specific phenotypic alterations. To identify new bone-related genes in osteoblasts we utilized the high-density mouse GEM1 microarray gene chip from IncyteGenomics, Inc. (St. Louis, Mo). We examined the expression profiles of over 8700 genes during the proliferation (day 3) and the mineralization (day 34) phases of MC3T3-E1 development. More than 8600 genes provided measurable signals. Of these genes, 252 were found to be differentially expressed on days 3 and 34. A large number of these genes have never been previously recognized in the context of osteoblast development. Approximately, 60% of the genes with expressions that were dominant in proliferating osteoblasts consisted of growth-related genes such as TACC3 and Pr22. The expressions of TIS21/BTG2, and a novel gene EST350, were found to peak during the differentiation phase (day 12), suggesting that they may play important roles in osteoblast differentiation. The majority of the genes with expressions that were dominant during the mineralization phase consisted of signal transduction genes and extracellular matrix (ECM) proteins such as lumican and cystatin-C. It is significant that lumican expression could not be detected on day 3, which indicates that this gene may serve as an important marker of postmitotic osteoblasts. The establishment of the expression profiles of these and other genes with various phases of MC3T3-E1 osteoblast development will allow us to distinguish the molecular events at different phases of osteoblast biology.

Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts

Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N-linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3- to 4-fold overexpressed in op versus normal bone. RT-PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non-osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast-specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function.

Compactin and Simvastatin, but Not Pravastatin, Induce Bone Morphogenetic Protein-2 in Human Osteosarcoma Cells

Bone morphogenetic protein (BMP)-2, a member of the BMP family, plays an important role in osteoblast differentiation and bone formation. To discover small molecules that induce BMP-2, a luciferase reporter vector containing the 5′-flanking promoter region of the human BMP-2 gene was constructed and transfected into human osteosarcoma (HOS) cells. By the screening of an in-house natural product library with stably transfected HOS cells, a fungal metabolite, compactin, known as an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was isolated. The stimulation of the promoter activity by compactin seemed to be specific for BMP-2 gene in HOS cells, since it had little effect on BMP-4 or SV40 promoter activity and the stimulation was not observed in Chinese hamster ovary (CHO) cells. RT-PCR analysis and alkaline phosphatase assay revealed that compactin induced an increase in the expression of BMP-2 mRNA and protein. Like compactin, simvastatin also activated the BMP-2 promoter, whereas pravastatin did not. The statin-mediated activation of BMP-2 promoter was completely inhibited by the downstream metabolite of HMG-CoA reductase, mevalonate, indicating that the activation was a result of the inhibition of the enzyme. These results suggest that statins, if they are selectively targeted to bone, have beneficial effects in the treatment of osteoporosis or bone fracture.