Chaitali Banerjee

Runt homology domain proteins in osteoblast differentiation: AML3/CBFA1 is a major component of a bone‐specific complex

The AML/CBFA family of runt homology domain (rhd) transcription factors regulates expression of mammalian genes of the hematopoietic lineage. AML1, AML2, and AML3 are the three AML genes identified to date which influence myeloid cell growth and differentiation. Recently, AML‐related proteins were identified in an osteoblast‐specific promoter binding complex that functionally modulates bone‐restricted transcription of the osteocalcin gene. In the present study we demonstrate that in primary rat osteoblasts AML‐3 is the AML family member present in the osteoblast‐specific complex. Antibody specific for AML‐3 completely supershifts this complex, in contrast to antibodies with specificity for AML‐1 or AML‐2. AML‐3 is present as a single 5.4 kb transcript in bone tissues. To establish the functional involvement of AML factors in osteoblast differentiation, we pursued antisense strategies to alter expression of rhd genes. Treatment of osteoblast cultures with rhd antisense oligonucleotides significantly decreased three parameters which are linked to differentiation of normal diploid osteoblasts: the representation of alkaline phosphatase–positive cells, osteocalcin production, and the formation of mineralized nodules. Our findings indicate that AML‐3 is a key transcription factor in bone cells and that the activity of rhd proteins is required for completion of osteoblast differentiation. J. Cell. Biochem. 66:1–8, 1997.

Expression and regulation of Runx2/Cbfa1 and osteoblast phenotypic markers during the growth and differentiation of human osteoblasts

The runt family transcription factor (AML‐3/PEBP2αA1/Cbfa1/RUNX2) plays a crucial role in formation of the mineralized skeleton during embryogenesis and regulates maturation of the osteoblast phenotype. Because steroid hormones and growth factors significantly influence growth and differentiation properties of osteoblasts, we addressed Cbfa1 as a target gene for regulation by dexamethasone (Dex), 1,25(OH)D3 (vitamin D3), 17β‐estradiol, and transforming growth factor‐β1 (TGF‐β1). The representation of functional protein levels by Western blot analyses and gel mobility shift assays was examined during the growth and mineralization of several conditionally immortalized human osteoblast cell lines HOB 04‐T8, 03‐CE6, and 03‐CE10, each representing different stages of maturation. In situ immunofluorescence demonstrates Cbfa1 is associated with nuclear matrix in punctate domains, some of which are transcriptionally active, colocalizing with phosphorylated RNA polymerase II. Although each of the cell lines exhibited different responses to the steroid hormones and to TGF‐β1, all cell lines showed a similar increase in Cbfa1 protein and DNA binding activity induced only by Dex. On the other hand, Cbfa1 mRNA levels were not altered by Dex treatment. This regulation of Cbfa1 by steroid hormones in human osteoblasts contrasts to modifications in Cbfa1 expression in primary rat calvarial osteoblasts and the mouse MC3T3‐E1 osteoblast cell line. Thus, these results reveal multiple levels of regulation of Cbfa1 expression and activity in osteoblasts. Moreover, the data suggest that in committed human osteoblasts, constitutive expression of Cbfa1 may be required to sustain the osteoblast phenotype. J. Cell. Biochem. 80:424–440, 2001.

Bone tissue-specific transcription of the osteocalcin gene: Role of an activator osteoblast-specific complex and suppressor Hox proteins that bind the OC box

Bone‐specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue‐specific expression. By gel mobility shift assays with wild‐type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence‐specific interactions with the primary transcriptional element, the OC box (nt −99 to −76). This OC box binding protein (OCBP) is present only in osteoblast‐like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2–3‐fold increase in transcription, while decreased binding results in transcription 30–40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non‐osseous cell lines. In summary, we demonstrate association of at least two classes of tissue‐restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity.

The osteocalcin gene promoter provides a molecular blueprint for regulatory mechanisms controlling bone tissue formation: Role of transcription factors involved in development

Characterization of regulatory sequences and their cognate binding factors in the bone-specific osteocalcin (OC) gene promoter has provided insight into mechanisms that control expression of the gene under diverse biological conditions. We present evidence for AP-1 motifs and two multipartite conserved regulatory sequences, the OC Box I (nt-99 to-76) and a site designated OC Box II (nt-136 to-130) in contributing to developmental and tissue-specific expression of osteocalcin. OC Box I is characterized by a homeodomain binding site and OC Box II is a recognition sequence for AML-1 (also called PEBP2 alpha), a runt homology-related DNA binding protein. Functional activity of the elements was established in osseous and non-osseous cell lines and is in part related to the binding of osteoblast-specific complexes which enhance OC transcription. The contribution of several elements and binding of multiple classes of transcription factors to independent elements in both these domains serve to illustrate the complexity of control required for tissue-specific OC expression.