Coordinate occupancy of AP-1 sites in the vitamin D-responsive and CCAAT box elements by Fos-Jun in the osteocalcin gene: model for phenotype suppression of transcription.

Abstract

Osteocalcin, a bone-specific protein and marker of the mature osteoblast, is expressed only in nonproliferating osteoblasts in a mineralizing extracellular matrix, while type I collagen is expressed in proliferating cells. The nuclear proteins encoded by the c-fos and c-jun protooncogenes are expressed during the proliferation period of osteoblast phenotype development. We present evidence that AP-1 (HeLa cell-activating protein 1) sites residing within two promoter elements of the osteocalcin gene bind the Fos-Jun protein complex: the osteocalcin box (OC box; nucleotides -99 to -76), which contains a CCAAT motif as a central element and influences tissue-specific basal levels of osteocalcin gene transcription, and the vitamin D-responsive element (VDRE; nucleotides -462 to -440), which mediates enhancement of osteocalcin gene transcription. Gel electrophoretic mobilityshift analysis demonstrated high AP-1 binding activity in proliferating osteoblasts and dramatic changes in this activity after the down-regulation of proliferation and the initiation of extraceilular-matrix mineralization in primary cultures of normal diploid osteoblasts. Methylation interference analysis established at single nucleotide resolution that purified recombinant Fos and Jun proteins bind in a sequence-specific manner to the AP-1 sites within the VDRE and OC box. Similarly, an AP-1 motif within a putative VDRE of the alkaline phosphatase gene, which is also expressed after the completion of proliferation, binds the Fos-Jun complex. These results support a model in which coordinate occupancy of the AP-1 sites in the VDRE and OC box in proliferating osteoblasts may suppress both basal level and vitamin D-enhanced osteocalcin gene transcription as well as transcription of other genes associated with osteoblast differentiation-a phenomenon we describe as phenotype suppression. This model is further supported by binding of the Fos-Jun complex at an AP-1 site in the type aI collagen promoter that is contiguous with, but not overlapping, the VDRE. Such a sequence organization in the collagen VDRE motif is compatible with vitamin D modulation of collagen but not with osteocalcin and alkaline phosphatase expression in proliferating osteoblasts.Development of the differentiated osteoblast phenotype both in culture and in vivo initially involves active proliferation, during which time genes associated with cell-cycle and cellgrowth control as well as those related to the biosynthesis of the type I collagen extracellular matrix (ECM) are expressed (1, 2). Following the completion of proliferative activity, genes related to ECM maturation and specialization are expressed, leading to competency of the ECM for mineralization and the initiation of osteocalcin gene expression (1, 2). Osteocalcin is a bone-specific calcium-binding protein that in normal diploid osteoblasts is synthesized only by mature nonproliferating cells (1, 2). Hence, it is necessary to account for how the osteocalcin gene is rendered transcribable only after the down-regul...

Preventing polyglutamine-induced activation of c-Jun delays neuronal dysfunction in a mouse model of SCA7 retinopathy

“…The series of events we describe in R7E mice, leading to c-Jun-mediated Nrl repression, appear to be very similar. Furthermore, c-Jun/AP-1 has been shown to repress expression of tissue-specific genes, including osteocalcin, a bone-specific marker and the pituitary-specific transcription factor (Pit-1/GHF-1) (Owen et al, 1990;Delhase et al, 1996). Interestingly, in both cases, the repression mechanism involves a mutually exclusive occupancy of AP-1 and tissue-specific transcription factors.…”

Cross-family interaction between the bHLHZip USF and bZip Fra1 proteins results in down-regulation of AP1 activity

“…Positive and negative regulatory interactions between Fos/Jun and nuclear receptors also have been reported (reviewed in Miner et al, 1991). Several gene promoters responsive to Fos/Jun are repressed by the glucocorticoid and retinoic acid receptors in cotransfection assays, and conversely, the activation of the promoters of nuclear receptorresponsive genes is repressed by overexpression of Fos and Jun (Jonat et al, 1990;Lucibello et al, 1990;Nicholson et al, 1990;Owen et al, 1990;SchuÈ le et al, 1990a,b;Yang-Yen et al, 1990a). Heterodimer formation can occur between a bHLHZip protein and a di erent class of proteins.…”

Functional Interference Between AP‐1 and the Vitamin D Receptor on Osteocalcin Gene Expression in Human Osteosarcoma Cells

“…Normal osteoblastic bone cells express the osteocalcin gene only after the proliferative stage (Owen et al, 1990) and its expression is regulated by calcitriol at the level of transcription, mRNA accumulation, and protein synthesis (Lian et al, 1989;Owen et al, 1991). We have previously shown by analysing mRNA from the human osteosarcoma cell lines MG-63, U2-0s, and SaOs-2 that osteocalcin gene expression is induced by calcitriol only in MG-63 cells (Mahonen et al, 1990).…”