Richard H. Kim

Transforming growth factor‐β1 regulation of bone sialoprotein gene transcription: Identification of a TGF‐β activation element in the rat BSP gene promoter

Transforming growth factor‐β (TGF‐β) increases steady‐state mRNA levels of several extracellular matrix proteins in mineralized connective tissues. Bone sialoprotein (BSP) is a major constituent of the bone matrix, thought to initiate and regulate the formation of mineral crystals. To determine the molecular pathways of TGF‐β1 regulation of bone proteins, we have analyzed the effects of the TGF‐β1 on the expression of the BSP in the rat osteosarcoma cell line (ROS 17/2.8). TGF‐β1 at 1 ng/ml, increased BSP mRNA levels in ROS 17/2.8 cells ∼8‐fold; the stimulation was first evident at 3 hr, reached maximal levels at 12 hr and slowly declined thereafter. Since the stability of the BSP mRNA was not significantly affected by TGF‐β1, and nuclear “run‐on” transcription analyses revealed only a ∼2‐fold increase in the transcription of the BSP gene, most of the increase in BSP mRNA appeared to involve a nuclear post‐transcriptional mechanism. Moreover, the effects of TGF‐β1 were indirect, since the increase in BSP mRNA was abrogated by cycloheximide (28 μg/ml). To identify the site of transcriptional regulation by TGF‐β1, transient transfection analyses were performed using BSP gene promoter constructs linked to a luciferase reporter gene. Constructs that included nt −801 to −426 of the promoter sequence were found to enhance transcriptional activity ∼1.8‐fold in cells treated with TGF‐β1. Within this sequence, ∼500 nt upstream of the transcription start site, a putative TGF‐β activation element (TAE) was identified that contained the 5′‐portion of the nuclearfactor‐1 (NF‐1) canonical sequence (TTGGC) overlapping a consensus sequence for activator protein‐2 (AP‐2). The functionality of the TAE was shown by an increased binding of a nuclear protein from TGF‐β1 stimulated cells in gel mobility shift assays and from the attenuation of TGF‐β1‐induced luciferase activity when cells were co‐transfected with a double‐stranded TAE oligonucleotide. Competition gel mobility shift analyses revealed that the nuclear protein that binds to the TAE has similar properties to, but is distinct from, NF‐1 nuclear protein. These studies have therefore identified a TGF‐β activation element (TAE) in the rat BSP gene promoter that mediates the stimulatory effects of TGF‐β1 on BSP gene transcription. J. Cell. Biochem. 65:501–512.

AP-1 regulation of the rat bone sialoprotein gene transcription is mediated through a TPA response element within a glucocorticoid response unit in the gene promoter.

Bone sialoprotein (BSP), a protein which has been implicated in the initial mineralization of newly-formed bone, provides an early phenotypic marker for differentiated osteoblasts. BSP expression is induced by glucocorticoids in association with osteoblast differentiation, and a glucocorticoid response element (GRE) overlapping a putative TRE (TPA, 12-O-tetradecanoyl-phorbol 13-acetate, response element) site has been identified in the rat BSP promoter (Ogata et al., 1995). Since AP-1 and the glucocorticoid receptor have a central role in regulating cell proliferation and differentiation, we have studied AP-1 activity, stimulated by 100 ng/ml TPA in normal fetal rat calvarial cells and in transformed rat osteosarcoma cells (ROS 17/2.8). A transient induction of both c-fos and c-jun mRNAs by TPA was observed in both cell populations, together with an associated suppression of BSP mRNA in the fetal rat calvarial cells. Rat BSP promoter constructs, transiently transfected into ROS 17/2.8 cells, were used to show that TPA suppressed transcription of a luciferase construct (-938/+60; pLUC6) that included the GRE/TRE, but not transcription of shorter contructs lacking this element. Notably, suppression of pLUC6 transcription by TPA was abrogated in the presence of the synthetic glucocorticoid, dexamethasone. Gel mobility shift analyses were performed using two double-stranded synthetic oligonucleotides. These encompassed the TRE and either the distal pair of GRE half-sites (-936/ -910; GRE3) or the proximal pair of GRE half-sites (-925/-899; GRE 4) that comprise the GRE/AP-1 element. The assay showed binding of both AP-1 complexes and recombinant c-Jun homodimers. Additionally, either the c-Jun or glucocorticoid receptor could displace its counterpart from the GRE/TRE but not from consensus GRE and TRE oligonucleotides, indicating that the abrogation of AP-1-mediated gene suppression by glucocorticoids could involve competitive binding. These studies, therefore, have identified a glucocorticoid response unit through which c-Fos and c-Jun can suppress the expression of BSP in proliferating pre-osteoblastic cells and through which glucocorticoids can ameliorate the effects of AP-1 and promote osteoblast differentiation and the associated expression of BSP.

Regulation of Bone Sialoprotein Gene Transcription by Steroid Hormones

During the initial formation of bone, dentine and cementum in tooth morphogenesis, fully differentiated osteoblasts, odontoblasts and cementoblasts express bone sialoprotein (BSP), a mineralized tissue-specific acidic glycoprotein that has been implicated in the nucleation of hydroxyapatite crystal growth. The expression of BSP is regulated by steroid hormones that modulate mineralized tissue formation. Thus, the transcription of the BSP gene is induced by glucocorticoids in association with osteoblast differentiation and glucocorticoids also stimulate the expression of BSP in differentiated osteoblasts. In contrast, however, vitamin D3 suppresses bone formation and abrogates the expression of BSP. Our studies, using the osteoblastic cell lines ROS 17/2.8 and UMR 106-06, have revealed that the glucocorticoid (10(-8) M dexamethasone; dex) effect on BSP mRNA involves both direct and indirect pathways. To determine the molecular basis of the direct pathway on transcriptional regulation of the BSP we have isolated and characterized the promoter regions of both the human and rat BSP genes. The promoters are characterized by a highly conserved region (BSP box) encompassing the immediate promoter region, which includes a unique inverted TATA box overlapped by a putative (DR3) vitamin D3 response element (VDRE). Possible glucocorticoid response elements are present approximately 1 kb and approximately 1.4 kb further upstream. Transient transfection analysis of chimeric constructs linked to a luciferase reporter gene have shown Dex-stimulated expression in constructs that include one or both GREs, whereas vit D3 suppresses expression in a short construct that includes the VDRE.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the Bone Sialoprotein (BSP) Gene Promoter

Bone sialoprotein is a 34 kDa phosphorylated and sulphated glycoprotein that is essentially unique to mineralizing connective tissues. Recent studies on the developmental expression of BSP mRNA and the temporo-spatial appearance of the protein during bone formation in vivo and in vitro have demonstrated that BSP is expressed by differentiated osteoblasts and that it may function in the initial nucleation of hydroxyapatite crystals in de novo bone formation. To study the cell-specific regulation of BSP we have isolated genomic clones that encompass the BSP promoter regions of both the human and rat genes. These promoters are characterized by a highly conserved region (BSP Box) that extends upstream from the transcription start site to nt -370. Within this region the immediate promoter is further characterized by a unique inverted TATA box and an inverted CCAAT box, both of which are required for basal transcriptional activity. The TATA box is overlapped by a vitamin D3 response element (VDRE) which appears to mediate vitamin D suppression of BSP gene transcription by competing with the TATA-binding protein (TBP) for occupancy of the site of the pre-initiation complex formation. Mutation of the inverted TATA box into a normal TATA sequence increases transcription slightly but does not affect the functionality of the VDRE indicating that the orientation of the TATA box is not critical for these functions. Further upstream an AP-1 site, overlapped by a steroid hormone response-like sequence, mediates down-regulation of BSP transcription induced by TPA that is abrogated by a complex interaction between Jun and the glucocorticoid receptor protein induced by dexamethasone. Thus, the characterization of approximately 3 kb of the BSP promoter and approximately 2 kb of the first intron has revealed several sites of transcriptional regulation that are important in regulating BSP expression and, consequently, bone formation.