Multiple and Essential Sp1 Binding Sites in the Promoter for Transforming Growth Factor-β Type I Receptor

Abstract

Maximal gene expression driven by the promoter for the transforming growth factor ␤ type I receptor (TGF-␤RI) occurs with a 1.0-kilobase pair fragment immediately upstream of exon 1. This region lacks a typical TATA box but contains CCAAT boxes, multiple Sp1, and PEBP2/CBF␣ binding sites among other possible cis-acting elements. Alterations within two CCAAT box sequences do not mitigate reporter gene expression driven by the basal promoter, and no nuclear factor binds to oligonucleotides encompassing these sites. In contrast, other deletions or site-specific mutations reveal an essential Sp1 site in the basal promoter and several dispersed upstream Sp1 sites that contribute to maximal reporter gene expression. The proportions of transcription factors Sp1 and Sp3, and their ratios of binding to consensus elements, are maintained in bone cells at different stages of differentiation. Finally, nuclear factor that binds to PEBP2/CBF␣-related cis-acting elements in the basal promoter sequence also occurs in osteoblasts. Our studies reveal that constitutive expression of TGF-␤RI may be determined by constitutive nuclear factor binding to Sp1 sites, whereas other elements may account for the variations in TGF-␤RI levels that parallel changes in bone cell differentiation or activity.Transforming growth factor-␤ (TGF-␤) 1 receptors occur on most cells, and a functional TGF-␤ type I receptor (TGF-␤RI) is required for all known TGF-␤-dependent effects. In some situations its activity is controlled by complex interactions with other cell surface components (1-3). However, in contrast to TGF-␤RII and the cell surface proteoglycan also termed TGF-␤RIII or betaglycan, expression of TGF-␤RI is maintained on differentiated bone cells (4). For these reasons, and because little is known about the molecular control of TGF-␤RI expression, we cloned the rat TGF-␤RI promoter and characterized several of its functional aspects in cultures of primary and continuous skeletal and nonskeletal cells derived from fetal rats. The rat TGF-␤RI promoter lacks a typical TATA box, but initiates transcription at multiple sites within a 220-bp span upstream of the initial methionine codon in differentiated bone cells. The 3Ј-terminal 300-bp sequence encompassing this region contains a GC-rich CpG island, seven consensus Sp1 binding sites, and two CCAAT boxes. Transfection studies using different fragments of TGF-␤RI promoter cloned upstream of the reporter gene luciferase demonstrated maximal activity by a 1.0-kb fragment that encompassed these and other possible cis-acting elements. Importantly, several dispersed elements appeared to cooperate for maximal reporter gene expression in osteoblast-enriched cultures (5). Coincident with this work, the human TGF-␤RI promoter was cloned, and its sequence reveals a similar organization with identically spaced CCAAT box motifs (6).These features suggested that the TGF-␤RI gene is driven by a constitutively active promoter that maintains expression of TGF-␤RI in many cells. Nevertheless, this promoter is part...

Runx2 Integrates Estrogen Activity in Osteoblasts

“…7A). Consistent with this, and the normal presence of Runx2 in osteoblasts (5,28,35), transfection with Runx2 antisense to limit endogenous Runx2 levels (37, 38) further increased ERE reporter gene expression by 2.5-3-fold (Fig. 7B).…”

“…Cells pooled from the last three digestions express high levels of nuclear factor Runx2, parathyroid hormone receptor, type I collagen synthesis, and alkaline phosphatase activity (28 -30). They exhibit an increase in osteocalcin expression in response to vitamin D 3 , differential sensitivity to TGF-␤, bone morphogenetic protein 2, and various prostaglandins, and form mineralized nodules in vitro (31)(32)(33)(34)(35). Cells were plated at 4000/cm 2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.…”

“…Plasmids SXN1C, containing two copies of a Runx RE from the rat TGF-␤ receptor I (T␤RI) gene promoter within pGL3-promoter (19,28), pEN1.0, containing a 1.0-kb fragment of the rat T␤RI gene promoter within pGL3-Basic (35,36), and antisense Runx2 expression plasmid, containing a 2.25-kb restriction fragment of murine Runx2 in reversed orientation within vector pSV7d (37, 38), were described previously. Our studies also benefited from the generous gifts of reporter and expression plasmids from other investigators.…”

“…To account for competition among plasmids for limiting transcriptional components, control cultures were transfected with a compensating amount of empty vector. Transfection efficiency was assessed in parallel in cells transfected with positive and negative reporter plasmids as described previously (16,23,(35)(36)(37).…”

Overlapping Sp1 and AP2 binding sites in a promoter element of the lens-specific MIP gene

“…A three zinc finger domain is highly conserved in members of this family, resulting in similar binding affinities to GC, GT and GA boxes. Sp1, Sp2 and Sp4 activate gene transcription, whereas Sp3 may function either as an activator or a repressor (49)(50)(51)(52)(53)(54)(55).…”