Sp3 Is a Transcriptional Repressor of Transforming Growth Factor-β Receptors

Abstract

. This report demonstrates that inappropriate overexpression of Sp3 is a mechanism that contributes to repression of TGF-␤ receptors. TGF-␤1 plays a vital role in the regulation of cell proliferation, differentiation, and extracellular matrix re-modeling in various cell types (1, 2). TGF-␤ carries out its biological effects through three cell surface receptors, which are referred to as type I (RI), type II (RII), and type III (RIII). RI and RII are serine/threonine kinases, and an active receptor complex consists of two molecules each of RI and RII, which are essential for TGF-␤ signal transduction and inhibition of cell growth (3-6).One of the crucial roles of TGF-␤ is the growth inhibition of normal epithelial cells as well as some cancer cells. Because RI and RII are necessary for TGF-␤-mediated growth arrest, mutational inactivation of either receptor has been reported to generate TGF-␤ resistance and hence the loss of the tumor suppressive function of TGF-␤ in cancer cells (7-9). DNA methylation of the RI promoter has been reported in a subset of gastric cancer cells (10). Another mechanism for loss of TGF-␤-mediated tumor suppression is transcriptional repression of RII due to decreased binding of stimulatory nuclear proteins to the RII promoter in keratinocytes and breast cancer cells (11,12). RI and RII replacement in cells that lack, or show reduced levels of, TGF-␤ receptors led to restoration of TGF-␤ response and subsequent reversal of malignancy, as seen in breast and colon cancer cells (13,14).The promoters for RI and RII have been characterized (15, 16). Both RI and RII promoters lack distinct TATA boxes and are highly GC-rich and depend on Sp1 transcription factor for the initiation of transcription. The RI promoter contains four consensus and several putative Sp1 sites, whereas the RII promoter contains two Sp1 sites. The Sp gene family of transcription factors consists of four members, which are referred to as Sp1-Sp4. Whereas Sp1, Sp2, and Sp4 are known to be activators of gene transcription, Sp3 is generally considered to be a repressor (17). Sp1 and Sp3 transcription factors recognize the same DNA element and have similar binding affinities. Sp3 has been shown to repress Sp1-mediated trans-activation of several genes (18 -20).MCF-7 early passage (MCF-7E) breast cancer cells express RI and RII and are responsive to the growth inhibitory effects of TGF-␤. However, MCF-7 late passage cells (MCF-7L) lack RII, show reduced levels of RI, and are TGF-␤-resistant. Loss or reduced expression of TGF-␤ receptors was due to low Sp1 protein levels in MCF-7L cells in comparison to 22). Sp1 deficiency was reversed by 5-aza-2Ј-deoxycytidine treatment of these cells, leading to the restoration of TGF-␤ receptor expression and signal transduction (12). We now show that in addition to Sp1 deficiency, MCF-7L breast cancer cells express higher levels of Sp3 than do MCF-7E cells, which express adequate amounts of both receptors and are consequently sensitive to TGF-␤. Sp3 acts as a transcriptional repressor of TG...

Restoration of Transforming Growth Factor-β Signaling through Receptor RI Induction by Histone Deacetylase Activity Inhibition in Breast Cancer Cells

“…The RI-Luc construct and control null vector without the RI promoter insert (pGL3) were transiently transfected into MCF-7L breast cancer cells using the FuGENE 6 method (Roche Applied Science) with a ␤-galactosidase plasmid for normalization (16). The cells were treated with 100 ng/ml SAHA 4 h following transfection.…”

“…Although Sp1 is known to be an activator of gene transcription, Sp3 is bifunctional and can either activate or repress target gene expression (15). For example, we reported previously that although unmodified Sp3 acts as a transcriptional repressor of TGF-␤ receptors, acetylated Sp3 acts as a transcriptional activator (16,21).…”

“…Although Sp1 is known to be an activator of gene transcription, Sp3 is bifunctional and can either activate or repress target gene expression (15). For example, we reported previously that although unmodified Sp3 acts as a transcriptional repressor of TGF-␤ receptors, acetylated Sp3 acts as a transcriptional activator (16,21).HDAC inhibitor (SAHA)-mediated RI expression resulted in the restoration of TGF-␤ response as evidenced by the enhanced activity of a TGF-␤-responsive plasminogen activator inhibitor promoter-luciferase reporter in the SAHA-treated cells. Consequently, this result suggests that TGF-␤ tumorsuppressive function can be regained following regeneration of RI expression in ERϩ breast cancer cells.…”

“…RI promoter activity is regulated by Sp1/Sp3 transcription factors (16). SAHA treatment inhibited Sp1/Sp3-associated HDAC activity leading to enhanced RI promoter activity and the concomitant RI expression.…”

“…Sp1 and Sp3 transcription factors recognize the same DNA sequence and have similar DNA-binding affinities. We have reported previously that Sp1/Sp3 proteins control RI promoter activities (16).…”