Transforming growth factor ␤ type II receptor (T␤RII) is a tumor suppressor gene that can be transcriptionally silenced by histone deacetylases (HDACs) in cancer cells. In this report, we demonstrated the mechanism by which trichostatin A (TSA), an inhibitor of HDAC, induces the expression of T␤RII in human pancreatic cancer cell lines by modulating the transcriptional components that bind a specific DNA region of the T␤RII promoter. This region of the T␤RII promoter possesses Sp1 and NF-Y binding sites in close proximity (located at ؊102 and ؊83, respectively). Treatment of cells with TSA activates the T␤RII promoter in a time-dependent manner through the recruitment of p300 and PCAF into a Sp1⅐NF-Y⅐HDAC complex that binds this DNA element. The recruitment of p300 and PCAF into the complex is associated with a concomitant acetylation of Sp1 and an overall decrease in the amount of HDAC associated with the complex. Transient overexpression of p300 or PCAF potentiated TSA-induced T␤RII promoter activity. The effect of PCAF was dependent on its histone acetyltransferase activity, whereas that of p300 was independent. Stable transfection of PCAF caused an increase in T␤RII mRNA expression, the association of PCAF with T␤RII promoter, and the acetylation of Sp1. Taken together, these results showed that TSA treatment of pancreatic cancer cells leads to transcriptional activation of the T␤RII promoter through modulation of the components of a Sp1⅐NF-Y⅐p300⅐PCAF⅐HDAC-1 multiprotein complex. Moreover, the interaction of NF-Y with the Sp1-associated complex may further explain why this specific Sp1 site mediates transcriptional responsiveness to TSA. TGF-␤1 plays a significant role in the growth inhibition of most normal epithelial and some cancer cells (1). TGF-␤ mediates its biological effects through cell surface receptors known as TGF-␤ type I receptor (T␤RI) and TGF-␤ type II receptor (T␤RII). Its intracellular signaling is initiated upon the selective binding of the active cytokine to the T␤RII homodimer. T␤RII is a ubiquitously expressed and constitutively active serine/threonine kinase. Ligand binding to T␤RII induces the assembly of a heterotetrameric complex consisting of T␤RI and T␤RII. Once the receptor complex is formed, T␤RII phosphorylates and thereby activates the T␤RI serine/threonine kinase. Activation of T␤RI propagates downstream signaling via Smad family proteins. T␤RI directly interacts with and phosphorylates Smad2 and Smad3. These Smads bind Smad4 and then result in the translocation of this complex to the nucleus and modulate TGF-␤-responsive gene expression (2-4).The TGF-␤ signaling pathway is inactivated in many tumors. Loss of negative growth regulation by TGF-␤ affords cells a selective growth advantage associated with decreased dependence of exogenous growth factor and increased tumorigenicity. Frequently, inhibition of TGF-␤ signaling occurs by either abolition of the function of a common mediator, Smad4, or interference with T␤RII function (5, 6). Smad4 and T␤RII are tumor suppressor gene...

Sp3 transcription factor can either activate or repress target gene expression. However, the molecular event that controls this dual function is unclear. We previously reported (Ammanamanchi, S., and Brattain, M. G.

. Northern analysis indicated that 5-aza-2-dC treatment did not affect the Sp1 transcript levels. Western blot analysis revealed an increase of Sp1 protein in the 5-aza-2-dCtreated MCF-7L cells, but there was no change in the c-Jun levels. Studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5-aza-2-dCtreated MCF-7L extracts compared with untreated control extracts. These results indicate that the transcriptional repression of RII in the ER ؉ breast cancer cells is caused by suboptimal activity of Sp1, whereas treatment with 5-aza-2-dC stabilizes the protein thus increasing steady-state Sp1 levels and thereby leads to enhanced RII transcription and subsequent restoration of TGF-␤ sensitivity.

. 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...

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