The Smad proteins are a family of transcriptional activators that are critical for transmitting the TGF-b superfamily signals from the cell surface to the nucleus (51,52). Based on distinct functions, Smads are grouped into three classes: the receptor-regulated Smads (R-Smads), Smad2, and Smad3; the common Smad (co-Smad), Smad4; and the inhibitory Smads (I-Smads), Smad6, and Smad7 (53-55). All TGF-b family members, including TGF - bs, activins, and bone morphogenetic proteins, use TGF-bRI and TGF-bRII receptors in a variety of cell types (53,56). After ligand binding, the activated TGF-bRII kinase phosphorylates the TGF-bRI receptor, which subsequently phosphorylates the R-Smads on a C-terminal SSXS motif. This induces dissociation of the R-Smad from the receptor, stimulates the assembly of a heteromeric complex between the phosphorylated R-Smads and Smad4, and results in the nuclear accumulation of this heteromeric Smad3/Smad4 complex (55-57). In the nucleus, Smads bind to a specific DNA site (GTCTAGAC) and cooperate with various transcription factors in regulating target gene expression (58,59).
We have recently demonstrated that Smad proteins are involved in the process mediating functions of cellular polyamines in IECs (60). Exposure of IEC-6 cells to DFMO for 4, 6, and 8 d significantly increased levels of both Smad3 and Smad4 proteins and induced their nuclear translocation. Smad3 is shown to be highly expressed in IECs and activation of this R-Smad is ligand-specific. It is not surprising that polyamine depletion increases Smad3 protein and enhances its nuclear translocation because decreased levels of cellular polyamines are known to stimulate expression of TGF-b/ TGF -b receptors in IEC-6 cells (10,11 ). Activated Smad3 results primarily from the increase in expression of TGF-b/TGF-b receptors in polyamine-deficient cells because inhibition of TGF-b by treatment with either immunoneutralizing anti-TGF-b antibody or TGF-b antisense oligomers prevents the increased Smad activation in the absence of cellular polyamines.
Polyamine depletion also induces Smad4 nuclear translocation in IEC-6 cells. Smad4 functions as a common mediator for all R-Smads, and forms heteromeric complexes with Smad3 after ligand activation (52,53). The observed change in Smad4 in polyamine-deficient cells, however, seems to be a secondary response to the activation of Smad3. In support of this possibility, treatment with exogenous TGF-b did not alter levels of Smad4 protein in normal IEC-6 cells (without DFMO), although it significantly increased Smad3 expression. Furthermore, exposure to immunoneutralizing anti-TGF-b antibody or TGF-b antisense oligomers did not prevent the increased levels of Smad4 protein in polyamine-deficient cells. The other possibility also exists that polyamine depletion induces Smad4 expression through a mechanism independent from the activated Smad3 in IECs. In addition, this increased Smad expression and nuclear translocation in the DFMO-treated cells are specifically related to polyamine depletion rather than to a nonspecific effect of DFMO, because the stimulatory effect of this compound on Smads was completely prevented by the addition of exogenous spermidine.
Importantly, polyamine depletion-induced Smad activity is associated with a significant increase in transcriptional activation of Smad-driven promoters. Using the electrophoretic mobility shift method and luciferase reporter assays, we have demonstrated that polyamine depletion significantly increases Smad sequence-specific DNA binding and induces luciferase reporter activity of Smad-dependent promoters (60). Our results are consistent with others that indicated that induced nuclear Smad proteins mediate transcription of Smad target genes in a variety of cell types (56,57). Our studies also show that increased transcriptional activation after polyamine depletion is primarily owing to the function of Smad3/Smad4 heteromeric complexes because ectopic expression of a dominant negative mutant, Smad4, prevented the increased Smad transcriptional activation in polyamine-deficient cells. Recently, Smad DNA-binding sites have been identified in various gene promoters, including PAI-1, JunB, c-Jun, and p21 by different approaches. In general, these sequences reported by different groups are essentially identical; regardless of whether one defines a Smad-binding site as the palindrome A G A C G T C T, as the CAGA box, or as repeats of GACA, all identified sites contain the Smad box, 5$-GTCT-3 <t, or its reverse complement, 5$-AGAC-3 <t. Our results clearly indicate that induced Smad3/Smad4 heteromeric complexes in polyamine-deficient cells are able to bind to this specific DNA site, suggesting that Smads mediate transcriptional activation, probably through a direct interaction with Smad-binding element (SBE) sites in target promoters.
Combining these findings with our previous studies (10,11 , 60), it has been shown that activation of the TGF-b/Smad signaling pathway plays a role in the inhibition of IEC proliferation after polyamine depletion (Fig. 6). Polyamines negatively regulate posttranscription of the TGF-b gene. Decreased cellular polyamines stabilize TGF-b mRNAs, increase TGF -b synthesis, and cause the activation of TGF -b receptors. Smads are the downstream intracellular effectors of activated TGF -b receptors after polyamine depletion. The resultant activation of TGF-b receptors induces Smad3/Smad4 levels and stimulates Smad nuclear translocation, resulting in the accumulation of nuclear Smad3/Smad4 complexes. In the nucleus, Smads bind to the specific DNA site and cooperate with their DNA-binding partners, such as JunD/AP-1, to activate or repress transcription of specific target genes, thus leading to the inhibition
Fig. 6. Schematic diagram depicting the proposed role of the TGF-b/Smad signaling pathway in the inhibition of cell proliferation after polyamine depletion. In this model, polyamines are the negative regulators for expression of the TGF-b gene, whereas Smad proteins are the downstream intracellular effectors of activated T GF -b receptors. Decreased levels of cellular polyamines increase expression of T GF -b through stabilization of T GF -b mRNA, enhance the release of TGF-b, and subsequently phosphorylate TGF-b type II receptor (R-II). The phospho-rylated R-II activates T GF - b type I receptor (R-I), induces the formation of Smad3/Smad4 heteromeric complexes, and stimulates their nuclear translocation. The activated Smads in the nucleus bind to the specific DNA site and cooperate with Smad DNA-binding partners, such as some AP-1 proteins, to activate or repress transcription of specific target genes, thus leading to
Fig. 6. Schematic diagram depicting the proposed role of the TGF-b/Smad signaling pathway in the inhibition of cell proliferation after polyamine depletion. In this model, polyamines are the negative regulators for expression of the TGF-b gene, whereas Smad proteins are the downstream intracellular effectors of activated T GF -b receptors. Decreased levels of cellular polyamines increase expression of T GF -b through stabilization of T GF -b mRNA, enhance the release of TGF-b, and subsequently phosphorylate TGF-b type II receptor (R-II). The phospho-rylated R-II activates T GF - b type I receptor (R-I), induces the formation of Smad3/Smad4 heteromeric complexes, and stimulates their nuclear translocation. The activated Smads in the nucleus bind to the specific DNA site and cooperate with Smad DNA-binding partners, such as some AP-1 proteins, to activate or repress transcription of specific target genes, thus leading to of IEC proliferation. In contrast, increased polyamines downregulate the TGF-b/Smad signaling pathway and enhance IEC proliferation.
Was this article helpful?
Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.