Submitted on April 1, 2003
Accepted on July 9, 2003
Activation of TGF-
/Smad signaling pathway following polyamine depletion in intestinal epithelial cells
Lan Liu1, Rachel Santora1, Jaladanki N. Rao1, Xin Guo1, Tongtong Zou1, Huifang M. Zhang1, Douglas J. Tuner1, and Jian-Ying Wang2*
1 Department of Sugery, University of Maryland School of Medicine, Baltimore, Maryland, USA; Baltimore Veterans Affairs Medical Center, Baltimore, Maryland, USA
2 Department of Sugery, University of Maryland School of Medicine, Baltimore, Maryland, USA; Baltimore Veterans Affairs Medical Center, Baltimore, Maryland, USA; Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland, USA
* To whom correspondence should be addressed. E-mail: jwang{at}smail.umaryland.edu.
Smad proteins are transcription activators that are critical for transmitting transforming growth factor-
(TGF-) superfamily signals from the cell-surface receptors to the nucleus. Our previous studies have shown that cellular polyamines are essential for normal intestinal mucosal growth and that a decreased level of polyamines inhibits intestinal epithelial cell proliferation, at least partially, by increasing expression of TGF-/TGF- receptors. The current study went further to determine the possibility that Smads are the downstream intracellular effectors of activated TGF-/TGF- receptor signaling following polyamine depletion. Studies were conducted in IEC-6 cells derived from rat small intestinal crypts. Depletion of cellular polyamines by 
-difluoromethylornithine (DFMO) increased basal levels of Smad3 and Smad4 proteins, induced their nuclear translocation, and stimulated Smad sequence-specific DNA-binding activity. Polyamine depletion-induced Smads also were associated with a significant increase in transcription activation as measured by luciferase reporter gene activity of Smad-dependent promoters. Inhibition of Smads by a dominant negative mutant Smad4 in the DFMO-treated cells prevented the increased Smad transcription activation. Polyamine-deficient cells highly expressed TGF- and were growth-arrested at the G1 phase. Inhibition of TGF- by treatment with either immunoneutralizing anti-TGF- antibody or TGF- antisense oligodeoxyribonucleotides not only blocked the induction of Smad activity, but also decreased the Smad-mediated transcriptional activation in polyamine-depleted cells. These findings suggest that Smads are involved in the downstream cellular processes mediated by cellular polyamines and that increased TGF-/TGF- receptor signaling following polyamine depletion activates Smads, thus resulting in the stimulation of Smad-target gene expression.
