Activation of TGF-beta-Smad signaling pathway following polyamine depletion in intestinal epithelial cells.
Smad proteins are transcription activators that are critical for transmitting transforming growth factor-beta (TGF-beta) 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-beta/TGF-beta receptors. The current study went further to determine the possibility that Smads are the downstream intracellular effectors of activated TGF-beta/TGF-beta receptor signaling following polyamine depletion. Studies were conducted in IEC-6 cells derived from rat small intestinal crypts. Depletion of cellular polyamines by alpha-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 were also 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-beta and were growth-arrested at the G1 phase. Inhibition of TGF-beta by treatment with either immunoneutralizing anti-TGF-beta antibody or TGF-beta 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-beta/TGF-beta receptor signaling following polyamine depletion activates Smads, thus resulting in the stimulation of Smad target gene expression.
Liu
Lan
L
Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 North Greene St., Baltimore, MD 21201, USA.
Santora
Rachel
R
Rao
Jaladanki N
JN
Guo
Xin
X
Zou
Tongtong
T
Zhang
Huifang M
HM
Turner
Douglas J
DJ
Wang
Jian-Ying
JY
D000818:Animals; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000518:Eflornithine / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D011073:Polyamines / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D051900:Smad3 Protein; D051901:Smad4 Protein; D014018:Tissue Distribution; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*
D004268:DNA-Binding Proteins; D011073:Polyamines; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D051901:Smad4 Protein; C490811:Smad4 protein, rat; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D004247:DNA; D000518:Eflornithine