TGFbeta induces apoptosis and EMT in primary mouse hepatocytes independently of p53, p21Cip1 or Rb status.
BACKGROUND
TGFbeta has pleiotropic effects that range from regulation of proliferation and apoptosis to morphological changes and epithelial-mesenchymal transition (EMT). Some evidence suggests that these effects may be interconnected. We have recently reported that P53, P21Cip1 and pRB, three critical regulators of the G1/S transition are variably involved in TGFbeta-induced cell cycle arrest in hepatocytes. As these proteins are also involved in the regulation of apoptosis in many circumstances, we investigated their contribution to other relevant TGFbeta-induced effects, namely apoptosis and EMT, and examined how the various processes were interrelated.


METHODS
Primary mouse hepatocytes deficient in p53, p21 and/or Rb, singly or in combination were treated with TGFbeta for 24 to 96 hours. Apoptosis was quantified according to morphology and by immunostaining for cleaved-capsase 3. Epithelial and mesenchymal marker expression was studied using immunocytochemistry and real time PCR.


RESULTS
We found that TGFbeta similarly induced morphological changes regardless of genotype and independently of proliferation index or sensitivity to inhibition of proliferation by TGFbeta. Morphological changes were accompanied by decrease in E-cadherin and increased Snail expression but the mesenchymal markers (N-cadherin, SMAalpha and Vimentin) studied remained unchanged. TGFbeta induced high levels of apoptosis in p53-/-, Rb-/-, p21cip1-/- and control hepatocytes although with slight differences in kinetics. This was unrelated to proliferation or changes in morphology and loss of cell-cell adhesion. However, hepatocytes deficient in both p53 and p21cip1were less sensitive to TGFbeta-induced apoptosis.


CONCLUSIONS
Although p53, p21Cip1 and pRb are well known regulators of both proliferation and apoptosis in response to a multitude of stresses, we conclude that they are critical for TGFbeta-driven inhibition of hepatocytes proliferation, but only slightly modulate TGFbeta-induced apoptosis. This effect may depend on other parameters such as proliferation and the presence of other regulatory proteins as suggested by the consequences of p53, p21Cip1 double deficiency. Similarly, p53, p21Cip1 and pRB deficiency had no effect on the morphological changes and loss of cell adhesion which is thought to be critical for metastasis. This indicates that possible association of these genes with metastasis potential would be unlikely to involve TGFbeta-induced EMT.
Sheahan
Sharon
S
Division of Pathology, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, EH16 4TJ, Edinburgh, UK. sheahan@hotmail.com
Bellamy
Christopher O
CO
Harland
Stephen N
SN
Harrison
David J
DJ
Prost
Sandrine
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis / Q000172:deficiency*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D005838:Genotype; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D008297:Male; D008648:Mesoderm / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008822:Mice, Transgenic; D016160:Retinoblastoma Protein / Q000096:biosynthesis / Q000172:deficiency*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000172:deficiency*
C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016160:Retinoblastoma Protein; D016212:Transforming Growth Factor beta; D016159:Tumor Suppressor Protein p53