Modulation of FAK, Akt, and p53 by stress release of the fibroblast-populated collagen matrix.
BACKGROUND
Fibroblast survival in a three-dimensional collagen matrix is dependent in part upon the rigid anchorage of the matrix to tissue culture plastic. We hypothesized that focal adhesion kinase (FAK) and protein kinase B (Akt) would be activated and that the p53 level would be low in the rigidly anchored (attached) collagen matrix; loss of anchorage (detachment) was hypothesized to have the opposite effects.


METHODS
Human foreskin fibroblasts were cultured in attached bovine collagen matrices for 48 h before detachment as free-floating matrices. At various time points postrelease, matrix lysates were blotted for the proteins of interest, and the terminal deoxynucleotidyltransferase-mediated dUTP nick-end label assay was performed on both whole matrices and cytospin preparations. Irradiated monolayer fibroblasts were used as positive controls for the amount of p53 protein.


RESULTS
Terminal deoxynucleotidyltransferase-mediated dUTP nick-end label positivity in attached versus detached matrices (at 24 h post detachment) was 0.7 +/- 03 versus 5.3 +/- 1.7% (P < 0.05, unpaired t test). FAK and Akt were phosphorylated (activated) in the attached matrix; there was a near complete of loss of both activated forms within 4 h of matrix detachment. Irradiated monolayer fibroblasts had increased levels of p53, mdm2, and p21. In contrast, the p53, mdm2, and p21 levels were just at the level of detection in the attached matrix, but were induced 5- to 10-fold within 2-4 h after matrix detachment.


CONCLUSIONS
FAK and Akt are activated in the attached fibroblast-populated collagen matrix whereas the p53 level is relatively low; matrix detachment downregulates FAK and Akt activity and induces p53. The state of mechanical anchorage of the collagen matrix regulates the survival of embedded fibroblasts through a mechanism which may involve FAK.
Carlson
Mark A
MA
Department of Surgery, University of Nebraska Medical Center and the Omaha VA Medical Center, 4101 Woolworth Avenue, Omaha, NE 68105, USA. macarlso@unmc.edu
Longaker
Michael T
MT
Thompson
Jon S
JS
D000268:Adhesiveness; D000818:Animals; D017209:Apoptosis; D002417:Cattle; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D003094:Collagen; D003584:Cytological Techniques / Q000295:instrumentation; D015536:Down-Regulation; D005109:Extracellular Matrix / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism* / Q000502:physiology / Q000528:radiation effects; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005720:Gamma Rays; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051736:Proto-Oncogene Proteins c-mdm2; D013314:Stress, Mechanical; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D003094:Collagen; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt