Modulation of FAK, Akt, and p53 by stress release of the fibroblast-populated collagen matrix

Mark Alan Carlson, Michael T. Longaker, Jon S Thompson

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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. Materials and 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.

Original languageEnglish (US)
Pages (from-to)171-177
Number of pages7
JournalJournal of Surgical Research
Volume120
Issue number2
DOIs
StatePublished - Aug 1 2004

Fingerprint

Focal Adhesion Protein-Tyrosine Kinases
Collagen
Fibroblasts
DNA Nucleotidylexotransferase
Foreskin
Proto-Oncogene Proteins c-akt
Plastics
Proteins
Down-Regulation
Survival

Keywords

  • Akt
  • fibroblast populated collagen matrix
  • focal adhesion kinase
  • mdm2
  • p21
  • p53
  • protein kinase B

ASJC Scopus subject areas

  • Surgery

Cite this

Modulation of FAK, Akt, and p53 by stress release of the fibroblast-populated collagen matrix. / Carlson, Mark Alan; Longaker, Michael T.; Thompson, Jon S.

In: Journal of Surgical Research, Vol. 120, No. 2, 01.08.2004, p. 171-177.

Research output: Contribution to journalArticle

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title = "Modulation of FAK, Akt, and p53 by stress release of the fibroblast-populated collagen matrix",
abstract = "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. Materials and 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.",
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T1 - Modulation of FAK, Akt, and p53 by stress release of the fibroblast-populated collagen matrix

AU - Carlson, Mark Alan

AU - Longaker, Michael T.

AU - Thompson, Jon S

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N2 - 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. Materials and 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.

AB - 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. Materials and 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.

KW - Akt

KW - fibroblast populated collagen matrix

KW - focal adhesion kinase

KW - mdm2

KW - p21

KW - p53

KW - protein kinase B

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