Cell attachment controls fibronectin and alpha 5 beta 1 integrin levels in fibroblasts. Implications for anchorage-dependent and -independent growth.
We have examined the effect of cell attachment on fibronectin and alpha 5 beta 1 integrin levels in three distinct anchorage-dependent fibroblast cell lines. Analysis of long-term biosynthetically labeled proteins from parallel cultures of adherent and nonadherent cells showed that the steady-state level of extracellular fibronectin is decreased upon loss of attachment. Pulse labeling studies and Northern blot analyses showed that the decrease occurs post-synthetically. A combined approach of surface radioiodination and biosynthetic labeling also demonstrated a selective post-synthetic decrease in cell-surface expression of the alpha 5 beta 1 integrin upon loss of cell attachment. Overall, we estimate that extracellular fibronectin and cell surface alpha 5 beta 1 integrin levels are reduced 5-7-fold in NIH-3T3, 15-20-fold in AKR-2B, and 50-fold in NRK fibroblasts. Finally, we find decreased total (serum- and cell-derived) fibronectin bound to the surface of nonadherent cells consistent with the reduced expression of alpha 5 beta 1 integrin. These results demonstrate a systematic down-regulation of fibronectin and its major receptor upon loss of attachment and suggest a potential mechanism involved in maintenance of the anchorage-dependent phenotype.
Dalton
S L
SL
Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Marcantonio
E E
EE
Assoian
R K
RK
D000818:Animals; D015152:Blotting, Northern; D002448:Cell Adhesion; D002460:Cell Line; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D011233:Precipitin Tests; D012313:RNA / Q000378:metabolism; D014158:Transcription, Genetic
D005353:Fibronectins; D016023:Integrins; D012313:RNA