Analysis of the suitability of calreticulin inducible HEK cells for adhesion studies: microscopical and biochemical comparisons.
Calreticulin is a Ca(2+)-buffering ER chaperone that also modulates cell adhesiveness. In order to study the effect of calreticulin on the expression of adhesion-related genes, we created a calreticulin inducible Human Embryonic Kidney (HEK) 293 cell line. We found that fibronectin mRNA and both intra- and extra-cellular fibronectin protein levels increased following calreticulin induction. However, despite this increase in fibronectin, HEK293 cells did not assemble an extracellular fibrillar fibronectin matrix regardless of the level of calreticulin expression. Furthermore, HEK293 cells exhibited a poorly organized actin cytoskeleton, did not have clustered fibronectin receptors at the cell surface, and did not form focal contacts. This likely accounts for the lack of fibronectin matrix deposition by these cells regardless of calreticulin expression level. Vinculin abundance did not appreciably increase upon calreticulin induction and the level of active c-Src, a regulatory kinase of focal contacts, was found to be abundant and unregulated by calreticulin induction in these cells. The inability to form stable focal contacts and to commence fibronectin fibrillogenesis due to high c-Src activity may be responsible for the poor adhesive phenotype of HEK 293 cells. Thus, we show here that HEK293 cells are not suitable for microscopical studies of cell-substratum adhesions, but are best suited for biochemical studies.
Papp
Sylvia
S
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
Fadel
Marc P
MP
Michalak
Marek
M
Opas
Michal
M
D000818:Animals; D037282:Calreticulin / Q000235:genetics / Q000502:physiology*; D002448:Cell Adhesion / Q000235:genetics; D002450:Cell Communication / Q000235:genetics*; D018929:Cell Culture Techniques; D002460:Cell Line; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000096:biosynthesis; D022001:Focal Adhesions / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism / Q000502:physiology; D051379:Mice; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
D037282:Calreticulin; D005353:Fibronectins; D039081:Integrin alpha5beta1; D016392:Proto-Oncogene Proteins pp60(c-src)