Abstract
We have shown that attachment to a fibronectin substrate stimulates two pathways of lipid biosynthesis in cultured human fibroblasts. Detachment of these cells (mechanically, with trypsin, or by RGDS peptides) caused a significant decrease in their 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and in their incorporation of [3H]acetate into fatty acids. This inhibition was substantially reversed by the reattachment of cells to fibronectin substrates, but not to poly-L-lysine substrates or to fibronectin in solution. Inhibiting phosphoprotein phosphatase activity with okadaic acid blocked the recovery of both biosynthetic activities. Both 3-hydroxy-3-methylglutaryl-coenzyme A reductase and fatty acid biosynthesis are known to be inhibited by the action of 5'-AMP-activated protein kinase, which is activated by an increase in the level of AMP relative to ATP. For example, in our system, sodium azide and 2-deoxy-D-glucose increased the ratio of cellular AMP to ATP and caused a decrease in lipid biosynthesis. We then verified the prediction that detachment of cells from substrates also caused an increase in the AMP/ATP ratio. We therefore conclude that the attachment of cells to fibronectin promotes lipid biosynthesis, presumably in coordination with the cellular growth response evoked by attachment to the extracellular matrix.
Highlights
Both 3-hydroxy-3-methylglutaryl-coenzyme A reductase and fatty acid biosynthesis are known to be inhibited by the action of 5*-AMP-activated protein kinase, which is activated by an increase in the level of AMP relative to ATP
We conclude that the attachment of cells to fibronectin promotes lipid biosynthesis, presumably in coordination with the cellular growth response evoked by attachment to the extracellular matrix
The predicted outcome is the inhibition of the biosynthesis of fatty acids, cholesterol, and other isoprenoid derivatives [24, 25]
Summary
Materials—[3H]Acetic acid (sodium salt, 100 mCi/mmol), [14C]acetic acid (sodium salt, 59 mCi/mmol), and DL-3-hydroxy-3-methyl[3-14C]glutaryl coenzyme A (57.7 mCi/mmol) were from NEN Life Science Products. [5-3H]Mevalonolactone (60 Ci/mmol) was from American Radiolabeled Chemicals. [4-14C]Cholesterol (53 mCi/mmol) and [N-methyl14C]sphingomyelin (58 mCi/mmol) were from Amersham Corp. [14C]Triglyceride and [14C]cholesteryl esters, used as recovery standards, were purified by TLC from hepatoma cells that had been incubated for 4 h with [14C]acetate. 2.5 mg/ml egg white trypsin inhibitor was added, and the cells were chilled on ice for 5 min and washed with cold phosphate-buffered saline. The suspended cells were either assayed immediately or, following a 10-min incubation at 37 °C, replated in buffer A in Petri dishes. The dishes had been precoated with 50 mg/ml poly-L-lysine or 5 mg/ml fibronectin overnight and blocked with 1 mg/ml bovine serum albumin for 30 min and rinsed with phosphate-buffered saline prior to use. [14C]Triglycerides, [14C]cholesteryl esters, and/or [14C]sphingomyelin were added as recovery standards.
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