CYTOKINES DECREASE GLUTAMINASE EXPRESSION IN HUMAN FIBROBLASTS

Background. Glutamine metabolism in fibroblasts is essential for energ y production, nucleotide biosynthesis, and growth during wound healing . Because cytokines can impair fibroblast proliferation, we tested the hypothesis that cytokines impair glutamine metabolism. We studied the influence of several cytokines on the expression of glutaminase, the major enzyme of intracellular glutamine metabolism in fibroblasts. Met hods. Human foreskin fibroblasts were incubated for 6 and 12 hours wit h varying doses (10, 100, or 1000 units/ml) of interleukin (IL)-1, IL- 6, tumor necrosis factor-alpha, or gamma-interferon. Cell lysates were assayed for glutaminase-specific activity, and glutaminase protein co ntent was measured by Western blotting with a polyclonal antibody. Tot al cellular RNA was extracted, and relative glutaminase messenger RNA levels were determined by Northern blotting with a P-32-labeled glutam inase complement DNA-derived probe. These mRNA levels were normalized by blotting with a beta-actin cDNA-derived probe as control. Cell nucl ei were isolated, and nuclear run-ons were used to determine relative glutaminase mRNA transcription rates. Results. IL-1, IL-6, tumor necro sis factor-alpha, and gamma-interferon decreased glutaminase activity and protein concentration after a 12-hour incubation in a dose-indepen dent fashion. No difference was noted at 6 hours. Western blot analysi s showed a 30% to 60% reduction in glutaminase protein zn treated cell s. These cytokines also decreased glutaminase mRNA levels, consistent with transcriptional regulation. This was confirmed by nuclear run-an assays that showed a decrease in the number of glutaminase transcripts . Conclusions. A variety of different pro-inflammatory cytokines decre ase glutaminase expression In cultured human fibroblasts. This cytokin e-mediated inhibition of glutamine metabolism may limit the availabili ty of key glutamine-derived intermediates and impair fibroblast prolif eration in certain patients.