Induction and Turnover of Fructose 1,6‐Bisphosphatase in HL‐60 Leukemia Cells by Calcitriol

Abstract

Fructose 1,6-bisphosphatase mRNA and enzyme activity in HL-60 cells were rapidly and markedly induced by calcitriol (formerly known as la,25-dihydroxyvitamin D3). The activity reached 70-80 times the basal level after 96 h. The enzyme activity in the cells incubated for 96 h with calcitriol decreased immediately after its withdrawal but after a 24-h incubation the activity in the cells continued to increase slightly and then decreased slowly. Calcitriol increased the enzyme activity dose-dependently with maximal stimulation at 10 nM and half-maximal at 2.1 nM. The rate of synthesis of fructose 1,6-bisphosphatase almost paralleled the increase in inRNA level during treatment with calcitriol. When calcitriol was removed from media after incubation for either 24 h or 96 h, fructose-1;6-bisphosphatase mRNA and fructose-l,6-bisphosphatase synthesis decreased rapidly to the basal level. The enzyme was only slightly degraded in the cells incubated with calcitriol for 24 h followed by the subsequent culture without calcitriol but it was degraded with a half-life estimated to be approximately 64 h in the same cells followed by culturing with calcitriol. In the cells incubated for 96 h, the same degradation rate (i.e. half-life =64 h) was observed irrespective of the following culture with or without calcitriol. Calcitriol did not affect the degradation rate of total soluble proteins.Fructose 1,6-bisphosphatase (FBPase) is one of the ratelimiting enzymes in hepatic gluconeogenesis. The in vivo level of FBPase is under nutritional and hormonal controls. The intracellular concentration of the enzyme in the liver increases under gluconeogenic conditions including fasting, a high-protein diet and alloxan diabetes [l]. The enzyme concentration is also increased by the injection of glucagon, glucocorticoids and thyroxine [2]. El-Maghrabi et al. showed that FBPase mRNA was increased in livers from adult diabetic rats and was reduced to control levels after insulin treatment [3]. Recently, they observed the accumulation of FBPase mRNA in primary hepatocyte cultures incubated with cAMP and showed the presence of CAMP-responsive elements in the 5'-flanking region of the rat FBPase gene [4].FBPase is present in fetal liver and increases exponentially during fetal development [ 5 ] . In organ culture from fetal mouse liver, the activity of FBPase is remarkably elevated in the presence of dibutyryl cAMP [6, 71. FBPase activity, however, increases during the culture even in the absence of an inducer, although at lower rates [6]. The increase in intracellular FBPase concentration, especially in fetal development, might also be regulated by another factor (s) in addition to elevation of CAMP.

Induction of fructose 1,6‐bisphosphatase in HL‐60 leukemia cells by retinoic acid

“…The duration until reaching the maximal expression of FBPase by retinoic acid coincided with the term necessary for the completion of granulocytic differentiation by the agent (13). FBPase induced with retinoic acid also reacted with the antisera raised against rat liver enzyme as the enzyme induced with 1,25-(OH)2D 3 did as described in a previous report (11).…”

“…In contrast to the rapid expression of FBPase in HL-60 cells cultured in the presence of 1,25-(OH)2D 3 (11), the enzyme in the cells was slowly induced by retinoic acid. When retinoic acid was added at a 1 gM concentration to the culture media, the enzyme activity began to increase after around 48 h, rapidly generated after 72 h, and then plateaued at a steady state level at 120 h (Fig.…”

“…FBPase activities were spectrophotometrically determined at 30~ by following the rate of NADP § reduction at 340 nm in a coupled enzyme assay system as previously described (11). One unit of enzyme activity is defined as that amount which catalyzes the hydrolysis of 1 gmol of FBPase per min.…”

“…The human promyelocytic cell line, HL-60 cells, do not express FBPase in their undifferentiated state. However, FBPase is markedly activated in these cells cultured in the presence of lc~,25-dihydroxyvitamin D 3 (1,25-(OH)2D3) (9,11). HL-60 cells can be induced to differentiate along either the granulocytic or the monocytic lineage, depending on the inducing agent (12).…”

“…1,25-(OH)2D 3 is an important compound in inducing monocyte-macrophage differentiation. The level of FBPase activity continues to increase over a few days from a trace amount of the enzyme until reaching a plateau during the monocytic differentiation by 1,25-(OH)2D 3 (11). The physiological significance of HL-60 cells were grown in RPMI1640 medium supplemented with 10%(v/v) heat-inactivated fetal bovine serum, 10 mM Hepes, 30 gg/ml penicillin, and 100 gg/ml streptomycin.…”