Ca2+-dependent translocation of hexose carrier in mouse fibroblast Swiss 3T3 cells

Ca2+-induced translocation of hexose carriers from microsomal membrane to plasma membrane was demonstrated in saponin-permeabilized Swiss 3T3 cells by a specific D-glucose-inhibitable cytochalasin B-binding assay. The number of hexose carriers in the plasma membrane and the hexose transport activity in intact cells were also compared. The incubation of permeabilized cells with 10 microM Ca2+ at 37 degrees C rapidly increased the number of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane from 13 to 40 pmol/mg protein and concomitantly decreased that in the microsomal membrane from 66 to 36 pmol/mg protein, each with a half-time of approx. 2 min. Furthermore, when Ca2+-stimulated cells were exposed to 50 microM EGTA, the effect of Ca2+ on the translocation of D-glucose-inhibitable cytochalasin B-binding sites was reversed with a half-time of approx. 5 min. The concentration of Ca2+ required for the half-maximal effect was approx 500 nM. The magnitude of the stimulatory effect of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane closely correlated with the magnitude of stimulatory action of Ca2+ on 3-O-methylglucose transport in the intact cells. These results suggest that Ca2+ regulates the activity of hexose transport across the plasma membrane through a rapid and reversible translocation of hexose carrier between microsomal and plasma membranes of mouse fibroblast Swiss 3T3 cells.     

Effect of protein kinase C activation and Ca2+ mobilization on hexose transport in Swiss 3T3 cells

Down-modulation of Ca²⁺-activated, phospholipid-dependent protein kinase (protein binase C), which was accomplished by pretreatment with phorbol-12,13-dibutyrate for 24 h, resulted in the loss of a phorbol ester-induced stimulation of hexose transport activity in Swiss 3T3 cells. In these cells, however, platelet-derived growth factor as well as Ca²⁺ ionophore A23187 were still able to induce stimulation of hexose transport activity accompanied by the elevation of intracellular free Ca²⁺ concentration. Since chelation of extracellular Ca²⁺ inhibited this stimulation, inflow of extracellular Ca²⁺ into cytoplasm seemed to be esential for the stimulatory effect of platelet-derived growth factor and A23187 on hexose transport. Epidermal growth factor and insulin also stimulated hexose transport activity regardless of the absence of protein kinase C. However, in the case of epidermal growth factor, intracellular Ca²⁺, but not extracellular Ca²⁺, was found to be necessary for the stimulation. On the other hand, insulin stimulated the hexose transport independent of both intra- and extracellular Ca²⁺.     

Tumor promoter-stimulated translocation of glucose transport system in mouse embryo fibroblast Swiss 3T3 cell

Assay for D-glucose-inhibitable 3H-cytochalasin B-binding was carried out to elucidate the action mechanism of the tumor promoter-induced enhancement of glucose transport activity in Swiss 3T3 cells. Incubation of the cells with 12-0-tetradecanoylphorbol-13-acetate (TPA) increased the amount of D-glucose-inhibitable cytochalasin B-binding sites in plasma membrane from 13.5 to 40.1 pmol/mg protein. On the other hand, TPA treatment resulted in the decrease of binding sites in microsomal membrane from 68.9 to 34.1 pmol/mg protein. The tumor promoter-induced translocation of hexose transport system from microsomal membrane to plasma membrane was inhibited by the treatment with 2,4-dinitrophenol before the addition of TPA but was not affected by the treatment with cycloheximide. By removal of the promoter from its receptor, the stimulatory effect of the promoter on the translocation of hexose transport system was decreased. The analysis by electrophoresis demonstrated that among the affinity labeled hexose transporter components of Mr 48,000 and Mr 55,000, the former was responsible for the TPA-induced increase in hexose transport activity in plasma membrane.     

Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B

The effect of simian virus 40 transformation on the hexose transport system in mouse embryo fibroblast Swiss 3T3 cells was examined. The concentration of hexose transporters was estimated by measuring D-glucose-inhibitable cytochalasin B binding. The binding of cytochalasin B to the plasma membranes of simian virus 40-transformed mouse 3T3 cells (SV3T3 cells) was significantly greater than that of 3T3 cells. On the other hand, cytochalasin B binding to the microsomal membranes of SV3T3 cells was decreased, and the total amount of binding to plasma and microsomal membranes was not significantly changed in both cell lines. The electrophoretic analysis demonstrated that both hexose-transporter components of Mr 46 000 and Mr 58 000 affinity labeled were responsible for an increase in the hexose transport by viral transformation. These results suggested that the higher hexose-transport activity of transformed cells is caused by a redistribution of transporter from intracellular membranes to plasma membranes.     

Ca2+-dependent stimulation of 3-O-methylglucose transport in mouse fibroblast Swiss 3T3 cells induced by phorbol-12,13-dibutyrate

Binding of phorbol-12,13-dibutyrate (PDBu), a tumor promoter, to quiescent Swiss 3T3 cells increased the number of hexose carriers, resulting in stimulation of membrane transport of 3-O-methylglucose (3MeGlu) in a Ca2+-dependent fashion. Extracellular Ca2+ was necessary to initiate the binding of PDBu to its receptor, and intracellular Ca2+ was required to maintain it. The loss of PDBu-binding, caused by elimination of Ca2+, was accompanied by a loss of stimulation of hexose transport. These results indicated that Ca2+-dependent, continuous binding of PDBu to its receptor was essential to induce the stimulation of hexose transport.     

Effect of tunicamycin on hexose transport in mouse embryo fibroblast Swiss 3T3 cells

The role of glycosylation of the carrier in the transporting activity was investigated in Swiss 3T3 cells. Inhibition of protein glycosylation by tunicamycin resulted in the decrease of hexose uptake in a dose- and time-dependent manner without a cytotoxic effect. From kinetic analysis, a decrease in the number or availability of hexose carriers in the plasma membrane was suggested. This was in good correlation with the decrease in the amount of photoaffinity cytochalasin B binding in the plasma membrane by the treatment with tunicamycin. The rate of phorbol 12,13-dibutyrate-induced translocation of the hexose carrier from microsomal to plasma membrane was reduced in tunicamycin-treated cells, which may be correlated with the decrease in the number of the completely glycosylated carrier translocatable from the microsomal membrane. In both tunicamycin-treated and untreated cells, the stimulation of hexose transport by phorbol 12,13-dibutyrate was abolished by the removal of phorbol 12,13-dibutyrate, and upon its readdition the stimulation recovered to the same degree as before the removal. Thus, the recycling of the functionally mature hexose carrier appeared not to be affected by the treatment with tunicamycin. These results suggested that complete glycosylation of the carrier may be necessary for the translocation of the carrier from microsomal to plasma membrane to accomplish its function on the cell surface.     

Possible involvement of protein kinase C and calcium ion in growth factor-induced expression of c-myc oncogene in Swiss 3T3 fibroblasts

The addition of platelet-derived growth factor and fibroblast growth factor to quiescent cultures of Swiss 3T3 fibroblasts rapidly induced protein kinase C activation and Ca2+ mobilization and afterwards markedly increased c-myc mRNA levels. 1-Oleoyl-2-acetylglycerol, a membrane-permeable synthetic diacylglycerol, and 12-O-tetradecanoylphorbol 13-acetate, a tumor-promoting phorbol ester, stimulated protein kinase C activation without Ca2+ mobilization. Inversely, Ca2+ ionophores, A23187 and ionomycin, elicited Ca2+ mobilization without protein kinase C activation. Both protein kinase C-activating and Ca2+-mobilizing agents were able to increase c-myc mRNA levels in an additive manner. Prolonged treatment of the cells with phorbol 12,13-dibutyrate, another protein kinase C-activating phorbol ester, led to the down-regulation and complete disappearance of protein kinase C. In these cells, 1-oleoyl-2-acetylglycerol and 12-O-tetradecanoylphorbol 13-acetate did not increase c-myc mRNA levels, but platelet-derived growth factor, fibroblast growth factor, and the Ca2+ ionophores, all of which still induced Ca2+ mobilization, stimulated the increase of c-myc mRNA levels. These results strongly suggest that both protein kinase C and Ca2+ may be involved in platelet-derived growth factor- as well as fibroblast growth factor-induced expression of the c-myc oncogene in Swiss 3T3 cells.     

Involvement of three intracellular messenger systems, protein kinase C, calcium ion and cyclic AMP, in the regulation of c-fos gene expression in Swiss 3T3 cells

In quiescent cultures of Swiss 3T3 cells, platelet-derived growth factor or fibroblast growth factor known to induce both protein kinase C activation and Ca2+ mobilization raised c-fos mRNA. This action of the growth factors was mimicked by the specific activators for protein kinase C, such as phorbol esters and a membrane-permeable synthetic diacylglycerol, and also by the Ca2+ ionophores, such as A23187 and ionomycin. Prostaglandin E1 known to elevate cyclic AMP also raised c-fos mRNA, and this action was mimicked by 8-bromo-cyclic AMP, dibutyryl cyclic AMP and forskolin. These results suggest that expression of the c-fos gene is regulated by three different intracellular messenger systems, protein kinase C, Ca2+ and cyclic AMP, in Swiss 3T3 cells.     

Purified plasma membranes inhibit polypeptide growth factor-induced DNA synthesis in subconfluent 3T3 cells

Plasma membranes derived from NR-6 cells, a variant line of Swiss mouse 3T3 cells that does not have cell surface receptors for epidermal growth factor (EGF), inhibited EGF-induced stimulation of DNA synthesis by 50% in serum-starved, subconfluent 3T3 cells. Membranes derived from SV3T3 cells were much less effective in inhibiting EGF-induced DNA synthesis. This inhibition on DNA synthesis by NR-6 membranes was not a direct effect of membranes on EGF, nor could it be overcome by high concentrations of EGF. NR-6 membranes were most effective when added 3 h before EGF addition and had little effect when added 2 h or more after EGF. NR-6 membranes also reduced the stimulation of DNA synthesis induced by platelet-derived growth factor or fibroblast growth factor in serum-starved 3T3 cells. These findings indicate that membrane- membrane interactions between nontransformed cells may diminish their ability to proliferate in response to serum polypeptide growth factors.     

Epidermal growth factor increases c-myc mRNA without eliciting phosphoinositide turnover, protein kinase C activation, or calcium ion mobilization in Swiss 3T3 fibroblasts

Incubation of quiescent cultures of Swiss 3T3 cells with epidermal growth factor (EGF) caused an increase in c-myc mRNA. Under these conditions, EGF did not induce phosphoinositide turnover, formation of diacylglycerol, formation of inositol tris-, bis-, and monophosphates, protein kinase C activation, or Ca2+ mobilization. Although it has been reported that both protein kinase C and Ca2+ may be responsible for the platelet-derived growth factor- and fibroblast growth factor-induced increases in c-myc mRNA in Swiss 3T3 cells (Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T., & Takai, Y. (1986) J. Biol. Chem. 261, 1187-1192), these results indicate that neither protein kinase C nor Ca2+ is involved in the EGF-induced increase in c-myc mRNA, and that an unidentified system may be involved in this reaction.     

Dihydrocytochalasin B disorganizes actin cytoarchitecture and inhibits initiation of DNA synthesis in 3T3 cells

Dihydrocytochalasin B (H2CB) disrupts the actin structure of Swiss/3T3 mouse fibroblasts and inhibits the ability of serum growth factors to stimulate DNA synthesis in quiescent cultures. Low doses of H2CB (2-10 X 10(-7) M) added to serum-arrested cells reversibly block initiation of DNA synthesis by serum; by epidermal growth factor and insulin; or by epidermal growth factor, fibroblast growth factor and insulin. H2CB is effective only when added to cells within 8-10 hr after stimulation. Low doses of H2CB cause cell rounding and a loss of actin microfilament bundles, but they do not interfere with glucose or thymidine transport. These results suggest that stimulation of 3T3 cells involves at least one obligatory actin-mediated step. Transformed cells appear to obviate this step, for H2CB does not inhibit the entry into S phase of SV40-transformed or Moloney murine sarcoma virus-transformed 3T3 cells synchronized by mitotic shake-off.     

Identification of the fibroblast growth factor receptor of Swiss 3T3 cells and mouse skeletal muscle myoblasts

Two distinct fibroblast growth factors (FGF) were purified to homogeneity from bovine brain on the basis of their ability to stimulate skeletal muscle myoblast proliferation. These growth factors are also mitogenic for Swiss 3T3 cells and appear to be closely related to or identical with previously isolated anionic and cationic fibroblast growth factors. The half-maximum concentrations (EC50) for stimulation of myoblast DNA synthesis by the anionic and cationic growth factors were 30pM and 1pM, respectively. In contrast, an EC50 of 45 pM was observed for stimulation of 3T3 cell DNA synthesis by both growth factors. Binding of 125I-labeled anionic FGF was saturable with apparent Kd values of 45 pM and 11 pM and approximately 60 000 and 2000 receptor sites per cell for 3T3 cells and MM14 murine myoblasts, respectively. Unlabeled anionic and cationic FGF equally displaced 125I-labeled anionic FGF from 3T3 cells while cationic FGF was more potent than anionic FGF for displacement from skeletal muscle myoblasts, demonstrating that a single receptor binds the two distinct growth factors. Binding was specific for these factors since platelet-derived growth factor, insulin, insulin-like growth factor 1, epidermal growth factor, and nerve growth factor were unable to displace bound 125I-labeled anionic FGF from Swiss 3T3 cells. Chemical cross-linking of specifically bound 125I-labeled anionic FGF to 3T3 cells and MM14 myoblasts identified a single detergent-soluble FGF receptor with an apparent molecular weight of 165 000.     

Induction of tumor promotor-inducible genes in murine 3T3 cell lines and tetradecanoyl phorbol acetate-nonproliferative 3T3 variants can occur through protein kinase C-dependent and -independent pathways.

We isolated a group of genes that are rapidly and transiently induced in 3T3 cells by tetradecanoyl phorbol acetate (TPA). These genes are called TIS genes (for TPA-inducible sequences). Epidermal growth factor (EGF), fibroblast growth factor (FGF), and TPA activated TIS gene expression with similar induction kinetics. TPA pretreatment to deplete protein kinase C activity did not abolish the subsequent induction of TIS gene expression by epidermal growth factor or fibroblast growth factor; both peptide mitogens can activate TIS genes through a protein kinase C-independent pathway(s). We also analyzed TIS gene expression in three TPA-nonproliferative variants (3T3-TNR2, 3T3-TNR9, and A31T6E12A). The results indicate that (i) modulation of a TPA-responsive sodium-potassium-chloride transport system is not necessary for TIS gene induction either by TPA or by other mitogens and (ii) TIS gene induction is not sufficient to guarantee a proliferative response to mitogenic stimulation.     

Protein phosphorylation in a tetradecanoyl phorbol acetate-nonproliferative variant of 3T3 cells.

The 3T3-TNR9 cell line is a variant of Swiss 3T3 cells which does not respond mitogenically to tumor promoters, but does respond mitogenically to epidermal growth factor, fibroblast growth factor, and serum. To elucidate differences between tumor promoters and polypeptide mitogens in the pathway(s) of mitogenesis which might be responsible for the nonresponsiveness of the 3T3-TNR9 cells, we have examined in these cells the early protein phosphorylation events known to be associated with mitogenesis in the parental 3T3 cells. We find that the 3T3-TNR9 cells display levels of tetradecanoyl phorbol acetate binding and of a calcium- and phospholipid-dependent protein kinase activity which are at least the equal of those seen in the parental 3T3 cells, implicating some postreceptor event in the nonmitogenic phenotype. In addition, we find that phosphorylation of the epidermal growth factor receptor and of 80-kDa and 22-kDa proteins, as well as the tyrosine phosphorylation of a 42-kDa protein, all proceed normally in the nonmitogenic variant, even though these phosphorylations must depend on the activation of different kinases. Thus, all these early phosphorylation reactions are intact in the 3T3-TNR9 cells. Although these phosphorylations may be necessary, they clearly are insufficient to trigger mitogenesis.     

Protein synthesis inhibitors activate glucose transport without increasing plasma membrane glucose transporters in 3T3-L1 adipocytes

In this study, we tested the hypothesis that hexose transport regulation may involve proteins with relatively rapid turnover rates. 3T3-L1 adipocytes, which exhibit 10-fold increases in hexose transport rates within 30 min of the addition of 100 nM insulin, were utilized. Exposure of these cells to 300 microM anisomycin or 500 microM cycloheximide caused a maximal, 7-fold increase in 2-deoxyglucose transport rate after 4-8 h. The effects due to either insulin (0.5 h) or anisomycin (5 h) on the kinetics of zero-trans 3-O-methyl[14C]glucose transport were similar, resulting in 2.5-3-fold increases in apparent Vmax values (control Vmax = 1.6 +/- 0.3 x 10(-7) mmol/s/10(6) cells) coupled with approximately 2-fold decreases in apparent Km values (control Km = 23 +/- 3.3 mM). Insulin elicited the expected increases in plasma membrane levels of HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) transporters (1.6- and 2.8-fold, respectively) as determined by protein immunoblotting. In contrast, neither total cellular contents nor plasma membrane levels of these two transporter isoforms were increased when 3T3-L1 adipocytes were treated with either anisomycin or cycloheximide. 3-[125I]Iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n labeling of glucose transporters in plasma membrane fractions of similarly treated cells was also unaffected by these agents. Thus, a striking discrepancy was observed between the marked increase in cellular hexose transport rates due to these protein synthesis inhibitors and the unaltered amounts of glucose transporter proteins in the plasma membrane fraction. These data indicate that short-term protein synthesis inhibition in 3T3-L1 adipocytes leads to large increases in the intrinsic catalytic activity of one or both of the GLUT1 and GLUT4 transporter isoforms.     

Cell surface-associated growth inhibitory proteins : Evidence for conservation between mouse and human cell lines

The ability of the normal human fibroblast line (IMR91) to exhibit density-dependent regulation of growth has been examined. The line exhibits density-dependent regulation of growth; saturation density in 15% fetal bovine serum is 2 × 10⁵ cells/cm². Membranes prepared from confluent monolayers of these cells contained growth inhibitory factors to both exponentially growing IMR91 and Swiss 3T3 cells. This factor(s) appears to be similar to a previously described factor found on the surface of Swiss 3T3 cells [14]. The inhibition of DNA synthesis in growing IMR91 cultures by membranes was both time- and concentration-dependent. The effect was reversible by high serum. Specificity experiments utilizing membranes prepared from Swiss 3T3 cells indicated some species specificity for inhibition by membranes, but this specificity was no longer exhibited by solubilized membrane preparations. These results are compatible with the suggestion that both the growth inhibitory factors and their receptors are conserved through evolution.     

Glucose as a regulator of insulin-sensitive hexose uptake in 3T3 adipocytes

In the present study we examined the role of glucose in the regulation of its own transport activity in the cultured 3T3 fat cell. A regulatory control of glucose became apparent after these cells were cultured in the absence of glucose. Glucose deprivation of the cells was accompanied by a specific time and protein synthesis-dependent increase in dGlc (2-deoxyglucose) uptake (up to 5-fold), which was due to an increase in the apparent Vmax of the transport system. Concomitantly, the stimulatory effect of insulin on hexose uptake almost completely disappeared. Addition of glucose to the glucose-deprived cells rapidly reversed the deprivation effects. Cycloheximide experiments revealed that the glucose deprivation-induced increase in hexose uptake required protein synthesis as well as a protein synthesis-independent response to glucose deprivation that retarded the turnover of hexose transport activity. Taken together, these data indicate that glucose deprivation is accompanied by retardation of the rate of degradation, internalization, or inactivation of hexose transporters while the increase in dGlc uptake requires at least the continuation of protein synthesis-dependent de novo synthesis, insertion, or activation of hexose transporters. Hexose competitively taken up with dGlc, including the nonmetabolizable glucose analogue 3-O-methylglucose, could replace glucose in the process of prevention and reversal of the deprivation effects, indicating that competitive transport but not the metabolism of hexose is a prerequisite for the regulatory effect of glucose on the activity of its own transport system. In conclusion, our results indicate that in cultured 3T3 fat cells glucose itself is involved in the regulation of the activity of its own transport system by influencing the rate of degradation, internalization, or inactivation of hexose transporters by a protein synthesis-independent mechanism.     

Hexose transport in undifferentiated and differentiated BALB/c 3T3 preadipose cells: Effects of 12–0-tetradecanoylphorbol-13-acetate and insulin

The effect of the phorbol diester 12-0-tetradecanoylphorbol-13-acetate (TPA) on hexose transport in undifferentiated and differentiated BALB/c 3T3 preadipose cells was studied. Additon of TPA to undifferentiated or fully differentiated cultures resulted in an increased rate of both 2-deoxyglucose uptake and 3-0-methylglucose transport; the time course and maximal stimulation differed for each type of culture and for each hexose. In confluent, undifferentiated cells, half-maximal stimulation of 2-deoxyglucose uptake occurred at 3 nM TPA, while the half-maximal stimulation of 3–0-methylglucose occurred at 30 nM. Epidermal growth factor and fetal bovine serum increased 2-deoxyglucose uptake in undifferentiated cells, while insulin did not. Insulin did, however, stimulate 3–0-methylglucose transport in differentiated cells. From dose-response curves in differentiated cells, halfmaximally effective concentrations were 0.17 nM for insulin and 30 nM for TPA. At optimal concentrations and incubation times for each, TPA was significantly more effective than insulin in stimulating hexose transport in differentiated cells. It was also shown that insulin could further increase hexose transport in maximally stimulated TPA-treated cells. Cycloheximide inhibited by 75% the increase in hexose transport by TPA in differentiated cells, while having no effect on the response of these cells to insulin. In differentiated cells, chronic exposure to insulin abolished the ability of these cells to respond acutely to insulin addition but they could still respond to TPA. On the other hand, differentiated cells exposed continuously to TPA for 5 days retained the ability to activate 3–0-methylglucose transport after either TPA or insulin addition. These results demonstrate that TPA can stimulate hexose transport directly in both undifferentiated and differentiated 3T3 cells and suggest that TPA and insulin affect transport by different mechanisms.     

Basic and acidic fibroblast growth factors modulate the epidermal growth factor receptor by a protein kinase C-independent pathway

Human acidic and basic fibroblast growth factors (aFGF and bFGF) inhibit epidermal growth factor (EGF) receptor binding in mouse Swiss 3T3 cells. Scatchard analysis indicates that aFGF and bFGF cause a decrease in the high affinity EGF receptor population, similar to that observed for activators of protein kinase C such as phorbol esters, platelet-derived growth factor (PDGF) and bombesin. However, unlike phorbol esters, aFGF and bFGF inhibit EGF binding in protein kinase C-deficient cells. The time course and dose response of inhibition of EGF binding by both aFGF and bFGF are very similar, with an ID50 of approximately 0.10 ng/ml. In contrast to bombesin but like PDGF, neither aFGF nor bFGF act on the EGF receptor through a pertussis toxin-sensitive G protein. These results indicate that both acidic and basic FGF depress high affinity EGF binding in Swiss 3T3 cells with similar potency through a protein kinase C/Gi-independent pathway.