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J Biol Chem, Vol. 274, Issue 36, 25785-25791, September 3, 1999


Coordinate Regulation of the alpha 2-Macroglobulin Signaling Receptor and the Low Density Lipoprotein Receptor-related Protein/alpha 2-Macroglobulin Receptor by Insulin*

Uma Kant Misra, Govind Gawdi, Mario Gonzalez-Gronow, and Salvatore V. PizzoDagger

From the Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have studied insulin-dependent regulation of macrophage alpha 2-macroglobulin signaling receptors (alpha 2MSR) and low density lipoprotein receptor-related protein/alpha 2M receptors (LRP/alpha 2MR) employing cell binding of 125I-alpha 2M*, inhibition of binding by receptor-associated protein (RAP) or Ni2+, LRP/alpha 2MR mRNA levels, and generation of second messengers. Insulin treatment increased the number of alpha 2M* high (alpha 2MSR) and low (LRP/alpha 2MR) affinity binding sites from 1,600 and 67,000 to 2,900 and 115,200 sites per cell, respectively. Neither RAP nor Ni2+ blocked the binding of 125I-alpha 2M* to alpha 2MSR on insulin- or buffer-treated cells, but they both blocked binding to LRP/alpha 2MR. Insulin significantly increased LRP/alpha 2MR mRNA levels in a dose- and time-dependent manner. Insulin-augmented 125I-alpha 2M* binding to macrophages was severely reduced by wortmannin, LY294002, PD98059, SB203580, or rapamycin. The increase in alpha 2MSR receptor synthesis was reflected by augmented generation of IP3 and increased [Ca2+]i levels upon receptor ligation. Incubation of macrophages with wortmannin, LY294002, PD98059, SB203580, rapamycin, or antibodies against insulin receptors before insulin treatment and alpha 2M* stimulation significantly reduced the insulin-augmented increase in IP3 and [Ca2+]i levels. Pretreatment of cells with actinomycin D or cycloheximide blocked the synthesis of new alpha 2MSR. In conclusion, we show here that insulin coordinately regulates macrophage alpha 2MSR and LRP/alpha 2MR, utilizing both the PI 3-kinase and Ras signaling pathways to induce new synthesis of these receptors.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human alpha 2-macroglobulin (alpha 2M)1 is a homotetrameric proteinase inhibitor present at high concentration in blood and tissue fluids (1, 2). alpha 2M reacts with endoproteinases of every mechanistic class in a reaction that induces a major conformational change in the inhibitor. This change exposes receptor recognition sites in the carboxyl terminus of each of its four subunits, leading to rapid clearance from the circulation and in vitro binding by cells expressing receptors for receptor-recognized forms of alpha 2M (alpha 2M*) (2, 3). Small nucleophiles such as methylamine directly attack internal beta -cysteinyl-gamma -glutamyl thiolesters present in each subunit causing a similar conformational change in the inhibitor with comparable exposure of receptor recognition sites (1-3). Low density lipoprotein receptor-related protein/alpha 2M receptor (LRP/alpha 2MR) is a high molecular weight cell surface receptor expressed by many cell types including macrophages, fibroblasts, hepatocytes, adipocytes, and dermal dendritic cells (2-5). LRP/alpha 2MR is a scavenger receptor (Kd ~5 nM) that binds multiple structurally and functionally diverse ligands besides alpha 2M*, including Pseudomonas exotoxin A, lipoproteinase lipase, apolipoprotein E-enriched lipoproteins, urokinase, and tissue-type plasminogen activator alone or in combination with plasminogen activator inhibitor-1, tissue factor pathway inhibitor, and lactoferrin (2, 5). Generally, these ligands do not compete with each other for binding to LRP/alpha 2MR, presumably because they bind to independent receptor domains; however, receptor-associated protein (RAP, Mr ~39,000) blocks the binding of all known ligands to this receptor (4, 5). In addition to LRP/alpha 2MR, alpha 2M* binds to a recently discovered alpha 2M signaling receptor (alpha 2MSR) (Kd ~50 pM) present on a more restricted range of cells than LRP/alpha 2MR (5-14). Binding of alpha 2M* to LRP/alpha 2MR is followed by uptake and degradation in lysosomes but not activation of a signaling cascade (6, 7, 12). By contrast, binding of alpha 2M* or its receptor binding fragment to alpha 2MSR triggers typical signaling cascades, which regulate cell proliferation (6-15). RAP and Ni2+ prevent alpha 2M* binding to LRP/alpha 2MR, but do not inhibit the ability of alpha 2M* to bind to alpha 2MSR, and they do not affect signal transduction (16). Based on a variety of evidence, we have proposed that alpha 2MSR behaves like various growth factor receptors and that it is involved in cellular growth regulation (6-14).

Insulin binding to its cognate receptor elicits a number of responses including glucose and amino acid transport, increased glycogen synthesis, gene transcription, growth regulation, and mitogenesis by activating a complex signaling cascade of lipid, protein-tyrosine, and serine/threonine kinases (17-22). Insulin-dependent signal transduction is controlled by PI 3-kinase and p21ras, respectively (17-22). Inhibition of PI 3-kinase by wortmannin or LY294002, expression of dominant inhibitory mutants of p21ras, or inhibition of farnesyl transferases suppress insulin-dependent glucose transport, gene expression, and mitogenesis (23, 24). Downstream targets of PI 3-kinase include the ribosomal p70s6k, some isoforms of protein kinase C, and the serine/threonine kinase akt, which is a direct target of PI 3-kinase (24, 25). p21ras GTP activates a cascade of protein-serine/threonine kinases, which include Raf, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase, ERK1, ERK2, MAPK, casein kinase II, p70s6k, and p90rsk, resulting in the phosphorylation of many cytosolic and nuclear proteins (24, 26-28). A number of these kinases play critical roles in the regulation of proliferation, differentiation, and cellular metabolism (27-33). We have recently reported that mitogenic effects observed upon ligation of macrophage alpha 2MSR are also accompanied by activation of the PI 3-kinase (34) and p21ras (35) signaling pathways.

A relationship between insulin and 125I-alpha 2M* receptors was first reported from this laboratory by Ney et al. (36), who showed that pretreatment of adipocytes and fibroblasts with insulin enhanced the binding of 125I-alpha 2M* to cell surface receptors and decreased receptor-mediated degradation of alpha 2M by fibroblasts. These observations were later confirmed by others (37-40). Certain growth factors, for example insulin and nerve growth factors as well as cytokinins like interleukin-Ibeta and tumor necrosis factor-alpha , also up-regulate the synthesis of LRP/alpha 2MR and low density lipoprotein receptors in neurons (41) and HepG2 cells (42). In the latter case, activation of ERK1 and -2 was required for cytokinin-induced increased expression of low density lipoprotein receptors (42). In the present study, we have studied the effect of insulin on expression of macrophage LRP/alpha 2MR and alpha 2MSR by the following parameters: 1) binding of 125I-alpha 2M* to high (alpha 2MSR) and low (LRP/alpha 2MR) affinity binding sites on the cells and generation of second messengers, namely IP3 and Ca2+; 2) RAP and Ni2+ sensitivity of binding of 125I-alpha 2M* to alpha 2MSR and LRP/alpha 2MR on buffer- or insulin-treated cells; 3) sensitivity of 125I-alpha 2M* binding and second messenger generation to PI 3-kinase inhibitors, wortmannin and LY294002, ERK1/2 inhibitor PD98059, p38MAPK inhibitor SB203580, and p70s6k inhibitor rapamycin; and 4) LRP/alpha 2MR receptor mRNA levels. Based upon these criteria, we report here that insulin appears to regulate the expression of macrophage LRP/alpha 2MR and alpha 2MSR in a coordinate manner.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Brewer's thioglycollate broth was purchased from Difco. Culture media were purchased from Life Technologies, Inc. Bovine serum albumin, pertussis toxin, staurosporin, insulin, cycloheximide, actinomycin D, genestein, HEPES, and rapamycin were purchased from Sigma. Fura-2/AM was obtained from Molecular Probes, Inc. (Eugene, OR). Myo[2-3H]inositol (specific activity 10-20 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Insulin receptor antibody was purchased from Immunotech (Westbrook, ME). A plasmid containing the RAP cDNA was a kind gift from Dr. Joachim Herz (University of Texas, Southwestern, Dallas, TX). It was used to produce RAP as described previously (11). [alpha -32P]dCTP and 125I were purchased from NEN Life Science Products. Iodobeads® were purchased from Pierce. Wortmannin, LY294002, PD98059, and SB203580 were obtained from Biomol (Plymouth Meeting, PA). Human alpha 2M was purified, converted to alpha 2M* with methylamine, and radiolabeled with 125I as described previously (6, 12). All other reagents used were of the highest grade available.

Insulin and Macrophage Binding of 125I-alpha 2M*-- Pathogen-free C57BI/6 mice (6 weeks old) were obtained from Charles River (Raleigh, NC). Binding of 125I-alpha 2M* to thioglycollate-elicited macrophages was studied as detailed earlier (12, 13, 16). 125I-alpha 2M* was added to cells in wells at varying concentrations and incubated for 2 h at 4 °C. Free ligand was separated from bound by aspirating the medium and monolayers carefully washed five times with ice-cold RPMI 1640 medium containing penicillin, streptomycin, and 2% bovine serum albumin (buffer A). The cells were lysed with 1 M NaOH at 40 °C, and bound activity was determined in a gamma -counter. The specific binding of 125I-alpha 2M* was calculated by subtracting nonspecific binding determined in the presence of 5 mM EDTA (12, 13, 16, 43). To study the effect of insulin on 125I-alpha 2M* binding, insulin (10 nM) was added to monolayers in buffer A, and monolayers were incubated for specific periods of time at 37 °C, transferred to ice, and washed five times with ice-cold buffer A. 125I-alpha 2M* was added to wells at the specified concentrations, monolayers were incubated for 2 h at 4 °C, and binding was determined as described above. The effects of PI 3-kinase inhibitor wortmannin (30 nM/30 min/37 °C) or LY294002 (20 µM/15 min/37 °C) on the binding of 125I-alpha 2M* to insulin- or buffer-treated macrophages was determined by addition to the monolayers prior to incubation with insulin (10 nM/1 h/37 °C). Cells were then washed, and binding of 125I-alpha 2M* was studied as outlined above. In experiments where the effects of PD98059 (50 µM/90 min/37 °C), SB203580 (15 µM/30 min/37 °C), or rapamycin (100 nM/5 min/37 °C) were studied on insulin-augmented cellular binding of 125I-alpha 2M*, these were added to monolayers prior to adding insulin (10 nM/1 h/37 °C), and binding of 125I-alpha 2M* was determined as outlined above. Protein in cell lysates was determined by the Bradford method (44). Kd values were calculated using the Sysstat Program as described previously (13).

Determination LRP/alpha 2MR mRNA Levels-- The effect of insulin on macrophage LRP/alpha 2MR mRNA was determined as described earlier (13). A partial human cDNA fragment ranging from base pair 188 to 6,179 inserted into plasmid pGEM 4 was used for hybridization with mRNA isolated from insulin-treated and -untreated macrophages. This plasmid was a kind gift of Dr. Joachim Herz.

Measurement of IP3 in Insulin-treated Cells-- The formation of IP3 in [3H]myoinositol-labeled macrophages under various experimental conditions was quantified essentially according to methods published earlier (6-10). In experiments where insulin effects were studied on alpha 2M*-induced changes in IP3 levels, the cells were treated with insulin at the specified concentrations or time periods, and insulin was washed out with HHBSS before stimulating with alpha 2M*. In studies where the effects of cycloheximide, actinomycin D, genestein, or staurosporin were studied on changes in IP3 in cells pretreated with insulin followed by stimulation with alpha 2M*, these agents were added at the specified concentration and incubated for the specified time period at 37 °C prior to the addition of insulin or buffer. The effects of wortmannin, LY294002, PD98059, SB203580, or rapamycin also were examined on insulin-augmented increased IP3 formation of alpha 2M*-stimulated cells. Macrophages labeled as above were incubated with the respective inhibitors, before the addition of insulin (10 nM/1 h/37 °C). The cells were incubated for 1 h, washed with HHBSS buffer containing 10 mM Li+, 1 mM Ca2+, and 1 mM Mg2+, followed by stimulation with alpha 2M* and quantification of IP3 as described above.

Insulin Receptor Antibody and Insulin-induced Changes in IP3-- These studies were performed as outlined above except that washed [3H]myoinositol-labeled macrophages (3 × 106 cells/4.5 cm2) in a volume of RPMI 1640 medium containing 0.25% bovine serum albumin, leupeptin (20 µg/ml), and phenylmethylsulfonyl fluoride (1 mM) were incubated with antibody against insulin receptors (10 µg/ml) for 30 min at 25 °C. At the end of incubation, insulin (10 nM) or buffer was added to monolayers, and the incubation continued for an additional 60 min at 37 °C. The monolayers were washed five times with ice-cold HHBSS buffer containing Li+, Ca2+, and Mg2+, and cells were incubated in this buffer for 5 min at 37 °C prior to stimulation with alpha 2M* followed by quantification of inositol phosphates (6-10).

Measurement of [Ca2+]i in Insulin-treated Cells-- Changes in [Ca2+]i levels in Fura-2/AM-loaded single cells were quantified using digital imaging microscopy as described earlier (6-10). Briefly, cells were incubated with the specified concentrations of insulin for the desired time periods and loaded with Fura-2/AM in the last 30 min of incubation. Monolayers were washed with HHBSS and stimulated with alpha 2M*, and changes in [Ca2+]i were measured as described (6-10). In experiments where the effects of insulin were studied on alpha 2M*-induced changes in [Ca2+]i levels, the cells were treated with insulin at specified concentrations or time periods, and insulin was washed out with HHBSS before stimulating with alpha 2M*. The effects of cycloheximide, actinomycin D, genestein, or staurosporin on changes in [Ca2+]i were studied in cells pretreated with insulin followed by stimulation with alpha 2M*. These agents were added at specified concentrations and incubated for the specified period of time at 37 °C prior to the addition of insulin or buffer.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin Enhances the Binding of 125I-alpha 2M* to both alpha 2MSR and LRP/alpha 2MR-- Insulin treatment increased the overall binding of 125I-alpha 2M* to macrophages by about 1.5-2-fold compared with buffer-treated cells (Fig. 1A). By Scatchard analysis, the Kd for ligand binding to the high affinity binding site (alpha 2MSR) was 50 pM, and the number of binding sites per cell was 1,600 (Fig. 1B). The Kd for ligand binding to the low affinity binding sites (LRP/alpha 2MR) was 25 nM, and the number of binding sites per cell was 67,000 (Fig. 1B). Incubation of macrophages with insulin prior to binding of ligand had little effect on the Kd of binding to either site, but it increased the number of both the high affinity (2,800) and the low affinity (115,200) sites, respectively. RAP (10-fold excess) did not block binding to the high affinity sites in buffer- or insulin-treated macrophages, but it reduced binding to the low affinity site for the ligand by about 50% in both buffer and insulin-treated cells (Fig. 1B). Like RAP, Ni2+, which inhibits the binding of alpha 2M* to LRP/alpha 2MR but not to alpha 2MSR (16), also inhibited the binding of 125I-alpha 2M* by about 60% in both buffer- and insulin-treated cells (Fig. 1C). The maximal binding of 125I-alpha 2M* occurred when cells were exposed to 10 nM insulin, while at lower or higher concentrations, insulin treatment markedly reduced ligand binding (Fig. 2).


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  		Fig. 1.   Direct binding of 125I-alpha 2M* to macrophage cell surface receptors and Scatchard analysis of binding. Details of binding studies are given under "Experimental Procedures." A, the binding of 125I-alpha 2M* was determined in buffer-treated (triangle ) or insulin-treated (black-triangle) macrophages (2-2.5 × 105 cells) at the specified ligand concentrations. The specific binding of the ligand was calculated by subtracting nonspecific binding determined in the presence of 5 mM EDTA from total binding. The values are mean ± S.E. from four independent experiments performed in quadruplicate. The effect of RAP (10-fold excess) on binding of 125I-alpha 2M* at various ligand concentrations in buffer-treated (open circle ) or insulin-treated () macrophages was determined. The specific binding was calculated as above. The values are mean ± S.E. from three independent experiments performed in quadruplicate. B, Scatchard analysis of the binding of 125I-alpha 2M* to buffer-treated () or insulin-treated (10 nM/1 h) (black-square) macrophages. C, effect of Ni2+ treatment (10 mM) on 125I-alpha 2M* binding to macrophages. The bars represent treatment with buffer (1), Ni2+ (2), insulin (10 nM/1 h) (3), and insulin followed by Ni2+ (4). Values are mean ± S.E. from two experiments performed in quadruplicate.


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  		Fig. 2.   Insulin concentration and binding of 125I-alpha 2M*. Macrophages (5-6 × 105 cells) were incubated with the indicated concentrations of insulin for 1 h at 37 °C and washed, and binding of 125I-alpha 2M* was determined as detailed under "Experimental Procedures." Values are mean ± S.E. from two experiments done in quadruplicate.

Insulin Increases mRNA Levels of LRP/alpha 2MR-- The maximum increase in LRP/alpha 2MR mRNA levels occurred when cells were treated with 10 nM insulin, but above or below this insulin concentration, a marked decrease in mRNA was observed (Fig. 3A). LRP/alpha 2MR expression was maximal at 1 h of incubation with 10 nM insulin (Fig. 3B). The presence of actinomycin D before and during insulin treatment nearly abolished the insulin-induced increase in receptor mRNA, demonstrating that the increased mRNA level observed in insulin-treated cells was due to insulin-induced new RNA synthesis (Fig. 3B).


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  		Fig. 3.   mRNA levels of LRP/alpha 2MR by Northern blot hybridization. A, effect of insulin concentration on receptor mRNA levels. Prior to extraction of mRNA, macrophages were incubated with various concentrations of insulin, alpha 2M*, or buffer at 37 °C, and mRNA was extracted. mRNA (0.75 µg in each case) was used for Northern blot hybridization. The slots are labeled with the insulin concentration employed. The last slot is alpha 2M* without insulin. B, modulation of insulin increase in LRP/alpha 2MR mRNA levels. Macrophages were treated with insulin (10 nM) for varying periods of time, and their mRNA was isolated and analyzed (0.75 µg in each case). In some experiments, cells were treated with actinomycin D (10 µg/ml/20 min) before adding insulin. The data presented are representative of two independent experiments. The slots are alpha 2M* (100 pM) (1), actinomycin D prior to insulin (10 nM/1 h) (2), reagent blank without mRNA (3), insulin (10 nM/1 h) (4), insulin (10 nM/2 h) (5), and insulin (10 nM/4 h) (6).

PI 3-Kinase and MAPK Inhibitors Reduce Insulin-induced Increased Binding of 125I-alpha 2M* to Cells-- Wortmannin (45) and LY294002 (46), both inhibitors of PI 3-kinase, reduced insulin-augmented increased cellular binding of 125I-alpha 2M* without significantly altering basal ligand binding (Fig. 4A). PI 3-kinase activity is required for activation of p70s6k, which plays a potential role in regulating protein synthesis by phosphorylation of ribosomal S6 kinase (47). Treatment of cells with rapamycin, an inhibitor of p70s6k (48), before insulin treatment reduced the insulin-augmented increased binding of 125I-alpha 2M* without significantly altering the basal binding of 125I-alpha 2M* to macrophages (Fig. 4B). Activation of the Ras pathway is the second route of signal transduction that has been implicated in the induction of gene expression by insulin (49). Components of the pathway that includes ERK1, ERK2, and p38MAPK are mediators of phosphorylation of intracellular substrates such as protein kinases and transcription factors as well as regulators of growth factor-induced cell growth and differentiation (50, 51). Treatment of cells with PD98059, a specific inhibitor of ERK1 and ERK2 (52), or SB203580, a specific inhibitor of p38MAPK (53), before insulin treatment, like the PI 3-kinase and p70s6k inhibitors significantly decreased the insulin-augmented increased binding of 125I-alpha 2M* to macrophages without significantly affecting basal binding of the ligand (Fig. 4B). These results support the hypothesis that insulin increased the binding of 125I-alpha 2M* to macrophages by activating both the PI 3-kinase and Ras signaling pathways to achieve this synthesis.


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  		Fig. 4.   Effect of PI 3-kinase, MAPK, and p70s6k inhibitors on insulin-induced increased binding of 125I-alpha 2M* to macrophages. Experimental details are given under "Experimental Procedures." A, effect of wortmannin and LY294002 treatment of cells. The bars represent treatment with buffer (1), insulin (10 nM/1 h) (2), wortmannin (30 nM/30 min) followed by insulin (10 nM/1 h) (3), and LY294002 (20 µM/15 min) followed by insulin (10 nM/1 h) (4). B, effect of MAPK and p70s6k inhibitor treatment of cells. The bars represent treatment with buffer (1), insulin (10 nM/1 h) (2), PD98059 (50 µM/90 min) followed by insulin (10 nM/1 h) (3), SB203580 (15 µM/30 min) followed by insulin (10 nM/1 h) (4), and rapamycin (100 nM/5 min) followed by insulin (10 nM)/1 h) (5). Values are mean ± S.E. from two independent experiments performed in quadruplicate.

Insulin-induced Increase in IP3 Synthesis in alpha 2M*-stimulated Cells Is Abolished by Actinomycin D-- Binding of alpha 2M* to alpha 2MSR, but not to LRP/alpha 2MR, generates a signaling cascade (6-15). Incubation of macrophages with insulin prior to stimulation with alpha 2M* increased IP3 levels in a biphasic manner (Fig. 5). In insulin-treated cells, a 2-4-fold increase in IP3 formation was observed in the initial 60 s of agonist stimulation, followed by another sustained increase of up to 5 min compared with buffer-treated cells (Fig. 5A). The generation of IP3 after alpha 2M* stimulation of insulin-pretreated macrophages was proportional to the concentration of insulin up to 10 nM, but incubation of macrophages with higher concentrations of insulin caused a gradual decrease in IP3 formation (Fig. 5B). The maximal stimulation of IP3 synthesis occurred after 1 h of insulin treatment, and a decrease in IP3 formation occurred after longer treatment. Insulin by itself showed no effect on the generation of IP3 in macrophages under the experimental conditions. Preincubation of macrophages with actinomycin D or cycloheximide before insulin treatment showed little effect on the alpha 2M*-induced early increase in IP3 levels, but this treatment abolished the insulin-induced sustained increase in IP3 levels (Fig. 6A). The addition of actinomycin D after incubation of macrophages with insulin followed by stimulation with alpha 2M* had no effect on insulin-induced increase in IP3. These results suggest that the potentiated generation of IP3 in insulin-treated cells arises as a consequence of the increased number of newly synthesized alpha 2MSR. Treatment of macrophages with genestein (a tyrosine kinase inhibitor) or staurosporin (a protein kinase C inhibitor) prior to incubation with insulin followed by stimulation with alpha 2M* abolished the augmented increase in IP3 production (Fig. 6B). Thus, the involvement of protein kinase C and tyrosine kinases in insulin-induced up-regulation of alpha 2MSR and second messenger events following alpha 2MSR activation is evident.


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  		Fig. 5.   Insulin-induced changes in IP3 levels in macrophages stimulated with alpha 2M*. Details of quantification of IP3 are described under "Experimental Procedures." A, shown are buffer followed by alpha 2M* (100 pM) (open circle ), insulin (10 nM/1 h) followed by alpha 2M* (100 pM) (), and insulin alone (triangle ). Values are mean ± S.E. from four independent experiments. B, effect of insulin concentration on IP3 formation. Values are mean ± S.E. from three independent experiments and are expressed percentage change in IP3 over basal levels.


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  		Fig. 6.   Modulation of insulin-induced increased synthesis of IP3 on alpha 2M* stimulation. Experimental details are given under "Experimental Procedures." A, shown are buffer followed by alpha 2M* (), insulin followed by alpha 2M* (black-square), actinomycin D prior to insulin followed by alpha 2M* (), insulin prior to actinomycin D followed by alpha 2M* (black-triangle), and cycloheximide prior to insulin followed by alpha 2M* (triangle ). Values are the mean ± S.E. from two or three independent experiments in each case. B, effect of genestein, staurosporin, and antibody against insulin receptor on insulin-induced increase in IP3 synthesis after alpha 2M* stimulation. The bars represent treatment with buffer followed by alpha 2M* (100 pM) (1), insulin (10 nM/1 h) followed by alpha 2M* (100 pM) (2), staurosporin (20 nM/16 h) prior to insulin (10 nM/1 h) followed by alpha 2M* (3), and genestein (20 µM/16 h) prior to insulin (10 nM/1 h) followed by alpha 2M* (100 pM) (4), and insulin receptor antibody (10 µg/ml 30 min/25 °C) prior to insulin (10 nM/1 h) followed by alpha 2M* (100 pM) (5). Values are mean ± S.E. from two or three experiments performed in duplicate and are expressed as percentage change in IP3 synthesis at 60 s after alpha 2M* stimulation.

Effect of Antibodies against Insulin Receptor on Insulin-induced Increase in IP3 Generation-- To demonstrate that the effects of insulin on the binding of alpha 2M* and generation of IP3 occur consequent to its binding to insulin receptors on macrophages, we next studied the effect of incubating macrophages with insulin receptor antibody before adding insulin (Fig. 6B). As expected, treatment of macrophages with antibody against membrane insulin receptors abolished the insulin-induced augmented increase in IP3 generation seen upon alpha 2M* stimulation, but it showed very little effect on IP3 generation in macrophages treated with buffer prior to stimulation with alpha 2M* (Fig. 6B). These results show that the increased level of IP3 observed on alpha 2M* stimulation of insulin-treated macrophages results from first binding of insulin to its receptor followed by sequential activation of downstream signal transduction events, resulting in up-regulation of alpha 2MSR.

Inhibition of Insulin-induced Increase in IP3 Formation by PI 3-Kinase and MAPK Pathways Inhibitors-- We next evaluated the involvement of the PI 3-kinase and MAPK pathways in the insulin-induced up-regulation of alpha 2MSR by quantifying the generation of IP3 in cells treated with PI 3-kinase pathway inhibitors wortmannin and LY294002, and MAPK pathway inhibitors PD98059 and SB203580, and the p70s6k inhibitor rapamycin before insulin treatment and alpha 2M* stimulation (Fig. 7). As expected, inhibitor treatment significantly reduced only the insulin-augmented increase in IP3 generation but not basal levels observed upon stimulation with alpha 2M* (Fig. 7). These results suggest that the potentiated increase in IP3 synthesis observed in insulin-treated macrophages occurs due to the availability of increased numbers of receptors and that insulin utilizes both PI 3-kinase and MAPK pathways downstream of insulin stimulation to induce this new synthesis of alpha 2MSR.


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  		Fig. 7.   PI 3-kinase, MAPK, and p70s6k inhibitors and insulin-induced increased synthesis of IP3 on alpha 2M* stimulation. Details are described under "Experimental Procedures." The bars represent treatment with buffer (1), alpha 2M* (100 pM) (2), insulin (10 nM/1 h) followed by alpha 2M* (100 pM) (3), wortmannin (30 nM/30 min) prior to insulin followed by alpha 2M* (100 pM) (4), LY294002 (20 nM/15 min) prior to insulin followed by alpha 2M* (100 pM) (5), PD98059 (50 µM/90 min) prior to insulin followed by alpha 2M* (100 pM) (6), SB203580 (15 µM/30 min) prior to insulin followed by alpha 2M* (100 pM) (7), and rapamycin (100 nM/5 min) prior to insulin followed by alpha 2M* (100 pM) (8). Values are average of two or three independent experiments and are expressed as percentage change in IP3 synthesis at 60 s after agonist stimulation.

Effect of Insulin on [Ca2+]i Levels in alpha 2M*-stimulated Macrophages-- Many extracellular stimuli cause an increase in cytosolic [Ca2+]i by stimulating formation of IP3, which then binds to the intracellular IP3 receptor, an ion channel. This causes its opening and release of Ca2+ from intracellular membrane-bound compartments. This is followed by the entry of Ca2+ from the extracellular medium by capacitative Ca2+ entry mechanisms (54). Treatment of macrophages with insulin prior to stimulation with alpha 2M* increased [Ca2+]i levels by 2-3-fold compared with buffer-treated macrophages (Fig. 8A). In a typical experiment, [Ca2+]i levels in unstimulated cells, alpha 2M*-stimulated cells, and insulin-treated and alpha 2M*-stimulated cells were 144.51 ± 4.58, 356.78 ± 13.79, and 836.18 ± 15.80 nM, respectively. The increase in [Ca2+]i levels was proportional to insulin concentration, being maximal at 10 nM and showing a slight decrease thereafter (Fig. 8B). As would be predicted, the effect of insulin concentration on [Ca2+]i levels upon alpha 2M* stimulation is very similar to the augmented increase in IP3 levels that was observed (Fig. 5). Increase in [Ca2+]i on alpha 2M* stimulation of insulin-treated macrophages was maximal at 1 h of insulin treatment, and longer incubation resulted in a slight decrease in [Ca2+]i levels. Incubation of macrophages with actinomycin D, cycloheximide, genestein, or staurosporin in separate experiments before treatment with insulin and stimulation with alpha 2M* suppressed the insulin-augmented increase in [Ca2+]i levels but only slightly affected alpha 2M*-induced increases in [Ca2+]i levels in buffer-treated macrophages (Fig. 8C).


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  		Fig. 8.   Insulin-induced changes in [Ca2+]i on alpha 2M* stimulation. A, shown are buffer-treated macrophages stimulated with alpha 2M* (100 M) (open circle ), macrophages incubated with insulin (10 nM/1 h/37 °C) and then stimulated with alpha 2M* (100 pM) as above (), and buffer-treated macrophages stimulated with insulin (10 nM) (×). The results shown are representative of 4-6 individual experiments using 20-25 cells per experiment in each case. The arrow indicates the time of addition. B, effect of insulin concentration on changes in [Ca2+]i on alpha 2M* stimulation. Macrophages were treated with indicated concentrations of insulin for 1 h and then stimulated with alpha 2M*. Values are mean ± S.E. performed in three independent experiments and are expressed as percentage change in [Ca2+]i levels over the basal value. C, modulation of insulin-induced increase in [Ca2+]i levels. Macrophages were treated with buffer followed by alpha 2M* (1), insulin (10 nM/1 h) followed by alpha 2M* (100 pM) (2), cycloheximide prior to insulin followed by alpha 2M* (100 pM) (3), actinomycin D prior to insulin followed by alpha 2M* (100 pM) (100 pM) (4), and staurosporin prior to insulin followed by alpha 2M* (5). The results are representative of three experiments using 25-30 cells in each case. Values are mean ± S.E. from three or four individual experiments using 20-25 cells in each case and are expressed as percentage change in [Ca2+]i levels over the basal value.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The primary observations of this study are that insulin treatment of peritoneal macrophages prior to stimulation with alpha 2M* are as follows. 1) Insulin treatment increases the number of both high (alpha 2MSR) and low (LRP/alpha 2MR) affinity binding sites by about 2-fold. 2) Treatment augments the increase in two second messengers, namely IP3 and [Ca2+]i, observed upon ligation of alpha 2MSR with alpha 2M*. 3) Increases in 125I-alpha 2M* binding, IP3, and [Ca2+]i levels observed upon alpha 2M* stimulation are drastically reduced by preincubation of macrophages with antibodies against insulin receptors, actinomycin D, cycloheximide, PI 3-kinase inhibitors, a p70s6k inhibitor, an ERK1/2 inhibitor, and a p38MAPK inhibitor. 4) increases in 125I-alpha 2M* binding to macrophages are to a large extent contributed by new synthesis of receptors. The results further demonstrate that insulin exerts a coordinate regulation of the synthesis of both LRP/alpha 2MR and alpha 2MSR. These results contrast to previous suggestions that the insulin-augmented increase in the binding of 125I-alpha 2M* to LRP/alpha 2MR occurred because of redistribution of receptors (36-39), decreased endocytosis (36-39, 55), or increased half-life of receptor-ligand complex (36).

Two mechanisms by which insulin exerts its metabolic and mitogenic effects after binding to receptors are activation of PI 3-kinase and its downstream signaling pathway and activation of the small GTP binding protein p21ras and its downstream protein kinases (17-22). Inhibition of PI 3-kinase activity by wortmannin and LY294002 attenuates the stimulation of glucose transport and mitogenesis by insulin (27). This lipid kinase is linked to the regulatory cascade that controls p70s6k and activation by insulin, which can be selectively blocked by rapamycin (26, 56). The p21ras pathway has been implicated in the induction of gene expression by insulin (17-21, 26, 57). The expression of dominant inhibitory mutants of p21ras inhibited insulin-stimulated gene expression (26). Selective inhibition of farnesyl protein transferase prevented the attachment of Ras proteins to plasma membranes and adversely affected their activation by growth factors (58). In macrophages, ligation of alpha 2MSR results in activation of the p21ras (35) and PI 3-kinase (34) signaling pathways, and we have proposed that these mechanisms are involved in alpha 2M*-induced mitogenesis (13, 14).

In most cells, insulin signaling extends to the nucleus with changes in transcription of a variety of specific genes (20, 21, 32, 59). Insulin receptors have been identified in nuclei (60, 61) causing stimulation of nuclear tyrosine kinase activity, which could be important in insulin action at the nuclear level (60-62). Insulin regulates the transcription and turnover of mRNAs that encode proteins involved both in growth and metabolic processes (17-22), stimulates phosphorylation of a number of transcription factors, and DNA-binding proteins (20-22, 32, 59), and increases the levels of several specific mRNAs including that of the p33 gene, c-fos, c-jun, early growth response genes, lipoprotein lipase (20, 21, 32, 59). The present work demonstrates that a coordinate up-regulation of LRP/alpha 2MR and alpha 2MR by insulin also occurs. The potentiated, biphasic increase in IP3 generation in cells treated with insulin first and subsequently stimulated with alpha 2M* is largely attributed to the availability of increased number of newly synthesized alpha 2MSR molecules available for occupancy.

When insulin-treated cells are exposed to a alpha 2M*, it binds to two pools of alpha 2MSR, one a constitutive/basal pool, unaffected by actinomycin D, and the second a newly synthesized pool, which is greatly reduced by actinomycin D, cycloheximide, genestein, staurosporin, PI 3-kinase inhibitors, MAPK pathway inhibitors, and antibodies against insulin receptors. The insulin-expanded and constitutive/basal receptor pools, upon alpha 2M* binding, trigger PIP2 hydrolysis, generating IP3 that, upon binding to IP3 receptors, causes higher levels of [Ca2+]i. These events trigger the onset of several intracellular signaling cascades that act in synergy with those activated by insulin consequent to its binding to receptors and thus enhance the cellular responses. Based upon the similarities in second messenger generation elicited upon ligation of alpha 2MSR with those elicited upon ligation of growth factor receptors with their cognate ligands, we have hypothesized that alpha 2MSR is a growth factor-like receptor whose ligation with receptor-recognized forms of alpha 2M generates a series of signaling events that play a role in growth and that this receptor may be involved in tissue repair. Thus, in the presence of a physiological concentration of insulin, the synergy or "cross-talk" between insulin and alpha 2MSR may be physiologically important.

    FOOTNOTES

* This work was supported by NHLBI, National Institutes of Health, Grant HL-24066.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: (919) 684-3528; Fax: (919) 684-8689; E-mail: Pizzo001@mc.duke.edu.

1 The abbreviations and trivial names used are: alpha 2M, alpha 2-macroglobulin; alpha 2M*, receptor-recognized forms of alpha 2M; alpha 2MSR, the alpha 2M* signaling receptor; LRP/alpha 2MR, the low density lipoprotein receptor-related protein/alpha 2M receptor; RAP, the receptor-associated protein; PI, phosphoinositide; IP3, inositol 1,4,5-trisphosphate; HHBSS, Hanks' balanced salt solution containing Hepes, pH 7.4, and 3.5 nM NaHCO3; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one; SB203580, [4-(4-flurophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H imidazole; PD98059, 2'-amino-3-methoxyflavone; ERK1/2, extracellular signal-regulated kinase, also termed p42/44MAPK; MAPK, mitogen-activated protein kinase; p70s6k, 70-kDa ribosomal S6 kinase; p90rsk, 90-kDa ribosomal S6 kinase.

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ABSTRACT
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RESULTS
DISCUSSION
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