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Endocrinology Vol. 143, No. 2 655-664
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

PLC-{gamma}1 Enzyme Activity Is Required for Insulin-Induced DNA Synthesis

Jens Eichhorn, Ayse G. Kayali, Laura Resor, Darrell A. Austin, David W. Rose and Nicholas J. G. Webster

Medical Research Service (J.E., A.G.K., L.R., D.A.A., N.J.G.W.), San Diego Veterans Affairs Healthcare System, San Diego, California 92161; and University of California San Diego/Whittier Diabetes Program (J.E., A.G.K., D.W.R., N.J.G.W.), Department of Medicine (J.E., A.G.K., D.W.R., N.J.G.W.), and University of California San Diego Cancer Center (N.J.G.W.), University of California, San Diego, California 92093

Address all correspondence and requests for reprints to: Nicholas J. G. Webster, Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: nwebster{at}ucsd.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previously, we had shown that inhibition of PLC activity impaired the ability of insulin to activate ERK in 3T3-L1 adipocytes. In this study, we confirmed that the insulin receptor and PLC-{gamma}1 are physically associated in hIRcB fibroblasts, insulin stimulates PLC-{gamma}1 enzyme activity, and inhibition of PLC activity impairs activation of ERK. We subsequently investigated whether PLC-{gamma}1 is required for insulin-stimulated mitogenesis. First, inhibition of PLC activity using U73122 impairs the ability of insulin to stimulate DNA synthesis. Second, disruption of the interaction of the insulin receptor with PLC-{gamma}1 by microinjection of SH2 domains derived from PLC-{gamma}1 or Grb2 but not Shc similarly blocks insulin-induced DNA synthesis. Third, microinjection of neutralizing antibodies to PLC-{gamma}1 blocks DNA synthesis, but nonneutralizing antibodies do not. The blockade in all three cases is rescued by synthetic diacylglycerols but not by inositol-1,4,5-trisphosphate, indicating a requirement for PLC enzyme activity. These experimental data point to a requirement for PLC-{gamma}1 in insulin-stimulated mitogenesis in hIRcB cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN'S MAJOR PHYSIOLOGICAL effects in the adult are the regulation of plasma glucose levels by stimulation of glucose uptake into target tissues and suppression of hepatic gluconeogenesis, and stimulation of lipid storage by inhibition of lipolysis and stimulation of triglyceride synthesis. All of these actions are mediated by the insulin receptor (IR) (1, 2). However, the IR also has important nonmetabolic effects. Homozygous deletion of the IR causes leprechaunism and growth retardation in humans (3). Insulin and IGF-II can stimulate the proliferation of a number of physiologically important cells via the IR. In the case of IGF-II, stimulation is via the type A insulin receptor, which lacks exon 11 (4). This particular isoform of the IR is expressed highly in the embryo. Knockout studies in mice have confirmed that IGF-II acts via both the classical IGF-IR, and the type A IR to regulate embryonic growth (5, 6). Hence, the IR has an important function to regulate mitogenesis and proliferation in addition to its metabolic effects. Recently, our laboratory has demonstrated that the insulin receptor and PLC-{gamma}1 physically interact in 3T3-L1 adipocytes and that insulin stimulates PLC-{gamma}1 enzyme activity in these cells (7). Attenuation of the PLC signaling pathway using pharmacological inhibitors or microinjection of inhibitory SH2 domains impairs the ability of insulin to stimulate maximal glucose transport and GLUT4 translocation leading us to propose that this pathway may modulate metabolic signaling. More importantly, we observed a diminution in the ability of insulin to activate ERK in these adipocytes. Because the ras-Raf-MEK-ERK cascade is intimately involved in mitogenesis, this latter result suggested that PLC activation might be involved in insulin-stimulated cellular proliferation.

Several studies have shown the importance of the PLC signaling pathway for proliferation in other cell systems (8, 9, 10, 11). In particular, the {gamma}1 and {gamma}2 isoforms of PLC are activated by receptor tyrosine kinases such as platelet-derived growth factor receptor, epidermal growth factor receptor, nerve growth factor receptor, and fibroblast growth factor receptor, and nonreceptor tyrosine kinases such as Src, Syk, and Jak/Tyk, respectively. These PLC{gamma} isoforms contain tandem SH2 domains and an SH3 domain in the linker region between the X and Y catalytic domains that serves to inhibit enzyme activity (12). Binding of the tandem SH2 domains to phosphotyrosine residues on tyrosine kinases relieves this repression and allows tyrosine phosphorylation of PLC-{gamma}1, which activates the enzyme further (13, 14, 15, 16). Disruption of this interaction between PLC-{gamma}1 and the upstream tyrosine kinases prevents growth factor-induced mitogenesis (17). Homozygous deletion of the PLC-{gamma}1 gene in mice causes embryonic lethality at embryonic d 9 (18). Mouse embryonic fibroblasts from null embryos have reduced PLC activity but do proliferate in culture in response to serum and growth factors, however, because they express low levels of PLC-{gamma}2 (19, 20). The {gamma}2 isoform is expressed predominantly in hematopoietic cells and can compensate for loss of PLC-{gamma}1 during early development until d 9 when cells commit to different lineages. Evidence also exists for a nuclear PLC cycle that is regulated by growth factor-induced phosphorylation of PLC-ß1, an isoform that lacks SH2 domains and is usually regulated by G protein signaling (21, 22, 23, 24, 25). Inhibition of this nuclear cycle also impairs the mitogenic response to growth factors.

The role for PLC-{gamma}1 in insulin-stimulated mitogenesis has not been established. Our finding that inhibition of PLC activity impaired that ability of insulin to activate ERK in 3T3-L1 adipocytes implicated PLC signaling; however, these cells are terminally differentiated, do not proliferate, and express the type B IR, which contains exon 11 and does not bind IGF-II (26). Consequently, we investigated whether PLC-{gamma}1 signaling is an integral part of insulin-stimulated DNA synthesis in a well-established model system, the hIRcB cells. These cells are derived from Rat-1 fibroblasts, overexpress the type A IR, and proliferate in response to insulin. In this paper, we provide evidence that enzymatic activity of PLC-{gamma}1 is required for insulin-induced activation of ERK and stimulation of DNA synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Porcine insulin was kindly provided by Eli Lilly \|[amp ]\| Co. (Indianapolis, IN). The PLC inhibitor U73122 and the control reagent U73343 were obtained from Calbiochem (San Diego, CA). Mixed monoclonal PLC-{gamma}1 antibodies (05–163) and recombinant protein A-agarose were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal PLC-{gamma}1 (sc-81) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). ERK1/2 antibodies (M12320) and antiphosphotyrosine antibodies (pY20) were from Transduction Laboratories, Inc. (Lexington, KY). Phospho-ERK antibodies (anti-ACTIVE MAPK) were from Promega Corp. (Madison, WI). Enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech (Piscataway, NJ). Polyvinylidene difluoride (PVDF) membranes (Immobilon-P) were from Millipore Corp. (Bedford, MA). Glutathione-Sepharose beads were from Amersham Pharmacia Biotech. Unless noted, all other reagents were supplied by Sigma (St. Louis, MO) or Fisher Scientific Co. (Springfield, NJ).

Cell culture
hIRcB fibroblasts were cultured in DMEM/F12 containing 10% FCS, 2 mM glutamax, and 500 nM methothrexate. Before experimental manipulations, the serum-containing medium was removed and replaced with fresh serum-free Cellgro DMEM (Mediatech Inc., Herndon, VA) to render cells quiescent.

Purification of glutathione S-transferase (GST)-fusion proteins and SH2 pull-down assay
The GST fusion proteins used in this article have been described previously (7). Proteins were expressed in Escherichia coli BL-21 cells, induced by isopropyl ß-D-thiogalactoside for 4 h, and purified on glutathione-Sepharose beads using standard procedures. Fusion proteins were eluted in 10 mM glutathione in PBS and then dialyzed extensively with microinjection buffer (5 mM Na3PO4/100 mM KCl). For the SH2 pull-down assay, wheat germ agglutinin-purified IRs were stimulated with insulin for 4 h on ice and then allowed to phosphorylate in the presence of 50 µM ATP at room temperature in kinase buffer (10 mM HEPES (pH 7.4), 10 mM MnCl2, 0.05% Triton X-100). The GST-fusion proteins (1–8 µg) were added and incubated for 60 min at room temperature and then precipitated on glutathione-Sepharose beads for an additional 30 min. The Sepharose beads were pelleted by centrifugation, washed three times in kinase buffer, and the associated proteins were boiled in SDS sample buffer and separated by SDS-PAGE and transferred to PVDF membranes. Tyrosine-phosphorylated proteins were detected by immunoblotting with anti-phosphotyrosine antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence.

Coprecipitation of IR and PLC-{gamma}1
The hIRcB cells were rendered quiescent by serum starvation for 36 h and stimulated with insulin or vehicle for 5 min. Cells were lysed on ice in lysis buffer (1% Triton X-100, 50 mM HEPES, pH 7.4, 10 mM EDTA, 150 mM NaCl, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10% glycerol, 4 mM sodium vanadate, 200 mM sodium fluoride, 20 mM sodium pyrophosphate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) for 30 min, and then extracts were clarified of nuclei and cell debris by centrifugation at 14,000 x g for 15 min at 4 C. Aliquots of extracts containing equal amounts of protein were immunoprecipitated with polyclonal antibodies to PLC-{gamma}1 for 4 h at 4 C followed by protein A/G agarose. PLC-{gamma}1 immunoprecipitates were washed extensively in lysis buffer and then boiled in SDS sample buffer. Precipitated proteins and portions of the remaining supernatants (10%) were separated by SDS-PAGE and transferred to PVDF membranes. Tyrosine phosphorylated proteins were detected by immunoblotting with an antiphosphotyrosine antibody followed by HRP-conjugated secondary antibodies and enhanced chemiluminescence.

Assay for PLC activity
The hIRcB cells were starved in serum-free DMEM for 36 h and treated with insulin (100 ng/ml) or vehicle for 1 min. Cells were solubilized in an extraction buffer containing 50 mM HEPES, 1% Triton X-100, 10 mM EDTA, 150 mM NaCl, 2 mM PMSF, 10% glycerol, 4 mM sodium vanadate, 200 mM sodium fluoride, 20 mM sodium pyrophosphate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. After centrifugation at 15,000 x g for 10 min, PLC-{gamma}1 was immunoprecipitated with either mouse monoclonal antibodies or rabbit polyclonal antibodies for 2 h followed by an overnight incubation with protein A agarose. The immunoprecipitates were washed three times in PLC assay buffer containing 5 mM Tris-HCL, pH 7.0, 75 mM KCl, 0.416 mM CaCl2, 0.4 mM EGTA, and 0.1 mM NaN3. The PLC activity was assayed as described previously for the purified enzyme with minor modifications (27, 28). Briefly, the immunoprecipitates were incubated at 37 C for 60 min in PLC assay buffer (100 µl final volume) containing 0.5 mM dithiothreitol and small unilamellar liposomes. The liposomes were prepared by sonication and composed of 30 µM cholesterol, 50 µM phosphatidylcholine, 30 µM phosphatidylethanolamine, 5 µM PI-(4,5)P2, 25,000 dpm of labeled [3H]-PI-(4,5)P2, and 10 µM profilin. Profilin was purified from human platelets by polyproline affinity chromatography. IP3 was separated from unhydrolyzed phosphatidylinositol 4,5-bisphosphate by organic extraction and counted.

MAPK phosphorylation and expression
The hIRcB cells were starved in serum-free DMEM medium for 36 h. Cells were pretreated with U73122, U73343, and PD98059 at the indicated concentrations and stimulated with insulin for 5 min. Cells were scraped into SDS-sample buffer containing 2 mM sodium orthovanadate, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM PMSF, 10 µg/ml aprotinin, 750 µM benzamidine, and 1 mM dichloroacetic acid. The samples were boiled and sonicated, and the proteins were resolved on a 10% SDS-PAGE gel. After being transferred to PVDF membranes, the proteins were immunoblotted with antibodies to the dually phosphorylated forms of ERK1 and 2 followed by an HRP-conjugated secondary antibody and chemiluminescent detection. Membranes were stripped and reblotted with a mixture of antibodies to native ERK1 and ERK2 to demonstrate equal loading.

Measurement of thymidine incorporation
The hIRcB cells were grown to 75% confluence in 6-well cluster plates. The growth medium was replaced with serum-free DMEM for 36 h. Cells were pretreated with U73122 or U73343 for 20 min and stimulated with insulin at the indicated concentrations for 18 h. Cells were pulsed with [3H]-thymidine, 2 µCi/ml, for 1 h at 37 C. The cells were washed five times in PBS, solubilized, the DNA precipitated with 10% trichloroacetic acid at 4 C, and counted in a scintillation counter.

Microinjection of hIRcB cells and staining for BrdU
The hIRcB cells were trypsinized and reseeded on acid-washed coverslips. Cells were injected as described previously using an Eppendorf (Brinkman, Westbury, NY) 5210 microinjector and micromanipulator (7). Briefly, cells were grown to 50% confluence and starved in serum-free DMEM for 36 h. Antibodies or GST fusion proteins were solubilized in a buffer consisting of 5 mM Na3PO4 and 100 mM KCl, pH 7.4, and then microinjected into cells along with sheep IgG as a marker, using glass capillary needles. Cells were allowed to recover for 1 h after microinjection and treated with the indicated agents and stimulated with 100 ng/ml insulin. GST fusion proteins were used at a concentration of 5 mg/ml. PLC-{gamma}1 antibodies were used at a concentration of 4 mg/ml. Bromodeoxyuridine (BrdU) was added after 12 h and the cells were incubated for an additional 4 h. Subsequently, cells were fixed with 3.7% formaldehyde in PBS. After washing, permeabilization, and blocking with 0.1% Triton X-100 and 2% FCS, cells were incubated with rat anti-BrdU antibodies, followed by incubation with fluorescein-conjugated antimouse or rabbit IgG to identify injected cells and rhodamine-labeled antirat IgG to detect BrdU. Immunofluorescence was detected using an Axiophot inverted fluorescence microscope (Carl Zeiss). Black-and-white images were recorded using a digital camera connected to a workstation running Inovision ISEE software (Inovision, Raleigh, NC). False colors were added using Adobe Photoshop (San José, CA) version 6.0.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PLC-{gamma}1 interacts with the IR in hIRcB cells
Previously we have shown that the IR and PLC-{gamma}1 are physically associated in 3T3-L1 adipocytes. We verified here that the two proteins also associate in another insulin-responsive cell, the hIRcB fibroblast. Wheat germ-agglutinin-purified IRs were stimulated with insulin and allowed to phosphorylate in the presence of ATP. Phosphorylated receptors were then precipitated with increasing amounts of GST-fusion protein containing the N-terminal SH2 domain from either PLC-{gamma}1 or the p85 subunit of PI-3Kinase for 1 h. The GST-fusion protein was precipitated using glutathione-Sepharose beads and associated proteins were detected by antiphosphotyrosine immunoblotting (Fig. 1Go, top panel). Both the PLC-{gamma}1 and p85 SH2 domains were able to precipitate the phosphorylated IR with similar efficiencies. An SH2 domain from Shc was unable to precipitate the IR (data not shown).



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  		Figure 1. PLC-{gamma}1 coprecipitates with the insulin receptor in hIRcB cells. Top panel, WGA-purified IRs were stimulated with insulin and allowed to phosphorylate in the presence of ATP. Phosphorylated receptors were precipitated with GST-fusion proteins containing the N-terminal SH2 domain from PLC-{gamma}1 or the p85 subunit of PI-3K for 60 min, and precipitated on glutathione-Sepharose beads. Precipitated proteins were separated by SDS-PAGE, immunoblotted with antiphosphotyrosine antibodies, and visualized by enhanced chemiluminescence. Panel shows a representative blot from three experiments. Middle panel, Quiescent hIRcB cells were stimulated with insulin (100 ng/ml) for 5 min, lysed, clarified, and immunoprecipitated with polyclonal antibodies to PLC-{gamma}1 or preimmune serum. Precipitated proteins were separated by SDS-PAGE, immunoblotted with antiphosphotyrosine antibodies, and visualized by enhanced chemiluminescence. An aliquot (10%) of the supernatant was run in parallel. The panel shows a representative blot from two experiments. Bottom panel, Quiescent hIRcB cells were stimulated with insulin (100 ng/ml) for 1 min, lysed, clarified, and immunoprecipitated with polyclonal antibodies against PLC-{gamma}1. Immunoprecipitates were washed three times in PLC assay buffer and PLC activity measured using [3H]-PI-(4,5)P2 in a mixed micelle assay. Released IP3 was separated by organic extraction and counted. Results are the mean and SEM of two experiments. Asterisks indicate statistical significance (***, P < 0.001).

 
We verified that the two proteins interact in the whole cell. The hIRcB cells were serum starved and then stimulated with insulin. Cell extracts were immunoprecipitated with antibodies to PLC-{gamma}1 or preimmune serum. Precipitated proteins and supernatants were immunoblotted with antiphosphotyrosine antibodies. Insulin causes tyrosine phosphorylation of the ß subunit of the IR and the endogenous substrate IRS-1 in the whole cell. Immunoprecipitation of PLC-{gamma}1 specifically brings down a single 95-kDa protein that corresponds to the tyrosine phosphorylated ß subunit of the IR (Fig. 1Go, middle panel). Phosphorylated IRS-1 is observed only in the supernatants, confirming our observations in adipocytes that PLC-{gamma}1 interacts with IR but not IRS-1.

Lastly, if PLC-{gamma}1 associates with the activated IR, then insulin stimulation might be expected to increase PLC activity. Consequently, cells were stimulated with insulin for 1 min, and PLC-{gamma}1 was immunoprecipitated from whole-cell lysates using a nonneutralizing rabbit polyclonal antibody for 2 h. A 1-min incubation with insulin was used in this experiment because we had previously found that IR-associated PLC activity is maximal at 1 min and declines over 30 min (28). PLC activity was measured on the immunoprecipitates using [3H]-PI-(4,5)P2 in a mixed micelle assay. Released [3H]-IP3 was extracted and counted. Insulin causes a 2.5-fold increase (P < 0.001) in phosphatidylinositol 4,5-bisphosphate hydrolysis in PLC-{gamma}1 immunoprecipitates (Fig. 1Go, bottom panel). Control experiments demonstrated that the same amount of PLC-{gamma}1 protein is precipitated in basal and insulin-stimulated cells (data not shown).

Inhibition of PLC impairs insulin-induced MAPK activation
To better understand whether PLC is required for insulin’s mitogenic effect, we investigated whether inhibition of PLC might regulate signaling pathways leading to cell cycle progression. The ras-MAPK cascade is intimately involved in cellular proliferation, so we investigated whether inhibition of PLC activity would block activation of MAPK in hIRcB cells. Cells were starved for 36 h in serum-free DMEM; pretreated with increasing doses of the PLC inhibitor U73122, the control compound U73343, the MEK inhibitor PD98059, or dimethylsulfoxide (DMSO) vehicle (0.33%); and stimulated with insulin (100 ng/ml) for 5 min. Whole-cell extracts were separated by SDS-PAGE and the proteins transferred to PVDF membranes. Activated ERK1 and 2 were detected using antibodies to the dually phosphorylated (pThr/pTyr) form of these kinases (Fig. 2Go, top panel). Protein loading was verified by stripping the membranes and reblotting for native unphosphorylated ERK1 and 2 (Fig. 2Go, middle panel). Phosphorylation of ERK was quantified by scanning densitometry of autoradiographs from multiple experiments and is corrected for ERK protein level. Insulin causes a 40-fold activation of ERK1 and 2 (Fig. 2Go, bottom panel). U73122 inhibits insulin-stimulated MAPK phosphorylation in a dose-dependent manner causing 48% inhibition at 10 µM U73122, whereas the inactive analog U73343 is without effect. Inhibition of MEK1 also causes a dose-dependent reduction of MAPK activation causing 75% inhibition at 20 µM PD98059. We also treated cells with both inhibitors before insulin stimulation. PD98059 (20 µM) and U73122 (10 µM) inhibit insulin-stimulated MAPK activation by 75% and 48%, respectively, and together cause 90% inhibition. The effect is more pronounced at submaximal doses of the inhibitors. PD98059 and U73122 both cause 25% inhibition at concentrations of 10 µM and 5 µM, respectively. Combining these two concentrations of inhibitor causes a 70% decrease in MAPK activation. These data suggest that U73122 is blocking insulin-stimulated MAPK phosphorylation via an MEK1-independent pathway.



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  		Figure 2. PLC inhibition impairs insulin-induced MAPK phosphorylation. Quiescent hIRcB cells were treated with the indicated concentrations (µM) of U73122, U73343, PD98059, or DMSO vehicle (0.33%) and then stimulated with 100 ng/ml insulin for 5 min. Cells were lysed, and equal volumes of extract were resolved by SDS-PAGE and transferred onto PVDF membranes. Top panel, Proteins were immunoblotted with antibodies specific for dually phosphorylated ERK1/2 (Anti-ACTIVE MAPK). Blot is representative of two experiments. Middle panel, Membranes were stripped and reblotted with native ERK1/2 antibodies to control for protein loading. Bottom panel, Results were quantified by densitometry; ERK phosphorylation was normalized to ERK protein and is presented as fold over basal (mean ± SEM).

 
Inhibition of PLC reduces insulin-stimulated thymidine incorporation
Because it was previously shown that the MAPK cascade is essential for mitogenesis and cellular proliferation, we investigated whether PLC mediates insulin’s effects on DNA synthesis. Insulin sensitivity was determined by generating insulin-dose response curves in the absence or presence of the PLC inhibitor. The hIRcB cells at 75% confluence were serum starved for 36 h; pretreated with 5 µM U73122, U73343, or DMSO for 20 min; and stimulated with increasing concentrations of insulin (0, 1, 10, 100, and 1000 ng/ml) for 18 h, after which time the cells were pulsed with [3H]-thymidine, the DNA precipitated with 10% trichloroacetic acid, and counted in a scintillation counter. Insulin stimulated thymidine incorporation 8-fold with an ED50 of 3 ng/ml (Fig. 3Go, top panel). The insulin dose response was severely blunted in the presence of U73122. Interestingly, the control compound U73343 caused a 10-fold decrease in insulin sensitivity (ED50 20 ng/ml) but no change in maximal effect. A dose response was performed with the PLC inhibitor U73122. Cells were serum starved for 36 h, pretreated with increasing concentrations of U73122 or U73343 for 20 min, and stimulated with insulin (100 ng/ml) for 18 h (Fig. 3Go, middle panel). Stimulation was significantly inhibited by U73122 with an IC50 of 2 µM, compared with control cells treated with U73343 (P < 0.05).



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  		Figure 3. PLC inhibition impairs insulin-stimulated DNA synthesis in hIRcB cells. Quiescent hIRcB cells were treated with U73122, U73343, or DMSO vehicle (0.33%) for 20 min and then stimulated with insulin for 18 h, after which the cells were pulsed with [3H]-thymidine. The cells were solubilized, and the DNA precipitated with 10% trichloroacetic acid at 4 C and counted in a scintillation counter. Results are means ± SEM from three different experiments. Asterisks indicate statistical significance from untreated cells (*, P < 0.05; ***, P < 0.001). Top panel, Cells were treated with 5 µM U73122, U73343, or DMSO vehicle (0.33%) and stimulated with increasing doses of insulin. Middle panel, Cells were treated with increasing doses of U73122 or DMSO vehicle and stimulated with 100 ng/ml insulin. Bottom panel, Cells were treated with 5 µM U73122, U73343, or DMSO vehicle (0.33%), and stimulated with 100 ng/ml insulin in the presence of increasing doses of OAG.

 
If insulin activation of PLC-{gamma}1 is truly required for stimulation of DNA synthesis, then the enzyme products of PLC-{gamma}1 should be able to substitute for the enzyme activity. Consequently, addition of diacylglycerol and/or inositol trisphosphate should rescue insulin-stimulated DNA synthesis that has been blocked by U73122. We have shown previously that a synthetic diacylglycerol, 1-oleyl-2-acetylglycerol (OAG), can rescue PLC-{gamma}1 signaling in 3T3-L1 adipocytes (28). In these experiments, we tested whether OAG could rescue DNA synthesis in hIRcB cells. Cells were serum starved for 36 h, pretreated with 5 µM U73122 or U73343, and stimulated with insulin (100 ng/ml) in the presence of increasing concentrations of OAG (0, 5, 10, and 25 µM). Insulin stimulated thymidine incorporation approximately 10-fold in these experiments (Fig. 3Go, bottom panel). Incorporation was inhibited 60% by blockade of PLC with U73122 (P < 0.05), but the control compound U73343 was without effect as before. OAG completely rescued DNA synthesis at all doses tested (P < 0.05) but has no effect on its own or in the absence of PLC inhibition. These results indicate that PLC activation is necessary but not sufficient for insulin stimulation of DNA synthesis.

Microinjection of PLC SH2 domains blocks BrdU incorporation
As an alternative to the use of a pharmacological inhibitor, we used the technique of single-cell microinjection to introduce isolated domains of PLC-{gamma}1 to disrupt association of PLC-{gamma}1 with the IR. The domains used in these experiments are diagrammed in Fig. 4Go (top panel). The domains were expressed as GST-fusion proteins in E. coli. The hIRcB cells were plated on acid-washed cover-slips for the microinjection experiments and were serum-starved for 36 h. GST-fusion proteins were injected at a concentration of 5 mg/ml along with sheep IgG as an injection marker. Cells were then stimulated with insulin (100 ng/ml). Twelve hours later, BrdU was added and the incubation continued for 4 h. Cells were fixed, permeabilized, and stained for the injection marker and for BrdU. In the basal, unstimulated state, 10% of the cells were positive for BrdU staining in the nucleus. After insulin stimulation, the number of positive cells increased to 70% (Fig. 4Go, bottom panel). Injection of PLC-{gamma}1 fusion proteins containing either the C- or N-terminal SH2 domain caused a reduction in the number of positive cells (P < 0.01). In contrast, the isolated SH3 domain was unable to inhibit BrdU incorporation. Control proteins containing the SH2 domain from SHC or Grb2 were injected in parallel coverslips. As expected, only the Grb2 SH2 was able to inhibit BrdU incorporation, demonstrating the specificity of the blockade.



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  		Figure 4. Microinjection of PLC-{gamma}1 SH2 domains into hIRcB cells blocks BrdU incorporation. Top panel, Structure of PLC-{gamma}1 and GST fusion proteins used in the microinjection experiments. The split catalytic lipase domain is shown in shaded boxes X and Y. The amino-terminal PH domain binds phosphatidylinositol 3,4,5-trisphosphate and the EF and C2 domains bind calcium and phospholipids. Two SH2 domains and an SH3 domain are located in the lipase insert region. The amino acids corresponding to the protein domains are indicated. Bottom panel, Quiescent hIRcB cells were microinjected with the indicated GST-fusion proteins (5 mg/ml) and sheep IgG as a marker. Cells were allowed to recover for 1 h and stimulated with 100 ng/ml insulin for 16 h. BrdU was added for the last 4 h. Cells were fixed in 3.7% paraformaldehyde and stained with an antibody to BrdU followed by a rhodamine-conjugated secondary antibody. Injected cells were visualized by staining with an antibody to sheep IgG followed by a fluorescein-labeled secondary antibody. Injected cells that were positive for nuclear rhodamine fluorescence were counted and results are presented as percent BrdU positive. Asterisks indicate statistical significance vs. insulin alone (**, P < 0.01).

 
Addition of a synthetic diacylglycerol (OAG) was able to overcome blockade of PLC by the pharmacological inhibitor U73122 in the thymidine incorporation assays earlier. Consequently, we tested whether OAG and/or IP3, the other PLC hydrolysis product, could rescue the blockade of BrdU caused by microinjection of PLC-{gamma}1 SH2 domains. Cells were injected with the PLC-{gamma}1 SH2-SH2-SH3 domain fusion protein, and stimulated with insulin, and labeled with BrdU as before. Typical fields of cells are shown in Fig. 5Go. In some experiments, cells were treated with the synthetic diacylglycerol OAG (5 µM), or coinjected with IP3 (20 µM). Nuclear BrdU staining is shown in red; injected cells can be identified by cytoplasmic green staining. Insulin treatment causes an increase in the number of BrdU-positive nuclei for both the uninjected cells and cells injected with IgG marker only. White arrows indicate injected cells that are positive for BrdU incorporation. Injection with the PLC-{gamma}1 SH2-SH2-SH3 fusion protein causes a reduction in the number of BrdU-positive nuclei. BrdU staining is restored in SH2-SH2-SH3 injected cells treated with OAG but not in cells coinjected with IP3.



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  		Figure 5. Treatment with DAG but not IP3 rescues hIRcB cells blocked by microinjection of the PLC-{gamma}1 SH2-SH2-SH3 fusion protein. Quiescent hIRcB cells were microinjected with combinations of sheep IgG, 5 mg/ml PLC-{gamma}1 GST-SH2-SH2-SH3 fusion protein (SH2), and 20 µM IP3. Cells were allowed to recover for 1 h and stimulated with 100 ng/ml insulin for 16 h in the presence or absence of 5 µM OAG. BrdU was added for the last 4 h. Cells were fixed in 3.7% paraformaldehyde and stained with an antibody to BrdU followed by a rhodamine-conjugated secondary antibody. Injected cells were visualized by staining with an antibody to sheep IgG followed by a fluorescein-labeled secondary antibody. White arrows indicate injected cells that have undergone DNA synthesis.

 
We also tested the ability of OAG and IP3 to rescue DNA synthesis in cell injected with other fusion proteins. Treatment of quiescent cells with OAG or microinjection of IP3 into quiescent cells was unable to cause BrdU incorporation above basal levels indicating that PLC activity per se is not sufficient for DNA synthesis (Fig. 6Go). Similarly in insulin-stimulated cells, treatment with OAG or microinjection of IP3 did not alter the number of BrdU-positive cells. Coinjection of IP3 with any of the fusion proteins was unable to rescue the blockade of DNA synthesis. However, treatment of cells with OAG, either alone or with coinjected IP3, was able to rescue completely the blockade of DNA synthesis because of microinjection of PLC-{gamma}1 SH2 domains. Neither OAG nor IP3 altered BrdU incorporation in cells injected with the PLC-{gamma}1 SH3 domain or the SHC and Grb2 SH2 domains, indicating that the rescue was specific for blockade of PLC. Disruption of Grb2-SHC and Grb2-IRS1 interactions, which are important for ras activation, with the Grb2 SH2 domain was not rescued by the diacylglycerol, so Grb2 must lie either downstream of PLC or in a parallel pathway.



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  		Figure 6. Treatment with DAG rescues hIRcB cells blocked by microinjection of the PLC-{gamma}1 but not the Grb2 or Shc SH2 fusion proteins. Quiescent hIRcB cells were microinjected with combinations of the indicated SH2 or SH3 fusion proteins (5 mg/ml), 20 µM IP3, and sheep IgG as a marker. Cells were allowed to recover for 1 h and stimulated with 100 ng/ml insulin for 16 h in the presence or absence of 5 µM OAG. BrdU was added for the last 4 h. Cells were fixed in 3.7% paraformaldehyde and stained with an antibody to BrdU followed by a rhodamine-conjugated secondary antibody. Injected cells were visualized by staining with an antibody to sheep IgG followed by a fluorescein-labeled secondary antibody. Injected cells that were positive for nuclear rhodamine fluorescence were counted and results are presented as percent BrdU positive. Asterisks indicate statistical significance vs. insulin alone (**, P < 0.01).

 
Microinjection of neutralizing PLC-{gamma}1 antibodies inhibits BrdU incorporation
Lastly, we determined the ability of microinjected antibodies to block DNA synthesis in response to insulin. Two antibodies were used, a mixture of mouse monoclonals and a rabbit polyclonal. The ability of these antibodies to precipitate PLC-{gamma}1 from insulin-stimulated cells was tested. Cells were serum-starved for 36 h and stimulated with insulin for 1 min and then lysed and PLC-{gamma}1 immunoprecipitated for 2 h. PLC activity was measured on the immunoprecipitates using unilamellar vesicles containing labeled [3H]-PI-(4,5)P2 as before. Both antibodies were able to precipitate PLC-{gamma}1 efficiently as judged by immunoblotting (data not shown). The monoclonal antibodies are neutralizing because no insulin-stimulated PLC activity was observed (Fig. 7Go, top panel). In contrast, the rabbit polyclonals, although immunoprecipitating very efficiently, are nonneutralizing because a 3-fold stimulation of PLC activity was observed in the precipitates. The basal PLC activity was also significantly lower in the immunoprecipitates with the monoclonal neutralizing antibodies than with the polyclonal antibodies despite equal protein.



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  		Figure 7. Microinjection of neutralizing antibodies to PLC-{gamma}1 blocks insulin-induced BrdU incorporation in hIRcB cells. Top panel, Quiescent hIRcB cells were stimulated with insulin (100 ng/ml) for 1 min, lysed, clarified, and immunoprecipitated with monoclonal antibodies or polyclonal antibodies against PLC-{gamma}1. Immunoprecipitates were washed three times in PLC assay buffer, then PLC activity measured using [3H]-PI-(4,5)P2 in a mixed micelle assay. Released IP3 was separated by organic extraction and counted. Results are the mean and SEM of two experiments. Asterisks indicate statistical significance (*, P < 0.05, ***, P < 0.001). Bottom panel, Quiescent hIRcB cells were microinjected with mouse IgG (mIgG), rabbit IgG (rIgG), rabbit polyclonal anti-PLC-{gamma}1 (rPLC), or mouse monoclonal anti-PLC-{gamma}1 (mPLC) antibodies as indicated, along with sheep IgG as marker. Cells were allowed to recover for 1 h and stimulated with 100 ng/ml insulin for 16 h. In some experiments, 20 µM IP3 was coinjected with the mPLC antibody and cells were incubated in the presence of 5 µM OAG. BrdU was added for the last 4 h. Cells were fixed in 3.7% paraformaldehyde and stained with an antibody to BrdU followed by a rhodamine-conjugated secondary antibody. Injected cells were visualized by staining with an antibody to sheep IgG followed by a fluorescein-labeled secondary antibody. Injected cells that were positive for nuclear rhodamine fluorescence were counted and results are presented as percent BrdU positive. Asterisks indicate statistical significance vs. insulin alone (**, P < 0.01).

 
The two antibodies were then injected into cells at a concentration of 4 mg/ml. In the basal, unstimulated state, 10% of cells were positive for nuclear BrdU staining. Insulin stimulation caused an increase in the number of BrdU-positive cells to 70%. Injection of preimmune mouse or rabbit IgG had no effect on the ability of insulin to stimulate BrdU incorporation (Fig. 7Go, bottom panel). Similarly, microinjection of the rabbit polyclonal anti-PLC-{gamma}1 antibodies had no effect. However, microinjection of the neutralizing monoclonal anti-PLC-{gamma}1 antibodies blocked insulin-stimulated BrdU incorporation by 50%. This blockade is rescued by OAG but not IP3 in agreement with the rescue of the SH2-mediated blockade. Thus, inhibition of PLC activity by introduction of the neutralizing antibodies prevents insulin stimulation of DNA synthesis.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We confirmed that PLC-{gamma}1 associates with the type A IR in hIRcB cells, as we had found in 3T3-L1 adipocytes, and that inhibition of PLC activity led to inhibition of insulin-stimulated MAPK phosphorylation and proliferation. This finding is similar to our previously published results on activation of MAPK by NGF via the trkA receptor in Chinese hamster ovary cells (29). In the case of both insulin and nerve growth factor, inhibition of both PLC with U73122 and MEK with PD98059 caused a greater inhibition of MAPK than either agent alone. This is in contrast to IGF-I signaling via the IGF-IR in CHO cells, in which PD98059 completely obliterates MAPK activation (IC50 < 1 µM). U73122 is without effect because IGF-I, unlike insulin and NGF, does not activate PLC-{gamma}1 (29). At the concentrations used in these experiments, PD98059 is specific for MEK1. At higher concentrations MEK2 is also inhibited, but the IC50 is 10-fold higher than for MEK1 although the two proteins show 90% amino acid homology (30). This suggests that IGF-I signals exclusively through MEK1. The IGF-IR can signal mitogenesis through both G{alpha}i via the Gß{gamma} activation of GRK2, src, Shc, SOS, and ras, and also through IRS-1 leading to the activation of PI3K, depending on the cell type (31, 32, 33, 34). PLC-{gamma}1, on the other hand, has been shown to activate ras via calcium-dependent activation of rasGRF, a ras guanine-nucleotide exchange factor, and PKC activation of Raf-1 by direct phosphorylation (35, 36). PKC also activates the Ral and Rap proteins that can interact with other isoforms of Raf, such as B Raf, which have differing abilities to activate MEK1 and MEK2 (37, 38, 39). Despite their high homology, MEK1 and MEK2 show differential regulation by growth factors and serum (40), so it is interesting to speculate that insulin and NGF activate MAPK through both MEK1 and MEK2; activation of MEK1 being via the classical Grb2-SOS pathway, leading to GTP exchange on ras, whereas activation of MEK2 being mediated by activation of PLC-{gamma}1, PKC and B-Raf. Further studies are planned to investigate this model.

The importance of PLC-{gamma}1 in insulin-stimulated mitogenesis is underscored by the microinjection of SH2 domains and PLC-{gamma}1 antibodies. The SH2 domains can bind to the activated IR and block interaction with endogenous PLC-{gamma}1, thereby acting in a dominant negative manner. Fusion proteins containing any SH2 domain from PLC-{gamma}1 were able to repress insulin-stimulated BrdU incorporation. Injection of the Grb2 SH2 also blocked mitogenesis in response to insulin, but the Shc SH2 was without effect. This should not be taken as evidence that Shc is dispensable because Shc has been shown to be essential for insulin-stimulated DNA synthesis in these cells (41, 42). However, Shc docks with the IR via its PTB domain rather than its SH2 domain. Furthermore, microinjection of the Shc or CrkII SH2 domains does block EGF stimulation of mitogenesis, even though they do not block insulin stimulation (43). This argues against nonspecific inhibition by the SH2 domains. The PLC-{gamma}1-SH3 domain alone was unable to block mitogenesis, even though it is known to interact with the proline-rich region of SOS. This result indicates that activation of ras is not mediated by the recruitment of SOS to PLC-{gamma}1 at the plasma membrane agreeing with a previous publication in EGF-stimulated MDCK cells (17). The SH3 domain of PLC-{gamma}1 is also responsible for the localization of the protein to the actin cytoskeleton (44, 45, 46). The lack of inhibition by injection of the isolated SH3 domain would also indicate that interaction of PLC-{gamma}1 with the actin network is not required for induction of DNA synthesis.

The ability to rescue cells that have been blocked either by treatment with the PLC inhibitor or by microinjection of SH2 domains argues strongly that PLC-{gamma}1 is functioning catalytically rather than as a scaffold for other signaling proteins. This notion is supported by the microinjection of PLC-{gamma}1 antibodies. Only those antibodies that neutralize PLC activity are able to block DNA synthesis. Treatment of blocked cells with synthetic diacylglycerols is able to restore insulin-stimulated BrdU incorporation to normal levels but microinjection of inositol triphosphate does not rescue. It is possible that the lack of effect of injected IP3 is related to the rapid degradation of IP3 in the cell before stimulation with insulin. However, the complete rescue with diacylglycerol (DAG) treatment alone suggests that IP3 signaling is not necessary. The rescue is specific for PLC-{gamma}1 because DAG has no effect on cells blocked by injection of the Grb2 SH2 domain. The target for DAG is not known. Diacylglycerol may be acting through classical or novel PKC isoforms. It is not a mitogen per se because addition of DAG to quiescent cells does not cause an increase in BrdU labeling. This is in contrast to phorbol esters that are commonly used to activate PKC signaling. A recent study has demonstrated that PKCßII activity is essential for insulin-stimulated mitogenesis in L6 skeletal muscle cells (47). Because this PKC isoform is DAG dependent, this result would suggest that activation of PLC signaling is also required in these cells.

The mitogenic signal may be PKC independent, however. A number of other proteins contain C1 domains, which bind diacylglycerol with high affinity. The {alpha} and ß chimaerins bind DAG and phorbol esters and function as GTP activating proteins for Rac, a small GTP-binding protein involved in insulin-stimulated cytoskeletal reorganization and prevention of apoptosis (48, 49, 50, 51, 52, 53). The effect of phorbol esters or DAG on Rac signaling is not known, but they may target Rac to different cellular locations. Activation of Rac has also been shown to cause nuclear translocation of PKC{partial} and activation of Stat3 transcriptional events (54). Other C1-containing proteins are the CalDAG-GEFs. These proteins are calcium- and diacylglycerol-dependent guanine-nucleotide exchange factors. CalDAG-GEF1 acts on Rap1, and CalDAG-GEFII (or RasGRP) and CalDAG-GEFIII acts primarily on ras proteins (55, 56, 57, 58). CalDAG-GEFI has been shown to link the mAChR to ERK activation via PLCß, Rap1, and B-Raf (59). Whether such proteins are important for mitogenic signaling in response to insulin remains to be determined.

In conclusion, we have shown that pharmacological inhibition of PLC activity, disruption of the interaction between the insulin receptor and PLC-{gamma}1, or introduction of neutralizing PLC-{gamma}1 antibodies all inhibit insulin-induced DNA synthesis. Thus, three independent pieces of experimental data point to a requirement for PLC-{gamma}1 in insulin-stimulated mitogenesis. Furthermore, we have shown that PLC-{gamma}1 enzyme activity is required and that the diacylglycerol generated is the essential second messenger. Additional studies are planned to identify the downstream targets for DAG in this pathway.


   Footnotes
 
This work was supported by a VA/JDF Diabetes Research Center Grant and a Pilot and Feasibility Grant from the UCSD/Whittier Diabetes Program. N.J.G.W. is a faculty member of the UCSD Biomedical Graduate Program. J.E. was supported by a grant from the Deutsche Forschungsgemeinschaft.

Abbreviations: BrdU, Bromodeoxyuridine; DAG, diacylglycerol; DMSO, dimethylsulfoxide; EGF, epidermal growth factor; GST, glutathione S-transferase; HRP, horseradish peroxidase; IR, insulin receptor; NGF, nerve growth factor; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride.

Received August 10, 2001.

Accepted for publication October 9, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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