Molecular species analysis of mitogen-stimulated 1,2-diglycerides in fibroblasts. Comparison of alpha-thrombin, epidermal growth factor, and platelet-derived growth factor

Recent studies have implicated the hydrolysis of phosphoinositides and phosphatidylcholine in agonist-stimulated events. The potent mitogen, alpha-thrombin, stimulates the generation of diglycerides in a biphasic and sustained manner in IIC9 fibroblasts (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). Using measurements of radiolabeled headgroup release and molecular species analysis, we previously determined that alpha-thrombin generates diglycerides through the hydrolysis of both the phosphoinositides and phosphatidylcholine at early times (15 s), and at later times (greater than or equal to 5 min) through the hydrolysis of primarily, if not exclusively, phosphatidylcholine (Pessin, M. S., and Raben, D. M. (1989) J. Biol. Chem. 264, 8729-8738). In contrast, IIC9 fibroblasts respond to the mitogenic treatments of (a) alpha-thrombin following chymotrypsin pretreatment or (b) epidermal growth factor by increasing their levels of diglycerides in a monophasic and sustained manner (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). In this report, we have analyzed the molecular species of the diglycerides generated by these two different treatments and have also examined the lipid response of IIC9 fibroblasts to platelet-derived growth factor. Based on both the molecular species analyses and the release of radiolabeled head-groups, all three of these different mitogenic treatments generate diglycerides primarily through the stimulation of phosphatidylcholine hydrolysis. However, while similar, the molecular species profiles of the diglycerides generated by these three treatments are not identical to the molecular species profile of total cellular phosphatidylcholine. In addition, the molecular species profiles of the diglycerides generated by these three mitogenic treatments greatly resemble each other, with significant differences between any two profiles occurring in at most one molecular species. This finding differs from that seen with alpha-thrombin stimulation alone, where the molecular species profile of the diglycerides generated following 5 min of alpha-thrombin stimulation is nearly identical to the molecular species profile of total cellular phosphatidylcholine. These data support the possibility of hormone-sensitive phosphatidylcholine pools or selective diglyceride metabolism.     

Kinetic and molecular species analyses of mitogen-induced increases in diglycerides: evidence for stimulated hydrolysis of phosphoinositides and phosphatidylcholine

A wide variety of agonist-induced events appear to be mediated through an increase in cellular diglyceride levels. With regard to the ability of diglycerides to mediate these events, three important parameters must be considered: a) the kinetics of diglyceride generation, b) the absolute mass levels, and c) their molecular species. While this increase is often due to a stimulated hydrolysis of phosphoinositides, there is increasing evidence that the stimulated hydrolysis of phosphatidylcholine also contributes to agonist-induced increases in diglyceride levels. The kinetics of mass increases in diglyceride levels stimulated in cultured fibroblasts are agonist-dependent. High concentrations of alpha-thrombin stimulate a biphasic increase in diglyceride levels with the first phase peaking at 15 s and the second phase peaking at 5 min. In contrast, stimulation with epidermal growth factor, or platelet-derived growth factor, results in a monophasic increase in cellular diglyceride levels. Furthermore, the molecular species and phospholipid source of the stimulated diglycerides are also agonist-dependent. While the hydrolysis of phosphoinositides is major source of diglycerides initially generated in response to some agonists (15 s with alpha-thrombin at 500 ng/ml), phosphatidylcholine is hydrolyzed as well. Following longer incubations, or at all times following stimulation by epidermal growth factor or platelet-derived growth factor, phosphatidylcholine hydrolysis is the principal source of the stimulated diglycerides.     

Nuclear diacylglycerol kinase-theta is activated in response to alpha-thrombin

Currently, there is substantial evidence that nuclear lipid metabolism plays a critical role in a number of signal transduction cascades. Previous work from our laboratory showed that stimulation of quiescent fibroblasts with alpha-thrombin leads to the production of two lipid second messengers in the nucleus: an increase in nuclear diacylglycerol mass and an activation of phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid. Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol to phosphatidic acid, making it an attractive candidate for a signal transduction component. There is substantial evidence that this activity is indeed regulated in a number of signaling cascades (reviewed by van Blitterswijk, W. J., and Houssa, B. (1999) Chem. Phys. Lipids 98, 95-108). In this report, we show that the addition of alpha-thrombin to quiescent IIC9 fibroblasts results in an increase in nuclear DGK activity. The examination of nuclei isolated from quiescent IIC9 cells indicates that DGK-theta and DGK-delta are both present. We took advantage of the previous observations that phosphatidylserine inhibits DGK-delta (reviewed by Sakane, F., Imai, S., Kai, M., Wada, I., and Kanoh, H. (1996) J. Biol. Chem. 271, 8394-8401), and constitutively active RhoA inhibits DGK-theta (reviewed by Houssa, B., de Widt, J., Kranenburg, O., Moolenaar, W. H., and van Blitterswijk, W. J. (1999) J. Biol. Chem. 274, 6820-6822) to identify the activity induced by alpha-thrombin. Constitutively active RhoA inhibited the nuclear stimulated activity, whereas phosphatidylserine did not have an inhibitory effect. In addition, a monoclonal anti-DGK-theta antibody inhibited the alpha-thrombin-stimulated nuclear activity in vitro. These results demonstrate that DGK-theta is the isoform responsive to alpha-thrombin stimulation. Western blot and immunofluorescence microscopy analyses showed that alpha-thrombin induced the translocation of DGK-theta to the nucleus, implicating that this translocation is at least partly responsible for the increased nuclear activity. Taken together, these data are the first to demonstrate an agonist-induced activity of nuclear DGK-theta activity and a nuclear localization of DGK-delta.     

Modulation of thrombin-stimulated lipid responses in cultured fibroblasts. Evidence for two coupling mechanisms

Treatment of cultured fibroblasts with thrombin results in the stimulation of cell division and lipid metabolism. Proteolytically active alpha-thrombin rapidly stimulates (a) release of arachidonic acid, (b) generation of inositol phosphates, and (c) increase in cellular diacylglycerol levels. Pretreatment of the fibroblasts with chymotrypsin before alpha-thrombin prevented the first two responses, (a) and (b), and reduced response c. Treatment of fibroblasts with gamma-thrombin, a proteolytic derivative of alpha-thrombin, produced a response indistinguishable from the alpha-thrombin treatment when preceded by chymotrypsin. These data support a model, similar to one for platelets [McGowan, E. B., & Detwiler, T. C. (1986) J. Biol. Chem. 261, 739-746], that fibroblasts possess two coupling mechanisms for the stimulation of lipid metabolism by thrombin. Similar to platelets, one mechanism, R1, mediates the stimulated release of arachidonic acid and is capable of activating Ni, a GTP-binding protein. R1 is inactivated by chymotrypsin and does not respond to gamma-thrombin. The other mechanism, R2, responds to gamma-thrombin and is not activated by chymotrypsin. In contrast to the mechanisms proposed for platelets, we demonstrate that the phospholipase C responsible for the hydrolysis of phosphoinositides is not activated by R2 but is activated via R1. Importantly, stimulation of either mechanism results in the elevation of cellular diacylglycerol. This indicates that the stimulated elevation of diacylglycerol, or those events dependent upon the elevation of diacylglycerol, is not a reliable indicator for establishing the hydrolysis of phosphoinositides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells.

The mechanism by which an agonist, binding to a cell surface receptor, exerts an effect on events in the nucleus is not known. We have previously shown (Leach, K. L., Ruff, V. A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266, 3215-3221) that alpha-thrombin treatment of IIC9 cells results in increased levels of cellular 1,2-diacylglycerol (DAG) and activation of protein kinase C (PKC). Here, we have examined whether changes in nuclear PKC and nuclear DAG also are induced following alpha-thrombin treatment. IIC9 cells were treated with 500 ng/ml alpha-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC alpha, but not PKC epsilon or zeta in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or alpha-thrombin. The increase in nuclear PKC alpha levels was accompanied by a 10-fold increase in nuclear PKC specific activity and stimulated phosphorylation of at least six nuclear proteins. The rise in nuclear PKC levels occurred rapidly and reached a maximum at 30-60 s, which was followed by a decline back to the control level over the next 15 min. In addition, alpha-thrombin treatment resulted in an immediate rise in DAG mass levels in the nuclear fractions. Kinetic analysis indicated that a maximum increase in DAG levels occurred 2.5-5 min after the addition of alpha-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, alpha-thrombin treatment induced an increase in radiolabeled nuclear diglycerides, suggesting that the stimulated nuclear DAGs are derived, at least in part, from phosphatidylcholine. Our results suggest that increases in both nuclear DAG levels and PKC activity following alpha-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.     

Differential dependence of early and late increases in 1,2-diacylglycerol on the presence of catalytically active alpha-thrombin: evidence for regulation at the level of 1,2-diacylglycerol generation.

alpha-Thrombin stimulates a biphasic increase in cellular 1,2-diacylglycerol mass in quiescent IIC9 fibroblasts. This report describes the use of hirudin, a high-affinity inhibitor of alpha-thrombin that renders it catalytically inactive, to investigate the dependence of elevated 1,2-diacylglycerol levels on the presence of catalytically active alpha-thrombin. When cultures were incubated in the presence of alpha-thrombin, 1,2-diacylglycerol levels remained elevated for greater than or equal to 4 h. Inactivation of alpha-thrombin after 15 s did not alter the kinetics of 1,2-diacylglycerol formation occurring over the next 1 h. However, sustained (1-4 h) increases in this lipid were eliminated. Inactivation of alpha-thrombin after 1 h of stimulation resulted in 1) an immediate and reversible decline in 1,2-diacylglycerol levels, 2) elimination of the sustained phase of 1,2-diacylglycerol production, 3) inhibition of the alpha-thrombin-stimulated generation of choline metabolites, and 4) a blunted mitogenic response to alpha-thrombin. These data indicate that early (0-1 h) and late (1-4 h) increases in 1,2-diacylglycerol are differentially dependent on the presence of catalytically active alpha-thrombin. Furthermore, sustained increases in 1,2-diacylglycerol in response to alpha-thrombin are regulated at least in part at the level of generation (via phosphatidylcholine hydrolysis). Our results also support a role for sustained 1,2-diacylglycerol levels in the mitogenic response.     

Dissociation of protein kinase C activation and sn-1,2-diacylglycerol formation. Comparison of phosphatidylinositol- and phosphatidylcholine-derived diglycerides in alpha-thrombin-stimulated fibroblasts

Diacylglycerols (DAGs) derived from phosphatidylcholine (PC) hydrolysis have been shown to activate protein kinase C (PKC) in vitro, but it is not known whether this event occurs in response to DAGs generated via agonist-induced PC hydrolysis in intact cells. In this report we have addressed this question directly, using alpha-thrombin stimulation of IIC9 fibroblasts. PKC activation in intact cells was assessed in two ways, by measuring: 1) PKC membrane association as determined by kinase activity and Western blot analysis and 2) the phosphorylation of an endogenous PKC substrate, an 80-kDa protein. Treatment with 500 ng/ml alpha-thrombin has been shown to stimulate both phosphoinositide and PC hydrolysis, whereas treatment with 100 pg/ml alpha-thrombin stimulates only PC breakdown. Using these two conditions, we show that DAG produced from phosphoinositide, but not PC hydrolysis, is associated with the activation of PKC.     

Biosynthesis of paf-acether. Activators of protein kinase C stimulate cultured mast cell acetyltransferase without stimulating paf-acether synthesis.

A property common to many growth factors is that they must be present for several hours before the commitment to DNA synthesis and cell division occurs. The intracellular signals that are relevant during this period are poorly defined. We examined the formation of 1,2-diacylglycerol in IIC9 fibroblasts after stimulation with epidermal growth factor (EGF), and found that the mass of this lipid remained elevated for at least four hours. The concentration-dependence of EGF-stimulated 1,2-diacylglycerol production and [3H]thymidine incorporation were similar. Studies of phospholipid metabolism strongly suggested that phosphatidylcholine was the source of the 1,2-diacylglycerol generated in response to EGF. EGF did not stimulate the hydrolysis of other phospholipids, including the phosphoinositides, nor did it increase synthesis de novo of 1,2-diacylglycerol. This pattern of sustained 1,2-diacylglycerol formation from phosphatidylcholine may be important in the mitogenic signalling of EGF and potentially other growth factors.     

Activation of protein kinase C by naturally occurring ether-linked diglycerides

Recent studies have demonstrated that ether-linked diglycerides are endogenous constituents of biologic tissues and accumulate during agonist stimulation (Daniel, L. W., Waite, M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132) and myocardial ischemia (Ford, D. A., and Gross, R. W. (1989) Circ. Res. 64, 173-177). Although protein kinase C previously had been thought to specifically require 1,2-diacyl-sn-glycerol (DAG) molecular species for activation, the present study demonstrates that purified rat brain protein kinase C is activated by naturally occurring ether-linked diglycerides (e.g. 1-O-hexadec-1'-enyl-2-octa-dec-9'-enoyl-sn-glycerol and 1-O-hexadecyl-2-octa-dec-9'-enoyl-sn-glycerol) with a similar dose response curve to that for DAG molecular species. Although in vitro assays demonstrated that DAG could partially activate protein kinase C in the absence of free calcium, activation by ether-linked diglycerides required free calcium concentrations found only in stimulated cells (greater than 1 microM [Ca2+]free). To substantiate these findings the alpha and beta isoforms of protein kinase C from rat brain cortical grey matter were resolved by hydroxylapatite chromatography. Although the beta isoform of protein kinase C was substantially activated by DAG in the absence of free calcium, activation by ether-linked diglycerides had an absolute requirement for physiologic increments in free calcium ion found in stimulated cells. Since ether lipids are localized in specific subcellular membrane compartments, accumulate during several pathophysiologic perturbations and are effective activators of protein kinase C with separate and distinct calcium requirements in comparison to DAG, these results suggest that ether-linked diglycerides are important and potentially specific biologic activators of one or more isoforms of protein kinase C.     

Alpha-thrombin-mediated phosphatidylinositol 3-kinase activation through release of Gbetagamma dimers from Galphaq and Galphai2

Chinese hamster embryonic fibroblasts (IIC9 cells) express the Galpha subunits Galphas, Galphai2, Galphai3, Galphao, Galpha(q/11), and Galpha13. Consistent with reports in other cell types, alpha-thrombin stimulates a subset of the expressed G proteins in IIC9 cells, namely Gi2, G13, and Gq as measured by an in vitro membrane [35S]guanosine 5'-O-(3-thio)triphosphate binding assay. Using specific Galpha peptides, which block coupling of G-protein receptors to selective G proteins, as well as dominant negative xanthine nucleotide-binding Galpha mutants, we show that activation of the phosphatidylinositol 3-kinase/Akt pathway is dependent on Gq and Gi2. To examine the role of the two G proteins, we examined the events upstream of PI 3-kinase. The activation of the PI 3-kinase/Akt pathway by alpha-thrombin in IIC9 cells is blocked by the expression of dominant negative Ras and beta-arrestin1 (Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046-18053, and Goel, R., Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2002) J. Biol. Chem. 277, 18640-18648), indicating a role for Ras and beta-arrestin1. Interestingly, inhibition of Gi2 and Gq activation blocks Ras activation and beta-arrestin1 membrane translocation, respectively. Furthermore, expression of the Gbetagamma sequestrant, alpha-transducin, inhibits both Ras activation and membrane translocation of beta-arrestin1, suggesting that Gbetagamma dimers from Galphai2 and Galphaq activate different effectors to coordinately regulate the PI 3-kinase/Akt pathway.     

Preferential inhibition of the platelet-derived growth factor receptor tyrosine kinase by staurosporine

Ligand stimulation of the platelet-derived growth factor receptor (PDGF-R) results in rapid activation of the receptor tyrosine kinase, stimulation of phosphoinositide hydrolysis, an increase in intracellular free Ca2+ concentration ([Ca2+]i), and, ultimately, cellular proliferation. In a previous study, we demonstrated that staurosporine, a known inhibitor of protein kinase C, blocked PDGF-induced [Ca2+]i increases in Swiss mouse 3T3 fibroblasts by a mechanism that appeared unrelated to inhibition of protein kinase activity (Olsen, R., Melder, D., Seewald, M., Abraham, R., and Powis, G. (1990) Biochem. Pharmacol. 39, 968-972). In the present study, we report that staurosporine inhibits ligand-dependent PDGF-R tyrosine kinase activation in cell-free receptor preparations and in intact Swiss 3T3 cells. At the same concentrations (10(-8)-10(-6) M), staurosporine suppressed both the tyrosine phosphorylation of phospholipase C activity and the hydrolysis of phosphoinositides induced by PDGF stimulation of intact cells. In contrast, guanine nucleotide-binding protein-dependent phospholipase C activation induced by bradykinin or fluoroaluminate anion was relatively insensitive to staurosporine. A preferential inhibitory effect of staurosporine on signal generation by the PDGF-R was indicated by findings that epidermal growth factor receptor (EGF-R) tyrosine kinase activity and EGF-dependent phospholipase C in A-431 carcinoma cells were approximately 100-fold less sensitive to this drug. These data indicate that submicromolar concentrations of staurosporine inhibit PDGF-dependent phosphoinositide hydrolysis and Ca2+ mobilization through a proximal inhibitory effect on ligand-induced activation of the PDGF-R tyrosine kinase.     

Characterization of two populations of statin and the relationship of their syntheses to the state of cell proliferation

Statin has previously been identified to be a 57-kD protein present in the nuclei of quiescent and senescent human fibroblasts, but not in their replicating counterparts (Wang, E. 1985. J. Cell Biol. 100: 545-551). In the present report we demonstrate by immunoprecipitation analysis of fractionated cellular extracts the existence of two populations of statin. The Triton X-100-soluble statin is found in replicating sparse cultures as well as in quiescent confluent cultures and quiescent serum-starved cultures of young human fibroblasts, but the Triton X-100-insoluble, nuclear envelope-localized statin is present only in the quiescent cultures. Two-dimensional gel analysis of the immunoprecipitated cellular fractions reveals that both populations of statin have an isoelectric point of 5.3. Pulse-chase experiments show that statin is synthesized as a 57-kD polypeptide and is not processed from a precursor of different molecular mass. Experiments on serum stimulation of quiescent cells show that synthesis of the Triton X-100-insoluble statin decreases rapidly during the transition from the G0 to S phase, and that this decrease is accompanied by a slower reduction in synthesis of the Triton X-100-soluble statin. These results suggest that the cellular expression of the two populations of statin may be associated with the mechanisms controlling the transition between the growing state and the quiescent state and confirm the previous finding that the Triton X-100-insoluble, nuclear envelope-localized statin could be used as a marker for cells arrested at the G0 phase of the cell cycle.     

Carbachol stimulates a different phospholipid metabolism than nerve growth factor and basic fibroblast growth factor in PC12 cells.

We have examined 1,2-diglycerides (DGs) generated in PC12 cells in response to the muscarinic agonist carbachol and compared them with those generated in response to the differentiation factors nerve growth factor and basic fibroblast growth factor. Whereas carbachol stimulates a greater release of inositol phosphates, all three agonists generate similar levels of DGs. In this report, we have analyzed the molecular species of PC12 DGs generated in response to these three agonists. Additionally, we have analyzed the molecular species of PC12 phospholipids. The data indicate that 1) after 1 min of either nerve growth factor or basic fibroblast growth factor stimulation, DGs arise primarily from phosphoinositide hydrolysis; 2) in contrast, after 1 min of carbachol stimulation, DG are generated equally by both phosphoinositide and phosphatidylcholine hydrolysis; and 3) after 15 min of stimulation by any of these agonists, DGs are generated largely by phosphatidylcholine hydrolysis, with a smaller component arising from the phosphoinositides. These results suggest that at least part of the mechanism by which PC12 cells distinguish between different agonists is via alterations in phospholipid sources and kinetics of DG generation.     

alpha-Thrombin induces rapid and sustained Akt phosphorylation by beta-arrestin1-dependent and -independent mechanisms, and only the sustained Akt phosphorylation is essential for G1 phase progression

In Chinese hamster embryonic fibroblasts (IIC9 cells) alpha-thrombin activates the MAPK(ERK) and phosphatidylinositol 3-OH-kinase (PI 3-kinase)/Akt pathways, and both are essential for progression through the G(1) phase of the cell cycle. We investigated in IIC9 cells, the role of beta-arrestin1 in alpha-thrombin signaling to these pathways. alpha-Thrombin stimulates rapid and sustained PI 3-kinase and Akt activities. Expression of a dominant negative beta-arrestin1 (beta-arrestin1(V53D)) inhibits rapid but not sustained PI 3-kinase and Akt activities. Surprisingly, expression of beta-arrestin1(V53D) does not block activation of the MAPK(ERK) pathway. PI 3-kinase and Akt activities are also inhibited by expression of a beta-arrestin1 mutant, which impairs binding to c-Src (beta-arrestin1(P91G-P121E)), indicating the involvement of c-Src in the rapid stimulation of the PI 3-kinase/Akt pathway. Consistent with these results, PP1, a selective inhibitor of c-Src family kinases, prevents alpha-thrombin-stimulated Akt phosphorylation. Expression of beta- arrestin1(V53D) does not prevent G(1) progression, as its expression has no effect on [(3)H]thymidine incorporation into DNA. In agreement with the ineffectiveness of beta-arrestin1(V53D) to block G(1) progression, cyclin D1 protein amounts and CDK4-cyclin D1 activity is unaffected by expression of beta-arrestin1(V53D). Thus in IIC9 cells, alpha-thrombin activates rapid beta-arrestin1-dependent and sustained beta-arrestin1-independent Akt activity, suggesting that two mechanisms are involved. Furthermore, although blocking the beta-arrestin1-independent PI 3-kinase/Akt pathway prevents G(1) progression, inhibition of the beta-arrestin1-dependent pathway does not, indicating different roles for the rapid and sustained activities.     

Activation of Cdk2/Cyclin E complexes is dependent on the origin of replication licensing factor Cdc6 in mammalian cells

Cyclin E-associated CDK2 activity is required for the initiation of DNA synthesis in human cells. CDK2 activity is tightly regulated; CDK2 must be in the nucleus, bound to a cyclin, phosphorylated on T160, and dephosphorylated on T14/Y15 for complete kinase activation. Nuclear localization exposes CDK2 to activating enzymes (CAK, Cdc25A) in stimulated cells. Previous studies from our lab indicate CDK2 nuclear localization and cyclin E co-expression are insufficient to cause CDK2 activation or T160 phosphorylation in stimulated IIC9 cells; these activities still require serum stimulation and ERK kinase activity. Recent studies have implicated a role for origin of replication (ORC) licensing proteins in the activation of G1/S Cdks. In this study, we show that CDK2 associates with chromatin and Cdc6 in an ERK-dependent manner following stimulation of IIC9 CHEF cells. We show that nuclear-localized CDK2 (CDK2-NLS) ectopically expressed with cyclin E requires mitogenic stimulation and ERK activation for chromatin association, in addition to previously shown kinase activation and T160 phosphorylation in IIC9 cells. Additionally, we show that expression of Cdc6 in stimulated IIC9 cells treated with ERK inhibitor rescues CDK2-NLS chromatin association, kinase activation, and T160 phosphorylation. From the above data, we deduce ERK-dependent CDK2 activation is due in part to ERK-dependent Cdc6 expression. To examine the role of Cdc6 directly in stimulated primary human fibroblasts, we used RNA interference to attenuate the expression of Cdc6. We show that Cdc6 expression is required for CDK2 chromatin association and kinase activation in stimulated primary human fibroblasts. Additionally, we show that Cdc6 expression is required for the initiation of DNA synthesis and S phase entry in stimulated primary human fibroblasts. Ultimately, this data implicates Cdc6 expression as an important mitogen-induced mechanism in the activation of CDK2/cyclin E, the initiation of DNA synthesis, and the regulation of G1-S phase progression.     

2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) activity in cultured nerve cell lines from central nervous system: Comparison of proliferating and resting growth states and cell cycle-dependent activity changes

1. The present communication is concerned with the expression and cell cycle-dependent regulation of the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in cultured nerve cell lines derived from the rat central nervous system (CNS). 2. The enzyme activity was measured in relation to two reversible serum-controlled growth states (exponentially growing/quiescent) including a comparison of the enzyme activities in cell lines of neuronal and glial origin as well as in fibroblasts. CNPase is present in all cell types tested, but the enzyme activity is very sensitive to changes in the cellular growth state. Nerve cell lines in exponentially growing cultures express a 3 to 15 times higher specific CNPase activity than the nonneural cell types. In serum-starved quiescent cultures, the differences in specific enzyme activity between the nerve cell lines and the fibroblasts are enlarged even more up to a ratio of about 50 to 150, indicating a specific function of this enzyme within the central nervous system. 3. Neuron-like B104 cells could be stimulated to synchronized growth by serum readdition to quiescent cultures. A series of ordered activity changes of CNPase has been observed after the reinitiation of cell growth. The enzyme is stimulated at two particular stages during the cell cycle, leading to a biphasic activity profile. Maximum stimulation of CNPase correlates with the G1 phase. 4. Hydroxyurea-induced blockage of synchronized B104 cells to traverse the S phase also prevents the subsequent stimulation of CNPase activity. Therefore, we conclude that a correlation exists between the periodic activity changes of CNPase and particular phases of the B104 cell cycle.     

Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate

Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I- thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.     

Hydrolysis of polyphosphoinositides by purified sheep seminal vesicle phospholipase C enzymes

Sheep seminal vesicles contain two immunologically distinct phospholipase C (PLC) enzymes that can hydrolyze phosphatidylinositol (PI) (Hofmann, S.L., and Majerus, P.W. (1982) J. Biol. Chem. 257, 6461-6469). One of these enzymes (PLC-I) has been purified to homogeneity; the second (PLC-II) has been purified 2600-fold from a crude extract of seminal vesicles. In the present study we have compared the ability of these purified enzymes to hydrolyze PI, phosphatidylinositol 4-phosphate (PI-4-P), and phosphatidylinositol 4,5-diphosphate (PI-4,5-P2). Using radiolabeled substrates in small unilamellar phospholipid vesicles of defined composition, the two enzymes were found to hydrolyze all three of the phosphoinositides. Hydrolysis of all three phosphoinositides by both enzymes was stimulated by Ca2+; however, in the presence of EGTA only the polyphosphoinositides were hydrolyzed. The two enzymes displayed substrate affinities in the order PI greater than PI-4-P greater than PI-4,5-P2, and maximum hydrolysis rates in the order PI-4,5-P2 greater than PI-4-P greater than PI. When present in the same vesicles, PI and the polyphosphoinositides competed for a limiting amount of either enzyme. Inclusion of phosphatidylcholine into vesicles containing the phosphoinositides resulted in greater inhibition of PI hydrolysis than polyphosphoinositide hydrolysis. When all three phosphoinositides were present in vesicles mimicking the cytoplasmic leaflet of cell membranes, there was preferential hydrolysis of the polyphosphoinositides over PI. We conclude that a single phospholipase C can account for the hydrolysis of all three phosphoinositides seen during agonist-induced stimulation of secretory cells. The cytoplasmic Ca2+ concentration and phospholipid composition of the membrane, however, may influence the relative rate of hydrolysis of the three phosphoinositides.     

Secreted proteins of quiescent,serum-stimulated and over-confluent mouse embryo fibroblasts

Quiescent and proliferating cultures of Swiss mouse embryo fibroblasts were pulse labelled with [¹⁴C]-amino acids and the newly synthesized proteins that were secreted into the medium were resolved by electrophoresis on Polyacrylafde gradient gels. Conditioned media obtained from quiescent cultures that were stimulated to grow by the addition of 20% fetal calf serum showed the presence of two unique polypeptides of molecular weights 48000 and 26000. A polypeptide of molecular weight 45000 was present in increased amounts in serum-stimulated cells than in quiescent cells. This protein was also superinduced in quiescent cells by cycloheximide treatment. Mouse embryo fibroblasts grown under over-crowded conditions secreted two proteins of molecular weights 35000 and 11000. The 35 K polypeptide was shown to be related to the major excreted protein of transformed cells, since it was immunoprecipitated by an antiserum to major excreted protein. These results indicate that the 48 K and 26 K proteins may be proliferation specific proteins, while the 35 K protein present in the conditioned media of over-confluent cells may be a marker of morphological transformation.     

Quantitative analysis of molecular species of diacylglycerol and phosphatidate formed upon muscarinic receptor activation of human SK-N-SH neuroblastoma cells.

Quantitative changes in the total mass and the molecular species of 1,2-diacyl-sn-glycerol (DAG) and phosphatidic acid (PA) formed upon muscarinic receptor activation were studied in cultured human SK-N-SH neuroblastoma cells. DAG was isolated from the total lipid extracts of carbachol (CCh)-stimulated and unstimulated cells and after benzoylation, was subjected to reverse phase high performance liquid chromatography to separate the component species. The molecular species of DAG were identified by analyzing the fatty acid composition of each separated fraction by gas chromatography, and their total and individual masses were quantified from the known amount of an internal standard, 1,2-distearoyl-sn-glycerol, added during the extraction of the lipid. Relatively high basal levels of DAG (1.5 nmol/mg protein) are present in these cells, and addition of CCh elicited a 50-60% increase in the total amounts of DAG within 5 min. The increase was biphasic: an initial major peak at 5 min was followed by a sustained increase that persisted for at least 30 min. An increase in DAG was elicited by both full and partial muscarinic agonists and was blocked by atropine. The presence of extracellular Ca2+ was necessary for muscarinic receptor-activated formation of DAG. To determine the source of the DAG, the molecular species of the major phospholipids present in SK-N-SH cells were also analyzed. The phospholipids were first enzymatically hydrolyzed to DAGs which were then analyzed as described above. A number of unusual fatty acids, the major one being 20:3 (n-9), were present in these lipids especially in the phosphoinositides and also in the DAG formed after CCh stimulation. Within 5 s of CCh stimulation there were transient increases in the DAG species representative of phosphoinositides. By 5 min the newly formed molecular species of DAG resembled a mixture of phosphoinositides and phosphatidylcholine (PC). Quantitative comparison of the molecular species compositions of phosphoinositides, PC, and newly formed DAGs indicated that at time periods up to 10 min, approximately 30% of the DAG originated from the phosphoinositides and the rest from PC. At longer intervals (greater than 20 min), most (85%) of DAGs originated from PC. Activation of muscarinic receptors in SK-N-SH cells also elicited an increase in PA (200% in 5 min). A quantitative molecular species analysis, using 1,2-distearoyl-sn-glycerol-3-P as internal standard, was performed by enzymatic (alkaline phosphatase) hydrolysis of PA to DAG and subsequent analysis.(ABSTRACT TRUNCATED AT 400 WORDS)