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INTRACELLULAR SIGNAL SYSTEMS |
, 
, 
) in T3-1 and LßT2 Gonadotrope Cell Lines
Centre National de la Recherche Scientifique UMR 6544, Université de la Méditerranée, Faculté de Médecine, 13916 Marseille, France
Address all correspondence and requests for reprints to: S. V. Drouva, Interactions Cellulaires Neuroendocrieniennes, UMR 6544 Centre National de la Recherche Scientifique, IFR Jean-Roche, Fac Med Nord, Bd P. Dramard, 13916 Marseille Cedex 20, France. E-mail: . drouva.sv{at}jean-roche.univ-mrs.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, 
, 
): 1) during 12-O-tetradecanoyl-phorbol-13 acetate (TPA) (10-7 M) or GnRH (10-7 M) desensitization conditions (2- to 6-h treatments) in two gonadotrope cell lines (T3-1, LßT2) and 2) in primary pituitary cell cultures from male rats during long-term phorbol ester administration. We demonstrated that, as in T3-1 cells, in a more differentiated gonadotrope cell line LßT2 the GnRH-receptor coupling (PLC, PLA2, PLD) generated second messengers essential for PKCs activation; the characterized isoforms (, ßII, 
, , ) were selectively and differentially down-regulated by TPA (, ßII, , ) or GnRH (, ). In whole cell lysates, proteasome inhibitors (proteasome inhibitor I and II, Lactacystin, ß-Lactone, Calpain inhibitor I) prevented in both gonadotrope cell lines the TPA-induced depletion of PKC , , and the GnRH-elicited PKC down-regulation; they counteracted in mixed pituitary cell cultures as well, the TPA-evoked PKC , depletion. In contrast, the inhibitors of calpain(s) and lysosomal proteases (Calpeptin, E64d, Calpain inhibitor II, and PD150606), were ineffective. As shown in T3-1 subcellular fractions, proteasome abrogation did not affect membrane translocation of TPA- and GnRH- target isoforms (, ) but, preventing their degradation, favored enzyme accumulation to the membrane compartment. Proteolysis processing of PKCs may be dependent upon their phosphorylated state and/or catalytic activity. Inhibition of PKC catalytic activity (GF109203X, Gö6976), selectively prevented the TPA-evoked PKC depletion in both mixed pituitary cells and T3-1 gonadotropes; in T3-1 subcellular fractions, PKC inactivation overcame the TPA-evoked isoenzyme degradation by inducing a pronounced membrane accumulation of the isoform without affecting its membrane relocalization. Thus, the proteasome system by adjusting PKC cellular levels, may represent a regulatory proteolytic pathway implicated in the adaptive mechanisms of the time dependent cell responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ßI, ßII, and 
) requiring for their activation both diacylglycerol (DAG) and calcium; 2) novel PKC (, , 
, and 
) not responding to Ca2+ but sensitive to DAG; 3) atypical PKCs ( and 
/
) mainly regulated through phosphoinositides (PI3K signaling pathway) the recently discovered PKCµ may represent another subgroup. When translocated, however, all PKC isoenzymes require the lipid phosphatidylserine as cofactor for their activation (3, 4, 5, 6, 7, 8). Activation of PKCs can be triggered by stimulating a variety of plasma membrane hormone, neurotransmitter, and growth factor receptors (2, 9, 10, 11, 12, 13). Receptor activation induces the formation of two important second messengers, inositol 1,4,5 triphosphate (IP3) and DAG (3). The binding of IP3 to its intracellular receptor results in a rise of intracellular Ca2+. Membrane DAG initiates activation of most isoenzymes by recruiting them to membranes, where they are further stimulated by interacting with phosphatidylserine. Binding of both lipids to the PKC results in a conformational change that removes the inhibitory pseudosubstrate domain from the active site of the enzyme thus allowing its catalytic function (7, 14). In addition to the natural activators, most of the isoenzymes (conventional and novel) are directly activated with high specificity by the tumor promoting phorbol esters (3, 7, 8, 14).
A number of recent studies have provided evidence indicating that some isoenzymes undergo multisite phosphorylation (trans- and autophosphorylations) before they become competent to respond to second messengers (4, 5, 6, 10, 14, 15, 16). These events combined with selective dephosphorylation, render the kinase in a catalytically active or inactive conformation, and direct its subcellular localization and metabolic processing as well (10, 14, 17, 18, 19).
Intracellular compartmentalization of individual PKC isoenzymes during cell cycle, as well as before or after stimulation, could be an important component in determining the biological activity of PKCs, by sequestering the corresponding PKC in the vicinity of the appropriate substrate(s) (8, 20, 21, 22). Thus, each PKC may display a distinct subcellular localization and bind to several intracellular proteins that serve as substrates and/or carriers such as RACK, RICK (receptors for activated or inactivated C kinase), myristoylated alanine-rich C kinase substrate (MARCKS), A kinase-anchoring protein 79 (AKAP 79), 14-3-3 proteins, annexins, or actin and cytoskeletal components (14, 20, 21, 22, 23, 24, 25).
Acute activation of PKCs is followed by desensitization of the isoenzymes and the associated cellular responses. Depending on the duration of the stimulus by either phorbol esters or neuropeptides, the isoenzymes are selectively down-regulated (7, 8, 11, 13, 26, 27). The cellular processes as well as the molecular mechanisms involved in this phenomenon are still elusive. It has been suggested that the phosphorylation state may predispose the enzyme for degradation (15, 18, 19, 28); the loss of protein might represent accelerated degradation (29), but modified synthesis for some isoenzymes is also reported (5). Proteolysis of PKC might implicate calpains, vesicle-dependent sorting mechanisms targeting the PKC isoenzymes for degradation in lysosomes, or proteasome-mediated degradation of ubiquitin-tagged isoenzymes (26, 28, 30, 31, 32, 33, 34). Thus PKC activation, phosphorylation/dephosphorylation, selective biding to the anchoring proteins, catalytic function of the enzyme and down-regulation might be associated events and physiologically significant in cellular signaling.
Among other groups we have previously demonstrated, that in T3-1 gonadotropes GnRH receptor coupled in a sequential manner to PLC, PLA2 and PLD, generates IP3, increases Ca2+i, and stimulates arachidonic acid release, phosphatidic acid (PA), and DAG production (35, 36, 37). These second messengers in coordination with Ca2+ via voltage dependent gated calcium channels stimulate PKC activity, which is a key enzyme in regulating the GnRH-dependent transient and sustained phases of gonadotropes functions (12, 27, 35, 36, 38, 39, 40).
We have further shown that T3-1 gonadotrope cell line possesses several PKC isoenzymes (, ßII, , , , ), which are activated and differentially down-regulated during 12-O-tetradecanoyl-phorbol-13 acetate (TPA) or GnRH desensitization conditions (12, 27). We have thus suggested that GnRH besides the rapid stimulation of specific PKC species during the early times of its action, might provide during long term desensitization of gonadotrope cells [situation exploited in the clinical use of GnRH analogs (41)], an additional mechanism in suppressing cellular responses, by inducing selective down-regulation of various PKC isoenzymes (, ).
The mechanism(s) involved in the down-regulation of GnRH target PKC isoenzymes are still unknown; they may be different from those concerning phorbol ester action. Thus, in the present study we investigated in two gonadotrope cell lines (T3-1, LßT2) as well as in normal pituitary cells: 1) to what extent different proteolytic activities such as calpains and proteasome may account for the selective down-regulation of PKC isoforms (, , ) induced after prolonged treatment (2–6 h) by TPA (10-7 M) or GnRH (10-7 M); 2) initial events predisposing the PKC for degradation.
We demonstrated that down-regulation of PKC isoforms induced by a physiological stimulus GnRH (PKC ), or by the phorbol ester TPA (PKC , ) involves mainly proteasome, an organelle that degrades selectively multiubiquitinylated proteins, and depending on the isoform (PKC ) may be initially relies upon the phosphorylated state and/or catalytic function of the isoenzyme. Thus, proteasome activity seems to be part of an adaptive process of the cell responding to chronic activation; by regulating PKC cellular level, it may play a crucial role in the PKC-dependent functions and might contribute to the modifications of gonadotrope responses.
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Materials and Methods |
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T3-1 as well as LßT2 cells (42, 43) were grown and passaged routinely in DMEM with 4.5 mg/ml glucose (Life Technologies, Inc.) supplemented with 10% FCS and penicillin/streptomycin (100 and 75 U/ml, respectively; Sigma, St. Louis, MO) (27, 36). Cultures were maintained at 37 C in a water-saturated air atmosphere (92–95% humidity) containing 5.8% CO2. When cells reached 60–70% confluency, they were cultured overnight with the culture medium containing the antibiotics and 1% FCS to avoid further divisions and thus obtain cells in a homogenous developing state for the following experiments.
b. Cell preparation and primary cultures of normal pituitary cells.
Anterior pituitary glands from adult male rats were removed and dispersed as previously described (44). Briefly, the anterior pituitaries after enzymatic digestion (consecutive incubation with trypsin and DNase; Sigma) were mechanically dispersed. After centrifugation at 300 x g for 10 min, the resuspended cells were plated and cultured in DMEM (1 g/liter glucose; Life Technologies, Inc.) supplemented with 10% FCS, glutamine, and antibiotics (penicillin/streptomycin, 100 and 75 U/ml, respectively). Cell cultures were maintained in a humidified atmosphere of 5.8% CO2 at 37 C for 4 d.
Measurement of inositol phosphates (IPs)
Inositol phosphates (IP1 + IP2 + IP3) were evaluated as previously described (36, 45). Briefly, LßT2 cells were prelabeled with myo-[3H]inositol (2.5 µCi/ml, 12.8 Ci/mmol; NEN Life Science Products, Boston, MA) 48 h before the experiment. On the day of the experiment, the cells were stimulated in the presence of 12 mM LiCl. Incubations were terminated by elimination of the culture medium and addition of ice-cold perchloric acid (5%). IPs were extracted by chromatography on anion exchange columns (AG1 x 8 formated form; Bio-Rad Laboratories, Inc., Richmond, CA). Results are expressed as count per min/dish (4 x 106 cells).
Measurement of [3H]arachidonic acid metabolites release
As previously described (36, 46) LßT2 cells were incubated with [3H]arachidonic acid (0.25 µCi/well; NEN Life Science Products, Boston, MA) to isotopic equilibrium (24 h). Before stimulation, the cells were extensively washed with Krebs-bicarbonate buffer (3.5 mM KCl; 0.75 mM CaCl2; 124 mM NaCl; 125 mM K2HPO4; 26.3 mM NaHCO3; 20 mM glucose, pH 7.4), supplemented with 0.1% fatty acid-free BSA (Sigma). After stimulation, the reaction was stopped by removing the incubation medium, and the released radioactivity was measured with a liquid scintillation spectrophotometer.
Measurement of PLD activity
The PLD activity was evaluated as previously described (36), by measuring the production of [3H]phosphatidylethanol ([3H]PEt) in the presence of ethanol. Briefly, LßT2 cells were preincubated overnight with [3H]myristic acid (5 µCi/ml, NEN Life Science Products) in DMEM containing fatty acid-free BSA (0.18%). Stimuli were applied for the indicated times in the presence of 0.5% ethanol. The extracted lipids (chloroform phases) dried under nitrogen, were redissolved in a mixture of chloroform/methanol (vol/vol) containing standards of PA (Sigma) and PEt (Avanti Polar Lipids Inc., Alabaster, AL) and applied on oxalate impregnated Silica Gel TLC plates (Whatman, Maidstone, UK). The plates were developed in the organic phase of a mixture of ethyl acetate/2,2,4-trimethyl pentane/acetic acid/water (13:2:3:10, vol/vol/vol/vol). The spots corresponding to the appropriate standards visualized by iodine vapor staining were extracted with 1 ml methanol/HCl (150:1, vol/vol) and counted in 9 ml of scintillation fluid ACSII (Amersham Pharmacia Biotech, Saclay, France).
Characterization of PKC isoenzymes
a. Preparation of whole cell extract.
Vehicle control and cells (4.106/dish) treated either with TPA (10-7 M) or GnRH analog (pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt, 10-7 M) for times indicated in Results or figures, were rinsed with ice-cold PBS (free Ca2+ and Mg2+) and harvested in ice-cold RIPA modified buffer (Tris-HCl 25 mM pH 7.4, NaCl 150 mM, Nonidet 1%, sodium deoxycholate 0,25%, EGTA 1 mM) containing 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM sodium orthovanadate. The solubilized cells were passed through a 21-gauge needle to sheer the DNA. The cell lysates were left for 40 min on ice and then microfuged for 20 min at 4 C. The resulting supernatant was the total cell lysate (27). Protein content was measured according to Bio-Rad Laboratories, Inc. detergent-compatible protein assay.
b. Subcellular fractionation.
For translocation experiments, the preparation of cytosolic and membrane fractions were adapted from methods previously described (36, 44). Briefly T3-1 were grown to approximately 70% confluence in 90-mm diameter culture dishes (8.106 cells) were treated as indicated in Results and then washed with ice-cold PBS (Ca2+ and Mg2+ free) and immediately scraped into 1 ml of homogenization buffer A (Tris-HCl 25 mM pH 7.5, containing 250 mM sucrose, 2 mM EDTA, 2 mM EGTA, 30 µg/ml aprotinin, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 30 µg/ml leupeptin, 1 mM sodium orthovanadate, 1 mM benzamidine, and 10 mM sodium pyrophosphate). Cells were then disrupted with 40 strokes in a Dounce homogenizer. The homogenates were first centrifuged at 150 x g for 3 min at 4 C. The supernatant was centrifuged at 4 C at 100,000 x g for 1 h. The resulting supernatant, cytosol fraction, was collected and kept at -80 C until assayed. The pellets were resuspended in cold buffer A containing Triton X-100 (1% wt/vol), gently rehomogenized by brief sonication, solubilized at 0 C for 45 min, and centrifuged at 100,000 x g for 30 min. The obtained supernatant was collected as membrane fraction. The fractions were then analyzed by western blotting. Protein content was measured according to corresponding Bio-Rad Laboratories, Inc. protein assay.
c. PAGE and Western blotting of PKC isoenzymes.
SDS-PAGE and Western blotting techniques were performed as previously described (27). Briefly, proteins (5 µg) from total cell lysates or subcellular fractions as well as prestained molecular weights markers were separated by SDS-PAGE on 10% polyacrylamide gels under denaturing conditions (heat at 100 C for 8 min) and previously diluted in electrophoresis buffer (8.7% glycerol, 2.6% SDS, 4.4% ß-mercaptoethanol, 108 mM Tris-HCl pH 6.7, bromophenol blue). Proteins were then transferred to a polyvinylidene difluoride membrane (Polyscreen, NEN Life Science Products) using a semidry electroblotter (Owl Separation Systems, Portsmouth, NH). Membranes after drying were washed with PBS for 5 min and successively incubated overnight at 4 C in blocking solution (0.2% highly purified casein, 0.1% Tween-20 in PBS) and then for 2 h at room temperature with affinity purified isoenzyme-specific antibody diluted in blocking solution. Polyclonal anti-PKC , ßII, , , , , were obtained from Santa Cruz Biotechnology, Inc. The antibodies were used in the following dilution: 1/10000 for , and ßII, 1/8000 for , 1/5000 for , 1/20000 for and 1/2500 for . Blots were then washed in blocking solution and incubated with the second antibody (1/5000 goat antirabbit IgG alkaline phosphatase conjugate, Tropix Inc., Bedford, MA) for 1 h at room temperature. They were developed with Tropix chemiluminescence reagents (CDP-Star chemiluminescent substrate for alkaline phosphatase) using NEN Life Science Products Reflexion films for the detection of light emission.
The intensity of bands on the films was quantified using a densitometric analysis system NIH Image program version 1.59 (Agfa Arcus II scanner, Agfa-Gevaert N.V., Mostel, Belgium) where background intensities have been substracted. The values given in histograms represent the mean ± SEM obtained after quantification of the specific bands from different experiments and are expressed as % OD control or as arbitrary OD units. The PKC predominantly migrated as a doublet accompanied in stimulation experiments by a band of a higher molecular mass; thus, the related immunoreactive bands, reflecting probably different phosphorylation states of the isoenzyme, were accordingly included in the quantification analysis and have been commented in the appropriate sections.
Statistical analysis
Statistical analysis between experimental groups was performed using ANOVA, followed by post-hoc Scheffé’s F statistic for multiple comparisons, with an overall two-tailed value of 0.05. The number of experimental points indicated in the figures, and used for statistical analysis represents individual harvested plates, from several independent experiments.
Pharmacooigical agents
Chemicals were purchased from Sigma unless otherwise stated.
GnRH (Neosystem, Strasbourg, France) was used for acute stimulation experiments; stable GnRH analog [des-Gly10,[D-Ala6]-LHRH ethylamide (Sigma)]: was applied for desensitization experiments (>2 h); GnRH antagonist: Dp-Glu1,D-Phe2,D-Try3,6-GnRH (Sigma).
The following agents were purchased from Calbiochem (San Diego, CA): Clasto-Lactasystin ß-Lactone; Lactacystin synthetic; Proteasome inhibitor II (Z-LLF-CHO); Proteasome inhibitor I (PSI); Calpain inhibitor II (ALLM); Calpain inhibitor I (ALLN); Bisindolylmaleimide I (GF109203X); E-64d (Loxastatin); Gö6976; PD150606 (3-(4-Iodophenyl)-2-mercapto-(Z)-2propenoic acid); Calpeptin (Z-Leu-Nle-CHO).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3-1 (nonsecretory) and LßT2 (secretory)] as well as in normal pituitary cell cultures, to 1) differentially evaluate the effects of selective inhibitors affecting several proteolytic activities (mainly calpains and proteasome) that might be involved in the process of the down-regulation of PKC isoenzymes (, , ) induced by the phorbol ester TPA or the neuropeptide GnRH; 2) investigate the importance of the PKC catalytic activity in the initial steps leading the individual isoenzymes to degradation.
A. T3-1 cells
1. Effect of calpain-inhibitors on TPA- and GnRH-induced down-regulation of PKC isoenzymes.
As previously shown (27), TPA (10-7 M) administration to T3-1 cells for 2–6 h down-modulated the PKC and the PKC isoenzymes (Fig. 1
); a biological inactive phorbol ester, 4-phorbol 12,13-didecanoate, used at 10-7 M was ineffective (data not shown). However, treatment of the cells by the GnRH analog (10-7 M) selectively depleted the PKC (Fig. 1). Interestingly, a slower mobility band related to PKC was revealed after GnRH or TPA stimulation; it was more important at earlier times (data not shown) and as the major and the faster migrating bands, it was progressively depleted under desensitization conditions (Fig. 1). The PKC remained resistant to both treatments. The down-regulation of the affected isoenzymes was progressive and became most evident at 4–6 h treatment; thus, we have chosen this timing to further evaluate the effects of the inhibitors applied.
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Calpeptin, a synthetic inhibitor of calpain(s) activity, is a dipeptide aldehyde that binds specifically to the critical cysteine residue in the active site of calpains, preventing the binding and subsequent proteolysis of calpain substrates (49). E64d affects both calpains and lysosomal proteases, whereas the PD150606 is a nonpeptide calpain inhibitor directed toward the calcium-binding sites of calpains (48, 50). The inhibitors were administrated to T3-1 cells 30 min before TPA or GnRH stimulation, at concentration of 10-5 M for Calpeptin, 10 or 25 µM for E64d, and 10 or 20 µM for PD150606. None of these substances affected the phorbol ester-induced down-modulation of PKC and PKC isoenzymes after either 2- and 4-h (data not shown) or 6-h stimulation (Fig. 2). Similar lack of effect was observed for the GnRH-evoked PKC depletion (Fig. 3). Because the dipeptide aldehyde might be reactive with amine groups containing in incubation media, similar experiments were performed in KREBS solution enriched with 4.5 g/liter glucose. Still, the calpain inhibitor calpeptin (10-5 M) remained ineffective to prevent the corresponding PKC-down-regulation induced by TPA (10-7 M) or GnRH (10-7 M) for 4 or 6 h (data not shown).
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. Small effect, but not consistent, however, was registered for the PKC disappearance evoked by TPA and GnRH (Fig. 4). Interestingly, the calpain inhibitor I (10-5 M) ALLN, which blocks calpains and cathepsin activities with similar potency as ALLM but with a more important efficiency proteasome peptidases (48, 51), significantly attenuated the TPA-induced down-regulation of PKC and PKC ; at the same concentration, it reversed the GnRH-evoked PKC depletion as well. In all cases, PKC remained insensitive (Fig. 4). None of the inhibitors applied, modified the immunoreactive bands of the PKC isoenzymes obtained under control conditions (data not shown).
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2. Proteasome involvement on TPA-and GnRH-induced depletion of target PKC and PKC isoforms in T3-1 cells
Whole lysates.
To investigate whether the down-regulation of PKC isoenzymes evoked by TPA or GnRH (4–6 h) is dependent on the proteasome pathway, T3-1 cells were incubated with cell-permeable highly selective proteasome inhibitors 15–30 min before the stimulus application.
Lactacystin and Clasto-Lactacystin ß-Lactone inhibit all three activities that are associated with the 20S proteasome (trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing activities) (51, 52); they were applied at 10 µM concentration. Proteasome inhibitor II inactivates the chymotrypsin-like activity (53); it was used at 10 µM concentration.
As shown in Fig. 5, all these three compounds effectively prevented with a comparable efficiency the corresponding TPA-induced depletion of PKC and PKC , as well as the GnRH -receptor mediated depletion of PKC . It is worthy to note that in the case of PKC isoenzyme, the effects of the inhibitors reflected not only increase of the major band of the isoform, but those concerning a lower and a higher molecular weight migrating species as well (Figs. 4
and 5); hypo and hyperphosphorylated forms? Do they have a common subcellular origin? As ß-Lactone (Fig. 5), the other proteasome blockers did not modify the immunoreactive bands of the PKC isoenzymes obtained under basal conditions (data not shown). The inhibitors had no effect on PKC , an isoform not responsive to the TPA or GnRH treatments (Fig. 5). Similar results were obtained with proteasome inhibitor I at 10 µM concentration (data not shown).
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T3-1 cell extracts were fractionated after acute (10 min) or prolonged (4 h) cell treatment by either TPA (10-7 M) or GnRH (10-7 M) in the presence or not of proteasome inhibitor I or proteasome inhibitor II (10 µM); they were then subjected to SDS-PAGE and the PKC content in cytosolic and detergent-soluble membrane fractions was analyzed by Western blot. As Fig. 6, A and B, shows, the phorbol ester (10 min) elicited an important membrane association of PKC and PKC isoenzymes. After 4 h of treatments, however, the cytosolic fraction of both PKCs was almost disappeared and the translocated-membrane levels of the isoenzymes were markedly diminished. Interestingly, pretreatment of the cells with the proteasome inhibitors did not affect the TPA-induced PKC and PKC translocation, but under desensitization conditions, highly increased the amount of the isoenzymes associated to the membrane fractions (Fig. 6, A and B). Under no treatment did PKC levels change (data not shown).
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isoenzyme toward membrane compartment (Fig. 6C); the translocation response remained unaffected by both proteasome inhibitors (Fig. 6C). As expected, 4-h treatment with the neuropeptide analog significantly down-regulated PKC in both subcellular fractions (compared with either basal or translocated-membrane levels). However, when the cells were pretreated with the proteasomal activity blockers, the amount of the isoenzyme was restored with predominant redistribution to the membrane compartment. Under extraction and fractionation conditions described, neither GnRH stimulation appeared to respectively relocalize or down-regulate the PKC and PKC isoenzymes (data not shown). Moreover, none of the inhibitors when applied alone influenced the distribution of PKC and PKC found under control conditions (Fig. 6, A and C, for P. Inh II, and data not shown for P. Inh I).
Of a great interest in these experiments is the migrating behavior of PKC , especially in membrane fractions. The acute cell stimulation by GnRH or TPA induced, along the redistribution of the major band of the isoenzyme, the formation of a slower mobility immunoreactive band that remained unaffected by the proteasome inhibitors. During respective desensitization conditions, however, it was strongly attenuated and further reappeared after inhibition of the proteasome peptidase activity. In addition, the fine band showing fast migration, was revealed usually in the membrane fractions (Fig. 6, B and C).
B. LßT2 cells
The gonadotrope cell line LßT2 has been generated by tumorogenesis in transgenic mice and expresses messages for both and ß subunits of LH as well as GnRH receptor mRNAs (42). These cells represent a more differentiated gonadotrope cell line than T3-1, with functional characteristics similar to those observed in normal gonadotropes (42).
Thus, the intent of the following studies was to compare functional similarities, between T3-1 and LßT2 cell lines, concerning PKC isoenzymes profiles during TPA- or GnRH-induced desensitization conditions and to provide a further insight about the proteasome implication in this process. Hence, we first characterized the couplings of GnRH-R (GnRH receptor) leading to second messengers that trigger PKC(s) activity.
1. Coupling of GnRH receptor
GnRH (10-7 M) applied to LßT2 cell cultures highly stimulated IPs production; the effect was abolished by the GnRH antagonist (DpGlu1,D-Phe2,D-Try3,6GnRH) and as in T3-1 gonadotropes, pretreatment of the cells with the neuropeptide for 1 h (10-7 M) significantly attenuated the GnRH- elicited IPs formation (Fig. 7A). Furthermore, stimulation of GnRH-R generated arachidonic acid release with similar time dependent kinetics as in T3-1 cells (36, 46) and induced 3[H]PEt accumulation, in a stereospecific manner (Fig. 7, B and C, respectively).
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T3-1 line (36), the phorbol ester TPA (10-7 M for 1 h) markedly stimulated PLD activity and further potentiated the GnRH-induced PLD activation (Fig. 7C). The effect was almost additive indicating either different mechanisms or different PKC isoforms involved in GnRH- and TPA-evoked PEt formation (36). These results demonstrate that in the LßT2 cell line the GnRH receptor is coupled with PLC, PLA2, and PLD activities and thus capable when activated, to generate different second messengers (IP3, DAG, AA, PA, Ca2+) essential for further stimulation of target PKC isoforms (3, 7, 14), key enzymes in the gonadotrope cell function.
2. PKC isoenzymes; desensitization profiles
The sensitivity of an individual PKC isoform toward stimuli is in part dependent upon the coexpression of another PKC isoenzyme (54, 55). Thus, the next step to our studies was to characterize the PKC isoforms and to further evaluate in this cell line as well, their modulation during desensitization conditions after long treatment (2–15 h) with either TPA 10-7 M or GnRH analog 10-7 M.
Specific immunoreactive bands of the appropriate molecular weight were identified for , , , ßII, and isoenzymes (Fig. 8). As in T3-1 cells PKC and were not expressed in LßT2 cell line. Interestingly and in contrast to T3-1 gonadotropes PKC , an isoenzyme associated with the differentiation state and proliferation capacity of some cells (56, 57), was not detected in this cell line. In addition, the PKC isoform was less expressed in LßT2 cells (for the same amount of protein, longer exposure of the film was necessary to reveal under identical conditions the immunoreactive band related to this isoenzyme).
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T3-1 gonadotropes (Fig. 8). PKC expression was not altered by either TPA (10-7 M) or GnRH (10-7 M) (2–15 h treatment). However, the levels of PKC , ßII, , isoforms were progressively diminished at different rates and to different extents for each isoenzyme after TPA (, , , ßII) or GnRH (, ) treatments (Fig. 8). In contrast to TPA, GnRH administration did not evoke down-regulation of PKC isoform at any time tested (Fig. 8).
Thus, as in T3-1 cells (27, 36), the PKC isoenzymes, which are activated and showed a distinct subcellular localization [(2) and Drouva, S. V., unpublished data], are subsequently and differentially down-regulated by the respective stimuli in LßT2 gonadotrope cell line as well.
3. Interaction of calpain and proteasome inhibitors with GnRH and TPA desensitizing effects on PKC , , and isoenzymes
As in T3-1 cells, both Calpeptin and ALLM (at 10 µM, respectively) had no effect on TPA-induced PKC and PKC , or on GnRH-elicited PKC depletion in LßT2 cells after 6 h treatment (Fig. 9). However, proteasome inhibitor II and ALLN applied at 10 µM for 30 min before TPA (10-7 M) or GnRH (10-7 M) treatments, significantly prevented and with comparable efficiency as in T3-1 cells, the down-regulation of TPA-induced PKC and PKC as well as the GnRH-evoked depletion of PKC (Fig. 9). Similar effects of the inhibitors were obtained on earlier time induced-depletion of the isoenzymes (2–4 h, data not shown). Furthermore, the migrating profiles of the lower mobility PKC isoform toward TPA and GnRH stimuli as well as proteasome inhibitors treatment, were comparable to those revealed in T3-1 cell line (Figs. 8 and 9).
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and PKC induced by a phorbol ester or a specific ligant (GnRH). This might further indicate, that ligant-regulated proteolysis of specific signal transduction proteins, is important for maintaining their appropriate cellular levels and hence, the corresponding cell function(s).
C. Normal pituitary cells
1. TPA-induced down-regulation of PKC isoenzymes.
We have previously shown, that PKC activity was recovered from all pituitary cell types was highly inducible by the phorbol esters and very responsive to steroid treatment (44); in addition, the phorbol ester TPA markedly increased the LH, PRL, and GH release from primary culture of mixed pituitary cells (44).
We thus pursued our studies, using normal pituitary cells in primary mixed cultures, to further validate in a physiological cell model the implication of the proteasome-mediated proteolysis in the degradation process of TPA-sensitive PKC isoenzymes and .
Several PKC isoforms (, , , ßII, ) were identified in the whole cell extracts of pituitary cells obtained from male rats (Fig. 10). With the exception of PKC , TPA (10-7 M) treatment progressively up to 15 h down-regulated the PKC , ßII, and isoenzymes, with approximately similar rates to those obtained in T3-1 (27) and LßT2 cells (Fig. 10).
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and isoenzymes sensitive to phorbol ester TPA, A and B, Calpeptin (10-5 M) and ALLM (10-5 M) failed to overcome the TPA-induced down-regulation of PKC and . However, the specific proteasome inactivation (Fig. 11, C and D) by proteasome inhibitor I and II at (10-5 M) effectively counteracted the PKC and PKC depletion evoked by the phorbol ester (4 or 6 h treatments). Comparable results (Fig. 11, A and B) were obtained with ALLN (10-5 M). Of interest as in T3-1 and LßT2 cells, the PKC showed comparable migrating behavior [the major band associated with a faster and a slower electrophoretic mobility ones; the latter being evident after stimulation or after slowing-down the proteasome activity (Figs. 10 and 11)].
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D. PKC down-regulation is dependent upon PKC kinase activity
Whole lysates (T3-1, primary pituitary cell cultures).
To begin investigating the initial mechanisms predisposing the PKC for further processing leading to down-regulation, we asked whether the kinase activity is important for the enzyme degradation (15, 18, 19). Indeed GF109203X [a competitive inhibitor of ATP binding site of PKC (58)] when administrated 15–30 min before the stimulus at 1 µM substantially prevented, in both T3-1 and normal pituitary cells, the TPA-induced depletion (4–6 h) of PKC (Fig. 12, A and C). Interestingly, under the same conditions, Gö6976 (1 µM) that specifically inhibits PKC activity (59) significantly attenuated the phorbol ester-elicited down-regulation of the enzyme in T3-1 cells, without affecting PKC and profiles (Fig. 13). In contrast, the proteolysis of PKC evoked by GnRH analog (10-7 M) or TPA (10-7 M) -stimuli with distinct mode of action-, was not markedly affected by GF109203X treatment (Fig. 12). Surprisingly, however, the kinase inhibitor induced a further decrease of the intensity of the immunoreactive bands, revealed particularly in T3-1 cells; as shown in histograms as well, it was slight but more evident at 6 h desensitization (Fig. 12, A and B). Modifications of the migrating species associated to PKC might contribute in fact to this effect (Fig. 12, A and B).
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T3-1 cells)T3-1 cells the TPA-induced membrane translocation of PKC and PKC (10-min stimulation, Fig. 14, A and C), as well as the GnRH-elicited membrane association of PKC (2- or 10-min treatment, Fig. 14D). Moreover, the inhibitors selectively preserved the PKC from down-regulation induced by 4-h phorbol ester administration; the enzyme was substantially accumulated in the membrane fraction (Fig. 14A). In contrast, the PKC -induced depletion by 4-h TPA or GnRH analog treatment was not markedly modified by either PKC kinase inactivation (Fig. 14, C and D).
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, but rather suggest mechanisms relaying on PKC catalytic activity involved in the proteolysis processing.
To further argument in favor to the specific behavior of PKC toward Gö6976, we show here exceptionally results concerning PKC ßII, another TPA-sensitive isoenzyme [translocated and down-regulated by the phorbol ester, Fig. 14B (2)]. As in the case of PKC , both inhibitors did not influence the TPA-induced translocation of the ßII isoform; however, GFX was the only one capable of blocking the down-regulation of the enzyme induced by the phorbol ester [once again the accumulation was detected in the membrane fractions (Fig. 14B)].
None of the inhibitors applied alone influenced the subcellular distribution of PKCs found under control conditions (Fig. 14, A–C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among others, we have shown that T3-1 gonadotrope cells possess several PKC isoenzymes (, ßII, , , , ), which are selectively activated by TPA or GnRH and differentially down-regulated during the corresponding desensitization conditions (2, 12, 27, 35). The neuropeptide modulates the PKC , (2, 27, 35) and probably (12) isoenzymes, whereas the phorbol ester affects PKC , , , ßII isoforms (12, 27). Hence, GnRH may modify signaling proteins through PKC isoenzymes different to those involved in TPA-induced modifications. For example, the neuropeptide- or the TPA-elicited PLD activation may implicate distinct PKC isoforms (27, 36). Moreover, although homologous rapid desensitization of GnRH-induced IP3 formation does not occur in these cells (27, 62), the TPA-induced rapid uncoupling of GnRH-evoked PLC stimulation (27, 36) may reflect activation of specific PKC isoenzymes (PKC , ß), known to be implicated in the phosphorylation of PLC signaling (45, 63); the latter may represent mechanisms involved in the interactions of GnRH-R with another receptor (heterologous desensitization) affecting similar PKC isoenzymes to those sensitive to phorbol ester TPA.
Futhermore, persistent stimulation of T3-1 cells with GnRH resulted in a time-dependent attenuation of GnRH elicited PLC, PLA2 and PLD activities through a process which does not implicate TPA sensitive PKC isoenzymes (, ßII, , ) (27). Interestingly, under such long term GnRH desensitization conditions, the neuropeptide provoked selective down-regulation of PKC and PKC isoforms; this might provide an additional mechanism in suppressing gonadotrope cell responses (27). This would seem to be part of an adaptive process responding to chronic activation. However the mechanism(s) underlying GnRH-induced PKC down-regulation are still unknown; they might represent increase of PKC degradation and/or decrease of PKC synthesis.
PKC isoenzymes are selectively regulated by multiple interdependent mechanisms including activation by a set of second messengers, phosphorylation/dephosphorylation process, translocation at specific subcellular sites by binding to anchoring proteins, where the enzymes at a precise time have access to their selective substrate(s), and finally proteolysis (7, 14, 16, 20, 21, 22, 24). In general, protein degradation is catalyzed by a diversity of enzymes such as calpains, lysosomal proteases or by the ubiquitin-proteasome system, to achieve an appropriate and time dependent intracellular protein level (34, 48, 64, 65).
Focusing our studies on PKC , , and isoenzymes, we provide here evidence demonstrating that in gonadotrope cell lines [T3-1, LßT2, reflecting distinct steps of gonadotrope cell differentiation (42)], the proteolysis of PKC isoenzymes induced by a physiological stimulus GnRH (PKC ) or by TPA (PKC , ) involves mainly proteasome, an organelle that degrades multiubiquitinylated proteins; the proteasome pathway is implicated in the phorbol ester-evoked PKC and depletion in normal pituitary cell cultures as well. Furthermore, depending on the PKC isoform, the phosphorylation state/catalytic activity of the isoenzyme may predispose it selectively to degradation. Thus, ligant-regulated proteolysis of specific signal transduction proteins may be important for maintaining their appropriate expression balance and hence, contribute to the modification of cell responsiveness.
Functional similarities of PKC isoenzymes in T3-1 and LßT2 cells
We found that, as T3-1 cells, the LßT2 gonadotrope cell line possesses several specific bands for PKC isoenzymes corresponding to PKC , ßII, , , and . Of interest is the absence of PKC in this cell line. Although there are studies suggesting an important role of PKC in the proliferation of glial cells and keratinocytes differentiation (56, 57), further work is needed to explore the involvement of this isoform in the gonadotrope cell development and proliferation, as well as its interference with the biological activity of other PKCs.
Moreover, lower expression of PKC was observed in LßT2 cells. It has been suggested that PKC , transiently overexpressed in GH4C1 cells, plays a crucial role in PRL secretory processes induced by either TPA or TRH (66). Overexpression experiments although sometimes indicative, may disturb, however, the preexisting functional interdependence between PKC isoenzymes in the cell (55). Contrary to T3-1, the LßT2 cells are secretory, more differentiated and exhibit functional characteristics consistent with those of normal gonadotrope cells (42).
In LßT2 cells the GnRH receptor is coupled, as in T3-1 cell line (35, 36, 37), to the PLC, PLA2 and PLD activities; consequently, it generates the plethora of second messengers essential for targeting specific PKC isoforms, key enzymes for gonadotrope cell functions. Furthermore, persistent stimulation of LßT2 cells by GnRH (2–15 h) selectively down-regulated the PKC and , whereas similar treatment by the phorbol ester TPA differentially down-modulated PKC , ßII, , and isoenzymes. The PKC remained resistant to both stimuli as in most cell models (8, 13, 27). However, it has been reported that this isoform, while not sensitive to TPA, was transiently translocated to membrane fraction by GnRH (12). Thus, although several cell types may differ with respect to their ability to down-regulate specific PKC isoforms in response to various stimuli (8), under our experimental conditions comparable profiles of PKC desensitization were obtained in both gonadotrope cell lines. Receptor-mediated down-regulation of PKCs has been reported in different cell types (9, 11, 13). For instance, exposure of GH4C1 cells to TRH resulted in a selective and time-dependent manner depletion of PKC isoenzyme (11, 26). Long-term treatments by bombesin and platelet-derived-growth factor induced down-regulation of PKC and isoforms in Swiss 3T3 cells; identical treatments by diacylglycerols reproduced the effect (13).
Proteasome-dependent proteolysis
We further investigated to what extend proteolysis and the related enzymes may account for the PKC depletion induced by TPA (PKC and ) or GnRH (PKC ) in T3-1 and LßT2 cells. Despite the fact that reports provided data indicating the involvement of calpains in the proteolysis of PKC enzymes, the evidence remains elusive and controversial (26, 28, 29, 30, 31, 34, 67).
The cell-permeant specific calpains inhibitors Calpeptin, PD150606, and E64d, which affects both calpains and lysosomal proteases, were unable to counteract the TPA-induced down-regulation of PKC and PKC , as well as the GnRH-evoked depletion of PKC . Only the ALLN, showing a stronger efficiency than the ALLM toward the 26S proteasome, significantly restored the respective TPA and GnRH-induced PKC(s) depletion. These findings suggest that calpains may not be involved in the down-regulation, for the time tested, of PKCs induced by either phorbol ester or the neuropeptide; instead the proteasome pathway may play a role in this process in both gonadotrope cell lines. The fact that ALLM slightly attenuated but only the PKC depletion induced by either TPA or GnRH, might indicate as well a partial sensitivity of the isoenzyme degradation to other compartment proteases affected by this inhibitor (48, 51, 54).
Previous studies have shown that the activated form of some isoenzymes, -, ß- and - PKC, are cleaved by calpains at specific sites in the V3 region of their molecule (30). However, others have demonstrated that PKC V3-domain mutants (m calpain-sensitive and -resistant mutant) expressed in COS-1 cells, remained sensitive to phorbol ester-induced down-regulation of the isoenzyme, suggesting the involvement of another proteolytic system than calpains in this process (67). However, µ-calpain-PKC complex in skeletal muscle has been reported (68). In addition, Eto and co-workers (26) have proposed a role of the calpain-Calpastatin system in the TRH-induced down-regulation of PKC in pituitary GH4C1 cells. Indeed, a 27-oligomer synthetic Calpastatin peptide blocked the neuropeptide-evoked depletion of PKC ; however, this peptide may independently interact with Ub/proteasome and calpains (32, 34). As in our experiments, only the ALLN restored the TRH-elicited PKC proteolysis, whereas the E64d was without effect (26).
Interestingly, Lactacystin, ß-Lactone, and proteasome inhibitor II, substances with a highly selective inhibitory potency toward proteasome (51, 52, 53), prevented the TPA-elicited PKC and PKC down-regulation, as well as GnRH-evoked PKC depletion in both gonadotrope cell lines. No detectable modifications concerning PKC were introduced by both stimuli or the inhibitors applied. These data are in favor to our hypothesis, suggesting a major contribution of the proteasome pathway to the down-modulation of PKC and during desensitization conditions induced by the phorbol ester or the neuropeptide in T3-1 and LßT2 cell lines; and may further indicate a physiological role of the proteasome-dependent proteolysis, because the proteolytic sensitivity was not unique toward the phorbol ester activation. As shown in T3-1 cells, the inhibitors overcame the stimuli-induced depletion of the respective target PKC(s) without affecting their membrane translocation (in most cases, used as an index of PKC activation). However, the accumulated enzymes were predominantly associated with the membrane fractions (more evident, as expected, in the case of phorbol ester), indicating that in the absence of proteasomal peptidase activity and under persistent stimulation, the non degraded isoenzymes are redistributed to membrane compartment. But, do they still retain their catalytic activity? If so, this might underline the importance of the protective role of a ligand-regulated proteasome activation during cell adaptive processes.
Pursuing our studies in primary cultures of pituitary cells, we further validated in a physiological cell model the implication of the proteasome-mediated proteolysis of TPA sensitive PKC isoenzymes and . Indeed, as in gonadotrope cell lines, the long-term application of TPA induced similar desensitization profiles of several PKC isoenzymes (, , ßII, ). Furthermore, the results imply that calpains are not responsible for the TPA-elicited down-regulation of PKC and PKC ; rather proteasome is involved in the mechanism(s) concerning their proteolysis because their degradation was highly sensitive only to proteasomal inhibitors.
The importance of protein degradation as a regulatory mechanism of cellular protein’s level has only recently been appreciated (34, 48, 64, 65). Accumulating evidence indicates that many cytosolic proteins (involved in the cell cycle, growth, signaling, transcription) or cell surface receptors undergo ubiquitin-proteasome proteolysis (69, 70, 71, 72, 73, 74). Polyubiquitination of proteins is for the most cases the substrate-targeting and recognition signal for the proteasome, albeit it may act independently (34, 64, 65). Recent studies have proposed that the ubiquitin conjugation system targets the PKC , , and for proteasomal degradation and thus contributes to their down-regulation induced by either bryostatin or phorbol esters in primary culture of human fibroblasts (32), in renal epithelial cells (31) as well as in 3Yi rat fibroblasts (28).
Besides, the selectivity by the proteasome, the entry of proteins for degradation into the ubiquitin-proteasome pathway might be regulated as well by modifications at the level of protein, such as phosphorylation/dephosphorylation, or by modifications of their respective chaperons (14, 16, 18, 19, 34, 70). Although little is known about the targeting signals, different recognition motifs for ubiquitin-dependent proteolysis (34, 64, 65) have been reported. Among them, Thr-Pro, Ser-Pro or PEST sequences (Pro, Glu, Ser, Thr) are targets for protein kinases or phosphatases. Indeed, PEST sequences have been identified in PKC and isoenzymes (32).
PKC phosphorylation state/catalytic activity
Recent studies have provided new insights for PKC phosphorylation as susceptible linker to its activation. However, the ligand-dependent regulation of how specific PKC isoenzyme becomes phosphorylated or dephophorylated, how these reactions modulate the stability of the enzyme and its compartmentation, how precise sites when modified render the isoenzyme accessible to phosphatases and potentially to different proteases, is not yet clearly established (14, 16).
Different trans- or autophosphorylation sites have been characterized for the PKC : Thr497 as essential for its catalytic activity, Thr638/Ser657 for keeping the kinase resistant to phosphatases (14, 16), and the recently Thr250 reported by Parker and co-workers (75) as a marker for its activated state. The latter represents an autophosphorylation site involved in the intracellular trafficking of the enzyme and is sensitive to PKC inhibitors (75, 76). The importance of autophosphorylation of PKC as a prerequisite for down-regulation has been initially suggested by Ohno et al. (15). Furthermore, dephosphorylation has been proposed as an active PKC-dependent process and thus by inactivating the enzyme may prime it to degradation (18, 19). Concerning PKC , C-terminal phosphorylation sites (Ser729, Thr703) are not essential for the catalytic activity of PKC ; they rather regulate the stability of the enzyme (77, 78). However, Thr566 phosphorylation (activation loop) is a prerequisite for the phosphorylation of Ser729 site.
Our results from pituitary cells and T3-1 cell line indicate that the processing leading to down-regulation of PKC requires the kinase activity and might involve autophosphorylation sites of the isoenzyme. Indeed, as further demonstrated in T3-1 subcellular fractions three TPA-sensitive PKC isoenzymes (, , ßII) showed selective behavior toward PKC inhibitors: the PKC catalytic activity inhibitor GFX (a common for several isoenzymes) markedly attenuated the TPA-induced down-regulation of PKC ; it had no evident effect on the sensitivity of PKC to both TPA or GnRH stimuli, but effectively restored the TPA-induced PKC ßII depletion. Of interest, a specific PKC inhibitor, Gö6976, showing no interference with the other PKC isoforms, selectively preserved the PKC isoenzyme from degradation without changing its translocation response. In 3Yi rat fibroblasts, however, PKC inhibitors prevented the depletion of both PKC and induced by the phorbol ester TPA (28).
PKC -activated-phosphatases through a dephosphorylation process might mediate the signal(s) for the isoenzyme degradation. If ubiquitination occurs before proteasomal proteolysis, would the PKC inhibitor block the isoenzyme ubiquitination as well? Studies are undertaken to investigate in the gonadotrope cell lines these particular points concerning PKC and to elucidate a differential dependency of PKC on the upstream kinase(s)/phosphatase(s) inputs responsible for possible associative process between ligand-induced phosphorylation/dephosphorylation and proteolysis.
Under our experimental conditions, PKC showed particular migrating behavior in whole cell lysates (T3-1, LßT2, mixed pituitary cell cultures) as well as in T3-1 subcellular fractions. The antibody used recognized a major band of 87 kDa and a very fine of faster mobility one; after acute stimulation by TPA or GnRH, a slower migrating band was revealed predominantly in membrane fraction [does it represent the Ser729 phosphorylated form of the isoenzyme? (77, 78)]; as seen also in whole extracts, it follows the progressive loss of the major band during desensitization conditions. Shift in mobility and down-regulation of PKC has been reported in Swiss 3T3 cells (13). Interestingly, inhibition of the proteasome favored not only the increase of the major band of PKC , but also the reappearance of this higher molecular weight one.
Blockade of the catalytic activity by GFX had no evident effect of PKC major band in both T3-1 and pituitary cells, however, selective effects toward other migrating PKC species are not excluded. Indeed, it has been reported that the serum-induced phosphorylation of PKC upon Ser729 or Thr566 was not affected by this inhibitor; only Thr710 was revealed as a sensitive site (78). Although preliminary results (Drouva, S. V., unpublished data) indicate that in gonadotrope cell lines, the higher molecular mass of PKC might concern a hyperphosphorylated form, this issue awaits further investigation. Furthermore, the subcellular sites of these events (77, 79, 80) as well as their relationship to the ubiquitin activating enzymes are currently evaluated. In addition, whether the activation of the PKC , isotypes or their induced depletion contribute to cell cycle progression on the gonadotrope cell lines remains to be established.
Thus, coordination between kinase and proteolytic proteasomal activities leading to selective PKC degradation, may constitute an important component of regulatory mechanisms involving timing control in response to different stimuli in gonadotrope cell lines; to what extent it mediates normal gonadotrope responsiveness requires further investigation.
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Acknowledgments |
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T3-1 and LßT2 cell lines. |
Footnotes |
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Abbreviations: ALLM, Calpain inhibitor II ; ALLN, Calpain inhibitor I; DAG, diacylglycerol; IP3, inositol 1,4,5 triphosphate; IPs, inositol phosphates; PA, phosphatidic acid; PEt, [3H]phosphatidylethanol; TPA, 12-O-tetradecanoyl-phorbol-13 acetate.
Preliminary reports of these data were presented at the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000 (1 ) and at the 27th FEBS Meeting, Lisbon 2001 (2 ).
Received September 6, 2001.
Accepted for publication December 14, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, < 0.05. *, < 0.01 vs. corresponding control group.
