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The Impact of the Human Genome on Endocrinology: Original Articles |
Institute of Reproductive and Developmental Biology (I.Y.H.M., J.J.B., M.C., F.A.H., L.R., J.O.W.), Wolfson and Weston Research Centre for Family Health, Faculty of Medicine, Imperial College of Science Technology and Medicine, Hammersmith Hospital, London W12 ONN, United Kingdom; and Department of Obstetrics and Gynecology (L.C.), National Women’s Hospital, University of Auckland, New Zealand
Address all correspondence and requests for reprints to: J. O. White, Institute of Reproductive and Developmental Biology, Imperial College, Hammersmith Hospital, London W12 ONN, United Kingdom. E-mail: . j.o.white{at}ic.ac.uk
Abstract
Differentiation of human endometrium during the secretory phase of the menstrual cycle is characterized by expression of a variety of genes implicated in the establishment and maintenance of pregnancy. An increased abundance of signal transducers and activators of transcription (Stats) in the secretory phase suggests Stat5 as a component of the differentiation of endometrium in response to ovarian hormone stimulation in vivo. Decidualization is initiated in a subset of endometrial stromal cells (ESC) in vivo during the secretory phase, but it is unclear whether regulated expression of Stat5 is a feature of these cells. Here, therefore, the abundance and subcellular distribution of Stat5 in ESC after a decidualization stimulus of cAMP plus medroxyprogesterone acetate (MPA) has been investigated in vitro. Western blotting revealed an increase in the apparent abundance of Stat5a and Stat5b, in the cytosolic and nuclear fractions, at 2, 3, and 4 d after stimulation. The potential functional relevance of this increase in Stat5 is suggested by the ability of transiently transfected Stat5a or Stat5b to significantly enhance the response of the decidual PRL promoter to cAMP/MPA and attenuation of the response to cAMP/MPA by dominant negative Stat5. Recent evidence suggests endometrial differentiation, including PRL production, as a possible target of antiphospholipid antibodies (aPL) prevalent in recurrent miscarriage. Monoclonal antibody, ID2, which has similar reactivity as human aPL, significantly decreased the apparent abundance of nuclear Stat5b in response to cAMP/MPA and was associated with decreased decidual PRL promoter activation and PRL secretion. Regulated expression of Stat5 is therefore a component of decidual differentiation of human ESC and contributes significantly to activation of the decidual PRL promoter. Alteration of this process by an aPL component suggests decidual differentiation as a potential clinical target in recurrent early miscarriages.
THE HUMAN ENDOMETRIUM undergoes morphological and biochemical change during the menstrual cycle in response to the circulating levels of E2 and progesterone secreted by the ovary. In the luteal phase, the secretory activity of glandular epithelium and predecidualization of endometrial stromal cells (ESC) are considered to be progesterone responses in an estrogen-primed tissue. The cellular response to E2 and progesterone is mediated by their respective receptors which are ligand-inducible transcription factors that upon activation acquire the ability to bind consensus DNA sequences and activate transcription (1). However, steroid hormone control of tissue-specific expression of inducible genes is more complex and dependent upon interaction with many other regulators of transcription. PRL and IGF binding protein-1 (IGFBP-1) are examples of genes whose expression increases during progesterone-induced decidualization in vivo and whose transcription is controlled by multiple regulatory elements in a tissue-specific manner (2, 3). Studies in vitro using human ESC suggest the involvement of PR and potentially interacting coactivators (4), CCAAT/enhancer-binding protein ß (5) and activator protein 1, (6) in activation of PRL gene transcription from the upstream promoter used in these cells (3).
Stats are latent transcription factors activated in the cytoplasm by diverse cell signaling molecules. Phosphorylation of a conserved tyrosine residue in all Stat family members induces their dimerization, translocation to the nucleus, and regulation of genes involved in growth and differentiation. Subsequently, Stats are dephosphorylated and return to their latent site in the cytoplasm (7). In addition to activation by phosphorylation in response to stimulus coupling, changes in abundance of Stat1, Stat3, and Stat5 have been reported during the differentiation of adipocytes, hepatocytes, and mammary epithelial cells (8, 9, 10, 11). In human endometrium, the selective expression of Stat5 in glandular epithelium and a subset of stromal cells during the secretory phase suggests a potential role in differentiation (12). The regulated expression of Stat5 in vivo in a subset of ESC during the secretory phase raises the possibility of its involvement in the pathway of decidualization. This study has therefore defined the abundance and subcellular distribution of Stat5 in ESC cells undergoing differentiation in response to a decidualizing stimulus [cAMP/medroxyprogesterone acetate (MPA)] in vitro and provides evidence of the ability of Stat5 to influence activation of the decidual PRL promoter. Recent evidence suggests that antiphospholipid antibodies (aPLs) associated with recurrent miscarriage may compromise decidual function (13) and inhibit PRL and IGFBP-1 production (14). Monoclonal antibody ID2 reacts with ß2-glycoprotein-1/phospolipid complexes considered to be a major target of aPL (15). Here, we demonstrate that ID2 inhibits PRL and IGFBP-1 production by ESC and that the effect on PRL correlates with reduced nuclear localization of Stat5 and attenuated activation of the decidual PRL promoter. Increased abundance of Stat5 is therefore a feature of decidual differentiation of human endometrium that has functional implications in the regulation of two major gene products of this process.
Materials and Methods
Materials
Phenol red-free DMEM-Ham’s F12 mixture (DMEM/F12), collagenase (type I), deoxyribonuclease (type I), 8-bromo-cAMP, MPA, Hoechst dye 33258 (bisbenzimide), calf thymus DNA, antibiotic-antimycotic solution containing penicillin, and streptomycin were all obtained from Sigma (Poole, UK). FBS and L-glutamine were from Life Technologies, Inc. (Uxbridge, UK). ProFection Mammalian Transfection and Luciferase Assay System were purchased from Promega Corp. (Southampton, UK). Plasmid Maxi-prep Kits were obtained from QIAGEN (Crawley, UK). Hybond P membrane and ECL-plus Western blotting detection reagents were obtained from Amersham Pharmacia Biotech (St. Albans, UK). The Janus kinase (JAK) inhibitor AG490 was purchased from Calbiochem (Nottingham, UK).
Primary culture of ESC
ESC were isolated from normal proliferative endometrial tissues obtained from cycling women by endometrial biopsy at the time of diagnostic laparoscopy and hysteroscopy. Hammersmith and Queen Charlotte’s Hospital Research and Ethics Committee approved the study, and patient consent was obtained before biopsy. Samples were collected in DMEM/F12 containing antibiotic-antimycotic solution. The tissues were washed twice in DMEM/F12, finely minced, and enzymatically digested with collagenase (134 U/ml) and deoxyribonuclease type I (156 U/ml) for 1 h at 37 C. After centrifugation at 400 x g for 4 min, the pellet was resuspended in maintenance medium of DMEM/F12, 10% wt/vol dextran-coated charcoal-treated FBS (DCC-FBS), 1% wt/vol L-glutamine, and 1% vol/vol antibiotic-antimycotic solution. ESC were separated from epithelial cells and passed into culture as described previously (4, 16). Proliferating ESC were cultured in maintenance medium until confluence. Confluent monolayers were treated in DMEM/F12 containing 2% vol/vol DCC-FBS with 0.5 mM 8-bromo-cAMP and/or 10-6 M MPA. All experiments were carried out before the third cell passage.
Antiphospholipid antibody
The characteristics and generation of the antibody ID2 have been previously described (15). Briefly, ID2 is a murine monoclonal IgG1 class antibody. It reacts with ß2-glycoprotein-I immobilized on irradiated polystyrene (ELISA) plates. It also reacts with ß2-glycoprotein-I immobilized on phosphatidylserine- or cardiolipin-coated ELISA plates. It is not inhibited in either of these reactions by liquid-phase ß2-glycoprotein-I. In all of these characteristics, ID2 has the same reactivity as human aPL (17). The hybridoma-secreting ID2 was grown in serum-free medium and purified by Protein G affinity chromatography. ID2 was used at a concentration of 5 µg/ml. An irrelevant control antibody (monoclonal anti-cytokeratin 19, DAKO Corp., Ely, UK) was used at the same concentration in control samples.
PRL, IGFBP-1, and DNA assays
PRL levels in supernatants were measured by microparticle enzyme immunoassay (AxSYM system, Abbott Laboratories, Abbott Park, IL). The coefficient of variation within assays was 2–3%, and that between assays was 6–8%. IGFBP-1 was determined by RIA as previously described (18). DMEM/F12 supplemented with DCC-FBS did not show measurable PRL concentrations. PRL and IGFBP-1 levels were normalized to the DNA content of each culture flask at the end of the treatment period. The DNA content was measured by quantitative fluorometric analysis at room temperature. Cells were solubilized with 0.02% wt/vol SDS. Aliquots were then mixed with 1 µg/ml Hoechst 33258 in 1x standard saline citrate, and fluorescence was measured in a fluorometer at excitation 344 nm and emission 460 nm. Calf thymus DNA was used as standard.
SDS-PAGE, Western blotting, and immunodetection
A modified method of Rittenhouse and Marcus (19) was used for protein analysis. Protein concentrations were determined by Bradford assay (Bio-Rad Laboratories, Inc., Hemel Hempstead, UK). Equal amounts of nuclear and cytosolic proteins (20 µg) were separated on a 7.5% SDS-polyacrylamide gel before electrotransfer at 80 V onto polyvinylidene difluoride (PVDF) membrane (Hybond-P, Amersham Pharmacia Biotech). Transfer efficiency was confirmed by Ponceau S staining. Nonspecific binding sites were blocked with 0.2% wt/vol I-Block (Tropix, Bedford, MA) in PBS with 0.1% vol/vol Tween at room temperature for 2 h. Rabbit polyclonal anti-Stat5a, rabbit polyclonal anti-Stat5b, and rabbit polyclonal antiphospho(Ser-726/Ser-731)-Stat5a/b were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Secondary antibody, peroxidase-conjugated goat antirabbit IgG was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Blots were exposed to primary antibodies diluted in PBS-Tween for 1 h at 4 C, and then incubated with secondary peroxidase-conjugated antibody for 1 h at 4 C. Protein bands were visualized by enhanced chemiluminescence (ECLplus Western Blotting Detection, Amersham Pharmacia Biotech).
Reporter constructs, expression vectors, and transfection studies
The decidual PRL promoter-luciferase reporter constructs dPRL-3000/luc and dPRL-332/luc, carrying 3000 and 332 bp, respectively, upstream of the major transcriptional start site, were provided by Dr. Birgit Gellersen (Hamburg, Germany) (5, 20). The 5'-deletion dPRL-913/luc promoter reporter containing -913 bp relative to the transcriptional start site was generated by exonuclease III digestion of the linearized(SacI/XhoI) dPRL-3000/luc using ExoIII/Mung Bean Deletion Kit (Stratagene, La Jolla, CA), according to the manufacturer’s instruction.
pCDNA-Stat5aWT, the expression vector for wild-type Stat5a, was generated by subcloning Stat5a excised from the retroviral vector pMX-Stat5a (EcoRI and NotI) into pCDNA3.1+ (5'-EcoRI, 3'-NotI). pMX-Stat5a (21) was a gift from Dr T. Kitamura (Tokyo, Japan). pCDNA-Stat5bWT expression vector was created by subcloning Stat5b excised from pEF-HM-plink (22) with XhoI into pCDNA3.1+ vector (5'XhoI, 3'XhoI). The original vector was a gift from Dr. I. Kerr (London, UK).
The specific dominant negative Stat5b expression vector, pCDNA-Stat5bDN, was generated by subcloning dominant negative Stat5b, excised from pBabeX-puro–Stat5bDN (23) with EcoRI/NotI, into pCDNA3.1+ vector (5'-EcoRI, 3'NotI). The encoded dominant negative Stat5b is a C-terminal truncated protein in which the major tyrosine phophorylation sites (after Tyr 683) have been removed (23). All of the constructs were confirmed by restriction digests and DNA sequencing.
Transient transfections of ESC plated at a density of 5 x 105 cells per well in 12-well plates were performed by calcium phosphate precipitation in medium supplement with 2% DCC-FBS. This method of transfection in ESC has been extensively described, and transfection efficiency has been optimized by us and others (4, 24, 25). Transfection efficiency, measured by transfected ß-galactosidase activity, was not altered by the treatments described in this paper (data not shown). Details of the transfection protocol and the treatments are indicated (see figure legends). Cell extracts were harvested, and luciferase activity was measured with the luciferase reagent kit (Promega Corp.) and expressed as relative light units. Transfections were performed in triplicate and repeated at least three times. Representative experiments are shown (means ± SD).
Results
Expression of Stat5 and Stat1 in decidualizing ESC
The temporal pattern of expression of Stat5a and Stat5b was determined in cytosolic and nuclear extracts of ESC exposed to a decidualizing stimulus of cAMP/MPA for 1–4 d. The apparent abundance of Stat5a and Stat5b, determined by Western blotting, was increased in cytosol and nuclear extracts at 2, 3, and 4 d after stimulation, with effects on Stat5b being more pronounced than effects on Stat5a (Fig. 1A
). As expected, nuclear Stat5 was phosphorylated and recognized by antiphospho-Stat5 (Fig. 1B). However, cytosolic phospho-Stat5 was below the threshold of detection. Apart from a transient increase in nuclear Stat1 2 d after stimulation (Fig. 1C), there was little apparent change in Stat1 abundance in response to cAMP/MPA. The increase in nuclear and cytosolic Stat5 abundance and the increase in the nuclear phospho-Stat5 was maintained when JAK activation was inhibited by AG490 (Fig. 1B). Therefore, coupling of the JAK/Stat5 pathway is not necessary for the observed changes in Stat5 abundance and subcellular distribution in ESC responding to stimulation by cAMP/MPA.
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Stat5 and Stat1 can be positive or negative regulators of target genes but are in competition in their regulation of the interferon-regulated factor-1 promoter (26). In ESC, Stat1 is a negative regulator of the decidual PRL promoter-reporter construct dPRL3000-Luc because its induction by interferon-
antagonizes the stimulatory effects of cAMP/MPA (25). The expression of Stat5 in ESC in response to cAMP/MPA (Fig. 1
) could possibly therefore contribute to regulation of the decidual PRL promoter. Two complementary approaches were therefore taken to test this possibility.
Transient transfection studies revealed the contribution of Stat5 to activation of the decidual PRL promoter. Expression of Stat5a (Fig. 2B) or Stat5b (Fig. 2C) was able to potentiate the response to cAMP/MPA. Neither Stat5a nor Stat5b affected the basal activity of dPRL-3000 in unstimulated cells, suggesting a requirement for cooperation with other factors induced in response to cAMP/MPA in ESC. Further evidence in support of the contribution of Stat5 to activation of the decidual PRL promoter was provided by the ability of transiently transfected mutant dominant negative Stat5 to almost completely abolish the inductive effects of cAMP/MPA on dPRL-3000 (Fig. 2A). The effect of dominant negative Stat5 was dose-dependent and reversible by coexpression of wild-type Stat5, suggesting the specificity of this effect (data not shown).
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). However, potentiation by Stat5b of the remaining response to cAMP/MPA was still observed using sequence to -913 and -332. Consistent with previous reports, the cAMP/MPA response was lost in the dPRL-32/luc reporter (5).
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The production of PRL and IGFBP-1 by term decidua is inhibited by aPLs (14) that are common in the serum of women who suffer recurrent miscarriage (27). To explore whether such direct effects on endometrial cells occurred early in the process of commitment to decidualization, we investigated the response of ESC to the monoclonal antibody, ID2, which reacts with ß2-glycoprotein-1/phospolipid complexes considered to be a major target of aPL (17). The induction of PRL and IGFBP-1 was used as a biochemical end-point of decidualization, and the nuclear accumulation of Stat5b was used as a candidate-signaling mediator of the differentiation response. PRL and IGFBP-1 levels, measured in the conditioned medium of ESC treated with cAMP/MPA, were significantly decreased after exposure to ID2 (Fig. 4, A and B). DNA assays carried out on ID2-treated and -untreated ESC showed no significant difference (data not shown), thus eliminating potential nonspecific effects of ID2 on cell viability. Similarly, activation of dPRL3000-Luc in response to cAMP/MPA was attenuated in the presence of ID2 (Fig. 4C), concomitant with a reduction in the abundance of nuclear Stat5b (Fig. 4D).
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Activation of Stats by tyrosine phosphorylation in response to cytokine and growth factor receptor stimulation is well studied, but there is relatively little information on regulation of abundance of these transcription factors. Expression of Stats is up-regulated during mammary gland differentiation in vivo, and aspects of this regulation are maintained in the regulation of expression of Stat5 in HC11 mammary epithelial cells responding to lactogenic stimuli in vitro (9). Similarly, the commitment of precursor preadipocyte fibroblasts to adipocytes in response to adipogenic stimuli is characterized by the tight coupling of Stat5 expression to the differentiated phenotype (8, 11, 28). The expression of Stat5 in vivo in human endometrium is restricted to secretory phase tissue being localized to glandular epithelium and a subset of stromal cells that also express PRL receptor (12, 29). This suggested that Stat5 expression was a feature of a subset of ESC undergoing decidualization and the possibility of paracrine and autocrine responses to PRL by these cells. The expression of Stat5 in ESC in vitro in response to a decidualization stimulus, described here, illustrates that expression of Stat5 is part of the differentiation response. An increase in Stat5 abundance in the cytoplasm and nucleus suggests that regulated expression of Stat5 in response to the decidualization stimulus is coupled to its activation and translocation to the nucleus. Apart from a transient increase in the apparent abundance of nuclear Stat1 after 2 d of treatment, the effects of cAMP/MPA were restricted to effects on Stat5. In vivo, the expression of Stat1 in human endometrial cells also suggested its regulation to be different from Stat5 in that its abundance was not sensitive to the hormonal changes of the menstrual cycle (12). The differential effects of cAMP/MPA on Stat expression in vitro, described here, are therefore consistent with the differential regulation of endometrial Stat5 and Stat1 in vivo. Stat family members show unique patterns of expression/activation related to their regulation of distinct tissue-specific genes (30, 31). The differential pattern of expression of Stat5 and Stat1 in endometrial tissues throughout the menstrual cycle and upon stimulation of ESC in vitro supports the possibility of such distinct physiological functions within the uterus.
Separated endometrial glandular epithelium responds to PRL stimulation in vitro by phosphorylation of JAK2, Stat1, and Stat5, consistent with activation of the PRL receptor and the downstream JAK/Stat pathway (12). In contrast, separated ESC respond to PRL by tyrosine phosphorylation of JAK2 but not of Stat1 or Stat5 (12). The increase in abundance and change in subcellular distribution of Stat5, reported here, occurred during a time period when ESC begin to secrete small but significant amounts of PRL in response to cAMP/MPA (4). This raised the possibility that changes in abundance and subcellular distribution of Stat5 could have been secondary to ligand-activated PRL receptor inducing recruitment of the JAK/Stat pathway. However, the increase in nuclear phospho-Stat5 was insensitive to the selective inhibitor AG490, which prevented phosphorylation of JAK2 (Fig. 1B), suggesting that the changes in Stat5 observed here were not dependent on activation of JAK. This does not necessarily exclude PRL in regulating the Stat5 response seen here because, for example, in Nb2 T lymphoma cells the response to PRL is independent of JAK-2 activation (32). The kinase(s) involved in such a response may include other PRL receptor-associated kinases, p59fyn (33) and c-src (34). In this regard, activation of c-src has been reported during decidualization of human ESC without any change in Fyn activity (35), which is difficult to explain if this is downstream of their recruitment to activated PRL receptor (36). Alternatively, the changes reported here may be related to other cytokines expressed during decidualization. For example, epidermal growth factor is induced during decidualization (37), regulates the expression and activation of Stat5 in breast epithelium (9) and in breast cancer cells (38), and recruits src to the epidermal growth factor receptor (39). The exact nature of the signaling pathway(s) activated by cAMP/MPA in ESC remains to be determined.
The potential functional relevance of the changes in abundance and subcellular distribution of Stat5 is suggested by effects on the decidual PRL promoter. The permissive role of Stat5 in the response of the decidual PRL promoter to cAMP/MPA is suggested by the antagonism of a dominant negative mutant form of Stat5 (Fig. 2A). This is reversible by expression of Stat5a/b (data not shown) and taken together with the separate evidence of the ability of Stat5a and Stat5b to enhance the activation of dPRL3000 by cAMP/MPA (Fig. 2, B and C) the data support a positive regulatory role for Stat5 on PRL gene expression. This contrasts completely with the negative effects of Stat1 on activation of dPRL3000 (25) and suggests these two members of the Stat family have opposing effects on PRL expression. Given the constant expression of Stat1 in ESC throughout the menstrual cycle (12), the elevation of Stat5 abundance in the secretory phase and in response to cAMP/MPA in vitro may favor expression of PRL by relieving the suppressive effects of Stat1 in the stromal compartment. Opposing effects of Stat1 and Stat5 in their regulation of the interferon-regulatory factor-1 gene (26, 40) may involve protein-protein interactions that result in competition for limiting amounts of regulatory coactivator CBP, N-myc interacting protein, or cytokine receptor adaptor protein. Alternatively, the opposing effects of Stat5 and Stat1 may be a function of their kinetics of nuclear translocation/retention and the role of the importin 
/ß transport pathway in this process (41).
Attenuation by aPL of the synergistic induction of PRL by cAMP/MPA was observed in ESC. This is consistent with reports of such effects of aPL on expression of differentiation markers by term decidua (14) but extends these effects to an early stage of commitment to decidual differentiation. The effect was not restricted to an effect on PRL but was also observed on the induction of expression of IGFBP-1. The concomitant decrease in nuclear Stat5 upon treatment with aPL suggests that a component of the signaling pathway to PRL expression in response to cAMP/MPA is compromised by this antibody. The mechanism underlying these effects of aPL is not known, but aPLs have been shown to affect cellular functions such as adhesion molecule expression and hormone secretion in both endothelial cells and placental trophoblast (42, 43, 44, 45, 46). The ß2-glycoprotein-I-phospholipid complex harbors a recognition site for the endocytic receptor megalin (47), and aPL interacts with late endosomes (48) suggesting the possibility of an endosomal signaling pathway as a target. In most cells, the plasma membrane maintains an asymmetric distribution of its major phospholipids by the action of a subfamily of ATPases with aminophospholipid-transporting activity (49). Virtually all the phosphatidylserine (PS) is on the inner leaflet, and its externalization to the outer leaflet, where it is bound by annexin V, is considered to represent an early stage of apoptosis (50). aPL has been suggested to displace annexin V from the cell surface (51), which raises the question why ESC with externalized PS (recognized by ID2 in combination with ß2-glycoprotein-I) (15) are not undergoing apoptosis but are capable of such positive responses to decidualization stimuli. Recent data obtained in B cells suggests that externalization of PS does not necessarily reflect apoptosis but instead plays a role in selection and differentiation (52, 53). In this case, annexin V binds PS on viable cells and colocalizes with lipid rafts, dynamic membrane microdomains thought to provide platforms for the optimal association of signaling molecules with their intracellular targets, involved in the cellular response to its microenvironment. This therefore raises the possibility that aPL disrupts annexinV-PS/ß2-glycoprotein-I interactions in ESC, thereby interfering with transmission of the decidualization signal in vitro and disorganization of the influence of the stromal microenvironment on this process, which is thought to be important in vivo (25). This is an area that is being further explored at present and that may provide a rational explanation for direct cellular effects of aPL on endometrium and decidua to compromise maintenance of pregnancy.
In summary, we demonstrate the importance of regulation of Stat5 expression to the control of PRL expression in differentiating ESC and illustrate that control of its abundance and biological activity contrasts with that of Stat1 (25). The mechanisms involved in regulation of Stat5 expression by cAMP/MPA and the means by which Stat5 potentiates the actions of cAMP/MPA at the decidual PRL promoter, in a region devoid of consensus binding sites, is an area of research that will further our understanding of the molecular endocrinology of decidual differentiation of human endometrium.
Acknowledgments
Footnotes
This work was supported by the Medical Research Council (Grant G84/5623), the Wellcome Trust (Grant 055214), and the TR Golden Charitable Trust. J.J.B. is the recipient of a Wellcome Clinician Scientist Fellowship (54043). L.C. is funded by the Health Research Council of New Zealand.
Abbreviations: aPL, Antiphospholipid antibodies; DCC-FBS, dextran-coated charcoal-treated FBS; DMEM/F12, DMEM-Ham’s F12 mixture; ESC, endometrial stromal cells; IGFBP-1, IGF binding protein-1; JAK, Janus kinase; MPA, medroxyprogesterone acetate; PS, phosphatidylserine; PVDF, polyvinylidene difluoride; Stat, signal transducer and activator of transcription.
Received September 19, 2001.
Accepted March 6, 2002.
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