Differential Activation of Pituitary Hormone Genes by Human Lhx3 Isoforms with Distinct DNA Binding Properties
Kyle W. Sloop,
Bradley C. Meier,
JeAnne L. Bridwell,
Gretchen E. Parker,
Amy McCutchan Schiller and
Simon J. Rhodes
Department of Biology Indiana University-Purdue University
Indianapolis Indianapolis, Indiana 46202-5132
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ABSTRACT
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Lhx3 is a LIM homeodomain transcription factor
essential for pituitary development and motor neuron specification in
mice. We identified two isoforms of human Lhx3, hLhx3a and hLhx3b,
which differ in their ability to trans-activate pituitary
gene targets. These factors are identical within the LIM domains and
the homeodomain, but differ in their amino-terminal sequences preceding
the LIM motifs. Both isoforms are localized to the nucleus and are
expressed in the adult human pituitary, but gene activation studies
demonstrate characteristic functional differences. Human Lhx3a
trans-activated the
-glycoprotein subunit
promoter and a reporter construct containing a high-affinity Lhx3
binding site more effectively than the hLhx3b isoform. In addition,
hLhx3a synergized with the pituitary POU domain factor, Pit-1, to
strongly induce transcription of the
TSHß-subunit gene, while hLhx3b did not. We
demonstrate that the differences in gene activation properties between
hLhx3a and hLhx3b correlate with their DNA binding to sites within
these genes. The short hLhx3b-specific amino-terminal domain inhibits
DNA binding and gene activation functions of the molecule. These data
suggest that isoforms of Lhx3 may play distinct roles during
development of the mammalian pituitary gland and other neuroendocrine
systems.
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INTRODUCTION
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The molecular mechanisms that regulate the development of complex
endocrine organ systems are poorly understood. The pituitary gland
represents an important model system to study how regulatory genes
direct the determination and differentiation of the individual cell
lineages that ultimately comprise a mature organ. During pituitary
development, neural ectoderm cells from the diencephalon, destined to
form the posterior lobe of the gland, associate with a fold of oral
ectoderm known as Rathkes pouch to form the primordial pituitary. Six
distinct cell types emerge from Rathkes pouch during organogenesis to
populate the mature anterior and intermediate lobes of the pituitary
(see Refs. 1, 2, 3 for reviews). These cells release characteristic
protein hormones that regulate growth, lactation, metabolism,
reproduction, and the response to stress.
Proper formation of Rathkes pouch and the subsequent differentiation
of pituitary cell types require the secretion of inductive signaling
molecules such as BMP-2, BMP-4, and FGF-8. These molecules are secreted
from the ventral diencephalon and the developing pituitary to initiate
cellular differentiation cascades within embryonic pituitary cells in a
spatial and temporal fashion (4, 5, 6). The protein signals induce
subsequent pituitary cellular specification by activating multiple
transcription factors. Based on their expression patterns, gene
knockout experiments, and the analysis of naturally occurring mutants,
many transcription factors have been implicated in pituitary
organogenesis. They include Lhx3 (also known as P-Lim or LIM3),
Lhx4/Gsh-4, Isl-1, Krox-24, T/ebp/TTF-1, Hesx1/Rpx, Pitx-1/P-Otx,
Pitx2, Prop-1, Pit-1/GHF-1, Brn-4, Six-3, SF-1, Pax-6, Msx-1, Nkx 3.1,
GATA-2, P-Frk, thyrotrope embryonic factor (TEF), and Zn-16 (Ref. 5 ;
reviewed in Refs. 1, 2, 3).
Mutations in pituitary transcription factors have been shown to cause
pituitary disease. Humans with mutations in the Pit-1 or
Prop-1 genes exhibit combined pituitary hormone deficiency
(CPHD). Pit-1 is a well characterized POU domain pituitary
transcription factor that directly activates the GH,
PRL, and TSHß genes, as well as its own gene
(reviewed by Refs. 1, 2, 3, 7). Patients with mutated Pit-1
genes have deficiencies in GH, PRL, and TSH. Many types of
Pit-1 gene mutations have been identified, including those
with autosomal dominant and recessive patterns of inheritance (see
Refs. 7, 8 for review). The Prop-1 (or Prophet of Pit-1)
transcription factor is upstream of Pit-1 in the pituitary
developmental cascade (9). In addition to deficiencies of GH, PRL, and
TSH, patients with mutations in the Prop-1 gene may lack LH
and FSH (8). Much effort continues to be devoted to the goal of
characterizing additional genetic lesions associated with human
pituitary disease.
During pituitary development, the actions of the LIM homeodomain
transcription factors Lhx3, Lhx4, and Isl-1 are essential for the
establishment of Rathkes pouch and the subsequent differentiation of
specialized hormone-secreting cell types. LIM homeodomain proteins
contain two amino-terminal LIM motifs and interact with DNA using a
characteristic homeodomain. The LIM domain is a conserved, zinc
finger-like structure that mediates interactions with other proteins,
and LIM homeodomain proteins have been demonstrated to be critical to
many developmental pathways (reviewed in Refs. 10, 11, 12). During early
embryogenesis, the Lhx3 gene is expressed in several regions
of the brain and spinal cord, and then it becomes restricted to the
primordial pituitary cells of Rathkes pouch and their descendents in
the adult gland (13, 14, 15, 16, 17). In the nervous system, the Lhx3,
Lhx4, and Isl-1 gene products are required for the
specification of motor neurons that emerge from the developing neural
tube (e.g. Refs. 18, 19, 20, 21). Elegant studies of mice with
ablated Lhx3 genes have revealed that pituitary development
is arrested after the formation of Rathkes pouch and that Lhx3 is
required for differentiation of the hormone-secreting cells of the
pituitary (22, 23). Lhx4 also is required for complete development of
Rathkes pouch; but unlike Lhx3, this factor is not essential for the
determination and specification of differentiated pituitary cell types
(23). Mice lacking both Lhx3 and Lhx4 genes do
not develop a rudimentary Rathkes pouch, indicating that at least one
of these genes is required during the initial stages of pituitary
development (23). Our laboratory and others have demonstrated that Lhx3
can activate pituitary trophic hormone genes, acting both alone and
with other pituitary transcription factors such as Pit-1 and
Pitx1/P-Otx (15, 24, 25). Additional functional analyses are required
to fully understand the role of products of the Lhx3 gene in
the developing nervous system, the early establishment of Rathkes
pouch, and in later pituitary gland function and maintenance.
To further characterize the molecular actions of Lhx3, we identified
two alternate forms of human Lhx3, hLhx3a and hLhx3b, which share the
LIM domains, homeodomain, and carboxyl terminus of Lhx3, but possess
distinct amino-terminal protein sequences. Both isoforms are nuclear
proteins and are detected in the pituitary gland, but
trans-activation assays revealed different abilities to
activate anterior pituitary hormone gene regulatory regions. Human
Lhx3a activated a reporter gene containing the
-glycoprotein
subunit (
GSU) promoter and a minimal reporter gene
containing consensus Lhx3 binding sites. Further, Lhx3a synergized with
Pit-1 in induction of the TSH ß-subunit
(TSHß) gene promoter. By contrast, Lhx3b was either
inactive or only weakly activated trophic hormone genes. We demonstrate
that the differences in trans-activation ability between
hLhx3a and Lhx3b correlate with their DNA binding to sites within these
target genes. Our results indicate that the amino terminus of hLhx3b
inhibits the ability of this factor to bind DNA and
trans-activate target genes compared with Lhx3a. To our
knowledge, this is the first description of different functional
properties for alternate forms of a LIM homeodomain class
transcriptional regulator. The hLhx3a-specific and hLhx3b-specific
amino-terminal domains may represent novel functional motifs derived
throughout evolution to confer properties unique to Lhx3 that are
important in mammalian development. These findings suggest that Lhx3
isoforms may perform distinct roles in the development of the pituitary
gland and during motor neuron differentiation.
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RESULTS
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Cloning of Human Lhx3 cDNAs
The PCR was used to amplify a region encoding the carboxyl
terminus of human Lhx3 (hLhx3) from pituitary cDNA (see Materials
and Methods). Rapid amplification of cDNA ends (5'-RACE) then was
performed to obtain longer cDNAs. Finally, partial hLhx3 cDNAs were
used as probes in screens of a pituitary cDNA library to obtain
full-length clones. Sequencing of hLhx3 clones obtained from RACE and
library screening revealed multiple, distinct hLhx3 cDNAs. Two
predominant species encoded hLhx3 proteins with identical LIM domains,
homeodomains, and carboxyl termini, but with distinct amino-terminal
protein sequences (Fig. 1A
). We named
these protein isoforms hLhx3a and hLhx3b. The GenBank accession numbers
of the reported sequences are: hLhx3a (AF156888), hLhx3b
(AF156889). Some hLhx3b cDNAs contained an approximately
200-bp insert in the 3'-untranslated region (nucleotide sequences
deposited in GenBank).

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Figure 1. Deduced Protein Sequences of Human Lhx3 Isoforms
A, Schematic depiction of the domain structures of hLhx3a and hLhx3b.
Hatched regions represent domains unique to each form;
L, LIM domain; HD, homeodomain; LSD, Lhx3/LIM3-specific domain. B,
Alignment of mammalian Lhx3 proteins. Comparison of human Lhx3a/b (Ha,
Hb), murine Lhx3 (Ma, accession number L38249; Mb, L38248), and porcine
Lhx3 (P, AF063245). The LIM domains, homeodomain, and
Lhx3/LIM3-specific domain are boxed/reversed.
Dots indicate identity; dashes denote
gaps introduced to optimize alignment. C, Similarity of Lhx3/LIM3
family proteins. Numbers are percentage identity to hLhx3 for
individual regions. C, Chicken (accession number 1708828); X,
Xenopus laevis (547856); z, zebrafish (2497671); mLhx4,
mouse Lhx4 (AF135415); d, Drosophila melanogaster
(AF109306).
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The unique amino-terminal region of hLhx3b is very similar to an
alternate form of murine Lhx3 (16) (94% identity, Fig. 1B
). By
contrast, the shorter amino-terminal sequence of hLhx3a is only 58%
similar to the amino terminus of murine Lhx3a (Fig. 1B
). The a- and
b-specific domains of hLhx3 do not significantly match other sequences
in the databases. The common regions of the hLhx3 proteins display
strong overall similarity to murine (94% identity) and porcine (95%
identity) Lhx3 sequences (Fig. 1B
). The LIM and DNA binding domains of
Lhx3 are strongly conserved in the mammalian species, with complete
identity in the LIM 2 domain and the homeodomain (Fig. 1B
). Comparison
of mammalian Lhx3 sequences with Lhx3/LIM3 family proteins from
nonmammalian species also reveals conservation of the LIM and
DNA-binding domains and the carboxyl-terminal LIM3-specific domain
(LSD) noted by Glasgow et al. (17) and Thor et
al. (18) (Fig. 1C
). The amino termini of nonmammalian Lhx3/LIM3
proteins display more similarity to the amino terminus of hLhx3a than
to that of hLhx3b, but overall are diverged in length, composition, and
sequence.
Analysis of the Human Lhx3 Gene and Its
Products
To determine whether the identified hLhx3 cDNAs
represented a single genomic locus, Southern analyses were performed.
Human Lhx3 cDNA probes hybridized to single DNA fragments (Fig. 2A
; and data not shown), indicating that
hLhx3 is encoded by a single gene. In Northern analyses, hLhx3 cDNA
probes hybridized to a rare mRNA of approximately 2.4 kb in samples of
adult human pituitary poly A+ RNA (Fig. 2B
). Signals were
not detected in samples of total pituitary RNA (Fig. 2B
). As controls,
RNA blots also were hybridized to a human Pit-1 cDNA probe (Fig. 2B
).
In vitro transcription/translation was performed using
rabbit reticulocyte lysates to generate radiolabeled hLhx3a and hLhx3b
proteins from the identified cDNAs. Analysis of these proteins by
electrophoresis revealed apparent molecular masses of approximately 60
kDa for both isoforms (Fig. 2C
). As we have previously described for
porcine Lhx3 (25), this apparent molecular mass is slightly larger than
that predicted from the hLhx3a/b open reading frames, suggesting
modification of the Lhx3 protein in these preparations or aberrant
migration during electrophoresis due to composition.

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Figure 2. Analysis of the Human Lhx3 Gene and
Its Products
A, Southern analysis demonstrates that hLhx3 is encoded by a single
gene. Human genomic DNA was digested with EcoRI and
blotted to a nylon membrane. The blot was probed with a hLhx3 cDNA
probe at high stringency. The migration position of mol wt standards
(kb) is given. B, Northern analysis of hLhx3 gene
expression. Human total (20 µg) or poly A+ RNA (1 µg)
was separated on a denaturing gel, transferred to a nylon membrane, and
probed with the indicated radiolabeled cDNA probes. The migration
positions of ribosomal RNAs are shown. Arrowhead
indicates hLhx3 RNA. C, Human Lhx3a and hLhx3b proteins. Radiolabeled
hLhx3a and hLhx3b isoforms and myc epitope-tagged derivatives (a-myc,
b-myc) were generated by in vitro
transcription/translation. Proteins were separated by SDS
electrophoresis, and dried gels were visualized by fluorography. The
migration positions of protein standards (in kilodaltons) are shown.
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To determine the intracellular localization of the hLhx3 isoforms,
vectors for expression of hLhx3 isoforms as fusions with a multimerized
green fluorescent protein (4xGFP) (26) were constructed. The 4xGFP
protein localized exclusively to the cytoplasm of transfected cells
(Fig. 3
, A and B). By contrast,
constructs containing hLhx3a or hLhx3b were detected in the nucleus
(Fig. 3
, CF), indicating that they are nuclear proteins.

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Figure 3. Nuclear Localization of hLhx3 Proteins
Human 293T cells were transiently transfected with expression vectors
for either multimerized GFP as a control (4xGFP, panels A and B),
hLhx3a-4xGFP (panel C and D), or hLhx3b-4xGFP (panel E and F). Cells
were observed under phase contrast (panels A, C, and E), or
fluorescence was visualized by krypton-argon laser scanning confocal
microscopy (panels B, D, and F). 4xGFP is restricted to the cytoplasm,
whereas hLhx3a-4xGFP and hLhx3b-4xGFP are detected in the nuclei of
transfected cells. Bar = 10 µm.
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Expression of Lhx3 RNAs in the Pituitary
To precisely measure expression levels of hLhx3 gene transcripts
in human pituitary samples, a "real time" quantitative PCR assay
was developed (see Materials and Methods). RNA was isolated
from human pituitary tissue, and cDNA was transcribed with a
hLhx3-specific primer. Quantitative PCR amplification reactions then
were performed using three hLhx3-specific oligonucleotides: two to
prime amplification of a region within the 3'-end of the mRNA and a
fluorescent internal oligonucleotide that hybridized to PCR products to
allow monitoring of the reaction. Serial dilutions of hLhx3 cDNA were
amplified simultaneously with patient samples to generate a standard
curve (Fig. 4A
, inset). The
standard curve was linear in a range from 100 to 10 million copies of
hLhx3. Seven normal adult pituitary samples were assayed: hLhx3 was
present at levels ranging from 2,000 to 11,000 copies/150 ng RNA with a
mean value of 7,200 copies/150 ng RNA (Fig. 4A
). This value is
consistent with hLhx3 being a rare message. In previous studies, we
have demonstrated that ubiquitously expressed genes such as ß-actin
are expressed at approximately 500,000 copies/150 ng RNA (27). In
specialized tissues such as adipose, we have shown that mRNAs for
hormones such as leptin are found at approximately 300,000 copies/150
ng RNA, and transcription factors such as CCAAT/enhancer binding
protein-
(C/EBP
) are found at about 150,000 copies/150 ng RNA
(27).

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Figure 4. Assay of Lhx3 Expression in Human Pituitary Glands
and Mouse Pituitary Cell Lines
A, A quantitative fluorescent assay for human Lhx3 gene
expression was developed. RNA was extracted from adult human
pituitaries and cDNA generated using a 3' gene-specific primer. PCR was
performed using gene-specific primers and reactions monitored in real
time using an internal fluorescent hLhx3-specific probe. Results are
the means of four independent experiments ± SEM.
Inset shows standard curve using hLhx3 cDNA as input.
Ct, PCR cycle number where the reporter dye fluorescent emission
increases above baseline emission (49 ). B, Pituitary expression of
transcripts of the hLhx3 gene encoding hLhx3a and hLhx3b
isoforms. RT-PCR was used to amplify the approximately 1.2-kb coding
regions of hLhx3a and hLhx3b using pituitary gland cDNA from two
representative patients. Reaction products were separated by agarose
gel electrophoresis. M, Mol wt markers; Control, negative control. C,
Expression of Lhx3 isoforms in pituitary cell lines. RT-PCR was used to
amplify specific regions of Lhx3a (139-bp product, a) and Lhx3b (165-bp
product, b) using cDNA from the indicated cell lines. Reaction products
were separated by acrylamide gel electrophoresis. Negative control
reactions (-) were performed in parallel in the absence of reverse
transcriptase.
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To examine whether hLhx3 RNAs encoding the hLhx3a and hLhx3b isoforms
are expressed in human pituitary samples, oligonucleotide primers were
designed to specifically amplify either the hLhx3a or hLhx3b protein
coding regions. RT-PCR experiments indicated that both forms are found
in the adult pituitary (Fig. 4B
). To investigate whether the Lhx3
isoforms were expressed in specific cell types, we determined
expression of Lhx3 isoforms in established mouse pituitary cell lines.
In the
-TSH thyrotrope tumor cell line (28), both mLhx3a and mLhx3b
isoforms were readily detected (Fig. 4C
). In AtT20 corticotrope cells,
low levels of mLhx3b were detected, and in some preparations, trace
levels of mLhx3a were also observed. Neither isoform was detected in
the GHFT-1 cell type: this Pit-1-expressing cell line represents an
intermediate stage of pituitary development before differentiation and
expression of trophic hormone genes (29). These data are consistent
with observations of the overall levels of Lhx3 gene
expression by RNA analyses of these cell types (Refs. 14, 15, 16 ; K.
W. Sloop and S. J. Rhodes, unpublished data).
Differential Activation of Pituitary Hormone Genes by Human Lhx3
Isoforms
We and others have previously demonstrated that Lhx3 can activate
anterior pituitary trophic hormone gene promoters (15, 24, 25). For
example, we have demonstrated that Lhx3 can induce transcription from
the
GSU gene by specifically binding to a site located at
-350 to -323 bp (known as the pituitary glycoprotein basal
element, PGBE) within the proximal region of the promoter (15, 25).
This gene encodes the common subunit of the LH, FSH, and TSH anterior
pituitary hormones. Other laboratories have shown that the Lhx2/LH-2
LIM homeodomain transcription factor also can recognize this element
(30), and that this element is required to correctly restrict
expression of the
GSU gene to pituitary gonadotropes and
thyrotropes in transgenic mice (31). To test the ability of the hLhx3
isoforms to activate the
GSU gene, we transiently
cotransfected human embryonic 293 cells with
GSU
luciferase reporter genes and expression vectors containing full-length
hLhx3a and hLhx3b cDNAs. 293 cells were used because they are of
human origin, efficiently transfected, and do not express Lhx3. In
these assays, hLhx3a activated the
GSU promoter (Fig. 5A
). Surprisingly, hLhx3b did not induce
transcription from this promoter (Fig. 5A
). Similar experiments using
expression vectors encoding hLhx3a or hLhx3b with carboxyl myc epitope
tags gave the same results: hLhx3a-myc activated the
GSU
promoter and hLhx3b-myc was inactive (Fig. 5A
). In these experiments,
the epitope-tagged constructs were generally more active: this is
likely due to the fact that, in these constructs, the 3'-untranslated
region of the cDNA is absent. The murine Lhx3 3'-untranslated
region contains ATTTA sequence motifs that may confer instability to
the RNA (32). These motifs are conserved in the human Lhx3 sequences
(data not shown), and the levels of both isoforms are therefore likely
to be somewhat lower in the full-length cDNA experiments. An alternate
explanation is that the single myc epitope confers an activation
function to the Lhx3 molecules. This has been noted in experiments
where multimers of five myc epitope tags are used (33). It is important
to note, however, that the relative activities observed for the two
Lhx3 isoforms are consistent for all types of expression constructs. To
confirm expression of the hLhx3 proteins in the transfected cells,
Western analysis was performed using a specific anti-myc antibody. Both
isoforms were detected at similar levels as protein species of
approximately 60 kDa (Fig. 5B
). This apparent molecular mass is similar
to that of the in vitro translated hLhx3 proteins described
above. Western blots were quantified to determine relative expression
levels of the hLhx3 isoforms. The observed expression levels of the two
isoforms were similar; on average, the hLhx3b isoform was expressed at
slightly higher levels (1.2-fold the level observed for hLhx3a).
In addition to its ability to trans-activate the
GSU promoter, we and others have demonstrated that Lhx3
can induce pituitary trophic hormone gene promoters in synergy with
pituitary transcription factors such as Pit-1 and Pitx1/P-Otx (15, 24, 25). To examine whether the hLhx3 isoforms also had distinct abilities
to cooperate with other factors in transcriptional induction, we
transfected a TSHß reporter gene with hLhx3a and hLhx3b
expression vectors. Pit-1 and hLhx3a moderately activated the
TSHß promoter and together strongly induced transcription
(Fig. 6A
). Human Lhx3b only weakly
activated the TSHß promoter and did not effectively
synergize with Pit-1 (Fig. 6A
). As controls, similar experiments were
performed with TEF. TEF is a PAR-bZIP transcription factor that
strongly induces the TSHß promoter (34). In our
experiments, TEF activated expression from the TSHß
promoter, and expression of hLhx3a or hLhx3b did not affect this
activity (Fig. 6B
), demonstrating that the observed synergy between
hLhx3a and Pit-1 was specific.

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Figure 6. Induction of the TSHß Promoter by
hLhx3 Isoforms and Other Pituitary Transcription Factors
Human 293 cells were transiently transfected with a mouse
TSHß promoter reporter gene plasmid and hLhx3a,
hLhx3b, Pit-1, and/or TEF expression vectors. Luciferase activity was
assayed after 48 h. Values are mean [light units/10 sec/µg
total protein] of triplicate assays ± SEM. A
representative experiment of at least four experiments is depicted.
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The
GSU and TSHß promoters are complex
regulatory structures containing recognition sites for multiple
transcription factors. For example, the
GSU PGBE Lhx3
binding site appears to cooperate with other elements in gene
regulation (31, 35). We therefore extended our studies by comparing the
abilities of hLhx3a and hLhx3b to activate a defined synthetic
luciferase reporter gene containing three copies of a consensus Lhx3
DNA binding element cloned upstream of a minimal promoter. Again,
expression vectors for hLhx3a and hLhx3a-myc activated transcription
from this reporter more effectively than the corresponding hLhx3b
vectors (Fig. 7
). These data further
support the hypothesis that hLhx3a is a more potent activator of
transcription than hLhx3b for target genes containing this type of
recognition element.

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Figure 7. Transactivation of a Luciferase Reporter Gene
Containing Lhx3 Binding Sites by hLhx3 Isoforms.
Human 293 cells were transiently transfected with a Lhx3 reporter gene
containing three Lhx3 consensus sites and hLhx3a or hLhx3b expression
vectors. Reporter gene activity was measured 48 h after
transfection. Activities are mean [light units/10 sec/µg total
protein] of triplicate assays ± SEM. A
representative experiment of at least five experiments is depicted.
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DNA Binding Properties of hLhx3 Isoforms
The different abilities of hLhx3 isoforms to activate
transcription from the tested reporter genes indicated that the hLhx3a-
and hLhx3b-specific domains conferred distinct transcriptional
properties upon the hLhx3 protein. Our data show that the two isoforms
were both expressed in the nucleus at similar levels (Figs. 3
and 5
).
Mechanisms for the different activities include the possibilities that
the a-specific and b-specific domains are trans-activation
or trans-repression domains, respectively; or that the a-
and b-specific domains may mediate interaction with distinct regulatory
factors; or that the a- and b-specific domains may differently modulate
the DNA binding properties of hLhx3. We have previously demonstrated
that the LIM domains of Lhx3 repress DNA binding by the homeodomain
(15, 25). To test the hypothesis that the a- and b-specific domains
conferred distinct DNA binding properties upon the hLhx3 molecule, we
performed electrophoretic mobility shift assays (EMSAs) to test binding
to the
GSU -350/-323 bp Lhx3 site and to the Lhx3
consensus binding site. Experiments using in vitro
translated native hLhx3 proteins revealed that hLhx3a bound to these
sites more effectively than hLhx3b (Fig. 8B
). Similar results were obtained with
hLhx3 proteins containing carboxyl-terminal myc epitope tags (Fig. 8B
).

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Figure 8. Expression of Recombinant hLhx3 Proteins and
Analysis of Binding to DNA Target Sequences
A, Coomassie brilliant blue stain of a SDS-polyacrylamide gel analysis
of GST fusion proteins containing either hLhx3a or hLhx3b. The
migration of mol wt standards is indicated. B, Electrophoretic mobility
shift assay using Lhx3 consensus binding site (lanes 112) or
GSU gene -350/-323 binding site (lanes 13 and 14)
oligonucleotide probes. Radiolabeled probes were incubated with the
indicated proteins and competitor DNAs and the resulting complexes were
separated from free probe (F) by electrophoresis. Lane 1, Unprogrammed
rabbit reticulocyte lysate as a negative control (lysate); lanes 25,
reactions contained in vitro translated hLhx3 proteins.
A nonspecific band is noted by an asterisk. Human Lhx3
protein/DNA complexes are indicated by an arrow. Lane 6,
GST as a negative control; lanes 714: recombinant hLhx3 proteins
expressed in E. coli. Comp, Reactions containing
approximately 1000-fold molar excess of unlabeled binding site as a
competitor.
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To confirm this result, we expressed recombinant hLhx3 proteins in
E. coli as fusions to the carboxyl terminus of
glutathione-S-transferase (GST). This approach allowed the
use of affinity-purified proteins of known concentration (Fig. 8A
).
EMSA reactions using the recombinant purified proteins again indicated
that hLhx3a bound more effectively than hLhx3b to both DNA sites (Fig. 8B
). Addition of excess unlabeled binding site DNA to hLhx3a EMSA
reactions reduced the amount of bound complex, whereas it abolished the
binding of hLhx3b (Fig. 7B
; and data not shown). These experiments
demonstrate that the b-specific domain inhibits the binding of hLhx3b
to this class of DNA site. Further, the b-specific domain can perform
this function when positioned at the amino terminus of the native
protein or when placed in an internal context such as in the GST fusion
proteins.
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DISCUSSION
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Through evolution, Lhx3-type proteins appear to have been largely
conserved in structure, expression pattern, and function. The
Lhx3/LIM3 gene is transiently expressed in the developing
nervous systems of Drosophila, Xenopus, zebrafish, chickens,
and mammals (13, 14, 15, 16, 17, 18, 19), and it has been demonstrated to play important
roles in the specification of motor neuron subtypes in some of these
species (18, 19, 20, 21). In addition, Lhx3 is critical for both early
development and terminal differentiation of the mammalian pituitary
(22, 23). Expression also is detected in other developing endocrine
structures including the pineal gland (13, 14, 15, 16, 17) and the
Drosophila ring gland (18), the major site of production of
developmental hormones in higher dipterans. As summarized in Fig. 1
, the LIM domains, the homeodomain, and the Lhx3/LIM3-specific
domain are conserved in Lhx3-type proteins from Drosophila
to humans.
In this study, we have demonstrated that proteins with distinct
functional properties are generated from the human Lhx3
gene. During evolution, the increased complexity of organisms has
required an extended number of regulatory factors with sufficient
capacity to control the development of organ systems comprised of
multiple, differentiated cell types. This increased molecular diversity
has been accomplished by mechanisms such as the formation of additional
genes by gene duplication; the generation of multiple proteins from
single genes by the use of distinct promoters; and posttranscriptional
processes. Posttranscriptional mechanisms include the use of alternate
translation initiation codons, alternative RNA splicing, the production
of protein isoforms from single RNAs by RNA editing, and
proteolytic processing. For Lhx3-type proteins, it appears that
both gene expansion (to produce genes encoding related proteins such as
Lhx4) and RNA-mediated mechanisms generating isoforms such as hLhx3a
and hLhx3b have been used to provide a battery of proteins with
important roles in the development of both neural and endocrine
structures, including the anterior pituitary.
Alternate isoforms of LIM homeodomain proteins, nonhomeodomain LIM
proteins, and several other types of transcription factors are known.
For example, the LIM homeodomain transcription factors, Lhx6.1a and
Lhx6.1b, appear to be generated by an alternative splicing event that
generates proteins with different carboxyl terminal amino acids (36).
These factors are expressed at different times in the developing mouse
brain, but unique functional properties of the isoforms have not been
demonstrated (36). Alternate forms of LMO7 and LIM-kinase proteins
lacking either the LIM domain or the kinase domain, respectively, have
also been described (37, 38). The isoforms of these proteins have
different expression patterns and distinct functional properties.
Interestingly, Skn-1a and Skn-1i are alternatively spliced POU domain
factors expressed in the epidermis that, like Lhx3, have different
amino-terminal domains (39). Similar to the hLhx3b-specific
domain, the i-specific domain of Skn-1 inhibits DNA binding and gene
trans-activation by this factor. However, primary
amino acid sequence comparison between these inhibitory domains reveals
no similarity. Alternate isoforms with distinct functions and/or
expression patterns are also seen in the nuclear receptor protein
superfamily. Examples include the distinct expression patterns of the
isoforms of the peroxisome proliferator-activated receptor
developmental regulatory protein (40, 41) and the unique
transcriptional properties of the ß2 thyroid receptor isoform
(42).
In the mouse, isoforms of Lhx3 also have been described (16, 32). We
suggest that the human Lhx3b clone described in this study is
equivalent to murine Lhx3b, because the two amino-terminal domains are
nearly identical (Fig. 1
). The human and murine Lhx3a amino-terminal
domains are much less alike, but some sequence features are similar
(Fig. 1
). We hypothesize that the Lhx3a forms are functionally related,
but this function does not require conservation of the Lhx3a-specific
domain. To date, other LIM homeodomain proteins with alternate
functional domains have not been described. The hLhx3a- and
hLhx3b-specific amino-terminal motifs may be novel functional domains
derived throughout evolution to confer properties unique to Lhx3a and
Lhx3b that allow specific roles in the development of the pituitary
gland and neural structures.
We have demonstrated that hLhx3a is able to activate specific target
genes, whereas hLhx3b is inactive and that these functional differences
correlate with a reduced binding of hLhx3b to DNA elements within these
genes (
Figs. 48



). These data, and the observation that the
Lhx3a-specific domain is poorly conserved in sequence and length
(Fig. 1
), together suggest that the Lhx3b-specific domain confers
intramolecular inhibition of DNA binding and
trans-activation functions. We have previously demonstrated
that the LIM domains of mammalian Lhx3 proteins inhibit DNA binding
(15, 25), and others have made similar observations for several LIM
homeodomain proteins (reviewed in Ref. 12). Therefore, the
Lhx3b-specific domain may inhibit DNA binding by configuring the LIM
domains such that they exert a greater inhibitory function upon the
homeodomain. Alternatively, the Lhx3b-specific domain may function
independently of the LIM domains, or it may exert both LIM-dependent
and independent effects.
Our data suggest that the alternate hLhx3 isoforms may have distinct
functions within the developing pituitary gland and neural structures
such as the embryonic spinal cord. Our results suggest that the
described Lhx3 isoforms are critical to thyrotrope differentiation and
maintenance because both are expressed in the
-TSH cell line, and
hLhx3a activated the
GSU and TSHß genes. The
low level of Lhx3 expression in AtT20 cells is consistent with
observations of Lhx3 knockout mice that have some corticotropes, but
lack other pituitary cell types (22). It is tempting to speculate that
the more abundant Lhx3b isoform in AtT20 cells plays a unique role in
differentiation of this cell type.
The described hLhx3 isoforms may have distinct target genes or,
dependent on its expression profile, hLhx3b may play a direct or
indirect dominant negative role. Experiments in the mouse have
suggested that in this species Lhx3a may be expressed earlier than
Lhx3b during development (32). Specification of differentiated
pituitary cell phenotypes appears to be controlled by the combinatorial
actions of multiple tissue-specific transcription factors and signaling
proteins (reviewed in Refs. 1, 2, 3). The differential activities or
expression patterns of alternate forms of essential members of this
program, such as Lhx3 and Pit-1, may play critical roles in the
determination of pituitary cell fates. Indeed, we and others have
described isoforms of Pit-1 that have distinct properties and
expression patterns (e.g. Refs. 43, 44 ; reviewed in Ref.
7). In addition, the described Lhx3 isoforms may interact differently
with broadly expressed regulatory proteins or transcriptional
coactivators/corepressors. For example, a conserved family of nuclear
LIM domain-interacting proteins, known as NLI/Ldb1/CLIM/Chip, has been
characterized (Refs. 45, 46, 47 and references therein). These proteins
appear to be regulatory partners for LIM homeodomain factors and can
mediate homo- and heterodimerization of these factors. Recently, a
RING-H2 zinc finger protein, RLIM, was identified and also described as
a regulatory partner for LIM homeodomain factors (48). This
coregulatory protein appears to recruit the Sin3a/histone deacetylase
corepressor complex to function as a LIM-associated inhibitory factor
(48). Coregulators such as NLI proteins and RLIM may differentially
modulate the ability of Lhx3 isoforms to regulate target genes.
The detection of hLhx3 in the pituitary gland and the demonstration of
its ability to regulate pituitary trophic hormone gene promoters
suggest a continued role for Lhx3 in the adult human pituitary gland in
maintenance of hormone gene expression. This study also provides tools
for future investigations examining the role of hLhx3 in pituitary
diseases such as combined pituitary hormone deficiency and pituitary
tumor disease. Further studies of hLhx3 and related factors will extend
our understanding of the developmental program that establishes the
hormone-releasing cells of the human pituitary gland and may facilitate
future protocols designed to treat pituitary diseases.
 |
MATERIALS AND METHODS
|
---|
RNA Extraction/cDNA Synthesis
Adult human pituitaries were obtained from the National Hormone
and Pituitary Program or were obtained at autopsy at the Indiana
University School of Medicine. As could best be assessed, pituitary
samples were normal and did not have adenomas. Patients ranged in age
from 36 to 65 yr, and weighed from 75 to 136 kg. Tissues were frozen in
liquid nitrogen and ground to a powder on dry ice/liquid nitrogen, and
total RNA was extracted using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) and bromochloropropane as a
chloroform substitute. cDNA was synthesized using Superscript II
reverse transcriptase (Life Technologies, Inc.,
Gaithersburg, MD) and oligo d(T), random hexamer, or gene-specific
primers as required.
DNA Cloning/DNA Sequencing/Plasmid Construction
Database searches revealed that a human expressed sequence
tag (EST) sequence displayed similarity to murine and porcine
Lhx3. The bacterial culture containing this plasmid could not be
retrieved by any of the distributors of EST clones. PCR was then used
to amplify this sequence from adult human pituitary cDNA using the
following primers: (5'-cggaattctacaacacctcgcccaagccgg-3',
5'-cggaattcggaacgaggggcccttgac-3'). RACE was then performed using the
5'-RACE 2.0 system (Life Technologies, Inc.) and adult
human pituitary cDNA. Finally, partial hLhx3 cDNAs were used as high
stringency probes (final wash = 0.5x SSC, 65 C) of a human
pituitary cDNA library (CLONTECH Laboratories, Inc., Palo
Alto, CA). From 1 million plaques, 12 positive bacteriophage were
identified, and cDNAs were subcloned into pBluescript KS II (-)
(Stratagene, La Jolla, CA) as described (25). Seven clones
were completely sequenced on both strands by automated DNA sequencing
using a Perkin Elmer Corp. (Norwalk, CT) DNA sequencer
(Biochemistry Biotechnology Facility, Indiana University School of
Medicine). Sequence analysis was performed using Wisconsin
Genetics/GCG and DNASIS (Hitachi, South San
Francisco, CA) software.
Expression vectors for hLhx3a and hLhx3b were generated by directly
cloning full-length cDNAs into pcDNA3 (Invitrogen). Myc
epitope-tagged hLhx3 expression vectors (hLhx3a-myc, hLhx3b-myc) were
made by cloning compatible cDNA fragments into
pcDNA3.1/Myc-His(-)C (Invitrogen). DNA
fragments were generated by PCR using the following primers:
5'-cgggatccgatcgcttcggcagcagctg-3' (5', hLhx3a-myc);
5'-cgggatccttgatatttaccccggaggc-3' (5', hLhx3b-myc); and
5'-gcgaagcttggaactgagcgtggtctacctca-3' (3', hLhx3a/b-myc). An
expression vector containing four copies of GFP was constructed by
removing the 4xGFP cassette from plasmid p713 (Ref. 26 ; a generous gift
of Dr. Ursula Stochaj, McGill University, Montreal, Quebec,
Canada) by digestion with KpnI and EcoRI
and cloning of this fragment into pcDNA3 to generate pcDNA34xGFP.
Compatible hLhx3 DNA fragments were generated by PCR using the
following primers: 5'-tacaagcttcgcgatgctgctggaaacgg-3' (5', hLhx3a);
5'-tacaagcttaccatggaggcgcgcgggga-3' (5', hLhx3b); and
5'-cccggtaccaactgagcgtggtctacctc-3' (3', hLhx3a/b). These fragments
then were digested with HindIII and KpnI and
cloned into pcDNA34xGFP to generate vectors expressing hLhx3a-4xGFP
and hLhx3b-4xGFP.
Southern and Northern Analyses
Human genomic DNA was extracted from peripheral blood using a
QIAamp Blood Maxi Kit (Qiagen, Chatsworth, CA). DNA was
digested to completion with restriction enzymes, electrophoresed
through 0.7% agarose gels, and transferred to nylon membranes
(Hybond-N+, Amersham Pharmacia Biotech,
Piscataway, NJ). Membranes were hybridized to radiolabeled hLhx3 cDNA
probes in ExpressHyb buffer (CLONTECH Laboratories, Inc.)
at 12 x 106 cpm/ml for 24 h. DNA fragments were
labeled by random priming using the Klenow fragment enzyme (Life Technologies, Inc.) and 32P-dCTP (Amersham Pharmacia Biotech) to a specific activity of >1 x
109 cpm/µg. After hybridization, membranes were washed in
0.5x SSC at 65 C, followed by exposure to MR film (Eastman Kodak Co., Rochester, NY) with intensifying screens.
Total human pituitary RNA was extracted as described above. Poly
A+ RNA was purchased from CLONTECH Laboratories, Inc. RNA was separated on denaturing, formaldehyde agarose gels
followed by transfer to Nytran Plus membranes using the Turboblotter
system (Schleicher & Schuell, Inc., Keene, NH). Probes
were a 0.4 kb hLhx3 cDNA fragment encoding the LIM domains of the
protein and a 1.2 kb cDNA encoding human Pit-1. Membranes were
hybridized to radiolabeled cDNA probes as described above.
In Vitro Transcription/Translation
Radiolabeled hLhx3 proteins were synthesized in vitro
from pcDNA3 expression vector substrates using T7 RNA polymerase, TnT
rabbit reticulocyte lysates (Promega Corp., Madison, WI),
and 35S-methionine (Amersham Pharmacia Biotech, Arlington Heights, IL). Proteins were analyzed using
SDS-PAGE followed by treatment with Amplify fluorography reagent
(Amersham Pharmacia Biotech) and exposure to MR film
(Eastman Kodak Co.) at -80 C.
Confocal Microscopy
Human 293T cells (1 x 105) were grown in
chamber slides (Nunc) and transfected with 4xGFP, hLhx3a-4xGFP, or
hLhx3b-4xGFP expression vectors as described below. After 48 h,
cells were washed with 1x PBS, and then either directly visualized
live or fixed with 2% paraformaldehyde, washed with 70% ethanol/30%
PBS, and stored in 1x PBS before visualization. Fluorescence and
trans-illumination images were collected using a MRC 1024
laser scanning confocal microscope (Bio-Rad Laboratories, Inc., Richmond, CA) with a 60x water immersion objective
(Nikon, Melville, NY) and a krypton-argon laser. Images
were captured with Metamorph software (Universal Imaging, West Chester,
PA).
Quantitative Assay of Gene Expression
"Real time" quantitative PCR utilizing the ABI PRISM 7700
Sequence Detection System (Perkin Elmer Corp.) was
performed as previously described (27, 49). Total RNA was isolated from
human pituitary tissue as described above. Reverse transcription of
total RNA with a hLhx3-specific reverse primer
(5'-ctcccgtagaggccattg-3') was performed using SuperScript II reverse
transcriptase (Life Technologies, Inc.). Quantitative PCR
amplification reactions were performed in triplicate and included: 2
µl of cDNA synthesis reaction, 1x TaqMan Buffer A, 300
nM dATP, dCTP, dGTP, and 600 nM dUTP, 3.5
mM MgCl2, 1.25 U AmpliTaq Gold DNA polymerase
(Perkin Elmer Corp.), 0.5 U of AmpErase uracil
N-glycosylase, 300 nM forward primer
(5'-ggacaaggacagcgttcag-3') and reverse primer, and 200 nM
hLhx3 fluorogenic probe (5'-ttccccgatgagccttccttggcggaa-3'). Reaction
parameters were as follows: 50 C, 2 min; 94 C, 10 min; and then 35
cycles of 94 C, 30 sec; 60 C, 1 min. Serial dilutions of hLhx3 cDNA
were amplified simultaneously with patient samples to generate a
standard curve. Values reported are averages of four independent
experiments.
RT-PCR Analysis
cDNA was synthesized from adult pituitary RNA as described
above using oligo-d(T) as a primer. PCR then was performed using
the following primers: 5'-cgggatccatgctgctggaaacggggct-3' (5', hLhx3a);
5'-cgggatccatggaggcgcgcggggagct-3' (5', hLhx3b); and
5'-cggaattctcagaactgagcgtggtcta-3' (3', hLhx3a/b). Cycling parameters
were as follows: 94 C, 30 sec; 60 C, 30 sec; 72 C, 1 min; for 30
cycles. Reaction products were analyzed on 1% agarose Tris-borate
gels. For analysis of mouse pituitary cell lines, cDNA was synthesized
from total RNA using 5'-tggtcacagcctgcacacat-3'. PCR then was performed
using the following primers: 5'-aaccactggattagtgactg-3' (5', mLhx3a);
5'-gaagttcagggtcggaggg-3' (5', mLhx3b); and 5'-tggtcacagcctgcacacat-3'
(3', mLhx3a/b). Cycling parameters were as follows: 94 C, 30 sec; 60 C,
30 sec; 72 C, 30 sec; for 30 cycles. Reaction products were analyzed on
11% acrylamide Tris-borate gels.
Cell Culture, Transfection Assays, Statistical Analysis
Human embryonic kidney 293 and 293T cells were cultured in DMEM
(Life Technologies, Inc.) with 10% FBS (Irvine
Scientific, Santa Ana, CA), 100 U/ml penicillin, and 100 µg/ml
streptomycin (Irvine Scientific); 1.5 x 105 cells per
60-mm dish were transfected with calcium phosphate/DNA precipitates
using the CalPhos system (CLONTECH Laboratories, Inc.).
Reporter plasmid (0.5 µg) and expression vector (0.11.0 µg of
expression vector) were added per 60-mm dish, and all groups received
equal final DNA concentrations. The murine
GSU promoter
luciferase plasmid (30) was a generous gift of Dr. Richard Maurer
(Oregon Health Sciences University, Beaverton, OR). The murine
TSHß -1.2-kb promoter luciferase plasmid was as described
(34). The Lhx3 consensus binding site reporter gene was constructed by
cloning three copies of 5'-cagaaaattaattaattgtaa-3' upstream of a
minimal PRL (-36 bp) promoter luciferase reporter gene (J. L.
Bridwell, J. R. Price, G. E. Parker, K. W. Sloop, A. McCutchan
Schiller, and S. J. Rhodes, in preparation). Control
cultures received empty expression vector DNA. Luciferase activity was
measured 48 h after transfection as described (25). All assays
were performed in triplicate. Total cell protein was determined by the
Bradford method (Bio-Rad Laboratories, Inc.) and
luciferase activity was normalized to protein concentration. Data
points were compared using a one-tailed Students t test
for paired samples using Sigma Plot 2.0 (Jandel Scientific, San Rafael,
CA). Values were considered significantly different when
P < 0.05.
Western Analysis
Western analysis of 293 or 293T cells transfected with
hLhx3a-myc, hLhx3b-myc, or control expression vectors was performed as
we described previously (25). The mouse anti-myc monoclonal antibody
9E10, developed by Evan et al. (50), was obtained from the
Developmental Studies Hybridoma Bank developed under the auspices of
the National Institute of Child Health and Human Development and
maintained by the Department of Biological Sciences, University of Iowa
(Iowa City, IA). Ascites fluid was used at a 1:5000 dilution.
The secondary antibody was a goat antimouse/horseradish peroxidase
(Sigma, St. Louis, MO) at 1:15,000. Results were
visualized using Supersignal chemiluminescence reagents (Pierce Chemical Co., Rockford, IL) and MR film (Eastman Kodak Co.). Data were quantified using a Bio-Rad Laboratories, Inc. imaging densitometer and Molecular Analyst software
(Bio-Rad Laboratories, Inc.).
Recombinant Protein Preparation/EMSA
Bacterial expression vectors for GST-hLhx3a and GST-hLhx3b
fusion proteins were generated by cloning
BamHI/EcoRI compatible fragments of the hLhx3a
and hLhx3b cDNAs into pGEX-KT (51). cDNA fragments were generated by
PCR using the following oligonucleotides:
5'-cgggatccatgctgctggaaacggggct-3',5'-cggaattctcagaactgagcgtggtcta-3'
(hLhx3a) and
5'-cgggatccatggaggcgcgcggggagct-3',5'-cggaattctcagaactgagcgtggtcta-3'
(hLhx3b). Recombinant proteins were expressed in E. coli
BL21 (DE3) pLysS and affinity-purified as we have previously described
(25). Proteins were analyzed on 12% SDS-PAGE gels. EMSAs were
performed as described (25). Oligonucleotides representing the -350 to
-323 bp region of the murine
GSU promoter
(5'-acattaggtacttagctaattaaatgtg-3' and
5'-cacatttaattagctaagtacctaatgt-3') were used or the Lhx3 consensus
binding site described above. In competition experiments, 1000-fold
molar excess of unlabeled binding site DNA was added to EMSA
reactions.
 |
ACKNOWLEDGMENTS
|
---|
We are very grateful to Drs. B. Azarelli, D. Crowell, R. Maurer,
L. Slieker, U. Stochaj, and the National Hormone and Pituitary Program
for materials and useful advice. We also thank T. Lahr, R. Sandoval,
and A. Showalter for assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Simon J. Rhodes, Ph.D., Department of Biology, Indiana University-Purdue University Indianapolis, 723 West Michigan Street, Indianapolis Indiana 46202-5132.
This work was supported by grants to S.J.R. from the National Science
Foundation, the National Research Initiative Competitive Grants
Program/US Department of Agriculture, and the Indiana
University-Purdue University Indianapolis Office of Faculty
Development. We dedicate this paper to the memory of Dr. Raymond
Russo.
Received for publication June 28, 1999.
Revision received August 24, 1999.
Accepted for publication September 8, 1999.
 |
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