(Received for publication, December 1, 1995 )
From the
The murine leukemia inhibitory factor receptor -chain
(mLIFR) exists in a membrane-bound and a soluble form. The two major
classes of mRNA transcript correspond to either the soluble or
membrane-bound form of the mLIFR. In this study we have identified a
complex and heterogeneous pattern of expression of mRNA transcripts for
this receptor in normal mouse tissues and cell lines. In order to
understand the molecular basis of these transcripts, genomic clones
encompassing the region of divergence from the soluble to the
membrane-bound form of the receptor were isolated. cDNAs encoding the
membrane-bound form of the mLIFR were generated by an alternative
splicing event where an exon that is specific to the soluble mLIFR was
skipped. The membrane-bound form of the mLIFR was heterogeneously
polyadenylated with at least five different sites of polyadenylation.
The mRNA transcript encoding the soluble form of the mLIFR contained a
region highly homologous to a murine B2 repetitive element, thus
providing a possible explanation for the genesis of this transcript.
The different forms of the mLIFR were analyzed in a wide range of mouse
tissues in pseudopregnant mice and in mice at various stages of
pregnancy. Only liver, placenta, and uterus showed an increase in the
levels of mLIFR mRNA expression during pregnancy, indicating an
important role for the LIFR in this process. However, somewhat
surprisingly, there was no detectable difference in mLIFR mRNA levels
or levels of soluble protein in leukemia inhibitory factor nullizygous
mice when compared with normal mice.
Leukemia inhibitory factor (LIF) ()is a
multifunctional cytokine that was initially purified and cloned on the
basis of its ability to induce differentiation and suppress
clonogenicity in the mouse leukemic cell line M1(1) . The
pleiotropic actions of LIF include its ability to maintain embryonic
stem cells in their pluripotent state, stimulate acute phase synthesis
by hepatocytes, inhibit adipogenesis, regulate nerve differentiation,
stimulate the function of osteoblasts and the proliferation of DA-1
hemopoietic cells, and potentiate the action of interleukin-3 on
megakaryocyte precursors (reviewed in (2) ).
The high
affinity LIF receptor complex is composed of a specific LIF-binding
component, the -chain, and an affinity converter, gp130 (3, 4) . Several other cytokines including ciliary
neurotrophic factor (5) and oncostatin-M (4, 6) require the LIFR
-chain to transduce a
biological signal. The recently characterized cytokine cardiotrophin-1
has also been shown to bind to the LIFR
-chain with low
affinity(7, 8) . The high affinity receptor complexes
of these cytokines as well as those for interleukin (IL)-6 (9) and IL-11 (10, 11) also contain gp130, and
this sharing of receptor components may account for the overlapping
actions and functional redundancy of this group of cytokines.
The
LIF receptor -chain (LIFR) and gp130 are members of the
hemopoietin family of receptors(12, 13) . The LIFR
contains in its extracellular region two hemopoietin domains that are
separated by an immunoglobulin-like module. In addition, three
fibronectin type III (FNIII) domains are located immediately N-terminal
to the transmembrane domain. cDNA clones that encode receptors
corresponding to a membrane-bound form of the LIFR were isolated from a
human placental cDNA library(3) . Further cDNA clones, that
have been isolated from murine liver cDNA libraries, encode stop codons
just after the second FNIII domain and are thus predicted to encode
soluble receptors (3, 14) . A naturally occurring
LIF-binding protein is present in high levels in normal mouse serum and
was shown to be a soluble form of the mLIFR(15, 16) .
The LIF-binding protein is able to bind mLIF with similar
characteristics to the membrane-bound form of the receptor, but unlike
the soluble form of the IL-6 receptor (17) it acts as an
inhibitor of LIF activity rather than as an agonist(18) . The
physiological function of this form of the mLIFR has not yet been
determined, but it may serve to sequester excess LIF in the
circulation.
The gene for the murine LIF receptor (mLIFR) has been mapped to the proximal region of chromosome 15 and is located within a cluster of cytokine receptor loci that includes growth hormone receptor, interleukin-7 receptor, and prolactin receptor(19) . Targeted disruption of the low affinity mLIFR results in a wide range of defects including abnormal placentation, severe osteopenia, various neural defects, and metabolic disorders of the liver(20) . mLIFR-deficient animals die during or shortly after birth. Mice in which the LIF gene has been disrupted have also been described(21, 22) , and although these mutant mice display retarded postnatal growth they are generally viable. Interestingly, blastocysts from LIF null females are viable but are only able to implant if transferred to heterozygous or wild type pseudopregnant females, indicating a requirement for LIF in the implantation process. An important role for LIF during pregnancy is also implied by studies of normal mice, as the levels of both LIF (23) and the soluble mLIFR (15, 16) increase dramatically during gestation. The maximum serum levels of soluble mLIFR occur several days after the transient burst of LIF expression on day 4 of pregnancy (23) .
In this study we have characterized the molecular basis for the soluble and membrane-bound forms of the mouse LIFR. Given the role of LIF and its receptor in pregnancy we have examined the mRNA levels of the soluble and membrane-bound forms of the mLIFR in a variety of tissues during gestation in normal mice. We have also investigated the levels of mLIFR mRNA expression in mice in which the LIF gene has been ablated.
In order to isolate
genomic clones encoding regions of the mLIFR gene a mouse 129SV genomic
library in FIXII (Stratagene) was screened under high stringency
conditions (0.2
SSC, 0.1% SDS at 65 °C) with radiolabeled
probes (a BglII-HindII fragment from the
extracellular domain that encompassed fibronectin III domains 1 and 2
plus a BglII-BstEII fragment from the cytoplasmic
domain) derived from cDNA clones. Positive plaques were purified, and
phage DNA was prepared using standard methods(25) . After
restriction mapping, and hybridization using exon-specific
oligonucleotide primers, genomic insert fragments were digested with
appropriate restriction enzymes and subcloned into the pBluescript SK
II vector.
Figure 1:
Northern blots of poly(A) RNA (3 µg/lane) from normal mouse tissues (A, B); peritoneal macrophages from granulocyte-macrophage
colony-stimulating factor transgenic mice, mouse cell lines (C); fetal tissues (D) showing the distribution mLIFR
mRNA isoforms. The blots were sequentially hybridized with a mLIFR cDNA
probe and a glyceraldehyde-3-phosphate dehydrogenase probe. The
locations of RNA size markers (kb) are shown on the right.
Figure 4:
Northern analysis of mLIFR transcripts.
The locations of RNA size markers (kb) are shown on the right in each panel. A, Northern blot of
poly(A) RNA (3 µg/lane) from mouse placenta and
liver hybridized with a cDNA probe to the extracellular region of the
mLIFR. B, Northern blot of poly(A)
RNA (3
µg/lane) from mouse placenta and liver hybridized with a cDNA probe
specific to the cytoplasmic domain of the membrane-bound mLIFR. C, Northern blot of poly(A)
RNA (3
µg/lane) from mouse placenta and liver hybridized with a cDNA probe
encoding the 3`-untranslated region of the membrane-bound mLIFR. D, Northern blot of poly(A)
RNA (3
µg/lane) from various mouse tissues and cell lines hybridized with
a cDNA probe containing the B2 element in the 3`-untranslated region of
the soluble mLIFR.
Fig. 1, A and C, shows that the
mLIFR was expressed in M1, F9, PC13, NIH3T3-L1, and embryonal stem
cells. Peritoneal macrophages from mice carrying a cDNA insert encoding
granulocyte-macrophage colony-stimulating factor as a transgene (30) also expressed mLIFR transcripts. mLIFR transcripts were
not detected in FD-CP1 and WEHI 3BD cells. Embryonal
stem cells showed a similar expression pattern of transcripts to
placenta, although the 5-kb band was absent. All three major forms of
transcript were present in M1 cells.
Expression of the mLIFR in fetal tissues was analyzed (Fig. 1D). In contrast to adult liver, in fetal liver the 3-kb band was expressed in lower amounts relative to the 10-kb band. A similar pattern was observed in liver samples from embryonic days 15 and 19 and newborn mice.
Figure 2: Nucleotide sequence of 3`-untranslated region of the membrane-bound mLIFR cDNA clone mLIFR52.4A. DNA sequence that has been previously described in (14) is shown in lowercase, and additional DNA sequence is shown in uppercase. The translational termination codon is indicated in boldface type. The putative poly(A) signal is underlined. The numbers shown on the left relate to and are a continuance of the nucleotide sequence reported in (14) .
Figure 3: A, nucleotide sequence comparison of the 3`-untranslated region of the membrane-bound mLIFR cDNA 52.4A with the corresponding region of the membrane-bound hLIFR cDNA. The numbers on the left of each DNA sequence refer to the nucleotide sequence for the mLIFR presented in Fig. 2and for the hLIFR sequence described in (3) as indicated. Identities are indicated by dashed lines. B, nucleotide sequence comparison of a 30-bp section on the minus strand of the 3`-untranslated region of the membrane-bound mLIFR with the mouse PCNA gene negative regulatory region. The numbers on the left of each DNA sequence refer to the nucleotide sequences of mLIFR cDNA clone 52.4A (see Fig. 2) and the mouse PCNA negative regulatory region (32) as indicated. Identities are indicated by dashed lines. C, nucleotide sequence comparison of the 3`-untranslated region of soluble mLIFR (sol. mLIFR) with a mouse B2 element. The translational termination codon and the sequence encoding the polyadenylation signal of the soluble mLIFR cDNA are underlined. Identities are indicated by dashed lines. The numbers on the left of each sequence are consistent with the partial nucleotide sequence described in (14) for the mLIFR and for the mouse B2 repeat sequence from clone MM14(33) .
The nucleotide sequence of clones encoding soluble mLIFRs obtained in this study corresponded with the sequence that has been previously reported(3, 14) . However, when the sequence of the 3`-untranslated region of the soluble receptor clones was compared with sequences in the GenBank data base there was a region, from nucleotide position 2336 to 2498, that was approximately 84% identical with the nucleotide sequence from a mouse B2 element (33) (Fig. 3C). Intriguingly, the poly(A) signal utilized by transcripts encoding soluble mLIFRs is part of the B2 element consensus sequence.
Figure 5:
Organization of the mLIFR gene at the
region of divergence between the soluble mLIFR to the membrane-bound
form. A, restriction map of overlapping phage clones
showing three phage clones and restriction enzyme sites. B, BamHI; H, HindIII; E = EcoRI. B, intron-exon organization of this section of
the mLIFR gene. The position of this section of the mLIFR gene relative
to the restriction map in panel A of this figure is
indicated by the dashed lines joining panels A and B. Exons are depicted by boxes, and the translational
termination codons (TAA) are indicated. The numbers above the
exons refer to the nucleotide positions in the mLIFR cDNA sequence
described in (14) . The intervening sequences (introns) are
depicted as narrow black lines interrupted by double
dashes to indicate that their lengths are not drawn to scale.
Noncoding regions that were present in cDNA clones are cross-hatched. The pattern of splicing that is required to
generate soluble or membrane-bound forms of the mLIFR is indicated by arrowed lines joining the relevant exons. The putative
polyadenylation signals (AATAAA and GATAAA) are indicated by curved
arrows. TM (black box) is the exon encoding the
transmembrane domain; CB1 refers to the exon encoding the
cytoplasmic box 1 motif; SS represents the exon specific to
transcripts encoding the soluble mLIFR. B2 is the section of
DNA in the 3`-untranslated region of the soluble mLIFR locus that shows
strong homology to the mouse B2 repetitive element. FNIII2b is
the second subdomain of the second fibronectin type three domain, which
is present in both soluble and membrane-bound forms of the mLIFR. FNIII3a and FNIII3b refer to the first and second
subdomains, respectively, of the third fibronectin type three domain
that is present only in the membrane-bound mLIFR. C, variant
mLIFR cDNA clone isolated from a mouse liver library. The abbreviations
used and the symbols for exons and intervening sequences are
as described for panel B, except that the regions of intronic
sequence (``INTRON'') that were included in this
variant clone are depicted as shaded boxes. The numbers
above the exons refer to the nucleotide positions in the mLIFR
cDNA sequence described in (14) . TAA refers to an
in-frame translation termination codon encoded in the
``intron'' sequence. The splicing pattern is indicated by the arrowed lines.
There were several different mRNA transcripts that corresponded to the membrane-bound mLIFR in the 4.5-10.2-kb range that were detected in placenta, liver, and several other tissues. Studies were performed to determine whether this might reflect variability in the 3`-untranslated regions. The membrane-bound mLIFR cDNA clone identified in (14) terminated at nucleotide position 3399, although it was unclear whether it was polyadenylated. Polyadenylated cDNA clones (mLIFR52.4A, mLIFR30.2, and mLIFR243) (Fig. 6B) extended a further 526 bp, 474 bp, and 1.4 kb, respectively, 3` of the sequence described in (14) . Thus three sites of polyadenylation were identified within this region of genomic DNA. All of the sequence constituting the 3`-untranslated regions from these clones was also identified in clones of genomic DNA (Fig. 6A).
Figure 6:
Location of polyadenylation sites and
genomic subclones. A, restriction map of the 3`-untranslated
region of the membrane-bound mLIFR gene. Only the HindIII (H) and BamHI (B) sites are shown. The open box indicates the final coding exon of the membrane-bound
mLIFR and the translational termination signal (TAA) is shown. The arrows indicate the positions of polyadenylation signals that
were identified by sequencing of cDNA clones. The relative positions
and identification numbers of subcloned genomic fragments are indicated below the restriction map. B, diagram of variant
forms of mLIFR transcript identified by cDNA cloning and sequencing.
cDNA clones (shaded boxes) are identified in italics and are further described under ``Results.'' Their
positions relative to the genomic map can be obtained by visual
alignment with the restriction map in panel A of this figure. cDNA clones that extend further 5` have a solid
line immediately preceding the shaded box, and those that
were polyadenylated are followed by A.
When a
Northern blot was probed with the genomic HindIII fragment
mLIFR319, (Fig. 6A), a signal corresponding to the
10-kb mLIFR transcript was detected. This was evident on
poly(A) mRNA from brain and salivary gland (Fig. 7). There was a significant amount of smearing in the
liver and placenta lanes, which is possibly due to the presence of
repetitive sequences. When a Northern blot was probed with genomic HindIII restriction fragments mLIFR316 and mLIFR318 (Fig. 6A), no signal was detected (data not shown).
This suggested that mLIFR transcripts did not extend beyond the HindIII fragment mLIFR319 but that additional sites of
polyadenylation remained to be defined. To identify these cDNA
sequences corresponding to this region of genomic DNA we screened a
mouse liver cDNA library with genomic HindIII fragment
mLIFR319. Of the large number of hybridizing clones, six were analyzed
by sequencing and then aligned using the GCG DNA sequence analysis
programs Pileup and Wordsearch. Two of the six clones (mLIFR329 and
mLIFR330, Fig. 6B), were polyadenylated. Sequence
analysis of cDNA clone mLIFR329 revealed a canonical poly(A) signal.
Sequence comparisons with genomic DNA showed that this poly(A) signal
was located approximately 160 bp upstream of the second HindIII site (Fig. 6A) identified in genomic
DNA (Fig. 6B). cDNA clone mLIFR330 was also positioned
by sequence comparison with genomic DNA, and its 5` end was located
approximately 145 bp downstream of the second genomic BamHI
site (Fig. 6A). Thus these cDNA clones utilized
polyadenylation signals up to 6 kb downstream of the clones described
above. Additional cDNA clones overlapped these two regions (Fig. 6B). Because we have not identified full-length,
complete cDNA transcripts it was not possible to definitively assign
the different 3`-untranslated regions of these cDNA clones to
particular mLIFR transcripts. However, at least five different poly(A)
signal sequences were identified for the membrane-bound form of the
mLIFR transcript.
Figure 7:
Northern blot of poly(A) RNA (3 µg/lane) from various mouse tissues hybridized with
genomic HindIII fragment mLIFR319 (See Fig. 6). The
locations of RNA size markers (kb) are shown on the right.
Figure 8:
Detection of mLIFR mRNA expression and
soluble mLIFR protein during gestation. For detection of mLIFR mRNA
expression, Northern blots of poly(A) mRNA (3 µg)
from various samples of mouse liver, uterus, or placenta were
hybridized sequentially with a mLIFR cDNA probe and a
glyceraldehyde-3-phosphate dehydrogenase probe and exposed to a
phosphor screen. The radioactivity in each band hybridizing to the
mLIFR probe was quantified using Imagequant version 3.0 software
(Molecular Dynamics) and then normalized to the signal obtained with
glyceraldehyde-3-phosphate dehydrogenase. A, quantitative
analysis of Northern blot expression of mLIFR transcripts (10 and 3 kb)
in the livers of nonpregnant, pseudopregnant mice and in mice at
various stages of pregnancy. B, levels of soluble LIFR protein
in the serum of nonpregnant, pseudopregnant mice and mice at various
stages of pregnancy. Data represent the mean ± S.D. of at least
two experiments. C, quantitative analysis of Northern blot
expression of mLIFR transcripts (10, 3, 4.5, 5, 7, and 8.5 kb) in
placentae of mice at various stages of pregnancy compared with
nonpregnant uterus and the uterus from a pseudopregnant mouse. The
sample at day 8 of pregnancy included the embryos. D,
quantitative analysis of Northern blot expression of mLIFR transcripts
(10 and 3 kb) in the uteri of mice at various stages of pregnancy
compared with nonpregnant uterus and the uterus from a pseudopregnant
mouse.
The levels of the 3-, 4.5-, 5-, 7-, 8.5-, and 10-kb bands were also analyzed in the mRNA from placentae of mice at varying stages of pregnancy (Fig. 8C). The placental sample taken at day 8 of pregnancy included the embryos. Nonpregnant uterus is shown for comparison. There was an increase of 6-9-fold in the levels of all the mLIFR isoforms (from day 8 of pregnancy) until day 15 of pregnancy, after which the levels of all the isoforms declined. There was an increase in LIFR mRNA in uterine tissue (Fig. 8D), although it is possible that this was partly due to contamination with placental tissue.
Figure 9: Expression of mLIFR and detection of soluble mLIFR protein in LIF nullizygous mice. Detection of mLIFR mRNA expression in mice that were genotypically LIF -/-, +/-, or +/+ was carried out as described in the legend to Fig. 8. A, levels of soluble LIFR protein in the serum of male (M) or female (F) mice that were genotypically LIF -/-, +/-, or +/+. Data represent the mean ± S.D. of at least two experiments. B, quantitative analysis of Northern blot expression of mLIFR transcripts (10 and 3 kb) in the livers from male (M) or female (F) mice that were genotypically LIF -/-, +/-, or +/+. The relative order of the samples analyzed is identical to that described in panel A of this figure.
The low affinity LIF receptor participates in a number of cytokine receptor signaling systems, including those of LIF, ciliary neurotrophic factor, oncostatin-M, and cardiotrophin-1, which are involved in processes regulating the growth and development of neurons, hemopoietic precursors, hepatocytes, adipocytes, myoblasts, cardiac myocytes, and osteoblasts(36) . Membrane-bound receptors for LIF have been found on a wide range of mouse cells(37) , and a protein present at relatively high levels in mouse serum was shown to be a truncated form of the LIFR(15) . cDNA clones encoding a predicted membrane-bound form and also potentially encoding a soluble form of the mLIFR have now been isolated. In this study we show that there are specific transcripts for both the predicted soluble and membrane-bound mLIFRs, and that they are both expressed in a wide range of mouse tissues and several mouse cell lines. The two transcript forms were differentially expressed in the tissues examined. Furthermore, the expression pattern of membrane-bound mLIFR transcripts was found to be highly complex and variable between different tissues. The presence of LIFR transcripts in liver, brain, bone marrow, muscle, placenta, fat, kidney, and spleen are in agreement with the LIF responsiveness of these tissues(2) . LIFR transcripts in tissues such as lung, intestine, and pancreas may indicate further roles for LIF or an alternative LIFR-associated cytokine involved in the growth and differentiation of these tissues. The LIFR has been shown to play a critical role in many important physiological processes since targeted disruption of the LIF receptor gene in mutant animals resulted in defects in bone formation, placentation, metabolic disorders in the liver, reduced numbers of astrocytes in the brain, and failure to survive beyond the day of birth (20) .
Transcripts encoding
the membrane-bound mLIFR were heterogeneous, and their expression
pattern was complex. In this study, we have shown that the
heterogeneity can be accounted for, at least in part, by differences in
the lengths of the 3`-untranslated region of the transcripts. The
function of these transcripts is unclear, however, as they are all
predicted to give rise to identical proteins. In order to determine the
molecular basis governing the expression of the soluble and
membrane-bound forms of the mLIFR, we have analyzed the corresponding
genomic regions and have shown that the different forms of the mLIFR
are derived from the same genetic locus by differential splicing. The
membrane-bound form of the receptor is generated by an alternative
splicing event and skips an exon that contains sequences specific to
the soluble receptor. The 3`-untranslated region of the soluble mLIFR
transcript contained part of a mouse B2 repetitive
element(33) , which provided the polyadenylation signal. There
was no similar element present in the immediate 3`-untranslated region
of membrane-bound mLIFR cDNAs. There are many examples of B2 elements
in the vicinity of expressed genes, and there is evidence that the
presence of these elements and similar elements near a polymerase
II-transcribed gene can affect its levels of expression(38) .
The presence of a B2-like element in the mouse but not the hLIFR gene
provides an explanation both for the mechanism and high levels of the
soluble LIFR transcript in the mouse and the failure to detect these
transcripts in the human(3) . The 3`-untranslated region of the
membrane-bound mLIFR mRNA transcripts was compared with other known
transcripts by searching the GenBank data base.
Interestingly, in the region just after the translational termination
codon there was a high degree of homology with the hLIFR cDNA. This may
point to an important regulatory role for this region of DNA in the
transcription of membrane-bound forms of the LIFR, as it was not
homologous to the 3`-untranslated region of the soluble mLIFR. A 30-bp
region on the DNA minus strand in the 3`-untranslated region of a
membrane-bound mLIFR transcript described in this study was homologous
to the negative regulatory region of the mouse PCNA gene(32) .
The significance of this homology, however, is unclear.
The fibronectin (FN) type III domain is a 90-amino acid residue unit that was originally noted in fibronectin, where it is repeated 15 times (39) . Domains homologous to this repeat have now been shown to be a feature of many other proteins with protein-protein interaction functions including contactin, N-CAM, and fasciculin(40) . Members of the hemopoietin family of receptors including the receptors for growth hormone, prolactin, IL-5, and LIF were shown to contain at least one copy of an FNIII domain in their extracellular regions. In receptors such as that for growth hormone the FNIII domain contains residues that constitute the binding site for its cognate ligand. Receptors such as those for G-CSF, gp130, and LIF contain additional FNIII domains that are C-terminal to the ``binding'' domains(41) . FNIII domains are typically flanked by phase 1 introns, which are defined as falling between the first and second base of a codon(42) . This phasing arrangement has also been conserved in the FNIII exons of this section of the mLIFR. The FNIII domains in the region of the mLIFR gene described in this study are encoded by two exons. The cytoplasmic domain of the LIFR has been shown to contain two functional motifs, box 1 and box 2, which are involved in signal transduction(35) . These two motifs are conserved among cytokine receptors including those for granulocyte-macrophage colony-stimulating factor(43) , G-CSF, and gp130 (44) . Box 1 is generally encoded by a single exon located immediately downstream of the transmembrane domain and has a phase 2 intron on its 5` side and a phase 0 intron on its 3` side. This arrangement has been conserved in the cytoplasmic region of the mLIFR. This conservation of intron-exon arrangement supports the notion that the domains in this group of receptors have a common evolutionary origin(43) .
LIF is known to have a critical function in the implantation process, as mice nullizygous for the LIF gene are viable but female mice are infertile due to failure of implantation(21) . The levels of transcripts encoding the soluble mLIFR in the liver have been previously shown to increase during pregnancy, and they correlate with increasing levels of soluble LIFR present in the serum(14, 15, 16) . The results from this study confirm these findings and also show that the levels of mRNA transcripts for the membrane-bound mLIFR increase in the liver during pregnancy. Several other tissues were examined for differences in mLIFR mRNA expression during pregnancy, but the only tissues to show an increase were liver, placenta, and uterus.
A large number of soluble receptors for cytokines and growth factors have been described. Although the level of soluble mLIFR in normal mouse serum is 1-2 µg/ml and increases 20-25-fold during pregnancy, the physiological levels of most of these receptors is generally low (45) . Some soluble forms of receptors e.g. the IL-6 receptor (17) are able, when complexed to their respective ligands, to act as agonists on responsive cells, while soluble mLIFR seems to act as an antagonist to LIF function. The transient burst of LIF expression during the implantation process precedes the rise in soluble LIFR levels. We therefore reasoned that the elevated levels of soluble LIFR might be a physiological response to elevated levels of LIF. The maintenance of higher levels of soluble LIFR mRNA transcript and protein in mutant mice lacking a LIF gene indicates that the LIFR is not regulated directly in response to the amount of LIF present in the serum or other tissues. Rather, it appears that the soluble mLIFR is constitutively expressed even in LIF nullizygous animals and is regulated at the transcriptional level by additional signaling processes associated with pregnancy.