(Received for publication, December 19, 1996, and in revised form, January 29, 1997)
From the Department of Medicine, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York, New York 10461
Expression of the
asialoglycoprotein receptor by the human hepatocellular carcinoma cell
line HuH-7 in response to intracellular cGMP concentrations was
previously shown to be regulated at the translational level. In a
cell-free system, initiation of asialoglycoprotein receptor mRNA
translation was dependent on the presence of the 7-methylguanylate cap
site and was independent of 8-bromo-cGMP levels in which the cells were
grown prior to RNA isolation. Stable transfection of COS-7 cells with
deletion constructs of the asialoglycoprotein receptor H2b subunit
localized the cGMP-responsive cis-acting element to the mRNA
5-untranslated region (UTR). Addition of biotin (an activator of
guanylate cyclase) induced the expression of
-galactosidase present
as a chimeric plasmid containing the H2b 187-nucleotide 5
-UTR. An RNA
gel retardation assay identified a 37-nucleotide cognate sequence
within this 187-nucleotide region. Titration of the 5
-UTR with a
cytosolic fraction isolated from HuH-7 grown in the presence or absence
of 8-bromo-cGMP or biotin provided direct evidence for an RNA-binding
protein responsive to intracellular levels of cGMP. Based on these
findings, it seems reasonable to propose that reduction of
intracellular levels of cGMP by biotin deprivation results in a
negative trans-acting factor associating with the 5
-UTR of
asialoglycoprotein receptor mRNAs, thereby inhibiting
translation.
Regulated expression of cell-surface lectins has been implicated in such diverse processes as endocytosis, bacterial and viral infection, regulation of cell proliferation, homing of lymphocytes, and metastasis of cancer cells (1). The asialoglycoprotein receptor (ASGR)1 is the hepatocellular prototype of a cell-surface lectin responsive to the differentiated state of the liver cell (for review, see Ref. 2). In addition to being a model of receptor-mediated endocytosis (3), the presence of ASGR on hepatocytes provides a membrane-bound active site for cell-to-cell interactions (4, 5), has made possible the selective targeting of chemotherapeutic agents (6) and foreign genes (7), and has also been implicated as a site that mediates hepatitis B virus uptake (8).
A human hepatoma cell line (HepG2) has provided a convenient model to investigate ASGR biosynthesis. When HepG2 cells were grown to confluence in a minimal essential medium or in a chemically defined medium containing a variety of hormones and growth factors supplemented with dialyzed fetal bovine serum, expression of ASGR was reduced by 60-70% (9). The low molecular weight factor required for the restoration of ASGR expression was isolated, purified, and identified as biotin (10). Similar results were obtained with a second hepatocellular carcinoma cell line, HuH-7 (11), indicating that the effect was not cell line-specific. Though usually not considered as part of an induction pathway, the effects of biotin upon the steady state expression of ASGR could be mimicked by the addition of the second messenger 8-bromo-cGMP (8-Br-cGMP), and these additions were not additive (11, 12). This suggested that the effect of biotin may have been mediated through changes in the cGMP level via biotin activation of the membrane-associated guanylate cyclase (13).
Estimates of the steady state level of ASGR mRNA suggested that cGMP-regulated expression of ASGR was at the posttranscriptional level (11, 12). Polysome analysis of ASGR subunits H1 and H2 mRNAs indicated that the addition of 8-Br-cGMP caused a shift of ASGR mRNA from the ribonucleoprotein fraction into a translationally active membrane-associated polysomal pool. Although the biochemical mechanisms have not been determined, cGMP has been suggested to regulate the expression of other proteins at a translational level (13, 14) and has been shown to increase total protein synthesis in isolated hepatocytes (15).
In mammalian cells, translation of most mRNA species appears to
occur by the association of the preinitiation complex at or near the
7-methylguanylate cap structure at the 5-untranslated region (UTR) of
mRNA and scanning downstream to the site of protein synthesis
initiation (16). The 60 S ribosomal subunit is subsequently recruited
to the complex, and translation begins. Recovery of the ASGR mRNA
in the ribonucleoprotein fraction during biotin deprivation suggested
that intracellular levels of cGMP may play a significant role in
modulating the initiation phase of ASGR mRNA translation. The
bimodal polysomal distribution of ASGR mRNA was characteristic of a
class of mRNAs that were inefficiently translated (17). Current
evidence suggests that mRNAs in these functionally distinct
fractions differ structurally or through the proteins they interact
with (18). Within this group of mRNAs, most interactions between
RNA and cytosolic proteins were defined by motifs localized to the
5
-UTR (19).
In the present study, the potential role of the 5-UTR as the
cis-acting element governing the cGMP-modulated expression of ASGR was
established. In vitro transcription coupled with an RNA gel
retardation assay defined the cis-acting element within a 37-nucleotide
region. In addition, the effective concentration of a cytoplasmic
protein trans-acting fraction was shown to be responsive to cGMP
and biotin deprivation.
The 5 and 3
-UTR regions of the H2b
cDNA of ASGR were deleted using polymerase chain reaction to
introduce unique restriction sites (XbaI, 6 nucleotides
upstream of ATG translation start site or BamHI, 9 nucleotides downstream of the translation stop site). The resulting
constructs were subcloned into either pcDNA3 for selection of
stable transfectants in COS-7 or pGEM-4Z for in vitro transcription. The 5
-UTR of the ASGR H2b was prepared by polymerase chain reaction with the addition of HindIII ends and cloned
into a
-galactosidase reporter vector (pSV-
-galactosidase,
Promega) 296 nucleotides upstream of the lacZ coding region
start site. The nucleotide sequences of the polymerase chain
reaction-generated 5
and 3
inserts were confirmed by the dideoxy
chain termination method with Sequenase (DNA Sequence Facility, Albert
Einstein College of Medicine).
COS-7 and HuH-7 cells were cultured in Eagle's minimum
essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) or dialyzed FBS with or without 8-Br-cGMP or biotin. Stable
transfectants of COS-7 cells (20) resistant to 400 µg/ml G418 were
subcloned, and those with the highest level of ASGR expression in MEM + 10% FBS as determined by Western blot (21) were utilized. Transient transfection of HuH-7 with the chimeric plasmid was mediated by LipofectAMINE (Life Technologies, Inc.) following the manufacturer's instructions. 5 hrs after transfection, cells were harvested by trypsinization and replated at a 1:3 ratio in MEM supplemented with
10% dFBS. 24 hrs later, the medium was changed to MEM + 10% dFBS with
or without 107 M biotin. Three days after
transfection, cells were harvested, and
-galactosidase activity was
measured following the manufacturer's instructions (Invitrogen).
Near-confluent HuH-7 cultures (1 × 106 cells) were
labeled with 200 µg/ml [35S]Met/Cys (Pro-mix, Amersham
Corp.) for 1 h, followed by a 2-h chase. ASGR was
immunoprecipitated, resolved on 10% SDS-PAGE, and the gel was
processed for fluorography. Western blot and immunoprecipitation protocols as well as antibodies used in this study have been previously described (11).
Total cytoplasmic RNA was isolated
by extraction with guanidine thiocycanate (22) from approximately
5 × 107 cells (HuH-7 or transfected COS-7). For
Northern blot analysis, RNA samples were resolved on 1%
agarose-formaldehyde gels and transferred to Nytran membrane. The blots
were hybridized at high stringency with random prime-labeled
([-32P]dCTP) H1- and H2-specific probes (12, 23).
Equal loading and transfer of RNA was verified by staining the membrane
with methylene blue.
Total RNA was isolated from HuH-7 cells grown in MEM supplemented with 10% FBS, 10% dFBS, or 10% dFBS plus 1.0 mM 8-Br-cGMP. The translation reaction was performed using a rabbit reticulocyte lysate as described by the manufacturer (Promega) in the presence or absence of 2 µg/assay of m7GpppG, an inhibitor of cap site recognition. Following a 90-min incubation at 30 °C, the [35S]-labeled (Pro-mix, Amersham Corp.) ASGR translation products were recovered by immunoprecipitation using a polyclonal antibody to affinity-purified human receptor (11). Samples were resolved on 10% SDS-PAGE, and gels were prepared for fluorography.
Gel Retardation AssayA nested set of RNA probes were
prepared by linearization of the full-length H2b cDNA using
restriction sites localized in the 5-UTR as templates for in
vitro transcription from the Sp6 promoter of pGEM-4Z. The
full-length (187-nucleotide) fragment was labeled by the incorporation
of [
-32P]UTP during transcription. The gel retardation
assay was adapted from that described by Leibold and Munro (24). HuH-7
cells were homogenized, the cytosol (S-100) was prepared by
centrifugation at 100,000 × g for 1 h, and the
aliquots were stored at
135 °C. S-100 was preincubated at 0 °C
for 10 min in assay buffer with a 100-fold molar excess or without
unlabeled transcripts prior to the addition of the labeled
187-nucleotide transcript, and incubation continued for an additional
10 min. Inclusion of proteinase K (100 µg/ml) in the assay mixture
completely abolished this protein-dependent assay. The
mixture was resolved on a 4% low cross-linked PAGE and prepared for
autoradiography.
Based on our
previous findings that biotin was required for expression of ASGR by
HepG2 and HuH-7 cell lines, we proposed that the mechanism of biotin
regulation was mediated by maintaining the intracellular level of cGMP
via the activation of guanylate cyclase (11). Our original experimental
protocol was modified from the steady state determination of receptor
concentrations via Western blot (11) to the measurement of biosynthesis
rate by pulse labeling with [35S]Met/Cys. This change in
protocol allowed the reduction of 8-Br-cGMP and atrial natriuretic
factor (ANF) concentrations used in the present experiments to the
level previously employed by others in short term protocols in cultured
or isolated hepatocytes (25-29). HuH-7 cells were grown to
near-confluence in MEM supplemented with either 10% FBS or 10% dFBS
to which 10 to 1000 µM 8-Br-cGMP, 10 nM ANF,
or 100 µM sodium nitroprusside (SNP) (shown to produce nitric oxide (25), an activator of soluble guanylate cyclase (29)) were
added (Fig. 1A). Within 1 h of the
addition of 500 µM 8-Br-cGMP and activators of both the
particulate (ANF) and soluble (SNP) guanylate cyclases (25-29), the
biosynthetic rate of ASGR was increased by 6.7-, 8.3-, and 4.2-fold,
respectively, when compared with untreated cells in dFBS alone. No
difference in the abundance of specific mRNAs was detected by
Northern blot analysis, regardless of whether cells were maintained in
MEM supplemented with FBS or dFBS with or without 8-Br-cGMP or its
inducers (Fig. 1B). These results strongly support our
hypothesis that intracellular levels of cGMP regulate the expression of
the ASGR at a posttranscriptional level. When taken together with our
earlier studies (11, 12), these results clearly demonstrated that the
molecular level of control was translational.
In Vitro Translation of ASGR
In mammalian cells, translation
of most mRNAs appears to occur by association of a preinitiation
complex at a 7-methylguanylate cap site and subsequent scanning to the
translation initiation site (16). To establish the cap site status of
ASGR mRNA in cells grown in FBS as compared with dFBS and dFBS
supplemented with 500 µM cGMP, the extent of
cap-dependent in vitro translation was
determined. Total mRNA was isolated from HuH-7 cells (5 × 107) and translated in a rabbit reticulocyte lysate in the
presence or absence of m7GpppG, an inhibitor of
cap-dependent initiation (30). The labeled ASGR translation
product was recovered by immunoprecipitation using a polyclonal
antibody to affinity-purified human receptor. Resolution on 10%
SDS-PAGE and subsequent fluorography indicated that inclusion of
m7GpppG reduced translation of ASGR mRNA isolated from
both control and biotin-deprived cells with or without 8-Br-cGMP to an
equal extent (>90%) (Fig. 2). These results indicated
that initiation of ASGR mRNA translation was
cap-dependent and that addition of a 7-methylguanylated cap
to ASGR mRNA was independent of biotin deprivation.
Localization of the Cis-acting H2 mRNA Cognate Sequence
To localize the cis-acting element, mutated H2 cDNAs
from which the entire 5 or 3
-UTR was deleted were constructed by
polymerase chain reaction amplification. These constructs, along with
the full-length H2 cDNA, were cloned into the eukaryotic expression vector pcDNA3 carrying a neomycin resistance gene. Stable
transfectants of COS-7 cells were selected with 400 µg/ml G418. As
shown in Fig. 3, deletion of the 5
-UTR resulted in loss
of the cGMP requirement for H2 expression. In contrast, deletion of the
3
-UTR was without effect. These results indicated that the
cGMP-responsive element was located in the 5
-UTR of the H2 mRNA.
Northern blot analysis indicated that there was no significant
difference in mRNA levels to account for this differential response
to biotin deprivation or supplementation with 8-Br-cGMP (Fig. 3),
supporting translational regulation in the transfected COS-7 cell
lines.
Transient transfection of HuH-7 with the chimeric plasmid confirmed
that the putative cis-acting element was localized within the 5-UTR
(Table I). Addition of biotin to the culture medium resulted in a two-fold increase in
-galactosidase activity.
Interestingly, the presence of the 5
-UTR in the plasmid reduced
-galactosidase expression when compared with the original or a
chimeric plasmid in which the 5
-UTR was inserted in the antisense
orientation by almost 35%, even when biotin was added. This finding
was consistent with the reduced level of H2b translation when compared
with the H1 ASGR subunit under normal physiologic conditions (2).
|
The 5-UTR (187-base pair) cDNA
fragment of the H2b of ASGR was directionally cloned into pGEM-4Z
vector for the generation of a nested set of 5
-UTR mRNA fragments
by in vitro transcription for an RNA-protein binding assay
(Fig. 4). RNA fragments of the 5
-UTR were added in
100-fold molar excess prior to the addition of the full-length
187-nucleotide-labeled RNA probe and resolution on 4% PAGE. As
illustrated in Fig. 5, the failure of the
Sp6-FokI transcript to inhibit the band shift assay
indicated that a cognate sequence lies between 70 and 110 nucleotides
relative to the Sp6 promoter. Since translational regulation due to
protein-protein interactions between two regions of a transcript has
been reported (33), a potential and equally critical role for the
upstream 1-70-nucleotide (Sp6-FokI) region cannot be
eliminated by the present study. Failure of the 208-nucleotide 3
-UTR
fragment of the H2 mRNA and the glyceraldehyde-3-phosphate
dehydrogenase open reading frame mRNA to inhibit the gel
retardation assay further supported the specificity of this assay (Fig.
5B).
As shown in Fig. 6, incubation of the 187-nucleotide
5-UTR probe with increasing amounts of the S-100 fraction isolated
from HuH-7 cells grown to confluence in MEM supplemented with FBS, dFBS + 1.0 mM 8-Br-cGMP (conditions required for normal ASGR
expression), or dFBS alone showed a concentration-dependent
gel retardation. As the concentration of S-100 protein was increased to
1 µg/assay, it became evident that the S-100 fraction isolated from
HuH-7 cells grown in dFBS had a higher effective concentration of
RNA-binding protein than cells grown in FBS and that inclusion of
8-Br-cGMP to the cell culture reduced the concentration to the control
level. This quantitative (as opposed to a qualitative difference) was consistent with the bimodal distribution of ASGR mRNA between the
translationally active polysomes isolated from cells grown in FBS and
the shift to the repressed state when cells were grown in dFBS (11,
12).
In mammalian cells, translation of most mRNA species appears to occur by the association of a preinitiation complex at a 7-methylguanylate cap site and subsequent scanning downstream to the site of protein synthesis initiation (16, 31). Our studies showed that cap site addition to ASGR mRNA was independent of biotin deprivation (Fig. 2). However, the recovery of the ASGR message in the ribonucleoprotein fraction during biotin deprivation (11, 12) suggested that intracellular levels of cGMP play a significant role in modulating the initiation phase of translation (31).
Perhaps the best-defined example of translational regulation is that of
ferritin synthesis in iron-deficient cells (16, 30). Analysis of the
cis-acting mRNA sequences led to the definition of the
iron-responsive element with a putative stem-loop structure in the
5-UTR (30). Modeling of the ASGR H2b 5
-UTR indicated the presence of
two potential regions of secondary structure. The free energy levels
(
7.4 and
8.5 kcal/mol) of these two stem-looped regions was far
below that usually considered necessary to prevent recognition of a cap
site (
50 kcal/mol). However, they might provide the loop structure
necessary for specific recognition by exposing the RNA backbone and
bases to interaction with protein groups (32, 33).
In the absence of a highly ordered 5-UTR stem-loop structure,
translation may be regulated by a short linear sequence (16). A highly
conserved CCAUCNN sequence localized within the 5
-UTR of both ASGR
subunit mRNAs isolated from either human or rat has been identified
as a conserved RNA-binding protein cognate sequence within the 5
-UTR
of ornithine decarboxylase (34). The presence of this conserved
sequence within the putative cis-acting element as indicated by gel
retardation assay (Fig. 5) supports the possibility that it may serve
as a recognition motif for the cGMP responsive trans-acting factor.
One plausible explanation for cGMP-regulated expression of ASGR would be modulation of a trans-acting factor phosphorylation status. Although the modulation of ASGR by cGMP was not liver-specific (Fig. 6), it should be viewed in the context of the original finding in HepG2 cells (11, 12). Since there was little, if any, cGMP-dependent protein kinase detected in hepatocytes (35, 36), the classic cGMP signal transduction pathways mediated by cGMP-dependent protein kinase was presumed to be absent in liver cells (36). Therefore, if a phosphorylation/dephosphorylation signal transduction pathway was involved in translational regulation of ASGR expression, one of the cGMP-binding phosphodiesterases (PDEs) would be the most likely effector target. As opposed to cGMP-dependent protein kinase, the various cGMP PDEs are regulated allosterically by the binding of cGMP to noncatalytic binding sites (37). Modulation of cGMP levels can either inhibit or stimulate PDE hydrolytic activity, increasing or decreasing intracellular cGMP itself or cAMP (38). Indeed, a number of recent studies have suggested that cGMP-stimulated PDE may play a central role in regulating the intracellular concentrations of cAMP (39, 40). Based on our previous findings that increased levels of cAMP resulted in the down-regulation of ASGR (41), induction of a cGMP-stimulated PDE resulting in a protein dephosphorylation via reduction of cAMP is a reasonable mechanism for cGMP-regulated expression of ASGR.
Presently, it may be premature to speculate that changes in the
phosphorylation state of any protein via PDE mediates the effects of
cGMP on translation of ASGR, especially in the light of the recent
discovery of new types of cyclic nucleotide receptors that include
other non-catalytic sites (37). Our understanding of the potential cGMP
cascade in liver is still in its infancy, and there may yet be other
schemes to account for cGMP action, such as Ca+2 ion flux
or induction of inositol triphosphate (42, 43). Whatever the
biochemical mechanism of the cGMP action may be, it is reasonable to
speculate that in the absence of cGMP, a negative trans-acting factor
associates with the 5-UTR of the ASGR mRNA, thereby inhibiting
translation. Purification of the ASGR mRNA-binding protein should
provide new insight into the physiologic affect and molecular target of
cGMP in the hepatocyte.