Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Biotin regulation of
asialoglycoprotein receptor expression and insulin receptor activity
has been established in two human hepatoblastoma cell lines, Hep G2 and
HuH-7. Second messenger cGMP mimics the effect of biotin on
asialoglycoprotein receptor expression at the translational level.
Metabolic labeling and subsequent immunoprecipitation indicate that the
loss of insulin receptor activity during biotin deprivation was due to
suppression of receptor synthesis. Evidence for posttranscriptional
regulation of insulin receptor synthesis was provided by rapid biotin
induction of receptor synthesis without an increase in gene transcript
number. Addition of a cGMP-dependent protein kinase (cGK) inhibitor
prevented biotin induction of the insulin and asialoglycoprotein
receptors, suggesting that protein phosphorylation propagates the cGMP
signal transduction cascade. Coatomer protein COPI was recently
identified as the trans-acting factor that regulates in
vitro translation of the asialoglycoprotein receptor. Biotin repletion
of the culture medium resulted in the hyperphosphorylation of -COP,
which was prevented by simultaneous addition of the cGK inhibitor.
These findings suggest that the end point of this cGMP signal cascade is modulated by cGK and that a phosphorylation reaction governs the
expression of both receptor proteins.
biotin induction; translational regulation; coatomer protein phosphorylation
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INTRODUCTION |
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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 (31). The asialoglycoprotein receptor (ASGR) is the hepatocellular prototype of a cell surface lectin that reflects the differentiated state of the liver cell (20, 32, 37). Expression of ASGR was reduced by 60-70%, and the binding of 125I-labeled insulin was reduced by >75% in human hepatoblastoma cell lines Hep G2 or HuH-7 grown to confluence in a minimal essential medium (MEM) supplemented with dialyzed fetal bovine serum (dFBS) (9). In contrast to the dramatic effect on receptor expression, the patterns of 35S-methionine-labeled cellular proteins resolved by two-dimensional electrophoresis were remarkably similar, as were the patterns of secreted glycoproteins isolated by solid-phase concanavalin A (10).
Reconstitution of dFBS supplemented medium with a 300- to 350-Da ultrafiltrate of FBS fully restored the expression of ASGR and insulin receptor (IR) (8). The low-molecular-weight factor required for restoration was identified as biotin (33), a water-soluble vitamin that acts as a cofactor of glucose and fatty acid biosynthesis. Although it is not usually considered part of a signal transduction pathway, biotin's effect on the steady-state expression of ASGR polypeptides could be mimicked in a nonadditive fashion by addition of the second messenger 8-bromo-cGMP (8-Br-cGMP) (33). This suggested that the effect of biotin was mediated through changes in cGMP levels via activation of guanylate cyclase (8, 39). Consistent with this hypothesis were the recent observations that the addition of atrial natriuretic factor or sodium nitroprusside, activators of particulate and soluble guanylate cyclase, resulted in normalization of ASGR biosynthesis (35). Although biotin induction of cGMP was indicated, it was not clear which of the potential cGMP target proteins propagated the cGMP signal transduction cascade: cGMP-dependent phosphodiesterases (activation or inhibition), cGMP-gated ion channels, or cGMP-dependent kinases.
Estimates of the steady-state levels of ASGR subunits H1 and H2 mRNAs
indicated that cGMP-regulated expression of the ASGR was at a
posttranscriptional level. Resolution of ASGR mRNA on sucrose gradients
demonstrated that the addition of cGMP shifts these mRNAs from the
ribonucleoprotein fraction into a translationally active
membrane-associated polysomal pool (9). These findings suggest that cGMP directly affects the translational regulation of both
H1 and H2, as opposed to mediating an alteration in their intracellular
processing. Gel shift assays indicated a specific cytoplasmic protein
interaction with the 5'-untranslated region (UTR) of the ASGR mRNA
(35). Protein purification led to the isolation of a
fraction highly enriched in RNA binding activity and the coatomer
protein COPI (14), a complex of seven unrelated subunits
(30). Northwestern analysis coupled with peptide sequence identified -COP as a potential RNA binding protein within this fraction. Antibody-induced RNA supershift confirmed
-COP as the trans-acting factor and indicated that
-COP was also part
of the protein-RNA complex (14).
In the present study, we demonstrate that the biotin-dependent loss of cell surface insulin binding was due to a reduction of IR polypeptide synthesis and that, as was the case for ASGR, repletion of the dFBS-supplemented medium with biotin fully restored IR expression. The absence of a significant difference in IR mRNA abundance, regardless of whether cells were maintained in MEM supplemented with FBS, dFBS, or dFBS plus biotin, suggests that this change in IR expression was regulated at a posttranscriptional level. In addition, cGMP-dependent kinase (cGK) was identified as a common downstream element in the biotin-induced signal transduction pathway, regulating both ASGR and IR expression. This, to our knowledge, is the first demonstration that activation of cGK by biotin regulates gene expression at the posttranscriptional level.
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EXPERIMENTAL PROCEDURES |
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Cell culture. The human hepatoblastoma cell line HuH-7, previously shown to require biotin for full expression of ASGR (34), was plated from confluent cultures onto 60-mm dishes (Falcon) in MEM containing 100 mg/dl glucose supplemented with 10% dFBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. This plating protocol initially deprived all cells of biotin. Medium was changed 24 h after plating to establish the various test conditions. Cells were allowed to grow to near confluence before the application of various experimental protocols.
Western blot.
Cells were suspended in lysis buffer (50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 30 µl/ml aprotinin from Sigma, A-6279,
and 0.1 mM phenylmethylsulfonyl fluoride) and passed 20 times through a
21-gauge needle. After centrifugation at 15,000 g at 4°C
for 10 min, the supernatants were used for protein assay and Western
blot. Forty micrograms of protein per lane from cell lysates were
resolved on 10% SDS-PAGE and transferred to nitrocellulose membrane.
The membrane was blocked for 1 h at room temperature in
Tris-buffered saline (TBS)-Tween buffer (150 mM NaCl, 50 mM Tris · HCl, pH 7.8, and 0.1% Tween 20) containing 10%
fat-free milk. The membrane was incubated for 1 h at room
temperature in either rabbit anti-human ASGR antibody diluted 1:5,000,
rabbit anti-human - and
-subunit specific IR (Upstate
Biotechnology) diluted to 1 µg/ml, mouse anti-human transferrin
receptor (TfR) (Zymed) diluted 1:2,000, or rabbit anti-
-COP (kindly
provided by Dr. Cordula Harter, Heidelberg University) diluted 1:1,000 in TBS-Tween containing 2% fat-free milk and processed for
chemiluminescence as previously described (8, 10).
Immunoprecipitation of IR and ASGR.
Cells were metabolically labeled with 200 µCi/ml of
[35S]Met-Cys (Pro-mix, Amersham) for 15 min in the
presence or absence of biotin (107 M) for an increasing
period of time as previously described (8, 10). Labeled
cells were washed with ice-cold 0.05 M PBS, pH 7.4, and harvested by
scrapping with a rubber policeman into 1 ml of PBS followed by
centrifugation at 1,000 g for 5 min at 4°C. The cell
pellet was resuspended in lysis buffer (described above) and incubated
at 4°C with constant mixing for 30 min followed by centrifugation at
10,000 g for 10 min. ASGR and IR were immunoprecipitated from aliquots of the supernatant containing equal amounts of
radiolabeled protein. The protein A/G ultralink (Pierce) recovered
immune complex was resolved on a 4-20% gradient SDS-PAGE, the
fixed gel was processed for fluorography with Amplify (Amersham Life
Science) and, after drying, was exposed to BioMax film with an
intensifying screen (Eastman Kodak) at
70°C. The extent of ASGR and
IR synthesis during the 15 min of labeling was quantified by
densitometric scanning of the exposed film.
Phosphate labeling and immunoprecipitation of COPI.
HuH-7 cells were incubated in phosphate-free MEM supplemented with 10%
dFBS with or without 10 µM
Rp-8(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate, a cGK inhibitor obtained from BioLog for 1 h
before labeling. Cells were treated with biotin (107 M)
or 8-Br-cGMP (1 mM) in the presence or absence of the inhibitor during
the 1 h of labeling with 200 µCi/ml
[32P]orthophosphate. Antisera to the
-COPI subunit was
added to cell lysates containing equal amounts of
32P-labeled proteins as determined by trichloroacetic acid
precipitation. To selectively immunoprecipitate the
-COP subunit,
anti-
-COP (kindly provided by Dr. Cordula Harter, Heidelberg
University) was used in high detergent concentration to dissociate the
COPI complex (24). Okadaic acid (1 µmol) (Sigma) was
added to the lysis buffer. The immunoprecipitated protein was
transferred to nitrocellulose and exposed to X-OMAT AR film with an
intensifying screen (Eastman Kodak) at
70°C.
Northern blot analysis.
Samples and gels were prepared according to protocols described
previously (42). Hybridization was carried out using
Sigma's PerfectHyb Plus buffer following manufacturer's instructions. [32P]dCTP-labeled probes used pT2IR (human insulin
receptor in pTZ19U vector kindly provided by Dr. Paul Pilch, Boston
University), ASGR H2b subunit, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). RNA abundance was quantified by densitometric
scanning of the exposed film.
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RESULTS |
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In conjunction with our previous finding that biotin was required
for maximum ASGR expression by Hep G2 and HuH-7 cells, a dramatic
reduction of insulin binding to the cell surface was also observed when
cells were grown in media deficient in biotin (i.e., supplemented with
dFBS) (34). To determine whether the effect of biotin
deprivation directly altered peptide-hormone binding or affected the
steady-state level of the IR expression, HuH-7 cells were grown to near
confluence in MEM supplemented with FBS, dFBS, or dFBS plus biotin
(Fig. 1). Consistent with our earlier
observations, Western blot analysis confirmed that ASGR expression was
markedly reduced in cells grown in MEM supplemented with dFBS and that
addition of biotin (107 M) fully restored
ASGR expression to the control level. As was the case for ASGR, when
cells were grown in medium supplemented with dFBS, the steady-state
level of IR expression was markedly reduced, and addition of biotin to
the culture medium fully restored IR polypeptide expression to the
level exhibited by cells grown in FBS. The reduction in the
steady-state level of IR exhibited by cells grown in dFBS was
sufficient to account for the 75% loss of cell-surface insulin binding
activity previously observed (8).
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In contrast to the specific requirement of ASGR and IR, the level of steady-state expression of the TfR was independent of biotin addition to the culture medium (Fig. 1). The lack of a TfR response to biotin was in line with earlier studies that indicated biotin deprivation did not significantly affect either total cellular protein synthesis or the overall pattern of 35S-labeled proteins resolved by two-dimensional electrophoresis (8, 10). Expression of characteristic TfR by cells grown in medium supplemented with dFBS suggests that biotin deprivation did not induce a global defect in protein glycosylation, which has the potential to reduce IR subunit processing and its ultimate expression at the cell surface (19).
Biotin is not usually considered part of an induction pathway, but its effect on the steady-state expression of ASGR can be mimicked in a nonadditive fashion by the second messenger cGMP and known activators of guanylate cyclase (33), such as atrial natriuretic factor and NO generating sodium nitroprusside (39). To determine whether the effect of biotin deprivation on IR expression can be reverted by cGMP, dFBS-containing media was supplemented with the membrane-permeable cGMP analog (8-Br-cGMP). Consistent with our earlier findings, the addition of 8-Br-cGMP fully substituted for the biotin requirement necessary for ASGR expression (33) (Fig. 1). The maintenance of normal IR expression by addition of 8-Br-cGMP to the culture medium (Fig. 1) suggests that a common or parallel signal transduction pathway exists for the induction of these two receptors.
The rate of biotin induction of ASGR and IR synthesis was determined by
immunoprecipitation of metabolically labeled receptor proteins. HuH-7
cells were labeled for 15 min with 35S-Met-Cys after
exposure to biotin for increasing amounts of time (Fig.
2). Due to the short labeling period,
only the pro-form of IR resolving at 190 kDa was detectable in the
immunoprecipitate. Consistent with the results obtained by immunoblot
(Fig. 1), biotin deprivation reduced ASGR and IR synthesis by >80%
compared with control cells maintained in medium supplemented with 10%
FBS. Within 15 min of biotin addition, the rate of ASGR synthesis
reached 87% and IR reached 72% of the FBS control. By 30 min, the
rate of ASGR synthesis was equal to that of the control cells, and the
rate of IR synthesis was just below the control value (97 and 84%,
respectively). No further increase in synthetic rate for either
receptor was observed, even 2 h after biotin addition (data not
shown). These findings support our original observation that the
reduction of specific plasma membrane protein expression due to biotin
deprivation was a direct consequence of peptide synthesis inhibition,
not accelerated degradation.
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To determine whether the induction of IR synthesis in the presence of
biotin was reflected by an increase of gene transcript number, the
level of IR mRNA was estimated by Northern blot analysis (Fig.
3). No significant difference in the
abundance of ASGR (1.35 kb) or the IR (11 and 8.5 kb) transcripts were
apparent, regardless of whether cells were maintained in MEM
supplemented with FBS, dFBS, or dFBS plus biotin. In contrast to the
four IR transcripts (11-kb major and 8.5-, 7.0-, 2.8-kb minor)
detectable in Hep G2 cells grown in medium containing a high glucose
concentration (450 mg/dl), when Hep G2 cells were grown in a more
physiological glucose concentration (100 mg/dl), as is necessary to
demonstrate the independent effect of biotin (41), only
the major 11-kb transcript was distinctly evident (3).
Expressions of IR transcripts by HuH-7 cells appear to respond to
glucose concentrations in a like manner. An extended exposure of the
Northern blot (2 wk) was required to detect the 8.5-kb IR transcript,
and, even after this long exposure, the 7.0- and 2.8-kb transcripts
were not evident. The dramatic decrease in the expression of the minor
transcripts in response to lower glucose concentrations is consistent
with previous findings in a wide variety of cell lines
(16). The lack of induction of the minor transcripts
points to biotin-regulated IR expression being independent of
carbohydrate metabolism.
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Induction of ASGR and IR by cGMP could, in principle, be mediated by
three different proteins: cGK (17), cGMP-gated ion channels (2), and cGMP-regulated phosphodiesterases
(1). To differentiate between the cGMP targets, HuH-7
cells were preincubated with a specific cGK inhibitor,
Rp-8(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Rp-cGMPS) (38) before
biotin and [35S]Met addition. After 15 min of metabolic
labeling, the cells were harvested, and the extent of ASGR and IR
synthesis determined by immunoprecipitation. As shown in Fig.
4, the addition of Rp-cGMPS (10 µM) during metabolic labeling inhibited biotin induction of both
receptor proteins by over 85%.
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Transfection studies with various deletion constructs of the cDNA
encoding the ASGR H2b subunit localized the cGMP responsive cis-acting element to a 187-nucleotide fragment of the mRNA
5'-UTR (14, 35). With a gel-shift assay, titration of the
5'-UTR with a cytosolic fraction isolated from HuH-7 cells grown in the presence of Br-cGMP or biotin provided direct evidence for an RNA-binding protein responsive to intracellular levels of cGMP (35). Recently, -COP, one of the seven unrelated
subunits of the coatomer protein COPI, was identified as the
trans-acting factor bound to the 5'-UTR responsible for in
vitro translational regulation of ASGR (14). To assess a
potential link between cGK activity and the trans-acting
factor, the phosphorylation status of
-COP following biotin or
Br-cGMP addition was determined. As illustrated in Fig.
5A, the addition of biotin or
Br-cGMP resulted in hyperphosphorlyation of
-COP, which was markedly
inhibited by the simultaneous addition of the cGK inhibitor
Rp-cGMP. To assure that biotin deprivation or treatment with
cGK inhibitor had no effect on the expression of
-COP, the
steady-state level of the coatomer subunit polypeptide was determined
by Western blot analysis. As shown in Fig. 5B, the
expression of
-COP was unaffected by the various growth conditions
after 24 h of treatment. On the basis of its reported turnover
rate (25), had biotin deprivation or Rp-cGMPS
inhibited
-COP synthesis, a 50% reduction in expression would have
been expected.
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DISCUSSION |
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Biotin is known to regulate the expression of hepatic proteins at both the transcriptional (7, 12) and translational levels (8). ASGR is the hepatocellular prototype of a cell surface glycoprotein that responds to a biotin-initiated signal transduction cascade. In conjunction with our previous finding that biotin was required for maximum ASGR expression by Hep G2 and HuH-7 cells, a dramatic effect on insulin binding to the cell surface was also observed (10). In the present study, we demonstrated that the loss of insulin binding activity as a result of biotin deprivation was due to a reduction in the steady-state level of IR and that addition of the second messenger cGMP prevented this loss (Fig. 1). In contrast to the many effects of biotin on hepatic enzyme expression thought to be secondary to glucokinase induction (26), this appears not to be the case for ASGR and IR due to the rapidity of the response to biotin addition (Fig. 2). The over sixfold induction of IR synthesis in the absence of an increase in gene transcript number (Fig. 3) supports posttranscriptional regulation of IR expression by cGMP. In the case of ASGR, similar findings initiated a line of investigation that ultimately led to the establishment of cGMP via biotin activation of guanylate cyclase as a regulator of translational regulation (14, 30, 35). In the absence of more rigorous proofs, we cannot yet confirm translational regulation of IR synthesis.
To date it has not been resolved which cGMP target protein or proteins
mediate the downstream effect of biotin. To differentiate between these
cGMP enzyme targets, cells were preincubated with a specific cGK
inhibitor before biotin induction and metabolic labeling (Fig. 4).
Rp-cGMPS inhibition of ASGR and IR synthesis pointed to cGK
as the mediator of the cGMP cascade. Two types of cGK have been
identified: cGK I, consisting of - and
-isoforms (5), and the membrane- associated cGK II (13,
23). Originally thought to be absent from hepatocytes
(22), cGK II mRNA has been detected in the Hep G2 cells
(29), the hepatoblastoma cell line in which cGMP
regulation of ASGR mRNA translation was originally demonstrated
(33, 35). Although it is difficult to distinguish between
the cGK isoforms, the cGK II isoform has been suggested to be more
sensitive than either of the cGK I isoforms to the chlorophenylthio
analog of the Rp-cGMPS inhibitor (13, 38). Although when purified from a baculovirus/Sf9 expression system, all
three isoforms have similar inhibition constants for the
chlorophenylthio analog (Ki = 0.5-0.6
µM) (27); in intact cells, other variables such as
intracellular localization and abundance of the kinase isoforms may be
more significant than the concentration of inhibitor necessary to
prevent biotin induction (23). With this caveat in mind,
the relatively low concentration of Rp-cGMPS (10 µM) necessary to block biotin induction of IR and ASGR expression compared
with the 200 µM needed to substantially inhibit calcium flux in
hepatocytes (28), a process usually associated with cGK I
(23), suggests that cGK II may be the cGMP target protein.
Unlike previous examples of cGK gene regulation at the transcriptional
level (11, 15), our data suggest that a phosphorylation step mediated by cGK regulates IR and ASGR synthesis at a
posttranscriptional level. Whether cGK is the end point of the cGMP
cascade is still an open question. There may be additional steps yet to
be resolved within the cGMP cascade regulating posttranscriptional
expression of ASGR and IR. For example, an interrelationship between
cGK activity and the Ras/MAP pathway at the level of c-Raf kinase phosphorylation and inhibition of MAP kinase phosphatase 1 expression was demonstrated in baby hamster kidney cells (36). More
recently, it has been shown that NO activation of the extracellular
signal-regulated kinase (ERK) pathway is dependent on the
production of cGMP and that cGK plays a role in the propagation of this
signal transduction cascade (18). In addition, cGK has
been implicated in the activation of p38 MAPK (4),
required for the translational regulation of TNF- (21).
The potential sites of cGK-mediated activation are not limited to
direct phosphorylation reactions. cGK has been shown to suppress
thrombin-stimulated phosphatidylinositol 3,4,5-trisphosphate [PtdIns
(3,4,5)P3] production and Ca elevation, two of the most common signal transduction elements in the cell. Resolution of our
findings at the biochemical level holds the promise of uncovering the
mechanism for discriminatory mRNA translation and the molecular targets
for the cGMP signal transduction cascade in liver.
In a previous study, gel-shift analysis using partially purified COPI
coatomer suggested that -COP was the most likely candidate for RNA
recognition. However, that result did not exclude the possibility of a
coatomer accessory molecule as the primary respondent to intracellular
cGMP. Interestingly, PtdIns(3,4,5)P3 has recently been
shown to specifically interact with
-COP (6). As a
result of the present findings (Fig. 5), we postulate that
phosphorylation of
-COP in response to cGMP induction of cGK
prevents high-affinity binding of the coatomer complex to the 5'-UTR of
the ASGR mRNA, thereby allowing ribosomal scanning to the site of
translation initiation. In an analogous reaction, cGK phosphorylation
of splicing factor 1 has recently been shown to prevent high-affinity
binding to U2AF65 crucial to the formation of the spliceosome with
snRNA (40). Unlike ASGR, there is no evidence for
-COP
playing a role in IR regulation. Indeed, at this time we have yet to
establish the molecular level at which cGK governs IR expression.
However, our present findings point to cGK as the physiological
mediator of cGMP induction of ASGR and IR and that a phosphorylation
reaction governs the expression of both receptor proteins, albeit by
potentially different mechanisms.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-32972 and DK-17702.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. J. Stockert, Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Ullmann 517, Bronx, NY 10461 (E-mail: stockert{at}aecom.yu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 May 2000; accepted in final form 27 July 2000.
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