(Received for publication, June 28, 1995)
From the
We have previously reported mouse SIP24 protein as a secreted
inducible protein produced by quiescent Balb/c 3T3 cells. SIP24 can be
produced in response to many factors, including serum, basic fibroblast
growth factor, prostaglandin F2, phorbol ester, and dexamethasone.
Here we present evidence to show that SIP24 is the product of mouse
24P3 mRNA. The 24P3 cDNA was originally cloned from an SV40-transformed
quiescent mouse primary kidney cell culture, and it has been classified
as a new member of the lipocalin protein family. We show that the
SIP24/24P3 protein and mRNA increase dramatically in mouse serum and
liver during the acute phase response induced by turpentine injection.
Injection of mice with dexamethasone caused a modest increase of
SIP24/24P3 mRNA in the liver. Tissue distribution studies revealed that
SIP24/24P3 is mainly expressed in liver during the acute phase
response. SIP24/24P3 was also detected in the brain and the uterus. In
mouse BNL (Balb/c normal liver) cells, the production of SIP24/24P3 is
stimulated by tumor necrosis factor
, which is a major regulator
of the expression of other acute phase proteins. From its pattern of
regulation, we conclude that SIP24/24P3 is a new type 1 acute phase
protein.
The acute phase response (APR) ()is a complex
reaction to various inflammatory or stressful stimuli such as surgery,
wounding, bacterial or virus infection, or elevated levels of stressful
and tissue-damaging agents. During this mammalian stress response, the
plasma levels of a group of proteins change rapidly. These proteins are
called the acute phase proteins (APPs; (1, 2, 3) ). Those proteins whose plasma
levels increase are called positive APPs; examples include C-reactive
protein,
-acid glycoprotein (AGP),
-macroglobulin, and haptoglobin. The plasma levels of
negative APPs decrease in response to inflammation or other invasive
stress; examples include retinol-binding protein and albumin. The APPs
are synthesized mainly in the liver and are secreted into the
bloodstream(4) . The precise functions of many APPs are still
largely unknown. It is generally believed that the APPs play an
anti-inflammatory role to prevent ongoing tissue damage and to return
the organism to normal function. The known functions of APPs can be
classified into three categories, including the maintenance of
homeostasis, the transport of a variety of factors, and defense against
infection(3) .
The regulation of hepatic APP production is
mediated by several classes of
factors(2, 3, 5) . First, the
interleukin-1-type cytokines, which include interleukin 1,
interleukin 1
, tumor necrosis factor
, and tumor necrosis
factor
, induce type-1 APPs such as human C-reactive protein and
rat haptoglobin. The interleukin-1-type like cytokines also decrease
the expression of type 2 APPs. Second, the interleukin 6-type
cytokines, which include interleukin 6 (IL-6), interleukin 11, leukemia
inhibitory factor, oncostatin M, and ciliary neurotrophic factor induce
primarily type 2 APPs such as rat
-macroglobulin.
Third, glucocorticoids are believed to play a permissive role during
the APR because by themselves they cause small increases in the
production of most APPs but strongly enhance the effect of cytokines on
most APPs. Fourth, growth factors such as FGF, insulin, transforming
growth factor
, and hepatocyte growth factor regulate APPs in a
way similar to glucocorticoids. Experimentally, the APR can be induced
by injection of animals with inflammatory agents such as
turpentine(1, 6) .
We have reported previously the
induction, characterization, purification, and partial peptide
sequencing of a superinducible protein, SIP24, produced by quiescent
Balb/c 3T3 mouse fibroblast cells (7, 8, 9) .
SIP24 is a 24-kDa secreted glycoprotein induced by serum, basic
fibroblast growth factor (bFGF), prostaglandin F2 (PGF2
),
phorbol ester, and dexamethasone. It is superinducible because it can
be further induced in a synergistic manner with growth factors if the
cells are pretreated with the protein synthesis inhibitor,
cycloheximide. We have identified SIP24 as the protein product of the
mouse 24P3 mRNA, whose cDNA was originally cloned from SV40-infected
mouse kidney primary cell cultures(10) . The 24P3 protein is
also a major secretory protein of cultured mouse PU5.1.8 macrophage
cells which have been stimulated by lipopolysaccharide(11) .
Based on the amino acid sequence deduced from its cDNA, Flower et al.(12) identified 24P3 as a new member of the
lipocalin protein family. Proteins closely related in sequence to 24P3
are rat -microglobulin-related protein and human
neutrophil gelatinase-associated lipocalin(12, 13) .
The lipocalin family is mainly composed of extracellular ligand binding
proteins with high specificity for small hydrophobic molecules.
Examples of lipocalin family members include
-acid
glycoprotein (AGP),
-microglobulin, plasma retinol
binding protein, and
-lactoglobulin. Some lipocalin proteins,
including AGP,
-macroglobulin, and retinol-binding
protein, are also APPs, and they can be regulated by glucocorticoids in vitro and in vivo(2, 3) . Here we
present evidence to show that SIP24/24P3 is highly induced during the
APR in vivo, and it is mainly expressed in liver during the
APR. SIP24/24P3 is also expressed in the brain and the uterus.
Moreover, the expression of SIP24/24P3 can be regulated in cultured
cells by TNF-
which is amajor mediator of the APR. Our results
also show that SIP24/24P3 is a type 1 APP.
The sequences of seven peptides obtained from a clostripain digestion of the purified SIP24 protein were compared with the sequences found in an updated SwissProt data base. All peptide sequences except peptide B were found to share identity with the deduced amino acid sequence of 24P3 cDNA (Fig. 1). The six SIP24 peptides that are identical with 24P3 cover 25% of the complete 24P3 sequence and are spread over the entire 24P3 coding region. The 24P3 cDNA was cloned by Hraba-Renevey et al.(10) from quiescent mouse kidney primary cell cultures infected by SV40. 24P3 mRNA can also be induced by serum in 3T3 fibroblast cells as can the SIP24 protein(7, 10) . These findings suggested that SIP24 is the product of the 24P3 mRNA, and that peptide B containing the cyclophilin-like sequence came from a contaminating protein in the SIP24 preparation. As judged by the area under the peptide B peak after reverse-phase high performance liquid chromatography, the molar ratio of the cyclophilin-like peptide to the other peptides in the clostripain digests was less than 0.4 (the sequencing of this peptide did not go to completion). Although such a molar ratio could have arisen because a larger proportion of peptide B was lost during the procedure, it is also consistent with peptide B having been derived from a contaminating protein.
Figure 1: Comparison between the SIP24 and 24P3 deduced amino acid sequences. The lower horizontal line represents the 24P3 sequence. All numbers except the first and the last indicate the amino acid position on the deduced 24P3 protein sequence at which the sequences of the peptides shown for SIP24 begin. Position 1 is the first amino acid in the 24P3 sequence that includes a putative signal sequence. Identities of sequence between the SIP24 and 24P3 are indicated by a line on the 24P3 map without interruptions of the amino acid letter code. The single location in which the sequence of one of the SIP24 peptides did not match the 24P3 sequence is indicated with a letter interrupting the line representing 24P3. The mismatch was found in peptide E in which a glycine was found in the SIP24 peptide sequence in the position of a lysine in the 24P3 sequence. The sequences of peptides A, C, and D have been reported previously(9) . Peptides E, F, and G are newly identified and sequenced peptides from a clostripain digest of SIP24. The symbol ? in the sequence of peptide G indicates that the amino acid in this position was not detected as a recognizable peak.
Figure 2:
Recognition of SIP24 by antisera raised
against 24P3 by Western blot analysis. Quiescent Balb/c 3T3 cells were
incubated with or without 1 mg/ml cycloheximide and 400 ng/ml
dexamethasone (DEX/CHX) in serum-free DME medium for
18 h. The media were concentrated and resolved by SDS-PAGE, and the
Western blots were stained with preimmune serum (PI),
anti-SIP24 (-SIP24), or two preparations of anti-24P3
sera (376, 377) either alone or as a 1:1 mixture. The
Western blot was visualized by using the Stratagene picoBlue
alkaline phosphatase kit. Arrows on the left indicate
the positions of the molecular weight markers expressed in thousands,
from top to bottom: bovine serum albumin (67,000),
ovalbumin (45,000), carbonic anhydrase (30,000), myoglobin (18,000),
and cytochrome c (12,500).
Figure 3:
SIP24/24P3 is an acute phase protein. A, Northern blot of saline- and turpentine-injected mouse
liver total RNA. Male and female mice were injected with 0.9% sterile
saline (Saline) or turpentine (Turpentine).
Twenty-four hours after injection, total RNAs were extracted from the
livers and resolved by agarose gel electrophoresis. The resolved RNAs
were transferred to nylon membranes and hybridized sequentially with P-labeled 24P3, mAGP, and rat 18 S rRNA cDNA probes. The
same experiment was performed twice, and a total of 12 mice (6 male and
6 female) were treated under each condition. Numbers on the right show the positions of the 18 S and 28 S rRNA markers and
the estimated molecular weight of the 24P3 mRNA. B, Western
blot analysis of sera from saline- and turpentine-injected mice. The
same male and female mice were used as for A. Serum samples
were prepared 12 and 24 h after injection of saline or turpentine.
Samples (30 µl) of 2.5% sera were resolved by SDS-PAGE and blotted
against preimmune serum (PI) and anti-SIP24 serum
(
-SIP24) as described under ``Experimental
Procedures.'' A sample of SIP24-enriched cell culture medium as
used in Fig. 2was included as positive control (C). Numbers on the left indicate molecular weight markers
in thousands.
To determine the effect of turpentine injection on the level of SIP24/24P3 in the bloodstream, sera from saline- and turpentine-injected mice were collected 12 and 24 h after injection, and SIP24 was detected by Western blot analysis. As in the results for the Northern blots of 24P3 mRNA, SIP24 protein was undetectable in sera from saline-injected mice. However, SIP24 was detected in sera from turpentine-injected mice. Sera collected at both 12 and 24 h after injection of turpentine had elevated levels of SIP24 (Fig. 3B). There was no observable difference between the sexes in the extent to which SIP24 was elevated. Thus, as for 24P3 mRNA in liver, turpentine treatment elevated the SIP24 protein level in the bloodstream.
Figure 4:
Northern blot analysis of total RNA
isolated from the livers of vehicle- and dexamethasone-injected mice.
Male and female mice were injected with
2-hydroxypropyl--cyclodextrin (C) or dexamethasone in
2-hydroxypropyl-
-cyclodextrin (DEX). Twenty-four hours
after injection, total liver RNAs were extracted and resolved by
agarose gel electrophoresis. The RNAs were then transferred to a nylon
membrane and hybridized sequentially with
P-labeled 24P3,
mAGP, and rat 18 S rRNA probes. The same experiment was performed
twice, and a total of 12 mice (6 male and 6 female) were used for each
condition.
Figure 5:
Tissue distribution of 24P3 mRNA in
saline- and turpentine-injected animals. Twenty-four hours after
injection of saline(-) or turpentine (+), total RNAs were
extracted from different tissues and resolved by agarose gel
electrophoresis. Three different mice (one male and two females) were
used as sources of the tissues. The RNAs were transferred to nylon
membranes and hybridized with P-labeled 24P3 and rat 18 S
rRNA probes. The figure shows representative results from one of each
of the tissues.
Figure 6:
24P3 mRNA expression during pregnancy.
Total RNAs from the maternal liver, uterus, placentas, and fetuses of
day 11 midgestation female mice were extracted and hybridized with P-labeled 24P3 and rat 18 S rRNA
probes.
Figure 7:
Regulation by cytokines of SIP24
expression in BNL cells. A, regulation by IL-6 and TNF-
of SIP24 synthesis and secretion. Quiescent BNL cells were incubated
for 24 h with IL-6, TNF-
, and the combination of both factors. The
culture media containing radiolabeled secreted proteins were resolved
by SDS-PAGE and analyzed by fluorography as described under
``Experimental Procedures.'' The concentrations used of IL-6
(10-1000 units/ml) and of TNF-
(0.1 to 10 ng/ml) are shown
for each lane. Numbers on the left show the positions
of the proteins used as molecular weight markers with their molecular
weights expressed in thousands. B, Western blot analysis of
secreted SIP24. Quiescent BNL cells were incubated for 24 h with IL-6
and TNF-
in serum-free DME medium. The culture media were
collected, centrifuged, lyophilized, and redissolved in distilled
H
O to 1/10 of the original volume. The concentrated media
were resolved by SDS-PAGE, and the Western blots were stained with
preimmune serum (PI) or anti-SIP24 (
-SIP24). The
same SIP24-enriched medium as used in Fig. 2was included as
positive control (DEX/CHX). Numbers on the left indicate the positions of the molecular weight markers in
thousands.
We have shown that 24P3 mRNA encodes a protein that is
identical in all known aspects with SIP24. Thus, we propose that SIP24
and the protein encoded by the 24P3 mRNA are the same protein. We base
our conclusion on the following structural evidence. First, the derived
amino acid sequence of 24P3 has 200 amino acid residues with a putative
signal peptide of 15 N-terminal hydrophobic residues, and its
calculated M = 22,800(10) , whereas
SIP24 is secreted, has approximately 180 amino acid residues, and its
estimated M
= 21,000 excluding the
polysaccharide moiety (9) . Second, six different peptides
derived from SIP24 showed identity with the 24P3 sequence. This
identity covers the length of the protein. Third, the deduced 24P3
protein sequence has a potential N-glycosylation
site(10) , and SIP24 is N-glycosylated(9) .
Fourth, antiserum raised against SIP24 purified from 3T3 cells
recognizes the same protein as do antisera raised against the 24P3
protein which had been expressed in and purified from E. coli.
Fifth, probes for 24P3 mRNA and SIP24 protein detected an mRNA and a
protein that were regulated in the same way in vivo. Based on
this information, which showed that SIP24 and the 24P3-encoded protein
are structurally and immunologically related and are regulated
identically in vivo and in cultured cells, we conclude that
the 24P3 mRNA encodes the SIP24 protein. Thus, we refer to this protein
as SIP24/24P3.
Our results of turpentine and dexamethasone injection
experiments have clearly shown that SIP24/24P3 is induced in the liver
in a manner indicative of a positive APP. Because SIP24/24P3 is
expressed at about the same levels in both males and females it is
unlikely that sex hormones play a major role in regulating its
expression. By comparison, the related lipocalin and APP rat
-microglobulin is synthesized only in male
liver(19) .
The results of our tissue distribution studies show that SIP24/24P3 is primarily expressed in liver during the APR. However, it is also expressed at lower levels in the brain and uterus during the APR. As reviewed by Aldred et al.(4) , the liver is not the only site of APP expression, even though it is the most important one in terms of magnitude of production. For example, the brain is an organ for which the extracellular compartment is separated by the blood-brain barrier from the main vascular/extravascular body compartment. So, it may be desirable for the brain to synthesize its own APPs when needed. The levels of messenger RNAs encoding several APPs are altered in the brains of various species during the APR. These proteins include rat transferrin, transthyretin, ceruloplasmin, and retinol-binding protein (4) .
SIP24/24P3 was also found expressed in the pregnant uterus in the unstressed animal. Besides liver, several APPs are also expressed in the tissues comprising the interface between the maternal and fetal body compartment in the pregnant animal; such tissues include uterus, placenta, and yolk sac(4) . Extensive tissue remodelling takes place during pregnancy with the constantly changing interaction between the fetus and the tissues of the maternal reproductive tract. The APPs may be needed to control the extent of tissue damage during pregnancy.
Although SIP24/24P3 mRNA was induced in mouse kidney primary cell culture after SV40 infection(10) , we did not detect any SIP24/24P3 mRNA expressed in kidney in either saline- or turpentine-treated mice. There are several possible explanations for these observations: 1) SIP24/24P3 mRNA may be expressed in the kidney at a level below the sensitivity of our assays; 2) conditions in vivo are different from those to which cultured cells are subjected; or 3) different factors may be required to induce SIP24/24P3 in nonparenchymal tissues such as the kidney. It has been reported that hepatic nonparenchymal cells produced IL-6 in response to intraperitoneal endotoxin (lipopolysaccharide) but not in response to intramuscular turpentine injection(20) .
The regulation of
APP expression is extremely complex with many regulatory humoral
factors being
involved(2, 3, 5, 20, 21) .
The regulation of SIP24/24P3 seems to be no exception. In Balb/c 3T3
cells, we have previously shown that SIP24/24P3 can be induced by
serum, bFGF, epidermal growth factor, prostaglandin F2, and
phorbol 12-myristate 13-acetate(7, 8, 9) .
Here we have shown that the two major APR regulating factors, IL-6 and
TNF-
, differently regulate SIP24/24P3 expression in BNL cells. BNL
cells are a Balb/c normal liver cell line that has been used to study
the regulation of the APP, rabbit serum amyloid A protein
gene(22) . The ability of SIP24/24P3 to be induced by TNF-
indicates that it is a type 1 APP(2) .
When the magnitudes of SIP24/24P3 induction after turpentine and dexamethasone injection are compared, it is clear that glucocorticoids cannot account for the entire increase in SIP24/24P3 during the APR. As for many other APPs, the interplay among various inducing factors may be necessary to achieve maximum induction of SIP24/24P3 in vivo and in cultured cells(2, 3, 5) . This possibility and the underlying mechanism of SIP24/24P3 regulation are currently under investigation in our laboratory.
Our current knowledge of the structure and regulation of SIP24/24P3 provides several clues relating to its possible function. Based upon the fact that SIP24/24P3 protein is produced both upon viral infection and lipopolysaccharide induction in cultured cells, Meheus et al.(11) have suggested that SIP24/24P3 could play a role in the defense mechanism against infection. Our identification of SIP24/24P3 as an APP suggests that it may be involved in homeostasis and have an anti-inflammatory role. A number of factors that induce SIP24/24P3, including bFGF, epidermal growth factor, and dexamethasone, also have been shown to have effects on cultured cells which would be anti-inflammatory in vivo(2, 23) . We have also shown that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate induces SIP24/24P3. 12-O-Tetradecanoylphorbol-13-acetate activates protein kinase C, which induces specific acute phase responses(2, 24) .
The identification of SIP24/24P3
as a member of the lipocalin protein family suggests that SIP24/24P3
might be a binding protein for small hydrophobic molecule(s). We have
reported that PGF2 also induces SIP24 production in 3T3
cells(7) . This prostaglandin is a mitogen for 3T3
cells(25) . Many prostaglandins, including PGF2
, are
released as a result of increased metabolism of arachidonic acid during
the APR(2) . Prostaglandins are also produced at different
rates throughout pregnancy(26) . So, it is possible that the
level of SIP24/24P3 is regulated during the APR and in pregnancy in
response to released PGF2
. We speculate that SIP24/24P3 may have
an anti-inflammatory role and that this role might involve the ability
of SIP24/24P3 to bind a prostaglandin-like molecule.