(Received for publication, September 18, 1995; and in revised form, November 22, 1995)
From the
Although all mouse mast cells are derived from a common
progenitor, these effector cells exhibit tissue-specific differences in
their expression of the chymase family of serine proteases whose genes
reside on chromosome 14. Immature bone marrow-derived mast cells
(mBMMC), developed in vitro with interleukin (IL) 3-enriched
medium, were cultured in the presence or absence of IL-10 to determine
at the molecular level how the expression of the individual chymases is
differentially regulated. As assessed by RNA blot analysis, mBMMC
contain high steady-state levels of the transcript that encodes mouse
mast cell protease (mMCP) 5, but not the homologous chymase transcripts
that encode mMCP-1, mMCP-2, or mMCP-4. Nevertheless, nuclear run-on
analysis revealed that these cells transcribe all four mast cell
chymase genes. IL-10 elicited high steady-state levels of the mMCP-2
transcript, and pulse-chase experiments revealed that the half-life of
the mMCP-2 transcript in mBMMC maintained in the presence of IL-10 is
4-fold longer than that in replicate cells subsequently cultured
in medium without IL-10. Reverse transcription-polymerase chain
reaction/nucleotide sequence analysis demonstrated that mBMMC cultured
in the absence or presence of IL-10 correctly process mMCP-2 pre-mRNA.
Experiments with cycloheximide and actinomycin D indicated that IL-10
induces expression of a trans-acting factor(s) that stabilizes
the mMCP-2 transcript or facilitates its processing. The discovery that
the expression of certain chymases in mBMMC is regulated primarily at
the post-transcriptional level provides a basis for understanding the
mechanism by which specific cytokines dictate expression of the
chromosome 14 family of serine proteases in cells that participate in
inflammatory processes.
Mast cells are derived from multipotential hematopoietic stem
cells, circulate in blood as immature cells, and undergo the final
stages of their differentiation and maturation after their agranular
progenitors become lodged in
tissues(1, 2, 3, 4, 5) . As
assessed by their expression of three different classes of granule
proteases, at least four phenotypically different types of mast cells
are present in the tissues of the BALB/c mouse. Mast cells express at
least four chymotrypsin-like neutral proteases (designated chymases)
whose genes reside at a complex on chromosome 14(6) , along
with the genes that encode cathepsin G and granzymes B, C, E, and
F(7, 8, 9) . Mast cells in the intestinal
mucosa preferentially express mouse mast cell protease (mMCP) ()1 and
mMCP-2(5, 10, 11, 12, 13, 14) ,
the mast cells in the peritoneal cavity and skin preferentially express
mMCP-4 and mMCP-5(15, 16, 17) , and those in
the spleen express every known granule chymase(5) .
Based on in vitro studies, it appears that much of the granule protease
pleiotropism of the mast cell is cytokine regulated. The immature mast
cells (mBMMC) generated by culturing BALB/c mouse bone marrow cells in
interleukin (IL) 3-enriched medium(18, 19, 20) contain high steady-state levels of the transcripts that
encode mMCP-5 (17) but not mMCP-1(13) ,
mMCP-2(12) , or mMCP-4(16) . However, these mBMMC will
also express high steady-state levels of the mMCP-1 and mMCP-2
transcripts when cultured in the presence of IL-9 (21) or IL-10 (14, 22) , or high steady-state levels of the mMCP-4
transcript when cultured in the presence of c-kit ligand(23) . Furthermore, the steady-state levels of the
mMCP-1 and mMCP-2 transcripts in BALB/c mBMMC can be reversibly altered
by adding or withdrawing IL-10 from the culture
medium(14, 22) . Half-maximal levels of both chymase
transcripts are achieved after the cells are cultured with IL-10 for
only 24 h, and plateau levels of the two chymase transcripts are
reduced to half-maximum within 24 h of IL-10 withdrawal. The rate by
which the steady-state levels of the two protease transcripts in these
nontransformed mast cells can be increased or decreased raised the
possibility that post-transcriptional control mechanisms might
contribute to mast cell protease heterogeneity.
We now demonstrate that the IL-10-regulated expression of the chymase mMCP-2 in BALB/c mBMMC occurs primarily by a post-transcriptional mechanism. This discovery has broad implications for understanding how the expression of the chromosome 14 superfamily of serine proteases is regulated in mast cells and other hematopoietic cells during inflammation.
RT-PCRs were performed with total or
cytoplasmic RNA isolated from 3-6-week-old BALB/c mBMMC. Samples
were incubated with RNase-free DNase I for 10 min at room temperature
to remove residual genomic DNA. Using the cDNA cycle kit from
Invitrogen (San Diego, CA), 2 µg of RNA and 0.2 µg of
oligo(dT) primer (18-mer) were placed in a mirocentrifuge tube and
incubated at 65 °C for 10 min to disrupt the secondary structure of
the transcript. Each RNA sample was reverse-transcribed at 42 °C
for 1 h in 20 µl of a solution containing 1
reverse
transcription buffer, 10 units of RNase inhibitor, 20 mM deoxynucleotide triphosphates, 20 mM sodium
pyrophosphate, and 5 units of avian myeloblastosis virus reverse
transcriptase. The reaction mixture was extracted with 20 µl of
phenol-chloroform, precipitated with 2 volumes of ethanol, and
resuspended in 20 µl of distilled water. A 2-µl sample of the
resulting cDNA preparation was mixed with 48 µl of 1
PCR
buffer containing 2 mM MgCl
, 2.5 units of AmpliTaq
polymerase (Perkin-Elmer), 0.2 µg of a primer corresponding to a 5`
region in exon 1 (5`-ACTGGCAAAATGCAGGCC-3`) of the mMCP-2 transcript,
and 0.2 µg of a primer corresponding to a 3` region in exon 5
(5`-CATCATCACAGACATGTG-3`) of the mMCP-2 transcript. Thirty to 35
cycles of PCR were performed. Each cycle consisted of a 1-min
denaturation step at 94 °C, a 2-min annealing step at 55 °C,
and a 3-min extension step at 72 °C. To verify the authenticity of
their nucleotide sequences, the RT-PCR products were purified by
electrophoresis on a 1% agarose gel. The appropriate bands were
excised, purified with Geneclean(TM) (BIO 101, Vista, CA), and
defined by Taq cycle-sequencing technology at the Biocore
Facility of the Dana Farber Cancer Institute.
mBMMC (1-5
10
) maintained in medium containing or lacking
IL-10 were washed with cold phosphate-buffered saline and disrupted by
two treatments with 5 ml of ice-cold lysis buffer (10 mM NaCl,
3 mM MgCl
, 0.5% Nonidet P-40, and 10 mM Tris-HCl, pH 7.4). After centrifugation at 800
g for 5 min at 4 °C, the nuclei-enriched pellets were
resuspended in 200 µl of storage buffer (40% glycerol, 5 mM MgCl
, 0.1 mM EDTA, and 50 mM Tris-HCl, pH 7.5). Each nuclear run-on reaction was initiated in a
400-µl reaction buffer containing 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 1 mM dithiothreitol, 10 units
of RNase inhibitor, 100 µCi of [
P]UTP, 20%
glycerol, 5 mM MgCl
, 150 mM KCl, and 30
mM Tris-HCl, pH 8.0. The reaction mixture was incubated at 30
°C for 30 min and then for
5 min at room temperature with 25
µg/ml DNase I (RNase-free). Tri Reagent (750 µl) and CHCl
(200 µl) were added, and the radiolabeled nascent nuclear
RNAs in the aqueous phase were precipitated with isopropanol,
resuspended in 100 µl of buffer (10 mM Tes, 10 mM EDTA, and 0.2% SDS), purified with a gel filtration spin column
(Clontech, Palo Alto, CA) and incubated with one of the DNA slot blots.
Each hybridization reaction was carried out at 65 °C for
36-48 h in a small vial containing 1-2 ml of hybridization
buffer (10 mM Tes, 10 mM EDTA, 0.2% SDS, 0.6 NaCl, 1
Denhardt's solution, and 300 µg/ml salmon sperm DNA)
and 1 to 5
10
cpm of radiolabeled nuclear RNA.
After hybridization, the blot was treated with 10 µg/ml RNase A for
20 min at 37 °C, washed three times (20 min each) with 2
SSC at 65 °C, and exposed to x-ray film for 1-3 days.
Plasmids containing either a full-length -actin, mMCP-2, or
mMCP-4 cDNA were also digested with a restriction enzyme, and the
digests (10 µg of DNA/lane) were electrophoresed in a 1.2% agarose
gel. The separated DNA was transferred to a nylon membrane, and the
resulting blot was evaluated for hybridization to the appropriately
sized insert with a replicate sample of radiolabeled nuclear RNA.
To
determine if an inhibitor of protein synthesis can block the
IL-10-induced expression of the mMCP-2 transcript, mBMMC
(10) were washed and resuspended in 50% WEHI-3
cell-conditioned medium containing IL-10 and varying doses (0, 1, 5,
and 20 µg/ml) of cycloheximide (Sigma). After
24 h of culture,
total RNA was isolated from the four populations of mBMMC, and the
amount of mMCP-2 mRNA in each was quantitated by blot analysis as
described above. To determine if cycloheximide can influence the
steady-state level of the established mMCP-2 transcript, 10
mBMMC were pretreated with IL-10, washed in enriched medium, and
resuspended in 50 ml of 50% WEHI-3 cell-conditioned medium containing 1
µg/ml cycloheximide with or without IL-10. At various time points,
total RNA was isolated and the steady-state levels of the mMCP-2
transcript in the resulting 10 samples were quantitated by blot
analysis.
Figure 1:
Transcription of chymase genes. A, for analysis of the steady-state levels of chymase
transcripts, a blot containing total RNA from BALB/c mBMMC cultured in
the absence(-) or presence (+) of IL-10 for 36 h was
analyzed sequentially with mMCP-1, mMCP-2, mMCP-4, mMCP-5, and
-actin gene-specific probes. B, for nuclear run-on
analysis, BALB/c mBMMC were cultured for 24 h in the absence(-)
or presence (+) of IL-10. Isolated nuclear run-on derived
transcripts were incubated with a membrane containing plasmid DNA alone (pBS) or plasmid DNA carrying a gene-specific probe for
mMC-CPA, type IX collagen (ColIX), mMCP-1, mMCP-2, mMCP-4,
mMCP-5, and
-actin. pBS and type IX collagen plasmids were used as
negative controls;
-actin and mMC-CPA plasmids were used as
positive controls. Similar findings were obtained in four other
experiments.
Figure 2: RT-PCR analysis of mMCP-2 transcripts. Samples of total RNA from BALB/c mBMMC cultured in the presence (lane 1) or absence (lane 2) of IL-10 were subjected to RT-PCR (30 cycles) with a set of primers specific for mMCP-2. The resulting products were then assessed for their relative size by agarose gel electrophoresis. The arrow on the left indicates the 910-bp product expected if the mature mMCP-2 transcript is present in a RNA sample. Size markers are indicated in lane 3 and on the right. Similar findings were obtained in four other experiments with other sets of mMCP-2-specific primers.
Figure 3:
Stability of the mMCP-2, mMCP-5, and
-actin transcripts. mBMMC were radiolabeled with
[
H]uridine in the presence of IL-10 and then were
washed and cultured in radioactive-free medium with (
) or without
(
) IL-10. The amounts of radiolabeled mMCP-2, mMCP-5, and
-actin transcripts in each culture were determined at the
indicated time points in the chase. Results are expressed as a percent
of the starting level of the transcript. In these experiments, the
specific binding of radiolabeled transcripts to the mMCP-2 probe was
17-81-fold higher than the background binding of radiolabeled
transcripts to pBS.
Figure 4: Effect of actinomycin D on the steady-state levels of the mMCP-2 transcript. BALB/c mBMMC, cultured in the presence of IL-10 to induce high steady-state levels of the mMCP-2 transcript, were washed and cultured in the presence of actinomycin D (Act D) with (+) or without(-) IL-10. At the indicated time points, RNA was isolated and analyzed for the presence of the mMCP-2 transcript (top panel). The change in the level of the mMCP-2 transcript is shown in the bottom panel as a percent of the starting level of the transcript. The RNA gel used in the depicted experiment was also stained with ethidium bromide (middle panel) to demonstrate that comparable amounts of 18 S ribosomal RNA were loaded into the lanes. Similar findings were obtained in two other experiments.
To determine whether or not the IL-10-regulated expression of the mMCP-2 transcript in BALB/c mBMMC requires de novo protein synthesis, cells were treated with IL-10 for 24 h in the absence or presence of 1 to 20 µg/ml cycloheximide. The IL-10-induced expression of the mMCP-2 transcript was effectively blocked by concomitant exposure of the cells to 1 µg/ml cycloheximide (Fig. 5A). In a control experiment, when mBMMC were cultured in WEHI-3 cell-conditioned medium containing IL-10 and 1 µg/ml cycloheximide for 24 h, washed, and then cultured in the presence of IL-10, but in the absence of cycloheximide for 48 h, the cells regained their ability to express high steady-state levels of the mMCP-2 transcript (data not shown). Thus, the cycloheximide effect on the steady-state level of the mMCP-2 transcript in mBMMC is reversible.
Figure 5: Effect of cycloheximide on the steady-state levels of mMCP-2 mRNA. A, BALB/c mBMMC were cultured for 24 h in medium containing IL-10 without (lane 1) or with 1 (lane 2), 5 (lane 3), or 20 (lane 4) µg/ml cycloheximide (CHX). An RNA blot prepared from these cells was analyzed for the presence of mMCP-2 mRNA (top panel) and 18 S ribosomal RNA (bottom panel). Similar findings were obtained in a preliminary experiment with 20 µg/ml cycloheximide. B, BALB/c mBMMC were cultured in medium containing IL-10 for 48 h and then were resuspended in medium containing both IL-10 and cycloheximide (lanes 4, 6, 8, and 10) or just cycloheximide (lanes 3, 5, 7, and 9). RNA was isolated from the various populations of mBMMC immediately (lanes 3 and 4), or at 8 h (lanes 5 and 6), 16 h (lanes 7 and 8), and 32 h (lanes 9 and 10) after the initiation of the cycloheximide treatment. For controls, replicate mBMMC were cultured in medium containing IL-10 but not cycloheximide (lane 1) or in medium containing neither IL-10 nor cycloheximide (lane 2). A blot was prepared and analyzed for the presence of the mMCP-2 transcript (top panel) and 18 S ribosomal RNA (middle panel). Results are expressed in the bottom panel as a percent of the starting level of the transcript. Similar findings were obtained in another experiment.
To determine if the half-life of the mMCP-2 transcript in IL-3-developed mBMMC depends on the synthesis of protein, mBMMC were exposed to IL-10 for 48 h to induce high steady-state levels of the mMCP-2 transcript. The cells were washed and then cultured for up to 32 h in WEHI-3 cell-conditioned medium containing 1 µg/ml cycloheximide with or without IL-10. The steady-state levels of the mMCP-2 transcript decreased in the two populations of mBMMC at comparable rates (Fig. 5B).
Mast cell heterogeneity is now well established in the mouse, rat, and human, and the chymase superfamily of granule proteases has served as the principal marker for identifying tissue-specific mast cell phenotypes in these three species. Because the individual members of the chymase superfamily are differentially regulated in cultured mouse mast cells by cytokines, we examined how the expression of the chymase mMCP-2 is controlled in BALB/c mBMMC. We now report that the expression of mMCP-2 in these mast cells is regulated primarily by a post-transcriptional mechanism.
Based on the amino acids in their putative substrate binding pockets (17, 34) , it is likely that each mast cell chymase whose gene resides at the chromosome 14 complex (6) has a preferred substrate in vivo. In terms of their deduced amino acid sequences and the nucleotide sequences of their genes and transcripts, mMCP-1, mMCP-2, and mMCP-4 are more similar to one another than to mMCP-5. BALB/c mBMMC developed with recombinant IL-3- or IL-3-enriched conditioned medium contain high steady-state levels of the mMCP-5 transcript, but the levels of the transcripts that encode the other members of the chymase superfamily are all below detection by RNA blot analysis (Fig. 1A and Refs. 12, 13, 16, and 17). Nonetheless, we now show by nuclear run-on analysis with gene-specific probes that all chymase genes are transcribed in these BALB/c mBMMC (Fig. 1B). Although it is possible that differential rates of transcription contribute somewhat to the overall steady-state levels of cytokine-inducible chymase transcripts, the nuclear run-on data indicate that the levels of these transcripts are controlled primarily by a post-transcriptional mechanism. When mBMMC cultured in the absence of IL-10 were compared to mBMMC cultured in the presence of IL-10, there was not much difference in the rates of transcription of the mMCP-1 and mMCP-2 genes despite the fact that there was a vast difference in the steady-state levels of these two chymase transcripts. Furthermore, even though the mMCP-4 gene is transcribed at a high rate with or without IL-10-stimulation, no mMCP-4 transcript could be detected in these cells by RNA blot analysis. RT-PCR analysis (Fig. 2) confirmed that the transcript that hybridizes to the mMCP-2 cDNA in the nuclear run-on analysis is mMCP-2. Moreover, nucleotide sequence analysis of the RT-PCR product revealed that the mMCP-2 transcript is properly processed in mBMMC whether these cells are cultured in the absence or presence of IL-10, a cytokine that induces high steady-state levels of mMCP-2 mRNA in these mast cells.
Because the levels of the mMCP-1, mMCP-2, or mMCP-4
transcripts in IL-3-developed mBMMC are below detection by blot
analysis (Fig. 1A), the post-transcriptional control of
any one of these mMCP transcripts can be studied only after its
expression has been induced by cytokine treatment of the cells. We
previously reported (14) that if IL-10-stimulated mBMMC are
subsequently cultured in the absence of IL-10, there is a slow, but
steady, decrease in the steady-state level of the mMCP-2 transcript.
Pulse-chase experiments revealed that the mMCP-2 transcript has an
4-fold longer half-life in IL-10-treated mBMMC that continue to be
cultured in the IL-10-enriched medium than in replicate cells that have
been transferred into IL-10-free medium (Fig. 3). IL-10 did not
influence the half-life of the mMCP-5 or
-actin transcripts in
these cells. However, the acquisition of high steady-state levels of
the mMCP-2 transcript in IL-10-treated cells was fully blocked by
cycloheximide (Fig. 5A), indicating that the inductive
phase of the regulation depends on the synthesis of new protein.
Inasmuch as the mMCP-2 gene is transcribed in mBMMC that have been
cultured in the absence of IL-10 (Fig. 1B and Fig. 2), IL-10 must induce synthesis of a trans-acting
protein that directly or indirectly stabilizes the mature mMCP-2
transcript or the processing of its precursor. If the mMCP-2 transcript
is already expressed in abundance in mBMMC, the half-life of the
transcript decreases from a rate of
34 h in nondrug-treated cells (Fig. 3) to 13-15 h in cells treated with actinomycin D (Fig. 4) or cycloheximide (Fig. 5B). These data
support the conclusion that IL-10 induces expression of a trans-acting factor that stabilizes the mMCP-2 transcript.
The fact that the half-life of the mMCP-2 transcript in cells
exposed to IL-10 and actinomycin D (Fig. 4) or cycloheximide (Fig. 5B) does not decrease to 8 h could be a
consequence of a secondary drug effect on the catabolism machinery
itself. Alternatively, both drugs could effect the movement and
processing of newly synthesized mMCP-2 pre-mRNA, as has been found with
the IL-2 precursor transcript in cycloheximide-treated tonsil
cells(35) . If IL-10 induces the expression of a protein that
stabilizes the mMCP-2 transcript, then the rate of turnover of the
mMCP-2 transcript in the pulse-chase assay (Fig. 3) depends, in
part, on the rate of turnover of the stabilizing protein. Because time
would be required to deplete the intracellular level of the stabilizing
protein following the removal of IL-10 from the culture medium, the
4-fold difference in the turnover of the mMCP-2 transcript in the
two populations of mBMMC is a minimum estimate. It, therefore, is
likely that the rate of decay of the mMCP-2 transcript is extremely
rapid in IL-3-developed mBMMC that have never been stimulated with
IL-10.
The half-life of a transcript, which can differ widely in cells, depends on the presence or absence of certain structural features in the transcript and trans-acting factors in the cell(36) . Some differentiating cells respond to developmental signals by changing the stability of their transcripts. For example, during erythrocyte development, the accumulation of globin is attributed mainly to the preferential stabilization of globin mRNA(37) . When progenitor cells differentiate into mature B lymphocytes, high levels of immunoglobulin are produced due to increased stabilization of the immunoglobulin transcript(38) . mBMMC are nontransformed mast cell-committed progenitors. The post-transcriptional regulatory pathway for protease expression in these cells may reflect their granule immaturity.
It is known that cis-acting elements in the 3`-UTRs of numerous transcripts regulate their half-life in cells. The most thoroughly studied RNA motif that controls the steady-state levels of mRNA in cells is the adenylate- and uridylate-rich cis-acting element (termed AU motif) (39) present in multiple copies in the 3`-UTRs of the transcripts that encode most cytokines and many early-response proteins. Because the mMCP transcripts do not possess an AU motif in their 3`-UTRs (Fig. 6), a different mechanism must control the steady-state levels of the varied chymase transcripts. The presence of the repetitive non-AU nucleotide sequence, ACCUACCUACCCAACUA, in 3`-UTRs of some Xenopus and Drosophila transcripts has been reported to influence their stability(40) . The mMCP-1, mMCP-2, and mMCP-4 transcripts all have multiple ``UGXCCCC'' sequences in their 3`-UTRs that are not present in the mMCP-5 transcript (Fig. 6). These non-AU repetitive sequences in the 3`-UTRs may be the cis-acting elements that regulate the steady-state levels of the mMCP-1, mMCP-2, and mMCP-4 transcripts.
Figure 6: Comparison of the 3`-UTRs of mast cell chymase transcripts. The nucleotide sequences of the 3`-UTRs of four mouse mast cell chymase mRNAs are depicted. The repetitive ``UGXCCCC'' sequence in mMCP-1(13, 27) , mMCP-2(6, 12) , and mMCP-4 (16) transcripts is underlined. The UAG and UAA translation stop codons are indicated in bold.
It has been established with use of
recombinant inbred mouse strains and interspecific backcrosses that the
genes that encode mMCP-1, mMCP-2, mMCP-4, mMCP-5, neutrophil cathepsin
G, and four cytotoxic lymphocyte granzymes all reside at a complex on
chromosome 14(6, 7, 8, 9) . Inasmuch
as no crossover has been detected by restriction-enzyme fragment length
polymorphism analysis of chromosomal DNA, either these 3-kb serine
protease genes reside close to one another or some physical restraint
in the particular region of chromosome 14 where they are located
hinders recombination. Pulsed field gel electrophoresis of genomic DNA
revealed that the mMCP-1, mMCP-2, mMCP-4, and mMCP-5 genes reside
within 850 kb of one another(6) . Nucleotide sequencing studies
revealed that another chymase gene, encoding mMCP-8, is located only
5-7 kb away from the mMCP-1 gene(41) . Recently, it was
reported that a mouse T cell line (derived with IL-3 and IL-9 but
maintained with IL-9 alone) expresses high steady-state levels of the
transcripts that encode granzyme B, mMCP-1, mMCP-2, and
mMCP-5(42) . Taken together, these findings raise the
possibility that a common cis-acting enhancer element induces
the transcription of many of the serine protease genes located at the
chromosome 14 complex and that the steady-state level of each protease
transcript is regulated by tissue-specific factors that instruct the
cell which protease transcripts should be processed and/or protected
from rapid degradation. Pertinent to this hypothesis is the fact that
many genes that reside on chromosome 11 in the mouse (43, 44) encode cytokines whose expression is also
post-transcriptionally regulated during inflammation.
Because the mast cell chymases are enzymatically active at neutral pH, it would be advantageous to limit their expression to a single cell type. Any cell could transcribe the mMCP-2 gene if it expresses the appropriate DNA binding protein that induces the transcription of the gene in mast cells or if a mutation was to occur in the promoter of the gene so that a new cis-acting motif is created that is recognized by a different transcription factor in that cell. Many hematopoietic effector cells (e.g. neutrophils, eosinophils, macrophages, natural killer cells, and cytotoxic lymphocytes) possess the chymase-zymogen processing enzyme dipeptidyl peptidase I (45) and therefore have the ability to convert any translated pro-mMCP-2 into mature, enzymatically active neutral protease. The post-transcriptional regulatory mechanism observed in the mast cell not only would accommodate cytokine regulation of protease expression during an inflammatory response but would also prevent the expression of the chymase family of mast cell proteases in other cell types if their genes were aberrantly transcribed.