From the
This study examines the molecular mechanisms of interaction
between tumor necrosis factor
Tumor necrosis factor
Cytokines characteristically possess multiple functions
with considerable overlap in their biological actions
(5) .
There are many examples of interaction between TNF
We have previously reported that
TNF
The object of this
study was to examine in detail the manner in which TNF
Total RNA from the
subfractions was prepared using the method described by Gough
(27) . Supernatant from the various RNA-containing subfractions
was added to 4 ml of protein-denaturing buffer containing 7
M urea, 350 m
M NaCl, 10 m
M EDTA, 1% SDS, and 10
m
M Tris-HCl, pH 7.4. RNA was extracted by adding 8 ml of
phenol/chloroform/isoamylalcohol (50:50:1) and precipitated with 8 ml
of isopropanol. The RNA pellet was washed once with 70% ethanol and
resuspended in TE buffer (10 m
M Tris-HCl, pH 8.0, 1 m
M EDTA, pH 8.0) at a concentration of 2 µg/µl. For Northern
blot analysis, total RNA was separated in a 1.5% agarose-formaldehyde
gel as described above. Equal loading was confirmed by staining the
gels with ethidium bromide.
To achieve a clearer
understanding of the mechanisms of action of TNF
UMR 201 cells were treated for 4, 12, and
24 h with 0.6 n
M TNF
Although we
have previously reported that co-treatment with cycloheximide
significantly inhibited the effect of retinoic acid on nuclear
processing
(20) , a similar experiment could not be performed in
this study because the combination of cycloheximide and TNF
We have used a non-transformed clonal rat cell line, UMR 201,
with features characteristic of pre-osteoblasts in our studies of
osteoblast differentiation. Retinoic acid significantly stimulates the
expression of mRNA for alkaline phosphatase, osteopontin, osteonectin,
matrix Gla protein, and type I collagen in these cells
(22, 32, 33) . Previous studies have shown that
retinoic acid and TNF
The alkaline phosphatase gene is constitutively transcribed in
control and TNF
We have previously described a novel action of
retinoic acid, in that it greatly increases the proficiency of nuclear
processing of newly transcribed alkaline phosphatase mRNA, enabling the
accumulation of mature mRNA in the non-matrix nuclear fractions prior
to translocation into the cytoplasm
(20) . The mechanism of
action of retinoic acid in enhancing nuclear processing is unclear.
Factors that will need to be considered include the role of RNA binding
proteins and their participation in regulation of the splicing
mechanism, stability of native and spliced RNA transcripts, as well as
translocation of the spliced product from the nucleus to the cytoplasm.
In contrast to our findings, another group has shown that in human
peripheral blood mononuclear cells, retinoic acid induced
interleukin-1
Although TNF
A
locally secreted cytokine such as TNF
In conclusion, this study has revealed that TNF
(TNF
) and retinoic acid on
the expression of the alkaline phosphatase gene by rat clonal
preosteoblastic cells. In this cell line, alkaline phosphatase mRNA was
not constitutively expressed but was progressively induced by treatment
with 1 µ
M retinoic acid, detectable by 6 h. Combining
retinoic acid with 0.6 n
M TNF
resulted in alkaline
phosphatase mRNA appearing by 2 h, as well as enhanced expression above
that observed with retinoic acid alone at 6, 12, and 24 h. Nuclear
run-on analysis showed constitutive transcription of the alkaline
phosphatase gene in control and TNF
-treated cells. At 4 h,
retinoic acid, alone or combined with TNF
, increased alkaline
phosphatase gene transcriptional rate by 2-fold. However, at 24 h,
while no retinoic acid effect was retained, retinoic acid plus TNF
resulted in a 5-fold increase in alkaline phosphatase
transcriptional rate. Examination of the distribution of nuclear
alkaline phosphatase mRNA demonstrated that pre-spliced precursor mRNA
was localized to the nuclear matrix in control and all treatment
groups. Retinoic acid caused a time-dependent accumulation of mature,
spliced alkaline phosphatase mRNA located in the non-matrix and
cytoplasmic fractions, implying a post-transcriptional action of
retinoic acid in nuclear processing and nucleocytoplasmic transport.
Adding TNF
with retinoic acid greatly enhanced this effect, which
was observed after 4 h, prior to any detectable interaction between TNF
and retinoic acid on gene transcription. In sharp contrast, only
a negligible amount of nuclear processing occurred in control and TNF
-treated cells. This study reveals distinct interactions between
TNF
and retinoic acid at post-transcriptional as well as
transcriptional levels to regulate expression of the alkaline
phosphatase gene in preosteoblasts.
(TNF
)
(
)
is a multifunctional cytokine produced chiefly by
infiltrating macrophages and monocytes at sites of inflammation to
regulate cell function locally. The actions of TNF
are mediated
by specific cell surface receptors present on virtually all cells
examined. Binding of TNF
followed by internalizaton of the
cytokine-receptor complex results in the activation of multiple signal
transduction pathways, transcription factors, and regulation of
transcription of a wide array of genes (for reviews, see Refs.
1-4).
and other
cytokines. For instance, many of its functions are synergistic with
interleukin 1. Together, they have pivotal roles in immune and
inflammatory responses, mediating host reactions to stimuli as diverse
as infections and tumor growth. Some of the mechanisms of cytokine
interaction have been studied in detail. Thus, it has been shown that
the induction of interleukin 6 by TNF
or interleukin 1 required
the participation of at least three different cis-acting elements in
the promoter region of the interleukin 6 gene, which, in turn, came
about through the activation of more than one signaling pathway or
transcription factor
(6, 7, 8) . TNF
also
cooperates with systemic factors such as
1,25(OH)
-dihydroxyvitamin D
or retinoic acid to
induce the differentiation of human leukemia-derived myeloid cell lines
(9, 10) , but the manner in which these interactions
occur remains largely unexplored.
potentiated retinoic acid-induced increase in alkaline
phosphatase activity in a rat clonal pre-osteoblastic cell line, UMR
201
(11) . Other investigators have also shown that osteoblasts
are targeted by TNF
. For instance, treatment with TNF
decreased collagen synthesis in fetal rat calvariae
(12) and
stimulated proliferation in human osteosarcoma as well as trabecular
bone cells
(13, 14) . TNF
has also been shown to
stimulate osteoclastic bone resorption in organ culture
(12) ,
possibly by stimulating the proliferation of marrow mononuclear cells
and enhancing the differentiation of committed osteoclast progenitors
(15) . The effects of TNF
on mature osteoclasts in
vitro are likely to be indirect, requiring the mediation of
cytokines secreted by co-cultured osteoblasts
(16, 17, 18, 19) .
interacted
with retinoic acid in the expression of the alkaline phosphatase gene
at transcriptional and post-transcriptional levels. We have recently
identified an important mechanism of action of retinoic acid whereby
the steady state mRNA levels of alkaline phosphatase were increased
primarily by enhancement of nuclear processing rather than by an
increase in gene transcription
(20) . The present results show
that TNF
regulates alkaline phosphatase gene expression by
facilitating the actions of retinoic acid at post-transcriptional as
well as transcriptional levels.
Materials
-Modified minimum
essential medium (
-MEM) was purchased from Flow Laboratories
Australia, Pty Ltd. (Mt. Waverley, Victoria, Australia). Fetal bovine
serum (FBS) was a product from Grand Island Biological Co. Laboratories
(Melbourne, Victoria, Australia). [
-
P]dCTP,
[
-
P]UTP, and
[
-
P]ATP were purchased from Amersham,
Australia, Pty. (Sydney, New South Wales, Australia). All- trans retinoic acid (retinoic acid) was purchased from Sigma. Dr. M.
Young (NIDR, National Insitutes of Health) provided the cDNA for
chicken actin. Dr G. Rodan (Merck Sharpe and Dohme) provided the cDNA
for rat alkaline phosphatase
(21) . Recombinant murine TNF
was obtained from Dr. R. G. Hammonds, Jr. (Genentech Inc., San
Francisco). Nuclease S1 and RNase-free DNase I were purchased from
Boehringer Mannheim GmbH (Mannheim, Germany). All other reagents were
of analytical grade obtained from standard suppliers.
Cell Cultures
UMR 201 cells were
routinely grown in -MEM containing 10% FBS
(11) .
Incubation was carried out at 37 °C in a humidified atmosphere
equilibrated with 5% CO
in air.
Northern Blot Analysis
Total RNA was
isolated with guanidine thiocyanate
(23) , separated in a 1.5%
agarose-formaldehyde gel, and transferred to nylon filters
(24) . Filters (Hybond-N, Amersham, UK) were hybridized
overnight in SSPE buffer (1 SSPE = 150 m
M NaCl,
10 m
M NaH
P0
H
0, 1 m
M sodium EDTA, pH 7.4) as previously described
(20) . cDNA
probes were nick-translated with [
-
P]dCTP
to a specific activity of 1
10
dpm/µg of DNA
(Boehringer Mannheim). The filters were washed sequentially in 2
SSPE with 0.1% SDS at 42 °C for 15 min, 1
SSPE with
0.1% SDS at 65 °C for 30 min, and finally 0.1
SSPE with
0.1% SDS at room temperature for 15 min. Specifically bound probe was
visualized by autoradiography and quantified by densitometry at
appropriate exposures (Molecular Dynamics, model 300A). Relative mRNA
levels were normalized for loading variability by comparison with actin
mRNA levels in the same filters.
Nuclear Run-on Analysis
Transcriptional
rates of alkaline phosphatase and actin genes were determined by
nuclear run-on analysis as previously described
(25) . UMR 201
cells were grown in 500 cmtrays in
-MEM containing
10% FBS until 80% confluent before the medium was changed to
-MEM
containing 2% vitamin A-deficient FBS, and treatment commenced with 1
µ
M retinoic acid, 0.6 n
M TNF
, or 1
µ
M retinoic acid together with 0.6 n
M TNF
for 4 and 24 h, respectively. The cDNA for alkaline phosphatase (10
µg) and actin (5 µg) were denatured and immobilized on
nitrocellulose (Hybond-C, Amersham, UK). Quantitation was carried out
by densitometry of submaximal signals and normalized with reference to
the actin signal on the same filter.
Subcellular Fractionation
Subcellular
fractions of UMR 201 cells, consisting of nuclear matrix with which
precursor mRNA is almost exclusively associated, pooled DNase I/salt
eluate enriched in mature mRNA, nuclear membrane, and cytoplasm were
obtained as previously described
(20) using a method originally
described by Leppard and Shenk
(26) . UMR 201 cells were grown
in 500-cmtrays in
-MEM containing 10% FBS until 80%
confluent before treatment with 1 µ
M retinoic acid, 0.6
n
M TNF
, or 1 µ
M retinoic acid together with
0.6 n
M TNF
in
-MEM containing 2% vitamin A-deficient
FBS for the indicated incubation times.
S1 Nuclease Protection Assay
S1 nuclease
protection assay was performed as previously described
(20, 28, 29) . A 27-nucleotide
(5`-CAATATAGCTGCCACATGCCTGCTCAC-3`) complementary to the start of the
second intron of the rat liver alkaline phosphatase gene
(30) was labeled with [-
P]ATP at the
5`-end. 50,000 cpm of the labeled nucleotide was hybridized to 20
µg of total RNA from each of the subcellular fractions obtained as
described above. The protected DNA-RNA hybrid was extracted with
phenol-chloroform, ethanol precipitated, and electrophoresed on a 20%
acrylamide gel containing 8
M urea; the dried gel was then
subjected to autoradiography.
Polymerase Chain Reaction Amplification of
Reverse-transcribed mRNA
First strand cDNA was synthesized
from 2 µg of total RNA by incubating for 1 h at 42 °C with 15
units of AMV reverse transcriptase (Promega) following oligo(dT)
priming. 2 µl of this reaction mixture was submitted to PCR to
amplify the region of the rat alkaline phosphatase gene transcript
across exons 4 and 5 to include intron 4 (see Fig. 5 A).
Primers were a 5`-primer (5`-AGAAAGAGAAAGACCCCAGTT-3`) representing
nucleotides 1-21 of exon 4 and a 3`-primer
(5`-CTTGGAGAGAGCCACAAAGG-3`) representing nucleotides 97-116 of
exon 5
(30) . 20 cycles of amplification were sufficient to
yield a PCR product corresponding to spliced mRNA, whereas 30 cycles of
amplification were required to demonstrate product corresponding to
intron-containing (unspliced) mRNA. Amplification using Taq DNA polymerase (Boehringer Mannheim) with an annealing temperature
of 55 °C was employed in a Perkin-Elmer Corp. 480 thermal cycler.
20 µl of each PCR reaction mixture was run on a 2% agarose gel and
transferred to nylon; products were authenticated by probing with a
mixture of 3 digoxigenin-labeled oligonucleotides corresponding to
sequences within exons 4 and 5 as well as in intron 4 (see
Fig. 5A). Oligonucleotides were labeled with
digoxigenin-dUTP using a 3`-tailing kit (Boehringer Mannheim).
Hybridization was carried out with 2 pmol/ml labeled oligonucleotide in
a buffer containing 5 SSC, 0.02% SDS, 0.1% sarcosine, and 100
µg/ml poly(A) at 55 °C for 14 h. Detection was by
chemiluminescence using Lumigen PPD (Boehringer Mannheim) according to
the manufacturer's instructions. Products of the expected sizes
were obtained, viz. 636 bp for unspliced and 236 bp for
spliced species.
Figure 5:
Subcellular localization of prespliced and
mature alkaline phosphatase mRNA in UMR 201 cells determined by reverse
transcriptase-PCR analysis. A, the alkaline phosphatase gene
comprises 13 exons (30). The locations of the oligonucleotides used for
reverse transcriptase-PCR primers are indicated by the arrows;
oligonucleotide probes to authenticate PCR products are indicated by
solid bars and labeled 1-3.
Oligonucleotide 4 was used for S1 nuclease analysis. Intron 4 is 400
bp. Splicing out intron 4 results in a PCR product of 236 bp; unspliced
mRNA yields a reverse transcriptase-PCR product of 636 bp. B,
subcellular fractions were prepared from control cells or cells treated
for 8 h with 0.6 n
M TNF ( T), 10
M retinoic acid ( R), or TNF
combined with
retinoic acid ( RT). C represents control (untreated)
cells. Fractions are as follows: Cyto, cytoplasmic;
Memb, nuclear membrane; DNase I, DNase
I/high salt eluate; and NM, nuclear matrix. 30 cycles of
amplification of reverse-transcribed mRNA from each fraction gave rise
to an amplified fragment of approximately 636 bp only in the nuclear
matrix fraction. The fragment hybridized to specific
digoxigenin-labeled oligonucleotides ( 1-3) complementary
to exons 4 and 5 and intron 4. Chemiluminescence was detected by
exposure of filters to x-ray films at room temperature. The
amplification reaction, which produced a 236-bp product corresponding
to exon 4 spliced to exon 5, was carried out for 20 cycles.
Reverse-transcribed mRNA was also amplified for 20 cycles using rat
GAPDH primers to produce the expected 414-bp fragment, which hybridized
to a specific digoxigenin-labeled internal oligonucleotide (see
``Experimental Procedures'').
To ensure equal starting quantities of RNA in each
sample, the reverse-transcribed material was also amplified using
oligonucleotide primers specific for rat GAPDH
(31) . A 414-bp
fragment was amplified using as 5`-specific olignucleotide, GAPDH-4
(5`-CATGGAGAAGGCTGGGGCTC-3`, representing nucleotides 306-325 of
rat GAPDH) and 3`-specific oligonucleotide, GAPDH-5
(5`-AACGGATACATTGGGGGTAG-3`, representing nucleotides 701-720).
Products were verified with a digoxigenin-labeled internal sense strand
oligonucleotide, GAPDH-1 (5`-GCTGTGGGCAAGGTCATCCC-3`, representing
nucleotides 640-659) using the hybridization conditions described
above.
Alkaline Phosphatase mRNA Steady State
Levels
We have previously reported that alkaline
phosphatase activity was highly induced in UMR 201 cells by retinoic
acid with a peak effect observed at 1 µ
M (32) .
Although TNF was without effect in inducing alkaline phosphatase
activity, retinoic acid-induced alkaline phosphatase activity was
greatly enhanced when UMR 201 cells were treated with TNF
and
retinoic acid together. This effect was dose dependent with respect to
TNF
, plateauing after 0.06 n
M (33) . In the
present study, UMR 201 cells were treated with a maximal concentration
of 0.6 n
M TNF
, 1 µ
M retinoic acid, or
retinoic acid together with TNF
. Total RNA prepared from the
cells after treatment for 2, 6, 12, or 24 h, respectively, was
separated in a 1.5% formaldehyde-agarose gel, transferred to nylon
filters, and probed for alkaline phosphatase mRNA in a Northern blot
analysis. The results show that retinoic acid progressively stimulated
the expression of alkaline phosphatase mRNA, detectable by 6 h (Fig.
1). Combined treatment with retinoic acid and TNF
resulted in
much earlier expression of alkaline phosphatase mRNA, now detectable at
2 h. In addition, an increase in the steady state level of alkaline
phosphatase mRNA, above that obtained with retinoic acid treatment
alone, was observed at all other time points. mRNA for alkaline
phosphatase was not expressed in control (untreated) UMR 201 cells, and
TNF
alone had no effect on alkaline phosphatase mRNA expression.
These results correlate very well with our previous report on the
action of TNF
and retinoic acid on alkaline phosphatase enzymatic
activity in these cells
(33) .
and the manner
in which it interacts with retinoic acid, the individual and combined
effects of these factors on the transcriptional and
post-transcriptional regulation of expression of the alkaline
phosphatase gene were examined.
Transcription of Alkaline Phosphatase
Gene
Transcription of the alkaline phosphatase gene was
examined by nuclear run-on analysis after UMR 201 cells were treated,
as described above, for 4 or 24 h, respectively (Fig. 2). The first
time point of 4 h was chosen to precede the earliest detectable
increase in alkaline phosphatase mRNA induced by retinoic acid (6 h,
Fig. 1
). The alkaline phosphatase gene was found to be
constitutively transcribed by untreated (control) UMR 201 cells and
those treated with TNF alone, despite the absence of detectable
alkaline phosphatase mRNA (Fig. 1). After 4 h, there was a small,
approximately 2-fold increase in the transcriptional rate of the
alkaline phosphatase gene in cells treated with 1 µ
M retinoic acid alone. There was also an increase in the rate of
transcription in cells treated with retinoic acid and 0.6 n
M TNF
but only to a similar extent as that induced by retinoic
acid alone. After treatment for 24 h, the significant feature was the
striking 5-fold increase in the rate of transcription of the alkaline
phosphatase gene in cells treated with retinoic acid and TNF
(Fig. 2, bottom row). The earlier effect of
retinoic acid alone in stimulating alkaline phosphatase gene
transcription was transient and no longer observed at 24 h, as
previously reported
(20) .
Figure 1:
Detection of mRNA for
alkaline phosphatase ( ALP) mRNA in UMR 201 in a Northern blot
analysis. C denotes control (untreated) cells. Cells were
treated with 0.6 n
M TNF ( T), 1 µ
M retinoic acid ( R), or 1 µ
M retinoic acid
combined with 0.6 n
M TNF
( RT) for the indicated
times. Total RNA (20 µg) was loaded into each lane. The
filter was initially probed with a plasmid containing a 2.4-kilobase
cDNA for rat alkaline phosphatase, washed, and reprobed with a chicken
actin cDNA for normalization. The arrows point to the
positions of the 28 and 18 S ribosomal bands, respectively. This is a
representative experiment performed three times with similar
results.
Figure 2:
Nuclear run-on analysis of transcriptional
rates of the alkaline phosphatase and actin genes in UMR 201 cells.
Cells were grown in 500-cmtrays either untreated
( C) or treated with 0.6 n
M TNF
( T), 1
µ
M retinoic acid ( R), or 1 µ
M retinoic acid combined with 0.6 n
M TNF
( RT) for 4 h ( top row) and 24 h ( bottom row), respectively. Nuclei were prepared, and run-on
transcription was allowed to proceed in the presence of 100 µCi of
[
-
P]UTP as described under
``Experimental Procedures.'' Newly synthesized labeled RNA
was hybridized to immobilized cDNAs as indicated. ALP,
alkaline phosphatase; pBS is the empty vector used to provide
a signal corresponding to nonspecific hybridization. Quantitation was
carried out by densitometry at appropriate exposures and normalized by
reference to the actin signal. This experiment was performed twice, in
duplicate, and a representative result is shown
here.
Nuclear Processing of Alkaline Phosphatase
mRNA
Clearly, there is a discrepancy between the small
increase in transcriptional rate of the alkaline phosphatase gene, as
well as the time course of those increases in response to the various
treatments, and the observed changes in the steady state levels of the
alkaline phosphatase mRNA in the Northern blot analysis shown in Fig.
1. To examine post-transcriptional events, therefore, UMR 201 cells
were subfractionated to isolate the nuclear matrix, DNase I/salt
eluate, nuclear membrane, and cytoplasmic fractions. The extent of
nuclear processing is indicated by the relative distribution of
precursor and mature alkaline phosphatase mRNA between the subnuclear
and cytoplasmic fractions.
, 1 µ
M retinoic acid,
or TNF
combined with retinoic acid, respectively, and compared
with untreated cells. The cells were subfractionated, and total RNA
from the various fractions was analyzed in two ways. First, to detect
pre-spliced precursor mRNA, solution hybridization to a 27-nucleotide
oligomer, complementary to the start of the second intron of the rat
alkaline phosphatase gene, was carried out before S1 nuclease digestion
of single-stranded nucleic acid was performed (Fig. 3). The protected
DNA-RNA hybrid was only detected in the nuclear matrix fraction of
control and treated cells, confirming that precursor alkaline
phosphatase mRNA was present specifically in that fraction. Although
the band in the nuclear matrix fraction of control cells was only
faintly visible, it was consistently present in repeated experiments.
Fig. 3
shows the results from an experiment where cells were
treated for 4 h, but similar results were obtained after 24 h (data not
shown).
Figure 3:
Subcellular localization of precursor mRNA
for alkaline phosphatase in UMR 201 cells using an intronic probe in a
S1 nuclease protection assay. Cells were grown in 500-cmtrays untreated (control, C), treated with 0.6 n
M TNF
, 1 µ
M retinoic acid ( R), or 1
µ
M retinoic acid combined with 0.6 n
M TNF
( RT) for 4 h. The isolated subcellular fractions, obtained as
described under ``Experimental Procedures,'' were cytoplasm,
nuclear membrane, DNase I/salt eluate, and nuclear matrix. Total RNA
was prepared from each fraction, and 20 µg were allowed to
hybridize in solution with a 27-nucleotide oligomer, complementary to a
sequence in the second intron of the alkaline phosphatase gene, as
described under ``Experimental Procedures.'' 50,000 cpm of
labeled oligomer was added to each reaction. The protected DNA-RNA
hybrid was electrophoresed on a 20% acrylamide gel containing 8
M urea, and the dried gel was subjected to autoradiography. This
experiment was performed three times, and a representative result is
shown here.
The second analysis was designed to determine whether the
precursor mRNA was being processed into mature mRNA, which can be
translocated from the nucleus into the cytoplasm. Total RNA from the
various subcellular fractions was separated in a 1.5%
formaldehyde-agarose gel, transferred to nylon filters, and probed with
a 2.4-kilobase full-length cDNA encoding rat alkaline phosphatase (Fig.
4). In contrast to the results obtained in Fig. 3, there was only
a minute amount of mature, spliced alkaline phosphatase mRNA in the
nuclear membrane fraction of control and TNF -treated cells,
despite the presence of precursor mRNA in the nuclear matrix fraction.
On the other hand, retinoic acid treatment resulted in the accumulation
of mature alkaline phosphatase mRNA mainly within the non-matrix and
cytoplasmic fractions, as previously reported
(20) . When the
cells were treated with the combination of TNF
and retinoic acid,
not only was there a considerable increase in the amount of mature
alkaline phosphatase mRNA located in the subcellular fractions, but it
occurred to such an extent that it was present even in the nuclear
matrix fraction. This enhancement of nuclear processing of alkaline
phosphatase mRNA transcripts with the combined treatment was evident at
all time points, beginning at 4 h. This early time point clearly
preceded the effect of the combined treatment on increasing the rate of
alkaline phosphatase gene transcription beyond that induced by retinoic
acid treatment alone (Fig. 2). Importantly, TNF
alone did
not have any notable influence on nuclear processing.
was
toxic to the cells.
Reverse Transcriptase-Polymerase Chain Reaction
Analyses
To verify these results and at the same time to
seek evidence of elongation of the nascent transcript, we employed
reverse transcriptase-PCR to amplify a sequence corresponding to intron
4 of the alkaline phosphatase gene, using primers complementary to
exons 4 and 5 (see Fig. 5 A). UMR 201 cells were
subfractionated after treatment with TNF , retinoic acid, or TNF
combined with retinoic acid for 8 h. Consistent with the S1
nuclease results, a 636-base pair amplified product corresponding to
RNA containing intron 4 was present in the nuclear matrix fractions
only of control and treated cells (Fig. 5 B). In
addition, a 236-base pair product corresponding to spliced mRNA was
seen in cells treated with retinoic acid alone or when combined with
TNF
. There was clearly a much greater abundance of spliced
alkaline phosphatase mRNA in all of the subcellular fractions in
response to the combined treatment (Fig. 5 B). DNase
treatment during the nuclear fractionation procedure ensured that this
product did not result from contaminating genomic DNA.
acted in concert to control the level of
alkaline phosphatase mRNA in UMR 201 cells
(11) . The present
results advance our understanding of this interaction. TNF
itself
had no effect on the transcriptional rate of the alkaline phosphatase
gene. When combined with retinoic acid, there was a significant
increase in transcriptional rate of this gene over and above the
increase caused by retinoic acid alone, but this was clearly evident
only when the cells had been treated for 24 h. The Northern blot
analysis, however, showed that the increase in steady state level of
alkaline phosphatase mRNA induced by the combination of TNF
and
retinoic acid was demonstrable after treatment for only 2 h, which
significantly preceded the induction of alkaline phosphatase mRNA by
retinoic acid treatment alone. These results point to the need to
consider a potential role for post-transcriptional regulatory events in
determining the steady state level of cytoplasmic alkaline phosphatase
mRNA.
-treated UMR 201 cells, and this was verified by
the demonstration of precursor alkaline phosphatase mRNA in the nuclear
matrix fraction of all treatment groups using the sensitive S1 nuclease
protection assay as well as reverse transcriptase-PCR. The detection of
only minute amounts of mature alkaline phosphatase mRNA in the nuclear
membrane fraction of control and TNF
-treated cells testifies to
the inefficiency of nuclear processing in cells grown under those
conditions. It further implies that newly transcribed precursor
alkaline phosphatase mRNA must be rapidly degraded within the nuclear
matrix fraction.
and interleukin-1
precursor mRNA, but these
were not processed into mature mRNA, requiring treatment with
cycloheximide to facilitate nuclear processing
(34) . These
recent findings may imply that the effect of retinoic acid on nuclear
processing might be cell-type and/or gene specific. An additional
implication of those results is that retinoic acid requires
collaboration with additional cofactor(s) to direct or enable
intranuclear processing of transcripts. In the present case, TNF
serves as one such cofactor to greatly enhance the retinoic
acid-induced nuclear processing.
alone appeared
not to influence either the transcription of the alkaline phosphatase
gene or the nuclear processing of the mRNA, it significantly enhanced
the post-transcriptional actions of retinoic acid early in the course
of the interaction and, later on, also with the transcriptional events.
Although it must still be suggested that the enhancement of retinoic
acid-induced nuclear processing by TNF
may be a consequence of
the greatly increased transcriptional rate of the alkaline phosphatase
gene induced by the combined treatment, the considerable delay before
this became evident is not consistent with this possibility.
or transforming growth
factor
, acting as an autocrine or paracrine regulator of cell
function, is ideally located to modulate the effects of systemic
agents. It is interesting to note that the mechanisms of action of TNF
in the regulation of alkaline phosphatase gene expression shown
in this study contrasted significantly with that of transforming growth
factor
(20) . Unlike TNF
, transforming growth factor
increased the transcriptional rate of alkaline phosphatase gene,
but that action was not accompanied by increased nuclear processing of
precursor mRNA.
interacts with retinoic acid at post-transcriptional as well as
transcriptional levels to regulate alkaline phosphatase gene
expression. Since alkaline phosphatase expression is a marker of
differentiation in osteoblasts, the enhancement of retinoic acid action
by TNF
suggests that this cytokine may further promote the
differentiation of preosteoblastic UMR 201 cells. Furthermore, this
system will be an excellent model in which to study the mechanisms
involved.
, tumor necrosis factor
; FBS, fetal bovine serum; PCR,
polymerase chain reaction; bp, base pair(s);
-MEM,
-modified
minimum essential medium; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.