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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3945-3950
NEOPLASIA
Differential expression of a novel C-terminally
truncated splice form of SMAD5 in hematopoietic stem cells
and leukemia
Yunfang Jiang,
Hong Liang,
Wei Guo,
Lazar
V. Kottickal, and
Lalitha Nagarajan
From the Department of Molecular Genetics, MD Anderson Cancer
Center, University of Texas, Houston, TX.
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Abstract |
SMADs are evolutionarily conserved transducers of the
differentiation and growth arrest signals from the transforming growth factor/BMP (TGF/BMP) family of ligands. Upon receptor activation, the
ligand-restricted SMADs1-35 are phosphorylated in the
C-terminal MH2 domain and recruit the common subunit SMAD4/DPC-4 gene
to the nucleus to mediate target gene expression. Frequent inactivating
mutations of SMAD4, or less common somatic mutations of
SMAD2 seen in solid tumors, suggest that these genes have a
suppressor function. However, there have been no identified mutations
of SMAD5, although the gene localizes to the critical region of loss in
chromosome 5q31.1 (chromosome 5, long arm, region 3, band
1, subband 1) in myelodysplasia (MDS) and acute myelogenous leukemia
(AML). A ubiquitously expressed novel isoform,
SMAD5 , encodes a 351 amino acid protein with a truncated MH2 domain and a unique C-terminal tail of 18 amino acids, which may be the functional equivalent of inactivating mutations. The levels of SMAD5 transcripts are
higher in the undifferentiated CD34+ hematopoietic stem
cells than in the terminally differentiated peripheral blood
leukocytes, thereby implicating the form in stem cell homeostasis.
Yeast 2-hybrid interaction assays reveal the lack of physical
interactions between SMAD5 and SMAD5 or SMAD4. The expression of
SMAD5 may represent a novel mechanism to
protect pluripotent stem cells and malignant cells from the growth
inhibitory and differentiation signals of BMPs.
(Blood. 2000;95:3945-3950)
© 2000 by The American Society of Hematology.
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Introduction |
The transforming growth factor- (TGF-)
superfamily of multifunctional cytokines regulates proliferation,
differentiation, and apoptosis in hematopoietic cells and a variety of
other tissues and cell types.1 The
TGF-/BMP/activin family of ligands signals by binding to specific
transmembrane receptors.2 In response to ligand binding,
the type II receptor, which is a serine/threonine kinase, initiates a
signaling cascade by phosphorylating the serine/threonine kinase type I
receptor. The type I receptor in turn phosphorylates the specific
ligand-restricted SMAD, which heterodimerizes with the common mediator
SMAD4/DPC4 and translocates into the nucleus to recruit transcriptional
coactivators. The multiprotein complex thus assembled induces target
gene expression.3-5
In the vertebrates, 8 highly conserved SMADs were identified and
categorized into 3 groups: (1) ligand-restricted SMADs (SMAD1 and SMAD5 respond to BMP, and SMAD2 and SMAD3
respond to activin and TGF-); (2) negative regulators (SMAD6
and SMAD7); and (3) a common mediator/subunit
(SMAD4/DPC4). The SMAD genes are modular because they
comprise 3 major domains: (i) the highly conserved N-terminal DNA
binding domain (MH1); (ii) the C-terminal regulatory domain (MH2),
which is involved in SMAD-receptor interaction, SMAD homodimerization,
SMAD-SMAD4 interaction, and SMAD-transcription cofactor interaction;
and (iii) a variable proline/serine-enriched linker region that is the
target of phosphorylation by mitogenic signals.3-5
The MH1 and MH2 domains inhibit each other in the basal state, and
tumor-specific mutations enhance autoinhibition.6 In response to receptor-mediated phosphorylation, this inhibition is
relieved, allowing the heterodimeric interactions through
the MH2 domain. X-ray crystallographic analysis of the SMAD1 MH1 domain reveals a novel DNA-binding motif consisting of a conserved 11-residue hairpin that can be embedded in the major groove of DNA to
bind the 4-base pair (4-bp) "SMAD box" motif.7
The physiological responses to the TGF- family of ligands, namely
growth inhibition and differentiation, render this pathway a critical
target for inactivation during neoplastic transformation. The common
subunit SMAD4/DPC4 was initially isolated as a tumor suppressor
that is homozygously deleted in patients with pancreatic cancer.8 Inactivating mutations of SMAD2 have also
been identified in patients with colon cancer.9
The SMAD5 gene is localized to human chromosome 5q31
(chromosome 5, long arm, region 3, band 1) to a region of invariant
allele loss (subband 1) in human myeloid leukemia. The lack of gross intragenic mutations suggests that SMAD5 is not a common target of somatic inactivation in leukemia and solid tumors.10-13
Nonetheless, microinjection of mouse SMAD5 transcripts into
Xenopus toad embryos induces embryonic globin synthesis
analogous to the response seen with BMP-4 transcripts.14
The human SMAD5 gene is transcribed into both major
(8.7-kilobase [8.7-kb]) and minor (4.2-kb) transcripts in several
hematolymphoid tissues including the spleen, fetal liver, appendix,
thymus, lymph node, bone marrow, and peripheral blood
leukocytes.10
Homozygous SMAD5 null mice undergo embryonic lethality with
abnormal vasculature and blood cells.15,16 Recent studies
demonstrate that SMAD1 and SMAD5, as well as
the type I receptors ALK3 and ALK6, are expressed in
CD34+CD38 lin human
hematopoietic stem cells, which are capable of giving rise to all the
lineages in nonobese diabetic/severe combined immune deficient
(NOD/SCID) mice.17 Furthermore, the
CD34+CD38 lin cells
show a unique biphasic response to BMP-4, with growth inhibition at low
concentrations and enhanced colony formation at higher concentrations.17
TGF-, a potent inducer of erythroid differentiation and inhibitor of
granulocytic macrophage progenitors, has a sparing effect on early
progenitors.18-20 Despite the growing body of evidence, very little is known about the mechanisms underlying the differential regulation of hematopoiesis by TGF-/BMP. Specifically, the precise regulation of hematopoietic stem cell, progenitor self-renewal, and
differentiation in the presence of circulating TGF-/BMP are poorly understood.
Here, we report an alternative splice form of SMAD5, designated
SMAD5, which encodes the entire MH1 and linker
domains, and a truncated MH2 domain. The stoichiometry between the
SMAD5 and SMAD5 transcripts is
higher in undifferentiated CD34+ hematopoietic bone marrow
stem cells than in terminally differentiated peripheral blood cells.
The SMAD5 isoform does not homodimerize or heterodimerize with SMAD5
or SMAD4. Thus, the enhanced expression of the alternative splice form
may explain, in part, the refractoriness of hematopoietic
stem cells to BMP-4.
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Materials and methods |
Genomic polymerase chain reaction
Long polymerase chain reactions (long-PCRs) were performed to obtain
genomic fragments spanning adjacent exons. We used the bacterial
artificial chromosome (BAC) b37i1610 as a template and the
ELONGase enzyme mix (Life Technologies, Gaithersburg, MD). The
following conditions of amplification were applied: initial denaturation (94°C for 2 minutes); then 30-35 cycles of
denaturation (94°C for 30 seconds), annealing (55-58°C for 30 seconds), and extension (68-70°C for 2-10 minutes); and a final
extension (68-70°C for 5-10 minutes). Intron 3 was amplified with
primers Ex1.F and Ex2.R; intron 4, JV5.1.F2 and JV5.1.R1; intron 5, Ex3.F and Ex4.R; intron 6, Ex4.F and Ex5.R; and intron 7, Ex5.F and
Ex6.R. Amplification of primers Ex1.F/Ex2.R, Ex4.F/Ex5.R, and
Ex5.F/Ex6.R were reported previously.10 The remaining
primers were amplified as follows: JV5.1.F2, 5'-AGC AAG TTC TGG
ACC AGG AAG-3'; JV5.1.R1, 5'-ATT ATT GCT TGT ATC CAT
AGG CT-3'; Ex3.F, 5'-CTA TCC TCA CTC CTA TCC TCA-3';
and Ex4.R, 5'-TAT AAA ATA CCG TAA GGC TGA-3'.
Hematopoietic cells, leukemia cells, and RNA isolation
Normal peripheral blood mononuclear cells (PBMCs) were enriched by
Ficoll Hypaque (Organon Teknika, Durham, NC) gradient. Bone marrow from healthy donors was sorted for CD34 expression by
fluorescence activated cell sorter (FACS). Several acute myelogenous leukemic cells with anomalies of chromosome 5q31 were used in the
study. Of these, AML193 (acute myelomonocytic leukemia), TF1 (erythroleukemia), and U937 (myelomonocytic leukemia) harbor
uncharacterized translocations involving the 5q31 chromosome. KG1
(acute myelogenous leukemia) cells are monosomic for chromosome 5, and
ML3 (acute myelogenous leukemia) cells carry a chromosome
deletion, del(5)(q13-q35), and a chromosome derivative, der(3), with
insertion of (5)(q13-35) material. Additionally, HEL (erythroleukemia)
and K562 (Ph+ chromosome, chronic myelogenous leukemia in
erythroid blast crisis) were also included. The cells were cultured
according to their growth requirements in humidified air containing 5%
carbon dioxide (CO2) at 37°C. ML-3, HEL, K562, and U937
cells were cultured in Roswell Park Memorial Institute medium (RPMI
1640) with 10% fetal bovine serum (FBS), and KG1 cells in Iscove's
Modified Dulbecco's medium (IMDM) with 20% FBS. The factor-dependent
TF1 cell line was grown in RPMI 1640 with 10% FBS plus 5 ng/mL
granulocyte macrophage-colony stimulating factor (GM-CSF), and the
factor-dependent cell line AML193 was grown in IMDM with 10% FBS plus
5 µg/mL transferrin, 5 µg/mL insulin, and 5 ng/mL GM-CSF.
The total RNA was prepared from PBMCs, bone marrow hematopoietic stem
cells from a normal donor flow sorted for the expression of the CD34
antigen, and leukemia cells in exponential growth by the TRIzol reagent
(Gibco BRL, Gaithersburg, MD) or guanidium isothiocyanate lysis.
Radioactive reverse transcriptase-PCR
A total of 3 µg RNA from each source was reverse transcribed
with oligo-dT primers, and the complimentary DNA (cDNA) pool was
diluted 40-fold. The following primer pairs were used for SMAD5: JV5.1.F4, 5'-TAA ACA ATC GTG TTG
GAG AAG C-3', and Ex4.RP, 5'-ATA CAT TCT TCA ATA TCG GCA
ACT-3'. The following primer pairs were used for full-length
SMAD5: JV5.1.F14, 5'-AAA TGT GTA CCA TTC GGA TGA
G-3', and JV5.1.R13, 5'-AAA CAG AAG ATA TGG GGT TCA G-3', and -2-microglobin (-2-m) was used as
an internal control. We pooled 5 pmol of each sense primer and
end-labeled them with 32P ATP (adenosine
5'-triphosphate) using polynucleotide kinase in the same
reaction. Unlabeled primer pairs (10 pmol) spiked with 0.1 pmol of the
end-labeled reverse primer were used in each amplification reaction,
and 30 cycles of amplifications were performed. The expected product
sizes were 307-bp SMAD5, 170-bp
SMAD5, and 114-bp -2-m. The
linear nature of the assay conditions was first established by
quantitating the products against a range of reference templates. Three
independent PCRs were performed for each sample and resolved on an 8%
polyacrylamide gel, and the amplifications were quantified in a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Competitor DNA constructs for quantitative PCR
To make a SMAD5 competitor construct, we designed a forward
primer that will anneal to the cDNA template 54 bases downstream of the
3' end of P1 (in the SMAD5 exon 6 at position 18 nucleotide [nt]). This was done by using a forward primer (P2,
5'-AGC CTA TGG ATA CAA GCA ATA ATT ATG AAG AGC CTA AAC ATT GGT
G-3') that contains the entire sequence of the native forward
primer (P1, 5'-AGC CTA TGG ATA CAA GCA ATA AT-3') at the
5' end (in the SMAD5 exon 5 at position 61 nt) followed
by a sequence downstream of P1. When used in PCR, this primer, together
with the 3' end reverse primer (P3') in the SMAD5 intron
6 at position 120 nt, generated a 373-bp fragment that was cloned into
pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) as a
SMAD5 competitor. The P1 and P3 primer pair yielded a 427-bp native
SMAD5 fragment. For the SMAD5
competitor construct, the primer P2 is the same as that for the
SMAD5 competitor, whereas the 3'
reverse primer (P3, 5'-GAT TAA CAT TTG ACA ACA AAC CC-3')
is complementary to SMAD5 exon 6 at position 158 nt. The P2 and P3 primer pair amplified a 196-bp fragment for
the SMAD5 competitor construct, and the primer P1 and P3 amplified a
250-bp native SMAD5 fragment.
Quantitation by competitive PCR
During PCR, a constant amount of synthesized cDNA (1:20 dilution, 2 µL for each reaction) was coamplified with varying concentrations of
competitor plasmid DNA (TA/SMAD5 or TA/SMAD5). PCR was performed with an initial denaturation at 94°C for 2 minutes; followed by 35 cycles of denaturation, annealing, and extension at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds,
respectively; and a final extension of 72°C for 5 minutes. At least
2 independent PCRs were performed for each sample. PCR products were
resolved by 2% agarose gel electrophoresis. The gel was stained with
ethidium bromide and then photographed. The ratio of intensity between native versus competitor bands was examined for each lane.
Yeast 2-hybrid interaction assays
To analyze protein-protein interactions, the LexA-based yeast
2-hybrid system (Invitrogen) was used. The full-length cDNA sequences
of SMAD5 and SMAD5 were subcloned into
pHyb/LexA and pYESTrp2 vectors, respectively.
SMAD5.N345S, corresponding to a common
polymorphism, as well as SMAD5.e6T, which is truncated at codon
333, were also cloned into the corresponding vectors. Sequences of all
constructs were confirmed to be accurate and in-frame. The plasmids
were transformed into the yeast L40 strain, and the expression was
verified by immunoblots with anti-LexA antibody, which recognizes the
epitope-tagged fusion proteins. The bait plasmids were initially
checked for nonspecific activation (histidine prototrophy and
-galactosidase activity). Protein interactions were detected by the
filter assay, which scores for -galactosidase activity on the filter
containing X-gal (5-bromo-4-chloro-3-indolyl--D-galactoside).
Databases
The following databases were employed in the study:
Whitehead Institute genome database,21 dbEST (IMAGE
consortium clones),22 and the Caenorhabditis
elegans genome database at Washington University, St Louis,
MO.23
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Results |
Characterization of the genomic organization of SMAD5 and
identification of an alternatively spliced form,
SMAD5
Previous studies concluded that the SMAD5 gene is made up of
8 exons, with the translation start at exon 3.13 An initial screen of the BAC b37i16 with sequence-tagged sites (STSs) derived from
the 5' and 3' untranslated regions (UTRs) of the
full-length SMAD5 cDNA were positive, indicating that the entire gene
resided within the 180-kb insert. The BAC b37i16 was used as a template to determine the size of the introns. Primers designed from the known
exonic and partial intronic sequences were used under multiple conditions to obtain amplifications of 1- to 10-kb genomic targets.
The genomic organization of SMAD5 shown in Figure
1 reveals that the coding sequences are
contained in a fragment of about 22.6 kb. Figure 1 also depicts the
modular nature of the SMAD5 gene. The entire MH1 domain is
encoded by exon 3, the unique linker region is encoded by
exons 4 and 5, and the MH2 domain is encoded by exons 6-8. As we could
not determine the exact size of exon 8 (3'UTR) nor identify a
polyadenylation signal, the Expressed Sequence Tagged Sequences (EST)
database (dbEST)22 was searched for cDNA clones with
polyadenylation signal. Surprisingly, a single cDNA clone from ovarian
cancer (IMAGE consortium clone ID594181) with a canonical
polyadenylation (poly-A) signal and a poly-A tail was identified. Upon
sequencing, the cDNA 594181 with an insert size of 770 bp was found to
encode a transcript that diversified within the MH2 domain at codon
334, although the upstream linker sequences were identical to
SMAD5. A comparison of the clone 594181 sequences with the
partial genomic sequences from BACb37i16 revealed that the EST clone
was an alternate splice form of SMAD5, in which the 5'
splice donor sequences of intron 6 were suppressed (Figure 2A). We therefore designated the novel
alternate splice form SMAD5.
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| Fig 1.
Organization of the human SMAD5 gene.
The boxes represent exons, and the lines denote introns. Filled boxes
with jagged lines represent the partial 5' and 3' UTR. The
SMAD5 coding region resides between the ATG start codon at exon 3 and
the TAA stop codon at exon 8. The sizes of the exons are as follows:
exon 3, 574 bp; exon 4, 252 bp; exon 5, 120 bp; exon 6, 222 bp; and
exon 7, 257 bp. The sizes of the introns are: intron 3, 6.5 kb; intron
4, 2.1 kb; intron 5, 8.0 kb; intron 6, 1.5 kb; and intron 7, 2.5 kb. The exons corresponding to the MH1, linker, and MH2 domains are
noted.
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| Fig 2.
Alternative splice forms of SMAD5.
(A) SMAD5 and SMAD5
diverge at the junction of exon 6. Arrows denote the exon 6/exon 7 junction of SMAD5 or the exon6/intron 6 junction of SMAD5. The
5' splice site (GT) is given in bold. SMAD5 is transcribed
through exon 6 and part of intron 6 to terminate at an alternative
poly-A site, which is underlined. The novel 18 amino acids are given in
bold. Note that codon 334, indicated with triangles, is a glycine (Gly)
in both isoforms. The common nucleotide polymorphism and the
corresponding amino acid polymorphism are italicized. (B) Comparison of
the open reading frames (ORFs) of both isoforms. Schematic depicts the
full-length ORFs of SMAD5 (top), which encode 465 amino acids, and
the C-terminally truncated splice form SMAD5 (bottom), which
encodes 351 amino acids. Both proteins are translated using the same
start site and have an identical amino acid sequence through the end of
exon 6 (333 amino acids).
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SMAD5 encodes a novel carboxyl-terminal
(C-terminal) tail of 18 amino acids, which includes a glycine (G) at
residue 334, a stop codon, and a short 3' UTR (119 nt) with a
canonical poly-A signal within intron 6. Furthermore, the splice donor
and acceptor sites at codon 333 are also conserved in the SMA-2
gene, the closest homolog of SMAD5 in C elegans,
although the much smaller intron (185 bp) of C elegans does not
contain a poly-A signal.23 In contrast to the full-length
SMAD5 that encodes for a protein of 465 amino acids,
SMAD5 would encode a protein of 351 amino acids with a truncated MH2 domain (Figure 2B). The novel 18 amino acid lysine-rich tail does not show any homology to sequences in the DNA and
protein databases. In addition, a single nucleotide polymorphism (A/G) at 37nt, intron 6 of SMAD5, which we
identified previously,10 is predicted to translate into an
amino acid polymorphism of asparagine/serine (N/S) at codon 345 of
SMAD5.
We further confirmed that the SMAD5 transcripts use the same
initiation methionine (M) codon as the full-length protein, and the
alternative splice form was also expressed in hematopoietic tissues.
RT-PCR performed using the cDNA pool from K562 cells with primers
spanning the initiation M of SMAD5 and the predicted stop codon
of SMAD5 yielded the expected product of 1053 bp (Y.J. and L.N., unpublished results, February, 1999).
DNA sequencing confirmed that the alternative transcript used identical
5' sequences.
Enhanced expression of SMAD5 in normal bone
marrow stem cells and in the erythroleukemia TF1 cell line
Full-length SMAD5 and the
SMAD5 isoform could not be readily detected
in normal and malignant cells by Northern blot analyses of total RNA.
Therefore, we employed a radio-label-coupled RT-PCR (RRT-PCR) with
unique primer pairs that detected either the full-length SMAD5 or the
alternative splice form to screen cDNA pools from CD34+
normal bone marrow stem cells and PBMCs. A panel of 7 acute myelogenous leukemia cell lines (ML3, TF1, AML193, HEL, KG1, K562, and U937), which
represented different lineages and stages of myeloid maturation with
and without anomalies of chromosome 5q31 (see "Materials and
methods"), were examined. Products specific for both the full-length form and the shorter isoform were detected in all the cell types examined. Interestingly, the intensity of the SMAD5 product was higher than the full-length SMAD5 in CD34+ hematopoietic
stem cells in contrast to the terminally differentiated PBMCs. Among
the leukemia cell lines, the erythroleukemia TF1 cell line expresses
the highest level of SMAD5 (Figure
3).
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| Fig 3.
Expression of SMAD5 in
hematopoietic and leukemic cells.
A representative autoradiograph of multiplexed RT-PCR products resolved
on a poly-A site. The upper band (307 bp) represents
SMAD5; the middle band (170 bp) represents the
full-length SMAD5; and the lower band (114 bp) represents the
internal control, -2-m. Templates used for amplification are as
indicated: peripheral blood mononuclear cells (PBMCs), normal bone
marrow progenitor cells (CD34+), acute myelogenous leukemia
(ML-3), erythroleukemia (TF1), acute myelogenous leukemia (AML193),
erythroleukemia (HEL), acute myelogenous leukemia (KG1), chronic
myelogenous leukemia in erythroid blast crisis (K562), and
myelomonocytic leukemia (U937).
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We further developed a quantitative competitive PCR (QC-PCR) to
quantify the absolute amount of transcripts present in the initial
cDNAs from PBMCs and CD34+ and TF1 cells. HEL cells were
also included as a control. The results in Figure
4 suggest that the steady-state levels of
SMAD5 transcripts may be similar in all these cells, as seen by equal amplification around 92.5 fmol competitor DNA. In contrast, the levels
of SMAD5 vary from one cell type to another. Thus, equimolar amplification is seen around 8.75 fmol PBMCs, whereas in the
CD34+ cells, equimolar products would be obtained between
43.75 and 87.5 fmol of the competitor. Similarly, the cDNA pool from
TF1 cells needs a competitor template between 43.75 and 87.5 fmol to
yield the same amount of products, unlike the HEL cells, which would
yield equimolar products between 17.5 and 43.75 fmol.
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| Fig 4.
Differential expression of SMAD5 in PBMC,
CD34+, TF1, and HEL cells.
Competitor plasmids, which would yield smaller products from the same
primer pair, were constructed. Serial dilutions of either the SMAD5 or
SMAD5 competitor were coamplified with cDNAs from PBMCs and
CD34+, TF1, and HEL cells. The positions of primers P1, P2,
P3, and P3' are indicated in Figure 2B. The 250-bp and 427-bp
bands correspond to the native SMAD5 and
SMAD5, respectively, and the 196-bp and 373-bp
bands correspond to the competitor templates. PCR products were
resolved in 2% agarose gel and stained with ethidium bromide. The
photographs were reversed to give a negative version.
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The results depicted in Figure 4 suggest that the molar ratio of the
full length to the form is higher in PBMCs and HEL cells than in
CD34+ and TF1 cells. Thus, the expression of the
alternative splice form appears to be differentially regulated during
normal hematopoietic maturation. The enhanced expression of the isoform in TF1 versus HEL erythroleukemic cells suggests that the
aberrant expression of the alternative splice form may be due to
genetic alterations that are unique to specific cases and are not
tightly correlated with the morphologic classification or stages of maturation.
SMAD5 does not homodimerize with itself
and heterodimerize with SMAD5 or SMAD4
The MH2 domain truncation in SMAD5 suggests that this isoform may
not readily heterodimerize with SMAD4/DPC4.6,21 However, the possibility of a homodimeric interaction due to the novel C-terminal tail or dimerization with the full-length SMAD5 could not be
ruled out. Interaction studies in the yeast 2-hybrid system, which
lacks endogenous SMAD, were performed to determine if this was indeed
the case. Physical interactions between the proteins were scored based
on the transcription activation of the -galactosidase reporter gene.
Homodimeric or heterodimeric interactions of SMAD5, SMAD5, and SMAD4
were examined. Another SMAD5 construct in which the MH2 domain was
truncated at codon 333 of exon 6 (SMAD5.e6t) and the polymorphic
variant SMAD5.N345S were also included. Results shown in Figure
5 demonstrate that the full-length SMAD5
molecules can both homodimerize and heterodimerize with SMAD4, with
heterodimerization being stronger than homodimerization. None of the
isoforms with the truncated MH2 domain, SMAD5,
SMAD5.N345S, or SMAD5.e6t, homodimerizes or interacts with the
full-length SMAD5 or SMAD4. These results confirm that the C-terminal
MH2 domain spanning amino acids 335-465 is involved in SMAD-SMAD
homodimeric or heterodimeric interactions.24
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| Fig 5.
SMAD5 does not heterodimerize or homodimerize.
The protein interactions of SMADs were assayed by the yeast 2-hybrid
system. Various SMAD forms were expressed in frame with the LexA DNA
binding domain in the pHyb-LexA plasmid (bait): the full-length forms,
SMAD4, SMAD5, and SMAD5; the polymorphic form SMAD5.N345S; and a
truncated form, SMAD5.e6t. Each SMAD was also expressed in frame with
the transcription activation domain B42 in the pYEST-B42AD plasmid
(prey). Transformants were tested for protein interactions using the
-galactosidase filter assay. The intensity of -galactosidase was
scored based on the intensity of the blue color, ranging from no color
( ) to strongly positive (+++).
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Discussion |
The genomic organization and expression of the human SMAD5
gene was studied in order to understand the function of BMPs in normal
hematopoiesis and leukemogenesis. Differential expression of a novel
C-terminal truncated splice form, SMAD5,
provides clues on how hematopoietic stem cells may override
constitutive differentiation and growth arrest cues from the BMP family
of ligands.
The C-terminal truncated SMAD5 isoform
The alternative splice form characterized here is the first example
of a physiological negative regulator of SMADs that may occur at the
level of DNA binding or protein-protein interactions necessary for
transcription. SMAD5 may act predominantly in the nucleus. This is
unlike SMAD6 and SMAD7, the well-characterized negative regulators of
SMAD signaling, which bind to the activated receptors and interfere
with the recruitment and phosphorylation of ligand-specific
SMADs.25-27 Absence of the receptor-mediated phosphorylation sites and heterodimerization domains render the isoform a ligand-independent regulator that can confer refractoriness. This can be achieved by competing with other SMADs to bind to the SMAD
response elements. The MH1 domain of the MAD gene of Drosophila exhibits 100-fold enhanced DNA binding in vitro
compared with the full-length protein.28 Alternatively, the
novel C-terminal tail of the isoform may interact with regulatory
proteins to activate and/or inactivate specific target genes that are
unique to stem cells. Thus, the possibility that SMAD5 is both a
transcriptional coactivator and corepressor, depending upon the
cellular context, cannot be excluded.
The evolutionary conservation of the splice donor and acceptor sites at
codon 333 between exons 6 and 7 of the human SMAD5 gene and the
SMA-2 gene of C elegans is noteworthy. Unlike the human intron, the intron in C elegans is much smaller (185 bp) and lacks a canonical poly-A signal. Thus, the SMAD5 isoform has
evolved to modulate the complexity of BMP responses in mammals.
Expression of alternative splice forms in normal
hematopoiesis and leukemogenesis
The high conservation of SMADs in evolution strongly suggests that
the SMAD5 pathway is central to mammalian hematopoiesis. SMAD5 is
implicated as a preferred transducer of signals from the BMP family of
ligands. Specifically, SMAD5 mimics the BMP-4 mediated hematopoietic
differentiation and globin synthesis in Xenopus laevis
embryos.14 Other studies of the mesenchymal precursor cell
line C2C1229,30 and the rat osteoprogenitor cells
ROB-C2631 have suggested that SMAD5 is a mediator of BMP-2
or BMP-7 responses.
The restricted number of hematopoietic stem cells in the normal adult
bone marrow makes this compartment a technical challenge for expression
studies. Although the estimation based on the competitive PCR assay may
not be absolute, Figure 4 is consistent with enhanced expression of the
isoform in undifferentiated hematopoietic stem cells. Thus,
SMAD5 may manifest a dominant negative function that counteracts
activation of the BMP-mediated differentiation program and may protect
self-replication of the hematopoietic stem cells. Future studies on
specific subpopulations of hematopoietic progenitors, as well as
hematopoietic cell lines (eg, TF1) amenable to in vitro
differentiation, will provide valuable clues to such a mechanism.
TGF- has a sparing effect on primitive progenitors, in contrast to
the inhibitory effect on committed GM-CSFs.18-20 TGF- is
also a potent inducer of erythroid differentiation. The role of
anti-SMADs in conferring refractoriness during hematopoietic differentiation is unknown at present. Antisense oligonucleotides to
SMAD5 reverse the inhibitory effects of TGF- on the
interleukin-3-mediated (IL-3-mediated) proliferative response of
hematopoietic progenitors.32 Our future studies will
elucidate whether the SMAD5 isoform confers refractoriness
specifically to BMP-4 or to the entire TGF-/BMP/activin family of
ligands. Finally, the regulation of alternative splicing may provide
clues to mechanisms governing hematopoietic stem cell homeostasis.
SMAD5, the functional equivalent of
inactivating mutations
Loss of sensitivity to the TGF- family-mediated growth arrest
represents an important step in neoplastic transformation. Refractoriness to TGF- is achieved by inactivation of the common subunit SMAD4 or by mutations in the type II receptor, which binds the
ligand. While mutations in SMAD4 appear to be associated with 50% of pancreatic and colon cancers,8 mutations in the
ligand-restricted SMAD2 gene are rare (6% in colon
cancer).9 Mice harboring targeted homozygous deletions of
the SMAD3 gene are predisposed to colonic neoplasms,33 although intragenic mutations of the human
ortholog have not been reported. Similarly, we and others have not
found inactivating mutations or homozygous deletions of SMAD5
in more than 200 malignant tissues and cell
lines.10-13
Inactivating mutations in SMAD4 and SMAD2 render them
incapable of homodimerization or heterodimerization.6 The
SMAD5 isoform is analogous to the inactivating mutations in that it does not exhibit homodimeric or heterodimeric interactions (Figure 5).
The aberrant expression of SMAD5 in the
erythroleukemia TF1 cell line is likely to alter the erythroid
differentiation response to TGF-/BMP ligands. Furthermore, the
ovarian cancer tissue from which the EST clone 594181 was isolated may
have acquired refractoriness to TGF-/BMPs by expressing the
alternative splice form. Absence of homozygous deletions for
SMAD5 in cancer may be in part due to the selective advantage
conferred by the isoform on the transformed phenotype. Nonetheless,
the short 3'UTR of the isoform may render the
transcript unstable, and the alternative splice form may be underrepresented in the cDNA libraries.
Finally, expression of the SMAD5 protein appears to be regulated at
multiple levels. Recently, we characterized low levels of
DAMS, an antisense transcript to SMAD5 that is expressed
in fetal tissues and a pancreatic tumor.34 Functional
studies on endogenous SMAD5 isoforms will elucidate their role in
normal cell growth and neoplastic transformation. Isolation and
characterization of the alternative splice form is a first step
toward these studies.
| |
Acknowledgments |
We thank Dr John Massague for the pCMV5/SMAD4-HA construct, Dr
Shourong Zhao for RNA from CD34+ cells, and Brian Johnson
and Monica Spears for manuscript preparation.
| |
Footnotes |
Submitted August 5, 1999; accepted February 17, 2000.
Reprints: Lalitha Nagarajan, the Department of Molecular
Genetics, 1515 Holcombe Blvd, Box 45, MD Anderson Cancer
Center, University of Texas, Houston 77030, TX; e-mail:
lalitha{at}odin.mdacc.tmc.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction