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Molecular and Cellular Biology, April 1999, p. 2495-2504, Vol. 19, No. 4
Department of Pharmacology and Cancer
Biology1 and Department of
Immunology,2 Duke University Medical Center,
Durham, North Carolina 27710
Received 18 September 1998/Returned for modification 26 October
1998/Accepted 22 December 1998
The Smads are a family of nine related proteins which function as
signaling intermediates for the transforming growth factor Transforming growth factor These effects of TGF- The highly related Smad2 and Smad3 serve as substrates for the type I
TGF- The role of the Smads as putative transcription factors was
strengthened by the finding that Smad3-Smad4 complexes and the Drosophila Mad are sequence-specific DNA binding proteins
which on binding DNA can activate transcription (11, 25, 63,
64). In addition to a direct DNA binding activity, the Smads can
be targeted to specific promoter sequences through their interaction with other transcription factors, as demonstrated by the finding that
Smad2-Smad4 complexes bind to the transcription factor FAST-1 in
response to activin and TGF- Apace with the rapid development of the understanding of the Smads on a
biochemical level, the role of the Smads in development and diseases is
beginning to be understood. Recently, mouse models for both Smad2 and
Smad4 function have been described (43, 52, 55, 61). Mice
with homozygous targeted disruptions of these genes are embryonic
lethal at day 9.5 and days 6.5 to 8.5, respectively. Thus, these Smads
play critical, nonredundant roles in early embryonic development. The
early embryonic lethality of these mice, however, renders the
functional analysis of these molecules in the adult animals impossible
in this system and makes their study on a cellular level difficult.
In humans, the role of Smad2 and Smad4 as tumor suppressor genes is now
well established (6, 14-16, 40, 44, 45, 48). Concurrent
with the identification of the Smads through genetic screens, Smad4 was
identified as a tumor suppressor gene, which is deleted in about 50%
of pancreatic carcinomas. In addition to pancreatic cancers, Smad4
mutations have also been discovered in breast, ovary, head and neck,
esophagus, colon, and lung cancers. Not only are Smad4 mutations found
in spontaneous cancers, but recent reports show that inherited juvenile
colon cancer can derive from the inheritance of a single mutant Smad4
allele (19). In addition, Smad2 is mutated in several types
of cancers, including colon cancers and head and neck cancers (14,
44). To date, Smad3 has not been reported to be mutated in human
cancers (2, 45). These data, together with their role as
intermediates in the TGF- The cellular functions of the Smads have largely been inferred from the
occurrence of mutations in human diseases and from cellular studies
employing the use of Smad dominant negatives and Smad overexpression in
Smad-deficient cell lines which likely harbor additional genetic
lesions. Thus, the physiological functions of Smad3, particularly its
potential involvement in mediating the TGF- Smad3 gene disruption.
The Smad3 gene was isolated from a
129/sv mouse genomic library by using the 5' end of the human Smad3
cDNA as a probe. An isolated 15-kb genomic clone was used for the
creation of a Smad3 targeting vector. Briefly, a 1.0-kb
EheI-HindIII fragment was cloned into the
XhoI site of the vector pPNT (54). A 6.0-kb BamHI fragment was next cloned into the resulting construct.
This produced a targeting vector which, when inserted into the genome, replaces the sequence between EheI and BamHI with
a neomycin expression cassette, as diagrammed in Fig.
1A. The resulting targeting vector was
linearized with HindIII and electroporated into 129/sv
embryonic stem (ES) cells. Screening of neomycin-resistant clones was
performed by PCR with the following primers: the common primer (P3;
GTC TTT GAG GCC CGT TTT CTG C), a primer from the targeted
sequence (P1; CTG GGG TGG TAA TGC ACT TGG), and a primer in
the PGK promoter (P2; CAT GCT CCA GAC TGC CTT GGG).
PCR of the wild-type allele results in a 1.2-kb product. PCR of
the targeted allele results in a 1.1-kb product. Positive clones were
confirmed by Southern blotting of EcoRI-digested genomic DNA
probed with an EcoRI-HindIII fragment
immediately adjacent to the sequences used in the targeting vector. The
wild-type allele of Smad3 produces a 5.6-kb fragment. The targeted
allele produces a 5.0-kb fragment.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Targeted Disruption of Smad3 Reveals an Essential
Role in Transforming Growth Factor
-Mediated Signal
Transduction
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(TGF-) superfamily of ligands. To discern the in vivo functions of
one of these Smads, Smad3, we generated mice harboring a targeted disruption of this gene. Smad3 null mice, although smaller than wild-type littermates, are viable, survive to adulthood, and exhibit an
early phenotype of forelimb malformation. To study the cellular functions of Smad3, we generated Smad3 null mouse embryonic fibroblasts (MEFs) and dermal fibroblasts. We demonstrate that null MEFs have lost
the ability to form Smad-containing DNA binding complexes and are
unable to induce transcription from the TGF--responsive promoter
construct, p3TP-lux. Using the primary dermal fibroblasts, we also
demonstrate that Smad3 is integral for induction of endogenous plasminogen activator inhibitor 1. We subsequently demonstrate that
Smad3 null MEFs are partially resistant to TGF-'s antiproliferative effect, thus firmly establishing a role for Smad3 in TGF--mediated growth inhibition. We next examined cells in which Smad3 is most highly
expressed, specifically cells of immune origin. Although no specific
developmental defect was detected in the immune system of the Smad3
null mice, a functional defect was observed in the ability of TGF-
to inhibit the proliferation of splenocytes activated by specific
stimuli. In addition, primary splenocytes display defects in
TGF--mediated repression of cytokine production. These data, taken
together, establish a role for Smad3 in mediating the antiproliferative
effects of TGF- and implicate Smad3 as a potential effector for
TGF- in modulating immune system function.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(TGF-) is a multifunctional polypeptide hormone which has diverse
effects on a variety of cell types to regulate many complex
multicellular systems (46). The complexity and diversity of
TGF-'s function is demonstrated through its multiple roles in
immune system suppression, wound healing, fibrosis, development, and
oncogenesis. Many of these global effects of TGF- stem from its
ability to regulate cellular proliferation, differentiation, and gene
expression (38). One of the most studied aspects of TGF-
function is its ability to inhibit the proliferation of many different
cell types, including cells of epithelial, endothelial, neuronal,
hematopoietic, and lymphoid origins (46, 31).
are mediated through its interaction with cell
surface receptors. By binding to its type I and type II
serine/threonine kinase receptors, TGF- induces the phosphorylation and activation of the type I receptor by the type II receptor (57,
58). The type I receptor kinase can then phosphorylate cytoplasmic substrates, including members of the Smad family of proteins, which function as intermediates in the signaling pathways for
the TGF- superfamily of ligands (3, 4, 12, 18, 39).
Originally identified in genetic screens for TGF- effectors in
Drosophila (49) and Caenorhabditis
elegans (47), the mammalian Smad family now consists of
nine structurally related proteins, Smad1 to Smad9. The identification
and characterization of these proteins has provided valuable insights
into the early events involved in TGF--mediated signal transduction.
receptor kinase (14, 30, 36, 42, 53, 62, 65). Upon
phosphorylation, these two Smads bind to their common partner, Smad4,
to form Smad2-Smad4 and Smad3-Smad4 complexes. These complexes then
translocate to the nucleus (1, 29, 33, 41, 66). Clues to the
nuclear function of these Smad complexes came from studies describing
an intrinsic transcriptional activity of the C-terminal domain of the
Smads (29, 59). Subsequently, overexpression of particular
combinations of Smads was shown to activate transcription from a number
of TGF--responsive promoters, including the plasminogen activator
inhibitor 1 (PAI-1) promoter and the reporter construct 3TP-lux
(11, 29, 65).
(7, 34). In addition, recent studies have implicated a functional interaction between the
Smad3-Smad4 complex and the AP1 family of transcription factors
(32, 63, 67).
signaling pathway, clearly implicate Smad2
and Smad4 as playing an important function in cell growth regulation.
antiproliferative
effect, remain speculative. To address the biological functions of
Smad3, we generated mice harboring a targeted disruption of the Smad3
gene. Unlike the Smad2 and Smad4 null mice, Smad3 null mice are viable
and survive to adulthood, demonstrating distinct roles for the three
Smad proteins during mouse development. In addition, Smad3 null mice
are smaller than wild-type littermates and have an incompletely
penetrant joint formation abnormality. At the cellular level, we
focused our study initially on defining the role of Smad3 in TGF-
signal transduction in the mouse embryonic fibroblast (MEF) and dermal
fibroblast model systems. Here we show that Smad3 is required for
activation of a TGF--responsive promoter, 3TP-lux, and the
endogenous PAI-1 gene and, more importantly, acts as an integral
effector of TGF--mediated inhibition of cellular proliferation. We
next focused on the cell types with highest Smad3 expression,
specifically cells of lymphoid origin, and found that under specific
conditions, the antiproliferative effects of TGF- on isolated Smad3
null splenocytes are lost. In addition, we found that the inhibition of
anti-CD3 (
CD3)-stimulated cytokine production by TGF- in primary
splenocytes is markedly blunted due to the absence of Smad3. Taken
together, these findings implicate Smad3 as a critical effector in
TGF--mediated inhibition of cellular proliferation and a potential
effector for TGF- regulation of immune system function.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Targeted disruption of Smad3. (A) Smad3 genomic
structure and targeting strategy. The Smad3 genomic clone used for the
creation of the targeting vector is diagrammed. The black box denotes
the first exon of Smad3. ATG denotes the initiating methionine. The
first exon coding sequence was replaced by a neomycin expression
cassette (NEO), creating an EcoRI digest size difference
between the wild-type and targeted loci. Ep denotes the
EcoRI-HindIII DNA fragment used as a
probe for Southern blotting. P1, P2, and P3 denote the locations of
primers used for PCR screening. Restriction sites are abbreviated as
follows: R, EcoRI; H, HindIII; E,
EheI; B, BamHI. HSV-TK, herpes simplex virus
thymidine kinase. (B) Southern and PCR detection of the targeted
allele. The targeted allele can be distinguished from the wild type
(W.T.) both by Southern blotting of EcoRI-digested genomic
DNA with the Ep probe and by PCR using the primers indicated in panel
A. (C) Northern blot analysis of Smad3 and Smad4. Northern analyses
were performed on RNA derived from multiple tissues of an adult
(2-month-old) C57BL/6 mouse and a 3' untranslated sequence probe for
Smad3 and a coding-sequence probe for Smad4. Organs are abbreviated as
follows: B, brain; H, heart; M, skeletal muscle; I, small intestine;
Lu, lung; K, kidney; T, thymus; S, spleen. (D) Western blot analysis of
Smad3 expression. The top panel shows Western analysis using an
antibody created against a peptide in the central linker domain of
Smad3 on thymic protein extract from wild-type, heterozygous, and
knockout mice. The bottom two panels demonstrate the specificity of
this antibody among overexpressed Smad family members. HA-tagged Smad1,
Smad2, and Smad4 and Flag-tagged Smad3 were overexpressed in COS cells
from which protein extract were isolated and used for Western analysis.
Identical blots were probed with the Smad3-specific antibody (middle
panel) and a mixture of HA and Flag (bottom panel). In all panels
of all figures, +/+ denotes Smad3 wild type, +/
denotes Smad3
heterozygous, and / denotes Smad3 null.
Primary fibroblast and immune cell culture. Primary fibroblasts were cultured from day 14 embryos. Embryos were mechanically disrupted by passage through an 18-gauge needle and plated on gelatin-coated 10-cm-diameter plates in Dulbecco modified Eagle medium (DMEM) with 20% heat-inactivated fetal bovine serum (FBS) and penicillin-streptomycin (P-S) (Gibco BRL, Gaithersburg, Md.). Confluent cells were trypsinized and further carried in medium containing 10% FBS. In all experiments, compared wild-type and null cells represent littermate embryos at the same passage number.
Primary dermal fibroblasts were isolated from 2-day-old mice. Trunk skin was removed, washed three times in phosphate-buffered saline (PBS) containing kanamycin, amphotericin, penicillin, and streptomycin (KAPS), and incubated overnight at 4°C in 0.25% trypsin (Worthington Biochemical, Freehold, N.J.) in PBS-KAPS. The skins were then incubated at 37°C for 20 min. The trypsin was next neutralized with 20% FBS, and the skins were then washed in DMEM-10% FBS. The trypsinized skins were next placed in individual 10-cm-diameter dishes, and the epidermis was peeled off and discarded. The resulting dermis layers were mechanically dissociated, and 10 ml of DMEM-10% FBS-P-S was added to each dish. Cells were then incubated at 37°C in 5% CO2 until dermal fibroblasts became confluent (3 to 4 days). The dermal fibroblasts were carried in DMEM-10% FBS-P-S and genotyped, and passage 5 cells were used in the PAI-1 assay. Primary splenocytes, thymocytes, and purified splenic B and T cells were generated from the spleens of 2- to 4-month-old mice. Lymphocytes were isolated by mechanical dissociation in the culture medium described below. Large debris was removed, and erythrocytes were lysed by complete resuspension of pelleted cells in 145 mM ammonium chloride-17 mM Tris (pH 7.5). Cells were subsequently washed in PBS and resuspended in their final culture medium for all experiments described: RPMI with the addition of 10% heat-inactivated FBS, P-S, and 0.1 mM-mercaptoethanol.
Northern and Western blotting. Northern blotting for Smad3 was performed on RNA prepared from adult C57BL/6 mouse organs by homogenizing the indicated tissues in Trizol reagent (Gibco BRL, Gaithersburg, Md.) as specified by the manufacturer. Ten micrograms of total RNA was resolved on a formaldehyde-agarose gel, which was subsequently transferred by capillary action to a nylon membrane (Hybond, Amersham Life Science) and visualized by methylene blue staining to confirm RNA loading and quality. Blots were probed with a mouse Smad3 cDNA probe created by random priming (Prime-It II, Stratagene, La Jolla, Calif.) of a HindIII-EcoRI fragment containing sequences entirely in the Smad3 3' untranslated region. The membrane was subsequently reprobed with a human Smad4 cDNA probe containing the entire coding sequence for human Smad4.
For Western blotting, all cell and organ lysates were prepared in a Nonidet P-40-based lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 50 mM NaF, 0.5% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM-glycerophosphate, 0.2 mM sodium molybdate, protease inhibitors). For Western blotting of thymus tissue, whole thymus was homogenized in
1 ml of lysis buffer and equal protein amounts from animals of each
genotype were analyzed. Western blotting of fibroblasts was performed
by lysis of 106 fibroblasts from 10-cm-diameter tissue
culture plates in 200 µl of lysis buffer and analyzing equal protein
amounts for each genotype. Western analyses of equal amounts of protein
extracts from splenic B cells purified by using Dynabeads- Mouse pan T (Dynal, Lake Success, N.Y.) and of splenic T cells purified by using
mouse T-cell enrichment columns (R&D Systems, Minneapolis, Minn.) were
performed with a Smad3-specific antibody. Western analyses of
CD3-stimulated splenocytes were performed on splenocytes isolated as
described above and cultured at a density of 107 cells in 2 ml of medium in six-well tissue culture plates in the presence 5 µg
of CD3 (01081D; PharMingen, San Diego, Calif.) with and without 100 pM TGF- for 24 (cyclin E cyclin-dependent kinase [Cdk2], and p27
western blots) or 48 h (retinoblastoma protein [Rb] Western
blots). Cells were lysed in 200 µl of lysis buffer, and equal protein
amounts were analyzed. Samples were resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western
analyses were performed with the following antibodies; cyclin E
(M-2), Cdk2 (M-2), p27 (C-19), p21 (C-19), p15 (C-20), and
CDC25A (144), all from Santa Cruz Biotechnology, Inc., and Rb
(14001A; PharMingen). Smad3 Western analyses were performed with a
rabbit polyclonal antibody created against the Smad3-specific peptide
DAGSPNLSPNPMSPAHNNLD. Crude rabbit serum was further purified on an
antigen affinity column. The specificity of this antibody was assessed
by Western analysis of equal protein amounts of COS cell extract in
which the following tagged human Smads were individually overexpressed:
Flag-tagged Smad1, Flag-tagged Smad2, hemagglutin epitope (HA)-tagged
Smad3, and Flag-tagged Smad4. Cytomegalovirus promoter-driven
constructs encoding the various Smads were transiently transfected into
COS cells by using a standard DEAE-dextran protocol as previously
described (10). Expression of the various Smads was
confirmed by Western analysis of equal amounts of appropriately
transfected COS extract, using a mixture of HA (Boehringer Mannheim,
Indianapolis, Ind.) and Flag (Eastman Kodak, New Haven, Conn.) antibodies.
Cdk2 kinase assays.
Splenocytes were isolated and cultured
as described above for Western blotting. Whole-cell lysates were
generated after 24 h of TGF- treatment in the same manner as
for Western analysis. Cdk2 kinase activity immunoprecipitated with a
polyclonal Cdk2 antibody (M-2; Santa Cruz Biotechnology) from equal
protein amounts for each condition described was measured on the
substrate histone H1 as previously described (10).
PAI-1 assay.
Dermal fibroblasts were plated at a density of
106 cells per 10-cm-diameter plate in the fibroblast
culture medium described above and incubated overnight at 37°C. Cells
were then incubated in methionine-free DMEM-0.5% FBS-P-S for 4 h and then treated with 100 pM TGF- for 6 h. During the last
2 h of TGF- treatment, the fibroblasts were labeled with
[35S]methionine (100 µCi/ml). The cells were then
washed with PBS and removed by lysis in three washes of 10 mM Tris-HCl
(pH 8.0)-0.5% sodium deoxycholate-1 mM phenylmethylsulfonyl
fluoride, and the resulting plate-bound extracellular matrix washed a
final time with PBS. The amount of matrix associated PAI-1 was assessed
by scraping the plates in SDS-PAGE loading buffer containing
dithiothreitol and resolving the protein on an 10% polyacrylamide gel.
Gels were subsequently dried, and autoradiography was performed.
Thymidine incorporation assays.
Fibroblasts of the indicated
genotypes were plated at a density of 20,000 cells/well in six-well
tissue culture plates in DMEM-10% FBS and incubated in the presence
or absence of 100 pM TGF- for 24 or 48 h as indicated. For the
last 4 h of culture, 5 µCi of [3H]thymidine was
added to the culture, and thymidine incorporation was assayed as
previously described (10). For mixed wild-type and Smad3
null experiments, the indicated percentage of each cell type was plated
in six-well plates to a total cell number of 20,000/ml. Thymidine
incorporation was assayed after 48 h as described above.
IgM; 5 µg/ml; Cappel, Durham,
N.C.) and interleukin-4 (IL-4; 12.5 U/well; PharMingen) or CD3 (2.5 µg/ml; PharMingen) in the presence or absence of 100 pM TGF- and
cultured for 48 h; 5 µCi of [3H]thymidine was
added to the culture for the last 4 h. Thymidine incorporation was
assayed by harvesting cells with a PHD cell harvester (Cambridge
Technologies, Inc.).
Luciferase assays.
Fibroblasts (200,000/well) from each
genotype were seeded into six-well tissue culture plates. Cells were
transfected by using a standard DEAE-dextran transfection protocol as
previously described (10) with the indicated amounts of DNAs
(Fig. 4B). Cells were cotransfected with 0.25 µg of a
cytomegalovirus-driven -galactosidase reporter vector to normalize
for transfection efficiency. Transfections with the expression and
reporter plasmids used here have been previously described
(63).
EMSAs.
For electrophoretic mobility shift assays (EMSAs),
nuclear extracts prepared from 106 fibroblasts of the
indicated genotype either treated or untreated with 100 pM TGF- for
1 h were incubated with a probe derived from an
NdeI-Sph1 fragment of 3TP-lux (63).
Nuclear extract preparation and gel shift conditions were exactly as
previously described (62).
RNase protection analysis of cytokine expression.
Primary
splenocytes were isolated as described above. Splenocytes (7.5 × 106) were plated in 2 ml of medium and stimulated with 5 µg of CD3 in the presence or absence of 100 pM TGF- for 48 h in individual wells of a six-well plate. The cells were harvested,
and total RNA was isolated (RNeasy; Qiagen, Santa Clarita, Calif.).
Cytokine RNA levels were assessed by RNase protection assays using a
RiboQuant multiprobe kit (45024K/mCK-1; PharMingen) on equal amounts of RNA (7.5 µg) for each culture condition as specified by the
manufacturer. Equal amounts and quality of RNA were confirmed through
the quantification of the protection fragments of two housekeeping
genes provided in the multiprobe template set, L32 and
GAPDH.
FACS analysis.
Fluorescence-activated cell sorting (FACS)
analysis was performed on live splenocytes and thymocytes isolated as
described above. Approximately 105 pelleted cells were
resuspended in 100 µl of PBS with 5% heat-inactivated FBS with the
inclusion of the antibodies indicated in each figure: phycoerythrin
(PE)-CD4 (01065B), fluorescein isothiocyanate (FITC)-CD8 (01044D), and FITC-B220 (01124D) (all from PharMingen), plus PE-IgM and 7-amino actinomycin D (7AAD; Molecular Probes, Eugene, Oreg.). After 30 min, these cells were washed once in PBS-5% FBS and
analyzed. The purity of the isolated splenic B and T cells used in the
thymidine incorporation assays was assessed by FACS analysis with the
use of PE-B220 and FITC-labeled anti-T-cell receptor beta chain
(01304D and 01125B, respectively; PharMingen). Viable cells were
identified by exclusion of 7AAD (Molecular Probes) staining.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Smad3 null mice are viable. To generate a targeted disruption of Smad3, we first screened a murine 129/sv genomic library with sequences in the amino terminus of Smad3 to obtain a 14-kb genomic clone. This clone contains the first exon of Smad3, including the initiating methionine and the first 69 amino acids. A targeting vector was created by replacing the first exon and part of the first intron with a PGK-neomycin expression cassette. Proper insertion of this targeting vector into the mouse genome removes the initiating ATG, making the production of full-length Smad3 impossible. In addition, this insertion does not disrupt any sequences 5' to the RNA transcriptional start site (Fig. 1A).
Using standard ES cell technology, Smad3 mutant heterozygous 129 ES lines were generated by transfection of the described targeting vector. Initial screening for proper insertion in neomycin-resistant clones was determined by Southern blotting and PCR as indicated in Fig. 1B. Three percent of neomycin-resistant ES cell clones had a properly targeted Smad3 allele. These ES cells were then used to create 129-C57BL/6 chimeric founder mice. When bred to C57BL/6 females, mice generated from one of these lines transmitted the mutant Smad3 allele at a frequency of 50%, with 100% of offspring being derived from the 129 stem cells. Heterozygous mice from these matings were subsequently mated to produce Smad3 null mice. Smad3 null mice are born to F1 heterozygotes at a frequency of 20.7%, the same frequency as for wild-type mice (297 Heterozygote, 106 wild-type, and 103 KO knockout mice). The near Mendelian inheritance of wild-type and targeted Smad3 alleles suggests no embryonic lethality of the Smad3 null mice. Thus, in sharp contrast to the Smad2 and Smad4 deficiencies, Smad3 is not essential for embryonic development. This F2 generation of mice was used for the experiments described below. To identify organs with highest Smad3 expression, we first performed multiple-tissue Northern analysis. Unlike Smad2 and Smad4, Smad3 has an expression pattern which varies with tissue types, with highest levels of expression in the spleen and thymus (Fig. 1C). Subsequently, the loss of Smad3 expression in the double-mutant animals was confirmed by Western analysis of thymus protein extracts, using a Smad3-specific antibody (Fig. 1D). The first noticeable phenotype in these null animals is a decrease in the size and growth rate of young mice. As shown in Fig. 2A, Smad3 null mice are smaller than both wild-type and heterozygous littermates. An additional early phenotype, which occurs in approximately 31% (32 of 103) of null mice, is the presence of medially torqued forepaws (Fig. 2B and C), with a smaller percentage of mice with noticeably torqued hind limbs. Mice with this phenotype can have either one or more limbs affected. In addition, mice with severely affected limbs often develop kyphosis and display marked rib cage malformation often resulting in a concave indentation at the base of the sternum (data not shown). Interestingly, this phenotype is remarkably similar to that of mice expressing a transgenic dominant negative type II TGF- receptor in bone (50), suggesting that the phenotype described here is intrinsic to the bone. In addition, the similarity between these two phenotypes suggests that the
previously described TGF- effects in bone development are at least
partially mediated by Smad3.
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TGF--mediated growth inhibition and gene responses are impaired
in Smad3 null fibroblasts.
One functional aspect of Smad3 that we
hoped to define through the generation of Smad3 null mice is its role
in TGF--mediated inhibition of cellular proliferation. To test this,
we isolated MEFs from both wild-type and Smad3 null mice. As shown in
Fig. 3A, Smad3 expression can be detected in wild-type fibroblast lines but not in lines derived from Smad3 null embryos. Using these fibroblasts, we first determined the proliferative responses of these
lines to TGF-. As shown in Fig. 3B,
the proliferation of wild-type fibroblasts is inhibited approximately
50% after 24 h and 80% after 48 h of TGF- treatment. In
null fibroblasts, this growth-inhibitory effect of TGF- is largely
lost. In addition, the basal proliferation rate of the null fibroblast
lines is approximately twofold higher than that of the wild type.
Similar results were obtained for two additional fibroblast lines of
each genotype (data not shown). Thus, these results firmly establish an
essential role for Smad3 in TGF--mediated inhibition of cellular
proliferation. Interestingly, none of the known mediators of the
growth-inhibitory effect of TGF- appear to be functioning in
fibroblasts. In these cells TGF- does not alter p21, p15, or CDC25A
protein levels, whereas p27 is undetectable (data not shown).
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in these
cultures is cell autonomous or due to inappropriately regulated production of paracrine factors, growth inhibition by TGF- of mixed
wild-type and null cultures was assayed. As shown in Fig. 3C, different
percentages of wild-type and null cells were seeded into the same well,
and TGF- mediated growth inhibition was assayed. The
growth-inhibitory effect of TGF- in these experiments is proportional to the amount of wild-type cells. This suggests that the
antiproliferative effect of TGF- in these cells is most likely cell
autonomous and not due to a Smad3-dependent production of growth-inhibitory or inhibition of growth-stimulatory paracrine factors.
As discussed above, the Smads have been characterized as DNA binding
transcription factors. To determine the requirement of Smad3 in the
activation of specific promoters, we studied the regulation of the
widely used TGF--responsive promoter 3TP-lux in our model fibroblast
system. In wild-type cells, the previously described TGF--induced,
Smad3-containing DNA binding complex forms on the concatemerized
tetradecanoyl phorbol acetate response elements (TREs) present in
this promoter. This DNA binding complex is lost in the Smad3 null
fibroblasts (Fig. 4A). In addition, transcription from this promoter in wild-type cells is activated 2.4-fold upon TGF- treatment. This activation is lost in the null
fibroblasts and can be restored by cotransfection of a Smad3 expression
vector (Fig. 4B). Thus, Smad3 is necessary not only for the
growth-inhibitory effects of TGF- in this system but also for the
induction of this specific promoter reporter construct.
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, we assayed the
TGF--mediated induction of PAI-1 in primary dermal fibroblasts. As
shown in Fig. 4C, the induction of PAI-1 by TGF- seen in Smad3
heterozygote dermal fibroblasts is greatly reduced in the null cells.
Analysis of the immune cells derived from Smad3 null mice reveals a
defect in TGF- signaling.
Having defined an essential role for
Smad3 in TGF- signaling in the fibroblast system, we next examined
the cell types with highest Smad3 expression, those of lymphoid origin.
We first examined the proliferation of splenocytes isolated from
wild-type and null animals in the presence and absence of TGF-.
Interestingly, the proliferation of unstimulated primary splenocytes,
consisting of a mixed B- and T-cell population, is inhibited by TGF-
regardless of mouse genotype when assayed by tritiated thymidine
incorporation (Fig. 5A). Thus, in
contrast to the MEF data presented above, Smad3 is not required for
TGF--mediated inhibition of cellular proliferation in unstimulated
splenocytes. In addition, the antiproliferative effects of TGF- in
primary splenocytes stimulated by LPS or IgM plus IL-4, which
specifically stimulate the proliferation of B lymphocytes through the
activation of IgM receptor expressed only on the surface of B cells, is
largely intact regardless of genotype. However, in primary splenocyte
cultures stimulated with CD3, an activator of the T-cell receptor
complex, inhibition of proliferation by TGF- is seen only in
wild-type cultures (Fig. 5B). This difference in Smad3-dependent
TGF- responsiveness of the mixed splenocytes to a specific stimulus
is not due to a difference in the expression pattern of Smad3, as
demonstrated by Smad3 Western blot analysis of isolated T and B cells
(Fig. 5C). Taken together, these data suggest that Smad3 plays a
specific role in the inhibition of immune cell proliferation by TGF-
dependent on the nature of stimulus.
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inhibits proliferation of activated B and T cells, making it difficult
to predict the role of Smad3 in this system. Concurrent with results
obtained for a variety of cell types, TGF- treatment of
CD3-stimulated wild-type spleen cultures leads to a decrease in Cdk2
kinase activity and a maintenance of Rb in a hypophosphorylated state.
These effects occur with minimal change in the levels of Cdk2 and
cyclin E and no change in the levels of p27. In contrast, TGF--mediated inhibition of Cdk2 kinase activity and maintained activation of Rb do not occur in the Smad3 null spleen cultures, further supporting the different growth properties of wild-type and
null immune cells (Fig. 6A). Western
analysis of various cell cycle components reveal no TGF--mediated
change in the levels of the CDC25A phosphatase or the
TGF--responsive Cdk inhibitors p15 and p21 (data not shown). Thus,
the TGF- growth-inhibitory pathway activated in CD3-stimulated
splenocytes represents a yet to be defined Smad3-dependent mechanism.
|
on cytokine production
in CD3-stimulated primary spleen cultures. In this system, TGF-
prevents the CD3-mediated increase in the production of a number of
different cytokines by the wild-type cells (Fig. 6B). This effect is
even more dramatic than the growth-inhibitory effects of TGF- on
these cultures. As shown in Fig. 6B, the production of several
cytokines, such as IL-2, IL-4, IL-5, IL-9, IL-13, and IL-15, is more
than 80% reduced by TGF- in wild-type splenocytes (Fig. 6C). In the
Smad3 null culture, however, TGF- clearly does not have the same
effect on the levels of these cytokines as seen in the wild-type
culture. This loss of TGF- responsiveness is most marked in gamma
interferon (IFN-
), IL-2, IL-13, and IL-15 production. In addition,
the non-TGF--treated levels of several cytokines are elevated in the
null cultures. These results strongly suggest abnormal regulation of
cytokine production in the absence of Smad3-mediated TGF- signal transduction.
Since lymphocyte proliferation abnormalities are observed in vitro in
Smad3 null cells, we next determined whether any abnormalities in the
profiles of lymphocyte distribution could be observed in vivo by
performing FACS analysis on Smad3 null and wild-type spleens and
thymuses. As shown in Fig. 7, the
thymuses of Smad3 null mice contain normal proportions of CD4 and CD8
single- and double-positive T cells, suggesting that thymic T-cell
maturation is normal in Smad3 null mice. Similarly, the spleens from
Smad3 null mice contain normal numbers and percentages of B cells and
CD4 and CD8 single-positive T cells, suggesting that there is not an
abnormal expansion of lymphocytes in the spleens of Smad3 null mice. In
addition, we performed FACS analysis on bone marrow and peripheral
lymph nodes for B- and T-cell populations and observed no difference
between wild-type and Smad3 null mice (data not shown). Finally, we
performed functional analysis of B and T cells by immunizing mice with
various antigens and measuring both T-cell-dependent and
T-cell-independent antibody production. Again, we did not observe any
significant differences in antibody production between wild-type and
Smad3 null mice (data not shown).
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
In an attempt to define the roles of Smad3 in TGF--mediated
signal transduction, we have created mice harboring a targeted disruption of Smad3. The first striking finding is that Smad3 null mice
are viable and survive to adulthood. The analysis of mice deficient in
other Smad genes, however, has firmly established the role of this
family of proteins in embryonic development. Mice with a targeted
disruption of Smad4 display an early embryonic lethal phenotype at
embryonic days 6.5 to 8.5. These embryos do not undergo gastrulation or
express mesodermal markers, and they show abnormal visceral endoderm
development (52, 61). Smad2-deficient mice also die early in
development, at embryonic day 9.5, primarily due to a loss of
anterior-posterior identity within the embryo. In the absence of
anterior-posterior identity, the entire epiblast develops a
extraembryonic mesodermal fate, failing to give rise to the three
primary germ layers (55). In a separate study, Smad2 was
found to play a role in mesoderm formation, left-right patterning, and
craniofacial development (43). Additional support for the
critical roles of the TGF- superfamily of ligands and the Smad
family of proteins in development has been established in studies of
the Xenopus oocyte developmental system (17, 24).
In sharp contrast to mice harboring a targeted disruption of Smad2 and
Smad4, the loss of Smad3 function, as we report here, has no
discernible effect on embryonic development. It is conceivable that
certain functions of Smad3 are redundant with, or compensated for by,
that of Smad2. These two proteins are 90% identical at the amino acid
level. Both proteins are inducibly phosphorylated by the TGF-
receptors, associate with Smad4, and undergo nuclear accumulation. One
main difference is that Smad2 may be expressed as two alternatively
spliced variants; one contains two inserts in the MH1 domain of the
protein, rendering it unable to bind to DNA (51, 60),
whereas the other, without the inserts, is structurally and
functionally virtually identical with Smad3 (60). Thus, the
molecular functions of Smad2 and Smad3 are most likely overlapping as
well as distinct, since functional differences in the DNA binding
properties and promoter activation by these molecules have been
reported (28, 63, 65). Although we still do not know the
expression patterns of the two variants of Smad2, clearly Smad3 cannot
fully compensate for the severe defect in Smad2 null mice which may
have lost the expression of both forms of Smad2. On the other hand,
Smad3 may play a more exclusive role as an effector for TGF- and
possibly activin in adult tissues, whereas Smad2 with its two forms may
function more globally in development and possibly in the adult as a
signaling mediator of these two ligands.
A role for Smad3 in TGF--mediated growth inhibition.
Our
initial goal in these studies was to define the role of Smad3 in the
regulation of cellular proliferation by TGF-. Since previous studies
on this topic have involved overexpression of Smads and the use of
various tumor lines which likely harbor additional mutations, a role
for the Smads in the regulation of proliferation remained uncertain. To
this end, we have demonstrated that Smad3 is required for
TGF--mediated growth inhibition in at least two cellular contexts:
CD3-stimulated primary splenocytes and primary MEFs.
-mediated growth inhibition is dependent on
Smad3 only under certain stimulated growth conditions. The proliferation of unstimulated, LPS-stimulated, and
IgM-IL-4-stimulated splenocytes is inhibited in response to TGF-
treatment in wild-type cells and to a nearly identical extent in Smad3
null cells. In contrast, a large reduction in TGF--mediated growth
inhibition is seen in the Smad3 null splenocytes specifically when they
are stimulated by CD3. Thus, there appear to be both Smad3-dependent and Smad3-independent growth-inhibitory signaling pathways for TGF-.
We have also observed a similar defect in TGF--mediated growth
inhibition in MEFs derived from Smad3 null mice. In these cells, the
growth-inhibitory effect of TGF- is largely absent, and this lack of
TGF- effect is most likely cell autonomous.
The molecular nature of the growth-inhibitory effects of TGF- is one
of its most studied properties. Through the work of a number of groups,
a model has been put forward in which TGF- regulates proliferation
by inhibiting the activity of Cdk complexes. This function of TGF-
is likely due, in part, to its ability to increase the expression of
the Cdk inhibitors p21 and p15, decrease the expression of a number of
different cyclins, Cdks, the phosphatase CDC25A, and c-Myc, as well as
regulate the activity of p27 (reviewed in reference
20). The signaling mechanisms of TGF--mediated
growth inhibition vary significantly from one cell type to another.
Unfortunately, none of the previously described TGF--mediated
growth-inhibitory pathways appear to be functioning in wild-type MEFs
or CD3-stimulated splenocytes. Specifically, MEFs and
CD3-stimulated splenocytes down regulate G1
cyclin-Cdk complex activity without significant changes in the levels
of p21, p15, p27, cyclin E, or Cdk2. Thus, Smad3 does not act through these defined downstream effectors to mediate the growth-inhibitory effects of TGF- in these cells. Consequently, these findings suggest
a novel Smad3-dependent growth-inhibitory pathway for TGF-.
The work presented here is complemented by a recent report by Zhu et
al., characterizing the phenotype of an independently created mouse
line with a targeted insertion into the second exon of Smad3
(68). This group describes a high prevalence of colon tumor
in the 129sv mouse background, and a lower prevalence of a less
aggressive tumor phenotype in the 129-C57BL/6 hybrid mouse background.
Although not experimentally addressed, it is an attractive hypothesis
that this tumor formation occurs due to defects in TGF--mediated
growth inhibition of the sort that we describe here. It remains to be
determined, however, if these tumors arise from some other
TGF--Smad3-dependent cellular effect or through a mechanism
unrelated to TGF- signaling. It is intriguing that we have not yet
observed the 30% prevalence of colon tumors in our 129-C57BL/6 hybrid
lines as in the reported study. This discrepancy may be due to
differences in genetic background of the Smad3 null animals or even
targeting strategies. It is also possible that a higher prevalence of
tumors may still occur in our lines with longer time or when the mice
with mixed genetic background are inbred into a pure 129 mouse line.
In addition to its antiproliferative role in the context of tumor
suppression, TGF- is a well-documented global inhibitor of immune
system function. This function of TGF- is evidenced by the phenotype
of TGF-1 null mice (9, 26). These mice present with a
multifocal inflammatory disease, with lymphocyte infiltration into
multiple organs and production of autoimmune antibodies (9,
13). The phenotype of these mice may be attributed to a loss of
the antiproliferative effect of TGF-1 on both B and T cells
(22, 23). Given the fact that Smad3 is most highly expressed
in the spleen and thymus, and the accumulating evidence that Smad3 is
regulated by TGF-, the development of an overactive inflammatory
phenotype similar to that of the TGF-1 knockout mice may have been
expected in the Smad3 null mice. This phenotype, however, is not observed.
These findings may be explained by the fact that under several
conditions for assay of B- and T-cell cultures in vitro, the antiproliferative effect of TGF- is intact in Smad3 null cells. Thus, under in vivo conditions, the proliferation of B and T cells may
be appropriately inhibited under most circumstances by endogenous TGF-. Since this is likely the case, a more subtle or incompletely penetrant inflammatory phenotype may still emerge in the Smad3 null
mice. These findings also support a model in which although Smad3 is
important in regulating the antiproliferative effects of TGF- under
certain conditions, TGF- can also activate or use other
Smad3-independent pathways to exert a growth-inhibitory effect.
A role for Smad3 in TGF--mediated gene responses.
TGF- can affect the expression of a number of different
genes of diverse functions (46). The identification of Smads
as sequence-specific DNA binding transcription factors
supports the notion that the regulation of specific genes by TGF-
may be through the functions of Smad2, Smad3, and Smad4. Both 3TP-lux,
a well-studied promoter reporter used for the analysis of TGF-
signaling, and the promoter of PAI-1, a highly TGF- inducible
extracellular matrix protein, contain Smad3-Smad4 DNA binding sites
(11, 21, 63). Although the Smads have been implicated in the
TGF--mediated induction of 3TP-lux and PAI-1, these studies are
based largely on Smad overexpression and dominant negative studies,
leaving the question of the physiological role for Smad3 in
TGF--mediated gene activation unresolved (29, 35, 65). In
addition, we have shown in a previous study that the Smad-DNA
interaction is dispensable for the activation of 3TP-lux by TGF-,
bringing into question the role of Smad3 in the regulation of this
promoter (63). Here we demonstrate that Smad3 is integral
for transactivation of 3TP-lux and PAI-1, as their induction by TGF-
is reduced in the absence of Smad3. Interestingly, although Smad2 has
been shown in the context of overexpression to activate 3TP-lux and
PAI-1 (29), no compensation by Smad2 is observed in the
Smad3 null fibroblasts.
.
Specifically we provide evidence that the TGF--mediated down
regulation of CD3-stimulated cytokine production is Smad3 dependent.
This suggests that Smad3 may play an important role in both the
activation as well as the repression of gene expression. We have also
evaluated the role of Smad3 in the regulation of several additional
promoters by TGF- in our Smad3 null model system. Specifically, we
describe the role of Smad3 in the induction of c-Jun by TGF- in a
separate study (56). In this study we find that the c-Jun
promoter contains a Smad3-Smad4 complex binding site and that Smad3 is
required for the induction of c-Jun in MEFs. Thus, Smad3 may be
specifically required for the activation of transcription from a subset
of TGF--responsive promoters. In this regard, the Smad3 null MEF
system provides a useful tool to define these genes which are regulated
by TGF- through Smad3. In doing so, we may be able to define novel
genes or pathways which are at the root of the Smad3-dependent, TGF-
antiproliferative effects which we have described.
| |
ACKNOWLEDGMENTS |
|---|
The first two authors contributed equally to this work.
We thank Cheryl Bock and the Duke Transgenic Mouse Facility for help in the generation of Smad3-deficient mice, Michael Cook and Lynn Martinek for flow cytometry services; Rik Derynk for Smad2 and Smad4 expression constructs, Allan Balmain and Sheelagh Frame for providing the dermal fibroblast isolation protocol, Yong Yu for technical help, and the members of the Wang lab for helpful scientific discussion.
This work was supported by grants from the NIH to X.-F.W. (DK45746 and CA75368) and to Y.Z. (CA72433). J.P.F. was supported by a fellowship (DAMD17-98-1-8067) from the Department of Defense Breast Cancer Research Program. X.-F.W. is a Leukemia Scholar, and Y.Z. is a Whitehead Fellow.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710. Phone: (919) 681-4861. Fax: (919) 681-7152. E-mail: wang{at}galactose.mc.duke.edu.
| |
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Materials and Methods
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Discussion
References
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