Deletion of Exon I of SMAD7 in Mice Results in Altered B Cell Responses

RESEARCH ARTICLE | JUNE 01 2006 Deletion of Exon I of SMAD7 in Mice Results in Altered B Cell Responses  Ronggui Li; ... et. al J Immunol (2006) 176 (11): 6777–6784. 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Heuchel4* T he TGF-␤ superfamily of growth and differentiation factors has ⬃34 members in humans, including TGF-␤ isoforms, activins, and bone morphogenetic proteins (BMP).5 They regulate a vast array of biological functions in the adult and are of great importance in governing cell fate determination and patterning of the developing embryo (1, 2). The TGF-␤ signal is initiated by the binding of ligand to type II and type I serine/threonine kinase receptors, resulting in hetero-oligomerization and subsequent phosphorylation and activation of the type I receptor. Receptor-regulated Smad proteins (R-Smads: Smad1, -2, -3, -5, and -8) are phosphorylated by type I receptors that trigger the association with a common partner Smad, Smad4; the complexes are then translocated to the nucleus, where the transcription of specific genes is affected (1). Smad6 and Smad7 are inhibitory Smads (I-Smads) that downregulate TGF-␤ superfamily signaling and thereby modulate biological responses. Both I-Smads recruit ubiquitin ligases of the *Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden; †AstraZeneca R&D Lund, Department of Biological Sciences, Lund, Sweden; ‡Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada; and §Department of Medicine, Washington University, School of Medicine, St. Louis, MO 63110 Received for publication July 19, 2005. Accepted for publication March 17, 2006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 R.L., A.R., and G.B. contributed equally to this work. 2 Current address: Jilin University, Key Laboratory of Pathobiology Ministry of Education, China. 3 Current address: Genentech, South San Francisco, CA 94080. 4 Address correspondence and reprint requests to Dr. Rainer L. Heuchel, Ludwig Institute for Cancer Research, Husargatan 3, Box 595, S-75124 Uppsala, Sweden. E-mail address: Rainer.Heuchel@LICR.uu.se 5 Abbreviations used in this paper: BMP, bone morphogenetic protein; ARBP, acidic ribosomal binding protein; CSR, class switch recombination; I-Smad, inhibitory Smad; MEF, mouse embryonic fibroblast; pSmad2, phosphorylated Smad2. Copyright © 2006 by The American Association of Immunologists, Inc. Smurf family to their respective receptors, causing their ubiquitination and degradation (3– 6). Ectopic expression studies revealed that Smad7 acts as a general inhibitor of the TGF-␤ family member pathway, whereas Smad6 preferentially blocks BMP signaling (7–10). Smad7 has also been implicated as a mediator or antagonist of TGF-␤-induced apoptosis depending on the cell type (10 –16). TGF-␤ ligands can modulate expression of adhesion molecules, provide chemotactic gradients for leukocytes and other cells participating in an inflammatory response, and inhibit the respective cells once they have become activated (17). Enhanced production and activation of latent TGF-␤ are associated with impaired immune regulation in malignancies, autoimmune disorders, opportunistic infection, and the fibrotic condition during chronic inflammation. In contrast with these roles in disease pathogenesis, TGF-␤ is a principal mediator of immune tolerance and as exemplified in mice lacking the type II receptor for TGF-␤ (18), or Smad3 (19), which both show clear signs of autoimmune disorders due to hyperproliferation and hyperactivity of immune cells. By this, TGF-␤ clearly provides regulatory signals that are shaped by the ongoing cellular interactions in the local cytokine milieu and by the relative state of differentiation. In addition, TGF-␤ signaling is essential for the development of Langerhans dendritic cells (20), proper thymic development (21), and is one of the most implicated pathways used by regulatory T cells to keep the immune system in balance (22). Gene targeting in mice has revealed specific developmental and physiological roles for individual Smad proteins (2, 23). Mice devoid of Smad1 (24), Smad2 (25–27), Smad4 (28, 29), and Smad5 (30, 31) die early during embryogenesis in utero. A less severe phenotype was observed in Smad3-deficient mice, which involved impaired immunity and chronic infection (19, 32), but also the occurrence of metastatic colorectal cancer at 4 – 6 mo of age (33). Moreover, lack of Smad3 was shown to 0022-1767/06/$02.00 Downloaded from http://journals.aai.org/jimmunol/article-pdf/176/11/6777/1213334/6777.pdf by guest on 02 January 2023 The members of the TGF-␤ superfamily, i.e., TGF-␤ isoforms, activins, and bone morphogenetic proteins, regulate growth, differentiation, and apoptosis, both during embryonic development and during postnatal life. Smad7 is induced by the TGF-␤ superfamily members and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. In addition, Smad7 is induced by other stimuli. Thus, it can fine-tune and integrate TGF-␤ signaling with other signaling pathways. To investigate the functional role(s) of Smad7 in vivo, we generated mice deficient in exon I of Smad7, leading to a partial loss of Smad7 function. Mutant animals are viable, but significantly smaller on the outbred CD-1 mouse strain background. Mutant B cells showed an overactive TGF-␤ signaling measured as increase of phosphorylated Smad2-positive B cells compared with B cells from wild-type mice. In agreement with this expected increase in TGF-␤ signaling, several changes in B cell responses were observed. Mutant B cells exhibited increased Ig class switch recombination to IgA, significantly enhanced spontaneous apoptosis in B cells, and a markedly reduced proliferative response to LPS stimulation. Interestingly, LPS treatment reverted the apoptotic phenotype in the mutant cells. Taken together, the observed phenotype highlights a prominent role for Smad7 in development and in regulating the immune system’s response to TGF-␤. The Journal of Immunology, 2006, 176: 6777– 6784. 6778 accelerate wound healing and increase resistance to bleomycininduced lung fibrosis (34, 35). Both I-Smads were initially identified in vascular endothelium subjected to laminar flow stress, predicting a functional role in the homeostasis of the vasculature (36). Indeed, mice targeted for the Smad6 gene displayed cardiovascular anomalies (37). To elucidate the functional role(s) of Smad7 in vivo, we have targeted the Smad7 gene in mice. We observed a significantly reduced body weight of mutant mice as well as altered B cell responses. These included increased Ig isotype switching to IgA, enhanced spontaneous apoptotic potential, and a significantly reduced proliferative response to LPS stimulation. Materials and Methods GENE TARGETING OF Smad7 tiMEM (Invitrogen Life Technologies) was added. A total of 200 ␮l of OptiMEM containing a total of 1.3 ␮g of DNA and 1.5 ␮l of LipofectAMINE was used for transfection. Cells were transfected with 0.25 ␮g of p3TPlux reporter plasmid (41), 0.1 ␮g of pCMV ␤-galactosidase (BD Clontech), and 0.75 ␮g of pCDNA3 (Invitrogen Life Technologies) expression vectors (containing a cDNA for constitutive active ALK-5, or empty) per well. After 48 h, the cells were washed twice with PBS and lysed in 125 ␮l of reporter lysis buffer (Promega E397A) for 20 min at room temperature. Luciferase assay was performed using Enhanced Luciferase Assay kit (BD Pharmingen), according to the manufacturer’s instructions. Absorbance at 405 nm and luciferase light detection were measured on the Victor II 1420 Multilabel counter. Variation in transfection efficiency was corrected by normalizing luciferase units with galactosidase activity from the same cell lysates. Luciferase assays were conducted in triplicates, indicated by error bars for SD in the figure. One representative of three experiments is shown. Western blot The Smad7 locus was isolated from a 129Sv genomic library (38). The targeting vector consisted of a 5.1-kb PvuII-EcoRI genomic 5⬘ fragment containing the Smad7 promoter and 5⬘-untranslated leader sequence, followed by a PGKneobpA expression cassette replacing the translated part of exon 1 and the exon I/intron I boundary, a 1-kb HindIII-NotI genomic 3⬘-fragment, and an HSV thymidine kinase expression cassette in pBluescript SK⫹. The construct was linearized for electroporation into 129Svderived R1-ES cells, and colonies were selected with G418 and gancyclovir. Homologous recombination events were screened by Southern blotting using a Smad7 genomic 5⬘ probe (1-kb PvuII-HindIII) or a genomic 3⬘ probe (700-bp NotI-HindIII), both situated outside the genomic targeting vector sequences. Tissue culture and morula aggregations were performed, as described previously (39). Mice were genotyped by PCR using a common 5⬘ primer (5⬘-GCGGGGGAGGGAGGGGTAGAGG-3⬘) and an exon 1 antisense primer (5⬘-GGGGCGAGGAGGCGAGGAGAAAAT-3⬘) for the wild-type allele (1.7 kb) or a neo antisense primer (5⬘-GCTACCGGT GGATGTGGAATGTGTGC-3⬘) for the mutant allele (1.1 kb). PCR conditions were as follows: predenaturation, 3⬘ at 95°C, 40 cycles at 93°C for 30 s, 61°C for 30 s, 65°C for 2 s, and a final extension at 65°C for 10 s. Heterozygous mutant mice were backcrossed five generations onto CD-1 mice, obtained from Charles River Laboratories. The mice were used between 4 and 6 mo of age, if not stated otherwise. All animal experimentation was approved by the local ethical committee. The 293T cells were maintained at 37°C in DMEM supplemented with 10% FBS (PAA Laboratories). Transfection was done using LipofectAMINE 2000 kit (Invitrogen Life Technologies). Cells (100,000/ well) were plated in 12-well tissue culture plates coated with 0.01% poly(L-lysine) (Sigma-Aldrich) and grown for 24 h. Fresh medium was added before transfection. Cells were transfected with 200 ␮l of OptiMEM (Invitrogen Life Technologies) containing 1.6 ␮g of DNA and 4 ␮l of LipofectAMINE 2000 and grown for another 2 days. Then, cells were washed twice with PBS and lysed in Laemmli sample buffer, sonicated, boiled for 5 min, and separated by SDS-gel electrophoresis using a 10% polyacrylamide gel (NuPAGE Bis-Tris; Invitrogen Life Technologies) using MOPS/SDS running buffer (Invitrogen Life Technologies). Proteins were transferred to Immobilon-polyvinylidene difluoride membrane (Millipore) using XCell II Blot Module (Invitrogen Life Technologies) with NuPAGE transfer buffer (Invitrogen Life Technologies). The membranes were blocked in 0.1% Tween 20, 5% BSA in TBS at room temperature for 1 h and incubated with a rabbit Ab (2 ␮g/ml in TBS with 0.1% Tween 20, 5% BSA) raised against the C terminus (aa 206 – 426) of Smad7 at 4°C overnight. After four washes at room temperature in TBS with 0.1% Tween 20, an HRP-conjugated secondary donkey anti-rabbit Ab (Amersham Biosciences) diluted 1/10,000 was used in combination with BM Chemiluminescence Blotting Substrate (Roche) to visualize immunocomplexes. Generation of mouse embryonic fibroblasts (MEFs) Real-time PCR of transcripts involved in Ig isotype switch The 3T3-like embryonic fibroblasts were derived from E13 embryos, according to standard procedures (40). Established MEFs were grown in DMEM (Invitrogen Life Technologies) supplemented with 10% FBS (Sigma-Aldrich), 0.1 mM 2-ME, and 2 mM L-glutamine. Northern blot analysis and cloning of transcriptional splice variants RNA was extracted from Smad7 mutant and wild-type MEFs stimulated by 1 ng/ml TGF-␤ for 90 min, unless indicated otherwise, using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. Poly(A)⫹ RNA was purified from the total RNA using Dynabeads Oligo-(dT)25 (Dynal Biotech), according to the manufacturer’s instructions. Following DNase I treatment, the RNA was reverse transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). cDNAs from reverse-transcribed reactions were specifically amplified with Pfu (Stratagene) or Pfx (Invitrogen Life Technologies) DNA polymerase using primer U5⬘397 (untranslated leader sequence upstream of gene-targeting event), 5⬘ACGGACGAGGAAAGACCAGAGAC-3⬘, and primer S7d (exon IV), 5⬘-TGCAGGCTGTAGGCTTTCTCA-3⬘. PCR products were cloned into the EcoRV site of pBluescript. After DNA sequencing, the fragments were inserted into pcDNA3-based vector containing the missing 3⬘ part of human Smad7. Northern blot analysis was done, according to standard procedures, using poly(A)⫹ RNA. The hybridization probe for Smad7 spanned 610 nt from within exon IV. Transfection of Mv1Lu cells Mv1Lu cells (ATCC: CCL64) were maintained at 37°C in MEM supplemented with 0.1 nM nonessential amino acids and 10% FBS. Transfection was performed using LipofectAMINE 2000 reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. Cells (160,000/ well) were plated in 12-well tissue culture plates and grown for 24 h. Before transfection, cells were washed twice with PBS, and 1 ml of Op- Total RNA was extracted from spleen cells prepared from Smad7⫺/⫺ and wild-type mice cultured in vitro, as described below. RNA was isolated from spleen cells using TRIzol (Invitrogen Life Technologies), according to manufacturer’s instructions. Following DNase I treatment, the RNA was reverse transcribed into cDNA. In a 20-␮l reaction mixture, 1 ␮g of total RNA, 4 ␮l of 5⫻ reaction buffer, 0.5 mM dNTP, 10 mM DTT, 0.5 ␮l of RNase inhibitor (Invitrogen Life Technologies), 25 ng of random hexamer primers, and 100 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies) were incubated for 90 min at 42°C. Primer sequences used to amplify postswitch ␣-transcripts (ImF and CaR) were as follows: ImF, 5⬘-CTCTGGCCCTGCTTATTGTTG-3⬘; CaR, 5⬘GAGCTGGTGGGAGTGTCAGTG-3⬘; and for germline ␣-transcripts (IaF and CaR), IaF, 5⬘-CCTGGCTGTTCCCCTATGAA-3⬘ (42). As internal control, the housekeeping gene acidic ribosomal binding protein (ARBP) was chosen: ARBPf, 5⬘-CACTGGTCTAGGACCCGAGAAG-3⬘ and ARBPr, 5⬘-GGTGCCTCTGGAGATTTTCG-3⬘. For quantitative PCR, 25␮l reaction mixtures contained 200 nM specific primers, 5 ␮l of template cDNA, and 12.5 ␮l of SYBR Green PCR Master Mix (2⫻ concentration) (Applied Biosystems). ABI Prism 7000 sequence detection system and ABI Prism 7000 SDS software (Applied Biosystems) were used to detect and quantitate amplified DNA fragments. The thermal program was as follows: 2 min at 50°C, 10 min at 95°C, then 40 cycles of 15 s at 95°C, and 1 min at 60°C. Cell culture, proliferation, and apoptosis assays For each spleen cell preparation, two to four spleens per genotype were pooled. Single-cell suspensions were made into RPMI 1640 supplemented with 2 nM L-glutamine, penicillin, streptomycin, nonessential amino acids, sodium pyruvate, 10 mM HEPES, and 10% heat-inactivated FCS in a petri dish. The cell solution was spun down and erythrocytes were lysed, as described previously (43). After rinsing twice, the cells were counted in a Burker chamber and examined for viability with trypan blue. Cells were resuspended at a density of 105 cells per 100 ␮l in 96-well plates. If not otherwise stated, 10 ␮g/ml LPS (LPS from Escherichia coli serotype 055: Downloaded from http://journals.aai.org/jimmunol/article-pdf/176/11/6777/1213334/6777.pdf by guest on 02 January 2023 Generation of mutant mice The Journal of Immunology 6779 Results Generation of Smad7 mutant mice To generate a mutant Smad7 allele, we used a targeting strategy designed to replace the coding region of the first exon of Smad7, including the starting ATG codon and a part of intron I, with a neomycin selection cassette (Smad7⌬exI; Fig. 1A). This approach would delete roughly the first half of the Smad7 protein, i.e., the N-terminal 204-aa residues (Fig. 1B). Mice heterozygous or homozygous for gene targeting were viable and fertile on a 129Sv/CD-1 mixed background, although we found slightly reduced numbers of homozygous offspring. Among 481 offspring from heterozygous matings (fifth backcross into CD1), we found 32% wild-type, 53% heterozygous, and 15% homozygous mice. The underrepresentation of homozygous offspring prompted us to investigate the feto-placental units, because numerous TGF-␤ signaling mutants display lethal vascular defects. No such defects were detected upon microscopical examination between embryonic days 10 –12. However, already at birth, homozygous mice appeared smaller than their wild-type or heterozygous siblings. A statistical analysis of the body weight of 7-wk-old mice indicated that homozygous mice were 16 –20% smaller compared with wild-type mice (Fig. 2). In addition, the average litter size from wild-type matings (9.8 ⫾ 2.2 pups; n ⫽ 11 litters) was significantly larger (t test: p ⬍ 0.0002) than the average litter size from homozygous mutant matings (6.1 ⫾ 1.4 pups; n ⫽ 12 litters). These initial findings pointed toward a clear disturbance of Smad7 function in homozygous mutant mice. Several differentially spliced mRNAs are generated from the Smad7⌬exI mutant locus To test whether the targeting strategy had resulted in a complete null allele, or in a truncated protein starting from an internal ATG in exon II, we generated MEFs from wild-type and Smad7⌬exI homozygous mutant E12.5 embryos. All efforts to unambiguously demonstrate endogenous wild-type or possible truncated, mutant Smad7 proteins with a series of commercial and homemade Abs were futile. We therefore decided to investigate the mRNA expression in MEFs by Northern analysis. Whereas there was only one major, TGF-␤-inducible transcript detectable in wild-type MEFs, at least three differentially FIGURE 1. Targeting of the Smad7 locus. A, Schematic representation of the targeting vector and the wild-type allele before and after the homologous recombination, which replaced the coding region of exon I and a part of intron I between the EcoRI and HindIII restriction sites with a neomycin selection cassette. Correct targeting was verified by Southern blot analysis of mouse tail DNA preparations after HindIII digestion using the 5⬘ probe for hybridization (bottom). [], Indicates restriction site destroyed during cloning process. B, Schematic representation of mouse Smad7. Upper part, Exon structure of mouse Smad7 according to ENSMUSG00000025880. EcoRI, 5⬘ break point of deletion of exon I in knockout (shaded area). Lower part, Domain structure of Smad7 protein with N domain (aa 1–204), linker region (aa 205–260), and MH2 domain (aa 261– 426). Bold star, major translational start site in exon I; small star, potential additional translational start site in exon II; TAG, translation stop codon; amino acid positions are in brackets next to the symbols. spliced mutant mRNAs were found in Smad7⌬exI homozygous mutant MEFs (Fig. 3A). We used RT-PCR to clone and examine the structure of the differentially spliced mutant transcripts, including the wild-type transcript as control. Sequencing analysis indicated that all mutant transcripts (M1 to M4) were spliced from either the remaining Smad7 5⬘-leader sequence, or different positions from within the neocassette to exon II, but not to the other protein coding exons III or IV (Fig. 3B). The neocassette is known to contain cryptic splice sites and has been used earlier to generate hypomorphic alleles (45). Smad7⌬exI mutant transcripts are translated into truncated proteins The cDNAs of the different mutant transcripts were cloned into ATG-less pcDNA3 expression vectors, including the missing Cterminal coding region of the MH2 domain of Smad7. The 293T cells were transfected with expression vectors containing cDNAs for mouse wild-type Smad7, differentially spliced Smad7⌬exI forms (M1-M4), or empty pcDNA3. As depicted in Fig. 3C, an affinity-purified Ab directed against the C terminus of Smad7 recognized overexpressed full-length Smad7 of a calculated mass of 46.3 kDa and two smaller proteins in lysates of M2-, M3-, and Downloaded from http://journals.aai.org/jimmunol/article-pdf/176/11/6777/1213334/6777.pdf by guest on 02 January 2023 B5; Sigma-Aldrich L2637) with or without neutralizing TGF-␤ Ab (MAB 1835; R&D Systems) at a final concentration of 20 ␮g/ml was added, and the cells were cultured for the indicated times. In some experiments, BMP7 and activin A were used at a concentration of 50 ng/ml each. Anti-IgM Abs (Jackson ImmunoResearch Laboratories) and anti-CD40 mAbs (clone 3/32; BD Pharmingen) were used to activate resting B cells at 20 and 10 ␮g/ml, respectively. Proliferation was measured by the BrdU Flow Kit (BD Biosciences 51-2354AK), according to manufacturer’s description. Briefly, 2 h before cell harvest, BrdU was added to a final concentration of 10 ␮M. The plates were then centrifuged at a speed of 300 ⫻ g for 10 min at 4°C. Unspecific Fc receptor binding was blocked with CD32/CD16 mAb (BD Pharmingen; BD 553140) at room temperature and B cells were labeled with CD19 Abs. Cells were fixed and permeabilized, and DNase was treated and stained with anti-BrdU Abs. To monitor induction of apoptosis, the annexin V apoptosis kit (BD Biosciences; 559763) was used according to manufacturer’s description. Briefly, B cells were stained with CD19 Abs, washed, and resuspended in binding buffer supplemented with annexin V and propidium iodide. Smad2 phosphorylation was analyzed using the phosphorylated Smad2 (pSmad2) Ab, which recognizes the C-terminal KKK-SSpMSp sequence (44), as described previously (21). FACS analysis was conducted on a FACSCalibur equipped with one blue laser, and analysis was done with CellQuest Pro software (BD Biosciences). All experiments were performed at least three times; one representative result is shown in each case. 6780 FIGURE 2. Smad7⌬ExI mutant mice are smaller than wild-type mice. A total of 113 offspring of heterozygous matings (N5F1 in CD-1) was weighed at the age of 7 wk, indicating that Smad7 homozygous female mice were 21% and homozygous male mice 16% smaller than their wildtype siblings. ⴱ, t test, p ⬍ 0.0001. from M1-transfected cells, which is most probably due to very inefficient reinitiation downstream of the translational stop signal of the neomycin phosphotransferase cassette. To test the biological functionality of the cloned mutant transcripts, Mv1Lu cells were cotransfected with the TGF-␤-responsive p3TP-lux reporter vector, expression constructs containing either Smad7 wild-type or mutant (M1-M4) cDNAs, and with or without a constitutive active form of the TGF-␤ type-I receptor (CA-ALK5). As expected, wild-type Smad7 had a strong suppressive effect on TGF-␤-induced luciferase activity compared with cotransfection of empty expression vector only. Interestingly, cDNAs M2 to M4 retained some suppressing effect, indicating that the targeted Smad7⌬exI allele produces truncated proteins, which cause a hypomorphic Smad7 allele, rather than a complete knockout (Fig. 3D). Increased class switch recombination (CSR) to IgA in Smad7⌬exI mutant B cells Ig CSR from IgM/D to IgA in mature naive B cells has been found to be induced by TGF-␤ signaling (47). In vitro, the combination of LPS and TGF-␤ induces the germline I␣ promoter via a concerted action of Smad, acute myeloid leukemia, and CREB transcription factors to produce germline transcripts (Gl␣), which are a prerequisite for CSR (48, 49). A following intrachromosomal DNA recombination results in the final postswitch transcripts (PS␣) coding for the IgA H chain (50). We speculated that if the hypomorphic mutation of Smad7 would lead to increased TGF-␤ signaling, then CSR to IgA should also be increased. Spleen cells prepared from wild-type and mutant mice were incubated with LPS alone, or LPS and FIGURE 3. Smad7 transcript analysis of mRNA from wild-type and Smad7⌬ExI mutant MEFs. A, Northern analysis of mRNA derived from wild-type (Wt) and Smad7⌬ExI mutant (Mut) cells either without stimulation, or after stimulation with TGF-␤ for different time periods, identified multiple differentially spliced transcripts from the mutant locus. The same filter was stripped and rehybridized with a probe for GAPDH to confirm equal loading. Transcript identity is indicated on the right side; hybridization probe and size marker in kilobases are indicated on the left side. B, Schematic representation of the sequence analysis of differentially spliced transcripts derived from TGF-␤-stimulated Smad7⌬ExI mutant MEFs. Bent arrow, transcriptional start site within neocassette; bold star, translational start of Neo gene; STOP, translational stop codon of Neo gene (left side) and Smad7 gene (right side); small star, translational start codon in exon II of Smad7; arrowheads indicate the positions of the PCR primers used to clone the wild-type cDNA (data not shown) and the spliced mutant cDNAs M1 to M4; dotted lines indicate splice donor and acceptor positions. C, Western analysis of wild-type or mutant Smad7⌬ExI cDNAs (M1 to M4) expressed in 293T cells. Molecular sizes in kDa are indicated on the right side. D, The Smad7⌬ExI mutant splice variants M2 to M4 retained a partially suppressive effect on transcriptional activity of a TGF-␤-sensitive reporter compared with wild-type Smad7. Mv1Lu cells were cotransfected with p3TP-lux reporter vector together with either empty pcDNA3 or pcDNA3 expression vectors containing cDNAs for wild-type and mutant forms of Smad7 and an expression vector coding for constitutive active ALK5 (CA-ALK5), as indicated. A CMV-␤-gal expression vector was used as control for transfection efficiency. Downloaded from http://journals.aai.org/jimmunol/article-pdf/176/11/6777/1213334/6777.pdf by guest on 02 January 2023 M4-transfected cells. Exon II contains a methionine codon with a slightly better Kozak consensus sequence than the actual exon I starting ATG (46). Translation commencing with the exon II ATG would result in a truncated Smad7 of 211 aa containing the complete MH2 domain. The splicing event in M2 transposes a perfect Kozak ATG consensus motif (46) from within the PGK-neo-promoter sequence in frame to exon II, predicting a 225-aa protein of a calculated mass of 25.4 kDa. Sequence analysis of M3 and M4 predicted a protein of 211 aa and a calculated molecular mass of 23.8 kDa, starting with the ATG in exon II, because no additional ATG was found in frame with exon II in the complete Smad7 5⬘-leader region. The Smad7 Ab recognized proteins of these expected sizes in lysates of cells transfected with M2, M3, and M4 (Fig. 3C). The Ab did not detect any specific protein in lysates GENE TARGETING OF Smad7 The Journal of Immunology TGF-␤ together, for 2 or 4 days. Real-time RT-PCR was performed on isolated RNAs using primers specific for germline ␣-transcripts, postswitch ␣-transcripts, and ARBP as an internal reference. Germline ␣-transcripts as well as postswitch ␣-transcripts were significantly elevated in Smad7 mutant B cells, even in the absence of TGF-␤ stimulation (Fig. 4). These findings are in agreement with the expectation of increased TGF-␤ signaling in the absence of fully functional Smad7. Smad7 mutant B cells have a TGF-␤-dependent proliferative defect upon LPS stimulation Increased phosphorylation of Smad2 in Smad7⌬exI mutant B cells To measure the intensity of TGF-␤ signaling in wild-type and Smad7 mutant B cells, we analyzed the amount of pSmad2. Overall, significantly more mutant B cells were pSmad2 positive compared with wild-type B cells, independent of treatment (Fig. 5C). LPS stimulation of the cell cultures significantly induced Smad2 phosphorylation in both wild-type and mutant B cells. The presence of neutralizing Abs to TGF-␤ during the FIGURE 4. Smad7⌬ExI mutant B cells show increased Ig isotype switching to IgA. RNA from wild-type or Smad7⌬ExI mutant spleen cell cultures, either stimulated with LPS alone or with LPS together with TGF-␤, were analyzed for relative amounts of A, germline ␣-transcripts (GL␣; 2-day stimulation) or B, postswitch ␣-transcripts (PS␣; 4 days of stimulation) by real time RT-PCR. Transcripts for ARBP were used as an internal reference. LPS stimulation significantly reduced the number of pSmad2positive cells. This clearly identified TGF-␤ as the main source for the increased Smad2 phosphorylation. Importantly, when the mutant B cells were stimulated with LPS in the presence of TGF-␤ neutralizing Abs, the number of pSmad2-positive cells was reduced below the basal level observed in untreated cells. Thus, the elevated number of pSmad2-positive mutant B cells inversely correlates with a reduced number of proliferating mutant B cells, due to increased TGF-␤ signaling. Apoptosis is not involved in the proliferative defect of LPSstimulated mutant spleen B cells Ectopic overexpression of Smad7 blocked TGF-␤/activin A/BMPinduced growth arrest and apoptosis in several tumor B cell lines (10, 15, 16). Thus, to investigate whether Smad7⌬exI mutant B cells show enhanced TGF-␤-induced apoptosis compared with wild-type B cells, we monitored annexin V by flow cytometric analysis. Indeed, the spontaneous level (day 0) of apoptosis in Smad7⌬exI mutant B cells was enhanced compared with wild-type B cells (Fig. 5D). Exogenous TGF-␤ induced apoptosis in both wild-type and Smad7⌬exI mutant B cells, especially after 5 days in culture. Importantly, addition of TGF-␤ significantly enhanced the apoptosis in the Smad7⌬exI mutant B cells compared with wild-type B cells at 2 and 5 days of culture. Surprisingly, however, LPS stimulation had only marginal effect on the apoptotic index of wild-type and Smad7⌬exI mutant B cells, excluding apoptosis as a factor responsible for the difference in the growth response to LPS. We also tested activin A and BMP for their proapoptotic effect on B cells; we only saw a minor increase in apoptosis upon activin A stimulation (Fig. 5D, right panel, and data not shown). This confirmed that, among the TGF-␤ family members, TGF-␤ is by far the most potent inducer of apoptosis in primary B cells. Discussion To investigate the in vivo role of Smad7, we inactivated the Smad7 gene in the mouse by replacing exon I of Smad7, coding for the 204 N-terminal amino acid residues of Smad7, with a PGK-neo selection cassette (Fig. 1). The significantly reduced body size of Smad7⌬exI mutant mice (Fig. 2) and the reduced litter size from homozygous matings compared with wild-type matings indicated a strong effect of the introduced mutation on mouse development. The biochemical analysis of the knockout using mouse embryonic fibroblasts indicated that the targeting approach has resulted rather in a partial knockout, because the differentially spliced transcripts arising from the mutant allele could be translated into N-terminally truncated forms of Smad7 when transfected as cDNAs into 293T cells (Fig. 3). The N domain of Smad7 had been shown earlier to be important for the suppressive function by assisting the MH2 domain in efficient binding of Smad7 to the TGF-␤ type I receptor (52). In agreement with this observation, we detected some residual suppressive activity of the mutant cDNAs in our TGF-␤-sensitive transcription assay. Dorsal injection of a similarly truncated Xenopus Smad7 RNA into Xenopus embryos led to eye defects, but no spina bifida, which are both caused by injection of fulllength Smad7 (53). These findings indicate that such truncated forms of Smad7 can retain activity also in complex biological systems. TGF-␤ is a very potent immunosuppressor (17). In line with these properties, TGF-␤ influences B cell growth and development both through direct stimulation and through effects on cells interacting with B cells (54). Among the direct effects are inhibition of proliferation, stimulation of apoptosis, and Ig class switch recombination to IgA. We analyzed germline ␣- and Downloaded from http://journals.aai.org/jimmunol/article-pdf/176/11/6777/1213334/6777.pdf by guest on 02 January 2023 The increased TGF-␤-dependent activation of the IgA H chain locus in Smad7 mutant B cells suggested that B cell function is altered in the knockout mice. To investigate whether other functional parameters in peripheral B cells were affected by the overactive TGF-␤ signaling, we monitored BrdU incorporation as a measurement of proliferation. The kinetic response to the B cell mitogen LPS was analyzed in spleen B cells isolated from wildtype or Smad7 mutant mice. Fig. 5A demonstrates that B cell proliferation was significantly lower in Smad7⌬exI mutants in day 1 and 2 LPS cultures. On the contrary, stimulation of wild-type and mutant B cells with a combination of Abs against the BCR and the costimulatory molecule CD-40 resulted in an equal growth response (Fig. 5A). LPS not only acts as a B cell mitogen, but it also leads to induction and secretion of active TGF-␤ by B cells and other leukocytes (17, 51). To investigate whether the difference in proliferative response was due to increased growth-suppressing TGF-␤ activity, we repeated the experiment and stimulated spleen cell cultures with LPS in the absence or presence of neutralizing pan-TGF-␤ Ab. Blocking TGF-␤ completely abolished the proliferative difference between wild-type and Smad7⌬exI mutant B cells at day 1 and 2 of LPS stimulation (Fig. 5B), clearly indicating that changes in the TGF-␤ signaling pathway were responsible for the different responses of wild-type and mutant B cells to LPS stimulation. 6781 6782 GENE TARGETING OF Smad7 postswitch ␣-transcripts in spleen cell cultures from wild-type or Smad7⌬ExI mutant mice after stimulation with LPS alone, or LPS together with TGF-␤. Both types of transcripts were significantly elevated in Smad7⌬ExI mutant B cells, even in the absence of TGF-␤ (Fig. 4, A and B). The observation that the IgA locus is activated even in the absence of exogenous TGF-␤ addition might be explained by the fact that LPS stimulates mouse spleen B cells to produce bioactive TGF-␤ (51). In addition, we also detected significantly elevated amounts of germline ␣-transcripts and postswitch ␣-transcripts in freshly prepared spleen tissue from mutant mice, indicating increased CSR activity in vivo (data not shown). Conversely, the B cell-specific knockout of the TGF-␤ type II receptor resulted in complete deficiency in serum IgA (55). Our findings are, therefore, in agreement with an expected increase in TGF-␤ signaling in the absence of fully functional Smad7. On one hand, LPS is a potent B cell mitogen, but, in contrast, it also induces bioactive TGF-␤ in B cells as well as the expression of Smad7 mRNA (51, 56). It was therefore intriguing to find that Smad7⌬ExI mutant B cells proliferated significantly less than wild-type B cells upon LPS stimulation (Fig. 5A). This difference was completely abolished by the addition of TGF-␤neutralizing Ab. This indicated that the mutant B cells have the same intrinsic ability to proliferate as wild-type B cells, provided growth-inhibitory TGF-␤ activity is blocked (Fig. 5B). In support of this idea, we found that the number of pSmad2positive B cells was significantly higher in mutant B cells compared with wild-type B cells and the presence of the TGF-␤neutralizing Ab during LPS stimulation dramatically reduced Smad2 phosphorylation in Smad7⌬ExI mutant B cells (Fig. 5C). Overexpression of Smad7 had been shown to counteract TGF␤-, activin A, and BMP-induced growth arrest and apoptosis in tumor B cell lines (10, 15, 16). We observed an inherent increased apoptosis rate in mutant B cells (Fig. 5D). The TGF␤-induced apoptosis rate was significantly higher after 5 days of stimulation in mutant B cells compared with wild-type B cells, in line with the idea that functional Smad7 reduces TGF-␤induced apoptosis in B cells. Unexpectedly, we saw only marginal to no apoptotic induction following activin A and BMP stimulation. This difference to previously published results (10, 15, 16) could be due to the fact that we used primary spleen cells compared with hybridoma B cells and B cell lymphoma cells. We saw only a very minute effect of LPS stimulation on the apoptosis rate in B cells, despite the fact that LPS induces TGF-␤ signaling (Fig. 5, B and C). LPS activates antiapoptotic signaling via NF-␬B and protects primary mouse spleen B cells from spontaneous and staurosporin-induced apoptosis (57). Therefore, the antiapoptotic activities induced by LPS seem to override the proapoptotic TGF-␤-induced signaling. In addition, LPS Downloaded from http://journals.aai.org/jimmunol/article-pdf/176/11/6777/1213334/6777.pdf by guest on 02 January 2023 FIGURE 5. Smad7⌬ExI mutant B cells show a reduced growth response upon LPS stimulation. A, Spleen cell cultures from wildtype and Smad7⌬ExI mutant mice were stimulated in the presence of BrdU with different concentrations of LPS, as indicated, or with a combination of Abs against IgM and CD40. Cultures were harvested after 1, 2, and 4 days. CD19-positive B cells were analyzed by FACS for the amount of incorporated BrdU. Statistical significance (wild-type vs mutant) was tested using paired t test, ⴱ, 0.05 ⬍ p ⬎ 0.01; ⴱⴱ, 0.01 ⬍ p ⬎ 0.001; NS, not significant. B, TGF-␤-neutralizing Ab abolished the proliferative difference between LPS-induced wildtype and Smad7⌬ExI mutant B cells. Spleen cell cultures from wild-type and mutant mice were either left untreated, stimulated with LPS, or stimulated with LPS in the presence of TGF-␤-neutralizing Ab for 1 or 2 days. Proliferating B cells were analyzed, as described for A. Statistical analysis, as in A. C, The amounts of Smad2 phosphorylation were significantly increased in Smad7⌬ExI mutant B cells. Spleen cell cultures from wild-type and mutant mice were either left untreated, stimulated with LPS, or stimulated with LPS in the presence of TGF-␤-neutralizing Ab for 0, 1, and 2 days. CD19-positive B cells were analyzed by FACS for the presence of pSmad2. D, TGF-␤ stimulation resulted in increased apoptosis of Smad7⌬ExI mutant B cells, whereas LPS stimulation only had a minor effect on apoptosis induction. Spleen cells from wild-type and mutant mice were grown either in medium ⫾ TGF-␤ (left panel), LPS, or activin A (right panel) for 0, 2, or 5 days, as indicated. The percentage of annexin V-positive apoptotic CD19-positive B cells was determined by FACS. Statistical analysis, as in A. The Journal of Immunology treatment resulted in a relatively higher rate of apoptosis in wildtype B cells at day 2, which coincides with the peak of proliferative response, indicating that the difference in growth rates cannot be attributed to apoptosis. Smad7 expression is not only up-regulated by TGF-␤, but also by LPS and other proinflammatory cytokines (56, 58). This puts Smad7 into a position in which its net activity modulates TGF-␤ signaling and determines, for instance, whether a cell becomes growth arrested and differentiates or proliferates. Our results demonstrate important roles for Smad7 during development and in B cell functions such as IgA class switching, apoptosis, and proliferation. Whether the Smad7 mutants also display aberrant T cell responses will be the focus of future investigations. The hypomorphic mutation we introduced into the Smad7 gene seems to interfere with its normal negative feedback activity on TGF-␤ signaling, and thus will allow the study of hyperactive TGF-␤ signaling in the adult mouse. We thank Pernilla Glader for technical assistance with the BrdU experiments. Disclosures The authors have no financial conflict of interest. References 1. Shi, Y., and J. Massague. 2003. 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Sideras. 2000. Smad and AML proteins synergistically confer transforming growth factor ␤1 responsiveness to human germ-line IgA genes. J. Biol. Chem. 275: 3552–3560. GENE TARGETING OF Smad7