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Cloning and Studies of the Mouse cDNA Encoding Smad3 Kiyoshi KANO, Azusa NOTANI, Sang-Yoon NAM, Masahiko FUJISAWA, Masamichi KUROHMARU and Yoshihiro HAYASHI Department of Veterinary Anatomy, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113–8657, Japan (Received 30 September 1998/Accepted 5 November 1998) ABSTRACT . Following stimulation by the transforming growth factor-β (TGF-β) family in the cytoplasm, the Smad family is phosphorylated and translocated to the nucleus and activates several gene transcriptions. In this study, the mouse Smad3 cDNA including the open reading frame (ORF) was cloned from the mouse brain using a RACE (rapid amplification of cDNA ends) technique, and its expression pattern was analyzed in mouse tissue using northern blot. The predicted amino acid (aa) sequences of mouse Smad3 showed a high homology with human Smad3 (99.3%) and mouse Smad2 (85.4%). It revealed that this protein may be highly conserved in different species of mammals. Northern blot analyses revealed that Smad3 was highly expressed in the brain and ovary, and that the size of major transcript was about 5.7 kb. In situ hybridization analyses revealed the high expression of Smad3 was detected in the pyramidal cells of the hippocampus, the granule cells of the dentate gyrus, the granular cells of the cerebral cortex and the granulosa cells of the ovary. Smad3 may be essential transducer of signals from TGF-β and activin in these cells.—KEY WORDS: in situ hybridization, mouse, northern blot analysis, open reading frame (ORF), Smad. J. Vet. Med. Sci. 61(3): 213–219, 1999

The transforming growth factor-β (TGF-β) family have a substantial roles in regulating cell growth, cell cycle, differentiation and apoptosis in various tissues. Recently, the signal transduction pathway in the cytoplasm following stimulation by the TGF-β family has been elucidated. The Smad family is regarded as one of signal transducers from the transmembrane receptor to the nucleus [11, 17]. At first, the Smad family was cloned in vertebrates as a homologue of Mothers against dpp (Mad) in Drosophila melanogaster [23] and sma in Caenorhabditis elegans [22]. These proteins of the Smad family have a highly conserved domain in their N- and C-terminal regions, namely Madhomology domains, MH1 and MH2, respectively, which are unique to these proteins. These domains are suggested as significant domains in signal transduction [5, 10, 15, 27]. In the C-terminal region, Smads have a characteristic SerSer-X-Ser (SSXS) motif, which are phosphorylated by the type I receptors of the TGF-β family [1, 13, 16, 24]. Among these Smads, Smad2 [3, 8, 13, 16, 19] and Smad3 [19, 26] were activated by the signal of TGF-β and activin, while Smad1 [12–15, 25] and Smad5 were activated by the bone morphogenetic protein 2 (BMP-2) [25]. A mutation in the C-terminal region has resulted in causing cancers such as pancreatic [9] and colorectal carcinoma [8], suggesting that this domain has an important role in the regulation of cells. Among these Smads, the function of Smad3 was less known compared with other Smads. In this study, the mouse Smad3 cDNA containing the complete open reading frame (ORF) was cloned from the mouse brain using a RACE technique, and its expression pattern in mouse was investigated using northern blot analyses and in situ hybridization.

MATERIALS AND METHODS

Isolation and sequencing of the mouse Smad3 cDNA: Total RNA was isolated from the brains of 8-week-old ICR mice (Japan Laboratory Animals, Inc.) using ultracentrifugation on a CsCl gradient. The first strand cDNA was prepared against 1 µ g of total RNA by reverse transcriptase (Superscript II; Gibco BRL) and processed to PCR using the degenerate primer set, which is expected to amplify the conserved regions of the Smad family. The sequences of the degenerate primer set, (5’-(AC)GITTCTGC (CT)TIGG(ACGT)TTG(TC)T(CG)TC-3’ and 5’-CCCCA (AGCT)CC(CT)TT(CG)AC(AG)AA(AG)CTC-3’), were designed based on the amino acid and nucleotide sequences of mouse [2, 3, 25] and human [8, 9, 15, 20, 26] Smad families reported. The result proved that the 331-bp DNA fragments contained a partial sequence of the mouse Smad3 as judged from the predicted amino acid sequence [7, 26]. Then, RACE was performed as described in the 5’/3’ RACE Kit (Boehringer-Mannheim) using primers, F1 (Foward 1; 5’-GCAGCCGTGGAACTTACAAGGC-3’), F2 (Forward 2; 5’-CTGCAACCAGCGCTATGGCTGG-3’), R1 (Reverse 1; 5’-GGTAGACAGCCTCAAAGCCCTG-3’), R2 (Reverse 2; 5’-TAGCGCTGGTTGCAGTTGGGAG-3’), and R3 (Reverse 3; 5’-CTCCGATGTAGTAGAGCCGCACA-3’). F2, R2 and R3 were used as primers for nested PCR. The directions and positions of each primer are shown in Fig. 1. All PCR amplifications were performed in a thermal cycler (Takara Shuzo) for 35 cycles at 95°C for 1 min, 58°C for 2 min, and 72°C for 2 min. Then, the PCR products of 3’ and 5’ RACEs were inserted into pGEM-T easy plasmid (Promega) and the sequences were determined using the fluorescence labeling method. Northern blot analysis: To prepare a specific cRNA probe

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Fig. 1. Nucleotide and deduced amino acid sequences of Smad3 derived from the mouse brain. The arrows indicate the sequences of the primers (F0, F1, F2, R0, R1, R2, and R3) used. MH1 is underlined and MH2 is double-underlined. The stop codon is at nt 1302–1304 (asterisk). The open-boxed aa sequence represents the Ser-Ser-X-Ser (SSXS) motif. The GenBank accession numbers for the nt sequence is AB008192*. * We have submitted the sequence data to GenBank on October 17, 1997. The sequence was also reported in GenBank as SMAD3 (accession number; AF016189) at present.

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for mouse Smad3 for northern blot analysis and in situ hybridization, PCR was carried out using the forward primer 5’-CCAGCCATGTCGTCCATCCTGC-3’ (F0: mouse Smad3 cDNA nt 21–42) and the reverse primer 5’CCCTTCCGATGGGACACCTGCA-3’ (R0: mouse Smad3 cDNA nt 250–271) (Fig. 1). The fragment was inserted into pGEM-T easy plasmid (Promega) and sequenced. DIGlabeled ribonucleotide probe for mouse Smad3 was synthesized using in vitro transcription in the presence of the Sp6 RNA polymerase (Boehringer-Mannheim) at 37°C for 90 min. For northern blot analyses, total RNA was electrophoresed on 1% agarose gel containing 2.2 M formaldehyde and transferred onto a Hybond N+ membrane (Amersham) by capillary blotting. The membrane was hybridized with the DIG-labeled RNA probe at 68°C for 16 hr. The membrane was washed twice in 2× SSC containing 0.1% SDS at room temperature for 5 min, then washed twice with 0.1× SSC containing 0.1% SDS at 68°C for 60 min. For signal detection, the membrane was incubated with an anti-DIG alkaline phosphatase-conjugated antibody and further processed to detect chemiluminescence of DIG as recommended by the manufacture (CDP-Star; Boehringer-Mannheim). In situ hybridization: In situ hybridization was performed using a new method introduced by Boehringer-Mannheim. After briefly washing with saline, the brain and ovary of 8week-old mouse were perfused with Bouin’s fixative through the left ventricle. They were dehydrated in a graded series of ethanol, embedded in paraffin and cut at 5 µm. Deparaffinized sections were postfixed in 4% paraformaldehyde for 15 min and incubated twice in 0.1% DEPC (Sigma) for 15 min. The sections were prehybridized in the hybridization mix (50% formamide/5× SSC containing 40 µg/ml sonicated salmon sperm DNA) at 58°C for 2 hr and hybridized at 58°C for 40 hr in the hybridization mix added the DIG-labeled antisense or sense riboprobe for Smad3. After hybridization, the sections were washed in 2 × SSC at room temperature for 30 min and at 58°C for 30 min. Then they were washed in 0.1× SSC at 58°C for 30 min. Thereafter, the sections were incubated for 2 hr at room temperature with alkaline phosphatase-coupled antidigoxigenin antibody (Boehringer-Mannheim) diluted 1:2,000 in the buffer 1 (Tris-HCl 100 mM, NaCl 150 mM, pH 7.5) containing 0.5% blocking reagent (BoehringerMannheim). After washing in buffer 1, the sections were developed at room temperature in buffer 2 (Tris-HCl 100 mM, NaCl 100 mM and MgCl2 50 mM, pH 9.5) containing NBT and BCIP (Boehringer-Mannheim).

26] Smad families. The sequence result revealed that these PCR products contained Smad1, Smad2 and Smad3 from the predicted amino acid sequence (data not shown). Between these Smads, the function of Smad3 was less known compared with other Smads. Therefore, Smad3 was chosen to determine the sequence of its full length cDNA by means of a RACE technique and analyze its distribution in various tissues. The cDNA nucleotide sequence and the predicted amino acid sequence of the mouse Smad3 were determined as shown in Fig. 1. Similar to Smad1 and Smad2, the amino acid sequence of mouse Smad3 contained 2 conserved regions at its N-and C-terminals, MH1 and MH2 (Fig. 1). The sequence showed a high homology with human Smad3 (89.1%) [26] in the nucleotide, with human Smad3 (99.3%) [26] and with mouse Smad2 (85.4%) [3] (Fig. 2) in the predicted amino acid. The high homology with human Smad3 and with mouse Smad2, suggested that this protein may be highly conserved in different species of mammals. Expression pattern and localization of Smad3: To clarify the expression pattern of Smad3 among various tissues of the adult mouse, northern blot analysis was performed. The high expression of Smad3 was observed in the brain and ovary (Fig. 3). The major size of Smad3 transcript in these tissues was about 5.7 kb, while minor transcript about 3.0 kb and 11 kb was detected. In order to detect cell type specificity of Smad3 expression in the brain and ovary, we hybridized these sections with DIG-labeled antisense using a new method of in situ hybridization. Intense signals of Smad3 mRNA were localized in the pyramidal cells of the hippocampus, the granule cells of the dentate gyrus (Fig. 4, A, B) and the granular layer cells of the cerebral cortex (Fig. 4, D, E), and in the granulosa cells of the ovary (Fig. 4, G, H). In these tissues, no signals were detected in the sense control (Fig. 4, C, F, I). In the adult rat, TGF- β type II receptor and activin type II receptor was localized in the hippocampal formation [4, 6, 18] and in the granulosa cells of ovary [6, 21]. These data support that Smad3 may be essential transducer of signals from TGF-β and activin in these cells. ACKNOWLEDGMENT. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan. REFERENCES 1.

RESULTS AND DISCUSSION

Nucleotide and deduced amino acid sequences of mouse Smad3: To search and determine the cDNA sequence of the mouse Smad family, degenerate primers were designed from the amino acid and nucleotide consensus primers were designed from the amino acid and nucleotide consensus sequences of the mouse [2, 3, 25] and human [8, 9, 15, 20,

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Fig. 2. Alignment of the deduced amino acid sequence of mouse Smad3 to human Smad3 (A), and mouse Smad2 (B). The asterisks indicate the conserved aa residues. 4.

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Fig. 3. Expression of mouse Smad3 in various tissues of the adult mouse. Northern blot analysis was performed on 20 µg of total RNA using a DIG-labeled cRNA probe specific for mouse Smad3. A major single transcript of about 5.7 kb is observed. BR, brain; HT, heart; LU, lung; LI, liver; SP, spleen; KD, kidney; SM, skeletal muscle; TS, testis; OV, ovary.

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Fig. 4. In situ hybridization analyses. Sections of the brain and ovary were hybridized with either DIG-labeled antisense (A, B, D, E, G, H) or sense (C, F, I) riboprobe for Smad3. In the brain, intense signals were detected in the pyramidal cells of the hippocampus (A, B), the granule cells of the dentate gyrus (A, B) and the granular layer cells of the cerebral cortex (D, E) and in the ovary, in the granulosa cells (G, H). P, pyramidal layer; G, granular layer. A, C, D, F, G, I) × 33, B, E, H) × 132.

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