Smad7 Inhibits Chondrocyte Differentiation at Multiple Steps during Endochondral Bone Formation and Down-regulates p38 MAPK Pathways
2008; Elsevier BV; Volume: 283; Issue: 40 Linguagem: Inglês
10.1074/jbc.m801175200
ISSN1083-351X
AutoresTakao Iwai, Junko Murai, Hideki Yoshikawa, Noriyuki Tsumaki,
Tópico(s)Osteoarthritis Treatment and Mechanisms
ResumoBone morphogenetic proteins (BMPs) play critical roles at various stages in endochondral bone formation. In vitro studies have demonstrated that Smad7 regulates transforming growth factor-β and BMP signals by inhibiting Smad pathways in chondrocytes. However, the in vivo roles of Smad7 during cartilage development are unknown. To investigate distinct effects of Smad7 at different stages during chondrocyte differentiation, we generated a series of conditional transgenic mice that overexpress Smad7 in chondrocytes at various steps of differentiation by using the Cre/loxP system. We generated Col11a2-lacZfloxed-Smad7 transgenic mice and mated them with three types of Cre transgenic mice to obtain Smad7Prx1, Smad711Enh, and Smad711Prom conditional transgenic mice. Smad7Prx1 mice overexpressing Smad7 in condensing mesenchymal cells showed disturbed mesenchymal condensation associated with decreased Sox9 expression, leading to poor cartilage formation. Smad711Enh mice overexpressing Smad7 in round chondrocytes showed decreased chondrocyte proliferation rates. Smad711Prom mice overexpressing Smad7 in flat chondrocytes showed inhibited maturation of chondrocytes toward hypertrophy. Micromass culture of mesenchymal cells showed that BMP-induced cartilaginous nodule formation was down-regulated by overexpression of Smad7, but not Smad6. Overexpression of Smad7, but not Smad6, down-regulated the phosphorylation of p38 MAPKs. Our data provide in vivo evidence for distinct effects of Smad7 at different stages during chondrocyte differentiation and suggest that Smad7 in prechondrogenic cells inhibits chondrocyte differentiation possibly by down-regulating BMP-activated p38 MAPK pathways. Bone morphogenetic proteins (BMPs) play critical roles at various stages in endochondral bone formation. In vitro studies have demonstrated that Smad7 regulates transforming growth factor-β and BMP signals by inhibiting Smad pathways in chondrocytes. However, the in vivo roles of Smad7 during cartilage development are unknown. To investigate distinct effects of Smad7 at different stages during chondrocyte differentiation, we generated a series of conditional transgenic mice that overexpress Smad7 in chondrocytes at various steps of differentiation by using the Cre/loxP system. We generated Col11a2-lacZfloxed-Smad7 transgenic mice and mated them with three types of Cre transgenic mice to obtain Smad7Prx1, Smad711Enh, and Smad711Prom conditional transgenic mice. Smad7Prx1 mice overexpressing Smad7 in condensing mesenchymal cells showed disturbed mesenchymal condensation associated with decreased Sox9 expression, leading to poor cartilage formation. Smad711Enh mice overexpressing Smad7 in round chondrocytes showed decreased chondrocyte proliferation rates. Smad711Prom mice overexpressing Smad7 in flat chondrocytes showed inhibited maturation of chondrocytes toward hypertrophy. Micromass culture of mesenchymal cells showed that BMP-induced cartilaginous nodule formation was down-regulated by overexpression of Smad7, but not Smad6. Overexpression of Smad7, but not Smad6, down-regulated the phosphorylation of p38 MAPKs. Our data provide in vivo evidence for distinct effects of Smad7 at different stages during chondrocyte differentiation and suggest that Smad7 in prechondrogenic cells inhibits chondrocyte differentiation possibly by down-regulating BMP-activated p38 MAPK pathways. The transforming growth factor-β (TGF-β) 2The abbreviations used are: TGF-β, transforming growth factor-β; BMP, bone morphogenetic protein; R-Smad, receptor-regulated Smad; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; dpc, day(s) postcoitus; rhBMP2, recombinant human BMP2; RT, reverse transcription; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. 2The abbreviations used are: TGF-β, transforming growth factor-β; BMP, bone morphogenetic protein; R-Smad, receptor-regulated Smad; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; dpc, day(s) postcoitus; rhBMP2, recombinant human BMP2; RT, reverse transcription; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. superfamily regulates cell growth and differentiation in a variety of tissues. This family includes three major subfamilies: TGFs-β, activins, and bone morphogenetic proteins (BMPs). Signaling by members of the TGF-β superfamily is transduced through type I and II serine/threonine kinase receptors (1.Miyazono K. Kusanagi K. Inoue H. J. Cell. Physiol. 2001; 187: 265-276Crossref PubMed Scopus (453) Google Scholar). Upon ligand binding, type II receptors phosphorylate type I receptors. Next, type I receptors phosphorylate downstream targets. Receptor-regulated Smads (R-Smads) are phosphorylated by type I receptors (2.Massague J. Seoane J. Wotton D. Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1908) Google Scholar). Smad1, Smad5, and Smad8 are R-Smads that transduce BMP signals, and Smad2 and Smad3 are R-Smads that transduce TGF-β and activin signals. Phosphorylated R-Smads form heteromers with Smad4, which is a common-partner Smad (referred to as Co-Smad), and translocate into the nucleus. There, they interact with transcription factors and activate gene transcription. Although the Smad pathway exists in most cell types and tissues, additional pathways are activated by BMP/TGF-β in certain cell types (3.Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4230) Google Scholar). BMP and TGF-β activate TAK1 (TGF-β-activated kinase 1), a member of the MAPK kinase kinase family. TAK1 is involved in the activation of several MAPKs, including JNK, p38, and ERK, which ultimately results in the activation of ATF2. TGF-β also activates Rho signaling pathways. Inhibitory Smads including Smad6 and Smad7 inhibit phosphorylation of R-Smads by competing with R-Smads for binding to phosphorylated type I receptors (2.Massague J. Seoane J. Wotton D. Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1908) Google Scholar). Smad6 inhibits BMP signaling, whereas Smad7 inhibits both TGF-β and BMP signaling. Smad6 has narrow specificity in its interaction with receptors (4.Goto K. Kamiya Y. Imamura T. Miyazono K. Miyazawa K. J. Biol. Chem. 2007; 282: 20603-20611Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Smad6 and Smad7 inhibit BMP-activated p38 MAPK pathways in neuronal cells (5.Yanagisawa M. Nakashima K. Takeda K. Ochiai W. Takizawa T. Ueno M. Takizawa M. Shibuya H. Taga T. Genes Cells. 2001; 6: 1091-1099Crossref PubMed Scopus (44) Google Scholar). Smad6 and Smad7 interact differently with TAK1 in PC12 cells. Smad6 and Smad7 are differentially expressed during development (6.Luukko K. Ylikorkala A. Makela T.P. Mech. Dev. 2001; 101: 209-212Crossref PubMed Scopus (42) Google Scholar). Mice deficient in exon 1 of Smad7 have abnormal B-cell responses and are small (7.Li R. Rosendahl A. Brodin G. Cheng A.M. Ahgren A. Sundquist C. Kulkarni S. Pawson T. Heldin C.H. Heuchel R.L. J. Immunol. 2006; 176: 6777-6784Crossref PubMed Scopus (73) Google Scholar). Data on skeletal tissues of these mice are not available. These mice have partial Smad7 function because N-terminally truncated forms of the Smad7 transcript may be produced (7.Li R. Rosendahl A. Brodin G. Cheng A.M. Ahgren A. Sundquist C. Kulkarni S. Pawson T. Heldin C.H. Heuchel R.L. J. Immunol. 2006; 176: 6777-6784Crossref PubMed Scopus (73) Google Scholar). During development, the limb skeleton is formed through endochondral bone formation, which consists of multiple steps of cellular differentiation (8.Kronenberg H.M. Ann. N. Y. Acad. Sci. 2006; 1068: 1-13Crossref PubMed Scopus (334) Google Scholar, 9.Olsen B.R. Reginato A.M. Wang W. Annu. Rev. Cell Dev. Biol. 2000; 16: 191-220Crossref PubMed Scopus (765) Google Scholar). Mesenchymal cells initially undergo condensation, which is followed by the differentiation of prechondrogenic cells within these condensations into round chondrocytes to form cartilage. Round chondrocytes in cartilage proliferate and produce cartilage extracellular matrix composed of collagen fibrils and proteoglycans. Proliferating chondrocytes in the central region of the cartilage then exit the cell cycle and differentiate into hypertrophic chondrocytes. The proliferating chondrocytes closest to the hypertrophic chondrocytes flatten out and form orderly columns of still proliferating flat chondrocytes. The zone of the hypertrophic chondrocytes is invaded by blood vessels along with osteoblasts, osteoclasts, and hematopoietic cells to form primary ossification centers. BMPs play critical roles at various stages in endochondral bone formation (10.Kobayashi T. Lyons K.M. McMahon A.P. Kronenberg H.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18023-18027Crossref PubMed Scopus (139) Google Scholar). Cartilage formation is severely disturbed in mice lacking BMP receptors (11.Yoon B.S. Ovchinnikov D.A. Yoshii I. Mishina Y. Behringer R.R. Lyons K.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5062-5067Crossref PubMed Scopus (341) Google Scholar) and in mice overexpressing Noggin, a BMP antagonist (12.Tsumaki N. Nakase T. Miyaji T. Kakiuchi M. Kimura T. Ochi T. Yoshikawa H. J. Bone Miner. Res. 2002; 17: 898-906Crossref PubMed Scopus (129) Google Scholar), in prechondrogenic cells; these results suggest that BMP signaling is necessary for cartilage formation. Smad7 is expressed in growth plate cartilage (13.Sakou T. Onishi T. Yamamoto T. Nagamine T. Sampath T. Ten Dijke P. J. Bone Miner. Res. 1999; 14: 1145-1152Crossref PubMed Scopus (128) Google Scholar) and osteoarthritic cartilage (14.Bauge C. Legendre F. Leclercq S. Elissalde J.M. Pujol J.P. Galera P. Boumediene K. Arthritis Rheum. 2007; 56: 3020-3032Crossref PubMed Scopus (63) Google Scholar, 15.Kaiser M. Haag J. Soder S. Bau B. Aigner T. Arthritis Rheum. 2004; 50: 3535-3540Crossref PubMed Scopus (41) Google Scholar). In vitro studies using cell culture systems or organ culture of mandibular explants have shown that Smad7 inhibits chondrocyte differentiation and/or proliferation induced by TGF-β (16.Ito Y. Bringas Jr., P. Mogharei A. Zhao J. Deng C. Chai Y. Dev. Dyn. 2002; 224: 69-78Crossref PubMed Scopus (65) Google Scholar, 17.Scharstuhl A. Diepens R. Lensen J. Vitters E. van Beuningen H. van der Kraan P. van den Berg W. Osteoarthritis Cartilage. 2003; 11: 773-782Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) or BMP (18.Fujii M. Takeda K. Imamura T. Aoki H. Sampath T.K. Enomoto S. Kawabata M. Kato M. Ichijo H. Miyazono K. Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (368) Google Scholar, 19.Valcourt U. Gouttenoire J. Moustakas A. Herbage D. Mallein-Gerin F. J. Biol. Chem. 2002; 277: 33545-33558Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). These in vitro studies have demonstrated down-regulation of R-Smad activation by Smad7 in chondrocytes. On the other hand, recent mouse genetic studies have revealed mild abnormalities in cartilage in mice lacking Smad4 (20.Zhang J. Tan X. Li W. Wang Y. Wang J. Cheng X. Yang X. Dev. Biol. 2005; 284: 311-322Crossref PubMed Scopus (80) Google Scholar) and in mice overexpressing Smad6 (21.Horiki M. Imamura T. Okamoto M. Hayashi M. Murai J. Myoui A. Ochi T. Miyazono K. Yoshikawa H. Tsumaki N. J. Cell Biol. 2004; 165: 433-445Crossref PubMed Scopus (95) Google Scholar) in chondrocytes, raising the possibilities that non-Smad pathways may also mediate BMP-induced cartilage formation. In addition, conditional inactivation of the TGF-β type II receptor gene (Tgfbr2) in prechondrogenic cells and chondrocytes results in mice without any long bone defects, leading to the conclusion that TGF-β signaling is not needed in the limb endochondral process (22.Baffi M.O. Slattery E. Sohn P. Moses H.L. Chytil A. Serra R. Dev. Biol. 2004; 276: 124-142Crossref PubMed Scopus (119) Google Scholar). In vivo effects of Smad7 on cartilage development are unknown and thus are worth examining. To examine the effects of Smad7 during cartilage development, we generated a series of conditional transgenic mice that overexpress Smad7 in chondrocytes at various steps of differentiation by using the Cre/loxP system. Smad7 overexpression in prechondrogenic cells disturbed mesenchymal condensation, leading to poor cartilage formation. Smad7 overexpression in round chondrocytes inhibited cell proliferation, and Smad7 overexpression in flat chondrocytes delayed hypertrophy. Micromass cultures of mesenchymal cells revealed that Smad7 inhibited cartilaginous nodule formation possibly by down-regulating p38 MAPK pathways activated by BMP. Construction of the Transgene—The α2(XI) collagen gene (Col11a2)-based expression vector, p742lacZInt, contains the Col11a2 promoter (-742 to +380), an SV40 RNA splice site, the β-galactosidase reporter gene (lacZ), the SV40 polyadenylation signal, and 2.3 kb of the first intron sequence of Col11a2 as an enhancer (23.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar). To create a Col11a2-lacZfloxed-Smad7 transgene plasmid, a loxP sequence was inserted into the 5′-untranslated region of lacZ p742lacZInt, and the NotI sites at both ends of the lacZ segment were mutated and abolished. Next, a sequence consisting of an SV40 RNA splice site, a loxP sequence, a NotI site, and the SV40 polyadenylation signal was inserted into the 3′-end of the SV40 polyadenylation signal of the plasmid. Finally, mouse Smad7 cDNA tagged with NotI sites at both ends was inserted into the NotI site. To create the Col11a2prom-Cre-enh plasmid, the lacZ sequence in p742lacZInt was replaced with a NotI-tagged Cre sequence at the NotI sites. To create the Col11a2prom-Cre plasmid, the lacZ sequence in p742lacZ (23.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar) was replaced with a NotI-tagged Cre sequence at NotI sites. Generation and Preparation of Transgenic Mice—The plasmids Col11a2-lacZfloxed-Smad7, Col11a2prom-Cre-enh, and Col11a2prom-Cre were digested with EcoRI and PstI to release the inserts. Transgenic mice were produced by microinjecting each of the inserts into the pronuclei of fertilized eggs from F1 hybrid mice (C57BL/6 × DBA) as described previously (23.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar). Transgenic embryos were identified by PCR assays of genomic DNA extracted from the placenta or skin. For Col11a2-lacZfloxed-Smad7 transgenic mice, primers derived from mouse Smad7 cDNA (5′-GGA TGG CGT GTG GGT TTA-3′) and from the SV40 poly(A) signal region (5′-GGT TTG TCC AAA CTC ATC AAT-3′) were used to amplify a 346-bp product. For Cre transgenic mice, primers derived from Cre (5′-CAA TTT ACT GAC CGT ACA CCA A-3′ and 5′-TCT TCA GGT TCT GCG GG-3′) were used to amplify a 187-bp product. Prx1-Cre transgenic mice were a kind gift from Dr. Malcolm Logan (24.Logan M. Martin J.F. Nagy A. Lobe C. Olson E.N. Tabin C.J. Genesis. 2002; 33: 77-80Crossref PubMed Scopus (679) Google Scholar). Col11a2-lacZfloxed-Smad7 transgenic mice were mated with Prx1-Cre, Col11a2prom-Cre-enh, or Col11a2prom-Cre transgenic mice to obtain various Smad7 conditional transgenic mice. Conventional Col11a2-Smad6 transgenic mice were described previously (21.Horiki M. Imamura T. Okamoto M. Hayashi M. Murai J. Myoui A. Ochi T. Miyazono K. Yoshikawa H. Tsumaki N. J. Cell Biol. 2004; 165: 433-445Crossref PubMed Scopus (95) Google Scholar). Staining of the Skeleton—Embryos were dissected, fixed in 100% ethanol overnight, and then stained with Alcian blue followed by alizarin red S solution according to standard protocols (25.Peters P.W.J. Neuberg H.J.M.D. Kwasigroch T.E. Methods in Prenatal Toxicology. Georg Thieme Verlag, Stuttgart, Germany1977: 153-154Google Scholar). Histological Analysis—Embryos were dissected with a stereomicroscope, fixed in 4% paraformaldehyde, processed, and embedded in paraffin. Serial sections were stained with hematoxylin and eosin or with safranin O/fast green/iron hematoxylin. Immunohistochemistry was performed with a rabbit polyclonal antibody against Smad7 (1:100 dilution; Santa Cruz Biotechnology, Inc.). Immune complexes were detected using streptavidin-peroxidase staining and Histofine SAB-PO kits (Nichirei, Tokyo, Japan). RNA in situ hybridization was performed using 35S-labeled antisense riboprobes as described previously (45.Pelton R.W. Dickinson M.E. Moses H.L. Hogan B.L. Development (Camb.). 1990; 110: 609-620PubMed Google Scholar). To detect proliferating cells in tissue sections, digoxigenin-11-UTP-labeled Hist2 (histone cluster 2) RNA probes were prepared (26.Muskhelishvili L. Latendresse J.R. Kodell R.L. Henderson E.B. J. Histochem. Cytochem. 2003; 51: 1681-1688Crossref PubMed Scopus (201) Google Scholar). Micromass Culture of Mesenchymal Cells—Micromass culture was performed according to previously described methods (27.Fujimaki R. Toyama Y. Hozumi N. Tezuka K. J. Bone Miner. Metab. 2006; 24: 191-198Crossref PubMed Scopus (56) Google Scholar). The distal quarters of limb buds from 12.0-day postcoitus (dpc) wild-type and transgenic mouse embryos were dissected and digested with 0.1% collagenase (Sigma) and 0.1% trypsin (Sigma) for 45 min in 5% CO2 at 37 °C. The dissociated cells were filtered through nylon mesh (40-μm pore size; Tokyo Screen, Tokyo) to generate a single cell suspension and were then adjusted to 2 × 107 cells/ml in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum. Cell suspensions (20-ml drops) were placed in the center of each well of 12-well plates. After the cells were allowed to attach for 90 min in 10% CO2 at 37 °C, they were overlaid with 2 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The medium was replaced by fresh medium every other day. To induce cartilaginous nodule formation, medium containing 50 or 100 ng/ml recombinant human BMP2 (rhBMP2) (Astellas Pharma, Tokyo) in 10% fetal bovine serum was used unless specifically described otherwise. For infection with adenoviral vectors for Smad6 and Smad7 (18.Fujii M. Takeda K. Imamura T. Aoki H. Sampath T.K. Enomoto S. Kawabata M. Kato M. Ichijo H. Miyazono K. Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (368) Google Scholar), cells were infected 1 day after plating. For Alcian blue staining, micromass cultures 4 days after plating were fixed with 4% paraformaldehyde for 10 min and stained overnight with 1% Alcian blue in 3% acetic acid at 37 °C. The cultures were then digitally photographed with a Nikon SMZ-U microscope. The size and number of nodules were measured using WinROOF software (Mitani Shoji, Fukui, Japan). For Western blot analysis, adenovirus-infected cells were starved for 16 h before replacement with Dulbecco's modified Eagle's medium in the presence or absence of rhBMP2. Cells were lysed 1 h after treatment with rhBMP2. For activation of MAPK pathways, 2 ng/ml anisomycin (Sigma) was added to the culture medium and used as a positive control. Total RNA extracted from micromass culture in the presence of 100 ng/ml rhBMP2 was digested with DNase to eliminate any contaminating genomic DNA before real-time quantitative reverse transcription (RT)-PCR. Real-time RT-PCR was performed as described previously (21.Horiki M. Imamura T. Okamoto M. Hayashi M. Murai J. Myoui A. Ochi T. Miyazono K. Yoshikawa H. Tsumaki N. J. Cell Biol. 2004; 165: 433-445Crossref PubMed Scopus (95) Google Scholar). The primer pair for Id1 was as follows: up, 5′-GCA TCT TGT GTC GCT GAG-3′; and down, 5′-TGG CTG CGG TAG TGT CTT-3′. The product size was 122 bp. ATDC5 Cell Culture—ATDC5 cells were a kind gift from Dr. Y. Hiraki. The cells were maintained at 20–80% confluency as described previously (28.Shukunami C. Shigeno C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (343) Google Scholar). After infection of ATDC5 cells with adenoviral vectors for LacZ, Smad6, Smad7, and constitutively active MKK3, the cells were washed and then starved for 16 h. The medium was replaced with Dulbecco's modified Eagle's medium in the presence or absence of rhBMP2. One hour after treatment with rhBMP2, the cells were lysed and subjected to Western blotting. For activation of MAPK pathways, 2 ng/ml anisomycin was added to the culture medium and used as a positive control. Western Blotting—Cells were cultured, lysed, subjected to SDS-PAGE, electroblotted, and immunostained. The antibodies used were anti-Smad7 antibody (1:200 dilution; Santa Cruz Biotechnology, Inc.); anti-Smad6 antibody (1:200 dilution; Zymed Laboratories Inc.), anti-Smad1 antibody (1:1000 dilution; Calbiochem); anti-phospho-Smad1/5/8 antibody (1:1000 dilution), anti-phospho-ATF2 antibody (1:1000 dilution), anti-ATF2 antibody (1:1000 dilution), anti-phospho-p38 MAPK antibody (1:1000 dilution), anti-p38 MAPK antibody (1:1000 dilution), anti-phospho-Smad2 antibody (1:1000 dilution), and anti-Smad2/3 antibody (1:1000 dilution) (Cell Signaling Technology). Proteins in the blots were visualized using an ECL Plus kit (Amersham Biosciences). Generation of Floxed lacZ-Smad7 Transgenic Mice—Because Smad7 inhibits various signaling pathways, we anticipated that its overexpression in chondrocytes might cause severe cartilage abnormalities in transgenic mice. To avoid lethality, we employed a conditional transgenic mouse system to express the Smad7 transgene. We first established floxed lacZ-Smad7 transgenic mouse lines bearing the Col11a2-lacZfloxed-Smad7 transgene (Fig. 1A). Col11a2 promoter/enhancer sequences direct expression to condensed mesenchymal cells and chondrocytes (23.Tsumaki N. Kimura T. Matsui Y. Nakata K. Ochi T. J. Cell Biol. 1996; 134: 1573-1582Crossref PubMed Scopus (68) Google Scholar). We expected that the mice bearing this construct would express lacZ but not Smad7 due to the poly(A) signal sequence that immediately follows the lacZ sequence. In the presence of Cre recombinase, the lacZ sequence would be deleted, and Smad7 would be expressed instead of lacZ under the control of the Col11a2 promoter/enhancer sequences. We obtained and analyzed two independent lines of Col11a2-lacZfloxed-Smad7 transgenic mice. Staining of transgenic embryos from one line with X-gal showed that lacZ was expressed in condensed mesenchyme at 12.5 dpc (Fig. 1B) and in primordial cartilage in limbs and ribs at 14.5 dpc (Fig. 1C). Histological analysis confirmed lacZ activities specifically in mesenchymal condensation at 12.5 dpc (Fig. 1D) and in chondrocytes at 14.5 dpc (Fig. 1E). lacZ activities were not recognized in cells in the perichondrium (Fig. 1F). Transgenic embryos from the other line showed a similar but weaker (Fig. 1G) pattern of lacZ activities than the first line (Fig. 1C) at 14.5 dpc. Both lines of mice developed normally. Generation of Cre Transgenic Mice—Next, we prepared three types of Cre transgenic mice. First, Prx1-Cre transgenic mice were obtained from Dr. Malcolm Logan (24.Logan M. Martin J.F. Nagy A. Lobe C. Olson E.N. Tabin C.J. Genesis. 2002; 33: 77-80Crossref PubMed Scopus (679) Google Scholar). Second, we linked the Cre sequence to the Col11a2 promoter plus intron enhancer to prepare the transgene construct 11Enh-Cre (Fig. 2A). Third, we prepared Cre linked to the Col11a2 promoter without an intron enhancer to prepare the transgene construct 11Prom-Cre (Fig. 2A). We injected 11Enh-Cre and 11Prom-Cre into fertilized ova and established transgenic lines. We examined the expression patterns of Cre by mating CAGCATfloxed-lacZ reporter transgenic mice (29.Sakai K. Miyazaki J. Biochem. Biophys. Res. Commun. 1997; 237: 318-324Crossref PubMed Scopus (425) Google Scholar) with these Cre transgenic mice. As reported previously (24.Logan M. Martin J.F. Nagy A. Lobe C. Olson E.N. Tabin C.J. Genesis. 2002; 33: 77-80Crossref PubMed Scopus (679) Google Scholar), Prx1-Cre mice showed Cre recombinase activity throughout the early limb bud mesenchyme at 12.5 dpc. (Fig. 2B). 11Enh-Cre transgenic mice started to show very weak Cre activities in mesenchymal condensation in forelimb buds at 12.5 dpc (Fig. 2C). 11Prom-Cre transgenic mice showed no lacZ activities in limb buds at 12.5 dpc (Fig. 2D). Aberrant Cre activities were recognized in forebrains. At 14.5 dpc, 11Enh-Cre transgenic mice showed Cre-mediated recombination in all primordial cartilage of limbs and ribs (Fig. 2E). In contrast, 11Prom-Cre transgenic mice showed recombination in a limited part of each primordial cartilage (Fig. 2F). X-gal staining was absent in the epiphyseal part of each primordial cartilage. Histological analysis of the distal ulnas of 11Enh-Cre transgenic mice at 14.5 dpc showed recombinase activities in all chondrocytes, including round proliferative chondrocytes, flat proliferative chondrocytes, prehypertrophic chondrocytes, hypertrophic chondrocytes, and perichondrial cells (Fig. 2G). Histological analysis of the distal ulnas of 11Prom-Cre transgenic mice at 14.5 dpc showed recombinase activities in flat chondrocytes, prehypertrophic chondrocytes, and hypertrophic chondrocytes, but not in round chondrocytes located at the ends of each primordial cartilage (Fig. 2H). These results suggest that 11Enh-Cre induces recombination from the step of round proliferative chondrocytes and that 11Prom-Cre induces recombination from the step of flat proliferative chondrocytes. The recombination pattern of our 11Enh-Cre transgenic mice was consistent with that of previously reported Cre transgenic mice containing identical promoter/enhancer sequences (30.Fujimaki R. Hayashi K. Watanabe N. Yamada T. Toyama Y. Tezuka K. Hozumi N. J. Bone Miner. Metab. 2005; 23: 270-273Crossref PubMed Scopus (6) Google Scholar). None of the Cre transgenic mice showed detectable abnormalities. Skeletal Abnormalities in Smad7 Conditional Transgenic Mice—We mated Col11a2-lacZfloxed-Smad7 transgenic mice with Prx1-Cre, 11Enh-Cre, and 11Prom-Cre transgenic mice, respectively, and generated double transgenic pups as follows: pups bearing Col11a2-lacZfloxed-Smad7 and Prx1-Cre transgenes (Smad7Prx1), those bearing Col11a2-lacZfloxed-Smad7 and 11Enh-Cre transgenes (Smad711Enh), and those bearing Col11a2-lacZfloxed-Smad7 and 11Prom-Cre transgenes (Smad711Prom). We examined the skeletons of Smad7 conditional transgenic mice. Staining with Alcian blue and alizarin red S revealed that the Smad7Prx1 double transgenic mice had very hypoplastic limb skeletons (Fig. 3B) compared with Col11a2-lacZfloxed-Smad7 control mice (Fig. 3A) at 16.5 dpc. The Smad7Prx1 axial skeleton was relatively preserved because the Prx1-Cre transgene directs Cre expression mainly in limbs. Hind limb skeletons were less affected than forelimb skeletons. Smad711Enh mice had moderately hypoplastic skeletons (Fig. 3C) compared with Col11a2-lacZfloxed-Smad7 control mice (Fig. 3A). Mineralization indicated by alizarin red staining was reduced in Smad711Enh mice. The cartilaginous components of Smad711Prom mice were slightly small (Fig. 3D) compared with those of Col11a2-lacZfloxed-Smad7 control mice (Fig. 3A). Mineralization was reduced slightly in Smad711Prom mice. We also mated the other line of Col11a2-lacZfloxed-Smad7 mice with Prx1-Cre mice, and the resultant Smad7Prx1 transgenic pups showed similar but milder abnormalities in limb skeleton (Fig. 3E) than the Smad7Prx1 mice from the first line of Col11a2-lacZfloxed-Smad7 mice (Fig. 3B). Overall, the skeletal lengths in each type of double transgenic pups varied with regard to cartilage size and mineralization depending on the types of Cre transgenic mice used for mating (Fig. 3F). Cre-mediated Recombination in Each Type of Conditional Transgenic Mice—The scheme in Fig. 4A shows the expected recombination events in this experiment. In Smad7Prx1 mice, recombination occurs in mesenchymal cells in limb buds before the onset of mesenchymal condensation when Col11a2 promoter/enhancer sequences start to direct expression. Thus, the expression of the Smad7 transgene in Smad7Prx1 mouse limbs may be controlled by Col11a2 promoter/enhancer sequences in the Col11a2-lacZfloxed-Smad7 transgene, and the Smad7 transgene may be gradually expressed from the step of condensed mesenchyme. The expression pattern of the Smad7 transgene in Smad7Prx1 conditional transgenic mouse limbs is considered to be similar to the expression pattern of the transgene expression in conventional transgenic mice bearing the Col11a2 promoter/enhancer. In Smad711Enh mice, recombination occurs at the step of round proliferative chondrocytes when the amount of Cre protein may be increased enough to catalyze recombination. In Smad711Prom mice, recombination occurs at the step of flat proliferative chondrocytes. We examined Cre-catalyzed recombination by monitoring the disappearance of lacZ activities. Col11a2-lacZfloxed-Smad7 mice showed LacZ expression in condensed mesenchyme at 12.5 dpc (Fig. 4B). Smad7Prx1 double transgenic mice did not exhibit LacZ expression (Fig. 4C), indicating that Prx1-Cre transgene products induced recombination of the Col11a2-lacZfloxed-Smad7 transgene in limb buds. Both Smad711Enh mice and Smad711Prom mice showed LacZ expression in condensed mesenchyme (Fig. 4, D and E), as did Col11a2-lacZfloxed-Smad7 control mice (Fig. 4B), indicating that recombination did not occur at this stage. At 14.5 dpc, Col11a2-lacZfloxed-Smad7 control mice showed LacZ expression in all primordial cartilage (Fig. 4F). Smad7Prx1 mice showed LacZ expression in primordial cartilage in the ribs and spine, but not in the limbs (Fig. 4G). LacZ expression in Smad711Enh mice disappeared in primordial cartilage in trunk and proximal segments in the limbs. LacZ expression remained in paws, which dev
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