Severe Intestinal Obstruction on Induced Smooth Muscle–Specific Ablation of the Transcription Factor SRF in Adult Mice
2007; Elsevier BV; Volume: 133; Issue: 6 Linguagem: Inglês
10.1053/j.gastro.2007.08.078
ISSN1528-0012
AutoresMeike Angstenberger, Jörg W. Wegener, Bernd J. Pichler, Martin S. Judenhofer, Susanne Feil, Siegfried Alberti, Robert Feil, Alfred Nordheim,
Tópico(s)Cell Adhesion Molecules Research
ResumoBackground & Aims: SRF (Serum Response Factor), a widely expressed transcription factor, controls expression of mitogen-responsive and muscle-specific genes, thereby regulating the contractile actin microfilament. Genetic Srf deletion studies showed SRF to be indispensable for in vivo skeletal and cardiac muscle cell development. We now investigated for the first time in vivo SRF functions in smooth muscle cells of adult mice. Methods: We conditionally deleted a floxed Srf allele (Srfflex1) in adult mice by inducible activation of the CreERT2 recombinase expressed specifically in smooth muscle cells. Tamoxifen-induced CreERT2 activity stimulated deletion of exon 1 coding sequences of Srfflex1, thereby abolishing full-length SRF protein expression in adult smooth muscle cells of the analyzed organs: colon, bladder, and stomach. Results: Smooth muscle cell–specific ablation of full-length SRF protein in adult mice showed impaired contraction of intestinal smooth muscle, resulting in defective peristalsis. Mutant mice died within 2 weeks of tamoxifen treatment, displaying clear symptoms of ileus paralyticus. Cultured primary SRF-deficient colon smooth muscle cells were viable, but displayed drastic structural alterations and elevated senescence, paralleled by degeneration of the actin microfilament and impaired expression of smooth muscle–specific genes. Conclusions: SRF plays a vital role in the contractile activity and cytoskeletal architecture of adult smooth muscle cells and is therefore essential for physiologic functions of the gastrointestinal tract in vivo. Our mouse genetic model may resemble features of human chronic intestinal pseudo-obstruction. Background & Aims: SRF (Serum Response Factor), a widely expressed transcription factor, controls expression of mitogen-responsive and muscle-specific genes, thereby regulating the contractile actin microfilament. Genetic Srf deletion studies showed SRF to be indispensable for in vivo skeletal and cardiac muscle cell development. We now investigated for the first time in vivo SRF functions in smooth muscle cells of adult mice. Methods: We conditionally deleted a floxed Srf allele (Srfflex1) in adult mice by inducible activation of the CreERT2 recombinase expressed specifically in smooth muscle cells. Tamoxifen-induced CreERT2 activity stimulated deletion of exon 1 coding sequences of Srfflex1, thereby abolishing full-length SRF protein expression in adult smooth muscle cells of the analyzed organs: colon, bladder, and stomach. Results: Smooth muscle cell–specific ablation of full-length SRF protein in adult mice showed impaired contraction of intestinal smooth muscle, resulting in defective peristalsis. Mutant mice died within 2 weeks of tamoxifen treatment, displaying clear symptoms of ileus paralyticus. Cultured primary SRF-deficient colon smooth muscle cells were viable, but displayed drastic structural alterations and elevated senescence, paralleled by degeneration of the actin microfilament and impaired expression of smooth muscle–specific genes. Conclusions: SRF plays a vital role in the contractile activity and cytoskeletal architecture of adult smooth muscle cells and is therefore essential for physiologic functions of the gastrointestinal tract in vivo. Our mouse genetic model may resemble features of human chronic intestinal pseudo-obstruction. See editorial on page 2052. See editorial on page 2052. Serum response factor (SRF) is a widely expressed MCM1/agamous/deficiens/serum–box transcription factor involved in multiple cellular processes. SRF binds a highly conserved 10-base pair DNA cis element, the CArG [CC(A/T)6GG] box.1Norman C. Runswick M. Pollock R. Treisman R. Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element.Cell. 1988; 55: 989-1003Abstract Full Text PDF PubMed Scopus (710) Google Scholar More than 200 SRF target genes are known, nearly half of them dedicated to actin cytoskeletal dynamics2Posern G. Treisman R. Actin’ together: serum response factor, its cofactors and the link to signal transduction.Trends Cell Biol. 2006; 16: 588-596Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar and cellular contractility.3Miano J.M. Long X. Fujiwara K. Serum response factor: master regulator of the actin cytoskeleton and contractile apparatus.Am J Physiol Cell Physiol. 2007; 292: C70-C81Crossref PubMed Scopus (379) Google Scholar SRF has been implicated in muscle development and postreplicative muscle gene expression in myocardial, skeletal, and smooth muscles (SMs). Many gene inactivation or knockdown studies in mice have consistently shown SRF to be indispensable for survival. Mouse embryos lacking SRF do not form any detectable mesoderm, show a drastic reduction in immediate-early gene expression, and die during early gastrulation.4Arsenian S. Weinhold B. Oelgeschlager M. Ruther U. Nordheim A. Serum response factor is essential for mesoderm formation during mouse embryogenesis.EMBO J. 1998; 17: 6289-6299Crossref PubMed Scopus (312) Google Scholar The Cre-loxP genomic recombination technology has been used for conditional, cell type-selective inactivation of SRF, so far primarily in neuronal systems (reviewed in Etkin et al5Etkin A. Alarcon J.M. Weisberg S.P. et al.A role in learning for SRF: deletion in the adult forebrain disrupts LTD and the formation of an immediate memory of a novel context.Neuron. 2006; 50: 127-143Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) and studies of muscle function.6Parlakian A. Tuil D. Hamard G. et al.Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality.Mol Cell Biol. 2004; 24: 5281-5289Crossref PubMed Scopus (162) Google Scholar, 7Parlakian A. Charvet C. Escoubet B. et al.Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart.Circulation. 2005; 112: 2930-2939Crossref PubMed Scopus (138) Google Scholar, 8Li S. Czubryt M.P. McAnally J. et al.Requirement for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice.Proc Natl Acad Sci U S A. 2005; 102: 1082-1087Crossref PubMed Scopus (232) Google Scholar, 9Charvet C. Houbron C. Parlakian A. et al.New role for serum response factor in postnatal skeletal muscle growth and regeneration via the interleukin 4 and insulin-like growth factor 1 pathways.Mol Cell Biol. 2006; 26: 6664-6674Crossref PubMed Scopus (64) Google Scholar, 10Miano J.M. Ramanan N. Georger M.A. et al.Restricted inactivation of serum response factor to the cardiovascular system.Proc Natl Acad Sci U S A. 2004; 101: 17132-17137Crossref PubMed Scopus (208) Google Scholar Cardiomyocyte-specific deletion of Srf during development results in embryonic lethality from myocardial defects,6Parlakian A. Tuil D. Hamard G. et al.Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality.Mol Cell Biol. 2004; 24: 5281-5289Crossref PubMed Scopus (162) Google Scholar whereas disruption of SRF in muscle of the adult heart leads to cardiac failure and death as a result of dilated cardiomyopathy.7Parlakian A. Charvet C. Escoubet B. et al.Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart.Circulation. 2005; 112: 2930-2939Crossref PubMed Scopus (138) Google Scholar SRF ablation in developing skeletal muscle results in perinatal lethality as a result of dysmyofibrillogenesis in skeletal muscle,8Li S. Czubryt M.P. McAnally J. et al.Requirement for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice.Proc Natl Acad Sci U S A. 2005; 102: 1082-1087Crossref PubMed Scopus (232) Google Scholar whereas defects in postnatal skeletal muscle growth and regeneration are observed in mice on Srf deletion in the adult skeletal muscle.9Charvet C. Houbron C. Parlakian A. et al.New role for serum response factor in postnatal skeletal muscle growth and regeneration via the interleukin 4 and insulin-like growth factor 1 pathways.Mol Cell Biol. 2006; 26: 6664-6674Crossref PubMed Scopus (64) Google Scholar Finally, ablation of SRF in the developing cardiovascular system results in defective myofibrillogenesis in the heart and cytoskeletal assembly in the dorsal aorta.10Miano J.M. Ramanan N. Georger M.A. et al.Restricted inactivation of serum response factor to the cardiovascular system.Proc Natl Acad Sci U S A. 2004; 101: 17132-17137Crossref PubMed Scopus (208) Google Scholar However, because of essential SRF functions in embryonic SM cell (SMC) development,10Miano J.M. Ramanan N. Georger M.A. et al.Restricted inactivation of serum response factor to the cardiovascular system.Proc Natl Acad Sci U S A. 2004; 101: 17132-17137Crossref PubMed Scopus (208) Google Scholar functions of SRF in adult SMCs has not been studied. The importance of SRF in gastrointestinal (GI) tract function was highlighted by observed SRF contributions to esophageal and gastric ulcer healing.11Chai J. Baatar D. Tarnawski A. Serum response factor promotes re-epithelialization and muscular structure restoration during gastric ulcer healing.Gastroenterology. 2004; 126: 1809-1818Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Chai J. Norng M. Tarnawski A.S. Chow J. A critical role of serum response factor in myofibroblast differentiation during experimental oesophageal ulcer healing in rat.Gut. 2007; 56: 621-630Crossref PubMed Scopus (48) Google Scholar In contrast to skeletal and cardiac muscle cells, mature SMCs are highly plastic and can modulate their phenotypes between proliferative and differentiated states (reviewed in Halayko and Solway13Halayko A.J. Solway J. Molecular mechanisms of phenotypic plasticity in smooth muscle cells.J Appl Physiol. 2001; 90: 358-368Crossref PubMed Scopus (237) Google Scholar and Yoshida and Owens14Yoshida T. Owens G.K. Molecular determinants of vascular smooth muscle cell diversity.Circ Res. 2005; 96: 280-291Crossref PubMed Scopus (251) Google Scholar). Many SRF target genes confer the unique contractile and physiologic properties of SMCs, eg, SM-myosin heavy chain (SM-MHC), SM α- and γ-actin, SM22α, SM-myosin light chain kinase (SM-MYLK), calponin, α-actinin, and smoothelin-A (reviewed in Miano15Miano J.M. Serum response factor: toggling between disparate programs of gene expression.J Mol Cell Cardiol. 2003; 35: 577-593Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). To determine the function of SRF in adult SM tissue in vivo, we conditionally mutated the Srf gene in SMCs by crossing mice carrying a floxed Srf allele (Srfflex1)16Wiebel F.F. Rennekampff V. Vintersten K. Nordheim A. Generation of mice carrying conditional knockout alleles for the transcription factor SRF.Genesis. 2002; 32: 124-126Crossref PubMed Scopus (33) Google Scholar with SM-CreERT2(ki) transgenic mice, expressing a tamoxifen-inducible Cre recombinase under control of the SM-specific SM22α promoter.17Kuhbandner S. Brummer S. Metzger D. Chambon P. Hofmann F. Feil R. Temporally controlled somatic mutagenesis in smooth muscle.Genesis. 2000; 28: 15-22Crossref PubMed Scopus (130) Google Scholar This enabled induced SRF ablation in adult murine SMCs. Dysfunction of intestinal contractility resulted in lethal intestinal obstruction. Mutant mice died prematurely within 16 days after tamoxifen-induced Srfflex1 deletion. SRF-depleted primary SMCs of the colon were viable but underwent senescence, simultaneously displaying disruption of the actin filament and impaired expression of SM-specific and immediate-early genes. Srfflex116 and SM-CreERT2(ki)/wt17Kuhbandner S. Brummer S. Metzger D. Chambon P. Hofmann F. Feil R. Temporally controlled somatic mutagenesis in smooth muscle.Genesis. 2000; 28: 15-22Crossref PubMed Scopus (130) Google Scholar mice were bred for Srfflex1 homozygosity and Cre transgene heterozygosity, using a mixed genetic background (C57BL/6N-NMRI). Srfflex1/flex1:SMCreERT2/wt mice were used in all experiments, when treated with tamoxifen for induced recombination, were referred to as mutant or SRF knockout mice. Cre-mediated recombination of the Srfflex1 allele generates the Srflx deletion allele.16Wiebel F.F. Rennekampff V. Vintersten K. Nordheim A. Generation of mice carrying conditional knockout alleles for the transcription factor SRF.Genesis. 2002; 32: 124-126Crossref PubMed Scopus (33) Google Scholar Control mice include tamoxifen-treated Srfflex1/wt:SMCreERT2/wt or Srfflex1/flex1:wt/wt animals, or Srfflex1/flex1:SMCreERT2/wt mice treated with solvent only (sunflower oil). DNA from tail biopsies was used for genotyping by polymerase chain reaction (PCR) using primers Srf-E and Srf-R for the Srf locus16Wiebel F.F. Rennekampff V. Vintersten K. Nordheim A. Generation of mice carrying conditional knockout alleles for the transcription factor SRF.Genesis. 2002; 32: 124-126Crossref PubMed Scopus (33) Google Scholar and primers RF67, RF90, and SC135 for the SM-CreERT2(ki) transgene.17Kuhbandner S. Brummer S. Metzger D. Chambon P. Hofmann F. Feil R. Temporally controlled somatic mutagenesis in smooth muscle.Genesis. 2000; 28: 15-22Crossref PubMed Scopus (130) Google Scholar Six- to 12-week-old mutant and control mice were given daily intraperitoneal injections of tamoxifen (1 mg/day; Sigma, St Louis, MO), or of solvent, on 5 consecutive days, with the day of the first injection counting as day 1. Animal experiments were in accordance with official guidelines. Cre-mediated excision of the Srfflex1 allele (generating the Srflx deletion allele) was detected by PCR on genomic DNA from different organs or primary colon SMCs using the following primers: Srf-L and Srf-R16Wiebel F.F. Rennekampff V. Vintersten K. Nordheim A. Generation of mice carrying conditional knockout alleles for the transcription factor SRF.Genesis. 2002; 32: 124-126Crossref PubMed Scopus (33) Google Scholar; Srf-KO-8-fw (ccggggaaatatgg-ggagaggggagat), Srf-KO-8-bw (cttcgcgcacaccaggacacagaggat), and Lox2 (gctcgcagcggcggccagatctataac). Protein lysates of different tissues were prepared by homogenization (Polytron; Kinematica, Littau-Lucerne, Switzerland) in lysis buffer [20-mmol/L Tris·Cl, pH 8.0, 100-mmol/L NaCl, 2.5-mmol/L 1,4-dithiothreitol (DTT), 2.5-mmol/L EDTA, 2.5-mmol/L benzamidin, 2.5-mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin]. Western blotting was done with overnight incubations at 4°C with antisera for SRF [SRF(G-20), sc-335; 1:1000; Santa Cruz Biotechnology, Heidelberg, Germany]. Total SRF was quantified by stripping the same membranes and reprobing with a monoclonal anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (HyTest 1:20,000). Relative levels of full-length SRF (±SD) were calculated from comparisons of 3 mutant and control mice, respectively. SM α-actin was Western blotted by using total protein extracts from primary colon SMCs and a monoclonal anti-SM α-actin antiserum (1:500; Chemicon, Ochsenhausen, Germany). For immunohistochemistry, the colon was removed, rinsed thoroughly, fixed for at least 2 days in 4% paraformaldehyde at 4°C, dehydrated in a graded series of alcohol/water solutions, embedded in paraffin, and sectioned in a microtome (Leica, Wetzlar, Germany). Sections (7-μm-thick) were dried overnight at 60°C and incubated overnight with anti-SRF antiserum (see above). Immunohistochemistry (Vectastain ABC system and peroxidase substrate diaminobenzidine; Vector Laboratories, Burlingame, CA) was performed according to the manufacturer’s instructions. Mice received 80 μL of Gastrolux CT contrast agent (Sanochemia Diagnostics, Neuss, Germany) diluted with 200 μL of isotonic NaCl by oral administration after a fasting period of 12 hours. Imaging with the micro-CAT-II high resolution animal x-ray computer tomography (CT; Siemens Preclinical Solutions, Knoxville, TN) was performed 2, 4, 6, 12, and 24 hours after administration of the contrast agent. For the scans, an x-ray voltage of 75 kVp and an anode current of 350 μA were used with an exposure time of 425 milliseconds per projection. A total of 360 projections over 360 degrees of rotation were acquired, resulting in a total acquisition time of 7 minutes 45 seconds. Projection data were rebinned by 4 and reconstructed, using a Shepp-Logan filter, into a 512 × 512 × 768 matrix having an isotropic voxel size of 77 μm. During imaging, mice were kept anesthetized with 1.5% isoflurane (0.8 L oxygen/minute). Between the scans, mice were awake and received regular food and water. Images were analyzed with the Amira software package (Mercury Computer Systems, Chelmsford, MA). Mice were killed by decapitation; detrusor muscles and segments from the jejunum and colon were quickly transferred to buffer solution [137-mmol/L NaCl, 5.4-mmol/L KCl, 1.8-mmol/L CaCl2, 1-mmol/L MgCl2, 12-mmol/L NaHCO3, 0.42-mmol/L NaH2PO4, 5.6-mmol/L glucose] bubbled with carbogen (95% O2, 5% CO2). Detrusor muscles and intestinal segments were washed and cleaned from connective tissue and mounted longitudinally into organ baths (Myograph 601; www.dmt.dk). Resting tension was set to 6–12 mN. Tension was recorded isometrically at 37°C ± 1°C. Results are presented as original recordings or expressed as means ± standard error of the means (SEMs). The amplitude of phasic contraction was measured as the peak value within 30 seconds and the amplitude of tonic contraction as the steady state value 10 minutes after carbachol (CCh) stimulation with respect to the baseline, respectively. Tension values (in N) were normalized to the wet weight (in g) of the preparation. The SM layer was separated from the colon, minced with scissors, and incubated for 30–45 minutes at 37°C in 1-mL digestion medium-A [0.7 mg/mL papain, 1.0 mg/mL albumin, 1 mg/mL DTT in isolation medium (60-mmol/L NaCl, 85-mmol/L L-glutamic acid, 5.6-mmol/L KCl, 2.0-mmol/L MgCl2, 10-mmol/L HEPES, pH 7.4)]. After collection by centrifugation at 600 ×g, cells were incubated for 15–30 minutes at 37°C in 1 mL of digestion medium-B (1.0 mg/mL collagenase, 1.0 mg/mL hyaluronidase, 1.0 mg/mL albumin in isolation medium). Cells were suspended by gentle pipetting, collected by centrifugation at 800 ×g, resuspended in culture medium (Dulbecco’s modified Eagle medium supplemented with 4500 mg/L D-glucose, 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin) and plated at a density of 2 × 104 cells/cm2. The cells were maintained at 37°C in a humidified, 5% CO2 atmosphere. Identity of the cells was confirmed by anti-SM α-actin staining (1:500; Chemicon). Primary colon SMCs were trypsinized and centrifuged for 5 minutes at 100 ×g, and cells were resuspended in 1 mL of phosphate-buffered saline (PBS). One part of the cell suspension was mixed with one part of 0.4% Trypan blue (Invitrogen, Frederick, MD) and incubated for 3 minutes at room temperature. Unstained (viable cells) and stained (nonviable) cells were counted separately in a hemacytometer. Primary colon SMCs were grown to confluency. Cells were washed with PBS before fixation for 10–15 minutes at room temperature with fixative solution (2% formaldehyde/0.2% glutaraldehyde in PBS). Cells were washed twice with PBS before the addition of the staining solution mix [composed of 930 μL staining solution (40-mmol/L citric acid/sodium phosphate, pH 6.0, 150-mmol/L NaCl, 2-mmol/L MgCl2), 10 μL staining supplement-A (5-mmol/L potassium ferrocyanide), 10 μL staining supplement-B (5-mmol/L potassium ferricyanide), and 50 μL of 20 mg/mL X-Gal in dimethylformamide; freshly prepared]. Incubation was overnight at 37°C. Cells were analyzed microscopically for development of blue color. Primary colon SMCs were seeded on coverslips, grown overnight, and fixed with 3% formaldehyde/PBS for 7–10 minutes. Cells were washed 3 times with PBST (PBS/0.1% TritonX-100). Nonspecific binding was blocked by incubation for 1 hour in 3% nonfat dry milk in PBST before staining with primary antibody (anti-SRF; 1:1000; Santa Cruz Biotechnology) for 1 hour at room temperature. Incubation with fluorescence-conjugated secondary antibody (1:250; Zymed, San Francisco, CA) was for 1 hour at room temperature. Staining for filamentous actin with Texas Red–Phalloidin (1:75 in PBST; Molecular Probes, Leiden, Netherlands) was for 20 minutes. Coverslips were washed 3 times with PBST and mounted with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Total RNA preparations (RNeasy Kit; QIAGEN, Hilden, Germany) from primary colon SMCs and first-strand cDNA synthesis (MMLV Reverse Transcriptase; Promega, Madison, WI) were done according to the manufacturer’s protocols. Total RNA (1 μg) treated with DNaseI (Roche, Indianapolis, IN) was used for reverse transcription–PCR. Quantitative analysis using SYBR Green technology (Applied Biosystems, Foster City, CA) was performed as previously described.18Schratt G. Philippar U. Berger J. Schwarz H. Heidenreich O. Nordheim A. Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells.J Cell Biol. 2002; 156: 737-750Crossref PubMed Scopus (156) Google Scholar Primers used were SM22α, fw(gatgtaggccgcccagatc) and bw(atcacaccattcttcagccaca); SM α-actin, fw(cagcaaacaggaatacgacgaa) and bw (tgtgtgctagaggcagagcag); SM-MHC, fw(catggacccgctaaatgaca) and bw(caatgcggtccacatccttc); smoothelin-A, fw(tccaatgatggcactcagacg) and bw(agcgcctcatgaaactggact); GAPDH, fw(tggcatggccttccgt) and bw(tctccaggcggcacgt); c-fos, fw(cctgccccttctcaacga) and bw(gctccacgttgctgatgct); egr-1, fw(gccg-agcgaacaaccctat) and bw(tccaccatcgccttctcatt); egr-2, fw(gttgactgtcactccaagaaatgg) and bw(agcgcagccctgtaggc). The data are presented as means ± standard deviations (SDs). The statistical significance was determined by using Student t test. Differences were considered statistically significant at P values <.05. To investigate the role of SRF in murine adult SMCs, we used the conditional Srfflex1 allele harboring loxP sites flanking the coding sequence of Srf exon 1, which is essential for nuclear localization and CArG box–specific DNA binding of SRF.16Wiebel F.F. Rennekampff V. Vintersten K. Nordheim A. Generation of mice carrying conditional knockout alleles for the transcription factor SRF.Genesis. 2002; 32: 124-126Crossref PubMed Scopus (33) Google Scholar Srfflex1 mice were bred with transgenic mice expressing a tamoxifen-inducible Cre recombinase, CreERT2, under the control of the SM-specific SM22α promoter [SM-CreERT2(ki)].17Kuhbandner S. Brummer S. Metzger D. Chambon P. Hofmann F. Feil R. Temporally controlled somatic mutagenesis in smooth muscle.Genesis. 2000; 28: 15-22Crossref PubMed Scopus (130) Google Scholar Thus, SM-CreERT2–mediated deletion of the floxed Srf exon 1 should occur only after application of tamoxifen. Srfflex1/flex1:SMCreERT2/wt mice, in the absence of tamoxifen treatment, were viable, had normal body weight, bred normally, and were indistinguishable from their control littermates. To induce deletion of the Srf gene, 6- to 12-week old Srfflex1/flex1:SMCreERT2/wt mice were given daily intraperitoneal injections of 1 mg of tamoxifen for 5 consecutive days. Control mice included Srfflex1/flex1:SMCreERT2/wt animals treated with solvent or Srfflex1/wt:SMCreERT2/wt animals treated with tamoxifen. Efficient deletion of exon 1 coding sequences on tamoxifen administration, as confirmed by genomic PCR analysis, was seen in organs with high content of SM tissue, ie, the GI tract, uterus, bladder, and aorta (Figure 1A). Exclusive, SMC-specific activation of the SM22α-CreERT2 transgene and resulting expression of the CreERT2 recombinase is evidenced by our study, as well as the independent works of Kuhbandner et al17Kuhbandner S. Brummer S. Metzger D. Chambon P. Hofmann F. Feil R. Temporally controlled somatic mutagenesis in smooth muscle.Genesis. 2000; 28: 15-22Crossref PubMed Scopus (130) Google Scholar and Boesten et al,19Boesten L.S. Zadelaar S.M. De Clercq S. et al.Mdm2, but not Mdm4, protects terminally differentiated smooth muscle cells from p53-mediated caspase-3-independent cell death.Cell Death Differ. 2006; 13: 2089-2098Crossref PubMed Scopus (51) Google Scholar which scored for potential nonmuscle genomic recombination by lacZ staining. Detection of low levels of Srflx in organs not containing SMCs per se, such as heart, liver, skeletal muscle, and brain, was likely due to the presence of vascular SMCs. Recombination in solvent-treated control mice, primarily in the bladder, indicated some leakiness of the tamoxifen-inducible CreERT2 system, at least in the context of recombination of the Srfflex1 locus. This “background” recombination over time was found to vary, depending on the genetic background of the mice. Animals in a congenic C57BL/6 background were more susceptible to background recombination, because, without tamoxifen induction, Srfflex1/flex1:SMCreERT2/wt mice died at the ages of 3–5 months, whereas mixed genetic background animals of this genotype survived beyond that age. Western blotting showed that, on tamoxifen-induced, Cre-mediated recombination, levels of full-length SRF protein were reduced by 70% in the colon and stomach or even by 90% in the bladder, compared with control mice. SRF protein levels were unaffected in organs not containing SMCs (Figure 1B and C). Unexpectedly, the anti-SRF antibody revealed a smaller protein (∼36 kDa), mainly in stomach and bladder (ie, in tissues which displayed high levels of recombination). Origin and identity of this ∼36-kDa protein are currently unresolved but are under investigation (see “Discussion”). Consistent with the Western blot data, strong nuclear staining of SRF protein could only be detected in nonrecombined colon tissue (Figure 1D, left), whereas nuclear SRF was absent in mutant colon SM (Figure 1D, right). Within 2–4 days after the last tamoxifen injection, mutant mice showed reduced physical body motility and were kyphotic, having a cramped posture. The lower abdomen started to bloat and increase in volume. At this stage mutant mice stopped food intake, and no excretion of feces could be observed any more. Strikingly, cachexiatic disease symptoms progressed rapidly, and first deaths of mutant animals were seen as early as 8 days after the first tamoxifen injection. There was 100% lethality within 16 days after the first application of tamoxifen (Figure 2A). In contrast, tamoxifen-treated Srfflex1/wt:SMCreERT2/wt or solvent-treated Srfflex1/flex1:SMCreERT2/wt control mice appeared normal and did not differ from their untreated littermates. The observation of 10% lethality among the control group after a 56-day time period is likely not due to SM-specific SRF depletion because the animals died without indications of obvious disease. In all mutant mice, gross necropsy showed severe intestinal situs with extremely dilated and food-filled caecum and colon, indicating the condition of ileus paralyticus (Figure 2B). We investigated the time course of intestinal food processing with x-ray CT. Intestinal processing of the contrast agent was followed over time (Figure 3). In control mice (Figure 3A–E), the contrast agent reached the stomach and the small intestine within 2 hours and the rectum within 4 hours (Figure 3A and B). Six hours after application, the signal of the contrast agent was much weaker but could still be observed in the rectum, whereas the contrast fluid had been excreted completely after 12 hours (Figure 3D). In the mutant mice (Srfflex1/flex1:SMCreERT2/wt; Figure 3F–J), however, a drastically delayed intestinal transit of the contrast agent was observed. In the initial phase, comparable to control animals, food was transported from the stomach into the small intestine within 2 hours (Figure 3F). Subsequently, however, contrast material was still retained in the small intestine of the mutant mice up to 6 hours (Figure 3G and H), whereas control animals had excreted contrast material within the initial 4 hours. Even after 12 and 24 hours, mutant mice still showed a strong signal in the massively dilated cecum and colon (Figure 3I and J). Twenty-four hours after application, the contrast fluid had reached the rectum of the mutant animals (Figure 3J), but no feces were excreted. These data show that impairment of intestinal food processing begins with the malfunctioning small intestine and worsens along the path of the digestive tract, as is mirrored by isometric tension recordings of SMC contractility (see below). Our CT study reveals severe food stasis along the GI tract of SM-specific mutant mice, resulting in intestinal obstruction and, ultimately, premature death. This type of visceral myopathy resembles symptoms of human patients with chronic intestinal pseudo-obstruction (CIP).20De Giorgio R. Sarnelli G. Corinaldesi R. Stanghellini V. Advances in our understanding of the pathology of chronic intestinal pseudo-obstruction.Gut. 2004; 53: 1549-1552Crossref PubMed Scopus (204) Google Scholar Contractile functions of the intestinal tunica muscularis were tested by tension recordings of SM tissues (jejunum, colon, and urinary bladder). Spontaneous contractile activity was observed in longitudinal muscles from the jejunum and colon of tamoxifen-treated control and mutant mice. The amplitude of this activity was smaller in muscles from the mutant mice than from the control mice (51% and 57% for jejunum and colon muscle, respectively), whereas the frequency did not differ between both groups of mice (data not shown). Stimulation of SM from the jejunum, colon, and urinary bladder with the muscarinic agonist CCh induced contractions, which consisted of a phasic component and a tonic component. The amplitude of both components in the muscles from mutant mice was roughly only 50% of that o
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