A Study Of The Role And Underlying Mechanisms Of Gsα In Smooth Muscle Dysfunctional Diseases

1.IntroductionAbdominal aortic aneurysm(AAA)is a common vascular degenerative disease with high risk of death,usually caused by damage to the normal structure of the abdominal aorta wall.When the damaged abdominal aorta expands more than 1.5 times the diameter of the normal abdominal aorta or exceeds 3cm,the AAA diagnosis can be made.The annual incidence of AAA is about 20-40/100,000 and according to the statistics,more than 11,000 patients die of AAA each year in the United States.Gender,age,smoking and genetic factors are the main risk factors for AAA.Among the 74-84 age groups,approximately 12.5%of men and 5.2%of women have AAA.More than 50%of AAA patients are diagnosed due to severe pain in the abdomen and back,and the mortality of AAA rupture is more than 80%.At present,the treatment of AAA is mainly based on surgery and interventional therapy,and there is no effective medical treatment.Therefore,further understanding the molecular mechanism of AAA is the key point to search the potential drug treatment targets.In terms of pathological features,AAA is mainly characterized by incomplete elastic fibers,reduced number of vascular smooth muscle cells(VSMCs)and inflammatory cells infiltration.Recent studies showed that the mechanisms of AAA mainly involved the synthesis/degradation imbalance of extracellular matrix(ECM)(mainly including elastic fibers and collagen),oxidative stress,angiogenesis.inflammatory factors,smooth muscle cell phenotypic switch and loss of VSMCs(including apotosis,autophagy and necrosis).Unlike other terminally differentiated cells,mature SMCs show strong plasticity and have ability to differentiate into other cell types.In the normal state,the mature VSMCs are contractile phenotype;its contractile function is mainly derived from the highly specific-expressed contractile markers,such as alpha smooth muscle actin(a-SMA),smooth muscle myosin heavy chain(SMMHC),SM22a.However,when the vascular injury occurred,the VSMCs lose its contractile phenotype and the expression of contractile proteins decreased;the VSMCs were stimulated to proliferate,migrate and secrete cytokines and transferred into synthetic phenotype which is characterized by up-regulation of various synthetic proteins such as Vimentin,Osteopontin,etc.Above functional state changes of VSMCs were called smooth muscle cell phenotypic switch.Various vascular diseases such as AAA,atherosclerosis(AS),and arterial calcification were closely related to smooth muscle cell phenotypic switch.For example,recent study discovered that mutations in the ACTA2 gene could result in the incidence and rupture of familial thoracic aortic aneurysms;another research revealed that SMCs-specific a-SMA knockout mice showed aortic dilation which was associated with increased NF-κB-dependent AngⅡsensitivity;SMCs-specific SM22a deficiency in mice aggravated AngⅡ-induced AAA.On the other hand,over-expression of SM22a in SMCs could inhibit AAA formation by suppressing smooth muscle cell phenotypic switch through inhibiting ROS/NF-κB pathway.In summary,maintaining the SMCs contractile phenotype is important for the normal function of blood vessels.A variety of signaling molecules such as KLF4,SRF,SP1,and TGF-β1 regulate the growth and differentiation of SMCs,and KLF4 has been widely studied as a key molecule regulating phenotypic switch of VSMCs.On the one hand,KLF4 directly inhibits the transcriptional activities of a-SMA,SM22a and SMMHC promoters;on the other hand,KLF4 competitively inhibits the binding of SRF to the gene promoters of a-SMA,SM22a and SMMHC,inhibits the expression of myocardin which is the cofactor of SRF and promotes SMCs phenotypic switch.KLF4 is widely involved in the progression of AAA,AS,arterial calcification,and vascular intimal hyperplasia by regulating phenotypic switch of VSMCs.In the smooth muscle layer of mice and human AAA tissues,the expression level of KLF4 is significantly increased.Previous studies demonstrated that KLF4 could increase the incidence of AAA by promoting the SMCs phenotypic switch and SMCs-specific KLF4 knock-out in mice attenuated aneurysm formation.Gsα refers to α subunit of the G protein stimulatory family and is widely expressed in a variety of cell types.The most important physiological function of Gsa is to activate the adenylate cyclase(AC),increase the level of second messenger cyclic adenosine monophosphate(cAMP)level and then activate the PKA signaling pathway.Gsa dysfunction is closely related to many diseases.For example,mice with Gsa specific knock-out in adipose showed a lean phenotype with impaired cold tolerance and increased diet-induced thermogenesis.While over-expression of Gsa in cardiomyocytes could increase myocardial contractility and lead to cardiac hypertrophy in mice.Our previous studies showed that SMCs-specific knockout of Gsa led to a phenotype of chronic intestinal pseudo-obstruction(CIP)in mice,and decreased expression of a-SMA and SMMHC protein in intestinal smooth muscle.However,the function of Gsa in VSMCs has not been studied.Whether Gsa could regulate VSMCs phenotypic switch through KLF4 signaling pathway has not been explored.The role of Gsa-deficiency in AAA formation is still unclear.In summary,this study proposes the hypothesis that Gsa in VSMCs can influence KLF4 signaling pathway to regulate VSMCs phenotype switch,thereby affecting AngⅡ-induced AAA formation.We generate the SMCs-specific Gsa knockout mice to explore the function of Gsa in SMCs phenotypic switch and establish the AAA model induced by subcutaneous infusion of AngⅡ to study the role and mechanism of Gsa in AAA formation.In the present study,we sought to supply experimental supports for searching potential drug targets of AAA.2.Objectives(1)To explore the role and mechanism of Gsa in aortic SMCs phenotypic switch;(2)To explore the role and mechanism of aortic SMCs-Gsa in AngⅡ-induced AAA.3.Methods3.1 Generate the SMC-specific Gsa knock-out miceGsαflox/flox mice were crossed with SM22-CreERT2 mice to generate Gsαflox/floxSM22-CreERT2 mice.Adult mice were injected with Tamoxifen(1 mg/d)intraperitoneally for 5 days and the Gsa will be deleted specificly in SMCs.These mice were named Adult GsαSMKO mice.Gsαflox/floxSM22-CreERT2 mice were crossed into ApoE-/-background to generate ApoE-/-/Gsαflox/floxSM22-CreERT2 mice.Tamoxifen(1 mg/d)was intraperitoneally injected for 5 days and the Gsa will be deleted specificly in SMCs.These mice were Gsa/ApoE double knock-out and named ApoE-/-GsαSMKO mice.Genomic DNA of mice was extracted,Gsa,Cre and ApoE sequences were amplified by PCR,and genotypes were identified by agarose gel electrophoresis.3.2 Cell culture and treatmentMouse primary aortic SMCs were cultured by substrate-attached explant methods,and 4-6 passage cells were used for experiments.(1)KLF4 mRNA stability:Smooth muscle cells were transfected with GFP adenovirus and HuR over-expressing adenovirus respectively,and D-actinomycin was added to inhibit transcription.The mRNA content of KLF4 was detected at Oh,0.5h,1h and 1.5h,and the half-life curve of KLF4 mRNA was calculated.(2)HuR inhibitor CMLD-2 was used to stimulate primary aortic smooth muscle cells.(3)HuR siRNA transfection:Primary aortic SMCs derived of Control and GsαSMKO mice were transfected with CTR siRNA and HuR siRNA respectively.(4)KLF4 siRNA transfection:Primary aortic SMCs derived of Control and GsαSMKO mice were transfected with CTR siRNA and KLF4 siRNA respectively.(5)Primary aortic SMCs derived of Control and GsαSMKO mice were stimulated with PBS and AngⅡ(0.5μM)for 25 min.(6)RIP assay:RIP assay was performed to verify the binding ability of HuR protein on KLF4 mRNA in aortic SMCs.3.3 Establish the AAA mice model10-week-old ApoE-/-/Gsαflox/flox and ApoE-/-/GsαSMKO male mice were divided into the following four groups:1)ApoE-/-/Gsαflox/flox+Saline group;2)ApoE-/-/Gsαflox/flox+AngⅡ group;3)ApoE-/-/GsaSMKO+Saline group;4)ApoE-/-/GsαSMKO+AngⅡ group.Group 2)and group4)were pumped with AngⅡ(1.44 mg/kg/d)for 28 days by subcutaneous implantation of mini-osmotic pumps;group 1)and group 3)were subcutaneously pumped with saline as control.All mice were fed with high-fat diet from two-weeks before the pump was buried until the mice were sacrificed.3.4 Western blotTotal proteins were lysised from aortic tissues or VSMCs and protein concentrations were detected with BCA protein assay kit.Western blots were used to analyze the protein expression levels of Gsα、HuR、KLF4、a-SMA、SM22、SMMHC、Calponin、Vimentin、Osteopontin、MMP2、Fibronectin、Collagen Ⅰ、Collagen Ⅲ、P65、p-P65、GAPDH and β-actin.3.5 RT-PCRTotal RNA was isolated from aortic tissues or VSMCs using Fastagen RNAfast200 kit.The mRNA levels of Gsα、HuR、KLF4、α-SMA、SMMHC、Vimentin、Osteopontin、TGF-β1、E-selectin、IL-6、IL-17C、ICAM-1、TNF-α、CCL-2、GAPDH and β-actin were detected by RT-PCR.3.6 Histological and pathological examination(1)Control and GsαSMKO aorta were extracted and the vascular structure was observed by HE staining.The expression of Gsa,a-SMA in smooth muscle layer of aorta was detected by immunofluorescence and nucleus was labeled by DAPI staining.The expressions of KLF4,HuR,a-SMA,Vimentin,SMMHC and Osteopontin were detected by immunohistochemistry.(2)Immunohistochemistry was used to detect the expression of Gsa in the aortas of normal mice and AAA mice.(3)Four groups of mice in the AAA experiment were sacrificed and the gross photographs of aorta were taken by SLR camera.The vascular structure was observed by HE staining.Elastic fiber was detected by Verhoeff-Van Gieson(VVG)staining.The expression of CD68,4-HNE,MMP2,Vimentin and Osteopontin was detected by immunohistochemistry.3.7 Detection of cAMP levelsAortic tissues were grinded in 0.IM hydrochloric acid and the cAMP contents were detected by Enzo Life Science Direct cAMP ELISA kit.3.8 Human AAA casesHuman AAA tissues were obtained from patients undergoing open operative repair.The normal aortic tissues were trimmed from the adjacent non-aneurysmal aorta from the same patients.Immunohistochemistry was used to detect the protein expression of Gsa,a-SMA,KLF4 and HuR in human AAA tissues and normal aortic tissues.3.9 Statistical analysisAll statistical data were expressed as mean± SEM.The data were first tested by normal distribution.The comparison between two groups was analyzed by independent sample t-test;the comparison of multiple groups was analyzed by one-way ANOVA.P<0.05 was considered as significantly different.4.Results4.1 Generation and Analysis of GsαSMKO miceGsαflox/flox mice were cross-bred with SM22-CreER72 mice to generate Gsαflox/+/Cre+ mice.These were further intercrossed to obtain Gsaflox/flox/Cre+ mice,which were injected with tamoxifen at age 8 weeks to generate GsαSMKO mice.Littermate Gsαflox/flox Cre-mice with the same dose of tamoxifen were controls.Immunofluorescence,Western blot and RT-PCR results showed that Gsa expression in GsαSMKO aorta decreased significantly compared with control group.Meanwhile,the cAMP content in the GsαSMKO aorta tissue was significantly lower than that in the control group.However,the protein levels of Gsa in macrophage and fibroblasts had no significant difference between GsαSMKO mice and control mice.Above data indicated that Gsa was specificly knocked out in SMCs.Compared with control mice,GsαSMKO mice showed a significant decrease in body weight,heart rate,and left ventricular ejection fraction;but there was no significant difference in systolic blood pressure and diastolic blood pressure.4.2 Gsa deficiency induces the smooth muscle cell phenotype switchImmunohistochemistry,Western blot and RT-PCR results showed that the expression of aortic smooth muscle contractile proteins(a-SMA and SMMHC)decreased,and the expression of synthetic proteins(Vimentin and Osteopontin)increased in GsαSMKO mice compared with control mice.Moreover,Gsα deficiency increased the phosphorylation of P65(the marker of NF-κB activation)and the levels of smooth muscle-derived cytokines.However,there was no significant difference in aortic diameter,media thickness,and number of smooth muscle cells4.3 Gsa deficiency increases KLF4 and HuR expressionWestern blot and RT-PCR results showed that the protein and mRNA levels of KLF4 and HuR in the aortic tissue of GsαSMKO mice were significantly higher than those of the control mice.4.4 KLF4 is the HuR target geneRIP results showed that HuR protein could bind to KLF4 mRNA.Over-expression of HuR protein could increase the stability of KLF4 mRNA and prolong its half-life.CMLD-2,the HuR inhibitor,could significantly reduce the expression of KLF4 protein.4.5 Gsa deletion induces the smooth muscle cell phenotype switch via the HuR/KLF4 pathwayCompared with control groups,the expression of HuR,KLF4,Vimentin were increased and a-SMA expression was decreased in GsaSMKO group.SMCs transfected with HuR siRNA decreased KLF4 and Vimentin expression,increased a-SMA expression significantly.SMCs transfected with KLF4 siRNA decreased Vimentin expression and increased a-SMA expression.Above results indicate that Gsa deletion induces the smooth muscle cell phenotypic switch through HuR/KLF4 pathway and HuR or KLF4 inhibition could rescue the smooth muscle cell phenotypic switch.4.6 Smooth muscle-specific Gsa deletion exaggerates AngⅡ-induced AAA formationTo determine the roles of smooth muscle-specific Gsa deletion in AAA,ApoE-/-/GsαSMKO mice were mgenerated and littermate ApoE-/-/Gsαflox/flox mice were used as controls.AAA model was induced by pumping of Angll subcutaneously.The AAA incidence,mortality rate and maximal diameter were increased significantly in ApoE-/-/GsαSMKO mice compared with ApoE-/-/Gsαflox/flox mice.The damage level of abdominal aortic wall was more severe in AAA tissues of ApoE-/-/GsαSMKO mice.Immunohistochemistry showed that CD68,MMP2,Vimentin and Osteopontin levels were up-regulated in AAA tissues of ApoE-/-/GsαSMKO mice compared with ApoE-/-/Gsαflox/flox mice,while 4-HNE expression had no significant difference.4.9 The levels of Gsa and a-SMA are reduced while those of HuR and KLF4 are increased in human AAA samplesImmunohistochemistry showed that the levels of Gsa and a-SMA in smooth muscle layer of AAA tissue were significantly decreased,while the levels of HuR and KLF4 were obviously increased in human AAA compared with controls.5.Conclusions(1)Gsa deficiency induces the smooth muscle cell phenotype switch;(2)Gsa regulates smooth muscle cell phenotypic switch through HuR/KLF4 signaling pathway;(3)Smooth muscle-specific Gsa deletion increases the maximal diameter,incidence,mortality and severity of AAA induced by Angll infusion;(4)The occurrence of human AAA may be related to smooth muscle cell phenotypic switch regulated by Gsa/HuR/KLF4 signaling pathway.1.IntroductionChronic intestinal pseudo-obstruction(CIP)is a rare,disabling disease with intestinal motility disorder.A recent national survey in Japan showed that there are approximately 3.7 children with CIP in every 1 million newborns,and the incidence rate in children under the age of 15 is about 1/270000.CIP patients generally have symptoms of intestinal obstruction such as bloating,vomiting,constipation and abdominal pain.CIP patients usually have no physical obstruction,and there is no occlusive disease in the intestine which is different from mechanical intestinal obstruction.The causes of CIP are complicated and CIP are usually affected by genetic factors,side effects of drug,or secondary to other diseases.At present,the most common form of CIP lesions in the clinic is neuronal and/or smooth muscle dysfunction in the intestinal wall,but the molecular mechanism is still unclear.Due to the long-term dysfunction of the digestive system.CIP patients often need total parenteral nutrition,so the life quality of patient is poor.Some studies have shown that the survival rate of CIP patients is only 1 9.7%.At present,CIP patients usually need surgery,and severe cases may require total gastrointestinal transplantation.Smooth muscle cells(SMCs)are involved in many important systems,for example,digestive system,cardiovascular system and genitourinary system.Maintaining normal contractile function of SMCs is a guarantee for the functions of these organs.SMCs dysfunction is closely related to the development of many human diseases,such as CIP,hypertension and asthma.These diseases are characterized by smooth muscle contractile dysfunction in the pathogenesis.Therefore,studying the regulation mechanism of SMCs contractile protein expression plays an important role in understanding the pathogenesis of smooth muscle contraction-related diseases,and is of great value for finding new effective therapeutic targets for these diseases.At present,many transcription factors such as SRF,Foxfl,and Myocardin are involved in the regulation of the expression of contractile proteins in SMCs.SRF is a ubiquitously expressed transcription factor that recognizes and binds to the CArG[CC(A/T)6GG]boxes in promoters of the target genes,activating the expression of various smooth muscle contraction-related proteins.SRF is essential for the growth and differentiation of gastrointestinal smooth muscle.SRF knock-out in SMCs can lead to decreased expression of a-SMA,SM22 and SMMHC,and the mice expressed CIP symptoms.Myocardin is a transcriptional coactivator of SRF;it could bind with SRF to form a Myocardin/SRF protein complex and enhances SRF function.Foxfl is essential for maintaining normal differentiation of SMCs.Recent study found that the heterozygous deletion and point mutation in the Foxfl gene locus exist in 40%of patients with Misalignment of Pulmonary Veins(ACD/MPV),which is characterized by severe gallbladder,lung and gastrointestinal dysplasia.In the mice with smooth muscle-specific knockout of Foxfl,the esophageal smooth muscle layer was significantly thinner and the expression of smooth muscle contractile protein was decreased.The colon of adult heterozygous smFoxfr-/+ mice also showed a thinner annular muscle layer with contractile abnormality.However,the mechanism by which Foxfl regulates the growth and differentiation of smooth muscle cells is not clear.On the one hand,Foxfl can directly bind to the promoter sequence of Telokin and activate its expression.On the other hand,Foxfl can bind to SRF,Myocardin or Myocardin-related transcription factors(MRTFs),regulate the expression of Telokin synergistically.As Foxfl has so close relationship with intestinal SMCs,we speculate that Foxfl dysfunction may participate in CIP progress.The heterotrimeric G protein is a much conserved protein family in animal and plant cells,and plays a major role in trans-membrane signaling transduction.The G protein is composed of three subunits(α,β,and y)and is classified into Gs,Gi,Gq,G12/13 according to different a subunit.The a subunit of the Gs protein,called Gsa,is capable of receiving extracellular signals transmitted by G protein-coupled receptor,activating the intracellular adenylate cyclase(AC)and generating the second messenger cAMP,thereby activating the downstream PKA signal.PKA exerts different effects by phosphorylating multiple downstream molecules.PKA could activate cAMP response element binding protein 1(CREB1)by phosphorylating its serine 133 site to regulate the growth,proliferation and migration of SMCs.Loss or dysfunction of Gsa usually leads to disability of many organs and even embryonic or postnatal death.At present,studies on Gsα protein mainly focus on metabolic diseases,endocrine diseases,and tumor-related diseases;while there have no reports of disease research using smooth muscle-specific Gsa-deficient mice.We established the smooth muscle-specific Gsa knockout mice for the first time.In our previous observation,the knockout mice showed CIP phenotypes such as gastrointestinal food delay and intestinal swelling.This study proposes the following scientific hypothesis:Gsa specific knock-out in mouse SMCs could down-regulate contraction-related proteins such as MYH11,ACTA2,CNN1 in intestinal smooth muscle layer,through affecting the Foxf1 signaling pathway,resulted in SMCs contractile dysfunction and leads to CIP eventually.This study will describe the phenotypic characteristics of the mice whose Gsa was specifically knocked out in SMCs for the first time,detect the contractile function of its small intestine,and explore the possible signaling pathways that cause CIP.This research will provide a reference for further understanding of CIP in molecular mechanism.2.Objectives(1)To explore the effect of Gsa-deficiency on intestinal smooth muscle contraction;(2)To identify the potential molecular mechanism of Gsa in regulating the expression of intestinal smooth muscle contractile proteins.3.Methods3.1 Generate the smooth muscle-specific Gsa knock-out miceGsαflox/flox mice were crossed with ACTA2-Cre mice to generate Gsαflox/flox ACTA2-Cre mice.The Gsa was knocked out specifically in SMCs from embryonic phase.Gsαflox/flox mice were crossed with SM22-CreERT2 mice to generate Gsαflox/flox SM22-CreERT2 mice.Adult mice were injected with Tamoxifen(1 mg/d)intraperitoneally for 5 days and Gsa will be deleted specificly in SMCs.These mice were named Adult GsαSMKO miceGenomic DNA of mice was extracted.Gsa and Cre sequences were amplified by PCR,and genotypes were identified by agarose gel electrophoresis.3.2 Cell experimentMouse primary small intestinal smooth muscle cells were extracted by patch method and 4-8 passage cells were used for experiments.1)Forskolin stimulates primary intestinal SMCs for 0,24,48h,respectively;2)Chromatin immunoprecipitation assay(ChIP):verify the binding ability of CREB1 protein to Foxfl promoter sequence;3)Luciferase Reporter Gene Assay:generate the wild-type luciferase plasmid(WT-luc)containing the Foxfl promoter sequence;generate another luciferase plasmid which also contains the promoter sequence of Foxfl,but the CRE site was mutated and the plasmid was called CRE-mutated luciferase plasmid(CRE-mutated-luc).The above two plasmids were transfected into mouse primary intestinal smooth muscle cells,and stimulated with Forskolin for 24 h to detect luciferase activity.3.3 Growth cu rve and survival curve1)Record body weights of newborn Control and GsαSMKO mice from 7 to 28 days,and plot the growth curves;2)Plot the survival curves of Control and GsαSMKO mice from birth to 28 days;3)After Tamoxifen injection,the body weights of male Control and Adult GsαSMKO mice were recorded for the following 70 days,and the growth curves were plotted;4)After Tamoxifen injection,draw the survival curve of Control and Adult GsαSMKO mice for the following 100 days.3.4 Whole-gut transmit time testThe mice were injected 100μL charcoal test meal by orogastric gavage and monitor the time interval when the black dye first appeared in the feces of the mice.3.5 Detection of contractile force of mouse small intestine smooth muscle stripsThe BL-420F bio-functional experimental system and tension transducer were used to record the spontaneous contraction waveform and contraction force of the small intestine muscle strips;KCl,ACh and electric field stimulation(EFS)were used to stimulate the contraction of the small intestine muscle strips,and the contraction waveform was recorded to calculate the contractile force.3.6 Detection of cAMP levelsEnzo Life Science’s Direct cAMP ELISA kit was used to detect cAMP levels in small intestine smooth muscle tissues.1)Measure the cAMP levels of Control and Adult GsαSMKO small intestine smooth muscle tissues in the basic state;2)Measure the cAMP levels in Control and Adult GsαSMKO small intestine smooth muscle tissues after isoproterenol(ISO)stimulation.3.7 Western blotThe total protein of small intestinal smooth muscle was extracted and the expression levels of Gsa,CREB1,p-CREB I,Foxf1,MYHI11,MLCK,ACTA2,CNN 1,ADRB1,ADRB2,GAPDH and β-actin were detected.3.8 RT-PCRTotal RNA of small intestinal smooth muscle was extracted and mRNA expression levels of ACTA2,MYH11,CNN1,MLCK,SRF,Myocardin,Foxfl andβ-actin were detected,respectively.3.9 Histopathological examination1)Immunohistochemistry was used to detect the expression of ACTA2,Gsa and Cre proteins in mice jej unum and ileum tissues;2)Immunohistochemistry was used to detect the expression of ACTA2 and Gsa proteins in mice aortic tissues;3)Immunohistochemistry was used to detect the protein expression of Gsa,Foxfl,CREB1 and p-CREB1 in human CIP and Control small intestine specimens;4)HE staining was performed to observe the structure of the small intestine,measure the diameter of the small intestine,and measure the thickness of the smooth muscle layer.3.10 SequencingThe total RNA of intestinal smooth muscles in Control and GsαSMKO mice,CIP patients and control ileal smooth muscle tissues were extracted,and the transcriptome sequences were analyzed by Nanjing Vazyme Biotech.3.11 Clinical CIP specimen collectionThe human CIP specimens were obtained from CIP patients;control specimens were obtained from age-matched patients with diseases unrelated to gastrointestinal motility disorders.3.12 Statistical analysisAll statistical data were expressed as mean±SEM.The data were first tested by normal distribution.The comparison between two groups was analyzed by independent sample t-test;the comparison of multiple groups was analyzed by one-way ANOVA.P<0.05 was considered as significantly different4.Results4.1 Gsα was specificly knocked out in smooth muscle cellsWestern blot and immunohistochemistry showed that compared with control group,Gsa protein levels in jejunum,ileum and aortic smooth muscles were decreased significantly in GsαSMKO mice.Immunohistochemistry showed that compared with control group,Gsa protein levels in jejunum,ileum and aortic smooth muscles were decreased significantly in Adult GsαSMKO mice.4.2 Gsa deficiency in smooth muscle results in postnatal lethalityThe body weight of male GsαSMKO mice was 34.1%less than their control mice 21 days after birth;GsαSMKO mice began to die at 14 days after birth,and all died at 28 days after birth,while all the control mice survived normally.4.3 Contraction was impaired in GsαSMKO smooth musclesIn the basal state,the contractile tension of jejunum and ileum of GsαSMKO mice was significantly lower than that of Control mice;KC1,ACh and EFS were used to stimulate contraction,and the contractile tension of jejunum and ileum of GsαSMKO mice was significantly lower than that of Control mice.4.4 Smooth muscle-specific Gsa deletion in adult mice confers severe intestinal obstructionThe body weight,food intake and fecal excretion of Adult GsαSMKO mice.significantly lower than those of the Control group.Within 3 months after Tamoxifen injection,Adult GsαSMKO mice had a mortality rate of more than 80%,and their ileum was found to have significant swelling and food residue accumulation.Mice were gavaged with charcoal test meal to monitor the intestinal mortility,the adult GsαSMKO mice showed about 3-fold longer whole-gut transit time than control mice.4.5 Contraction was impaired in Adult GsαSMKO smooth musclesHE staining analysis showed that the mean diameter of the ileum in adult GsαSMKO mice increased about 1.6±0.3 times than that of the control group(3566±1011 μm vs.2254 ± 562μm);the cross section thickness of ileal smooth muscle layer in adult GsαSMKO mice also increased significantly,the thickness of longitudinal muscle layer increased 2.4±0.5 times and the thickness of the annular muscle layer increased 2.1±0.4 times.KCl was used to stimulate smooth muscle contraction;the mean contractile force of adult Gs1qSMKO mice was only about 25%of that observed in control mice.The jejunal contractile force responses to KCl in adult GsαSMKO mice accounted for 34.3%of that in control mice;the ileal contractile force responses to KCl in adult GsαSMKO mice accounted for 15.4%of that in control mice.Similar conclusions were obtained when stimulated with Ach and EFS.4.6 Gsa deletion reduced the expression of contractile proteins and cAMP/CREB1 signalingWestern blot and RT-PCR showed that the expressions of contractile proteins MYH11,ACTA2,CNN1 and MLCK were significantly decreased in adult GsαSMKO smooth muscles,the expressions of the transcription factors Myocardin and Foxf1 which regulate smooth muscle contractile protein expression were significantly decreased.Gsa is responsible for generating cAMP and activating CREB1,so we measured the cAMP levels and the CREB1 activity in intestinal smooth muscle.In basal state or stimulated with isoproterenol(ISO),the cAMP levels in GsαSMKO was significantly lower than that of the control group.Both the total CREB1 and the phosphor-CREB1 at Ser133 were significantly decreased in adult GsαSMKO smooth muscles compared with Controls.4.7 CREB1 binds with Foxfl promoter and regulates its expressionSMCs were stimulated with Forskolin and western blot showed that Foxfl expression was significantly increased.Using the JASPAR database analysis,we found that in the promoter sequence of Foxfl there is a CRE sequence which could be recognized by CREB1.The ChIP results indicated that CREB1 could bind to the promoter sequence of Foxfl.Luciferase Reporter Gene Assay results showed that CREB1 could activate the Foxfl promoter.4.8 Levels of Gsa,Foxfl,p-CREB1 and CREB1 were reduced in the intestinal muscle layer of patients with chronic intestinal pseudo-obstruction.Genome-wide microarray analysis showed that the levels of Gsa,CREB1 and Foxf1 was significantly down-regulated in human CIP tissues compared with control groups.Immunohistochemistry showed that the levels of Gsa,Foxfl,p-CREB1 and CREB1 in the small intestinal smooth muscle layer of CIP patients were significantly lower than that of the control groups.5.Conclusions(1)GsαSMKO mice showed growth retardation and postnatal lethality;(2)Adult GsαSMKO mice showed CIP phenotype;(3)CREB1 binds with Foxf1 promoter and promotes its expression;(4)Gsa in SMCs regulates the expression of contractile-related proteins by affecting the CREB1/Foxfl signaling pathway;(5)The Gsα/CREB1/Foxf1 signaling pathway may be involved in human CIP disease progression.