TMEFF2基因敲除导致老鼠的生长发育迟缓和白色脂肪组织严重的缺失
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摘要
Tmeff2（transmembrane protein with EGF-like and two follistatin-likedomains）基因编码一种由一个类表皮生长因子区域（Epidermal growthfactor-like），两个类卵泡抑制素（follistatin-like）区域，一个跨膜结构域组成的蛋白。其也被称为（tomoregulin2， TPEF和HPP1）。自从这个基因在上个世纪被Tohru Uchida等人首次发现以来到现今人们对Tmeff2功能的研究报道十分矛盾也非常有限。 Horie M等人的研究发现Tmeff2的胞外区域能以可溶的形式促进多巴胺能神经元的存活。Ali N和Knauper V发现在HEK293细胞系中过量表达Tmeff2基因可以促进细胞的增殖。Glynne-Jones等人在非雄激素依赖型前列腺癌细胞中检测到Tmeff2的大量表达，证明这种癌症细胞的增殖和存活可能与Tmeff2的促进细胞存活功能相关联。然而，Gery S等人的实验研究证明Tmeff2基因对非雄激素依赖性前列腺癌细胞系有抑制作用。并且，大量实验证明在很多癌症细胞中检测到的Tmeff2基因的启动子区域经常会超甲基化，表明其有可能具有抑制癌症的功能。此外，Chen X等人认为Tmeff2基因是依靠其在细胞质的末端部分与肌氨酸脱氢酶的相互作用来发挥抑制肿瘤活性的作用的。 Tmeff2基因的在活体内的生理学功能至今还无人知晓。
我们通过使用人的胎盘碱性磷酸酶（human placental alkaline phosphatase，hPLAP）代替Tmeff2基因的第一个外显子来使其变成无效等位基因，同时hPLAP可以被AP染色检测到。纯和的Tmeff2基因突变的老鼠显示出生长发育迟缓，体重增长缓慢的特质，并且在断奶期发生死亡。通过对濒临死亡的老鼠进行检测，没有检测到肿瘤组织和器官的异常。之前的研究发现Tmeff2广泛的表达在中枢神经系统（central nerve system，CNS）和外周神经系统（peripheral nervesystem，PNS），我们通过AP染色实验对Tmeff2表达情况的追踪确认了这一结论。然而，我们通过各种手段检测中枢神经系统和外周神经系统并没有发现任何结构，功能和相关分子表达的异常。最终，我们发现Tmeff2表达在白色脂肪组织里，而且Tmeff2基因敲除老鼠的白色脂肪组织明显减少。但这些Tmeff2基因敲除老鼠保留有正常的棕色脂肪组织。因此，体内实验表明白色脂肪组织的形成选择性的需要Tmeff2基因而棕色脂肪组织不需要Tmeff2基因来进行脂肪合成。
本文创新点：（1）通过对Tmeff2基因的第一个外显子用人的胎盘碱性磷酸酶（human placental alkaline phosphatase，hPLAP）cDNA进行替换，第一次完成了对Tmeff2基因的老鼠体内敲除实验。（2）通过检测胎盘碱性磷酸酶活性的染色实验确定了Tmeff2基因在体内的表达位置。（3）首次发现Tmeff2基因在体内敲除后的行为表征和其在活体内的作用，为将来进一步对此基因进行研究奠定基础。
Tmeff2gene encodes a protein with one epidermal growth factor (EGF) likedomain, two follistatin-like domains, a single transmembrane domain, and a shortcytoplasmic tail (also known as tomoregulin2, TPEF and HPP1). Previous studiesreported conflicting functions of Tmeff2. Soluble form of Tmeff2extracellulardomain was shown to promote the survival of dopaminergic neurons and cell growthin HEK293cell line culture. Consistent with the pro-survival role, elevated Tmeff2expression has been associated with androgen-independent prostate cancers. Incontrast, others reported that Tmeff2exhibited anti-proliferative effects onandrogen-independent prostate cancer cell lines. Furthermore, the promoter-region ofTmeff2gene was frequently found to be hypermethylated in many cancers, suggestinga possible role of Tmeff2as a tumor suppressor. Additionally, the tumor suppressoractivity of Tmeff2was thought to depend on its cytoplasmic tail interacting withsarcosine dehydrogenase. The in vivo physiological function of Tmeff2remainselusive.
We generated a null allele of Tmeff2gene by replacing the first coding exon ofTmeff2with human placental alkaline phosphatase (hPLAP). Homozygous Tmeff2mutant mice (designated as Tmeff2-KO mice) show growth retardation, fail to gainweight properly, and die around weaning age. No tumors were found in the dyingmutants. AP staining confirmed previous reports that it is widely expressed in thecentral and peripheral nervous system (CNS and PNS). However, despite our besteffort, we did not find any structural or molecular abnormalities of CNS and PNS.Instead, we found that Tmeff2is expressed in the adipocytes, and Tmeff2-KO mice have very little white adipose tissues (WAT), but contain normally developed brownadipose tissues (BAT). Thus, Tmeff2is selectively required in vivo for adipogenesisin WAT but not in BAT.
我们通过使用人的胎盘碱性磷酸酶（human placental alkaline phosphatase，hPLAP）代替Tmeff2基因的第一个外显子来使其变成无效等位基因，同时hPLAP可以被AP染色检测到。纯和的Tmeff2基因突变的老鼠显示出生长发育迟缓，体重增长缓慢的特质，并且在断奶期发生死亡。通过对濒临死亡的老鼠进行检测，没有检测到肿瘤组织和器官的异常。之前的研究发现Tmeff2广泛的表达在中枢神经系统（central nerve system，CNS）和外周神经系统（peripheral nervesystem，PNS），我们通过AP染色实验对Tmeff2表达情况的追踪确认了这一结论。然而，我们通过各种手段检测中枢神经系统和外周神经系统并没有发现任何结构，功能和相关分子表达的异常。最终，我们发现Tmeff2表达在白色脂肪组织里，而且Tmeff2基因敲除老鼠的白色脂肪组织明显减少。但这些Tmeff2基因敲除老鼠保留有正常的棕色脂肪组织。因此，体内实验表明白色脂肪组织的形成选择性的需要Tmeff2基因而棕色脂肪组织不需要Tmeff2基因来进行脂肪合成。
本文创新点：（1）通过对Tmeff2基因的第一个外显子用人的胎盘碱性磷酸酶（human placental alkaline phosphatase，hPLAP）cDNA进行替换，第一次完成了对Tmeff2基因的老鼠体内敲除实验。（2）通过检测胎盘碱性磷酸酶活性的染色实验确定了Tmeff2基因在体内的表达位置。（3）首次发现Tmeff2基因在体内敲除后的行为表征和其在活体内的作用，为将来进一步对此基因进行研究奠定基础。
Tmeff2gene encodes a protein with one epidermal growth factor (EGF) likedomain, two follistatin-like domains, a single transmembrane domain, and a shortcytoplasmic tail (also known as tomoregulin2, TPEF and HPP1). Previous studiesreported conflicting functions of Tmeff2. Soluble form of Tmeff2extracellulardomain was shown to promote the survival of dopaminergic neurons and cell growthin HEK293cell line culture. Consistent with the pro-survival role, elevated Tmeff2expression has been associated with androgen-independent prostate cancers. Incontrast, others reported that Tmeff2exhibited anti-proliferative effects onandrogen-independent prostate cancer cell lines. Furthermore, the promoter-region ofTmeff2gene was frequently found to be hypermethylated in many cancers, suggestinga possible role of Tmeff2as a tumor suppressor. Additionally, the tumor suppressoractivity of Tmeff2was thought to depend on its cytoplasmic tail interacting withsarcosine dehydrogenase. The in vivo physiological function of Tmeff2remainselusive.
We generated a null allele of Tmeff2gene by replacing the first coding exon ofTmeff2with human placental alkaline phosphatase (hPLAP). Homozygous Tmeff2mutant mice (designated as Tmeff2-KO mice) show growth retardation, fail to gainweight properly, and die around weaning age. No tumors were found in the dyingmutants. AP staining confirmed previous reports that it is widely expressed in thecentral and peripheral nervous system (CNS and PNS). However, despite our besteffort, we did not find any structural or molecular abnormalities of CNS and PNS.Instead, we found that Tmeff2is expressed in the adipocytes, and Tmeff2-KO mice have very little white adipose tissues (WAT), but contain normally developed brownadipose tissues (BAT). Thus, Tmeff2is selectively required in vivo for adipogenesisin WAT but not in BAT.
引文
[1] Horie M, Mitsumoto Y, Kyushiki H, Kanemoto N, et al. Identificationand characterization of TMEFF2, a novel survival factor forhippocampal and mesencephalic neurons. Genomics，2000,67(2):146-152.
[2] Uchida T, Wada K, Akamatsu T, et al. A novel epidermal growthfactor-like molecule containing two follistatin modules stimulatestyrosine phosphorylation of erbB-4in MKN28gastric cancer cells.Biochemical and biophysical researchcommunications,1999,266(2):593-602.
[3] Lin H, Wada K, Yonezawa M, et al. Tomoregulin ectodomain sheddingby proinflammatory cytokines. Life sciences,2003,73(13):1617-1627.
[4] Heanue TA and Pachnis V. Expression profiling the developingmammalian enteric nervous system identifies marker and candidateHirschsprung disease genes. Proceedings of the National Academy ofSciences of the United States of America,2006,103(18):6919-6924.
[5] Afar DE, Bhaskar V, Ibsen E, et al. Preclinical validation ofanti-TMEFF2-auristatin E-conjugated antibodies in the treatment ofprostate cancer. Molecular cancer therapeutics,2004,3(8):921-932.
[6] Gery S, Sawyers CL, Agus DB, et al. TMEFF2is an androgen-regulatedgene exhibiting antiproliferative effects in prostate cancer cells.Oncogene,2002，21(31):4739-4746.
[7] Glynne-Jones E, Harper ME, Seery LT, et al. TENB2, a proteoglycanidentified in prostate cancer that is associated with diseaseprogression and androgen independence. International journal ofcancer. Journal international du cancer,2001,94(2):178-184.
[8] Ali N and Knauper V. Phorbol ester-induced shedding of the prostatecancer marker transmembrane protein with epidermal growth factor andtwo follistatin motifs2is mediated by the disintegrin andmetalloproteinase-17. The Journal of biologicalchemistry,2007,282(52):37378-37388.
[9] Jones PA and Laird PW. Cancer epigenetics comes of age. Naturegenetics,1999,21(2):163-167.
[10] Jones PA. DNA methylation errors and cancer. Cancerresearch,1996,56(11):2463-2467.
[11] Baylin SB, Makos M, Wu JJ, et al. Abnormal patterns of DNAmethylation in human neoplasia: potential consequences for tumorprogression. Cancer Cells,1991,3(10):383-390.
[12] Laird PW and Jaenisch R. DNA methylation and cancer. Humanmolecular,1994,genetics3Spec No:1487-1495.
[13] Sakai T, Toguchida J, Ohtani N, et al. Allele-specifichypermethylation of the retinoblastoma tumor-suppressor gene.American journal of human genetics,1991,48(5):880-888.
[14] Zion M, Ben-Yehuda D, Avraham A, et al. Progressive de novo DNAmethylation at the bcr-abl locus in the course of chronic myelogenousleukemia. Proceedings of the National Academy of Sciences of theUnited States of America,1994,91(22):10722-10726.
[15] Herman JG, Latif F, Weng Y, et al. Silencing of the VHLtumor-suppressor gene by DNA methylation in renal carcinoma.Proceedings of the National Academy of Sciences of the United Statesof America,1994,91(21):9700-9704.
[16] Katzenellenbogen RA, Baylin SB and Herman JG. Hypermethylationof the DAP-kinase CpG island is a common alteration in B-cellmalignancies. Blood,1999,93(12):4347-4353.
[17] Tanaka H, Shimada Y, Harada H, et al. Methylation of the5' CpGisland of the FHIT gene is closely associated with transcriptionalinactivation in esophageal squamous cell carcinomas. Cancerresearch,1998,58(15):3429-3434.
[18] Esteller M, Hamilton SR, Burger PC, et al. Inactivation of theDNA repair gene O6-methylguanine-DNA methyltransferase by promoterhypermethylation is a common event in primary human neoplasia. Cancerresearch,1999,59(4):793-797.
[19] Bachman KE, Herman JG, Corn PG, et al. Methylation-associatedsilencing of the tissue inhibitor of metalloproteinase-3gene suggesta suppressor role in kidney, brain, and other human cancers. Cancerresearch,1999,59(4):798-802.
[20] Young J, Biden KG, Simms LA, et al. HPP1: a transmembraneprotein-encoding gene commonly methylated in colorectal polyps andcancers. Proceedings of the National Academy of Sciences of the UnitedStates of America,2001,98(1):265-270.
[21] Liang G, Robertson KD, Talmadge C, et al. The gene for a noveltransmembrane protein containing epidermal growth factor andfollistatin domains is frequently hypermethylated in human tumorcells. Cancer research,2000,60(17):4907-4912.
[22] Powell DW, Mifflin RC, Valentich JD, et al. Myofibroblasts. II.Intestinal subepithelial myofibroblasts. The American journal ofphysiology,1999,277(2Pt1):C183-201.
[23] Esteller M, Corn PG, Baylin SB, et al. A gene hypermethylationprofile of human cancer. Cancer research,2001,61(8):3225-3229.
[24] Herman JG. Hypermethylation of tumor suppressor genes in cancer.Seminars in cancer biology,1999,9(5):359-367.
[25] Kang GH, Shim YH and Ro JY. Correlation of methylation of the hMLH1promoter with lack of expression of hMLH1in sporadic gastriccarcinomas with replication error. Laboratory investigation; ajournal of technical methods and pathology,1999,79(7):903-909.
[26] Leung SY, Yuen ST, Chung LP, et al. hMLH1promoter methylationand lack of hMLH1expression in sporadic gastric carcinomas withhigh-frequency microsatellite instability. Cancerresearch,1999,59(1):159-164.
[27] Jass JR, Young J and Leggett BA. Hyperplastic polyps and DNAmicrosatellite unstable cancers of the colorectum.Histopathology,2000,37(4):295-301.
[28] Rhyu MG, Park WS and Meltzer SJ. Microsatellite instabilityoccurs frequently in human gastric carcinoma.Oncogene,1994,9(1):29-32.
[29] Bevilacqua RA and Simpson AJ. Methylation of the hMLH1promoterbut no hMLH1mutations in sporadic gastric carcinomas with high-levelmicrosatellite instability. International journal of cancer. Journalinternational du cancer,2000,87(2):200-203.
[30] Fleisher AS, Esteller M, Wang S, et al. Hypermethylation of thehMLH1gene promoter in human gastric cancers with microsatelliteinstability. Cancer research,1999,59(5):1090-1095.
[31] Wheeler JM, Loukola A, Aaltonen LA, et al. The role ofhypermethylation of the hMLH1promoter region in HNPCC versus MSI+sporadic colorectal cancers. Journal of medicalgenetics,2000,37(8):588-592.
[32] Shibata DM, Sato F, Mori Y, et al. Hypermethylation of HPP1isassociated with hMLH1hypermethylation in gastric adenocarcinomas.Cancer research,2002,62(20):5637-5640.
[33] Geddert H, Kiel S, Iskender E, et al. Correlation of hMLH1andHPP1hypermethylation in gastric, but not in esophageal and cardiacadenocarcinoma. International journal of cancer. Journalinternational du cancer,2004,110(2):208-211.
[34] Wynter CV, Walsh MD, Higuchi T, et al. Methylation patterns definetwo types of hyperplastic polyp associated with colorectal cancer.Gut,2004,53(4):573-580.
[35] Patel K. Follistatin. The international journal of biochemistry&cell biology,1998,30(10):1087-1093.
[36] Lin SY, Morrison JR, Phillips DJ, et al. Regulation of ovarianfunction by the TGF-beta superfamily and follistatin.Reproduction,2003,126(2):133-148.
[37] Sato F, Shibata D, Harpaz N, et al. Aberrant methylation of theHPP1gene in ulcerative colitis-associated colorectal carcinoma.Cancer research,2002,62(23):6820-6822.
[38] Suzuki M, Shigematsu H, Shames DS, et al. DNAmethylation-associated inactivation of TGFbeta-related genesDRM/Gremlin, RUNX3, and HPP1in human cancers. British journal ofcancer,2005,93(9):1029-1037.
[39] Takahashi T, Shivapurkar N, Riquelme E, et al. Aberrant promoterhypermethylation of multiple genes in gallbladder carcinoma andchronic cholecystitis. Clinical cancer research: an official journalof the American Association for Cancer Research,2004,10(18Pt1):6126-6133.
[40] Elahi A, Zhang L, Yeatman TJ, et al. HPP1-mediated tumorsuppression requires activation of STAT1pathways. Internationaljournal of cancer. Journal international ducancer,2008,122(7):1567-1572.
[41] Chen X, Overcash R, Green T, et al. The tumor suppressor activityof the transmembrane protein with epidermal growth factor and twofollistatin motifs2(TMEFF2) correlates with its ability to modulatesarcosine levels. The Journal of biologicalchemistry,2011,286(18):16091-16100.
[42] Mohler JL, Morris TL, Ford OH, et al. Identification ofdifferentially expressed genes associated with androgen-independentgrowth of prostate cancer. The Prostate,2002,51(4):247-255.
[43] Burns AJ and Douarin NM. The sacral neural crest contributesneurons and glia to the post-umbilical gut: spatiotemporal analysisof the development of the enteric nervous system. Development，1998，125(21):4335-4347.
[44] Gershon MD. Genes and lineages in the formation of the entericnervous system. Current opinion in neurobiology，1997，7(1):101-109.
[45] Sang Q and Young HM. Chemical coding of neurons in the myentericplexus and external muscle of the small and large intestine of themouse. Cell and tissue research，1996，284(1):39-53.
[46] Newgreen D and Young HM. Enteric nervous system: development anddevelopmental disturbances--part1. Pediatric and developmentalpathology: the official journal of the Society for PediatricPathology and the Paediatric Pathology Society，2002，5(3):224-247.
[47] Brooks AS, Oostra BA and Hofstra RM. Studying the genetics ofHirschsprung's disease: unraveling an oligogenic disorder. Clinicalgenetics，2005，67(1):6-14.
[48] Pachnis V, Mankoo B and Costantini F. Expression of the c-retproto-oncogene during mouse embryogenesis. Development，1993，119(4):1005-1017.
[49] Schuchardt A, D'Agati V, Larsson-Blomberg L, et al. Defects inthe kidney and enteric nervous system of mice lacking the tyrosinekinase receptor Ret. Nature，1994，367(6461):380-383.
[50] Durbec PL, Larsson-Blomberg LB, Schuchardt A, et al. Commonorigin and developmental dependence on c-ret of subsets of entericand sympathetic neuroblasts. Development，1996，122(1):349-358.
[51] Taraviras S, Marcos-Gutierrez CV, Durbec P, et al.() Signallingby the RET receptor tyrosine kinase and its role in the developmentof the mammalian enteric nervous system. Development，1999，126(12):2785-2797.
[52] Young HM, Hearn CJ, Farlie PG, et al. GDNF is a chemoattractantfor enteric neural cells. Developmental biology，2001，229(2):503-516.
[53] Natarajan D, Marcos-Gutierrez C, Pachnis V, et al. Requirementof signalling by receptor tyrosine kinase RET for the directedmigration of enteric nervous system progenitor cells during mammalianembryogenesis. Development，2002，129(22):5151-5160.
[54] Heldin CH and Westermark B. Mechanism of action and in vivo roleof platelet-derived growth factor. Physiological reviews，1999，79(4):1283-1316.
[55] Hoch RV and Soriano P. Roles of PDGF in animal development.Development，2003，130(20):4769-4784.
[56] Raines EW, Lane TF, Iruela-Arispe ML, et al. The extracellularglycoprotein SPARC interacts with platelet-derived growth factor(PDGF)-AB and-BB and inhibits the binding of PDGF to its receptors.Proceedings of the National Academy of Sciences of the United Statesof America，1992，89(4):1281-1285.
[57] Patthy L and Nikolics K. Functions of agrin and agrin-relatedproteins. Trends in neurosciences，1993，16(2):76-81.
[58] Kupprion C, Motamed K and Sage EH. SPARC (BM-40, osteonectin)inhibits the mitogenic effect of vascular endothelial growth factoron microvascular endothelial cells. The Journal of biologicalchemistry，1998，273(45):29635-29640.
[59] Chang C, Eggen BJ, Weinstein DC, et al. Regulation of nodal andBMP signaling by tomoregulin-1(X7365) through novel mechanisms.Developmental biology，2003，255(1):1-11.
[60] Harms PW and Chang C. Tomoregulin-1(TMEFF1) inhibits nodalsignaling through direct binding to the nodal coreceptor Cripto.Genes&development，2003，17(21):2624-2629.
[61] Lin K, Taylor JR, Jr., et al. TMEFF2is a PDGF-AA binding proteinwith methylation-associated gene silencing in multiple cancer typesincluding glioma. PloS one，2011，6(4):e18608.
[62] Zylka MJ, Rice FL and Anderson DJ. Topographically distinctepidermal nociceptive circuits revealed by axonal tracers targetedto Mrgprd. Neuron，2005，45(1):17-25.
[63] Leighton PA, Mitchell KJ, Goodrich LV, et al. Defining brainwiring patterns and mechanisms through gene trapping in mice. Nature，2001，410(6825):174-179.
[64] Erlander MG, Tillakaratne NJ, Feldblum S, et al. Two genes encodedistinct glutamate decarboxylases. Neuron，1991，7(1):91-100.
[65] Watanabe M, Maemura K, Kanbara K, et al. GABA and GABA receptorsin the central nervous system and other organs. International reviewof cytology，2002，213:1-47.
[66] Cates MS, Berry MB, Ho EL, et al. Metal-ion affinity andspecificity in EF-hand proteins: coordination geometry and domainplasticity in parvalbumin. Structure，1999，7(10):1269-1278.
[67] Cates MS, Teodoro ML and Phillips GN, Jr.. Molecular mechanismsof calcium and magnesium binding to parvalbumin. Biophysical journal，2002，82(3):1133-1146.
[68] Verret L, Mann EO, Hang GB, et al. Inhibitory interneuron deficitlinks altered network activity and cognitive dysfunction in Alzheimermodel. Cell，2012，149(3):708-721.
[69] Zubrzycka M and Janecka A. Substance P: transmitter ofnociception (Minireview). Endocrine regulations，2000，34(4):195-201.
[70] De Felipe C, Herrero JF, O'Brien JA, et al. Altered nociception,analgesia and aggression in mice lacking the receptor for substanceP. Nature，1998，392(6674):394-397.
[71] Ebner K and Singewald N. The role of substance P in stress andanxiety responses. Amino acids，2006，31(3):251-272.
[72] Huston JP, Hasenohrl RU, Boix F, et al. Sequence-specific effectsof neurokinin substance P on memory, reinforcement, and braindopamine activity. Psychopharmacology，1993，112(2-3):147-162.
[73] Massari VJ, Shults CW, Park CH, et al. Deafferentation causes aloss of presynaptic bombesin receptors and supersensitivity ofsubstance P receptors in the dorsal horn of the cat spinal cord. Brainresearch，1985，343(2):268-274.
[74] Bonham AC. Neurotransmitters in the CNS control of breathing.Respiration physiology，1995，101(3):219-230.
[75] Hesketh PJ. Potential role of the NK1receptor antagonists inchemotherapy-induced nausea and vomiting. Supportive care in cancer:official journal of the Multinational Association of Supportive Carein Cancer，2001，9(5):350-354.
[76] Woolsey TA and Van der Loos H. The structural organization oflayer IV in the somatosensory region (SI) of mouse cerebral cortex.The description of a cortical field composed of discretecytoarchitectonic units. Brain research，1970，17(2):205-242.
[77] Petersen CC. The functional organization of the barrel cortex.Neuron，2007，56(2):339-355.
[78] Wilson MA, Johnston MV, Goldstein GW, et al. Neonatal leadexposure impairs development of rodent barrel field cortex.Proceedings of the National Academy of Sciences of the United Statesof America，2000，97(10):5540-5545.
[79] Clem RL, Celikel T and Barth AL. Ongoing in vivo experiencetriggers synaptic metaplasticity in the neocortex. Science，2008，319(5859):101-104.
[80] Takasaki C, Okada R, Mitani A, et al. Glutamate transportersregulate lesion-induced plasticity in the developing somatosensorycortex. The Journal of neuroscience: the official journal of theSociety for Neuroscience，2008，28(19):4995-5006.
[81] Michalik L, Auwerx J, Berger JP, et al. International Union ofPharmacology. LXI. Peroxisome proliferator-activated receptors.Pharmacological reviews，2006，58(4):726-741.
[82] Belfiore A, Genua M and Malaguarnera R. PPAR-gamma agonists andtheir effects on IGF-I receptor signaling: Implications for cancer.PPAR research，2009，2009:830501.
[83] Berger J and Moller DE. The mechanisms of action of PPARs. Annualreview of medicine，2002，53:409-435.
[84] Feige JN, Gelman L, Michalik L, et al. From molecular action tophysiological outputs: peroxisome proliferator-activated receptorsare nuclear receptors at the crossroads of key cellular functions.Progress in lipid research，2006，45(2):120-159.
[85] Kodera Y, Takeyama K, Murayama A, et al. Ligand type-specificinteractions of peroxisome proliferator-activated receptor gammawith transcriptional coactivators. The Journal of biologicalchemistry，2000，275(43):33201-33204.
[86] Picard F, Gehin M, Annicotte J, et al.(2002) SRC-1and TIF2control energy balance between white and brown adipose tissues. Cell111(7):931-941.
[87] Smas CM, Chen L, Zhao L, et al. Transcriptional repression ofpref-1by glucocorticoids promotes3T3-L1adipocyte differentiation.The Journal of biological chemistry，1999，274(18):12632-12641.
[88] Rieusset J, Andreelli F, Auboeuf D, et al. Insulin acutelyregulates the expression of the peroxisome proliferator-activatedreceptor-gamma in human adipocytes. Diabetes，1999，48(4):699-705.
[89] Sasaki M, Jordan P, Welbourne T, et al. Troglitazone, a PPAR-gammaactivator prevents endothelial cell adhesion molecule expression andlymphocyte adhesion mediated by TNF-alpha. BMC physiology，2005，5(1):3.
[90] Camp HS, Li O, Wise SC, et al. Differential activation ofperoxisome proliferator-activated receptor-gamma by troglitazoneand rosiglitazone. Diabetes，2000，49(4):539-547.
[91] Balemans W and Van Hul W. Extracellular regulation of BMPsignaling in vertebrates: a cocktail of modulators. Developmentalbiology，2002，250(2):231-250.
[92] Gumienny TL and Padgett RW. The other side of TGF-beta superfamilysignal regulation: thinking outside the cell. Trends in endocrinologyand metabolism: TEM，2002，13(7):295-299.
[93] Li C, Garland JM and Kumar S. Re: Role of transforming growthfactor-beta signaling in cancer. Journal of the National CancerInstitute，2001，93(7):555-557.
[94] Teicher BA. Malignant cells, directors of the malignant process:role of transforming growth factor-beta. Cancer metastasis reviews，2001，20(1-2):133-143.
[95] Roberts AB and Wakefield LM. The two faces of transforming growthfactor beta in carcinogenesis. Proceedings of the National Academyof Sciences of the United States of America，2003，100(15):8621-8623.
[96] de Caestecker MP, Piek E and Roberts AB. Role of transforminggrowth factor-beta signaling in cancer. Journal of the NationalCancer Institute，2000，92(17):1388-1402.
[97] Pearce JJ, Penny G and Rossant J. A mouse cerberus/Dan-relatedgene family. Developmental biology，1999，209(1):98-110.
[98] Hsu DR, Economides AN, Wang X, et al. The Xenopus dorsalizingfactor Gremlin identifies a novel family of secreted proteins thatantagonize BMP activities. Molecular cell，1998，1(5):673-683.
[99] Topol LZ, Marx M, Laugier D, et al. Identification of drm, a novelgene whose expression is suppressed in transformed cells and whichcan inhibit growth of normal but not transformed cells in culture.Molecular and cellular biology，1997，17(8):4801-4810.
[100] Zuniga A, Haramis AP, McMahon AP, et al. Signal relay by BMPantagonism controls the SHH/FGF4feedback loop in vertebrate limbbuds. Nature，1999，401(6753):598-602.
[101] Ghosh Choudhury G, Kim YS, Simon M, et al.() Bone morphogeneticprotein2inhibits platelet-derived growth factor-induced c-fos genetranscription and DNA synthesis in mesangial cells. Involvement ofmitogen-activated protein kinase. The Journal of biologicalchemistry，1999，274(16):10897-10902.
[102] Shi W, Zhao J, Anderson KD and Warburton D. Gremlin negativelymodulates BMP-4induction of embryonic mouse lung branchingmorphogenesis. American journal of physiology. Lung cellular andmolecular physiology，2001，280(5):L1030-1039.
[2] Uchida T, Wada K, Akamatsu T, et al. A novel epidermal growthfactor-like molecule containing two follistatin modules stimulatestyrosine phosphorylation of erbB-4in MKN28gastric cancer cells.Biochemical and biophysical researchcommunications,1999,266(2):593-602.
[3] Lin H, Wada K, Yonezawa M, et al. Tomoregulin ectodomain sheddingby proinflammatory cytokines. Life sciences,2003,73(13):1617-1627.
[4] Heanue TA and Pachnis V. Expression profiling the developingmammalian enteric nervous system identifies marker and candidateHirschsprung disease genes. Proceedings of the National Academy ofSciences of the United States of America,2006,103(18):6919-6924.
[5] Afar DE, Bhaskar V, Ibsen E, et al. Preclinical validation ofanti-TMEFF2-auristatin E-conjugated antibodies in the treatment ofprostate cancer. Molecular cancer therapeutics,2004,3(8):921-932.
[6] Gery S, Sawyers CL, Agus DB, et al. TMEFF2is an androgen-regulatedgene exhibiting antiproliferative effects in prostate cancer cells.Oncogene,2002，21(31):4739-4746.
[7] Glynne-Jones E, Harper ME, Seery LT, et al. TENB2, a proteoglycanidentified in prostate cancer that is associated with diseaseprogression and androgen independence. International journal ofcancer. Journal international du cancer,2001,94(2):178-184.
[8] Ali N and Knauper V. Phorbol ester-induced shedding of the prostatecancer marker transmembrane protein with epidermal growth factor andtwo follistatin motifs2is mediated by the disintegrin andmetalloproteinase-17. The Journal of biologicalchemistry,2007,282(52):37378-37388.
[9] Jones PA and Laird PW. Cancer epigenetics comes of age. Naturegenetics,1999,21(2):163-167.
[10] Jones PA. DNA methylation errors and cancer. Cancerresearch,1996,56(11):2463-2467.
[11] Baylin SB, Makos M, Wu JJ, et al. Abnormal patterns of DNAmethylation in human neoplasia: potential consequences for tumorprogression. Cancer Cells,1991,3(10):383-390.
[12] Laird PW and Jaenisch R. DNA methylation and cancer. Humanmolecular,1994,genetics3Spec No:1487-1495.
[13] Sakai T, Toguchida J, Ohtani N, et al. Allele-specifichypermethylation of the retinoblastoma tumor-suppressor gene.American journal of human genetics,1991,48(5):880-888.
[14] Zion M, Ben-Yehuda D, Avraham A, et al. Progressive de novo DNAmethylation at the bcr-abl locus in the course of chronic myelogenousleukemia. Proceedings of the National Academy of Sciences of theUnited States of America,1994,91(22):10722-10726.
[15] Herman JG, Latif F, Weng Y, et al. Silencing of the VHLtumor-suppressor gene by DNA methylation in renal carcinoma.Proceedings of the National Academy of Sciences of the United Statesof America,1994,91(21):9700-9704.
[16] Katzenellenbogen RA, Baylin SB and Herman JG. Hypermethylationof the DAP-kinase CpG island is a common alteration in B-cellmalignancies. Blood,1999,93(12):4347-4353.
[17] Tanaka H, Shimada Y, Harada H, et al. Methylation of the5' CpGisland of the FHIT gene is closely associated with transcriptionalinactivation in esophageal squamous cell carcinomas. Cancerresearch,1998,58(15):3429-3434.
[18] Esteller M, Hamilton SR, Burger PC, et al. Inactivation of theDNA repair gene O6-methylguanine-DNA methyltransferase by promoterhypermethylation is a common event in primary human neoplasia. Cancerresearch,1999,59(4):793-797.
[19] Bachman KE, Herman JG, Corn PG, et al. Methylation-associatedsilencing of the tissue inhibitor of metalloproteinase-3gene suggesta suppressor role in kidney, brain, and other human cancers. Cancerresearch,1999,59(4):798-802.
[20] Young J, Biden KG, Simms LA, et al. HPP1: a transmembraneprotein-encoding gene commonly methylated in colorectal polyps andcancers. Proceedings of the National Academy of Sciences of the UnitedStates of America,2001,98(1):265-270.
[21] Liang G, Robertson KD, Talmadge C, et al. The gene for a noveltransmembrane protein containing epidermal growth factor andfollistatin domains is frequently hypermethylated in human tumorcells. Cancer research,2000,60(17):4907-4912.
[22] Powell DW, Mifflin RC, Valentich JD, et al. Myofibroblasts. II.Intestinal subepithelial myofibroblasts. The American journal ofphysiology,1999,277(2Pt1):C183-201.
[23] Esteller M, Corn PG, Baylin SB, et al. A gene hypermethylationprofile of human cancer. Cancer research,2001,61(8):3225-3229.
[24] Herman JG. Hypermethylation of tumor suppressor genes in cancer.Seminars in cancer biology,1999,9(5):359-367.
[25] Kang GH, Shim YH and Ro JY. Correlation of methylation of the hMLH1promoter with lack of expression of hMLH1in sporadic gastriccarcinomas with replication error. Laboratory investigation; ajournal of technical methods and pathology,1999,79(7):903-909.
[26] Leung SY, Yuen ST, Chung LP, et al. hMLH1promoter methylationand lack of hMLH1expression in sporadic gastric carcinomas withhigh-frequency microsatellite instability. Cancerresearch,1999,59(1):159-164.
[27] Jass JR, Young J and Leggett BA. Hyperplastic polyps and DNAmicrosatellite unstable cancers of the colorectum.Histopathology,2000,37(4):295-301.
[28] Rhyu MG, Park WS and Meltzer SJ. Microsatellite instabilityoccurs frequently in human gastric carcinoma.Oncogene,1994,9(1):29-32.
[29] Bevilacqua RA and Simpson AJ. Methylation of the hMLH1promoterbut no hMLH1mutations in sporadic gastric carcinomas with high-levelmicrosatellite instability. International journal of cancer. Journalinternational du cancer,2000,87(2):200-203.
[30] Fleisher AS, Esteller M, Wang S, et al. Hypermethylation of thehMLH1gene promoter in human gastric cancers with microsatelliteinstability. Cancer research,1999,59(5):1090-1095.
[31] Wheeler JM, Loukola A, Aaltonen LA, et al. The role ofhypermethylation of the hMLH1promoter region in HNPCC versus MSI+sporadic colorectal cancers. Journal of medicalgenetics,2000,37(8):588-592.
[32] Shibata DM, Sato F, Mori Y, et al. Hypermethylation of HPP1isassociated with hMLH1hypermethylation in gastric adenocarcinomas.Cancer research,2002,62(20):5637-5640.
[33] Geddert H, Kiel S, Iskender E, et al. Correlation of hMLH1andHPP1hypermethylation in gastric, but not in esophageal and cardiacadenocarcinoma. International journal of cancer. Journalinternational du cancer,2004,110(2):208-211.
[34] Wynter CV, Walsh MD, Higuchi T, et al. Methylation patterns definetwo types of hyperplastic polyp associated with colorectal cancer.Gut,2004,53(4):573-580.
[35] Patel K. Follistatin. The international journal of biochemistry&cell biology,1998,30(10):1087-1093.
[36] Lin SY, Morrison JR, Phillips DJ, et al. Regulation of ovarianfunction by the TGF-beta superfamily and follistatin.Reproduction,2003,126(2):133-148.
[37] Sato F, Shibata D, Harpaz N, et al. Aberrant methylation of theHPP1gene in ulcerative colitis-associated colorectal carcinoma.Cancer research,2002,62(23):6820-6822.
[38] Suzuki M, Shigematsu H, Shames DS, et al. DNAmethylation-associated inactivation of TGFbeta-related genesDRM/Gremlin, RUNX3, and HPP1in human cancers. British journal ofcancer,2005,93(9):1029-1037.
[39] Takahashi T, Shivapurkar N, Riquelme E, et al. Aberrant promoterhypermethylation of multiple genes in gallbladder carcinoma andchronic cholecystitis. Clinical cancer research: an official journalof the American Association for Cancer Research,2004,10(18Pt1):6126-6133.
[40] Elahi A, Zhang L, Yeatman TJ, et al. HPP1-mediated tumorsuppression requires activation of STAT1pathways. Internationaljournal of cancer. Journal international ducancer,2008,122(7):1567-1572.
[41] Chen X, Overcash R, Green T, et al. The tumor suppressor activityof the transmembrane protein with epidermal growth factor and twofollistatin motifs2(TMEFF2) correlates with its ability to modulatesarcosine levels. The Journal of biologicalchemistry,2011,286(18):16091-16100.
[42] Mohler JL, Morris TL, Ford OH, et al. Identification ofdifferentially expressed genes associated with androgen-independentgrowth of prostate cancer. The Prostate,2002,51(4):247-255.
[43] Burns AJ and Douarin NM. The sacral neural crest contributesneurons and glia to the post-umbilical gut: spatiotemporal analysisof the development of the enteric nervous system. Development，1998，125(21):4335-4347.
[44] Gershon MD. Genes and lineages in the formation of the entericnervous system. Current opinion in neurobiology，1997，7(1):101-109.
[45] Sang Q and Young HM. Chemical coding of neurons in the myentericplexus and external muscle of the small and large intestine of themouse. Cell and tissue research，1996，284(1):39-53.
[46] Newgreen D and Young HM. Enteric nervous system: development anddevelopmental disturbances--part1. Pediatric and developmentalpathology: the official journal of the Society for PediatricPathology and the Paediatric Pathology Society，2002，5(3):224-247.
[47] Brooks AS, Oostra BA and Hofstra RM. Studying the genetics ofHirschsprung's disease: unraveling an oligogenic disorder. Clinicalgenetics，2005，67(1):6-14.
[48] Pachnis V, Mankoo B and Costantini F. Expression of the c-retproto-oncogene during mouse embryogenesis. Development，1993，119(4):1005-1017.
[49] Schuchardt A, D'Agati V, Larsson-Blomberg L, et al. Defects inthe kidney and enteric nervous system of mice lacking the tyrosinekinase receptor Ret. Nature，1994，367(6461):380-383.
[50] Durbec PL, Larsson-Blomberg LB, Schuchardt A, et al. Commonorigin and developmental dependence on c-ret of subsets of entericand sympathetic neuroblasts. Development，1996，122(1):349-358.
[51] Taraviras S, Marcos-Gutierrez CV, Durbec P, et al.() Signallingby the RET receptor tyrosine kinase and its role in the developmentof the mammalian enteric nervous system. Development，1999，126(12):2785-2797.
[52] Young HM, Hearn CJ, Farlie PG, et al. GDNF is a chemoattractantfor enteric neural cells. Developmental biology，2001，229(2):503-516.
[53] Natarajan D, Marcos-Gutierrez C, Pachnis V, et al. Requirementof signalling by receptor tyrosine kinase RET for the directedmigration of enteric nervous system progenitor cells during mammalianembryogenesis. Development，2002，129(22):5151-5160.
[54] Heldin CH and Westermark B. Mechanism of action and in vivo roleof platelet-derived growth factor. Physiological reviews，1999，79(4):1283-1316.
[55] Hoch RV and Soriano P. Roles of PDGF in animal development.Development，2003，130(20):4769-4784.
[56] Raines EW, Lane TF, Iruela-Arispe ML, et al. The extracellularglycoprotein SPARC interacts with platelet-derived growth factor(PDGF)-AB and-BB and inhibits the binding of PDGF to its receptors.Proceedings of the National Academy of Sciences of the United Statesof America，1992，89(4):1281-1285.
[57] Patthy L and Nikolics K. Functions of agrin and agrin-relatedproteins. Trends in neurosciences，1993，16(2):76-81.
[58] Kupprion C, Motamed K and Sage EH. SPARC (BM-40, osteonectin)inhibits the mitogenic effect of vascular endothelial growth factoron microvascular endothelial cells. The Journal of biologicalchemistry，1998，273(45):29635-29640.
[59] Chang C, Eggen BJ, Weinstein DC, et al. Regulation of nodal andBMP signaling by tomoregulin-1(X7365) through novel mechanisms.Developmental biology，2003，255(1):1-11.
[60] Harms PW and Chang C. Tomoregulin-1(TMEFF1) inhibits nodalsignaling through direct binding to the nodal coreceptor Cripto.Genes&development，2003，17(21):2624-2629.
[61] Lin K, Taylor JR, Jr., et al. TMEFF2is a PDGF-AA binding proteinwith methylation-associated gene silencing in multiple cancer typesincluding glioma. PloS one，2011，6(4):e18608.
[62] Zylka MJ, Rice FL and Anderson DJ. Topographically distinctepidermal nociceptive circuits revealed by axonal tracers targetedto Mrgprd. Neuron，2005，45(1):17-25.
[63] Leighton PA, Mitchell KJ, Goodrich LV, et al. Defining brainwiring patterns and mechanisms through gene trapping in mice. Nature，2001，410(6825):174-179.
[64] Erlander MG, Tillakaratne NJ, Feldblum S, et al. Two genes encodedistinct glutamate decarboxylases. Neuron，1991，7(1):91-100.
[65] Watanabe M, Maemura K, Kanbara K, et al. GABA and GABA receptorsin the central nervous system and other organs. International reviewof cytology，2002，213:1-47.
[66] Cates MS, Berry MB, Ho EL, et al. Metal-ion affinity andspecificity in EF-hand proteins: coordination geometry and domainplasticity in parvalbumin. Structure，1999，7(10):1269-1278.
[67] Cates MS, Teodoro ML and Phillips GN, Jr.. Molecular mechanismsof calcium and magnesium binding to parvalbumin. Biophysical journal，2002，82(3):1133-1146.
[68] Verret L, Mann EO, Hang GB, et al. Inhibitory interneuron deficitlinks altered network activity and cognitive dysfunction in Alzheimermodel. Cell，2012，149(3):708-721.
[69] Zubrzycka M and Janecka A. Substance P: transmitter ofnociception (Minireview). Endocrine regulations，2000，34(4):195-201.
[70] De Felipe C, Herrero JF, O'Brien JA, et al. Altered nociception,analgesia and aggression in mice lacking the receptor for substanceP. Nature，1998，392(6674):394-397.
[71] Ebner K and Singewald N. The role of substance P in stress andanxiety responses. Amino acids，2006，31(3):251-272.
[72] Huston JP, Hasenohrl RU, Boix F, et al. Sequence-specific effectsof neurokinin substance P on memory, reinforcement, and braindopamine activity. Psychopharmacology，1993，112(2-3):147-162.
[73] Massari VJ, Shults CW, Park CH, et al. Deafferentation causes aloss of presynaptic bombesin receptors and supersensitivity ofsubstance P receptors in the dorsal horn of the cat spinal cord. Brainresearch，1985，343(2):268-274.
[74] Bonham AC. Neurotransmitters in the CNS control of breathing.Respiration physiology，1995，101(3):219-230.
[75] Hesketh PJ. Potential role of the NK1receptor antagonists inchemotherapy-induced nausea and vomiting. Supportive care in cancer:official journal of the Multinational Association of Supportive Carein Cancer，2001，9(5):350-354.
[76] Woolsey TA and Van der Loos H. The structural organization oflayer IV in the somatosensory region (SI) of mouse cerebral cortex.The description of a cortical field composed of discretecytoarchitectonic units. Brain research，1970，17(2):205-242.
[77] Petersen CC. The functional organization of the barrel cortex.Neuron，2007，56(2):339-355.
[78] Wilson MA, Johnston MV, Goldstein GW, et al. Neonatal leadexposure impairs development of rodent barrel field cortex.Proceedings of the National Academy of Sciences of the United Statesof America，2000，97(10):5540-5545.
[79] Clem RL, Celikel T and Barth AL. Ongoing in vivo experiencetriggers synaptic metaplasticity in the neocortex. Science，2008，319(5859):101-104.
[80] Takasaki C, Okada R, Mitani A, et al. Glutamate transportersregulate lesion-induced plasticity in the developing somatosensorycortex. The Journal of neuroscience: the official journal of theSociety for Neuroscience，2008，28(19):4995-5006.
[81] Michalik L, Auwerx J, Berger JP, et al. International Union ofPharmacology. LXI. Peroxisome proliferator-activated receptors.Pharmacological reviews，2006，58(4):726-741.
[82] Belfiore A, Genua M and Malaguarnera R. PPAR-gamma agonists andtheir effects on IGF-I receptor signaling: Implications for cancer.PPAR research，2009，2009:830501.
[83] Berger J and Moller DE. The mechanisms of action of PPARs. Annualreview of medicine，2002，53:409-435.
[84] Feige JN, Gelman L, Michalik L, et al. From molecular action tophysiological outputs: peroxisome proliferator-activated receptorsare nuclear receptors at the crossroads of key cellular functions.Progress in lipid research，2006，45(2):120-159.
[85] Kodera Y, Takeyama K, Murayama A, et al. Ligand type-specificinteractions of peroxisome proliferator-activated receptor gammawith transcriptional coactivators. The Journal of biologicalchemistry，2000，275(43):33201-33204.
[86] Picard F, Gehin M, Annicotte J, et al.(2002) SRC-1and TIF2control energy balance between white and brown adipose tissues. Cell111(7):931-941.
[87] Smas CM, Chen L, Zhao L, et al. Transcriptional repression ofpref-1by glucocorticoids promotes3T3-L1adipocyte differentiation.The Journal of biological chemistry，1999，274(18):12632-12641.
[88] Rieusset J, Andreelli F, Auboeuf D, et al. Insulin acutelyregulates the expression of the peroxisome proliferator-activatedreceptor-gamma in human adipocytes. Diabetes，1999，48(4):699-705.
[89] Sasaki M, Jordan P, Welbourne T, et al. Troglitazone, a PPAR-gammaactivator prevents endothelial cell adhesion molecule expression andlymphocyte adhesion mediated by TNF-alpha. BMC physiology，2005，5(1):3.
[90] Camp HS, Li O, Wise SC, et al. Differential activation ofperoxisome proliferator-activated receptor-gamma by troglitazoneand rosiglitazone. Diabetes，2000，49(4):539-547.
[91] Balemans W and Van Hul W. Extracellular regulation of BMPsignaling in vertebrates: a cocktail of modulators. Developmentalbiology，2002，250(2):231-250.
[92] Gumienny TL and Padgett RW. The other side of TGF-beta superfamilysignal regulation: thinking outside the cell. Trends in endocrinologyand metabolism: TEM，2002，13(7):295-299.
[93] Li C, Garland JM and Kumar S. Re: Role of transforming growthfactor-beta signaling in cancer. Journal of the National CancerInstitute，2001，93(7):555-557.
[94] Teicher BA. Malignant cells, directors of the malignant process:role of transforming growth factor-beta. Cancer metastasis reviews，2001，20(1-2):133-143.
[95] Roberts AB and Wakefield LM. The two faces of transforming growthfactor beta in carcinogenesis. Proceedings of the National Academyof Sciences of the United States of America，2003，100(15):8621-8623.
[96] de Caestecker MP, Piek E and Roberts AB. Role of transforminggrowth factor-beta signaling in cancer. Journal of the NationalCancer Institute，2000，92(17):1388-1402.
[97] Pearce JJ, Penny G and Rossant J. A mouse cerberus/Dan-relatedgene family. Developmental biology，1999，209(1):98-110.
[98] Hsu DR, Economides AN, Wang X, et al. The Xenopus dorsalizingfactor Gremlin identifies a novel family of secreted proteins thatantagonize BMP activities. Molecular cell，1998，1(5):673-683.
[99] Topol LZ, Marx M, Laugier D, et al. Identification of drm, a novelgene whose expression is suppressed in transformed cells and whichcan inhibit growth of normal but not transformed cells in culture.Molecular and cellular biology，1997，17(8):4801-4810.
[100] Zuniga A, Haramis AP, McMahon AP, et al. Signal relay by BMPantagonism controls the SHH/FGF4feedback loop in vertebrate limbbuds. Nature，1999，401(6753):598-602.
[101] Ghosh Choudhury G, Kim YS, Simon M, et al.() Bone morphogeneticprotein2inhibits platelet-derived growth factor-induced c-fos genetranscription and DNA synthesis in mesangial cells. Involvement ofmitogen-activated protein kinase. The Journal of biologicalchemistry，1999，274(16):10897-10902.
[102] Shi W, Zhao J, Anderson KD and Warburton D. Gremlin negativelymodulates BMP-4induction of embryonic mouse lung branchingmorphogenesis. American journal of physiology. Lung cellular andmolecular physiology，2001，280(5):L1030-1039.