self - renewal of escs is maintained by culture with the cytokine leukemia inhibitory factor ( lif ) . actually , lif is always provided to the culture medium of mouse escs and the removal of lif causes rapid differentiation of mouse escs . upon binding to the lif receptor ( lifr ) , lif causes heterodimerization of the low - affinity lifr and gp130 , which acts as the main receptor for intracellular signal transduction ( fig .  1 ) . briefly , ligand binding leads to phosphorylation and activation of janus kinase ( jak ) , which is tethered to the intracellular region of gp130 . activated jak phosphorylates gp130 at four sites of tyrosine , and the phosphorylated tyrosine interacts with sh2-bearing proteins such as signal transducer and activator of transcription 3 ( stat3 ) . then stat3 is also tyrosine - phosphorylated by jak and is homodimerized via its src - homology-2 ( sh2 ) domains . consequently , activation of several genes by translocated stat3 plays a role in suppression of the differentiation of mouse escs . jak - stat3 activates klf4 , which maintains oct3/4 expression via sox2 and nanog . the molecules including oct3/4 , sox2 and nanog are known to be crucial for maintenance of pluripotency in escs . therefore , stat3 is one of the main players in the signaling pathway dependent on lif in escs to maintain a pluripotent state . importantly , it has been reported that activation of stat3 is regulated by the ubiquitin system and translocation from the cytosol to the nucleus via molecular chaperones such as hsp90 . lif causes phosphorylation and activation of jak , which is tethered to the intracellular region of gp130 . activated jak phosphorylates gp130 and its tyrosine phosphorylations causes interaction with stat3 . then stat3 is also tyrosine - phosphorylated by jak and is homodimerized via its sh2 domains . the jak - stat pathway is negatively regulated by several inhibitory systems : dephosphorylation by tyrosine phosphatases including shp and physical inhibition or ubiquitin - mediated degradation of jak by socs . jak phosphorylates shp and then shp interacts with the grb2/sos complex to activate the mapk pathway , leading to inhibition of tbx3 and nanog . slim is an e3 ubiquitin ligase that negatively regulates the jak - stat signal pathway , but slim is expressed at a low level in mouse escs . lif - mediated signaling regulates proliferation or differentiation depending on the cell type or differentiated stage of the cell . past evidence showed that stage of the cell and uncontrolled secretion of lif are associated with pathological conditions . lif is also a key cytokine for maintaining self - renewal and pluripotency of mouse escs . therefore , it is important to clarify molecular mechanism of lif signaling in mouse escs . in contrast to mouse escs , fibroblast growth factor 2 ( fgf2 ) , also called basic fgf ( bfgf ) , and activin a are used to maintain self - renewal in human escs . recent studies have shown that human escs are slightly differentiated cells in the  primed state  and that mouse escs are fully undifferentiated cells in the  nave state ,  suggesting that lif maintains the actually , transcriptome analyses showed differences in the expression profiles of genes in mouse and human escs , indicating that these cells may not be derived from the same lineages of early embryos and that human escs are likely to be lif - independent because of the establishment from cell lineages later than early epiblast . lif is a key cytokine for maintaining self - renewal and pluripotency of mouse escs . in contrast to mouse escs , fibroblast growth factor 2 ( fgf2 ) , also called basic fgf ( bfgf ) , and activin a are used to maintain self - renewal in human escs . human escs are likely to be slightly differentiated cells in the  primed state  and mouse escs are fully undifferentiated cells in the  nave state . the activity of lif was first identified in 1969 by ichikawa , and it was shown that its activity could inhibit proliferation and induce macrophage differentiation of the m1 leukemic myeloid cell line . this biological activity , called  differentiation factor ( d - factor ) ,  was partially purified from a cell - conditioned medium prepared from mouse embryos . purified mouse d - factor / lif from a krebs ii ascites cell - derived conditioned medium and isolated a mouse d - factor / lif cdna clone . human lif and mouse lif are 180-amino - acid glycoproteins and belong to the interleukin-6 ( il-6 ) subfamily . lif proteins have at least three isoforms : a soluble form called lif - d ( soluble form ) , lif - i / lif - t ( intracellular form ) and lif - m ( a form localized in the extracellular matrix ) . lif - m and lif - d are synthesized as alternative transcripts differing in the first exon . lif - t is a truncated isoform because of the initiation of translation at the first atg codon of exon 2 . although homozygous lif mice are viable , they have many defects including decrease in cell lineages such as hematopoietic stem cells , primordial germ cells and motor neurons . notably , female lif mice are fertile , but their blastocysts can not implant in the uterine epithelium because of the absence of lif secretion as a nidation hormone from the uterus . lif - related cytokines , including lif , oncostatin m ( osm ) , cardiotrophin ( ct-1 ) and ciliary neurotrophic factor ( cntf ) , function within a gp130 receptor complex and can sustain esc self - renewal . furthermore , escs can be maintained by using a combination of interleukin-6 and soluble interleukin-6 receptor ( il-6/sil-6r ) and their intracellular signaling is engaged via formation of gp130 homodimers even without lifr . therefore , intracellular signals from gp130 are sufficient for self - renewal of mouse escs . in addition to esc self - renewal , activation of gp130 receptor complexes causes differentiation and growth inhibition in m1 myeloid leukemic cells , survival and differentiation of neurons , astrocytes and hypertrophy in cardiomyocytes . lifr mice showed decrease in bone volume ( osteopenia ) , reduction in the numbers of motor neurons and astrocytes , abnormality of placenta and metabolic liver diseases . gp130 mice die between 12 to 16 d of embryogenesis , due to hematopoietic , neuronal and heart disorders . these findings suggest that lifr and gp130 are critical receptors for early embryogenesis and organogenesis . ligand - induced dimerization of the receptors leads to phosphorylation at tyrosine 1022 ( y1022 ) of jak1 and activation of associated jak1 . four tyrosine residues ( y765/812/904/914 ) of the cytoplasmic domain of gp130 and three tyrosine residues ( y976/996/1023 ) of lifr are phosphorylated by the activated jaks . jak then phosphorylates stat3 at tyrosine 705 ( y705 ) , leading to homodimerization of stat3 via its sh2 domain and its translocation to the nucleus to transcribe target genes . homodimerized stat3 is imported into the nucleus through interaction with importin-3 and importin-6 and binds to the consensus sequence ttccsgggaa ( s = c or g ) at the promoter or enhancer regions of target genes . a previous study showed that dominant interfering mutants of stat3 inhibit macrophage differentiation of myeloid m1 cells after stimulation with lif . furthermore , studies using knockout mice have shown that homologous disruption of the stat3 gene causes early embryonic lethality , while escs in which both stat1 genes have been deleted are phenotypically normal . stat5a and stat5b knockout mice fail to response to prolactin and growth hormone , respectively . however , stat5a / b double knockout mice develop a full complement of hematopoietic lineages and display subtler defects in embryonic hematopoietic development . these findings suggest that stat3 is important for maintenance of esc proliferation and pluripotency , whereas stat1 and stat5 are dispensable for maintenance of pluripotency . the jak - stat pathway is negatively regulated by several inhibitory systems : dephosphorylation by tyrosine phosphatases including sh2-containing tyrosine phosphatase ( shp ) , protein tyrosine phosphatase 1b ( ptp1b ) and protein tyrosine phosphatase basophil - like ( ptp - bl ) , inhibition by sumo-1 conjugation via protein inhibitor of activated stat ( pias ) , and physical inhibition or ubiquitin - mediated degradation by socs3 ( fig .  1 ) . as a second signal via lif , jak activates phosphoinositide 3-kinase ( pi3k ) through tyrosine - phosphorylation of the regulatory subunit p85 , which acts as an activator for akt ( fig .  1 ) . akt inhibits glycogen synthase kinase 3 ( gsk3 ) by direct phosphorylation of gsk3 at serine 9 ( s9 ) and nuclear export of gsk3 independent of phosphorylation . hence , a gsk3 inhibitor supports self - renewal of mouse escs in the absence of lif . moreover , akt upregulates tbx3 as another pluripotency gene and causes acetylaton of stat3 at lysine 686 ( k686 ) , which induces more stable homodimer formation of stat3 , probably followed by the activation of klf4 and oct3/4 . actually , a constitutively active form of akt is sufficient for self - renewal of mouse escs even without feeder cells and lif . as a third signal via lif , jak phosphorylates shp2 recruited by binding to tyrosine 757 ( y757 ) of gp130 and tyrosine 969 ( y969 ) of lifr ( fig .  1 ) . shp2 then interacts with the grb2/sos complex and activates the mapk pathway , leading to downregulation of tbx3 and nanog probably via nuclear export . therefore , the mek inhibitor pd98059 inhibits differentiation of mouse escs and promotes esc self - renewal . ubiquitination is one of the post - translational modifications used by eukaryotic cells , and the ubiquitin - mediated proteolytic pathway plays an important role in the degradation of short - lived regulatory proteins including those that contribute to cellular signaling , transcriptional regulation , cell cycle , dna repair and protein quality control . ubiquitin modification is mediated by a ubiquitin - activating enzyme ( e1 ) , ubiquitin - conjugating enzyme ( e2 ) and ubiquitin ligase ( e3 ) . e3 is thought to be the component of the ubiquitin conjugation system that is most directly responsible for substrate recognition . on the basis of structural similarity , e3 enzymes have been classified into two major families : the hect ( homologous to e6-ap cooh terminus ) family and the ring - finger protein family . members of the superfamily of tripartite motif ( trim)-containing proteins are defined as e3 ubiquitin ligases by the presence of a ring - finger domain . trim proteins are characterized by the presence of a ring - finger domain , one or two zinc - binding motifs named b - boxes , and an associated coiled - coil region and are classified in subfamilies i to xi on the basis of differences in c - terminal domains . comparative analysis has shown that vertebrates have many trim family proteins with an spry domain in the c - terminal region , whereas invertebrate species have only a small number of trim family proteins with the spry domain . so far , trim proteins have not been identified in arabidopsis thaliana , whereas proteins belonging to the u - box type e3 ligase family , another type of e3 ubiquitin family , have evolutionally been vastly amplified in a. thaliana genomes . however , there are 32 proteins with n - terminal b - boxes such as col , sto and sth1/2 in a. thaliana . trim family proteins are involved in several biological processes including cell proliferation / differentiation and diverse pathological conditions such as cancer , developmental disorders , neurodegenerative diseases , inflammation and autoimmune diseases . most of the trim proteins have roles as e3 ubiquitin ligases in the ubiquitination process , and several trim family members are involved in various biological processes , such as transcriptional regulation , cell proliferation and differentiation . the jak - stat signal is negatively regulated by several mechanisms ( fig .  1 ) . the socs family has eight proteins ( socs1~7 and cis ) , which are induced by activation via cytokines , growth factors and hormones , and they were originally identified as negative molecules that form negative feedback loops . all socs proteins have sh2 domain that is involved in binding to phosphorylated tyrosine , and a socs - box that functions as a binding motif for cullin-2 as a component of e3 ubiquitin ligase . first , the n - terminal domain of socs likely becomes a pseudosubstrate for jak to inhibit activated signals . second , socs competes with downstream signal molecules by binding to tyrosine - phosphorylated receptor proteins . third , socs proteins form e3 ubiquitin ligase complexes with cullin-2 , elongin b , elongin c and rbx1 and ubiquitinate jak and receptor molecules , followed by their degradation and internalization , respectively . socs1 mediates ubiquitination of jak2 and tel - jak2 oncogene dependent on a socs - box . socs3 induces proteasome - dependent degradation of target receptors including cd33 and sialic - binding immunoglobulin - like lectin ( siglec ) 7 . it has been reported that stat - interacting lim protein ( slim ) , which is a nuclear protein with both pdz and lim domains , functions as an e3 ubiquitin ligase for stat proteins ( fig .  1 ) . slim overexpression suppressed stat1/4-mediated transcription by degradation of stat proteins , whereas slim knockout caused increased stat expression , followed by enhancement of interferon- ( ifn- ) production from th1 cells . these findings suggest that slim is a bona fide e3 ubiquitin ligase that negatively regulates the jak - stat signal pathway . however , based on results of transcriptome analysis , slim is weakly expressed in mouse escs , indicating that slim may not be a main negative regulator for mouse escs ( personal communications ) . further detailed analysis of slim expression in mouse escs is needed to clarify the importance of jak - stat signals in escs . trim8 is highly expressed in a variety of cancers including anaplastic oligodendroglioma and its gene maps to human chromosome 10q24.3 . hence , trim8 is also called glioblastoma - expressed ring finger protein ( gerp ) . it has been reported that trim8 is induced by ifn- and acts as a socs1-interacting protein . trim8 overexpression suppresses the negative effect of pias3 on stat3 , either by degradation of pias3 via the ubiquitin - proteasome pathway or exclusion of pias3 from the nucleus . although we checked more than 30 different types of cancer cell lines , we failed to detect endogenous trim8 in those cell lines . finally , we checked escs because trim8 regulates pias3 and stat3 is a crucial regulator for the lif - dependent signaling pathway . we found that endogenous trim8 is highly expressed in mouse undifferentiated escs and that differentiated escs cultured without lif do not express endogenous trim8 , suggesting that trim8 is necessary for maintenance of pluripotency and self - renewal of escs . actually , we showed that trim8 interacts with hsp90 , which interacts with stat3 and selectively downregulates transcription of nanog in escs ( fig .  1 ) . trim8 inhibits translocation of stat3 into the nucleus through interaction with hsp90 and consequently inhibits transcription of nanog in escs , suggesting that trim8 regulates self - renewal or differentiation of escs . recently , we found that trim6 , another of the trim family ubiquitin ligases , is also highly expressed in escs and binds to c - myc and that trim6 overexpression causes decrease in c - myc - mediated transcription . the proto - oncogene product c - myc is known to be a master regulator of cell proliferation by specific binding to the e - box motif in promoter regions of target genes . it has also been reported that c - myc plays an important role in the proliferation and maintenance of pluripotency of escs and that the transcriptional activity of c - myc is regulated by several post - translational modifications including phosphorylation and ubiquitination . it has been reported that c - myc overexpression maintains the pluripotency of mouse escs even without lif , but sustained activation of c - myc induces differentiation of human escs , suggesting that c - myc has different functions in mouse and human escs . we showed that trim6 overexpression in mouse escs inhibits c - myc - mediated transcription , resulting in induction of the differentiation of escs ( fig .  1 ) . these findings suggest that trim6 controls the transcriptional activity of c - myc for maintaining self - renewal and pluripotency of escs . in addition to escs , induced pluripotent stem cells ( ipscs ) are established by the expression of four genes , oct3/4 , sox2 , klf4 and c - myc , into several cell lineages of mice and humans . however , the carcinogenic potential of c - myc inhibits its use in ipscs for clinical application . however , the efficiency of ipsc establishment without c - myc expression is very low . therefore , appropriate regulation of the expression level of c - myc at an inducing stage of stem cells should be important to establish ipscs . trim6 is expressed at higher level in undifferentiated escs than in differentiated cells , and c - myc expression level is robustly elevated in trim6-knockdown escs . once escs enter the differentiation stage , the expression of trim6 is rapidly decreased . taken together , the findings indicate that trim6 may regulate c - myc expression level within optimal ranges to maintain pluripotency of escs . it is likely that trim6 strictly modulates the expression level or activity of c - myc to prevent dysregulation of cell proliferation by c - myc in escs . recently , it has been reported that lif can be replaced by chemical compounds to inhibit several signaling pathways . the cocktail of three inhibitors , a fgf - r tyrosine kinase inhibitor ( su5402 ) , mek inhibitor ( pd184352 ) and gsk3 inhibitor ( chir99021 ) , is called 3i . if a more potent mek inhibitor ( pd325901 ) is used , the cocktail ( 2i ) including pd325901 and chir99021 is sufficient for maintaining self - renewal of mouse escs without lif ( fig .  3 ) . this recent technology has been established on the basis of results of studies on the lif - mediated signal pathway at the detailed molecular level . further studies to clarify related molecules and signal networks should lead to novel technologies for developmental engineering and tissue engineering . moreover , activation of gsk3 causes phosphorylation of c - myc followed by degradation of c - myc and also inhibits nanog expression , resulting in the differentiation of escs . lif - mediated akt activation causes phosphorylation of gsk3 , followed by inactivation of gsk3 ( a ) . without lif , mouse escs are differentiated through unphosphorylated gsk3 and mapk activated by autocrine fgf4 ( b ) . the combination ( 2i ) of a mek inhibitor ( pd184352/pd325901 ) and gsk3 inhibitor ( chir99021 ) is sufficient for maintaining self - renewal and pluripotency of mouse escs without lif ( c ) .
lif activates several intracellular signaling pathways including jak - stat , pi3k / akt and mapk pathways . 
 lif is an important cytokine for maintenance of pluripotency and self - renewal of mouse es cells . 
 the jak - stat signal plays a key role in maintenance of the pluripotency of escs . 
 recent evidence shows that several post - translational modifications regulate activation or inhibition of intracellular signal transductions . 
 the jak - stat signal is also modulated by several modifications including phosphorylation , acetylation and ubiquitination . in this review , 
 we discuss regulation of the lif - mediated - jak - stat signaling pathway that contributes to self - renewal of pluripotent escs .