STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells.

Embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). LIF acts through a receptor complex composed of a low affinity LIF receptor (LIFRbeta) and gp130. We reported that the intracellular domain of gp130 plays an important role in self-renewal of ES cells. In the present study, we examined the signaling pathway through which gp130 contributes to the self-renewal of ES cells. Mutational analysis of the cytoplasmic domain of gp130 revealed that the tyrosine residue of gp130 responsible for STAT3 activation is necessary for self-renewal of ES cells, while that required for SHP2 and MAP kinase activation was dispensable. Next, we constructed a fusion protein composed of the entire coding region of STAT3 and the ligand binding domain of the estrogen receptor. This construction (STAT3ER) induced expression of junB (one of the targets of STAT3) in ES cells in the presence of the synthetic ligand 4-hydroxytamoxifen (4HT), thereby indicating that STAT3ER is a conditionally active form. ES cells transfected with STAT3ER cultured in the presence of 4HT maintained an undifferentiated state. Taken together, these results strongly suggest that STAT3 activation is required and sufficient to maintain the undifferentiated state of ES cells.     

Differential regulation of leukemia inhibitory factor-stimulated neuronal gene expression by protein phosphatases SHP-1 and SHP-2 through mitogen-activated protein kinase-dependent and -independent pathways

The neurally active cytokine leukemia inhibitory factor (LIF) signals through a bipartite receptor complex composed of LIF receptor alpha (LIFR) and gp130. gp130 and LIFR contain consensus binding motifs for the protein tyrosine phosphatase SHP-2 surrounding tyrosines 118 and 115 (Y118 and Y115) of their cytoplasmic domains, respectively. These sites are necessary for maximal activation of mitogen-activated protein kinase (MAPK). Coexpression of catalytically inactive, but not wild-type, SHP-2 reduced LIFR- and gp130-mediated activation of MAPK up to 75%. Conversely, coexpression of the wild-type, but not catalytically inactive, SHP-1, a related phosphatase, reduced activity up to 80%, demonstrating that SHP-2 and SHP-1 have opposing effects on the MAPK pathway. Mutation of Y115 of the cytoplasmic domain of LIFR eliminates receptor-mediated tyrosine phosphorylation of SHP-2. In contrast, SHP-1 association with gp130 and LIFR is constitutive and independent of Y118 and Y115, respectively. SHP-1 has a positive regulatory role on LIF-stimulated vasoactive intestinal peptide (VIP) reporter gene expression in neuronal cells, whereas the effect of SHP-2 is negative. Furthermore, LIF-stimulated MAPK activation negatively regulates this VIP reporter gene induction. SHP-2 also negatively regulates LIF-dependent expression of choline acetyltransferase, but this regulation could be dissociated from its effects on MAPK activation. These data indicate that SHP-1 and SHP-2 are important regulators of LIF-dependent neuronal gene expression via both MAPK-dependent and -independent pathways.     

白血病抑制因子与胚胎干细胞

白血病抑制因子对细胞的生长和分化有多种作用,通过与其受体结合传导信号,gp130与LIF受体β链的结合激活JAK激酶(JAK1和JAK2),JAK激酶磷酸化STAT信号转录子,STAT3的磷酸化对于阻止体外培养的干细胞的分化具有十分重要的作用。     

The Pleiotropic role,functions and targeted therapies of LIF/LIFR axis in cancer: Old spectacles with new insights

The dysregulation of leukemia inhibitory factor (LIF) and its cognate receptor (LIFR) has been associated with multiple cancer initiation, progression, and metastasis. LIF plays a significant tumor-promoting role in cancer, while LIFR functions as a tumor promoter and suppressor. Epithelial and stromal cells secrete LIF via autocrine and paracrine signaling mechanism(s) that bind with LIFR and subsequently with co-receptor glycoprotein 130 (gp130) to activate JAK/STAT1/3, PI3K/AKT, mTORC1/p70s6K, Hippo/YAP, and MAPK signaling pathways. Clinically, activating the LIF/LIFR axis is associated with poor survival and anti-cancer therapy resistance. This review article provides an overview of the structure and ligands of LIFR, LIF/LIFR signaling in developmental biology, stem cells, cancer stem cells, genetics and epigenetics of LIFR, LIFR regulation by long non-coding RNAs and miRNAs, and LIF/LIFR signaling in cancers. Finally, neutralizing antibodies and small molecule inhibitors preferentially blocking LIF interaction with LIFR and antagonists against LIFR under pre-clinical and early-phase pre-clinical trials were discussed.     

Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.     

Solution structure of the C-terminal domain of the ciliary neurotrophic factor (CNTF) receptor and ligand free associations among components of the CNTF receptor complex

The functional receptor complex of ciliary neurotrophic factor (CNTF), a member of the gp130 family of cytokines, is composed of CNTF, the CNTF receptor alpha (CNTFR), gp130, and the leukemia inhibitory factor receptor (LIFR). However, the nature of the receptor-mediated interactions in this complex has not yet been resolved. To address this issue we have determined the solution structure of the C-terminal or BC domain of CNTFR and studied the interactions of CNTFR with LIFR and gp130. We reported previously that the membrane distal cytokine-binding domain (CBD1) of LIFR could interact in vitro with soluble CNTFR (sCNTFR) in the absence of CNTF. Here we show that the CBD of human gp130 can also bind in vitro to sCNTFR in the absence of CNTF. In addition, the gp130 CBD could compete with the LIFR CBD1 for the binding of sCNTFR. Substitution of residues in the gp130 CBD, the LIFR CBD1, and the CNTFR BC domain that are expected to be involved in receptor-receptor interactions significantly reduced their interactions. An NMR chemical shift perturbation study of the interaction between the BC domains of CNTFR and gp130 further mapped the interaction surface. These data suggest that both the gp130 CBD and the LIFR CBD1 interact with CNTFR in a similar way and provide insights into the nature of the CNTF receptor complex.     

SHP2 and SOCS3 contribute to Tyr-759-dependent attenuation of interleukin-6 signaling through gp130

Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kinase cascade. Tyrosine 759 of the IL-6 signal-transducing receptor subunit gp130 has been identified as being involved in negative regulation of IL-6-induced gene induction and activation of the Jak/STAT pathway. Because this site is known to be a recruitment motif for the protein-tyrosine phosphatase SHP2, it has been suggested that SHP2 is the mediator of tyrosine 759-dependent signal attenuation. We recently observed that the suppressor of cytokine-signaling SOCS3 also acts through the tyrosine motif 759 of gp130. However, the relative contributions of SHP2 and SOCS3 to the repression of IL-6 signaling are not understood. Therefore, we designed experiments allowing the independent recruitment of each of these proteins to the IL-6-receptor complex. We show that receptor- and membrane-targeted SHP2 counteracts IL-6 signaling independent of SOCS3 binding to gp130. On the other hand, SOCS3 inhibits signaling in cells expressing a truncated SHP2 protein, which is not recruited to gp130. These data suggest, that there are two, largely distinct modes of negative regulation of gp130 activity, despite the fact that both SOCS3 and SHP2 are recruited to the same site within gp130.     

Regulation of neurokine receptor signaling and trafficking

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are neurally active cytokines, or neurokines. LIF signals through a receptor consisting of gp130 and the low affinity LIF receptor (LIFR), while the CNTF receptor consists of gp130, LIFR, and the low affinity CNTF receptor (CNTFR). Ser1044 of the LIFR is phosphorylated by Erk1/2 MAP kinase. Stimulation of neural cells with growth factors which strongly activate Erk1/2 decreases LIF-mediated signal transduction due to increased degradation of the LIFR as a consequence of Erk1/2-dependent phosphorylation of the receptor at Ser1044.     

Gene expression of receptors for IL-6, LIF, and CNTF in regenerating skeletal muscles.

The biological actions of interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF) are mediated via respective functional receptor complexes consisting of a common signal-transducing component, gp130, and other specific receptor components, IL-6 receptor alpha (IL-6R), LIF receptor beta (LIFR), and CNTF receptor alpha (CNTFR). IL-6, LIF, and CNTF are implicated in skeletal muscle regeneration. However, the cell populations that express these receptor components in regenerating muscles are unknown. Using in situ hybridization histochemistry, we examined spatiotemporal expression patterns of gp130, IL-6R, LIFR, and CNTFR mRNAs in regenerating muscles after muscle contusion. At the early stages of regeneration (from 3 hr to Day 2 post contusion), significant signals for gp130 and LIFR mRNAs were detected in myonuclei and/or nuclei of muscle precursor cells (mpcs) and in mononuclear cells located in extracellular spaces between myofibers after muscle contusion, but IL-6R mRNA was expressed only in mononuclear cells. At Day 7 post contusion, signals for gp130, LIFR, and IL-6R mRNAs were not detected in newly formed myotubes, whereas the CNTFR mRNA level was upregulated in myotubes. These findings suggest that the upregulation of receptor subunits in distinct cell populations plays an important role in the effective regeneration of both myofibers and motor neurons. (J Histochem Cytochem 48:1203-1213, 2000)     

Differences between human and mouse embryonic stem cells

We compared gene expression profiles of mouse and human ES cells by immunocytochemistry, RT-PCR, and membrane-based focused cDNA array analysis. Several markers that in concert could distinguish undifferentiated ES cells from their differentiated progeny were identified. These included known markers such as SSEA antigens, OCT3/4, SOX-2, REX-1 and TERT, as well as additional markers such as UTF-1, TRF1, TRF2, connexin43, and connexin45, FGFR-4, ABCG-2, and Glut-1. A set of negative markers that confirm the absence of differentiation was also developed. These include genes characteristic of trophoectoderm, markers of germ layers, and of more specialized progenitor cells. While the expression of many of the markers was similar in mouse and human cells, significant differences were found in the expression of vimentin, beta-III tubulin, alpha-fetoprotein, eomesodermin, HEB, ARNT, and FoxD3 as well as in the expression of the LIF receptor complex LIFR/IL6ST (gp130). Profound differences in cell cycle regulation, control of apoptosis, and cytokine expression were uncovered using focused microarrays. The profile of gene expression observed in H1 cells was similar to that of two other human ES cell lines tested (line I-6 and clonal line-H9.2) and to feeder-free subclones of H1, H7, and H9, indicating that the observed differences between human and mouse ES cells were species-specific rather than arising from differences in culture conditions.     

Coexpression of oncostatin M and its receptors and evidence for STAT3 activation in human ovarian carcinomas

The expression of oncostatin M and leukemia inhibitory factor (LIF), JAK-STAT activators and members of the interleukin-6 family of cytokines, were examined in a series of primary ovarian carcinomas using immunohistochemistry. The malignant epithelial cells of all 29 ovarian carcinomas examined expressed oncostatin M; none expressed LIF. Oncostatin M can activate two related receptors, one consisting of a low-affinity LIF receptor subunit, LIFR beta, which forms a heterocomplex with the gp130 signal transducing protein and can recognize both oncostatin M and LIF, and a second heterocomplex consisting of a subunit that specifically recognizes oncostatin M, OSMR beta, and the gp130 protein. By immunohistochemistry, 25 of 25 ovarian carcinomas examined expressed the LIFR beta subunit in the malignant epithelial cells (all samples express gp130), and two-thirds the ovarian carcinomas studied expressed OSMR beta mRNA as determined by RT-PCR. Thus oncostatin M and its receptors are commonly coexpressed in malignant ovarian epithelial cells, and represent a potential autocrine loop in this tumor type. STAT3, of one the signaling proteins downstream of the oncostatin M/LIF receptors, was found in its phosphorylated, activated form (phosphotyrosine 705 STAT3) in the malignant epithelial cells of 17 of 23 ovarian carcinomas examined (74%) as determined by immunohistochemistry; this suggests that this protein is constitutively activated in most ovarian carcinomas, as it is in many other human malignancies. Recombinant human Oncostatin M (rhOSM) can induce the transient tyrosine 705 phosphorylation of STAT3 in serum-starved LIFR beta/OSMR beta expressing ovarian carcinoma cell lines, but does not alter cell growth and effects only a modest increase in the apoptotic rate in these cultured cells. Oncostatin M and its receptors may be part of a network of cytokine systems within ovarian carcinomas that may act to maintain STAT3 in its activated form, a phenomenon associated with the malignant phenotype.