Neuropoietin activates STAT3 independent of LIFR activation in adipocytes

Neuropoietin (NP) is a member of the gp130 cytokine family that is closely related to cardiotrophin-1(CT-1) and shares functional and structural features with other family members, including ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC). Studies have shown that NP can play a role in the development of the nervous system, as well as affect adipogenesis and fat cell function. However, the signaling mechanisms utilized by NP in adipocytes have not been examined. In our present studies, we demonstrate that NP-induced activation of STAT3 tyrosine phosphorylation is independent of leukemia inhibitory factor receptor (LIFR) phosphorylation and degradation. Although it is widely accepted that NP signals via the LIFR, our studies reveal that NP results in phosphorylation of gp130, but not LIFR. These observations suggest that the profound effects that NP has on adipocytes are not mediated via LIFR signaling.     

Neuropoietin attenuates adipogenesis and induces insulin resistance in adipocytes

Recent findings have implicated gp130 receptor ligands, particularly ciliary neurotrophic factor (CNTF), as potential anti-obesity therapeutics. Neuropoietin (NP) is a recently discovered cytokine in the gp130 family that shares functional and structural features with CNTF and signals via the CNTF receptor tripartite complex comprised of CNTFRalpha, LIF receptor, and gp130. NP plays a role in the development of the nervous system, but the effects of NP on adipocytes have not been previously examined. Because CNTF exerts anti-obesogenic effects in adipocytes and NP shares the same receptor complex, we investigated the effects of NP on adipocyte development and insulin action. Using cultured 3T3-L1 adipocytes, we observed that NP has the ability to block adipogenesis in a dose- and time-dependent manner. We also observed that cultured adipocytes, as well as murine adipose tissue, are highly responsive to acute NP treatment. Rodents injected with NP had a substantial increase in STAT3 tyrosine phosphorylation and ERK 1 and 2 activation. We also observed the induction of SOCS-3 mRNA in 3T3-L1 adipocytes following NP treatment. Unlike CNTF, our studies have revealed that NP also substantially attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes. In addition, NP blocks insulin action in adipose tissue in vivo. These observations are supported by data demonstrating that NP impairs insulin signaling via decreased activation of both IRS-1 and Akt. In summary, we have observed that both adipocytes in vitro and in vivo are highly responsive to NP, and this cytokine has the ability to affect insulin signaling in fat cells. These novel observations suggest that NP, unlike CNTF, may not be a viable obesity therapeutic.     

The critical role of Shc in insulin-like growth factor-I-mediated mitogenesis and differentiation in 3T3-L1 preadipocytes

Insulin-like growth factor-I (IGF-I) stimulates mitogenesis in proliferating preadipocytes, but when cells reach confluence and become growth arrested, IGF-I stimulates differentiation into adipocytes. IGF-I induces signaling pathways that involve IGF-I receptor-mediated tyrosine phosphorylation of Shc and insulin receptor substrate 1 (IRS-1). Either of these adaptor proteins can lead to activation of the three-kinase cascade ending in activation of the extracellular signal-regulated kinase 1 and -2 (ERK-1 and -2) mitogen-activated protein kinases (MAPKs). Several lines of evidence suggest that activation of MAPK inhibits 3T3-L1 preadipocyte differentiation. We have shown that IGF-I stimulation of MAPK activity is lost as 3T3-L1 preadipocytes begin to differentiate. This change in MAPK signaling coincides with loss of IGF-I-mediated Shc, but not IRS-1, tyrosine phosphorylation. We hypothesized that down-regulation of MAPK via loss of proximal signaling through Shc is an early component in the IGF-I switch from mitogenesis to differentiation in 3T3-L1 preadipocytes. Treatment of subconfluent cells with the MEK inhibitor PD098059 inhibited both IGF-I-activation of MAPK as well as 3H-thymidine incorporation. PD098059, in the presence of differentiation-inducing media, accelerated differentiation in subconfluent cells as measured by expression of adipocyte protein-2 (aP-2), peroxisome proliferator-activated receptor gamma (PPARgamma) and lipoprotein lipase (LPL). Transient transfection of subconfluent cells with Shc-Y317F, a dominant-negative mutant, attenuated IGF-I-mediated MAPK activation, inhibited DNA synthesis, and accelerated expression of differentiation markers aP-2, PPARgamma, and LPL. We conclude that signaling through Shc to MAPK plays a critical role in mediating IGF-I-stimulated 3T3-L1 mitogenesis. Our results suggest that loss of the ability of IGF-I to activate Shc signaling to MAPK may be an early component of adipogenesis in 3T3-L1 cells.     

Desensitization of signaling by oncostatin M in human vascular cells involves cytoplasmic Tyr residue 759 in gp130 but is not mediated by either Src homology 2 domain-containing tyrosine phosphatase 2 or suppressor of cytokine signaling 3

Oncostatin M (OnM) signals through cell surface receptors, which utilize the gp130 subunit. In cultured human umbilical vein endothelial cells (HUVEC), OnM transiently elevates mRNA encoding for suppressor of cytokine signaling-3 (SOCS-3). By 1 h of OnM treatment, HUVEC become refractory to the restimulation by OnM, measured as failure to reinduce SOCS-3 mRNA. OnM-induced desensitization also prevents responses to other gp130-signaling cytokines (e.g. leukemia inhibitory factor and interleukin 11). OnM treatment does not affect gp130 expression levels and desensitizes signaling mediated by a transduced chimeric receptor containing extracellular domains of platelet-derived growth factor receptor-beta (PDGFRbeta) and the cytoplasmic region of gp130. Interestingly, a chimeric PDGFRbeta-gp130 mutant receptor, in which intracellular Tyr residue 759 of gp130 is replaced by a Phe residue, mediates prolonged signaling and is not cross-desensitized by OnM. Phospho-Tyr759 is the binding site for both SOCS-3 and for Src homology domain 2-containing tyrosine phosphatase 2 (SHP-2). In human aortic smooth muscle cells, neither prevention of SOCS-3 protein induction, using STAT3 or SOCS-3 antisense, nor prevention of SHP-2 expression, also with antisense, ablates desensitization. These data suggest that desensitization of vascular cells to OnM is mediated in trans and involves Tyr residue 759 in gp130 but is not mediated by either SOCS-3 or SHP-2, the only two proteins currently known to bind to gp130 at this site.     

Activation of SOCS-3 by resistin

Resistin is an adipocyte hormone that modulates glucose homeostasis. Here we show that in 3T3-L1 adipocytes, resistin attenuates multiple effects of insulin, including insulin receptor (IR) phosphorylation, IR substrate 1 (IRS-1) phosphorylation, phosphatidylinositol-3-kinase (PI3K) activation, phosphatidylinositol triphosphate production, and activation of protein kinase B/Akt. Remarkably, resistin treatment markedly induces the gene expression of suppressor of cytokine signaling 3 (SOCS-3), a known inhibitor of insulin signaling. The 50% effective dose for resistin induction of SOCS-3 is approximately 20 ng/ml, close to levels of resistin in serum. Association of SOCS-3 protein with the IR is also increased by resistin. Inhibition of SOCS function prevented resistin from antagonizing insulin action in adipocytes. SOCS-3 induction is the first cellular effect of resistin that is independent of insulin and is a likely mediator of resistin's inhibitory effect on insulin signaling in adipocytes.     

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.     

Exchange protein activated by cyclic AMP (Epac)-mediated induction of suppressor of cytokine signaling 3 (SOCS-3) in vascular endothelial cells

Here, we demonstrate that elevation of intracellular cyclic AMP (cAMP) in vascular endothelial cells (ECs) by either a direct activator of adenylyl cyclase or endogenous cAMP-mobilizing G protein-coupled receptors inhibited the tyrosine phosphorylation of STAT proteins by an interleukin 6 (IL-6) receptor trans-signaling complex (soluble IL-6Ralpha/IL-6). This was associated with the induction of suppressor of cytokine signaling 3 (SOCS-3), a bona fide inhibitor in vivo of gp130, the signal-transducing component of the IL-6 receptor complex. Attenuation of SOCS-3 induction in either ECs or SOCS-3-null murine embryonic fibroblasts abolished the inhibitory effect of cAMP, whereas inhibition of SHP-2, another negative regulator of gp130, was without effect. Interestingly, the inhibition of STAT phosphorylation and SOCS-3 induction did not require cAMP-dependent protein kinase activity but could be recapitulated upon selective activation of the alternative cAMP sensor Epac, a guanine nucleotide exchange factor for Rap1. Consistent with this hypothesis, small interfering RNA-mediated knockdown of Epac1 was sufficient to attenuate both cAMP-mediated SOCS-3 induction and inhibition of STAT phosphorylation, suggesting that Epac activation is both necessary and sufficient to observe these effects. Together, these data argue for the existence of a novel cAMP/Epac/Rap1/SOCS-3 pathway for limiting IL-6 receptor signaling in ECs and illuminate a new mechanism by which cAMP may mediate its potent anti-inflammatory effects.     

A functional role of the membrane-proximal extracellular domains of the signal transducer gp130 in heterodimerization with the leukemia inhibitory factor receptor.

gp130 is the common signal transducing receptor subunit of interleukin (IL)-6-type cytokines. gp130 either homodimerizes in response to IL-6 and IL-11 or forms heterodimers with the leukemia inhibitory factor (LIF) receptor (LIFR) in response to LIF, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) or cardiotrophin-like cytokine resulting in the onset of cytoplasmic tyrosine phosphorylation cascades. The extracellular parts of both gp130 and LIFR consist of several Ig-like and fibronectin type III-like domains. The role of the membrane-distal domains of gp130 (D1, D2, D3) and LIFR in ligand binding is well established. In this study we investigated the functional significance of the membrane-proximal domains of gp130 (D4, D5, D6) in respect to heterodimerization with LIFR. Deletion of each of the membrane-proximal domains of gp130 (Delta 4, Delta 5 and Delta 6) leads to LIF unresponsiveness. Replacement of the gp130 domains by the corresponding domains of the related GCSF receptor either restores weak LIF responsiveness (D4-GCSFR), leads to constitutive activation of gp130 (D5-GCSFR) or results in an inactive receptor (D6-GCSFR). Mutation of a specific cysteine in D5 of gp130 (C458A) leads to constitutive heterodimerization with the LIFR and increased sensitivity towards LIF stimulation. Based on these findings, a functional model of the gp130-LIFR heterodimer is proposed that includes contacts between D5 of gp130 and the corresponding domain D7 of the LIFR and highlights the requirement for both receptor dimerization and adequate receptor orientation as a prerequisite for signal transduction.     

Signaling pathways recruited by the cardiotrophin-like cytokine/cytokine-like factor-1 composite cytokine: specific requirement of the membrane-bound form of ciliary neurotrophic factor receptor alpha component

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of a number of neural cell types. Its receptor complex consists of a ligand-binding component, CNTF receptor (CNTFR), associated with two signaling receptor components, gp130 and leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second developmentally important ligand. We recently demonstrated that cardiotrophin-like cytokine (CLC) associates with the soluble orphan receptor cytokine-like factor-1 (CLF) to form a heterodimeric cytokine that displayed activities only on cells expressing the tripartite CNTF receptor on their surface. In this present study we examined the membrane binding of the CLC/CLF composite cytokine and observed a preferential interaction of the cytokine with the CNTFR subunit. Signaling pathways recruited by the CLC/CLF complex in human neuroblastoma cell lines were also analyzed in detail. The results obtained showed an activation of Janus kinases (JAK1, JAK2, and TYK2) leading to a tyrosine phosphorylation of the gp130 and LIFR. The phosphorylated signaling receptors served in turn as docking proteins for signal transducing molecules such as STAT3 and SHP-2. In vitro analysis revealed that the gp130-LIFR pathway could also stimulate the phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathways. In contrast to that reported before for CNTF, soluble CNTFR failed to promote the action CLC/CLF, and an absolute requirement of the membrane form of CNTFR was required to generate a functional response to the composite cytokine. This study reinforces the functional similarity between CNTF and the CLC/CLF composite cytokine defining the second ligand for CNTFR.