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.     

Retinoids arrest breast cancer cell proliferation: retinoic acid selectively reduces the duration of receptor tyrosine kinase signaling

Retinoic acid (RA) induces cell cycle arrest of hormone-dependent human breast cancer (HBC) cells. Previously, we demonstrated that RA-induced growth arrest of T-47D HBC cells required the activity of the RA-induced protein kinase, protein kinase Calpha (PKCalpha) [J. Cell Physiol. 172 (1997) 306]. Here, we demonstrate that RA treatment of T-47D cells interfered with growth factor signaling to downstream, cytoplasmic and nuclear targets. RA treatment did not inhibit epidermal growth factor (EGF) receptor activation but resulted in rapid inactivation. The lack of sustained EGFR activation was associated with transient rather than sustained association of the EGFR with the Shc adaptor proteins and activation of Erk 1/2 and with compromised induction of expression of immediate early response genes. Inhibiting the activity of PKCalpha, a retinoic acid-induced target gene, prevented the effects of RA on cell proliferation and EGF signaling. Constitutive expression of PKCalpha, in the absence of RA, decreased cell proliferation and decreased EGF signaling. RA treatment increased steady-state levels of the protein tyrosine phosphatase PTP-1C and all measured effects of RA on EGF receptor function were reversed by the tyrosine phosphate inhibitor orthovanadate. These results indicate that RA-induced target genes, particularly PKCalpha, prevent sustained growth factor signaling, uncoupling activated receptor tyrosine kinases and nuclear targets that are required for cell cycle progression.     

SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation

Suppressor of cytokine signaling 3 (SOCS3) binds cytokine receptors and thereby suppresses cytokine signaling. Deletion of SOCS3 causes an embryonic lethality that is rescued by a tetraploid rescue approach, demonstrating an essential role in placental development and a non-essential role in embryo development. Rescued SOCS3-deficient mice show a perinatal lethality with cardiac hypertrophy. SOCS3-deficient placentas have reduced spongiotrophoblasts and increased trophoblast secondary giant cells. Enforced expression of SOCS3 in a trophoblast stem cell line (Rcho-1) suppresses giant cell differentiation. Conversely, SOCS3-deficient trophoblast stem cells differentiate more readily to giant cells in culture, demonstrating that SOCS3 negatively regulates trophoblast giant cell differentiation. Leukemia inhibitory factor (LIF) promotes giant cell differentiation in vitro, and LIF receptor (LIFR) deficiency results in loss of giant cell differentiation in vivo. Finally, LIFR deficiency rescues the SOCS3-deficient placental defect and embryonic lethality. The results establish SOCS3 as an essential regulator of LIFR signaling in trophoblast differentiation.     

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。     

A MAPK-positive feedback mechanism for BLR1 signaling propels retinoic acid-triggered differentiation and cell cycle arrest

MAPK signaling is required for retinoic acid (RA)-triggered G(0) cell cycle arrest and cell differentiation, but the mechanism is not well defined. In this study, RA is found to cause MAPK activation with sustained association of RAF to MEK or ERK, leading to a MAPK-dependent accumulation of p21(Waf1/Cip1) and binding to CDK2 blocking G(1)/S transition. BLR1, a chemokine receptor, was found to function as a critical component of RA-triggered MAPK signaling. Unlike wild-type parental cells, RA-treated BLR1 knock-out cells failed to show RAF and consequential MEK and ERK phosphorylation, failed to accumulate CDK inhibitors that control G(1)/S transition, and failed to differentiate and arrest in response to RA, whereas ectopically overexpressing BLR1 enhanced MAPK signaling and caused accelerated RA-induced differentiation and arrest. Ectopic overexpression of RAF enhanced BLR1 expression in response to RA, whereas inhibition of RAF or MEK by inhibitors or knockdown of RAF by short interfering RNA diminished RA-induced BLR1 expression and attenuated differentiation and growth arrest. Ectopic expression of the RAF CR3, the catalytically active domain, in the BLR1 knock-out restored RA-induced MAPK activation and the ability to differentiate and arrest, indicating that RAF effects MAPK signaling by BLR1 to propel differentiation/arrest. Taken together, RA induces cell differentiation and growth arrest through activation of a novel MAPK pathway with BLR1 as a critical component in a positive feedback mechanism that may contribute to the prolonged MAPK signaling propelling RA-induced cell cycle arrest and differentiation.     

Retinoic acid induces expression of SLP-76: expression with c-FMS enhances ERK activation and retinoic acid-induced differentiation/G0 arrest of HL-60 cells

Retinoic acid (RA) is known to cause MAPK signaling which propels G0 arrest and myeloid differentiation of HL-60 human myeloblastic leukemia cells. The present studies show that RA up-regulated expression of SLP-76 (Src-homology 2 domain-containing leukocyte-specific phospho-protein of 76 kDa), which became a prominent tyrosine-phosphorylated protein in RA-treated cells. SLP-76 is a known adaptor molecule associated with T-cell receptor and MAPK signaling. To characterize functional effects of SLP-76 expression in RA-induced differentiation and G0 arrest, HL-60 cells were stably transfected with SLP-76. Expression of SLP-76 had no discernable effect on RA-induced ERK activation, subsequent functional differentiation, or the rate of RA-induced G0 arrest. To determine the effects of SLP-76 in the presence of a RA-regulated receptor, SLP-76 was stably transfected into HL-60 cells already overexpressing the colony stimulating factor-1 (CSF-1) receptor, c-FMS, from a previous stable transfection. SLP-76 now enhanced RA-induced ERK activation, compared to parental c-FMS transfectants. It also enhanced RA-induced differentiation, evidenced by enhanced paxillin expression, inducible oxidative metabolism and superoxide production. RA-induced RB tumor suppressor protein hypophosphorylation was also enhanced, as was RA-induced G0 cell cycle arrest. A triple Y to F mutant SLP-76 known to be a dominant negative in T-cell receptor signaling failed to enhance RA-induced paxillin expression, but enhanced RA-induced ERK activation, differentiation and G0 arrest essentially as well as wild-type SLP-76. Thus, SLP-76 overexpression in the presence of c-FMS, a RA-induced receptor, had the effect of enhancing RA-induced cell differentiation. This is the first indication to our knowledge that RA induces the expression of an adapter molecule to facilitate induced differentiation via co-operation between c-FMS and SLP-76.     

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.     

Convergent Regulation of Neuronal Differentiation and Erk and Akt Kinases in Human Neural Progenitor Cells by Lysophosphatidic Acid,Sphingosine 1-Phosphate,and LIF: Specific Roles for the LPA1 Receptor

The bioactive lysophospholipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have diverse effects on the developing nervous system and neural progenitors, but the molecular basis for their pleiotropic effects is poorly understood. We previously defined LPA and S1P signaling in proliferating human neural progenitor (hNP) cells, and the current study investigates their role in neuronal differentiation of these cells. Differentiation in the presence of LPA or S1P significantly enhanced cell survival and decreased expression of neuronal markers. Further, the LPA receptor antagonist Ki16425 fully blocked the effects of LPA, and differentiation in the presence of Ki16425 dramatically enhanced neurite length. LPA and S1P robustly activated Erk, but surprisingly both strongly suppressed Akt activation. Ki16425 and pertussis toxin blocked LPA activation of Erk but not LPA inhibition of Akt, suggesting distinct receptor and G-protein subtypes mediate these effects. Finally, we explored cross talk between lysophospholipid signaling and the cytokine leukemia inhibitory factor (LIF). LPA/S1P effects on neuronal differentiation were amplified in the presence of LIF. Similarly, the ability of LPA/S1P to regulate Erk and Akt was impacted by the presence of LIF; LIF enhanced the inhibitory effect of LPA/S1P on Akt phosphorylation, while LIF blunted the activation of Erk by LPA/S1P. Taken together, our results suggest that LPA and S1P enhance survival and inhibit neuronal differentiation of hNP cells, and LPA1 is critical for the effect of LPA. The pleiotropic effects of LPA may reflect differences in receptor subtype expression or cross talk with LIF receptor signaling.     

All-trans-retinoid acid induces the differentiation of P19 cells into neurons involved in the PI3K/Akt/GSK3β signaling pathway

The pluripotent mouse embryonal carcinoma cell line P19 is widely used as a model for research on all-trans-retinoid acid (RA)-induced neuronal differentiation; however, the signaling pathways involved in this process remain unclear. This study aimed to reveal the molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells. Real-time quantitative polymerase chain reaction and Western blot analysis were used to determine the expression of neuronal-specific markers, whereas flow cytometry was used to analyze cell cycle and cell apoptosis. The expression profiles of messenger RNAs (mRNAs) in RA-induced neuronal differentiation of P19 cells were analyzed using high-throughput sequencing, and the functions of differentially expressed mRNAs (DEMs) were determined by bioinformatics analysis. RA induced an increase in both class III β-tubulin (TUBB3) and neurofilament medium (NEFM) mRNA expression, indicating that RA successfully induces neuronal differentiation of P19 cells. Cell apoptosis was not affected; however, cell proliferation decreased. We found 4117 DEMs, which were enriched in the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, Wnt signaling pathway, and cell cycle. Particularly, a few DEMs could be identified in the PI3K/Akt signaling pathway networks, such as PI3K, Akt, glycogen synthase kinase-3β (GSK3β), cyclin-dependent kinase 4 (CDK4), P21, and Bax. RA significantly increased the protein expression of PI3K, Akt, phosphorylated Akt, GSK3β, phosphorylated GSK3β, CDK4, and P21, but it reduced Bax protein expression. The Akt inhibitor affected the increase of TUBB3 and NEFM mRNA expression in RA-induced P19 cells. The molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells is potentially involved in the PI3K/Akt/GSK3β signaling pathway. The decreased cell proliferation ability of neuronally differentiated P19 cells could be associated with the expression of cell cycle proteins.     

Exogenous Nanog alleviates but is insufficient to reverse embryonic stem cells differentiation induced by PI3K signaling inhibition

PI3K signaling pathway plays a significant role in embryonic stem cells (ES cells) self‐renewal. Overexpression of Nanog maintains mouse ES cells pluripotency independent of leukemia inhibitory factor (LIF). However, little is known about the effect of PI3K signaling pathway on ES cells with Nanog overexpression. Our experiments aimed to explore the relationship between PI3K signaling pathway and Nanog expression in ES cells. We observed the effect of LY294002, a specific inhibitor of PI3K pathway, on wild‐type J1 cells and Nanog overexpressing (Ex‐Nanog) J1 cells in the presence or absence of LIF. With LY294002 treatment, both of them lost their ES features even in the presence of LIF. But the differentiation induced by LY294002 on Ex‐Nanog J1 cells was slighter lower than that on wild‐type J1 cells. These results indicate that inhibition of PI3K pathway induces mouse ES cells differentiation. Exogenous Nanog sustains mouse ES cells pluripotency independent of LIF, and alleviates the differentiation induced by LY294002. But it is insufficient to totally reverse the differentiation. J. Cell. Biochem. 106: 1041–1047, 2009. © 2009 Wiley‐Liss, Inc.