Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044.

Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission.

The high-affinity interleukin 2 (IL-2) receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two members of the Janus kinase family, Jak1 and Jak3, are associated with IL-2R beta c and IL-2R gamma c, respectively, and they are activated upon IL-2 stimulation. The cytokine-mediated Jak kinase activation usually results in the activation of a family of latent transcription factors termed Stat (signal transducer and activator of transcription) proteins. Recently, the IL-2-induced Stat protein was purified from human lymphocytes and found to be the homologue of sheep Stat5/mammary gland factor. We demonstrate that the human Stat5 is activated by IL-2 and that Jak3 is required for the efficient activation. The cytoplasmic region of the IL-2R beta c chain required for activation of Stat5 is mapped within the carboxyl-terminal 147 amino acids. On the other hand, this region is not essential for IL-2-induced cell proliferation.

Reduced surface expression of transforming growth factor beta receptor type II in mitogen-activated T cells from Sézary patients.

Sézary syndrome (SzS), the leukemic form of cutaneous T-cell lymphoma, is characterized by clonal proliferation of CD4+ T cells and immune dysfunctions, raising the possibility of cytokine-related abnormalities. We previously described a decreased response to the growth-inhibitory effects of transforming growth factor type beta (TGF-beta) in SzS T cells accompanied by apparent loss of surface type II TGF-beta receptor (TGF beta RII). To specifically determine if defects exist in TGF beta RII protein expression and/or transport in SzS patients, we developed a sensitive flow cytometric method to detect TGF beta RII on the surface and intracellularly in the CD4+ T cells. Our results indicate that unlike normal CD4+ T cells, CD4+ T cells from 9 of 12 SzS patients expressed little, if any, surface TGF beta RII in response to mitogen stimulation. At the intracellular level, however, pools of TGF beta RII were comparable to those in normal CD4+ T cells. This indicates that defective trafficking of this inhibitory cytokine receptor may contribute significantly to the development of this disease.

Cell-surface-expressed T-cell antigen-receptor zeta chain is associated with the cytoskeleton.

The T-cell antigen receptor zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. zeta chain can associate with certain protein tyrosine kinases and retains the capacity to transduce signals independently of the other receptor subunits. Thus, zeta chain could couple cell-surface-expressed T-cell antigen receptors to the intracellular signal-transduction apparatus by its association with various intracellular molecules in addition to tyrosine kinases. In the process of searching for zeta chain-associated molecules we observed that after lysis of resting T cells with Triton X-100, zeta chain is localized in the detergent-insoluble fraction, in addition to its presence in the detergent-soluble fraction. Treatment of T cells with cytochalasin B, an actin-depolymerizing agent, leads to the complete dissociation of zeta chain from the Triton-insoluble fraction, suggesting a linkage between zeta chain and the cytoskeletal matrix. We have also determined that cytoskeletal-associated zeta chain is expressed on the cell surface. Furthermore, a tyrosine-phosphorylated 16-kDa zeta chain was detected only in the Triton-insoluble cytoskeletal fraction of resting T cells. zeta chain also maintains its association with the cytoskeleton when expressed in COS cells, inferring that the cytoskeletal elements involved in this linkage may be ubiquitous. Finally, we have localized a 42-amino acid region in the intracytoplasmic domain of zeta chain, which is crucial for maximal interaction between zeta chain and the cytoskeleton. Anchorage of cell-surface-expressed zeta chain to the cytoskeleton in resting T cells may facilitate recycling of receptor complexes and/or allow the transduction of external stimuli into the cell.

Ganglioside GM1 binds to the Trk protein and regulates receptor function.

Several lines of evidence have suggested that ganglioside GM1 stimulates neuronal sprouting and enhances the action of nerve growth factor (NGF), but its precise mechanism is yet to be elucidated. We report here that GM1 directly and tightly associates with Trk, the high-affinity tyrosine kinase-type receptor for NGF, and strongly enhances neurite outgrowth and neurofilament expression in rat PC12 cells elicited by a low dose of NGF that alone is insufficient to induce neuronal differentiation. The potentiation of NGF activity by GM1 appears to involve tyrosine-autophosphorylation of Trk, which contains intrinsic tyrosine kinase activity that has been localized to the cytoplasmic domain. In the presence of GM1 in culture medium, there is a > 3-fold increase in NGF-induced autophosphorylation of Trk as compared with NGF alone. We also found that GM1 could directly enhance NGF-activated autophosphorylation of immunoprecipitated Trk in vitro. Monosialoganglioside GM1, but not polysialogangliosides, is tightly associated with immunoprecipitated Trk. Furthermore, such tight association of GM1 with Trk appears to be specific, since a similar association was not observed with other growth factor receptors, such as low-affinity NGF receptor (p75NGR) and epidermal growth factor receptor (EGFR). Thus, these results strongly suggest that GM1 functions as a specific endogenous activator of NGF receptor function, and these enhanced effects appear to be due, at least in part, to tight association of GM1 with Trk.

Replication factor encoded by a putative oncogene, set, associated with myeloid leukemogenesis.

DNA replication of the adenovirus genome complexed with viral core proteins is dependent on the host factor designated template activating factor I (TAF-I) in addition to factors required for replication of the naked genome. Recently, we have purified TAF-I as 39- and 41-kDa polypeptides from HeLa cells. Here we describe the cloning of two human cDNAs encoding TAF-I. Nucleotide sequence analysis revealed that the 39-kDa polypeptide corresponds to the protein encoded by the set gene, which is the part of the putative oncogene associated with acute undifferentiated leukemia when translocated to the can gene. The 41-kDa protein contains the same amino acid sequence as the 39-kDa protein except that short N-terminal regions differ in both proteins. Recombinant proteins, which were purified from extracts of Escherichia coli, expressing the proteins from cloned cDNAs, possessed TAF-I activities in the in vitro replication assay. A particular feature of TAF-I proteins is the presence of a long acidic tail in the C-terminal region, which is thought to be an essential part of the SET-CAN fusion protein. Studies with mutant TAF-I proteins devoid of this acidic region indicated that the acidic region is essential for TAF-I activity.

Heat shock protein hsp90 regulates dioxin receptor function in vivo.

The dioxin (aryl hydrocarbon) receptor is a ligand-dependent basic helix-loop-helix (bHLH) factor that binds to xenobiotic response elements of target promoters upon heterodimerization with the bHLH partner factor Arnt. Here we have replaced the bHLH motif of the dioxin receptor with a heterologous DNA-binding domain to create fusion proteins that mediate ligand-dependent transcriptional enhancement in yeast (Saccharomyces cerevisiae). Previously, our experiments indicated that the ligand-free dioxin receptor is stably associated with the 90-kDa heat shock protein, hsp90. To investigate the role of hsp90 in dioxin signaling we have studied receptor function in a yeast strain where hsp90 expression can be down-regulated to about 5% relative to wild-type levels. At low levels of hsp90, ligand-dependent activation of the chimeric dioxin receptor construct was almost completely inhibited, whereas the activity of a similar chimeric construct containing the structurally related Arnt factor was not affected. Moreover, a chimeric dioxin receptor construct lacking the central ligand- and hsp90-binding region of the receptor showed constitutive transcriptional activity in yeast that was not impaired upon down-regulation of hsp90 expression levels. Thus, these data suggest that hsp90 is a critical determinant of conditional regulation of dioxin receptor function in vivo via the ligand-binding domain.

Basic fibroblast growth factor can mediate the early inductive events in renal development.

The earliest characterized events during induction of tubulogenesis in renal anlage include the condensation or compaction of metanephrogenic mesenchyme with the concurrent upregulation of WT1, the gene encoding the Wilms tumor transcriptional activator/suppressor. We report that basic fibroblast growth factor (FGF2) can mimic the early effects of an inductor tissue by promoting the condensation of mesenchyme and inhibiting the tissue degeneration associated with the absence of an inductor tissue. By in situ hybridization, FGF2 was also found to mediate the transcriptional activation of WT1 and of the hepatocyte growth factor receptor gene, c-met. Although FGF2 can induce these early events of renal tubulogenesis, it cannot promote the epithelial conversion associated with tubule formation in metanephrogenic mesenchyme. For this, an undefined factor(s) from pituitary extract in combination with FGF2 can cause tubule formation in uninduced mesenchyme. These findings support the concept that induction in kidney is a multiphasic process that is mediated by more than a single comprehensive inductive factor and that soluble molecules can mimic these inductive activities in isolated uninduced metanephrogenic mesenchyme.

Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol.

Antisense oligodeoxyribonucleotides targeted to the epidermal growth factor (EGF) receptor were encapsulated into liposomes linked to folate via a polyethylene glycol spacer (folate-PEG-liposomes) and efficiently delivered into cultured KB cells via folate receptor-mediated endocytosis. The oligonucleotides were a phosphodiester 15-mer antisense to the EGF receptor (EGFR) gene stop codon (AEGFR2), the same sequence with three phosphorothioate linkages at each terminus (AEGFR2S), a randomized 15-mer control of similar base composition to AEGFR2 (RC15), a 14-mer control derived from a symmetrized Escherichia coli lac operator (LACM), and the 5'-fluorescein-labeled homologs of several of the above. Cellular uptake of AEGFR2 encapsulated in folate-PEG-liposomes was nine times higher than AEGFR2 encapsulated in nontargeted liposomes and 16 times higher than unencapsulated AEGFR2. Treatment of KB cells with AEGFR2 in folate-PEG-liposomes resulted in growth inhibition and significant morphological changes. Curiously, AEGFR2 and AEGFR2S encapsulated in folate-PEG-liposomes exhibited virtually identical growth inhibitory effects, reducing KB cell proliferation by > 90% 48 hr after the cells were treated for 4 hr with 3 microM oligonucleotide. Free AEGFR2 caused almost no growth inhibition, whereas free AEGFR2S was only one-fifth as potent as the folate-PEG-liposome-encapsulated oligonucleotide. Growth inhibition of the oligonucleotide-treated cells was probably due to reduced EGFR expression because indirect immunofluorescence staining of the cells with a monoclonal antibody against the EGFR showed an almost quantitative reduction of the EGFR in cells treated with folate-PEG-liposome-entrapped AEGFR2. These results suggest that antisense oligonucleotide encapsulation in folate-PEG-liposomes promise efficient and tumor-specific delivery and that phosphorothioate oligonucleotides appear to offer no major advantage over native phosphodiester DNA when delivered by this route.

Platelet-activating factor downregulates the expression of liver X receptor-α and its target genes in human neutrophils

Liver X receptors (LXRs) are ligand-activated members of the nuclear receptor superfamily that regulate the expression of genes involved in lipid metabolism and inflammation, although their role in inflammation and immunity is less well known. It has been reported that oxysterols/LXRs may act as anti-inflammatory molecules, although opposite actions have also been reported. In this study, we investigated the effect of platelet-activating factor (PAF), a proinflammatory molecule, on LXRα signalling in human neutrophils. We found that PAF exerted an inhibitory effect on mRNA expression of TO901317-induced LXRα, ATP-binding cassette transporter A1, ATP-binding cassette transporter G1, and sterol response element binding protein 1c. This negative action was mediated by the PAF receptor, and was dependent on the release of reactive oxygen species elicited by PAF, as it was enhanced by pro-oxidant treatment and reversed by antioxidants. Current data also support the idea that PAF induces phosphorylation of the LXRα molecule in an extracellular signal-regulated kinase 1/2-mediated fashion. These results suggest that a possible mechanism by which PAF exerts its proinflammatory effect is through the downregulation of LXRα and its related genes, which supports the notion that LXRα ligands exert a modulatory role in the neutrophil-mediated inflammatory response.

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca2+, but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca2+. This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca2+, absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized anti-phospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (645R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q660) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.

Y-box binding protein 1 (YB-1) relevance in estrogen receptor-positive (ER+) breast cancer

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2016

Implicating the mechanisms of ADP-ribosylation factor activation in the resistance of invasive breast cancer cells to EGFR tyrosine kinase inhibitors

ADP-ribosylation factor-1 (ARF1) est une petite GTPase principalement connue pour son rôle dans la formation de vésicules au niveau de l’appareil de Golgi. Récemment, dans des cellules de cancer du sein, nous avons démontré qu’ARF1 est aussi un médiateur important de la signalisation du récepteur du facteur de croissance épidermique (EGFR) contrôlant la prolifération, la migration et l'invasion cellulaire. Cependant, le mécanisme par lequel l’EGFR active la GTPase ainsi que le rôle de cette dernière dans la régulation de la fonction du récepteur demeure inconnue. Dans cette thèse, nous avions comme objectifs de définir le mécanisme d'activation de ARF1 dans les cellules de cancer du sein hautement invasif et démontrer que l’activation de cette isoforme de ARF joue un rôle essentiel dans la résistance de ces cellules aux inhibiteurs de l'EGFR. Nos études démontrent que les protéines d’adaptatrices Grb2 et p66Shc jouent un rôle important dans l'activation de ARF1. Alors que Grb2 favorise le recrutement d’ARF1 à l'EGFR ainsi que l'activation de cette petite GTPase, p66Shc inhibe le recrutement du complexe Grb2-ARF1 au récepteur et donc contribue à limiter l’activation d’ARF1. De plus, nous démontrons que ARF1 favorise la résistance aux inhibiteurs des tyrosines kinases dans les cellules de cancer du sein hautement invasif. En effet, une diminution de l’expression de ARF1 a augmenté la sensibilité descellules aux inhibiteurs de l'EGFR. Nous montrons également que de hauts niveaux de ARF1 contribuent à la résistance des cellules à ces médicaments en améliorant la survie et les signaux prolifératifs à travers ERK1/2, Src et AKT, tout en bloquant les voies apoptotiques (p38MAPK et JNK). Enfin, nous mettons en évidence le rôle de la protéine ARF1 dans l’apoptose en réponse aux traitements des inhibiteurs de l’EGFR. Nos résultats indiquent que la dépletion d’ARF1 promeut la mort cellulaire induite par gefitinib, en augmentant l'expression de facteurs pro-apoptotiques (p66shc, Bax), en altérant le potentiel de la membrane mitochondriale et la libération du cytochrome C. Ensemble, nos résultats délimitent un nouveau mécanisme d'activation de ARF1 dans les cellules du cancer du sein hautement invasif et impliquent l’activité d’ARF1 comme un médiateur important de la résistance aux inhibiteurs EGFR.

Environmental Enteropathy and Fibroblast Growth Factor 21 are associated with early childhood growth in Bangladesh

Thesis (Ph.D.)--University of Washington, 2016-06

The role of insulin-like growth factor II and its receptor in mouse preimplantation development

Insulin-like growth factor II (IGF-II) and its receptor, the IGF-II/mannose-6-phosphate (IGF-II/M6P) receptor, are first expressed from the zygotic genome at the two-cell stage of mouse development. However, their role is not clearly defined. Insulin-like growth factor II is believed to mediate growth through the heterologous type 1 IGF and insulin receptors, whereas the IGF-II/M6P receptor is believed to act as a negative regulator of somatic growth by limiting the availability of excess levels of IGF-II. These studies demonstrate that IGF-II does have a role in growth regulation in the early embryo through the IGF-II/M6P receptor. Insulin-like growth factor II stimulated cleavage rate in two-cell embryos in vitro. Moreover, this receptor is required for the glycaemic response of two-cell embryos to IGF-II and for normal progression of early embryos to the blastocyst stage. Improved development of embryos in crowded culture supports the concept of an endogenous embryonic paracrine activity that enhances cell proliferation. These responses indicate that the IGF-II/M6P receptor is functional and likely to participate in such a regulatory circuit. The functional role of IGF-II and its receptor is discussed with reference to regulation of early development.