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L M Pfeffer

Publications and source records attributed to L M Pfeffer.

At least 19 recordsLinked to original sources
PubMed1999

EGF induces nuclear translocation of STAT2 without tyrosine phosphorylation in intestinal epithelial cells.

Signal transducers and activators of transcription (STATs) are cytoplasmic proteins that bind to activated membrane receptors, undergo ligand-dependent phosphorylation on tyrosine residues, and translocate to the nucleus, where they induce transcription of specific genes in response to a variety of ligands, including cytokines and some growth factors. Using immunocytochemical and biochemical techniques, we investigated the localization and responses of STAT1 and STAT2 to epidermal growth factor (EGF) stimulation in IEC-6 intestinal epithelial cells and HeLa cells. These studies provide the first description of STAT activation and localization in response to EGF in intestinal epithelial cells and some novel findings regarding the activation and localization of STATs in general. These include the following. First, EGF promoted the tyrosine phosphorylation of STAT1 in IEC-6 cells and caused its translocation to the nucleus. Second, in the absence of EGF stimulation both STAT1 and STAT2 were localized to the Golgi apparatus in IEC-6 cells. Third, EGF caused the translocation of STAT2 to the nucleus in both IEC-6 and HeLa cells without inducing the tyrosine phosphorylation of STAT2.

Animals↗
PubMed1998

Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons.

IFNs were first described as potent antiviral agents 40 years ago, and recombinant IFN-alpha2a and IFN-alpha2b were approved for the treatment of hairy cell leukemia just 11 years ago. Today, alpha-IFNs are approved worldwide for the treatment of a variety of malignancies and virologic diseases. Although the exact mechanism of action of IFN-alpha in the treatment of such diseases is not fully understood, many advances have been made in the characterization of the physicochemical and diverse biological properties of this highly pleiotropic cytokine. Here we review recent developments in our understanding of the antiviral and immunoregulatory properties of IFN-alpha, the nature of the multisubunit IFN-alpha receptor, and the molecular mechanisms of signal transduction. Where available, we have included comparative data on recombinant alpha-IFNs derived from both naturally occurring and nonnaturally occurring synthetic genes. We also review clinical data and data on the side effects and antigenicity of different sources of recombinant alpha-IFNs in humans. These latter topics are of clinical interest, because they may potentially affect the efficacy of these various products. Hopefully, what is already known about IFN will prompt further exploration into the mechanism(s) of action of IFN-alpha and thus deliver new applications for this prototypic cytokine, whose full therapeutic potential is yet to be realized.

Antineoplastic Agents↗
PubMed1998

STAT3 complements defects in an interferon-resistant cell line: evidence for an essential role for STAT3 in interferon signaling and biological activities.

STAT proteins play critical roles in the signal transduction pathways for various cytokines. The type I interferons (IFNalpha/beta) promote the DNA-binding activity of the transcription factors STAT1, STAT2, and STAT3. Although the requirement for STAT1 and STAT2 in IFNalpha/beta signaling and action is well documented, the biological importance of STAT3 to IFN action has not yet been addressed. We found that STAT3 plays a critical role in signal transduction by IFNalpha/beta. A human cell line that is resistant to the antiviral and antiproliferative activities of IFN but is still IFN-responsive by virtue of STAT1 and STAT2 activation was found to be defective in STAT3 activation and in induction of NF-kappaB DNA-binding activity. Expression of STAT3 in these resistant cells complemented these signaling defects and also markedly increased cellular sensitivity to the antiviral and antiproliferative effects of IFN. Because STAT3 is involved in the induction of NF-kappaB DNA-binding activity and in the induction of antiviral and antiproliferative activity, our results place STAT3 as an important upstream element in type I IFN signal transduction and in the induction of biological activities. Therefore, our results indicate that STAT1 and STAT2 are not the only STATs required for the expression of the key biological activities of IFNalpha/beta.

Antiviral Agents↗
PubMed1998

Induction of Jak/STAT signaling by activation of the type 1 TNF receptor.

Cellular responses to TNF are initiated by either of two cell surface receptors, the type 1 TNF receptor (TNFR1) and the type 2 TNF receptor (TNFR2). Although neither receptor contains an intrinsic protein tyrosine kinase, such activity has been implicated in TNF action. In this study, we show that murine TNF induces the tyrosine phosphorylation and activation of the intracellular Janus tyrosine kinases Jak1, Jak2, and Tyk2 in murine 3T3-L1 adipocytes. Activation of Jak kinases by TNF was associated with tyrosine phosphorylation of STAT1, STAT3, STAT5, and STAT6, but not STAT2 or STAT4, showing that TNF acts on a specific subset of these latent cytoplasmic transcription factors in 3T3-L1 adipocytes. Agonist antiserum to TNFR1 induced Jak kinase and STAT protein phosphorylation. Phosphorylation of Jak proteins was also induced by human TNF, which selectively binds to TNFR1 on murine cells. 35S-labeled Jak kinases were precipitated from a cell-free system and from lysates of 3T3-L1 adipocytes by a glutathione S-transferase fusion protein containing the cytoplasmic domain of TNFR1. These results suggest that the cytoplasmic domain of TNFR1 can directly interact with and form signaling complexes with Jak kinases. Jak2 was precipitated from HeLa cells by antiserum to TNFR1, directly demonstrating their association in vivo. Thus, TNF activates a Jak/STAT signal-transduction cascade by acting through TNFR1.

3T3 Cells↗
PubMed1998

The antiviral action of interferon is potentiated by removal of the conserved IRTAM domain of the IFNAR1 chain of the interferon alpha/beta receptor: effects on JAK-STAT activation and receptor down-regulation.

The first cloned chain (IFNAR1) of the human interferon-alpha (IFN alpha) receptor acts as a species-specific transducer for type 1 IFN action when transfected into heterologous mouse cells. Stably transfected mouse L929 cell lines expressing truncation mutants of the intracellular domain of the human IFNAR1 chain were tested for biological responses to human IFN alpha. Deletion of the intracellular domain resulted in a complete loss of sensitivity to the biological activity of human IFN but markedly increased IFNAR1 cell surface expression, demonstrating that the intracellular domain is required for biological function and contains a domain that negatively regulates its cell surface expression. Removal of the conserved membrane distal 16-amino-acid IRTAM (Interferon Receptor Tyrosine Activation Motif) sequence: (1) increased sensitivity to IFN alpha's antiviral activity, (2) increased the rapid IFN alpha-dependent formation of STAT-containing DNA-binding complexes, (3) prolonged tyrosine phosphorylation kinetics of the JAK-STAT pathway, and (4) blocked the IFN-dependent down-regulation of the IFNAR1 chain. These results indicate that the IRTAM negatively regulates signalling events required for the induction of IFN's biological actions via regulating receptor down-regulation.

Amino Acid Sequence↗
PubMed1997

A type I interferon signaling factor, ISF21, encoded on chromosome 21 is distinct from receptor components and their down-regulation and Is necessary for transcriptional activation of interferon-regulated genes.

The type I interferons (IFNs) are a family of cytokines, comprising at least 17 subtypes, which exert pleiotropic actions by interaction with a multi-component cell surface receptor and at least one well characterized signal transduction pathway involving JAK/STAT (Janus kinase/signal transducer and activator of transcription) proteins. In a previous report, we showed that a signaling factor, encoded by a gene located on the distal portion of chromosome 21, distinct from the IFNAR-1 receptor, was necessary for 2'-5'-oligoadenylate synthetase activity and antiviral responses, but not for high affinity ligand binding. In the present studies using hybrid Chinese hamster ovary cell lines containing portions of human chromosome 21, we show that the type I IFN signaling molecule, designated herein as ISF21, is distinct from the second receptor component, IFNAR-2, which is expressed in signaling and non-signaling cell lines. The location of the gene encoding ISF21 is narrowed to a region between the 10;21 and the r21 breakpoints, importantly eliminating the Mx gene located at 21q22.3 (the product of which is involved in IFN-induced antiviral responses) as a candidate for the signaling factor. To characterize the action of this factor in the type I IFN signaling pathway, we show that it acts independently of receptor down-regulation following ligand binding, both of which occur equally in the presence or absence of the factor. In addition, we demonstrate that ISF21 is necessary for transcriptional activation of 2'-5'-oligoadenylate synthetase, 6-16, and guanylate-binding protein gene promoter reporter constructs, which are mediated by several signaling pathways. ISF21 represents a novel factor as the localization to chromosome 21, and the data presented in this study exclude any of the known type I IFN signal-transducing molecules.

Animals↗
PubMed1997

STAT3 as an adapter to couple phosphatidylinositol 3-kinase to the IFNAR1 chain of the type I interferon receptor.

STAT (signal transducers and activators of transcription) proteins undergo cytokine-dependent phosphorylation on serine and tyrosine. STAT3, a transcription factor for acute phase response genes, was found to act as an adapter molecule in signal transduction from the type I interferon receptor. STAT3 bound to a conserved sequence in the cytoplasmic tail of the IFNAR1 chain of the receptor and underwent interferon-dependent tyrosine phosphorylation. The p85 regulatory subunit of phosphatidylinositol 3-kinase, which activates a series of serine kinases, bound to phosphorylated STAT3 and subsequently underwent tyrosine phosphorylation. Thus, STAT3 acts as an adapter to couple another signaling pathway to the interferon receptor.

Acute-Phase Proteins↗
PubMed1997

The short form of the interferon alpha/beta receptor chain 2 acts as a dominant negative for type I interferon action.

We have characterized the functional properties of the short form of the human interferon alpha/beta receptor chain 2 (IFNAR2), denoted IFNAR2.1. IFNAR2.1 contains a shortened cytoplasmic domain when compared with the recently cloned full-length IFNAR2 chain (IFNAR2. 2). We show that IFNalpha8 and IFNbeta1b induce antiviral and antiproliferative activity in mouse cell transfectants expressing the human IFNAR1 chain of the receptor and induce the formation of STAT1/STAT2 dimers in IFN-stimulated response element (ISRE)-dependent gel shift assays. In contrast, coexpression of IFNAR2.1 with IFNAR1 reduces the IFN-induced antiviral, antiproliferative and ISRE-dependent gel shift binding activity conferred by IFNAR1 alone. No antiviral or antiproliferative response to IFN, nor IFN-induced ISRE-dependent gel shift binding activity, was observed when IFNAR2.1 was expressed alone in murine cells. Therefore, IFNAR2.1 acts as a dominant negative for these IFN-induced activities. Our results suggest that IFNAR2.1 represents a nonfunctional version of the full-length chain (IFNAR2.2).

Animals↗
PubMed1997

Biologic activities of natural and synthetic type I interferons.

Because alpha-interferon (IFN-alpha) has a number of therapeutic applications in the treatment of various human cancers and diseases of viral origin, an understanding of how this family of proteins interacts with cells to induce their pleiotropic biologic activities is essential. Available data suggest that recombinant IFN-alphas from both natural and synthetic genes bind to a common cell surface receptor and induce antiviral activity in a variety of cell lines. IFN-alphas were found to differ significantly in their abilities to bind to cells; this difference varied with the types of IFN-alpha and cell type used. Consensus interferon (IFN-con1), a nonnaturally occurring synthetic IFN, and IFN-alpha2b competed about equally well for receptor binding sites on Daudi and CaKi cells and were followed by IFN-alpha8 in the ability to compete. Results of affinity cross-linking experiments indicated that all three IFN-alphas interacted similarly with the multichain IFN-alpha receptor. IFN-alpha7, however, competed poorly for binding sites on both cell lines. Each of the IFN-alphas tested displayed discrete biologic differences, which also varied with the assay system used. IFN-con1 and IFN-alpha2b displayed similar antiviral activities on CaKi cells using vesicular stomatitis virus; the viral activities of these IFNs were significantly greater than those of IFN-alpha7 or IFN-alpha8. Studies with murine transfectants demonstrated significant differences in the various IFNs to interact with the IFN-alpha receptor-1 chain of the type I IFN receptor. It is yet to be established, however, that these various in vitro distinctions result in differences in clinical benefit or toxicity between the various subtypes.

Animals↗
PubMed1996

The human type I interferon receptor. Identification of the interferon beta-specific receptor-associated phosphoprotein.

We used specific antibodies recognizing the receptor 1 (IFNAR1) and the recently cloned receptor 2.2 (IFNAR2.2) chains of the human type I interferon receptor complex to demonstrate that the interferon beta (IFN-beta)-specific receptor-associated phosphoprotein is IFNAR2.2 and not an unknown or additional receptor component. Immunoprecipitation experiments demonstrated that IFNAR2.2 is present in Daudi cells as a cell surface protein of approximately 90-100 kDa, which is tyrosine-phosphorylated and associated with IFNAR1, upon stimulation of cells with IFN-beta. IFNAR2.2 was not detected associated with IFNAR1 in cells stimulated with IFN-alpha, suggesting differences in receptor interaction between the two type I interferons. Both IFNAR1 and IFNAR2.2 undergo tyrosine phosphorylation upon induction by either IFN-alpha or IFN-beta. Therefore, it is unclear as to why IFNAR2.2 is not detectable in IFNAR1 immunoprecipitates in IFN-beta-treated cells. These data suggest that, although IFN-alpha and IFN-beta may utilize similar receptor chains, they interact with IFNAR1 and IFNAR2.2 in different ways.

Baculoviridae↗
PubMed1996

Direct association of STAT3 with the IFNAR-1 chain of the human type I interferon receptor.

Based on the reports of the activation of the transcription factor known as STAT3 (for signal transducers and activators of transcription) or APRF (for acute phase response factor) by various cytokines, we investigated the possible role of STAT3 in type I interferon (IFN) receptor signaling. We show that STAT3 undergoes IFNalpha-dependent tyrosine phosphorylation and IFNalpha treatment induces protein-DNA complexes that contain STAT3. In addition, STAT3 associates with the IFNAR-1 chain of the type I receptor in a tyrosine phosphorylation-dependent manner upon IFNalpha addition. The binding of STAT3 to the IFNAR-1 chain occurs through a direct interaction between the SH2 domain-containing portion of STAT3 and the tyrosine-phosphorylated IFNAR-1 chain. Furthermore, tyrosine-phosphorylated STAT3 bound to the IFNAR-1 chain also undergoes a secondary modification involving serine phosphorylation. This phosphorylation event is apparently mediated by protein kinase C, since it was blocked by low concentrations of the protein kinase inhibitor H-7. The biological relevance of IFN activation of STAT3 is further illustrated by the finding that STAT3 is not activated by IFN in a cell line resistant to the antiviral and antiproliferative actions of IFN alpha but in which other components of the JAK-STAT pathway are activated by IFNalpha.

Base Sequence↗
PubMed1996

Human renal cancers resistant to IFN's antiproliferative action exhibit sensitivity to IFN's gene-inducing and antiviral actions.

PURPOSE: Although treatment with interferon-alpha (IFN alpha) results in tumor regression in a subset (< 20%) of patients with renal cell carcinoma, the underlying mechanisms for the resistance of renal cancer (RC) cells to IFN alpha is unknown. MATERIALS AND METHODS: We examined 5 RC lines resistant and 5 RC lines sensitive to the antiproliferative effects of IFN alpha for differences in: 1) the number of IFN binding sites, 2) the number of signal-transducing IFNAR-1 chains of the IFN alpha receptor, 3) IFN alpha receptor structure, 4) IFN-stimulated gene (ISG) expression and 5) IFN alpha sensitivity in antiviral assays. RESULTS: No structural alterations in the IFN alpha receptor were detected in any RC line examined, although varying numbers of ligand binding sites and IFNAR-1 signal transducer chains were present. All 5 IFN-sensitive, and 4 of 5 IFN-resistant RC lines were sensitive to the antiviral and gene-inducing actions of IFN alpha. CONCLUSIONS: The resistance of RC lines to IFN's antiproliferative action is not due to defects in ligand binding or in IFN-receptor structure. Our results indicate that the defective antiproliferative response in most RC cells is not due to their failure to induce the gene-inducing and antiviral effects of IFN alpha.

Antineoplastic Agents↗
PubMed1995

Expression and signaling specificity of the IFNAR chain of the type I interferon receptor complex.

The IFNAR chain of the type I interferon (IFN) receptor (IFNIR) undergoes rapid ligand-dependent tyrosine phosphorylation and acts as a species-specific transducer for type I IFN action. Using the vaccinia/T7 expression system to amplify IFNAR expression, we found that human HeLa-S3 cells transiently express high levels of cell surface IFNAR chains (approximately 250,000 chains per cell). Metabolic labeling and immunoblot analysis of transfected HeLa cells show that the IFNAR chain is initially detected as 65-kDa and 98-kDa precursors, and then as the 130-kDa mature protein. Due to variation in N-glycosylation, the apparent molecular mass of the mature IFNAR chain varies from 105 to 135 kDa in different cells. IFNIR structure was characterized in various human cell lines by analyzing 125I-labeled IFN cross-linked complexes recognized by various antibodies against IFNIR subunits and JAK protein-tyrosine kinases. Precipitation of cross-linked material from Daudi cells with anti-IFNAR antibodies showed that IFNAR was present in a 240-kDa complex. Precipitation of cross-linked material from U937 cells with anti-TYK2 sera revealed a 240-kDa complex, which apparently did not contain IFNAR and was not present in IFN-resistant HEC1B cells. The tyrosine phosphorylation and down-regulation of the IFNAR chain were induced by type I IFN in several human cell lines of diverse origins but not in HEC1B cells. However, of type I IFNs, IFN-beta uniquely induced the tyrosine phosphorylation of a 105-kDa protein associated with the IFNAR chain in two lymphoblastoid cell lines (Daudi and U266), demonstrating the specificity of transmembrane signaling for IFN-beta and IFN-alpha through the IFNAR chain.

Animals↗
PubMed1995

An Alu cassette in the cytoplasmic domain of an interferon receptor subunit.

All the cloned subunits of interferon receptors (IFNRs) belong to the type II cytokine receptor family (CRF2). Although three members of CRF2 encoded on human chromosome 21 share a 50 amino acid cytoplasmic homology domain (the IRH2 domain), a fourth subunit, the second cloned chain of the type I IFNR (IFNIR-2), contains a juxtamembrane 20 amino acid stretch of high similarity to the IRH2 domain that stops abruptly. Comparison of the membrane-distal portion of the IFNIR-2 cytoplasmic domain with sequence databases revealed a very high similarity to Alu repeat sequences. We provide evidence that all but 18 amino acids of the predicted cytoplasmic domain of the IFNIR-2 chain are encoded by an Alu cassette in its antisense orientation. Incorporation of an Alu cassette into the receptor chain is proposed to occur by a splicing mechanism. All previous well-characterized examples of insertion of an antisense Alu cassette into an open reading frame have involved alternative splicing. Thus, we predict the existence of an alternatively spliced product of the IFNIR-2 chain with a substantially different cytoplasmic domain.

Amino Acid Sequence↗
PubMed1994

Role of interferon alpha/beta receptor chain 1 in the structure and transmembrane signaling of the interferon alpha/beta receptor complex.

A previously cloned cDNA encodes one subunit of the human interferon alpha/beta receptor (IFN alpha R), denoted IFN alpha R1. To study the expression and signaling of IFN alpha R1, we used monoclonal antibodies (mAbs) generated against the baculovirus-expressed ectodomain of IFN alpha R1. Immunoprecipitation and immunoblotting of lysates from a variety of human cell lines showed that IFN alpha R1 has an apparent molecular mass of 135 kDa. Binding analysis with 125I-labeled mAb demonstrated high levels of cell surface expression of IFN alpha R1 in human cells and in mouse cells transfected with IFN alpha R1 cDNA, whereas no cross-reactivity was observed in control mouse L929 cells expressing only the endogenous mouse receptor. The subunit was rapidly down-regulated by IFN alpha (80% decrease within 2 hr) and degraded upon internalization. The IFN alpha R1 chain appeared to be constitutively associated with the 115-kDa subunit of the IFN alpha/beta receptor, since the mAbs coprecipitated this protein. IFN alpha/beta treatment induced tyrosine phosphorylation of IFN alpha R1 within 1 min, with kinetics paralleling that of the IFN-activated protein-tyrosine kinases Jak1 and Tyk2. Ligand-induced tyrosine phosphorylation of IFN alpha R1 was blocked by the kinase inhibitors genistein or staurosporine. Although IFN alpha R1 cDNA-transfected mouse cells expressed high levels of this subunit when compared with empty vector-transfected cells the number of binding sites for human IFN alpha (50-75 sites per cell) was not increased. Human IFN alpha induced the expression of a mouse IFN alpha/beta-responsive gene (the 204 gene) in mouse L929 cells transfected with the IFN alpha R1 cDNA, but not in mock-transfected cells. These results suggest that the IFN alpha R1 subunit acts as a species-specific signal transduction component of the IFN alpha/beta receptor complex.

Animals↗
PubMed1994

Complementation of the interferon alpha response in resistant cells by expression of the cloned subunit of the interferon alpha receptor. A central role of this subunit in interferon alpha signaling.

A subunit of the interferon alpha receptor (IFN alpha R) that confers biologic response to and specific "binding" for IFN alpha 8 has recently been cloned. We have explored the biological consequences of expressing the cloned IFN alpha R subunit in human cells resistant to IFN alpha and in mouse cell lines nonresponsive to human IFN alpha. The expression of the cloned IFN alpha R subunit in the human IFN alpha-resistant K-562 cell line restored sensitivity to the antiviral effect of not only IFN alpha 8 but also IFN alpha 2 and IFN alpha Con1. In mouse L-929 cells the expression of the cloned receptor subunit markedly increased antiviral sensitivity to human type I IFNs. In either human K-562 or mouse L-929 cells these effects were observed without a detectable increase in the binding for any of the subtypes of IFN alpha tested. We propose that the cloned IFN alpha R subunit functions as a transducer subunit for the IFN alpha R. This concept is supported by the finding that the cloned receptor protein, when it is expressed in Cos cells, has an M(r) of 75 kDa, which is different from the main IFN alpha-binding proteins, the alpha and beta subunits of the IFN alpha R. This report also suggests that alterations at the receptor level could be involved in IFN alpha resistance in some cell lines.

Animals↗