Biomedical subjects

P Leder

Publications and source records attributed to P Leder.

At least 73 records · Page 4Linked to original sources

PubMed1996

RIP and FADD: two "death domain"-containing proteins can induce apoptosis by convergent, but dissociable, pathways.

With use of the yeast two-hybrid system, the proteins RIP and FADD/MORT1 have been shown to interact with the "death domain" of the Fas receptor. Both of these proteins induce apoptosis in mammalian cells. Using receptor fusion constructs, we provide evidence that the self-association of the death domain of RIP by itself is sufficient to elicit apoptosis. However, both the death domain and the adjacent alpha-helical region of RIP are required for the optimal cell killing induced by the overexpression of this gene. By contrast, FADD's ability to induce cell death does not depend on crosslinking. Furthermore, RIP and FADD appear to activate different apoptotic pathways since RIP is able to induce cell death in a cell line that is resistant to the apoptotic effects of Fas, tumor necrosis factor, and FADD. Consistent with this, a dominant negative mutant of FADD, lacking its N-terminal domain, blocks apoptosis induced by RIP but not by FADD. Since both pathways are blocked by CrmA, the interleukin 1 beta converting enzyme family protease inhibitor, these results suggest that FADD and RIP can act along separable pathways that nonetheless converge on a member of the interleukin 1 beta converting enzyme family of cysteine proteases.

Apoptosis↗

PubMed1996

Tal-1 induces T cell acute lymphoblastic leukemia accelerated by casein kinase IIalpha.

Ectopic activation of the TAL-1 gene in T lymphocytes occurs in the majority of cases of human T cell acute lymphoblastic leukemia (T-ALL), yet experiments to date have failed to demonstrate a direct transforming capability for tal-1. The tal-1 gene product is a serine phosphoprotein and basic helix-loop-helix (bHLH) transcription factor known to regulate embryonic hematopoiesis. We have established a transgenic mouse model in which tal-1 mis-expression in the thymus results in the development of clonal T cell lymphoblastic leukemia/lymphoma. Thus, overexpression of tal-1 alone can be transforming, verifying its pathogenic role in human T-ALL. In addition, leukemogenesis is accelerated dramatically by transgenic co-expression of tal-1 and the catalytic subunit of casein kinase IIalpha (CKIIalpha), a serine/threonine protein kinase known to modulate the activity of other bHLH transcription factors. Although tal-1 is a substrate for CKII, the synergy of the tal-1 and CKIIalpha transgenes appears to be indirect, perhaps mediated through the E protein heterodimeric partners of tal-1. These studies prove that dysregulated tal-1 is oncogenic, providing a direct molecular explanation for the malignancies associated with TAL-1 activation in human T-ALL.

Animals↗

PubMed1996

Genetic evidence that formins function within the nucleus.

The murine limb deformity (ld) locus encodes a set of proteins, termed formins, that are required for embryonic limb and kidney development. Previous studies had indicated that these proteins are located in the nucleus and cytoplasm and have biochemical properties consistent with an action within the nucleus. To test the notion that nuclear localization is crucial for formin function, we carried out molecular and biochemical studies on three ld alleles. We show that two transgene-induced alleles, ldTgHd and ldTgBri, generate similar COOH-truncated formins that lack the terminal 110 amino acids, while a third allele, ldIn2, generates a less extensively truncated formin that lacks the terminal 42 amino acids. Using subcellular fractionation analysis, we find that wild-type formin is detected in both nuclear and cytosolic fractions; in contrast, the truncated formins encoded by ldTgHd and ldTgBri are strictly cytosolic. The less extensively truncated ldIn2 formin shows a similar, but less complete, localization defect. Consistent with this weaker cellular phenotype, hind limbs from ldIn2 mice have milder skeletal defects than those of ldTgBri mice. These observations define a small region in the carboxyl terminus that is required for nuclear localization and suggest that nuclear localization plays a role in formin action.

Abnormalities, Multiple↗

PubMed1996

NDF/heregulin induces persistence of terminal end buds and adenocarcinomas in the mammary glands of transgenic mice.

Neu differentiation factor (NDF), a member of the neuregulin family of ligands of erbB receptors, induces both differentiative and mitogenic effects on cultured human mammary epithelial cells. Since members of the epidermal growth factor receptor family, including Neu/erbB2, have been implicated in mammary carcinoma, we wished to know whether a potential ligand of this family, NDF, could induce such effects in the mammary gland in vivo. We therefore targeted expression of NDF to the mammary gland of transgenic mice using the mouse mammary tumor virus (MMTV) promoter in a fusion construct. There was a clear, but subtle effect on development of the adult virgin gland of female transgenic animals. Terminal end bud structures (TEBs), which normally disappear from the mammary gland at the age of approximately 8 weeks in wild type mice, persist in glands of virgin MMTV-NDF transgenic females, suggesting that NDF inhibits signals that normally lead to the terminal differentiation of these structures. Further, female mice, bred continuously to maximize expression of the transgene in the mammary gland, develop mammary adenocarcinomas at a median age of 12 months. Since these tumors arise in a solitary fashion, we infer that NDF is necessary, but not sufficient for their formation. In order to explore the signal transduction pathways potentially activated by NDF, we examined expression of the receptors erbB2, erbB3 and erbB4 in mammary epithelial cells established from an NDF-induced tumor. All three receptors were expressed, though only the erbB3 receptor was phosphorylated, suggesting that overexpression of NDF might operate through this receptor. Additionally, about 50% of MMTV-NDF transgenic mice developed Harderian (lachrymal) gland hyperplasia, a benign tumor that does not progress to frank malignancy.

Adenocarcinoma↗

PubMed1996

Fibroblast growth factor receptor 3 is a negative regulator of bone growth.

Endochondral ossification is a major mode of bone that occurs as chondrocytes undergo proliferation, hypertrophy, cell death, and osteoblastic replacement. We have identified a role for fibroblast growth factor receptor 3 (FGFR-3) in this process by disrupting the murine Fgfr-3 gene to produce severe and progressive bone dysplasia with enhanced and prolonged endochondral bone growth. This growth is accompanied by expansion of proliferating and hypertrophic chondrocytes within the cartilaginous growth plate. Thus, FGFR-3 appears to regulate endochondral ossification by an essentially negative mechanism, limiting rather than promoting osteogenesis. In light of these mouse results, certain human disorders, such as achondroplasia, can be interpreted as gain-of-function mutations that activate the fundamentally negative growth control exerted by the FGFR-3 kinase.

Animals↗

PubMed1996

Formin binding proteins bear WWP/WW domains that bind proline-rich peptides and functionally resemble SH3 domains.

The formins, proteins involved in murine limb and kidney development, contain a proline-rich region that matches consensus sequences for Src homology 3 (SH3) ligands. To identify proteins that interact with formins, we used this proline-rich region to screen mouse limb bud expression libraries for formin binding proteins (FBPs). As expected, we found one class of FBPs that contains SH3 domains, including two novel members of this class. In addition, however, we also found a novel class of FBPs that contains one or two copies of a 26 amino acid homology region that has been recently termed the WWP or WW motif. We demonstrate that WWP/WW domains as short as 26 amino acids can act as modular protein-binding interfaces that bind with high affinity to proline-rich sequences that are similar and, in some cases, identical to SH3 ligands. Furthermore, we find that the WWP/WW domain can compete with the Abl SH3 domain in binding a proline-rich peptide present in formin. Our results suggest that these novel protein interaction domains can perform functions similar to those of SH3 domains and, thus, might regulate SH3 interactions with target proteins through competitive binding.

Amino Acid Sequence↗

PubMed1996

The protein tyrosine phosphatase epsilon gene maps to mouse chromosome 7 and human chromosome 10q26.

We have mapped the mouse protein tyrosine phosphatase epsilon (PTP epsilon, gene symbol Ptpre) gene to the distal region of chromosome 7 by linkage analysis using two sets of multilocus genetic crosses. The human PTP epsilon gene (gene symbol PTPRE) was mapped to chromosome 10q26 by fluorescence in situ hybridization. We have previously documented the existence of two isoforms of PTP epsilon--a transmembranal, receptor-type isoform and a shorter, cytoplasmic one. Both isoforms have been suggested to arise from a single gene through the use of alternative promoters and 5' exons. The identification of a single PTP epsilon locus in both organisms is consistent with this suggestion.

Animals↗

PubMed1996

Polydactyly in the Strong's luxoid mouse is suppressed by limb deformity alleles.

The study of limb development has provided insight into pattern formation during vertebrate embryogenesis. Genetic approaches offer powerful ways to identify the critical molecules and their pathways of action required to execute a complex morphogenetic program. We have applied genetic analysis to the process of limb development by studying two mouse mutants, limb deformity (ld) and Strong's luxoid (lst). These mutations confer contrasting phenotypic alterations to the anteroposterior limb pattern. The six mutant ld alleles are fully recessive and result in oligosyndactyly of all four limbs. By contrast, the two mutant lst alleles result in a mirror-image polydactylous limb phenotype inherited in a semidominant fashion. Morphological and molecular analysis of embryonic limbs has shown that the ld and lst alleles affect the extent and distribution of two key signaling centers differentially: the apical ectodermal ridge and the zone of polarizing activity. Molecular characterization of the ld gene has defined a new family of evolutionarily conserved proteins termed the formins. The underlying molecular defect in the lst mutation has not been identified; however, both loci are tightly linked on mouse chromosome 2, suggesting the possibility that they may be allelic. In this study, we have used genetic analysis to examine the epistatic and allelic relationships of ld and lst. We observed that in + ld/lst + double heterozygotes, a single mutant ld allele is able to suppress the semi-dominant polydactylous lst limb phenotype. By segregating the lst and ld loci in a backcross, we observed that these loci recombine and are separated by a genetic distance of approximately 6 cM. Therefore, while our observations demonstrate a genetic interaction between ld and lst, it is probable that ld and lst are not allelic. Instead, lst and ld may be operating either in a linear or in a parallel (bypass) genetic pathway to affect the limb signaling centers.

Alleles↗

PubMed1996

Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia.

Eotaxin is an eosinophil-specific chemoattractant that has been recently identified in rodent models of asthma and host response against tumors. To determine whether a similar molecule might play a role in human inflammatory diseases characterized by eosinophilia, we isolated the human eotaxin gene. We demonstrate that human eotaxin is an early response gene of cytokine-stimulated epithelial and endothelial cells, and is induced in peripheral blood eosinophils by interleukin-3. Eotaxin is directly chemotactic for eosinophils, but not mononuclear cells or neutrophils. Eotaxin messenger RNA accumulates markedly in the lesions of patients with inflammatory bowel disease (ulcerative colitis and Crohn's disease), but not in the lesions of patients with diverticulitis. These results now provide a mechanism involving eotaxin to explain the eosinophil infiltration seen in a variety of human disease; as such, an eotaxin antagonist may be a novel therapy for certain human diseases characterized by tissue eosinophilia.

Amino Acid Sequence↗

PubMed1996

Eotaxin triggers eosinophil-selective chemotaxis and calcium flux via a distinct receptor and induces pulmonary eosinophilia in the presence of interleukin 5 in mice.

BACKGROUND: Understanding the processes that control selective eosinophilia is of fundamental importance in a variety of human diseases (e.g., allergies, parasitic infections, malignancy). Interleukin 5, an eosinophil-specific growth and activating factor, and eotaxin appear to collaborate in this process. Eotaxin is a recently described chemotactic factor that belongs to the C-C (or beta) chemokine family and has been implicated in animal and human eosinophilic inflammatory states. We have recently reported the molecular characterization of murine eotaxin and now report the biological properties of purified recombinant murine eotaxin in vitro and in vivo in the presence or absence of interleukin 5 (IL-5) in mice. MATERIALS AND METHODS: Murine eotaxin was expressed in bacteria and purified by affinity chromatography and HPLC. Activity was tested in vitro by examining chemotactic and calcium flux responses of purified murine leukocytes. Additionally, desensitization of calcium flux responses to other chemokines, eosinophil survival assays, and basophil histamine release were examined. Finally, eotaxin was delivered to wild-type or IL-5 transgenic mice and the host response was examined. RESULTS: Eotaxin had activity only when the recombinant molecule had the native mature amino terminus and contained the first 25 amino acids of the mature protein. It was active in vitro at an effective concentration between 10 and 100 ng/ml in both chemotaxis and calcium flux assays toward eosinophils, but not macrophages or neutrophils. Furthermore, intranasal or subcutaneous application of eotaxin selectively recruited large numbers of eosinophils into the mouse lung and skin, respectively, only in the presence of interleukin 5. Macrophage inflammatory protein-1 alpha, a related C-C chemokine active on eosinophils, and eotaxin were not able to cross-desensitize. Eotaxin had no affect on the in vitro survival of eosinophils and did not induce basophil histamine release. CONCLUSIONS: Mouse eotaxin is an eosinophil specific chemoattractant that has a markedly enhanced effect in vivo in the presence of another eosinophil selective cytokine IL-5, and utilizes a signal transduction receptor pathway that is distinct from that utilized by macrophage inflammatory protein-1 alpha. This data suggests that the development of tissue eosinophilia in vivo involves a two-step mechanism elicited by interleukin 5 and eotaxin.

Animals↗

PubMed1995

Identification of a cytoplasmic, phorbol ester-inducible isoform of protein tyrosine phosphatase epsilon.

The protein-tyrosine phosphatase epsilon (PTP epsilon) is a transmembranal, receptor-type protein that possesses two phosphatase catalytic domains characteristic of transmembranal phosphatases. Here we demonstrate the existence of a nontransmembranal isoform of PTP epsilon, PTP epsilon-cytoplasmic. PTP epsilon-cytoplasmic and the transmembranal isoform of PTP epsilon have separate, nonoverlapping expression patterns. Further, the data clearly indicate that control of which of the two isoforms is to be expressed is initiated at the transcriptional level, suggesting that they have distinct physiological roles. PTP epsilon-cytoplasmic mRNA is the product of a delayed early response gene in NIH 3T3 fibroblasts, and its transcription is regulated through a pathway that requires protein kinase C. The human homologue of PTP epsilon-cytoplasmic has also been cloned and is strongly up-regulated in the early stages of phorbol 12-tetradecanoate 13-acetate-induced differentiation of HL-60 cells. Sequence analysis indicates and cellular fractionation experiments confirm that this isoform is a cytoplasmic molecule. PTP epsilon-cytoplasmic is therefore the initial example to our knowledge of a nontransmembranal protein-tyrosine phosphatase that contains two tandem of catalytic domains.

Amino Acid Sequence↗

PubMed1995

Protein-tyrosine phosphatase epsilon. An isoform specifically expressed in mouse mammary tumors initiated by v-Ha-ras OR neu.

Transgenic mice that overexpress v-Ha-ras, c-myc, c-neu or int-2 proto-oncogenes in the mammary epithelium develop breast tumors with morphologies that are characteristic of each initiating oncogene. Since these morphological differences reflect distinctive patterns of tumor-specific gene expression, the identification of the products of these genes might shed light on the mechanisms of transformation and/or the identity of target cells that are transformed by specific classes of oncogenes. By focusing on the tyrosine phosphorylation pathway, we have found that the transmembranal protein-tyrosine phosphatase epsilon (PTP epsilon) is highly expressed in murine mammary tumors initiated by c-neu and v-Haras, but not in mammary tumors initiated by c-myc or int-2. This difference is striking and occurs both in primary tumors and in epithelial cells cultured from them. Moreover, PTP epsilon overexpression appears to be mammary tumor-specific in that it is not found in other ras-based tumors and cell lines. These observations suggest that PTP epsilon either plays a role in ras- and neu-mediated transformation of mammary epithelium or marks mammary epithelial cells particularly susceptible to transformation by these oncogenes. Because of its distinctive expression in these mammary tumors, we have further characterized murine PTP epsilon, cloning and determining the complete structures of its cDNAs and showing that it is a glycoprotein that is N-glycosylated in a tissue-specific manner.

Aging↗

PubMed1995

Expression of Brca1 is associated with terminal differentiation of ectodermally and mesodermally derived tissues in mice.

We have isolated genomic and cDNA clones of Brca1, a mouse homolog of the recently cloned breast cancer-associated gene, BRCA1. Brca1 encodes an 1812-amino-acid protein with a conserved zinc finger domain and significant homology to the human protein. Brca1 maps to Chromosome 11 within a region of conserved synteny with human chromosome 17, consistent with the mapping of the human gene to 17q21. Brca1 transcripts are expressed in a variety of cultured cells but reveal a specific and dynamic expression pattern during embryonic development. For example, expression is observed first in the otic vesicle of embryonic day 9.5 (E9.5) embryos. This expression diminishes and is replaced by expression in the neuroectoderm at E10.5. By E11-12.5, higher levels are observed in differentiating keratinocytes and in whisker pad primordia. Transcripts also become evident in epithelial cells of the E14-17 kidney. Brca1 expression occurs in differentiating epithelial cells of several adult organs as well, suggesting a general role in the functional maturation of these tissues. Consistent with this, Brca1 transcripts are expressed in both alveolar and ductal epithelial cells of the mammary gland. During pregnancy, there is a large increase in Brca1 mRNA in mammary epithelial cells, an increase that parallels their functional differentiation. Because high rates of breast cancer are associated with loss of BRCA1 in humans, it is possible that this gene provides an important growth regulatory function in mammary epithelial cells. In addition, increased transcription of mammary Brca1 during pregnancy might contribute, in part, to the reduced cancer risk associated with exposure to pregnancy and lactation.

Amino Acid Sequence↗

PubMed1995

Murine eotaxin: an eosinophil chemoattractant inducible in endothelial cells and in interleukin 4-induced tumor suppression.

Guinea pig eotaxin is a recently described member of the Cys-Cys family of chemokines and is involved in a guinea pig model of asthma. To determine whether eotaxin is a distinctive member of this family and to understand its physiologic role, we have cloned the mouse eotaxin gene and determined its structure and aspects of its biologic function. The sequence relationship between the mouse and guinea pig genes indicates that eotaxin is indeed a distinct member of the chemokine family. Moreover, murine eotaxin maps to a region of mouse chromosome 11 that encodes other Cys-Cys chemokines. In addition, recombinant murine eotaxin protein has direct chemoattractant properties for eosinophils. The eotaxin gene is widely (but not ubiquitously) expressed in normal mice and is strongly induced in cultured endothelial cells in response to interferon gamma. Eotaxin is also induced locally in response to the transplantation of interleukin 4-secreting tumor cells, indicating that it likely contributes to the eosinophil recruitment and antitumor effect of interleukin 4. Such responses suggest that eotaxin may be involved in multiple inflammatory states.

Amino Acid Sequence↗

PubMed1995

Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control.

p21CIP1/WAF1 is a CDK inhibitor regulated by the tumor suppressor p53 and is hypothesized to mediate G1 arrest. p53 has been suggested to derive anti-oncogenic properties from this relationship. To test these notions, we created mice lacking p21CIP1/WAF1. They develop normally and (unlike p53-/- mice) have not developed spontaneous malignancies during 7 months of observation. Nonetheless, p21-/- embryonic fibroblasts are significantly deficient in their ability to arrest in G1 in response to DNA damage and nucleotide pool perturbation. p21-/- cells also exhibit a significant growth alteration in vitro, achieving a saturation density as high as that observed in p53-/- cells. In contrast, other aspects of p53 function, such as thymocytic apoptosis and the mitotic spindle checkpoint, appear normal. These results establish the role of p21CIP1/WAF1 in the G1 checkpoint, but suggest that the anti-apoptotic and the anti-oncogenic effects of p53 are more complex.

Animals↗

PubMed1995

The MMTV/c-myc transgene and p53 null alleles collaborate to induce T-cell lymphomas, but not mammary carcinomas in transgenic mice.

A number of properties of the cancer-related genes c-myc and p53 suggest that they might collaborate to induce tumorigenesis. To test this notion, we produced doubly heterozygotic mice bearing disrupted p53 alleles and a fusion transgene consisting of the mouse mammary tumor virus (MMTV) LTR and the oncogene c-myc. Mice bearing both the MMT/c-myc transgene and a single p53- allele develop very aggressive pre-T- and T-cell lymphomas with a significantly shorter latency than mice carrying either the p53- allele or the c-myc transgene alone. Moreover, every lymphoma occurring in these animals has lost or suffers an inactivation of its wild type p53 allele indicating that loss of p53 activity is necessary for this c-myc-accelerated lymphomagenesis. Nonetheless, p53 inactivation and expression of the MMTV/c-myc transgene are not sufficient for lymphoid transformation. Tumors that arise in homozygous p53- mice carrying the c-myc transgene are monoclonal, suggesting that at least one additional event is necessary for their transformation. Moreover, since mice bearing only the MMTV/c-myc transgene predominantly develop mammary carcinomas, it was surprising that the p53- allele failed to accelerate the incidence of mammary carcinomas. Further, in contrast to the lymphomas, only one in four mammary tumors that arose in the double heterozygotic mice had lost its wild type p53 allele. Apparently cell context influences the ability of c-myc and p53- to cooperate in inducing oncogenesis.

Alleles↗

PubMed1995

The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation.

IP-10 is a member of the chemokine family of cytokines and is induced in a variety of cells in response to interferon gamma and lipopolysaccharide. The self-aggregation common to many chemokines, including IP-10, has hindered the identification of a specific IP-10 receptor. Using an IP-10 alkaline phosphatase fusion protein that fortuitously blocks this self-aggregation, we have identified an IP-10 binding site on a variety of cells including endothelial, epithelial, and hematopoietic cells. This binding site has a Kd of 25 nM, is inhibited by recombinant murine or human IP-10, and is dependent on the presence of cell surface heparan sulfate proteoglycans (HSPG). This conclusion is based on the findings that IP-10 binding to cells is: (a) inhibited by heparin and heparan sulfate; (b) sensitive to a 1 M NaCl wash; (c) eliminated by treatment with heparinase and trypsin; and (d) absent on mutant CHO cells that do not express cell surface HSPG. Platelet factor 4 (PF4), but not IL-8, monocyte chemoattractant protein-1, RANTES, monocyte inflammatory protein (MIP)-1 alpha, or MIP-1 beta, can compete effectively with IP-10 for binding to the cell surface. Furthermore, IP-10 shares with PF4 the ability to inhibit endothelial cell proliferation (IC50 = 150 nM). These studies demonstrate specificity in the interaction of chemokines and HSPG, and they define IP-10 and PF4 as a distinct subset of chemokines sharing an HSPG-binding site and angiostatic properties.

Animals↗

PubMed1995

RIP: a novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death.

Ligation of the extracellular domain of the cell surface receptor Fas/APO-1 (CD95) elicits a characteristic programmed death response in susceptible cells. Using a genetic selection based on protein-protein interaction in yeast, we have identified two gene products that associate with the intracellular domain of Fas: Fas itself, and a novel 74 kDa protein we have named RIP, for receptor interacting protein. RIP also interacts weakly with the p55 tumor necrosis factor receptor (TNFR1) intracellular domain, but not with a mutant version of Fas corresponding to the murine lprcg mutation. RIP contains an N-terminal region with homology to protein kinases and a C-terminal region containing a cytoplasmic motif (death domain) present in the Fas and TNFR1 intracellular domains. Transient overexpression of RIP causes transfected cells to undergo the morphological changes characteristic of apoptosis. Taken together, these properties indicate that RIP is a novel form of apoptosis-inducing protein.

Amino Acid Sequence↗