Douglas Pat Cerretti

An Essential Role for Ectodomain Shedding in Mammalian Development

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo

The Eph family of receptor tyrosine kinases and their ligands, known as ephrins, play a crucial role in vascular development during embryogenesis. The function of these molecules in adult angiogenesis has not been well characterized. Here, we report that blocking Eph A class receptor activation inhibits angiogenesis in two independent tumor types, the RIP-Tag transgenic model of angiogenesis-dependent pancreatic islet cell carcinoma and the 4T1 model of metastatic mammary adenocarcinoma. Ephrin-A1 ligand was expressed in both tumor and endothelial cells, and EphA2 receptor was localized primarily in tumor-associated vascular endothelial cells. Soluble EphA2-Fc or EphA3-Fc receptors inhibited tumor angiogenesis in cutaneous window assays, and tumor growth in vivo. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor vascular density, tumor volume, and cell proliferation, but increased cell apoptosis. However, EphA2-Fc had no direct effect on tumor cell growth or apoptosis in culture, yet inhibited migration of endothelial cells in response to tumor cells, suggesting that the soluble receptor inhibited blood vessel recruitment by the tumor. These data provide the first functional evidence for Eph A class receptor regulation of pathogenic angiogenesis induced by tumors and support the function of A class Eph receptors in tumor progression.

An antibody against the colony-stimulating factor 1 receptor depletes the resident subset of monocytes and tissue- and tumor-associated macrophages but does not inhibit inflammation

The development of the mononuclear phagocyte system requires macrophage colony-stimulating factor (CSF-1) signaling through the CSF-1 receptor (CSF1R, CD115). We examined the effect of an antibody against CSF1R on macrophage homeostasis and function using the MacGreen transgenic mouse (csf1r-enhanced green fluorescent protein) as a reporter. The administration of a novel CSF1R blocking antibody selectively reduced the CD115(+)Gr-1(neg) monocyte precursor of resident tissue macrophages. CD115(+)Gr-1(+) inflammatory monocytes were correspondingly increased, supporting the view that monocytes are a developmental series. Within tissue, the antibody almost completely depleted resident macrophage populations in the peritoneum, gastrointestinal tract, liver, kidney, and skin, but not in the lung or female reproductive organs. CSF1R blockade reduced the numbers of tumor-associated macrophages in syngeneic tumor models, suggesting that these cells are resident type macrophages. Conversely, it had no effect on inflammatory monocyte recruitment in models, including lipopolysaccharide-induced lung inflammation, wound healing, peritonitis, and severe acute graft-versus-host disease. Depletion of resident tissue macrophages from bone marrow transplantation recipients actually resulted in accelerated pathology and exaggerated donor T-cell activation. The data indicate that CSF1R signaling is required only for the maturation and replacement of resident-type monocytes and tissue macrophages, and is not required for monocyte production or inflammatory function.

Surface densities of ephrin‐B1 determine EphB1‐coupled activation of cell attachment through αvβ3 and α5β1 integrins

Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. Here, EphB1 regulated cell attachment by discriminating the density at which ephrin‐B1 was displayed on a reconstituted surface. EphB1–ephrin‐B1 engagement did not promote cell attachment through mechanical tethering, but did activate integrin‐mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen was mediated through αvβ3 integrin. EphB1 transfection conferred ephrin‐B1‐responsive activation of α5β1 integrin‐mediated cell attachment in human embryonic kidney cells. Activation‐competent but signaling‐defective EphB1 point mutants failed to stimulate ephrin‐B1 dependent attachment. These findings lead us to propose that EphB1 functions as a ‘ligand density sensor’ to signal integrin‐mediated cell–matrix attachment.

Molecular characterization of a family of ligands for eph-related tyrosine kinase receptors

A family of tyrosine kinase receptors related to the product of the eph gene has been described recently. One of these receptors, elk, has been shown to be expressed only in brain and testes. Using a direct expression cloning technique, a ligand for the elk receptor has been isolated by screening a human placenta cDNA library with a fusion protein containing the extracellular domain of the receptor. This isolated cDNA encodes a transmembrane protein. While the sequence of the ligand cDNA is unique, it is related to a previously described sequence known as B61. Northern blot analysis of human tissue mRNA showed that the elk ligands mRNA is 3.5 kb long and is found in placenta, heart, lung, liver, skeletal muscle, kidney and pancreas. Southern blot analysis showed that the gene is highly conserved in a wide variety of species. Both elk ligand and B61 mRNAs are inducible by tumour necrosis factor in human umbilical vein endothelial cells. In addition, both proteins show promiscuity in binding to the elk and the related hek receptors. Since these two ligand sequences are similar, and since elk and hek are members of a larger family of eph‐related receptor molecules, we refer to these ligands as LERKs (ligands for eph‐related kinases).

Nck Recruitment to Eph Receptor, EphB1/ELK, Couples Ligand Activation to c-Jun Kinase*

Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and itsDrosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK.

Human macrophage-colony stimulating factor: Alternative RNA and protein processing from a single gene

Macrophage-colony stimulating factor (M-CSF, CSF-1) has been reported to be required for the proliferation and differentiation of macrophages from hematopoietic progenitor cells. Recently, two human M-CSF cDNA clones were isolated encoding proteins of 256 and 554 amino acids. We report here the isolation of a third M-CSF cDNA that encodes a protein of 438 amino acids. The coding regions for the three cDNA clones share a common amino-terminus of 149 amino acids and a common carboxyl-terminus of 75 amino acids including a membrane spanning region. In addition, we isolated a genomic clone of human M-CSF. When each of the cDNA clones or the genomic clone were transfected into COS-7 monkey kidney cells, biologically active M-CSF was expressed as judged by the ability of transfected cell supernatants to stimulate proliferation and colony formation of murine bone marrow cells, as well as formation of monocytic colonies from human bone marrow cells. Surprisingly, proliferation of human bone marrow cells was not induced by recombinant human M-CSF. Analysis of the M-CSF proteins released by COS-7 cells revealed that monomer subunit proteins of 44 or 28 kDa were produced. In addition, we found that the membrane spanning region, present in all three forms of M-CSF cDNA, was not required for the synthesis of a biologically active protein. However, when the membrane spanning region was present in the three M-CSF cDNAs, cell surface associated forms of M-CSF could be readily detected.

Molecular characterization of receptors for human interleukin-8, GRO/melanoma growth-stimulatory activity and neutrophil activating peptide-2

Interleukin-8 (IL-8), neutrophil activating peptide-2 (NAP-2), and growth regulated gene (GRO, also known as melanoma growth stimulatory activity) are members of a family of peptides which are chemotactic agents for inflammatory cells such as neutrophils. Receptors have been identified for IL-8, GRO and NAP-2 on human neutrophils and granulocytic cell lines, and it has been observed that these cytokines can cross-compete for binding to a common receptor. Using the recently characterized rabbit IL-8 receptor as a probe, two classes of cDNAs, termed type 1 and type 2, were isolated from a human neutrophil library. The type 1 receptor binds only IL-8 while the type 2 receptor binds IL-8, GRO and NAP-2 at high affinity when respective cDNAs are expressed in COS-7 cells. The two cDNAs encode proteins that have an amino acid sequence identity of 77% while the type 1 and 2 receptors have an identity of 84 and 74% with the rabbit IL-8 receptor. These receptors also show significant homology with receptors for other chemotactic agents and with potential coding regions from the human cytomegalovirus genome.

EphB1 recruits c-Src and p52Shc to activate MAPK/ERK and promote chemotaxis.

Eph receptors and their ligands (ephrins) play an important role in axonal guidance, topographic mapping, and angiogenesis. The signaling pathways mediating these activities are starting to emerge and are highly cell- and receptor-type specific. Here we demonstrate that activated EphB1 recruits the adaptor proteins Grb2 and p52Shc and promotes p52Shc and c-Src tyrosine phosphorylation as well as MAPK/extracellular signal–regulated kinase (ERK) activation. EphB1-mediated increase of cell migration was abrogated by the MEK inhibitor PD98059 and Src inhibitor PP2. In contrast, cell adhesion, which we previously showed to be c-jun NH2-terminal kinase (JNK) dependent, was unaffected by ERK1/2 and Src inhibition. Expression of dominant-negative c-Src significantly reduced EphB1-dependent ERK1/2 activation and chemotaxis. Site-directed mutagenesis experiments demonstrate that tyrosines 600 and 778 of EphB1 are required for its interaction with c-Src and p52Shc. Furthermore, phosphorylation of p52Shc by c-Src is essential for its recruitment to EphB1 signaling complexes through its phosphotyrosine binding domain. Together these findings highlight a new aspect of EphB1 signaling, whereby the concerted action of c-Src and p52Shc activates MAPK/ERK and regulates events involved in cell motility.

Cloning, sequence and expression of bovine interleukin 1α and interleukin 1β complementary DNAs

Abstract Interleukin 1 (IL-1) is a cytokine which mediates a variety of immunoregulatory and inflammatory activities. Using human IL-1α and IL-1β probes, cDNAs for the corresponding bovine genes were isolated from an alveolar macrophage library. The open reading frames of the bovine IL-1α and IL-1β cDNAs encode proteins of 268 and 266 amino acids, respectively, each with a predicted mol. wt of approx. 31,000. Both forms of bovine IL-1 exhibit a high degree of sequence homology with IL-1 gene products from other mammalian species. Based upon comparisons with human IL-1 amino acid sequences, the post-translationally processed, mature forms of bovine IL-1 would occur as 17–18,000 mol. wt proteins. Sequences encoding mature bovine IL-1α and IL-1β were inserted into E. coli expression plasmids and biologically active proteins were synthesized as judged by the ability of the recombinant proteins to induce proliferation of bovine thymocytes. Both IL-1α and IL-1β exist as single genomic copies. In addition, bovine IL-1β mRNA is approx. 10-fold more abundant than IL-1α mRNA in stimulated alveolar macrophages.