Laszlo G. Komuves

A Novel Interleukin-17 Receptor-like Protein Identified in Human Umbilical Vein Endothelial Cells Antagonizes Basic Fibroblast Growth Factor-induced Signaling

We have previously utilized a combination of high throughput sequencing and genome-wide microarray profiling analyses to identify novel cell-surface proteins expressed in human umbilical vein endothelial cells. One gene identified by this approach encodes a type I transmembrane receptor that shares sequence homology with the intracellular domain of members of the interleukin-17 (IL-17) receptor family. Real-time quantitative PCR and Northern analyses revealed that this gene is highly expressed in human umbilical vein endothelial cells and in several highly vascularized tissues such as kidney, colon, skeletal muscle, heart, and small intestine. In addition, we also found that it is also highly expressed in the ductal epithelial cells of human salivary glands, seminal vesicles, and the collecting tubules of the kidney by in situ hybridization. This putative receptor, which we have termed human SEF (hSEF), is also expressed in a variety of breast cancer tissues. In co-immunoprecipitation assays, this receptor is capable of forming homomeric complexes and can interact with fibroblast growth factor (FGF) receptor 1. Overexpression of this receptor inhibits FGF induction of an FGF-responsive reporter gene in human 293T cells. This appears to occur as a result of specific inhibition of p42/p44 ERK in the absence of upstream MEK inhibition. This inhibitory effect is dependent upon a functional intracellular domain since deletion mutants missing the IL-17 receptor-like domain lack this inhibitory effect. These findings are consistent with the recent discovery of the zebrafish homologue, Sef (similar expression to fgf genes), which specifically antagonizes FGF signaling when ectopically expressed in zebrafish or Xenopus laevis embryos. Based on sequence and functional similarities, this novel IL-17 receptor homologue represents a potential human SEF and is likely to play critical roles in endothelial or epithelial functions such as proliferation, migration, and angiogenesis.

PDGF C Is A Selective α Platelet-Derived Growth Factor Receptor Agonist That Is Highly Expressed in Platelet α Granules and Vascular Smooth Muscle

Objective—The platelet-derived growth factor (PDGF) family consists of four members, PDGF A, PDGF B, and 2 new members, PDGF C and PDGF D, which signal through the &agr; and &bgr; PDGF receptor (PDGFR) tyrosine kinases. This study was performed to determine the receptor specificity and cellular expression profile of PDGF C. Methods and Results—PDGF C growth factor domain (GFD) was shown to preferentially bind and activate &agr; PDGFR and activate &bgr; PDGFR when it is co-expressed with &agr; PDGFR through heterodimer formation. An investigation of PDGF C mRNA and protein expression revealed that during mouse fetal development, PDGF C was expressed in the mesonephric mesenchyme, prefusion skeletal muscle, cardiac myoblasts, and in visceral and vascular smooth muscle, whereas in adult human tissues expression was largely restricted to smooth muscle. Microarray analysis of various cell types showed PDGF C expression in vascular smooth muscle cells, renal mesangial cells, and platelets. PDGF C mRNA expression in platelets was confirmed by real-time polymerase chain reaction, and PDGF C protein was localized in &agr; granules by immuno-gold electron microscopy. Western blot analysis of platelets identified 55-kDa and 80-kDa PDGF C isoforms that were secreted on platelet activation. Conclusions—Taken together, our results demonstrated for the first time to our knowledge that like PDGF A and B, PDGF C is likely to play a role in platelet biology.

Enhancing the antitumor efficacy of a cell-surface death ligand by covalent membrane display

Significance A recombinant soluble version of the transmembrane death ligand Apo2L/TRAIL has shown compelling preclinical results as a potential cancer therapeutic, but studies in cancer patients have demonstrated little efficacy. Supported membrane display of Apo2L/TRAIL, to mimic the endogenous ligand more faithfully, markedly augments receptor clustering and apoptosis stimulation in cancer cells. Covalent attachment of Apo2L/TRAIL to the surface of liposomes offers a therapeutically tractable approach to membrane display that substantially increases tumor exposure, caspase activation, and antitumor potency. These findings open new avenues for clinical investigation of Apo2L/TRAIL as a cancer therapeutic and may apply to other members of the TNF superfamily, such as FasL and CD70, which are expressed on immune-cell surfaces and are important candidates for cancer immunotherapy. TNF superfamily death ligands are expressed on the surface of immune cells and can trigger apoptosis in susceptible cancer cells by engaging cognate death receptors. A recombinant soluble protein comprising the ectodomain of Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) has shown remarkable preclinical anticancer activity but lacked broad efficacy in patients, possibly owing to insufficient exposure or potency. We observed that antibody cross-linking substantially enhanced cytotoxicity of soluble Apo2L/TRAIL against diverse cancer cell lines. Presentation of the ligand on glass-supported lipid bilayers enhanced its ability to drive receptor microclustering and apoptotic signaling. Furthermore, covalent surface attachment of Apo2L/TRAIL onto liposomes—synthetic lipid-bilayer nanospheres—similarly augmented activity. In vivo, liposome-displayed Apo2L/TRAIL achieved markedly better exposure and antitumor activity. Thus, covalent synthetic-membrane attachment of a cell-surface ligand enhances efficacy, increasing therapeutic potential. These findings have translational implications for liposomal approaches as well as for Apo2L/TRAIL and other clinically relevant TNF ligands.

Membrane display and functional analysis of juxtacrine ligand-receptor signaling.

We developed a strategy for identifying modulators of juxtacrine signaling, triggered by a cell-surface ligand displayed on synthetic lipid bilayers, via cognate receptors on apposed cells. Using readouts for receptor lateral transport and intracellular signaling, we screened a small interfering RNA (siRNA) library and identified specific receptor tyrosine kinases (RTKs) that directly or indirectly modulate apoptosis signaling by a model death ligand through its cognate death receptors. This approach may be broadly useful for studying juxtacrine cell-cell signaling systems.