Barbara Moepps

Collective Chemotaxis Requires Contact-Dependent Cell Polarity

Summary Directional collective migration is now a widely recognized mode of migration during embryogenesis and cancer. However, how a cluster of cells responds to chemoattractants is not fully understood. Neural crest cells are among the most motile cells in the embryo, and their behavior has been likened to malignant invasion. Here, we show that neural crest cells are collectively attracted toward the chemokine Sdf1. While not involved in initially polarizing cells, Sdf1 directionally stabilizes cell protrusions promoted by cell contact. At this cell contact, N-cadherin inhibits protrusion and Rac1 activity and in turn promotes protrusions and activation of Rac1 at the free edge. These results show a role for N-cadherin during contact inhibition of locomotion, and they reveal a mechanism of chemoattraction likely to function during both embryogenesis and cancer metastasis, whereby attractants such as Sdf1 amplify and stabilize contact-dependent cell polarity, resulting in directional collective migration.

Molecular Interaction between Projection Neuron Precursors and Invading Interneurons via Stromal-Derived Factor 1 (CXCL12)/CXCR4 Signaling in the Cortical Subventricular Zone/Intermediate Zone

Most cortical interneurons are generated in the subpallial ganglionic eminences and migrate tangentially to their final destinations in the neocortex. Within the cortex, interneurons follow mainly stereotype routes in the subventricular zone/intermediate zone (SVZ/IZ) and in the marginal zone. It has been suggested that interactions between invading interneurons and locally generated projection neurons are implicated in the temporal and spatial regulation of the invasion process. However, so far experimental evidence for such interactions is lacking. We show here that the chemokine stromal-derived factor 1 (SDF-1; CXCL12) is expressed in the main invasion route for cortical interneurons in the SVZ/IZ. Most SDF-1-positive cells are proliferating and express the homeodomain transcription factors Cux1 and Cux2. Using MASH-1 mutant mice in concert with the interneuron marker DLX, we exclude that interneurons themselves produce the chemokine in an autocrine manner. We conclude that the SDF-1-expressing cell population represents the precursors of projection neurons during their transition and amplification in the SVZ/IZ. Using mice lacking the SDF-1 receptor CXCR4 or Pax6, we demonstrate that SDF-1 expression in the cortical SVZ/IZ is essential for recognition of this pathway by interneurons. These results represent the first evidence for a molecular interaction between precursors of projection neurons and invading interneurons during corticogenesis.

Expression and Regulation of the Orphan Receptor RDC1 and Its Putative Ligand in Human Dendritic and B Cells

Based on phylogenetic analysis and chromosomal mapping, the orphan receptor RDC1 was proposed to be a chemokine receptor. In this study we examined the expression of RDC1 on leukocytes by measuring mRNA levels and receptor expression using a new specific mAb. Both mRNA and protein levels were high in monocytes and B cells, relatively low on immature dendritic cells (DC), and up-regulated during final stages of maturation. Strikingly, in mature plasmacytoid DC the mRNA was up-regulated, but did not correlate with protein surface expression. We indeed report that CpG-activated plasmacytoid DC produce a putative ligand for RDC1, which selectively down-regulates RDC1, but not CXCR4 on primary human B cells. RDC1 expression was found to be tightly regulated during B cell development and differentiation. In blood-derived switch memory B cells, the expression of RDC1 appeared to correlate with the ability to differentiate into plasma cells upon activation, suggesting that RDC1 is a marker for memory B cells, which are competent to become Ab-secreting cells.

A New Missense Mutation in the Leptin Gene Causes Mild Obesity and Hypogonadism without Affecting T Cell Responsiveness

OBJECTIVE Leptin, a protein product of adipocytes, plays a critical role in the regulation of body weight, immune function, pubertal development, and fertility. So far, only three homozygous mutations in the leptin gene in a total of 13 individuals have been found leading to a phenotype of extreme obesity with marked hyperphagia and impaired immune function. DESIGN Serum leptin was measured by ELISA. The leptin gene (OB) was sequenced in patient DNA. The effect of the identified novel mutation was assessed using HEK293 cells. RESULTS We describe a 14-yr-old child of nonobese Austrian parents without known consanguinity. She had a body mass index of 31.5 kg/m(2) (+2.46 SD score) and undetectable leptin serum levels. Sequencing of the leptin gene revealed a hitherto unknown homozygous transition (TTA to TCA) in exon 3 of the LEP gene resulting in a L72S replacement in the leptin protein. RT-PCR, Western blot, and immunohistochemical analysis indicated that the mutant leptin was expressed in the patients adipose tissue but retained within the cell. Using a heterologous cell system, we confirmed this finding and demonstrated that the side chain of Leu72 is crucial for intracellular leptin trafficking. Our patient showed signs of a hypogonadotropic hypogonadism. However, in contrast to the literature, she showed only mild obesity and a normal T cell responsiveness. CONCLUSIONS These findings shed a new light on the clinical consequences of leptin deficiency. Congenital leptin deficiency should be considered possible in pediatric patients with mild obesity even if parents are lean and unrelated.

Identification of the First Nonpeptidergic Inverse Agonist for a Constitutively Active Viral-encoded G Protein-coupled Receptor

Human cytomegalovirus (HCMV) encodes a G protein-coupled receptor (GPCR), named US28, which shows homology to chemokine receptors and binds several chemokines with high affinity. US28 induces migration of smooth muscle cells, a feature essential for the development of atherosclerosis, and may serve as a co-receptor for human immunodeficiency virus-type 1 entry into cells. Previously, we have shown that HCMV-encoded US28 displays constitutive activity, whereas its mammalian homologs do not. In this study we have identified a small nonpeptidergic molecule (VUF2274) that inhibits US28-mediated phospholipase C activation in transiently transfected COS-7 cells and in HCMV-infected fibroblasts. Moreover, VUF2274 inhibits US28-mediated HIV entry into cells. In addition, VUF2274 fully displaces radiolabeled RANTES (regulated on activation normal T cell expressed and secreted) binding at US28, apparently with a noncompetitive behavior. Different analogues of VUF2274 have been synthesized and pharmacologically characterized, to understand which features are important for its inverse agonistic activity. Finally, by means of mutational analysis of US28, we have identified a glutamic acid in transmembrane 7 (TM 7), which is highly conserved among chemokine receptors, as a critical residue for VUF2274 binding to US28. The identification of a full inverse agonist provides an important tool to investigate the relevance of US28 constitutive activity in viral pathogenesis.

Mice deficient in the chemokine receptor CXCR4 exhibit impaired limb innervation and myogenesis.

The chemokine CXCL12/SDF-1 and its receptor CXCR4 regulate the development and the function of the hematopoietic system and control morphogenesis of distinct brain areas. Here, we demonstrate that inactivation of CXCR4 results in a massive loss of spinal cord motoneurons and dorsal root ganglion neurons and, subsequently, in a reduced innervation of the developing mouse fore- and hindlimbs. However, only the death of sensory neurons seems to be a direct consequence of receptor inactivation as suggested by the observations that DRG neurons, but not motoneurons, of wild-type animals express CXCR4 and respond to CXCL12 with an increase in cell survival. In contrast, the increased death of motoneurons in CXCR4-deficient animals seems to result from impaired limb myogenesis and a subsequent loss of muscle-derived neurotrophic support. In summary, our findings unravel a previously unrecognized complex role of CXCL12/CXCR4 in the control of limb neuromuscular development.

Interleukin-6 and cAMP Induce Stromal Cell-derived Factor-1 Chemotaxis in Astroglia by Up-regulating CXCR4 Cell Surface Expression IMPLICATIONS FOR BRAIN INFLAMMATION

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 control the migration of neurons and microglial cells in the central nervous system. Although functional CXCR4 is also expressed by astroglia, recent studies have failed to observe a chemotactic response of these cells to SDF-1. Here, we demonstrate that SDF-1-dependent chemotaxis can be induced by treating cultured cortical astroglia with either dibutyryl cyclic AMP (dbcAMP; 10−4 m) or interleukin-6 (IL-6; 10 ng/ml). Flow cytometric analysis revealed that both the dbcAMP- and IL-6-induced onset of SDF-1-dependent chemotaxis of astroglia are due to the increased cell surface expression of CXCR4. In addition, dbcAMP and IL-6 also increased CXCR4 transcript levels, further suggesting that both treatments primarily affect CXCR4 surface expression in astroglia by stimulation of gene expression. Moreover, unlike the case with IL-6 and dbcAMP, which allowed for an optimal chemotactic response to SDF-1 only after 48 h, a similar chemotactic response, associated with an increase in CXCR4 cell surface expression, already occurred after 24 h when astroglial cultures were maintained with medium conditioned by IL-6- or dbcAMP-pretreated astrocytes, indicating that the stimulatory effects of IL-6 and cAMP on CXCR4 cell surface expression involve a secondary mechanism. The findings that elevated extracellular levels of IL-6 or factors positively coupled to cAMP result in increased CXCR4 cell surface expression levels and subsequent SDF-1-dependent chemotaxis in central nervous system astrocytes point to a crucial role of this chemokine during reactive gliosis and human immunodeficiency virus-mediated dementia.

Isozyme-specific Stimulation of Phospholipase C-γ2 by Rac GTPases

The regulation of the two isoforms of phospholipase C-γ, PLCγ1 and PLCγ2, by cell surface receptors involves protein tyrosine phosphorylation as well as interaction with adapter proteins and phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) generated by inositol phospholipid 3-kinases (PI3Ks). All three processes may lead to recruitment of the PLCγ isozymes to the plasma membrane and/or stimulation of their catalytic activity. Recent evidence suggests that PLCγ may also be regulated by Rho GTPases. In this study, PLCγ1 and PLCγ2 were reconstituted in intact cells and in a cell-free system with Rho GTPases to examine their influence on PLCγ activity. PLCγ2, but not PLCγ1, was markedly activated in intact cells by constitutively active Rac1G12V, Rac2G12V, and Rac3G12V but not by Cdc42G12V and RhoAG14V. The mechanism of PLCγ2 activation was apparently independent of phosphorylation of tyrosine residues known to be modified by PLCγ2-activating protein-tyrosine kinases. Activation of PLCγ2 by Rac2G12V in intact cells coincided with a translocation of PLCγ2 from the soluble to the particulate fraction. PLCγ isozyme-specific activation of PLCγ2 by Rac GTPases (Rac1 ≈ Rac2 > Rac3), but not by Cdc42 or RhoA, was also observed in a cell-free system. Herein, activation of wild-type Rac GTPases with guanosine 5′-(3-O-thio)triphosphate caused a marked stimulation of PLCγ2 but had no effect on the activity of PLCγ1. PLCγ1 and PLCγ2 have previously been shown to be indiscriminately activated by PtdInsP3 in vitro. Thus, the results suggest a novel mechanism of PLCγ2 activation by Rac GTPases involving neither protein tyrosine phosphorylation nor PI3K-mediated generation of PtdInsP3.

Biologically Inactive Leptin and Early-Onset Extreme Obesity

Mutations in the gene encoding leptin (LEP) typically lead to an absence of circulating leptin and to extreme obesity. We describe a 2-year-old boy with early-onset extreme obesity due to a novel homozygous transversion (c.298G→T) in LEP, leading to a change from aspartic acid to tyrosine at amino acid position 100 (p.D100Y) and high immunoreactive levels of leptin. Overexpression studies confirmed that the mutant protein is secreted but neither binds to nor activates the leptin receptor. The mutant protein failed to reduce food intake and body weight in leptin-deficient ob/ob mice. Treatment of the patient with recombinant human leptin (metreleptin) rapidly normalized eating behavior and resulted in weight loss.

Constitutive Inositol Phosphate Formation in Cytomegalovirus-Infected Human Fibroblasts Is due to Expression of the Chemokine Receptor Homologue pUS28

ABSTRACT An open reading frame (ORF), US28, with homology to mammalian chemokine receptors has been identified in the genome of human cytomegalovirus (HCMV). Its protein product, pUS28, has been shown to bind several human CC chemokines, including RANTES, MCP-1, and MIP-1α, and the CX3C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxin-sensitive increase in the concentration of cytosolic free Ca2+. Recently, pUS28 was shown to mediate constitutive, ligand-independent, and pertussis toxin-insensitive activation of phospholipase C via Gq/11-dependent signaling pathways in transiently transfected COS-7 cells. Since these findings are not easily reconciled with the former observations, we analyzed the role of pUS28 in mediating CC chemokine activation of pertussis toxin-sensitive G proteins in cell membranes and phospholipase C in intact cells. The transmembrane signaling functions of pUS28 were studied in HCMV-infected cells rather than in cDNA-transfected cells. Since DNA sequence analysis of ORF US28 of different laboratory and clinical strains had revealed amino acid sequence differences in the amino-terminal portion of pUS28, we compared two laboratory HCMV strains, AD169 and Toledo, and one clinical strain, TB40/E. The results showed that infection of human fibroblasts with all three HCMV strains led to a vigorous, constitutively enhanced formation of inositol phosphates which was insensitive to pertussis toxin. This effect was critically dependent on the presence of the US28 ORF in the HCMV genome but was independent of the amino acid sequence divergence of the three HCMV strains investigated. The constitutive activity of pUS28 is not explained by expression of pUS28 at high density in HCMV-infected cells. The pUS28 ligands RANTES and MCP-1 failed to stimulate binding of guanosine 5′-O-(3-[35S]thiotriphosphate to membranes of HCMV-infected cells and did not enhance constitutive activation of phospholipase C in intact HCMV-infected cells. These findings raise the possibility that the effects of CC chemokines and pertussis toxin on G protein-mediated transmembrane signaling previously observed in HCMV-infected cells are either independent of or not directly mediated by the protein product of ORF US28.