Netrin-1 inhibits leukocyte migration in vitro and in vivo.

Cell migration plays important roles in embryonic development and inflammation, and this process is highly regulated to ensure tissue homeostasis. A number of barriers exist to prevent the inappropriate migration of leukocytes into healthy peripheral tissues, including retention of these cells in the inactive state and maintenance of the integrity and charge of the vascular endothelium. However, active signals also are likely to exist that can repulse cells or abolish existing cell migration. One such paradigm exists in the developing nervous system, where neuronal migration is mediated by a balance between chemoattractive and chemorepulsive signals. The ability of the guidance molecule netrin-1 to repulse or abolish attraction of neuronal cells expressing the UNC5b receptor makes it an attractive candidate for the regulation of inflammatory cell migration. Here, we show that netrin-1 is expressed on vascular endothelium, where it is regulated by infection and inflammatory cytokines. The netrin-1 receptor UNC5b is strongly expressed by leukocytes, upon which netrin-1 acts as a potent inhibitor of migration to different chemotactic stimuli both in vivo and in vitro. These data suggest that endothelial expression of netrin-1 may inhibit basal cell migration into tissues and that its down-regulation with the onset of sepsis/inflammation may facilitate leukocyte recruitment.

Retinoic acid decreases fetal lung mesenchymal cell proliferation in vivo and in vitro.

Retinoic acid (RA) is an important coordinator of mammalian organogenesis. RA is implicated in critical lung developmental events. Cell proliferation is precisely regulated during development. We investigated the effect of RA on proliferating mesenchymal cells in both whole organ lung cultures and cell cultures. The potential pathways required for the response were studied in cultures of lung mesenchymal cells from embryonic day (e) 12. We observed an RA-dependent reduction in proliferation of mesenchymal cells in both whole organ and in cell culture. In mesenchymal cell cultures, RA decreased proliferation in lung mesenchymal cells by 72%. This was associated with a decrease of erk-1/2 activity by 68%. Mesenchymal cell proliferation is erk-1/2 dependent. Erk-1/2 can be activated by G-protein coupled receptors (GPCR) or tyrosine kinase receptors (RTK). RA treatment altered both the RTK and the GPCR pathways in primary lung mesenchymal cells. The Epidermal Growth Factor (EGF) dependent erk-1/2 activation was increased by 35% whereas the G(i)-protein cascade was inhibited by 44% in cells treated with RA. Our results suggest that RA decreases proliferation of lung mesenchyme via a G(i)-protein and the erk-1/2 signaling cascade.

Expression of Netrin-1 and its two receptors DCC and UNC5H2 in the developing mouse lung.

The ligand Netrin-1 and its receptors DCC and UNC5H2 are critical for the regulation of neuronal migration in nervous system development. Here we demonstrate expression of these molecules in lung development. The mRNA expression profiles of Netrin-1, DCC and UNC5H2 are developmentally regulated during embryonic mouse lung formation. Netrin-1 shows a bimodal expression pattern with elevated mRNA levels early followed by a second peak in late gestation. Peak expression of DCC occurs early in development whereas expression of UNC5H2 peaks late in development. We also demonstrate localization of Netrin-1, DCC and UNC5H2 during the stages of lung development. We present evidence that these proteins are modulated spatially in the mesenchyme and epithelium during lung organogenesis.

Slit and robo: expression patterns in lung development.

First described as an axonal guidance cue through its repulsive effect on neurons expressing its receptor Roundabout (Robo), the Slit ligand has effects on cell migration, axon branching and elongation. Indirect evidence implicates Slit and Robo in lung development. We now demonstrate that Slit-2 and Slit-3 are developmentally regulated in embryonic murine lung. Immunohistochemistry demonstrates Slit-2 and Slit-3 expression by the pulmonary mesenchyme and airway epithelium. Robo-1 and Robo-2 are also expressed by the developing mesenchyme and airway epithelium. As lung development progresses, Robo-1 and Robo-2 expression localizes to only the airway epithelium. We conclude Slit/Robo are expressed in temporo-spatially adjacent domains suggesting interactive roles in pulmonary bronchiolar development.

Modulation of G(ialpha(2)) signaling by the axonal guidance molecule UNC5H2.

The G protein, G(ialpha(2)), regulates a number of cellular functions including cell migration, proliferation, and differentiation. The transduction of signal depends on the ability of the alpha subunit to cycle between a GDP bound and an active GTP bound state capable of interacting with intracellular enzymes. Here, we now report the novel interaction of gip2 (constitutively activated G(ialpha(2))) with the cytoplasmic domain of UNC5H2. Like G(ialpha(2)), we found that UNC5H2 is widely expressed particularly in cells which migrate. UNC5H2 binds G(ialpha(2)) when it is charged with GTP. The interaction of G(ialpha(2)) and UNC5H2 liberated adenylyl cyclase from G(ialpha(2)) inhibition. Thus, by sequestering the alpha subunit, UNC5H2 is a novel inhibitor of G(ialpha(2)) thereby increasing intracellular cAMP levels. The expression of UNC5H2 in the brain and immune system suggests that this novel inhibitor of G protein signaling may have broad significance for axonal guidance and chemotaxis.