No abstract

Axon extension during development is guided by many factors, but the signaling mechanisms responsible for its regulation remain largely unknown. We have now investigated the role of the transmembrane protein CD47 in this process in N1E-115 neuroblastoma cells. Forced expression of CD47 induced the formation of neurites and filopodia. Furthermore, an Fc fusion protein containing the extracellular region of the CD47 ligand SHPS-1 induced filopodium formation, and this effect was enhanced by CD47 overexpression. SHPS-1-Fc also promoted neurite and filopodium formation triggered by serum deprivation. Inhibition of Rac or Cdc42 preferentially blocked CD47-induced formation of neurites and filopodia, respectively. Overexpression of CD47 resulted in the activation of both Rac and Cdc42. The extracellular region of CD47 was sufficient for the induction of neurite formation by forced expression, but the entire structure of CD47 was required for enhancement of filopodium formation by SHPS-1-Fc. Neurite formation induced by CD47 was also inhibited by a mAb to the integrin ␤3 subunit. These results indicate that the interaction of SHPS-1 with CD47 promotes neurite and filopodium formation through the activation of Rac and Cdc42, and that integrins containing the ␤3 subunit participate in the effect of CD47 on neurite formation. INTRODUCTIONThe extension of axons from neurons to their target cells during development of the nervous system is guided by a variety of environmental cues, which include diffusible chemoattractants and chemorepellents, extracellular matrix proteins, and cell adhesion molecules (Tessier-Lavigne and Goodman, 1996;Dickson, 2002). In response to these guidance cues, the growth cone of the axon produces and retracts filopodia and lamellipodia, processes that require the temporal and spatial regulation of the actin cytoskeleton. Members of the Rho family of small G proteins, including Rho, Rac, and Cdc42, are implicated as key mediators that link guidance signals to rearrangement of the actin cytoskeleton (Ridley et al., 1992;Nobes and Hall, 1995;Kozma et al., 1997;Luo et al., 1997;Hirose et al., 1998;Takai et al., 2001;Arakawa et al., 2003). Although Rac and Cdc42 promote neurite extension, Rho inhibits it or induces growth cone collapse. These proteins are also implicated in dendritic development (Threadgill et al., 1997). Although certain guidance factors, including slit, semaphorin, and ephrin, have been shown to regulate Rho family proteins (Wahl et al., 2000;Whitford and Ghosh, 2001;Wong et al., 2001), it remains unclear how other extracellular cues control neurite extension through these small G proteins.CD47, also known as integrin-associated protein (IAP), was originally identified in association with the integrin ␣v␤3 (Brown et al., 1990). It is a member of the immunoglobulin (Ig) superfamily, possessing an Ig-V-like extracellular region, five putative transmembrane domains, and a short cytoplasmic tail (Brown and Frazier, 2001). CD47 is implicated in the regulation of multiple cellular processe...

The development of axons and dendrites is controlled by small GTP-binding proteins of the Rho family, but the upstream signaling mechanisms responsible for such regulation remain unclear. We have now investigated the role of the transmembrane protein cluster of differentiation 47 (CD47) in this process with hippocampal neurons. CD47-deficient neurons manifested markedly impaired development of dendrites and axons, whereas overexpression of CD47 promoted such development. Interaction of SH2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) with CD47 also induced the formation of dendritic filopodia and spines. These effects of CD47 were prevented by inhibition of either cell division cycle 42 (Cdc42) or Rac. In CD47-deficient neurons, autophosphorylation of Src was markedly reduced. In addition, overexpression of CD47 promoted the autophosphorylation of Src. Inhibition of Src family kinases indeed prevented CD47-promoted dendritic development. Inhibition of either FGD1-related Cdc42-guanine nucleotide exchange factor (GEF) (FRG) or Vav2, which is a GEF for Cdc42 and Rac and is activated by Src, also prevented the effects of CD47 on dendritic development. These results indicate that CD47 promotes development of dendrites and axons in hippocampal neurons in a manner dependent, at least in part, on activation of Cdc42 and Rac mediated by Src as well as by FRG and Vav2.

Treg are increased in proportion in the circulation of patients with SCCHN. These cells appear to downregulate cytokine expression in both Tc1 and Tc2 subsets of CD8+ effector T cells, which may be responsible for antitumor responses.

Several previous investigators have reported that long-term administration of certain macrolides is efficacious in patients with persistent pulmonary Pseudomonas aeruginosa infections, even though the clinically achievable concentrations of these medications are far below their MICs. In the present study, we examined how sub-MICs of macrolide antibiotics affect the viability of and protein synthesis in several strains of P. aeruginosa. We report that 48 h, but not 12 or 24 h, of growth on agar containing a clinically achievable concentration of azithromycin (0.5 microgram/ml, 1/128 the MIC) significantly reduces the viability of strain PAO-1. Similar effects were seen with erythromycin and clarithromycin at 2 micrograms/ml (1/128 and 1/64 the respective MICs), whereas josamycin, oleandomycin, ceftazidime, tobramycin, minocycline, and ofloxacin had no effect on viability, even following 48 h of incubation with concentrations representing relatively high fractions of their MICs. The bactericidal activity of azithromycin seen following 48 h of incubation was not limited to strain PAO-1 but was also seen against 13 of 14 clinical isolates, including both mucoid and nonmucoid strains. Although viability was not decreased prior to 48 h, we found that 4 micrograms of azithromycin per ml inhibits protein synthesis after as little as 12 h and that protein synthesis continues to decrease in a time-dependent manner. We likewise found that P. aeruginosa accumulates azithromycin intracellulary over the period from 12 to 36 h. These results suggested that sub-MICs of certain macrolides are bactericidal to P. aeruginosa when the bacteria are exposed to these antibiotics for longer periods. Exposure-dependent intracellular accumulation of the antibiotic and inhibition of protein synthesis may partially account for the antipseudomonal activity of macrolides over relatively prolonged incubation periods.