Full-length and fragmented netrin-1 in multiple sclerosis plaques are inhibitors of oligodendrocyte precursor cell migration.

Oligodendrocytes exhibit a limited capacity to remyelinate in multiple sclerosis. Factors present in multiple sclerosis lesions are thought to inhibit oligodendrocyte precursor cell migration, limiting their recruitment to axons requiring remyelination; however, few inhibitors have been identified. A candidate inhibitor is netrin-1, a secreted protein that repels migrating oligodendrocyte precursor cells during neural development and is expressed by myelinating oligodendrocytes in the mature rodent central nervous system. Herein, we examined the distribution of netrin-1 in adult human white matter and multiple sclerosis lesions. We detected full-length netrin-1 protein and shorter netrin-1 fragments in samples of normal white matter and of multiple sclerosis lesions from adult human brain. We demonstrate that peptides corresponding to amino terminal domains VI and V of netrin-1 repel migrating oligodendrocyte precursor cells, but lack the chemoattractant activity of full-length netrin-1. Furthermore, recombinant domains VI-V of netrin-1 disrupt the chemoattractant activity of full-length netrin-1, consistent with a competitive mechanism of action. These findings indicate that full-length and fragmented forms of netrin-1, found in multiple sclerosis lesions, have the capacity to inhibit oligodendrocyte precursor migration, identifying netrin-1 as a potential target for therapies that promote remyelination.

Rb/E2F regulates expression of neogenin during neuronal migration.

The Rb/E2F pathway has long been appreciated for its role in regulating cell cycle progression. Emerging evidence indicates that it also influences physiological events beyond regulation of the cell cycle. We have previously described a requirement for Rb/E2F mediating neuronal migration; however, the molecular mechanisms remain unknown, making this an ideal system to identify Rb/E2F-mediated atypical gene regulation in vivo. Here, we report that Rb regulates the expression of neogenin, a gene encoding a receptor involved in cell migration and axon guidance. Rb is capable of repressing E2F-mediated neogenin expression while E2F3 occupies a region containing E2F consensus sites on the neogenin promoter in native chromatin. Absence of Rb results in aberrant neuronal migration and adhesion in response to netrin-1, a known ligand for neogenin. Increased expression of neogenin through ex vivo electroporation results in impaired neuronal migration similar to that detected in forebrain-specific Rb deficiency. These findings show direct regulation of neogenin by the Rb/E2F pathway and demonstrate that regulation of neogenin expression is required for neural precursor migration. These studies identify a novel mechanism through which Rb regulates transcription of a gene beyond the classical E2F targets to regulate events distinct from cell cycle progression.

CrkI and CrkII function as key signaling integrators for migration and invasion of cancer cells.

Crk adaptor proteins play an important role during cellular signaling by mediating the formation of protein complexes. Increased levels of Crk proteins are observed in several human cancers and overexpression of Crk in epithelial cell cultures promotes enhanced cell dispersal and invasion, implicating Crk as a regulator of invasive responses. To determine the requirement of Crk for invasive signals, we targeted the CRKI/II gene by RNA interference. Consistent knockdown of CrkI/II was observed with two small interfering RNA targeting sequences in all human cancer cell lines tested. CrkI/II knockdown resulted in a significant decrease in migration and invasion of multiple malignant breast and other human cancer cell lines (MDA-231, MDA-435s, H1299, KB, and HeLa). Moreover, CrkI/II knockdown decreased cell spreading on extracellular matrix and led to a decrease in actin stress fibers and the formation of mature focal adhesions. Using immunohistochemistry, we show elevated CrkI/II protein levels in patients with breast adenocarcinoma. Together, these studies identify Crk adaptor proteins as critical integrators of upstream signals for cell invasion and migration in human cancer cell lines and support a role for Crk in metastatic spread.

Inhibition of Salmonella typhimurium enteropathogenicity by piperidine, a metabolite of the polyamine cadaverine.

Piperidine is a 1-ring heterocyclic compound formed from the polyamine cadaverine in the human intestine. Because heterocyclic compounds are routinely used in the promotion of antimicrobial treatment strategies, it was considered whether piperidine could be used against infection with enteric pathogens. This study demonstrates that piperidine treatment prevented the invasion of Salmonella typhimurium into model intestinal epithelium by nearly 95%. In vivo studies also revealed that it increased mouse survival and reduced S. typhimurium translocation into and colonization of various organs and tissues. Initial evaluations demonstrated that piperidine reduced the S. typhimurium-induced polymorphonuclear leukocyte transepithelial migration response in vitro by inhibiting activation of protein kinase C. Piperidine did not affect the ability of S. typhimurium to elicit interleukin-8 secretion by epithelial cells or to activate extracellular-regulated kinase signal transduction pathways. These results show that piperidine does not exhibit paninhibitory activity and suggest that piperidine may be useful in down-regulating active inflammation at mucosal surfaces.

Shigella flexneri Interactions with the Basolateral Membrane Domain of Polarized Model Intestinal Epithelium: Role of Lipopolysaccharide in Cell Invasion and in Activation of the Mitogen-Activated Protein Kinase ERK.

An early step governing Shigella flexneri pathogenesis is the invasion of the colonic epithelium from the basolateral surface followed by disruption of the colonic epithelial barrier. Despite recent insight into S. flexneri-host interactions, much remains to be determined regarding the nature of the initial contact between S. flexneri and the host epithelial basolateral membrane domain. Since the lipopolysaccharide (LPS) is located at the outermost part of the bacterial membrane, we considered that this component might be used by S. flexneri to attach to the basolateral surface of the intestinal epithelium and promote a proinflammatory response. Therefore, polarized human T84 intestinal epithelial cells were infected from the basolateral surface with either wild-type S. flexneri or one of its isogenic LPS-defective strains with mutations in either rfc, rfaL, or galU. We found that both adherence to and internalization into the basolateral surface of a polarized intestinal epithelium with S. flexneri were highly dependent on the length of the LPS (i.e., rfc > rfaL > galU). Furthermore, the addition of the anti-inflammatory LPS (RsDPLA) considerably decreased the invasion profile of wild-type S. flexneri by nearly 50%. Since LPS is associated with host inflammation, we further examined whether this molecule was involved in Shigella-induced inflammatory events. We found that S. flexneri LPS plays an important role in mediating epithelial-derived signaling, which leads to directed migration of polymorphonuclear leukocytes across model intestinal epithelium. This signaling most likely involves the activation of the mitogen-activated protein kinase extracellular regulated kinase. Thus, our findings have important implications on the understanding of the mechanisms by which S. flexneri can elicit mucosal inflammation.