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LIM kinases 1 and 2 (LIMK1/2) are centrally positioned regulators of actin cytoskeleton dynamics. Using siRNA-mediated knockdown or a novel small molecule inhibitor, we show LIMK is required for path generation by leading tumor cells and nontumor stromal cells during collective tumor cell invasion. LIMK inhibition lowers cofilin phosphorylation, F-actin levels, serum response factor transcriptional activity and collagen contraction, and reduces invasion in three-dimensional invasion assays. Although motility was unaffected, LIMK inhibition impairs matrix protein degradation and invadopodia formation associated with significantly faster recovery times in FRAP assays indicative of reduced F-actin stability. When LIMK is knocked down in MDA-MB-231 cells, they lose the ability to lead strands of collectively invading cells. Similarly, when LIMK activity is blocked in cancer-associated fibroblasts, they are unable to lead the collective invasion of squamous carcinoma cells in an organotypic skin model. These results show that LIMK is required for matrix remodeling activities for path generation by leading cells in collective invasion.

Replicative senescence and oxidative stress have been implicated in ageing, endothelial dysfunction and atherosclerosis. Replicative senescence is determined primarily by telomere integrity. In endothelial cells the glutathione redox-cycle plays a predominant role in the detoxification of peroxides. The aim of this study was to elucidate the role of the glutathione-dependent antioxidant system on the replicative capacity and telomere dynamics of cultured endothelial cells. Human umbilical vein endothelial cells were serially passaged while exposed to regular treatment with 0.1 microM tert-butyl hydroperoxide, a substrate of glutathione peroxidase, or 10 microM L-buthionine-[S,R]-sulphoximine, an inhibitor of glutathione synthesis. Both treatments induced intracellular oxidative stress but had no cytotoxic or cytostatic effects. Nonetheless, treated cultures entered senescence prematurely (30 versus 46 population doublings), as determined by senescence-associated beta-galactosidase staining and a sharp decrease in cell density at confluence. In cultures subjected to oxidative stress terminal restriction fragment (TRF) analysis demonstrated faster telomere shortening (110 versus 55 bp/population doubling) and the appearance of distinct, long TRFs after more than 15-20 population doublings. Fluorescence in situ hybridisation analysis of metaphase spreads confirmed the presence of increased telomere length heterogeneity, and ruled out telomeric end-to-end fusions as the source of the long TRFs. The latter was also confirmed by Bal31 digestion of genomic DNA. Similarly, upregulation of telomerase could not account for the appearance of long TRFs, as oxidative stress induced a rapid and sustained decrease in this activity. These findings demonstrate a key role for glutathione-dependent redox homeostasis in the preservation of telomere function in endothelial cells and suggest that loss of telomere integrity is a major trigger for the onset of premature senescence under mild chronic oxidative stress.

Objective: Telomerase plays a major role in the control of replicative capacity, a critical property for successful angiogenesis and maintenance of endothelial integrity. In this study, we examined the relationship between telomerase activity and endothelial cell proliferation as well as the regulation of this enzyme by fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF)., Methods and Results: Telomerase was repressed in endothelial cells freshly derived from intact endothelium, whereas activity was present during logarithmic growth in culture. In cultured human umbilical vein endothelial cells (HUVECs), mRNA levels of hTERT-the catalytic subunit of telomerase-and enzyme activity decreased reversibly on induction of quiescence. Treatment of quiescent HUVECs with FGF-2 restored telomerase activity in a time- and dose-dependent manner, whereas VEGF had no such effect, although both factors induced comparable mitogenic responses. FGF-2, but not VEGF, upregulated the mRNA levels for hTERT and for the hTERT gene transactivation factor Sp1. Serial passage in the presence of individual growth factors accelerated the accumulation of senescent cells in VEGF-treated cultures compared with cultures treated with FGF-2., Conclusions: FGF-2, but not VEGF, restores telomerase activity and maintains the replicative capacity of endothelial cells.

RYK is an atypical orphan receptor tyrosine kinase that lacks detectable kinase activity. Nevertheless, using a chimeric receptor approach, we previously found that RYK can signal via the mitogen-activated protein kinase pathway. Recently, it has been shown that murine Ryk can bind to and be phosphorylated by the ephrin receptors EphB2 and EphB3. In this study, we show that human RYK associates with EphB2 and EphB3 but is not phosphorylated by them. This association requires both the extracellular and cytoplasmic domains of RYK and is not dependent on activation of the Eph receptors. It was also previously shown that AF-6 (afadin), a PDZ domain-containing protein, associates with murine Ryk. We show here that AF-6 does not bind to human RYK in vitro or in vivo. This suggests that there are significant functional differences between human and murine RYK. Further studies are required to determine whether RYK modulates the signaling of EphB2 and EphB3.

Coffin-Lowry syndrome (CLS) is an X-linked disorder characterized by facial dysmorphism, digit abnormalities and severe psychomotor retardation. CLS had previously been mapped to Xp22.2. Recently, mutations in the ribosomal S6 kinase (Rsk-2) gene were shown to be associated with CLS. We have tested five unrelated individuals with CLS for mutations in nine exons of Rsk-2 using Single Strand Conformation Polymorphism (SSCP) analysis. Two patients had the same missense mutation (C340T), which causes an arginine to tryptophan change (R114W). This mutation falls just outside the N-terminal ATP-binding site in a highly conserved region of the protein and may lead to structural changes since tryptophan has an aromatic side chain whereas arginine is a 5 carbon basic amino acid. The third patient also had a missense mutation (G2186A) resulting in an arginine to glutamine change (R729Q). The fourth patient had a 2bp deletion (AG) of bases 451 and 452. This creates a frameshift that results in a stop codon 25 amino acids downstream, thereby producing a truncated protein. This deletion also falls within the highly conserved amino-catalytic domain of the protein. The fifth patient has a nonsense mutation (C2065T) which results in a premature stop codon, thereby producing a truncated protein. These mutations further confirm Rsk-2 as the gene involved in CLS and may help in understanding the structure and function of the protein.

The Coffin-Lowry syndrome (CLS), an X-linked disorder, is characterized by severe psychomotor retardation, facial and digital dysmorphisms, and progressive skeletal deformations. Genetic linkage analysis mapped the CLS locus to an interval of 2-3 megabases at Xp22.2. The gene coding for Rsk-2, a member of the growth-factor-regulated protein kinases, maps within the candidate interval, and was tested as a candidate gene for CLS. Initial screening for mutations in the gene for Rsk-2 in 76 unrelated CLS patients revealed one intragenic deletion, a nonsense, two splice site, and two missense mutations. The two missenses affect sites critical for the function of Rsk-2. The mutated Rsk-2 proteins were found to be inactive in a S6 kinase assay. These findings provide direct evidence that abnormalities in the MAPK/RSK signalling pathway cause Coffin-Lowry syndrome.

The gene involved in juvenile retinoschisis (RS) has previously been localized, by genetic linkage analyses, to Xp22.1-p22.2, between DXS274 and DXS43/DXS207; it is closely linked to the latter markers. From our recent data, this interval represents a genetic distance of approximately 10 cM. In the present study, we have studied 14 French families with X-linked juvenile RS by using four CA polymorphisms that are closely linked to the RS locus and that have recently been included in an Xp22.1-p22.2 high-resolution map. Complete cosegregation with the disease locus was observed for three of them, DXS207, DXS418, and DXS999, which further confirms the locus homogeneity for RS and the close linkage to this region. One recombinant was found with the most proximal marker, AFM291wf5, thereby defining this marker as the new proximal boundary of the candidate region for RS. Under the assumption that DXS207 and DXS43 constitute the distal boundary, the present study further reduces the region containing the disease gene to a interval of 3-4 cM. The results reported here should facilitate the eventual cloning of the RS gene.

The genes responsible for two X-linked diseases, the Coffin-Lowry syndrome (CLS) and juvenile retinoschisis (RS), have been previously mapped, through linkage studies, to an 8-cM region, in Xp22.1-p22.2, flanked distally by two tightly linked markers, DXS207 and DXS43, and proximally by DXS274. In the present study, five Genethon markers have been assigned to the (DXS207, DXS43)-DXS274 interval using somatic cell hybrids and a meiotic breakpoint panel and ordered together with three markers previously mapped to this region. A genetic map, which includes 13 loci and spans a distance of approximately 13 cM, was derived from linkage analysis using the CEPH families. The most likely locus order and map distances (in centimorgans) are Xpter-DXS16-(3.4)-(DXS207, DXS43, DXS1053)-(2.0)-(DXS999, DXS257)-(1.7)-AFM291 wf5-(1.4) - DXS443 - (2.0) - (DXS1229, DXS365) - (2.1) - (DXS1052, DXS274, DXS41)-Xcen. Analysis of multiply informative crossovers established AFM291 wf5 and DXS1052 as new flanking markers for CLS, which significantly reduces the candidate region for this disease gene to a 4- to 5-cM interval. Three markers, DXS443, DXS1229, and DXS365, mapping within this interval showed complete cosegregation with the disease phenotype, giving a multipoint lod score of 14.2. The present map provides the framework for constructing a YAC contig for the CLS and RS region and should be useful for refining the localization of other disease genes mapping to this region. The panel of somatic cell hybrids characterized for the present study has also allowed us to refine the localization of five genes (CALB3, GRPR, PDHA1, GLRA2, and PHKA2) and two expressed sequence tags (DXS1118E and DXS1006E) previously assigned to the Xp22 region.