Transcriptomic analysis of adult zebrafish heart and brain in response to 2, 6-dichloro-1, 4-benzoquinone exposure.

Halobenzoquinones (HBQs) are emerging and widespread disinfection byproducts (DBPs), but their toxicological mechanisms to aquatic organisms remain elusive. Herein, we evaluated oxidative stress, cardiac toxicity, and cerebral toxicity after 2, 6-dichloro-1, 4-benzoquinone (2,6-DCBQ) exposure in zebrafish. Adult zebrafish were respectively exposed to 0.25, 0.5, and 1 µM 2,6-DCBQ for 96 h. The mortality rate of 2,6-DCBQ (1 µM) was 10%, while the LC50 value was 1.532 µM. Besides, 2,6-DCBQ exposure caused irregularity and elimination of myocardial fiber in the heart, and the pyknosis of nuclears and the agglutination of chromatin in the brain. We measured the 2,6-DCBQ-induced oxidative stresses in the heart and brain. Additionally, the glutathione (GSH) content, superoxide dismutase (SOD) activity, catalase (CAT) activity, and total antioxidant capacity (T-AOC) were significantly inhibited. To better understand the potential toxicity of 2,6-DCBQ, transcriptomic analysis was performed in the control and 1 µM group after 96 h exposure. As a result, 545 and 1228 differentially expressed genes (DEGs) were detected in the heart and brain, respectively. GO analysis revealed that these DEGs were primarily enriched in blood vessel development, vasculature development, and oxidoreductase activity in the heart; response to stimulus, nervous system development, and oxidoreductase activity in the brain. KEGG enrichment analysis indicated that the DEGs were mainly enriched in VEGF signaling pathway and vascular smooth muscle contraction pathway in the heart; neuroactive ligand-receptor interaction, and NOD-like receptor signaling pathway in the brain. These findings exposed the underlying toxicity mechanism of 2,6-DCBQ exposure on zebrafish cardiovascular and brain systems.

Active MT1-MMP is tethered to collagen fibers in DDR2-containing remnants.

Type I collagen is a major extracellular matrix (ECM) component in the interstitial stroma of solid tumors, and it represents the first barrier against tumor cell invasion after basement-membrane degradation. The collagen receptors that convey molecular signals into the cells are collagen-binding discoidin domain receptors (DDRs) and integrins. Collagen-activated DDR2 clusters form DDR2-containing remnants in an integrin-dependent manner in three-dimensional (3D) collagen matrix. Although DDR2-containing remnants in the collagen matrix may generate sustained perturbation to ECM remodeling, the molecular components and function of the remnants are largely unknown. Here we determined the interaction and co-localization between DDR2 and membrane type I-matrix metalloproteinase (MT1-MMP) in the cells and the DDR2-containing remnants on collagen fibers, and we found that MT1-MMP was co-tethered to collagen fibers in the remnants. These collagen fiber-associated MT1-MMP remained active. Furthermore, DDR2 enhanced MT1-MMP proteolytic activity. These results demonstrate that DDR2 ensures the remnant-associated MT1-MMP to continue the degradation of ECM in addition to pericellular ECM degradation mediated by cell surface tethered MT1-MMP. Thus, our findings reveal a new alternative ECM degradation mechanism mediated by MT1-MMP in the DDR2-containing remnants.

110 Patients with adenosquamous carcinomas of the pancreas (PASC): imaging differentiation of small (≤ 3 cm) versus large (> 3 cm) tumors.

OBJECTIVE: This study examined radiological imaging features of small (≤ 3 cm) and large (> 3 cm) adenosquamous carcinomas of the pancreas (PASC) lesions to better understand the morphology of these lesions. METHODS: Images from 110 patients with pathologically proven PASC (80 males and 30 females, mean age: 62.6 years) were retrospectively reviewed. Two radiologists analyzed images and reached a consensus regarding the following features: location, shape, margins, presence of solid and necrotic components, rim enhancement, density/intensity during the portal venous phase, invasion of surrounding organs, vascular invasion, venous tumor thrombus formation, and enlarged lymph nodes. Differences in the imaging features between the two groups were evaluated with the Chi-square test or Fisher's exact test. RESULTS: There were 41 small PASC lesions (mean age: 60.59 years) and 69 large PASC lesions (63.74 years). Statistical analysis demonstrated significant differences in the location, shape, adjacent organ and vessel invasion, and venous tumor thrombus formation (P < 0.05). Small PASC lesions were more frequently detected in the pancreatic head and had an ovoid shape. There was no significant difference in the presence of solid and necrotic components (P = 0.090), including approximately 3/4 of the lesions with necrosis and 1/4 purely solid lesions, enlarged lymph nodes (P = 0.068) and other features. CONCLUSION: Regardless of the tumor size, 75% of PASC lesions present with central necrosis while 25% are purely solid. Small PASC lesions can be associated with lymph node metastasis at a relatively early stage. Large PASC lesions are likely to invade adjacent tissues and be associated with venous tumor thrombus formation.

Incorporation of DDR2 clusters into collagen matrix via integrin-dependent posterior remnant tethering.

Cell-matrix interactions play critical roles in cell adhesion, tissue remodeling and cancer metastasis. Discoidin domain receptor 2 (DDR2) is a collagen receptor belonging to receptor tyrosine kinase (RTK) family. It is a powerful regulator of collagen deposition in the extracellular matrix (ECM). Although the oligomerization of DDR extracellular domain (ECD) proteins can affect matrix remodeling by inhibiting fibrillogenesis, it is still unknown how cellular DDR2 is incorporated into collagen matrix. Using 3-dimentional (3D) imaging for migrating cells, we identified a novel mechanism that explains how DDR2 incorporating into collagen matrix, which we named as posterior remnant tethering. We followed the de novo formation of these remnants and identified that DDR2 clusters formed at the retracting phase of a pseudopodium, then these clusters were tethered to fibrillar collagen and peeled off from the cell body to generate DDR2 containing posterior remnants. Inhibition of ß1-integrin or Rac1 activity abrogated the remnant formation. Thus, our findings unveil a special cellular mechanism for DDR2 clusters incorporating into collagen matrix in an integrin-dependent manner.

Author Correction: Immune-checkpoint protein VISTA critically regulates the IL-23/IL-17 inflammatory axis.

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Immune-checkpoint protein VISTA critically regulates the IL-23/IL-17 inflammatory axis.

V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an inhibitory immune-checkpoint molecule that suppresses CD4+ and CD8+ T cell activation when expressed on antigen-presenting cells. Vsir -/- mice developed loss of peripheral tolerance and multi-organ chronic inflammatory phenotypes. Vsir -/- CD4+ and CD8+ T cells were hyper-responsive towards self- and foreign antigens. Whether or not VISTA regulates innate immunity is unknown. Using a murine model of psoriasis induced by TLR7 agonist imiquimod (IMQ), we show that VISTA deficiency exacerbated psoriasiform inflammation. Enhanced TLR7 signaling in Vsir -/- dendritic cells (DCs) led to the hyper-activation of Erk1/2 and Jnk1/2, and augmented the production of IL-23. IL-23, in turn, promoted the expression of IL-17A in both TCRγδ+ T cells and CD4+ Th17 cells. Furthermore, VISTA regulates the peripheral homeostasis of CD27- γδ T cells and their activation upon TCR-mediated or cytokine-mediated stimulation. IL-17A-producing CD27- γδ T cells were expanded in the Vsir -/- mice and amplified the inflammatory cascade. In conclusion, this study has demonstrated that VISTA critically regulates the inflammatory responses mediated by DCs and IL-17-producing TCRγδ+ and CD4+ Th17 T cells following TLR7 stimulation. Our finding provides a rationale for therapeutically enhancing VISTA-mediated pathways to benefit the treatment of autoimmune and inflammatory disorders.

PLEKHM1/DEF8/RAB7 complex regulates lysosome positioning and bone homeostasis.

Mutations of the Plekhm1 gene in humans and rats cause osteopetrosis, an inherited bone disease characterized by diminished bone resorption by osteoclasts. PLEKHM1 binds to RAB7 and is critical for lysosome trafficking. However, the molecular mechanisms by which PLEKHM1 regulates lysosomal pathways remain unknown. Here, we generated germline and conditional Plekhm1-deficient mice. These mice displayed no overt abnormalities in major organs, except for an increase in trabecular bone mass. Furthermore, loss of PLEKHM1 abrogated the peripheral distribution of lysosomes and bone resorption in osteoclasts. Mechanistically, we indicated that DEF8 interacts with PLEKHM1 and promotes its binding to RAB7, whereas the binding of FAM98A and NDEL1 with PLEKHM1 connects lysosomes to microtubules. Importantly, suppression of these proteins results in lysosome positioning and bone resorption defects similar to those of Plekhm1-null osteoclasts. Thus, PLHKEM1, DEF8, FAM98A, and NDEL1 constitute a molecular complex that regulates lysosome positioning and secretion through RAB7.

Mst1 Kinase Regulates the Actin-Bundling Protein L-Plastin To Promote T Cell Migration.

Exploring the mechanisms controlling lymphocyte trafficking is essential for understanding the function of the immune system and the pathophysiology of immunodeficiencies. The mammalian Ste20-like kinase 1 (Mst1) has been identified as a critical signaling mediator of T cell migration, and loss of Mst1 results in immunodeficiency disease. Although Mst1 is known to support T cell migration through induction of cell polarization and lamellipodial formation, the downstream effectors of Mst1 are incompletely defined. Mice deficient for the actin-bundling protein L-plastin (LPL) have phenotypes similar to mice lacking Mst1, including decreased T cell polarization, lamellipodial formation, and cell migration. We therefore asked whether LPL functions downstream of Mst1. The regulatory N-terminal domain of LPL contains a consensus Mst1 phosphorylation site at Thr(89) We found that Mst1 can phosphorylate LPL in vitro and that Mst1 can interact with LPL in cells. Removal of the Mst1 phosphorylation site by mutating Thr(89) to Ala impaired localization of LPL to the actin-rich lamellipodia of T cells. Expression of the T89A LPL mutant failed to restore migration of LPL-deficient T cells in vitro. Furthermore, expression of T89A LPL in LPL-deficient hematopoietic cells, using bone marrow chimeras, failed to rescue the phenotype of decreased thymic egress. These results identify LPL as a key effector of Mst1 and establish a novel mechanism linking a signaling intermediate to an actin-binding protein critical to T cell migration.

Three-dimensional matrix fiber alignment modulates cell migration and MT1-MMP utility by spatially and temporally directing protrusions.

Multiple attributes of the three-dimensional (3D) extracellular matrix (ECM) have been independently implicated as regulators of cell motility, including pore size, crosslink density, structural organization, and stiffness. However, these parameters cannot be independently varied within a complex 3D ECM protein network. We present an integrated, quantitative study of these parameters across a broad range of complex matrix configurations using self-assembling 3D collagen and show how each parameter relates to the others and to cell motility. Increasing collagen density resulted in a decrease and then an increase in both pore size and fiber alignment, which both correlated significantly with cell motility but not bulk matrix stiffness within the range tested. However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment. Cellular protrusion rate, protrusion orientation, speed of migration, and invasion distance showed coupled biphasic responses to increasing collagen density not predicted by 2D models or by stiffness, but instead by fiber alignment. The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified. Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.

ß-Catenin serves as a clutch between low and high intercellular E-cadherin bond strengths.

A wide range of invasive pathological outcomes originate from the loss of epithelial phenotype and involve either loss of function or downregulation of transmembrane adhesive receptor complexes, including Ecadherin (Ecad) and binding partners ß-catenin and α-catenin at adherens junctions. Cellular pathways regulating wild-type ß-catenin level, or direct mutations in ß-catenin that affect the turnover of the protein have been shown to contribute to cancer development, through induction of uncontrolled proliferation of transformed tumor cells, particularly in colon cancer. Using single-molecule force spectroscopy, we show that depletion of ß-catenin or the prominent cancer-related S45 deletion mutation in ß-catenin present in human colon cancers both weaken tumor intercellular Ecad/Ecad bond strength and diminishes the capacity of specific extracellular matrix proteins-including collagen I, collagen IV, and laminin V-to modulate intercellular Ecad/Ecad bond strength through α-catenin and the kinase activity of glycogen synthase kinase 3 (GSK-3ß). Thus, in addition to regulating tumor cell proliferation, cancer-related mutations in ß-catenin can influence tumor progression by weakening the adhesion of tumor cells to one another through reduced individual Ecad/Ecad bond strength and cellular adhesion to specific components of the extracellular matrix and the basement membrane.

α-actinin1 and 4 tyrosine phosphorylation is critical for stress fiber establishment, maintenance and focal adhesion maturation.

In polarized, migrating cells, stress fibers are a highly dynamic network of contractile acto-myosin structures composed of bundles of actin filaments held together by actin cross-linking proteins such as α-actinins. As such, α-actinins influence actin cytoskeleton organization and dynamics and focal adhesion maturation. In response to environmental signals, α-actinins are tyrosine phosphorylated and this affects their binding to actin stress fibers; however, the cellular role of α-actinin tyrosine phosphorylation remains largely unknown. We found that non-muscle α-actinin1/4 are critical for the establishment of dorsal stress fibers and maintenance of transverse arc stress fibers. Analysis of cells genetically depleted of α-actinin1 and 4 reveals two distinct modes for focal adhesion maturation. An α-actinin1 or 4 dependent mode that uses dorsal stress fiber precursors as a template for establishing focal adhesions and their maturation, and an α-actinin-independent manner that uses transverse arc precursors to establish focal adhesions at both ends. Focal adhesions formed in the absence of α-actinins are delayed in their maturation, exhibit altered morphology, have decreased amounts of Zyxin and VASP, and reduced adhesiveness to extracellular matrix. Further rescue experiments demonstrate that the tyrosine phosphorylation of α-actinin1 at Y12 and α-actinin4 at Y265 is critical for dorsal stress fiber establishment, transverse arc maintenance and focal adhesion maturation.

Actin cap associated focal adhesions and their distinct role in cellular mechanosensing.

The ability for cells to sense and adapt to different physical microenvironments plays a critical role in development, immune responses, and cancer metastasis. Here we identify a small subset of focal adhesions that terminate fibers in the actin cap, a highly ordered filamentous actin structure that is anchored to the top of the nucleus by the LINC complexes; these differ from conventional focal adhesions in morphology, subcellular organization, movements, turnover dynamics, and response to biochemical stimuli. Actin cap associated focal adhesions (ACAFAs) dominate cell mechanosensing over a wide range of matrix stiffness, an ACAFA-specific function regulated by actomyosin contractility in the actin cap, while conventional focal adhesions are restrictively involved in mechanosensing for extremely soft substrates. These results establish the perinuclear actin cap and associated ACAFAs as major mediators of cellular mechanosensing and a critical element of the physical pathway that transduce mechanical cues all the way to the nucleus.

Dimensional and temporal controls of three-dimensional cell migration by zyxin and binding partners.

Spontaneous molecular oscillations are ubiquitous in biology. But to our knowledge, periodic cell migratory patterns have not been observed. Here we report the highly regular, periodic migration of cells along rectilinear tracks generated inside three-dimensional matrices, with each excursion encompassing several cell lengths, a phenotype that does not occur on conventional substrates. Short hairpin RNA depletion shows that these one-dimensional oscillations are uniquely controlled by zyxin and binding partners α-actinin and p130Cas, but not vasodilator-stimulated phosphoprotein and cysteine-rich protein 1. Oscillations are recapitulated for cells migrating along one-dimensional micropatterns, but not on two-dimensional compliant substrates. These results indicate that although two-dimensional motility can be well described by speed and persistence, three-dimensional motility requires two additional parameters, the dimensionality of the cell paths in the matrix and the temporal control of cell movements along these paths. These results also suggest that the zyxin/α-actinin/p130Cas module may ensure that motile cells in a three-dimensional matrix explore the largest space possible in minimum time.

Curcumin inhibits gene expression of receptor for advanced glycation end-products (RAGE) in hepatic stellate cells in vitro by elevating PPARγ activity and attenuating oxidative stress.

BACKGROUND AND PURPOSE: Diabetes is characterized by hyperglycaemia, which facilitates the formation of advanced glycation end-products (AGEs). Type 2 diabetes mellitus is commonly accompanied by non-alcoholic steatohepatitis, which could lead to hepatic fibrosis. Receptor for AGEs (RAGE) mediates effects of AGEs and is associated with increased oxidative stress, cell growth and inflammation. The phytochemical curcumin inhibits the activation of hepatic stellate cells (HSCs), the major effectors during hepatic fibrogenesis. The aim of this study was to explore the underlying mechanisms of curcumin in the elimination of the stimulating effects of AGEs on the activation of HSCs. We hypothesize that curcumin eliminates the effects of AGEs by suppressing gene expression of RAGE. EXPERIMENTAL APPROACH: Gene promoter activities were evaluated by transient transfection assays. The expression of rage was silenced by short hairpin RNA. Gene expression was analysed by real-time PCR and Western blots. Oxidative stress was evaluated. KEY RESULTS: AGEs induced rage expression in cultured HSCs, which played a critical role in the AGEs-induced activation of HSCs. Curcumin at 20 µM eliminated the AGE effects, which required the activation of PPARγ. In addition, curcumin attenuated AGEs-induced oxidative stress in HSCs by elevating the activity of glutamate-cysteine ligase and by stimulating de novo synthesis of glutathione, leading to the suppression of gene expression of RAGE. CONCLUSION AND IMPLICATIONS: Curcumin suppressed gene expression of RAGE by elevating the activity of PPARγ and attenuating oxidative stress, leading to the elimination of the AGE effects on the activation of HSCs. LINKED ARTICLE: This article is commented on by Stefanska, pp. 2209-2211 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01959.x.

The LIMD1 protein bridges an association between the prolyl hydroxylases and VHL to repress HIF-1 activity.

There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O(2) tension. In high O(2) tension (normoxia) the PHDs hydroxylate two conserved proline residues on HIF-1α, which leads to binding of the von Hippel-Lindau (VHL) tumour suppressor, the recognition component of a ubiquitin-ligase complex, initiating HIF-1α ubiquitylation and degradation. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIF-1α or as a multiprotein complex. Here we show that the tumour suppressor protein LIMD1 (LIM domain-containing protein) acts as a molecular scaffold, simultaneously binding the PHDs and VHL, thereby assembling a PHD-LIMD1-VHL protein complex and creating an enzymatic niche that enables efficient degradation of HIF-1α. Depletion of endogenous LIMD1 increases HIF-1α levels and transcriptional activity in both normoxia and hypoxia. Conversely, LIMD1 expression downregulates HIF-1 transcriptional activity in a manner depending on PHD and 26S proteasome activities. LIMD1 family member proteins Ajuba and WTIP also bind to VHL and PHDs 1 and 3, indicating that these LIM domain-containing proteins represent a previously unrecognized group of hypoxic regulators.

LIS1 regulates osteoclast formation and function through its interactions with dynein/dynactin and Plekhm1.

Microtubule organization and lysosomal secretion are both critical for the activation and function of osteoclasts, highly specialized polykaryons that are responsible for bone resorption and skeletal homeostasis. Here, we have identified a novel interaction between microtubule regulator LIS1 and Plekhm1, a lysosome-associated protein implicated in osteoclast secretion. Decreasing LIS1 expression by shRNA dramatically attenuated osteoclast formation and function, as shown by a decreased number of mature osteoclasts differentiated from bone marrow macrophages, diminished resorption pits formation, and reduced level of CTx-I, a bone resorption marker. The ablated osteoclast formation in LIS1-depleted macrophages was associated with a significant decrease in macrophage proliferation, osteoclast survival and differentiation, which were caused by reduced activation of ERK and AKT by M-CSF, prolonged RANKL-induced JNK activation and declined expression of NFAT-c1, a master transcription factor of osteoclast differentiation. Consistent with its critical role in microtubule organization and dynein function in other cell types, we found that LIS1 binds to and colocalizes with dynein in osteoclasts. Loss of LIS1 led to disorganized microtubules and aberrant dynein function. More importantly, the depletion of LIS1 in osteoclasts inhibited the secretion of Cathepsin K, a crucial lysosomal hydrolase for bone degradation, and reduced the motility of osteoclast precursors. These results indicate that LIS1 is a previously unrecognized regulator of osteoclast formation, microtubule organization, and lysosomal secretion by virtue of its ability to modulate dynein function and Plekhm1.

Regulation of U6 promoter activity by transcriptional interference in viral vector-based RNAi.

The direct negative impact of the transcriptional activity of one component on the second one in cis is referred to as transcriptional interference (TI). U6 is a type III RNA polymerase III promoter commonly used for driving small hairpin RNA (shRNA) expression in vector-based RNAi. In the design and construction of viral vectors, multiple transcription units may be arranged in close proximity in a space-limited vector. Determining if U6 promoter activity can be affected by TI is critical for the expression of target shRNA in gene therapy or loss-of-function studies. In this research, we designed and implemented a modified retroviral system where shRNA and exogenous gene expressions were driven by two independent transcriptional units. We arranged U6 promoter driving shRNA expression and UbiC promoter in two promoter arrangements. In primary macrophages, we found U6 promoter activity was inhibited by UbiC promoter when in the divergent arrangement but not in tandem. In contrast, PKG promoter had no such negative impact. Instead of enhancing U6 promoter activity, CMV enhancer had significant negative impact on U6 promoter activity in the presence of UbiC promoter. Our results indicate that U6 promoter activity can be affected by TI in a proximal promoter-specific and arrangement-dependent manner.

A comparative investigation on absorption performances of three expanded graphite-based complex materials for toluene.

Three kinds of expanded graphite-based complex materials were prepared to absorb toluene by dispersing plant oil, animal oil and mineral oil on the surface of expanded graphite, respectively. These complex materials were characterized by scanning electronic micrograph, contact angle meter and Brunauer-Emmett-Teller surface area. And their absorption capacities for toluene were comparatively investigated. The results showed that the surfaces of the three types of sorbents were very hydrophobic and nonporous, but they all had excellent absorption capacities for toluene. And their absorption capacities were proportional to the toluene concentration in streams and decreased differently with increasing the absorption temperature. It was noteworthy that the absorption capacities varied with the unsaturated degree of the complex materials and kept unchanged under different relative humidities of streams. Moreover, the regeneration experiments showed that after 15-run regeneration the absorption capacities of expanded graphite modified by mineral oil almost kept unchanged, while that of expanded graphite loaded plant oil and animal oil dropped by 157 and 93.6 mg g(-1), respectively. The losses of their absorption capacities were ascribed to the destruction of their unsaturated carbon bounds.

A distinctive role for focal adhesion proteins in three-dimensional cell motility.

Focal adhesions are large multi-protein assemblies that form at the basal surface of cells on planar dishes, and that mediate cell signalling, force transduction and adhesion to the substratum. Although much is known about focal adhesion components in two-dimensional (2D) systems, their role in migrating cells in a more physiological three-dimensional (3D) matrix is largely unknown. Live-cell microscopy shows that for cells fully embedded in a 3D matrix, focal adhesion proteins, including vinculin, paxillin, talin, alpha-actinin, zyxin, VASP, FAK and p130Cas, do not form aggregates but are diffusely distributed throughout the cytoplasm. Despite the absence of detectable focal adhesions, focal adhesion proteins still modulate cell motility, but in a manner distinct from cells on planar substrates. Rather, focal adhesion proteins in matrix-embedded cells regulate cell speed and persistence by affecting protrusion activity and matrix deformation, two processes that have no direct role in controlling 2D cell speed. This study shows that membrane protrusions constitute a critical motility/matrix-traction module that drives cell motility in a 3D matrix.

Prediction of sphingosine 1-phosphate-stimulated endothelial cell migration rates using biochemical measurements.

The ability to predict endothelial cell migration rates may aid in the design of biomaterials that endothelialize following implantation. However, the complexity of the signaling response to migration-promoting stimuli such as sphingosine 1-phosphate (S1P) makes such predictions quite challenging. A number of signaling pathways impact S1P-mediated cell migration, including the Akt and Src pathways, which both affect activation of the small GTPase Rac. Rac activation promotes the formation of lamellipodia, and thus should be intimately linked to cell migration rates. In immortalized endothelial cells, expression of proteins that inhibit Akt, Src, and Rac (PTEN, CSK, and beta2-chimaerin, respectively) was decreased using RNA interference, resulting in increases in the basal level of activation of Akt, Src, and Rac. Cells were scrape-wounded and stimulated with 1 microM S1P. The timecourse of Akt, Src, and Rac activation was followed over 2 h in the perturbed cells, while migration into the scrape wound was measured over 6 h. Rac activation at 120 min post-stimulation was highly correlated with the mean migration rate of cells, but only in cells stimulated with S1P. Using partial least squares regression, the migration rate of cells into the scrape wound was found to be highly correlated with the magnitude of the early Akt peak (e.g., 5-15 min post-stimulation). These results demonstrated that biochemical measurements might be useful in predicting rates of endothelial cell migration.