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For seven years Clark worked part-time selling cowboy clothing and waitressing. During , following the advice of Nashville manager Woody Bowles The Judds , she enrolled in vocal and songwriting sessions. Two years later she secured a writing and publishing deal with Sony Tree.

In Mercury Records offered her a recording contract. Clark's songwriting and performances reflect country music's ability to tell real-life stories in an honest way. Fans especially know her for her upbeat songs about empowerment. In hit songs, such as "Better Things to Do" and "A Little Gasoline," Clark sings of the survivor who refuses to be victimized by any life experience. Whereas country artists often cross over into popular music territory, Clark's music remains rooted in country music tradition. Her debut release Terri Clark , Mercury achieved triple-platinum sales in Canada and platinum in the US.

Clark's next recording, Fearless , , offered introspective songwriting and a sound more influenced by folk and bluegrass. The recording, which Clark called her "art piece," was not aimed at commercial radio; it did however reach gold status in Canada. Attempting to resurrect past commercial success, the next album, Pain to Kill , Mercury , blended Clark's earlier upbeat style with her recent introspective approach.

Since Terri Clark has toured extensively throughout North America. She has headlined her own show and shared the stage with major country music artists such as Reba McEntire and Brad Paisley. In Clark toured Australia. Billboard magazine named Clark top new female country artist in Clark won Juno awards in for best new solo artist and in for best country female artist. For four successive years she received the Canadian Country Music Association Fans' Choice of the Year Award, and in was named its female artist of the year.

In recognition of her work to promote country music, in she was also awarded the prestigious Country Music Association's Connie B. Gay Award. Search The Canadian Encyclopedia. Remember me. I forgot my password. Why sign up? The results suggest that S1P causes activation of RhoA at the cell periphery, stimulating local activation of the actin cytoskeleton and focal adhesions, and resulting in endothelial barrier enhancement. S1P-induced Rac1 activation, however, does not appear to have a significant role in this process. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. The endothelial cells of capillaries and postcapillary venules form a semi-permeable barrier that is crucial for normal blood-tissue exchange and tissue homeostasis. Compromised endothelial barrier function which occurs during inflammation [ 1 ] significantly contributes to a wide variety of pathologies, including the systemic inflammatory response syndrome [ 2 ], ischemia-reperfusion injury [ 3 , 4 ], atherosclerosis [ 5 ], and cancer cell metastasis [ 6 ].

The mechanisms that control endothelial barrier function have long been a key focus of investigation, yet remain incompletely understood. Continuous cytoskeleton maintenance is critical for normal endothelial barrier function [ 7 , 8 ], and significant cytoskeletal rearrangements often accompany changes in permeability.

For example, actin stress fibers are typically elicited by inflammatory agents that compromise barrier function [ 9 ]. In contrast, strengthening of cortical actin at the cell periphery has been postulated to enhance endothelial barrier function [ 10 , 11 ]. In addition, we recently reported that dynamic changes in the normal cycling of actin-rich local lamellipodia in endothelial cells correlated with alterations in barrier function [ 12 — 14 ]. Rho family small GTPases strongly influence the actin cytoskeleton and have been shown to be important for controlling endothelial barrier integrity.

Several studies have shown that RhoA activation correlates with increased permeability of the endothelium [ 13 , 15 , 16 ]. In contrast, activation of Rac1 has been correlated with endothelial barrier enhancement [ 12 , 17 , 18 ]. Collectively these data have led to the general notion that Rac1 activation enhances endothelial barrier enhancement, while RhoA activation disrupts integrity of the endothelium [ 19 ].

Terri Clark

Some recent data have challenged to this paradigm, however. An elegant study by Szulcek and colleagues demonstrated that RhoA activation at the cell periphery correlates with barrier integrity, while its activation in the perinuclear area of the endothelial cell contributes to barrier disruption [ 20 ]. Another observation challenging this paradigm is our recent finding that sphingosinephosphate S1P elicits a strong increase in the GTP-bound, activated forms of both RhoA and Rac1 [ 12 ]. These findings raise the question about the relative involvement of RhoA and Rac1 in S1P-induced endothelial barrier enhancement, and whether spatiotemporal activation of RhoA is a key factor.

For this study we focused on the endothelial barrier enhancement elicited by S1P, an endogenously released, bioactive lipid that is a potent endothelial barrier enhancer at its physiological concentration [ 22 , 23 ]. After binding to its receptors, S1P induces dynamic cytoskeletal, junctional and adhesion changes and decreases permeability [ 24 ]. In our previous observation of S1P-induced activation of both Rac1 and RhoA, the Rac1 activation was relatively short lived, while RhoA activation was strong and sustained [ 12 ].

In addition, we and other groups have reported that S1P causes cortical Myosin light chain-2 MLC-2 phosphorylation [ 10 — 12 , 25 ], which is thought to help stabilize barrier integrity [ 17 , 26 ]. In the current study, we investigated the roles of Rac1 and RhoA in S1P-induced endothelial barrier enhancement, the spatiotemporal activation of RhoA in response to S1P, and the potential roles MLC2 phosphorylation, actin fiber formation, and localization of the focal adhesion protein vinculin.

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Spingosinephosphate was purchased from Tocris Bristol, UK. All other drugs and chemicals were purchased from Sigma-Aldrich St. Louis, MO. Santa Cruz, CA. Mouse anti vinculin ab was purchased from Abcam Cambridge, UK.

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For all studies, passage 1—5 cells were used. Cells were later distributed evenly onto gelatin-coated mm dishes for protein extraction, gelatin-coated MatTek mm 1 glass bottom dishes for time-lapse microscopy, or 96W1E ECIS arrays Applied Biophysics, Troy, NY for determination of barrier function. Cell protein lysates were obtained as previously described [ 12 , 28 ]. Proteins were transferred onto 0. Afterwards, secondary antibodies were applied at room temperature for 1 h, followed by three washes with TBST.

The next day, medium was changed to EBM at least 1 h before the experiment. Total impedance was reported by monitoring the voltage across the electrodes and its phase relative to the applied current. The cell-covered electrode unit was treated as an RC circuit, from which impedance data was later converted into monolayer resistance and capacitance, respectively representing barrier function and membrane capacitance [ 29 ]. The medium was changed to EBM 3 h before the experiment.

Briefly, images were first cropped, background subtracted and converted to bit images. Immunofluorescence labeling and confocal microscopy was performed as previously described [ 12 , 28 ].

The cells were then incubated with Texas Red-phalloidin at room temperature for 30 min. For two group comparisons, student t-tests were used. For comparisons of 3 or more groups, one-way ANOVA was used, with Tukey's multiple comparisons test for post-hoc analysis. For multiple group comparisons over time, repeated measures two-way ANOVA followed by Tukey's multiple comparisons test was used.

We previously observed that overexpression of Rac1 can reduce permeability of endothelial monolayers [ 12 ]. We also used a second approach, which was to knock down Rac1 expression using siRNA.

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Taken together, our data indicate that Rac1 has a critical role in the maintenance of baseline endothelial barrier integrity. S1P has a well-known role in the enhancement of endothelial barrier function, although recently it has been reported that high concentrations of S1P can disrupt endothelial barrier integrity [ 31 ].

When examining the maximum increase in TER that could be elicited by each concentration of S1P, we observed that treatment with S1P significantly increased TER compared to vehicle control in a concentration-dependent manner Fig 2A. However, it is worth noting that while all concentrations tested caused a rapid, significant, initial rise in TER, with higher concentrations of S1P this elevation in TER was typically not sustained, and sometimes fell below the baseline TER within 30 min Fig 2B.

Rac1 has been previously reported to mediate the barrier protective effect of S1P [ 23 ]. We tested the extent to which the selective Rac1 inhibitor Z would attenuate S1P-induced endothelial barrier enhancement.

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These data suggest that the endothelial barrier enhancement elicited by S1P may not require Rac1 activation. S1P Vehicle pretreated groups. These data provide additional evidence that S1P is able to enhance endothelial barrier function independently of Rac1 activation. The TER is normalized to the time point just prior to addition of S1P for a more direct comparison of the magnitude of the response. Vehicle treated groups.

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A third approach we used was to test the extent to which siRNA-induced Rac1 knockdown would inhibit S1P-induced endothelial barrier enhancement Fig 5. These data demonstrate that reduction of Rac1 expression does not impair the ability of S1P to enhance endothelial barrier function. The TER is normalized to the time point just prior to the addition of S1P, for more direct comparisons of the responses to S1P between the groups. The recent report of differential localized RhoA activation in endothelial cells during endothelial barrier maintenance and disruption [ 20 ] prompted us to investigate the localization of RhoA activation after stimulation with S1P.

During baseline, RhoA activation at any given point in the cell was low and oscillatory in nature, primarily located in the outer peri-nuclear region.


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S1P treatment significantly increased RhoA activity, shifting the maximal activity primarily near cell borders Fig 7 and S1 Movie. The data suggest that S1P elicits a specific spatiotemporal activation of RhoA near the borders in order to promote enhanced endothelial barrier function.

The entire time course can be viewed in S1 Movie. The results show that S1P rapidly increased MLC-2 phosphorylation within 1 min, demonstrating a peak at 10 min that returned to baseline level by 30 min. Each image represents three replicates for each time point. Quantification of phosphorylated MLC-2 intensity for each time point. Both RhoA activation and MLC-2 phosphorylation has been implicated in the reorganization of the actin cytoskeleton and formation of focal adhesions [ 33 ].

We tested if the role of RhoA in the formation of actin fibers and vinculin-containing focal adhesions near intercellular junctions, Pretreatment with Rhosin and Y16 abrogated S1P-induced F-actin and vinculin recruitment to the cell periphery Fig 9. These data suggest that S1P-induced F-actin and vinculin assembly at the cell periphery involves RhoA activation. The results showed that S1P increases F-actin and vinculin labeling in the peripheral areas of cells 10 min after the addition of S1P.

The inhibitors alone had no impact. All images are representative of 3 separate experiments. We were initially surprised by these results based on the well-established role of Rac1 in maintaining baseline endothelial barrier function [ 17 , 18 ] and reports that have suggested its role in S1P-induced enhancement of the endothelial barrier [ 12 , 34 ].

However, closer investigation of the literature revealed that the data directly supporting the role of Rac1 in S1P-induced endothelial barrier enhancement were quite limited. To our knowledge there have been no previous investigations that rigorously coupled siRNA knockdown of Rac1, DN Rac1 expression, and pharmacologic strategies all in one model to test the role of Rac1 in S1P-induced endothelial barrier enhancement.

To understand discrepancies between our study and reports in the literature, it is important to discuss the time course of the endothelial barrier response to S1P. Our current results indicate that all concentrations of S1P tested 0. Other groups have reported similar results [ 15 , 31 , 38 ]. It is clear that different S1P concentrations have distinct impacts on whether the initial endothelial barrier enhancement is sustained. While this study focused mainly on the mechanism responsible for the early stage endothelial barrier enhancement by S1P, a potential limitation is that higher concentrations of S1P may activate additional receptor subtypes and differentially affect Rho family small GTPases, or stimulate additional signals, resulting in a less sustained response and eventual reduction in barrier function, explaining the findings reported in other studies [ 31 , 37 ].

We confirmed that Rac1 is critical for baseline barrier integrity, as shown by other investigators [ 19 , 39 ]. However, we found no evidence that inhibition of Rac1 activation, whether by pharmacologic agents, overexpression of dominant-negative Rac1, or depletion of Rac1 with siRNA, could inhibit S1P-induced barrier enhancement. Possible explanations for the differences between the current study and previous reports are the use of different endothelial cell types and experimental conditions [ 28 ].

In that study, normalized data were presented, less frequent time points were obtained, and a different detection system Ussing chamber method was used to determine TER, which is less sensitive than ECIS [ 29 ], With the less frequent time points measured, it is possible that the initial peak increase in TER caused by S1P was missed.

However, it is difficult to compare these results with ours as only one single time point was shown and it is unclear whether NSC had any impact on the baseline barrier function. These data indicate reductions in barrier function elicited by inhibition of Rac1 must be taken into consideration in the overall data analysis. Based on the current data, we think that the rapid, S1P-induced, early rise in TER occurs independently of Rac1 activation. The apparent discrepancy of our data from reports in the literature may be summarized by differences in the time points of data collected, whether baseline changes are reported, and perhaps to some extent the endothelial cell types or other experimental conditions.

A current limitation is the lack of studies with Rac1 deletion or specific inhibition in intact postcapillary venules, which represents a future step that will help resolve this issue. Previously we showed that in addition to a transient Rac1 activation, RhoA activity was greatly increased and sustained for at least 10 min upon S1P treatment [ 12 ]. We observed that RhoA activity during our baseline measurements oscillates in the outer peri-nuclear regions.