The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states.
Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines.
Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli. As the interface between blood and all tissues, the vascular endothelium is critically involved in the biological response to inflammation 1.
Proinflammatory stimuli such as cytokines induce endothelial dysfunction and confer a proadhesive and prothrombotic phenotype 2 , 3. Although these events are important in certain adaptive responses such as wound healing, sustained endothelial activation can lead to deleterious consequences as seen in several chronic inflammatory disease states such as rheumatoid arthritis, inflammatory bowel disease, and atherosclerosis 4.
One of the initial and key events in the endothelium's response to inflammatory stimuli is the expression of adhesion molecules such as vascular cell adhesion molecule VCAM -1 and E-selectin. These proteins mediate early leukocyte attachment and rolling on the endothelial surface 5. Subsequent events such as firm adhesion and transmigration across the endothelial lining then set the stage for a developing inflammatory response within tissues 5.
Given the importance of these adhesion molecules in inflammation, the molecular mechanisms regulating their expression has been the subject of considerable investigation. Several studies have shown that the induction of VCAM-1 and E-selectin by inflammatory stimuli occurs principally at the level of transcription 6 , 7. Accumulating evidence also suggests that in addition to biochemical stimuli such as inflammatory cytokines, fluid mechanical forces regulate key aspects of endothelial function 9.
Previous studies show that LSS induces specific gene products such as endothelial nitric oxide synthase eNOS , which confers antiinflammatory and antithrombotic properties to the endothelium 10 , In addition, eNOS is an essential regulator of vascular reactivity and tone 12 , The ability of laminar flow to induce genetic factors that impart favorable properties to the endothelium is particularly relevant in certain inflammatory disease states such as atherosclerosis 14 , Indeed, atherosclerotic lesion development is accentuated at branch points that do not experience LSS 2 , These observations suggest that hemodynamic forces such as LSS may be protective against atherosclerotic disease development Kruppel-like factors KLFs are a subclass of the zinc finger family of transcription factors which regulates cellular differentiation and tissue development Furthermore, within the blood vessel wall KLF2 expression is exclusively within endothelial cells, and KLF2-null mice exhibit abnormal blood vessel formation, resulting in embryonic hemorrhage and death embryonic day Recently, KLF2 expression was shown to be increased when cultured endothelial cells were exposed to sustained shear stress However the targets and function of KLF2 in endothelial cell biology have not been elucidated.
Human umbilical vein endothelial cells, human umbilical arterial endothelial cells, human aortic endothelial cells, and the tissue culture components were obtained from Cambrex Company. Gimbrone Jr. Adenoviral constructs were generated by the Harvard Gene Therapy Group. The eNOS promoter constructs were provided by C.
PCR reactions were performed under conditions established previously in a GeneAmp sequence detection system Applied Biosystems. The scanned images were processed using Agilent feature extraction software.
The resulting feature intensities, background intensities, and artificial flags were imported into Argus microarray analysis software for further data analysis as described previously 30 , Northern blot studies were performed as described previously After experimental treatment of HUVECs, cellular protein extraction was performed as described, and Western blot analyses were performed Cells were harvested 48 h after transfection, assayed for luciferase activity, and normalized to total protein as analyzed by BCA kit Pierce Chemical Co.
All transfections were performed in triplicate for at least three independent experiments. Aliquots of these lysates were withdrawn to determine total cellular protein abundance and 3 H incorporation. L-[ 3 H] citrulline was isolated from the remaining lysate by anion exchange chromatography with AG 50W-X8 resin Bio-Rad Laboratories and quantitated by liquid scintillation counting.
Cells in the two groups were individually sorted based on equal levels of GFP expression and plated on fibronectin-coated coverslips for adhesion and rolling experiments. The laminar flow chamber used has been described previously in detail 34 , To better understand how biochemical and biomechanical stimuli modulate endothelial function, high throughput genomic transcriptional profiling studies were undertaken.
Using real-time PCR, we verified our transcriptional profiling results. As shown in Fig. In contrast, KLF2 expression was induced 4. Previous studies have identified several genes that are induced by LSS such as eNOS, a central regulator of endothelial cell function This effect was specific, since no induction was observed on the expression of another LSS-inducible gene COX-2 not depicted.
Furthermore, we consistently noted that exogenous KLF2 Fig. To determine if the increase in eNOS expression translates into an increase in catalytically active protein, we assayed for enzymatic activity by formation of L-[ 3 H] citrulline from L-[ 3 H] arginine 33 , For these studies we used the —1. Using a series of deletion constructs, we found that KLF2-mediated transactivation is partially reduced with deletion from —0. The ability to transactivate the —0. The region between —0.
To assess the importance of this site, the sequence was mutated in the context of the full-length and the —0. Furthermore, mutation of this site within the —0. Inflammatory cytokines are known to induce several effects on endothelial cells, such as the expression of key adhesion molecules like VCAM-1, E-selectin, and ICAM-1 41 , 5 , This effect was dose dependent, since no significant effect was observed at a dose of 1 MOI.
Adhesion molecules such as VCAM-1 mediate lymphocyte and leukocyte attachment to endothelial cells 41 , 5. To determine the functional consequence of KLF2's effect on adhesion molecule expression, in vitro flow assays were performed 42 , In contrast, T cell attachment and rolling was markedly attenuated in KLF2-overexpressing cells. These data demonstrate that KLF2 can inhibit adhesion molecule expression and T cell attachment and rolling to activated endothelial cells.
This factor normally exists in the cytoplasm as a heterodimer of the p50 and p65 subunits. As a first step, we assessed the effect of adenovirally expressed KLF2 versus control virus; Fig. However, as shown in Fig. In the presence of KLF2 right , a nearly identical binding pattern was observed.
Previous studies have demonstrated that nitric oxide NO can partially inhibit VCAM-1 and E-selectin induction by inflammatory cytokines Thus, although the production of NO may contribute in small part to the inhibitory effect of KLF2 on adhesion molecule expression, NO does not account for the large majority of KLF2's inhibitory effect. Cotransfection with KLF2 markedly attenuated this induction.
Similar results were seen in the heterologous cell line, COS 7. The data presented above demonstrate that KLF2 can induce eNOS and inhibit cytokine-mediated induction of endothelial adhesion molecules. In addition, previous studies demonstrate that the interaction of other KLF family members with p is important for the ability of these factors to transactivate reporter genes 49 — These data suggest that KLF2 and p can work in a cooperative fashion.
To verify that this interaction occurs in cells, coimmunoprecipitation experiments were performed. Together, these data suggest that KLF2 and p can physically interact in cells. The appreciation that inflammatory cytokines and fluid mechanical forces can modulate endothelial gene phenotype served here as the basis for the use of transcriptional profiling approaches to assess for global patterns of gene expression. Furthermore, we suggest that competition for rate-limiting amounts of the coactivator p is the underlying mechanism for these distinct effects of KLF2.
As such, these studies identify KLF2 as a novel regulator of proinflammatory endothelial activation. Recruitment of immune cells is a critical feature common to most inflammatory disease states.
This occurs through endothelial expression of certain adhesion molecules such as VCAM-1 and E-selectin 41 , 5 , 14 , These molecules allow for the attachment of leukocytes and lymphocytes to the blood vessel wall. Subsequently, these cells migrate across the endothelial barrier, invade adjacent tissues, and thereby sustain the inflammatory response.
As a functional consequence, T cell adhesion to an endothelial cell monolayer is impaired Fig. This is an intriguing observation, although the basis for this differential inhibition has not been fully elucidated by our studies. Finally, our observations may be particularly relevant in the context of atherosclerosis. Recent studies by Dekker et al. Previous studies from several laboratories demonstrate that LSS can confer antiinflammatory, antithrombotic, and antiproliferative properties 10 , Indeed, to our knowledge the magnitude of eNOS induction by KLF2 exceeds that observed by any other factor or stimulus.
This enzyme is a well-established central regulator of vascular function 52 — Mice deficient in eNOS are hypertensive, lack endothelium-dependent vasodilation, respond poorly to inflammatory challenge, and exhibit enhanced atherosclerotic lesion formation in susceptible mouse strains 55 — 59 , 12 , Therefore, the KLF2-mediated induction of eNOS expression may have important functional consequences in vascular health and disease.
However, despite its central importance our understanding of eNOS gene regulation remains incomplete. Transgenic studies suggest that the proximal 1.
Previous studies examining the human —1. More recently, members of the Smad family have been shown to induce eNOS expression and promoter activity Our dissection of the —1. Whether some of these previously identified factors may cooperate with KLF2 has yet to be determined. It is also noteworthy that this KLF2 binding site lies next to an AP-1 element, which has been implicated recently as mediating the LSS induction of the eNOS promoter, raising the possibility that these factors may cooperate to regulate eNOS gene expression Finally, our studies do not exclude the possibility that KLF2 may also bind to regulatory regions outside of the —1.
For example, Laumonnier et al. Indeed, a potential KLF binding site is present within this enhancer, raising the potential that KLF2 may bind additional regions in regulating endogenous eNOS expression. Previous studies demonstrate that NO can partially inhibit inflammatory cytokine-mediated induction of endothelial adhesion molecules such as VCAM-1 and E-selectin These results are consistent with previous studies 46 but do suggest that mechanism s independent of NO production account for the vast majority of KLF2's inhibitory effects.
This mechanism has been implicated in other systems such as the mutual antagonism observed between nuclear hormone receptors and AP-1 pathways These results suggest that KLF2 recruits p away from p65 to a target gene such as eNOS and thereby promotes opposing effects on endothelial gene expression.
In summary, we have identified KLF2 as a cytokine-inhibited and LSS-induced transcription factor that regulates the expression of key genes involved in the endothelial response to inflammation. Jain , HL to T. Michel , and HL to F. Garcia-Cardena and M. KLF2 expression in endothelial cells in response to biochemcial and biomechanical stimuli. C Deletion and mutational analyses of eNOS promoter. Effect of KLF2 on cytokine-mediated induction of adhesion molecules.
Experiments were performed as described in A except cells were harvested for total protein and Western blot analysis was performed. HUVECs infected with the adenovirus at the indicated dose, stimulated with cytokine for 4 h, and total RNA was assessed for adhesion molecule expression. NE, nuclear extracts; Cyto, cytoplasmic extracts.
Specificity was verified by competition and supershift studies. Transient transfection studies were performed in BAECs with the indicated constructs. Transient transfection studies were performed in COS-7 cells with the indicated constructs.
KLF2 interacts directly with p A p rescues KLF2-mediated inhibition. Transient transfection studies with the indicated plasmids were performed in COS-7 cells. Cotransfection studies were performed in COS-7 cells. C KLF2 and p interact. D KLF2 and p interact in cells. Sign In or Create an Account. Advanced Search. User Tools. Sign In. Skip Nav Destination Article Navigation. Article May 10 This Site. Google Scholar. Zhiyong Lin , Zhiyong Lin.
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Thomas M. Michel , Thomas M. Michael A. Gimbrone, Jr. Mukesh K. Jain Mukesh K. Author and Article Information. Sucharita SenBanerjee. Zhiyong Lin. Ajay Kumar. Zhiping Chen. Address correspondence to Mukesh K. Phone: ; Fax: ; email: mjain rics. SenBanerjee and Z. Lin contributed equally to this work. Received: July 09 Accepted: January 29 Online Issn: The Rockefeller University Press. J Exp Med 10 : — Article history Received:.
Cite Icon Cite. Kiely, M. Lee, M. Perrella, and J. Search ADS. Topper, T. Nagel, K. Anderson, and G. Abe, W. Min, J. Suprapisitchat, and C. Williams, H. Palmer, M. Whitley, and T. Whitley, A. Williams, and T. Rose, Z. Haque, R. Kurokawa, E. McInerney, S. Westin, D. Thanos, M. Rosenfeld, C. Glass, and T. Nagel, and J. Cai, D. Falb, and M. Hsu, B. Chen, S. The molecular mechanism by which tumor cells reduce their sensitivity to therapeutic radiation remains to be elucidated.
As shown in Fig. As described above, the molecular mechanisms causing the adaptive response to IR by pre-exposure to low levels of radiation is poorly understood yet critical for adaptive protection against DNA damage and carcinogenesis. Scheme 2 shows the signaling network causing the adaptive resistance in immortalized human HK18 keratinocytes by LDIR low dose ionizing radiation; 5—20 cGy of X-ray manuscript in preparation.
Their primary function appears to help cells to deal with the genotoxic stress; especially DSBs. We appreciate the collaborators, postdoctoral fellows, and graduate students at the School of Health Sciences, Purdue University for their perceptive discussion and support. National Center for Biotechnology Information , U.
Curr Cancer Drug Targets. Author manuscript; available in PMC Sep 5. Author information Copyright and License information Disclaimer. Copyright notice. The publisher's final edited version of this article is available at Curr Cancer Drug Targets. See other articles in PMC that cite the published article. Abstract Ionizing radiation IR plays a key role in both areas of carcinogenesis and anticancer radiotherapy.
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