TNFα/IFNγ Mediated Intestinal Epithelial Barrier Dysfunction Is Attenuated by MicroRNA-93 Downregulation of PTK6 in Mouse Colonic Epithelial Cells.

Since inflammatory bowel diseases (IBD) represent significant morbidity and mortality in the US, the need for defining novel drug targets and inflammatory mechanisms would be of considerable benefit. Although protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) has been primarily studied in an oncogenic context, it was noted that PTK6 null mice exhibited significantly enhanced colonic epithelial barrier function. Considering that the inflammatory functions of PTK6 have not yet been explored, we hypothesized that cytokines responsible for mediating IBD, such as TNFα/IFNγ, may solicit the action of PTK6 to alter barrier function. After first assessing critical mediators of TNFα/IFNγ driven epithelial barrier dysfunction, we further explored the possibility of PTK6 in this inflammatory context. In this report, we showed that PTK6 siRNA and PTK6 null young adult mouse colonic epithelial cells (YAMC) exhibited significant attenuation of TNFα/IFNγ induced barrier dysfunction as measured by electric cell-substrate impedance sensing (ECIS) assay and permeability assays. In addition, PTK6 null cells transfected with PTK6 cDNA displayed restored barrier dysfunction in response to TNFα/IFNγ, while the cells transfected with vector alone showed similar attenuation of barrier dysfunction. Furthermore, using subcellular fractionation and immunocytochemistry experiments, we found that PTK6 plays a role in FoxO1 nuclear accumulation leading to down-regulation of claudin-3, a tight junction protein. Moreover, we searched for relevant miRNA candidates putative for targeting PTK6 in order to identify and assess the impact of microRNA that target PTK6 with respect to TNFα/IFNγ induced barrier dysfunction. Subsequently, we assayed likely targets and determined their effectiveness in attenuating PTK6 expression as well as cytokine induced barrier dysfunction. Results showed that miR-93 reduced PTK6 expression and attenuated TNFα/IFNγ imposed decrease in transepithelial electrical resistance (TER), as well as excluded FoxO1 from the nucleus. Our results indicate that PTK6 may act as a novel mediator of intestinal epithelial permeability during inflammatory injury, and miR-93 may protect intestinal epithelial barrier function, at least in part, by targeting PTK6.

MicroRNA-147b regulates vascular endothelial barrier function by targeting ADAM15 expression.

A disintegrin and metalloproteinase15 (ADAM15) has been shown to be upregulated and mediate endothelial hyperpermeability during inflammation and sepsis. This molecule contains multiple functional domains with the ability to modulate diverse cellular processes including cell adhesion, extracellular matrix degradation, and ectodomain shedding of transmembrane proteins. These characteristics make ADAM15 an attractive therapeutic target in various diseases. The lack of pharmacological inhibitors specific to ADAM15 prompted our efforts to identify biological or molecular tools to alter its expression for further studying its function and therapeutic implications. The goal of this study was to determine if ADAM15-targeting microRNAs altered ADAM15-induced endothelial barrier dysfunction during septic challenge by bacterial lipopolysaccharide (LPS). An in silico analysis followed by luciferase reporter assay in human vascular endothelial cells identified miR-147b with the ability to target the 3' UTR of ADAM15. Transfection with a miR-147b mimic led to decreased total, as well as cell surface expression of ADAM15 in endothelial cells, while miR-147b antagomir produced an opposite effect. Functionally, LPS-induced endothelial barrier dysfunction, evidenced by a reduction in transendothelial electric resistance and increase in albumin flux across endothelial monolayers, was attenuated in cells treated with miR-147b mimics. In contrast, miR-147b antagomir exerted a permeability-increasing effect in vascular endothelial cells similar to that caused by LPS. Taken together, these data suggest the potential role of miR147b in regulating endothelial barrier function by targeting ADAM15 expression.

Non-muscle Mlck is required for ß-catenin- and FoxO1-dependent downregulation of Cldn5 in IL-1ß-mediated barrier dysfunction in brain endothelial cells.

Aberrant elevation in the levels of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) contributes to neuroinflammatory diseases. Blood-brain barrier (BBB) dysfunction is a hallmark phenotype of neuroinflammation. It is known that IL-1ß directly induces BBB hyperpermeability but the mechanisms remain unclear. Claudin-5 (Cldn5) is a tight junction protein found at endothelial cell-cell contacts that are crucial for maintaining brain microvascular endothelial cell (BMVEC) integrity. Transcriptional regulation of Cldn5 has been attributed to the transcription factors ß-catenin and forkhead box protein O1 (FoxO1), and the signaling molecules regulating their nuclear translocation. Non-muscle myosin light chain kinase (nmMlck, encoded by the Mylk gene) is a key regulator involved in endothelial hyperpermeability, and IL-1ß has been shown to mediate nmMlck-dependent barrier dysfunction in epithelia. Considering these factors, we tested the hypothesis that nmMlck modulates IL-1ß-mediated downregulation of Cldn5 in BMVECs in a manner that depends on transcriptional repression mediated by ß-catenin and FoxO1. We found that treating BMVECs with IL-1ß induced barrier dysfunction concomitantly with the nuclear translocation of ß-catenin and FoxO1 and the repression of Cldn5. Most importantly, using primary BMVECs isolated from mice null for nmMlck, we identified that Cldn5 repression caused by ß-catenin and FoxO1 in IL-1ß-mediated barrier dysfunction was dependent on nmMlck.

Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function.

Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regulates AS and eNOS transcription to support NO production. Moreover, we show that insulin enhances NO production in response to physiological cues such as bradykinin. To translate these results to an in vivo model, we show that AS transcription is diminished in coronary endothelial cells isolated from rats with streptozotocin (STZ)-induced diabetes. Importantly, we demonstrate restoration of AS and eNOS transcription by insulin treatment in STZ-diabetic rats, and show that this restoration was accompanied by improved endothelial function as measured by endothelium-dependent vasorelaxation. Overall, this report demonstrates, both in cell culture and whole animal studies, that insulin maintains vascular function, in part, through the maintenance of AS transcription, thus ensuring an adequate supply of arginine to maintain vascular endothelial response to physiological cues.

Argininosuccinate synthase: at the center of arginine metabolism.

The levels of L-arginine, a cationic, semi-essential amino acid, are often controlled within a cell at the level of local availability through biosynthesis. The importance of this temporal and spatial control of cellular L-arginine is highlighted by the tissue specific roles of argininosuccinate synthase (argininosuccinate synthetase) (EC 6.3.4.5), as the rate-limiting step in the conversion of L-citrulline to L-arginine. Since its discovery, the function of argininosuccinate synthase has been linked almost exclusively to hepatic urea production despite the fact that alternative pathways involving argininosuccinate synthase were defined, such as its role in providing arginine for creatine and for polyamine biosynthesis. However, it was the discovery of nitric oxide that meaningfully extended our understanding of the metabolic importance of non-hepatic argininosuccinate synthase. Indeed, our knowledge of the number of tissues that manage distinct pools of arginine under the control of argininosuccinate synthase has expanded significantly.