BACKGROUND & AIMS-Diarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na + /H + exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na + /H + exchanger activity and Na + -dependent fluid absorption in the intestine.

Background & Aims-Chronic inflammation is a risk factor for colon cancer (CC). Lysophosphatidic acid (LPA), a naturally produced phospholipid, mediates multiple effects that are vital to disease process, including inflammation and cancer. The expression of LPA receptor 2 (LPA 2 ) is up-regulated in several types of cancer, including ovarian and colon cancer, but the importance of LPA and LPA 2 in the development and progression of CC is unclear. In this study, we sought to determine whether LPA and LPA 2 regulate the progression of CC in vivo.

P2Y1 purinergic receptors (P2Y1Rs) mediate rises in intracellular Ca 2؉ in response to ATP, but the duration and characteristics of this Ca 2؉ response are known to vary markedly in distinct cell types. We screened the P2Y1R carboxyl terminus against a recently created proteomic array of PDZ (PSD-95͞Drosophila Discs large͞ZO-1 homology) domains and identified a previously unrecognized, specific interaction with the second PDZ domain of the scaffold NHERF-2 (Na ؉ ͞H ؉ exchanger regulatory factor type 2). Furthermore, we found that P2Y1R and NHERF-2 associate in cells, allowing NHERF-2-mediated tethering of P2Y 1R to key downstream effectors such as phospholipase C␤. Finally, we found that coexpression of P2Y1R with NHERF-2 in glial cells prolongs P2Y1R-mediated Ca 2؉ signaling, whereas disruption of the P2Y1R-NHERF-2 interaction by point mutations attenuates the duration of P2Y1R-mediated Ca 2؉ responses. These findings reveal that NHERF-2 is a key regulator of the cellular activity of P2Y 1R and may therefore determine cellspecific differences in P2Y1R-mediated signaling.G protein-coupled receptor ͉ purinergic ͉ ATP ͉ proteomic array A denine-based nucleotides such as ATP and ADP are prominent extracellular signaling molecules that mediate a wide variety of physiological actions in tissues throughout the body. Many of the physiological effects evoked by ATP and ADP are mediated by metabotropic P2Y receptors (P2YRs) (1), which are members of the G protein-coupled receptor (GPCR) superfamily. To date, seven distinct mammalian P2YR subtypes have been cloned: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , and P2Y 13 . Most P2YRs are coupled to G␣ q proteins and thus to the activation of phospholipase C␤ (PLC␤) and generation of diacylglycerol and inositol-3,4,5-trisphosphate, ultimately leading to the activation of PKC and release of Ca 2ϩ from internal stores (1). Some P2YRs, including P2Y 1 , P2Y 2 , P2Y 12 , and P2Y 13 , are also known to couple to G␣ i and the inhibition of adenylyl cyclase (2-5). The purinergic P2YR type 1 (P2Y 1 R) subtype is abundantly expressed in a number of tissues, including the CNS (6, 7), where it plays a key role in the transmission of astrocytic Ca 2ϩ waves (8), activation of mitogenic responses in astrocytes to brain trauma (9), inhibition of neuronal N-type voltage-activated Ca 2ϩ channels (10), and embryonic brain development (11). P2Y 1 R also plays critical roles in the cardiovascular system, including the regulation of coronary vasodilation (12) and platelet aggregation (13).Signaling by P2Y 1 Rs is known to be heavily dependent on cellular context. For example, stimulation of P2Y 1 Rs in some cell types is known to strongly promote cell proliferation (14), whereas P2Y 1 R stimulation in other cell types is known to induce apoptosis (15). Furthermore, P2Y 1 Rs can exert cellular effects that are quite different from those exerted by other P2YRs expressed in the same cell type and that couple to similar G proteins. For example, in astrocytes, both P2Y 1 Rs and P2Y 2 Rs can cou...

Background Metastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4) and its ligand stromal cell-derived factor-1 (CXCL12) interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites. Methodology/Principal Findings We describe the actions of N,N′-(1,4-phenylenebis(methylene))dipyrimidin-2-amine (designated MSX-122), a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using 18 F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles. Conclusions/Significance We developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for long-term blockade of metastasis than other reported CXCR4 antagonists.

Among the multiple cellular effects mediated by lysophosphatidic acid (LPA), the effect on cell proliferation has extensively been investigated. A recent study showed that LPA-mediated proliferation of colon cancer cells requires activation of ␤-catenin. However, the majority of colon cancer cells have deregulation of the Wnt/␤-catenin pathway. This prompted us to hypothesize the presence of additional pathway(s) activated by LPA resulting in an increase in the proliferation of colon cancer cells. Krüppel-like factor 5 (KLF5) is a transcriptional factor highly expressed in the crypt compartment of the intestinal epithelium. In this work, we investigated a role of KLF5 in LPA-mediated proliferation. We show that LPA stimulated the expression levels of KLF5 mRNA and protein in colon cancer cells and this stimulation was mediated by LPA 2 and LPA 3 . Silencing of KLF5 expression by small interfering RNA significantly attenuated LPA-mediated proliferation of SW480 and HCT116 cells. LPA-mediated KLF5 induction was partially blocked by inhibition of the mitogen-activated protein kinase kinase and protein kinase C-␦. Moreover, we observed that LPA regulates KLF5 expression via eukaryotic elongation factor 2 kinase (eEF2k). Inhibition of calmodulin or silencing of eEF2k blocked the stimulation in KLF5 expression. Knockdown of eEF2k specifically inhibited KLF5 induction by LPA but not by fetal bovine serum or phorbol 12-myristate 13-acetate. These results identify KLF5 as a target of LPA-mediated signaling and suggest a role of KLF5 in promoting proliferation of intestinal epithelia in response to LPA.

A major of Na+ absorptive process in the proximal part of intestine and kidney is electroneutral exchange of Na+ and H+ by Na+/H+ exchanger type 3 (NHE3). During the past decade, significant advance has been achieved in the mechanisms of NHE3 regulation. A bulk of the current knowledge on Na+/H+ exchanger regulation is based on heterologous expression of mammalian Na+/H+ exchangers in Na+/H+ exchanger deficient fibroblasts, renal epithelial, and intestinal epithelial cells. Based on the reductionist's approach, an understanding of NHE3 regulation has been greatly advanced. More recently, confirmations of in vitro studies have been made using animals deficient in one or more proteins but in some cases unexpected findings have emerged. The purpose of this paper is to provide a brief overview of recent progress in the regulation and functions of NHE3 present in the luminal membrane of the intestinal tract.

Lin S, Lee S, Shim H, Chun J, Yun CC. The absence of LPA receptor 2 reduces the tumorigenesis by Apc Min mutation in the intestine.

Glucocorticoids stimulate the intestinal absorption of Na(+) and water partly by regulation of the Na(+)/H(+) exchanger 3 (NHE3). Previous studies have shown both genomic and nongenomic regulation of NHE3 by glucocorticoids. Serum and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be part of this cascade, where phosphorylation of NHE3 by SGK1 initiates the translocation of NHE3 to the cell surface. In the present work, we examined a series of changes in SGK1 and NHE3 induced by glucocorticoids using human colonic Caco-2 and opossum kidney cells. We found that dexamethasone rapidly stimulated SGK1 mRNAs, but a significant change in protein abundance was not detected. Instead, there was an increase in SGK1 kinase activity as early as at 2 h. An increase in NHE3 protein abundance was not detected until 12 h of dexamethasone exposure, although the transport activity was significantly stimulated at 4 h. These data demonstrate that the changes of SGK1 precede those of NHE3. Chronic regulation (24 h) of NHE3 was blocked completely by prevention of protein synthesis with cycloheximide or actinomycin D and by the glucocorticoid receptor blocker RU486. The acute effect of dexamethasone was similarly abrogated by RU486, but was insensitive to cycloheximide and actinomycin D. Similarly, the stimulation of SGK1 activity by dexamethasone was blocked by RU486 but not by actinomycin D. Together, these data show that the acute effect of glucocorticoids on NHE3 is mediated by a glucocorticoid receptor dependent mechanism that activates SGK1 in a nongenomic manner.