Leukotriene D4 activates distinct G-proteins in intestinal epithelial cells to regulate stress fibre formation and to generate intracellular Ca2+ mobilisation and ERK1/2 activation

2005 ◽  
Vol 302 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Christian Kamp Nielsen ◽  
Ramin Massoumi ◽  
Maria Sonnerlind ◽  
Anita Sjölander
Keyword(s):  
Epithelial Cells ◽  
G Proteins ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Leukotriene D4 ◽  
Stress Fibre ◽  
Fibre Formation ◽  
Stress Fibre Formation

scholarly journals Leukotriene D4 induces stress-fibre formation in intestinal epithelial cells via activation of RhoA and PKCδ

2002 ◽  
Vol 115 (17) ◽  
pp. 3509-3515 ◽  
Author(s):  
Ramin Massoumi ◽  
Christer Larsson ◽  
Anita Sjölander
Keyword(s):  
Intestinal Epithelial Cells ◽  
Dominant Negative ◽  
Intestinal Epithelial ◽  
Regulatory Domain ◽  
Leukotriene D4 ◽  
Stress Fibre ◽  
Fibre Formation ◽  
Induced Stress ◽  
C3 Exoenzyme ◽  
Stress Fibre Formation

The intestinal epithelial barrier, which is regulated by the actin cytoskeleton, exhibits permeability changes during inflammation. Here we show that activation of the CysLT1 receptor by the inflammatory mediator leukotriene D4 (LTD4) causes a rapid increase in stress-fibre formation in intestinal epithelial cells. This effect was mimicked by cytotoxic necrotising factor-1 (CNF-1)-induced activation of RhoA,overexpression of constitutively active RhoA (L63-RhoA) and phorbol-ester-induced activation of protein kinase C (PKC). In accordance,inhibition of RhoA, by C3 exoenzyme or by dominant-negative RhoA (N19-RhoA),as well as GF109203X-induced inhibition of PKC, suppressed the LTD4-induced stress-fibre formation. Introduction of the dominant-negative regulatory domain of PKCδ, but not the corresponding structures from PKCα, βII or ϵ, blocked the LTD4-induced stress-fibre formation. Evaluating the relationship between PKCδ and RhoA in LTD4-induced stress-fibre formation,we found that C3 exoenzyme inhibited the rapid LTD4-elicited translocation of PKCδ to the plasma membrane. Furthermore, CNF-1-induced stress-fibre formation was blocked by GF109203X and by overexpression of the regulatory domain of PKC-δ, whereas PKC-induced stress-fibre production was not affected by N19-RhoA. We conclude that PKC-δ is located downstream of RhoA and that active RhoA and PKCδ are both necessary for LTD4-induced stress-fibre formation.

scholarly journals The anti-apoptotic effect of leukotriene D4 involves the prevention of caspase 8 activation and Bid cleavage

2003 ◽  
Vol 371 (1) ◽  
pp. 115-124 ◽  
Author(s):  
Katarina WIKSTRÖM ◽  
Maria JUHAS ◽  
Anita SJÖLANDER
Keyword(s):  
Colon Cancer ◽  
Epithelial Cells ◽  
Cytochrome C ◽  
Intestinal Epithelial Cells ◽  
Caspase 8 ◽  
Intestinal Epithelial ◽  
Leukotriene D4 ◽  
Apoptotic Protein ◽  
Increased Risk ◽  
Cox 2

We have shown in a previous study that leukotriene D4 (LTD4) signalling increases cell survival and proliferation in intestinal epithelial cells [Öhd, Wikström and Sjölander (2000) Gastroenterology 119, 1007—1018]. This is highly interesting since inflammatory conditions of the bowel are associated with an increased risk of developing colon cancer. The enzyme cyclo-oxygenase 2 (COX-2) is important in this context since it is up-regulated in colon cancer tissues and in tumour cell lines. Treatment with the COX-2-specific inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)methane sulphonamide has been shown previously to cause apoptosis in intestinal epithelial cells. In the present study, we attempted to elucidate the underlying mechanisms and we can now show that a mitochondrial pathway is employed. Inhibition of COX-2 causes release of cytochrome c, as shown by both Western-blot and microscopy studies, and as with apoptosis, this is significantly decreased by LTD4. Since previous studies showed increased Bcl-2 levels on LTD4 stimulation, we further studied apoptotic regulation at the mitochondrial level. From this we could exclude the involvement of the anti-apoptotic protein Bcl-XL as well as its pro-apoptotic counterpart Bax, since they are not expressed. Furthermore, the activity of the pro-apoptotic protein Bad (Bcl-2/Bcl-XL-antagonist, causing cell death) was completely unaffected. However, inhibition of COX-2 caused cleavage of caspase 8 into a 41kDa fragment associated with activation and caused the appearance of an activated 15kDa fragment of Bid. This indicates that N-(2-cyclohexyloxy-4-nitrophenyl)methane sulphonamide-induced apoptosis is mediated by the activation of caspase 8, via generation of truncated Bid, and thereafter release of cytochrome c. Interestingly, LTD4 not only reverses the effects induced by inhibition of COX-2 but also reduces the apoptotic potential by lowering the basal level of caspase 8 activation and truncated Bid generation.

scholarly journals Molecular Mechanism of Stimulation of Na-K-ATPase by Leukotriene D4 in Intestinal Epithelial Cells

2021 ◽  
Vol 22 (14) ◽  
pp. 7569
Author(s):  
Niraj Nepal ◽  
Subha Arthur ◽  
Molly R. Butts ◽  
Soudamani Singh ◽  
Balasubramanian Palaniappan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Atpase Activity ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Leukotriene D4 ◽  
Intestinal Crypt ◽  
Calphostin C ◽  
Chronic Intestinal Inflammation ◽  
Crypt Cells ◽  
Stimulation Of

Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and β1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and β1 subunits, mediated via the Ca-activated PKC pathway.

scholarly journals Leukotriene D4 induces association of active RhoA with phospholipase C-γ1 in intestinal epithelial cells

2002 ◽  
Vol 365 (1) ◽  
pp. 157-163 ◽  
Author(s):  
Charles Kumar THODETI ◽  
Ramin MASSOUMI ◽  
Lene BINDSLEV ◽  
Anita SJÖLANDER
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Phospholipase C ◽  
Fluorescent Protein ◽  
Intestinal Epithelial Cells ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Leukotriene D4 ◽  
Green Fluorescent ◽  
Constitutively Active

It has been previously suggested that leukotriene-induced Ca2+ signalling is mediated through a Rho-dependent process, but neither direct activation of Rho nor a mechanism underlying such signalling has been reported. Accordingly, we used the Rhotekin binding assay to assess RhoA activation in intestinal epithelial cells and observed that RhoA was activated by leukotriene D4 (LTD4). We also found that, within 15s, activation of RhoA by LTD4 led to an increased association of RhoA with G-protein βγ (Gβγ) and phospholipase C-γ1 (PLC-γ1) in the plasma membrane, as evidenced by the results of co-immunoprecipitation, glutathione S-transferase (GST) pulldown assays, and confocal microscopy. Amounts of RhoA increased in both Gβ and PLC-γ1 immunoprecipitates within 15s of LTD4 treatment. An interaction between RhoA, Gβγ and PLC-γ1 is supported by our finding that a GST fusion protein of constitutively active RhoA (GST-RhoAV14) precipitated Gβγ and PLC-γ1 from cell lysates in an agonist-dependent manner. Such an association is also substantiated by our confocal immunofluorescence results, which revealed that LTD4 induction increased co-localization of constitutively active RhoA and PLC-γ1 to the plasma membrane of cells transfected with enhanced green fluorescent protein L63RhoA. Furthermore, microinjection of neutralizing RhoA antibodies, but not control antibodies, significantly reduced LTD4-induced Ca2+ mobilization. Our results are the first to demonstrate a LTD4-induced activation of RhoA and more importantly its association with PLC-γ1, which are essential for the PLC-γ1-mediated calcium mobilization.

Leukotriene D4 activates MAPK through a Ras-independent but PKCϵ-dependent pathway in intestinal epithelial cells

2002 ◽  
Vol 115 (9) ◽  
pp. 1883-1893
Author(s):  
Sailaja Paruchuri ◽  
Bengt Hallberg ◽  
Maria Juhas ◽  
Christer Larsson ◽  
Anita Sjölander
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Proliferative Response ◽  
Egf Receptor ◽  
Intestinal Epithelial Cells ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Intestinal Epithelial ◽  
Leukotriene D4 ◽  
Mitogen Activated Protein

We have recently shown that leukotriene D4 (LTD4)increases cell survival in intestinal epithelial cells. Here we report and explore the complementary finding that LTD4 also enhances proliferation in these cells. This proliferative response was approximately half of that induced by epidermal growth factor (EGF) and its required activation of protein kinase C (PKC), Ras and the mitogen-activated protein kinase (MAPK) Erk-1/2. EGF also activated Erk-1/2 in these cells; however the EGF-receptor inhibitor PD153035 did not affect the LTD4-induced activation of Erk-1/2. In addition, LTD4 did not induce phosphorylation of the EGF receptor, nor did pertussis toxin (PTX) block EGF-induced activation of Erk-1/2, thus refuting a possible crosstalk between the receptors. Furthermore, LTD4-induced, but not EGF-induced,activation of Erk-1/2 was sensitive to PTX, PKC inhibitors and downregulation of PKCϵ. A definite role for PKCϵ in LTD4-induced stimulation of Erk-1/2 was documented by the inability of LTD4 to activate Erk-1/2 in cells transfected with either the regulatory domain of PKCϵ (an isoform specific dominant-negative inhibitor) or a kinase-dead PKCϵ. Although Ras and Raf-1 were both transiently activated by LTD4, only Raf-1 activation was abolished by abrogation of the PKC signal. Furthermore, the LTD4-induced activation of Erk-1/2 was unaffected by transfection with dominant-negative N17 Ras but blocked by transfection with kinase-dead Raf-1. Consequently, LTD4 regulates the proliferative response by a distinct Ras-independent, PKCϵ-dependent activation of Erk-1/2 and a parallel Ras-dependent signaling pathway.

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