RhoA regulation of NF-κB activation is mediated by COX-2-dependent feedback inhibition of IKK in kidney epithelial cells

2007 ◽  
Vol 293 (3) ◽  
pp. C1160-C1170 ◽  
Author(s):  
William W. Polk ◽  
Maureen E. Ellis ◽  
John V. Kushleika ◽  
P. Lynne Simmonds ◽  
James S. Woods
Keyword(s):  
Dna Binding ◽  
Epithelial Cells ◽  
Feedback Inhibition ◽  
Rat Kidney ◽  
Constitutive Expression ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cox 2 ◽  
Kidney Epithelial Cells ◽  
Iκbα Phosphorylation

Numerous studies have demonstrated a central role of renal tubular epithelial cells in the etiology of kidney injury and disease through the elaboration of inflammatory mediators. However, little is known about the cellular signaling mechanisms involved in this process. In this study we employed normal rat kidney epithelial (NRK52E) cells to identify a novel LPS-induced signaling pathway in which RhoA-mediated AP-1 activity promotes expression of cyclooxygenase-2 (COX-2) with consequent feedback inhibition of NF-κB activation through IKKβ. Inhibition of RhoA signaling using either the RhoA kinase inhibitor Y-27632 or a dominant negative mutant of RhoA (RhoA-DN) dramatically extended the duration of p65-DNA binding, IκBα phosphorylation, and IKKβ activity following LPS treatment. Prolongation of events associated with NF-κB activation was also observed in cells pretreated and/or cotransfected with the JNK inhibitor SP600125 or deletion mutants of MEKK1 (MEKK1-KD) or Jun (Jun-DN). Conversely, constitutive expression of RhoA prevented NF-κB activation by LPS, and this effect was reversed by cotransfection with MEKK1-KD. In addition, we found that the RhoA/AP-1 signaling axis plays a necessary role in COX-2 expression by LPS and that this effect is independent of NF-κB activation. Moreover, inhibition of COX-2 activity results in persistent p65-DNA binding, IκBα phosphorylation, and IKKβ activity, similar to that observed after prevention of RhoA/AP-1 axis signaling. These findings suggest that COX-2 links the RhoA/AP-1 signaling cascade to NF-κB activation, thereby defining a novel integrated model for regulation of the inflammatory response of kidney epithelial cells to LPS and potentially other external stimuli.

scholarly journals Atypical Protein Kinase C Is Involved in the Evolutionarily Conserved Par Protein Complex and Plays a Critical Role in Establishing Epithelia-Specific Junctional Structures

2001 ◽  
Vol 152 (6) ◽  
pp. 1183-1196 ◽  
Author(s):  
Atsushi Suzuki ◽  
Tomoyuki Yamanaka ◽  
Tomonori Hirose ◽  
Naoyuki Manabe ◽  
Keiko Mizuno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Polarity ◽  
Protein Complex ◽  
Mdck Cells ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Surface Polarity ◽  
C Elegans ◽  
Evolutionarily Conserved

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

Prevention of TNF-α-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

2004 ◽  
Vol 286 (3) ◽  
pp. G479-G490 ◽  
Author(s):  
Sujoy Bhattacharya ◽  
Ramesh M. Ray ◽  
Leonard R. Johnson
Keyword(s):  
Epithelial Cells ◽  
Cytochrome C ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cytochrome C Release ◽  
Erk Phosphorylation ◽  
Tnf Α ◽  
Induced Apoptosis ◽  
Jnk Activation

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-α/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by α-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-α/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-α/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-α/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-α/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-α/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-α/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-α/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.

scholarly journals DNA microarray analysis of normal rat kidney epithelial cells treated with cadmium

2011 ◽  
Vol 36 (1) ◽  
pp. 127-129 ◽  
Author(s):  
Maki Tokumoto ◽  
Tomoaki Ohtsu ◽  
Akiko Honda ◽  
Yasuyuki Fujiwara ◽  
Hisamitsu Nagase ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Microarray Analysis ◽  
Dna Microarray ◽  
Rat Kidney ◽  
Dna Microarray Analysis ◽  
Normal Rat ◽  
Kidney Epithelial Cells

scholarly journals An IFN regulatory factor-2 DNA-binding domain dominant negative mutant exhibits altered cell growth and gene expression

Oncogene ◽  
2000 ◽  
Vol 19 (11) ◽  
pp. 1411-1418 ◽  
Author(s):  
Y R Rubinstein ◽  
P H Driggers ◽  
V V Ogryzko ◽  
A M Thornton ◽  
K Ozato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Dna Binding ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dna Binding Domain ◽  
Regulatory Factor ◽  
Altered Cell ◽  
Negative Mutant

scholarly journals Perturbation of β1-Integrin Function in Involuting Mammary Gland Results in Premature Dedifferentiation of Secretory Epithelial Cells

2002 ◽  
Vol 13 (10) ◽  
pp. 3521-3531 ◽  
Author(s):  
Marisa M. Faraldo ◽  
Marie-Ange Deugnier ◽  
Sylvie Tlouzeau ◽  
Jean Paul Thiery ◽  
Marina A. Glukhova
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Dominant Negative Mutant ◽  
Β1 Integrin ◽  
Mrna Levels ◽  
Pregnancy And Lactation

To study the mechanism of β1-integrin function in vivo, we have generated transgenic mouse expressing a dominant negative mutant of β1-integrin under the control of mouse mammary tumor virus (MMTV) promoter (MMTV-β1-cyto). Mammary glands from MMTV-β1-cyto transgenic females present significant growth defects during pregnancy and lactation and impaired differentiation of secretory epithelial cells at the onset of lactation. We report herein that perturbation of β1-integrin function in involuting mammary gland induced precocious dedifferentiation of the secretory epithelium, as shown by the premature decrease in β-casein and whey acidic protein mRNA levels, accompanied by inactivation of STAT5, a transcription factor essential for mammary gland development and up-regulation of nuclear factor-κB, a negative regulator of STAT5 signaling. This is the first study demonstrating in vivo that cell–extracellular matrix interactions involving β1-integrins play an important role in the control of milk gene transcription and in the maintenance of the mammary epithelial cell differentiated state.

scholarly journals trans-dominant inhibition of poly(ADP-ribosyl)ation sensitizes cells against gamma-irradiation and N-methyl-N'-nitro-N-nitrosoguanidine but does not limit DNA replication of a polyomavirus replicon.

1995 ◽  
Vol 15 (6) ◽  
pp. 3154-3163 ◽  
Author(s):  
J H Küpper ◽  
M Müller ◽  
M K Jacobson ◽  
J Tatsumi-Miyajima ◽  
D L Coyle ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Gamma Irradiation ◽  
Cellular Proliferation ◽  
Molecular Genetic ◽  
Genetic System ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dna Binding Domain

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30), with NAD+ serving as the substrate. PARP is strongly activated upon recognition of DNA strand breaks by its DNA-binding domain. Experiments with low-molecular-weight inhibitors of PARP have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evidence for nonspecific inhibitor effects prompted us to develop a molecular genetic system to inhibit PARP in living cells, i.e., to overexpress selectively the DNA-binding domain of PARP as a dominant negative mutant. Here we report on a cell culture system which allows inducible, high-level expression of the DNA-binding domain. Induction of this domain leads to about 90% reduction of poly(ADP-ribose) accumulation after gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In contrast, induction does not affect normal cellular proliferation or the replication of a transfected polyomavirus replicon. Thus, trans-dominant inhibition of the poly(ADP-ribose) accumulation occurring after gamma-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflicted damage.

Pneumococci induced TLR- and Rac1-dependent NF-κB-recruitment to the IL-8 promoter in lung epithelial cells

2006 ◽  
Vol 290 (4) ◽  
pp. L730-L737 ◽  
Author(s):  
Bernd Schmeck ◽  
Sylvia Huber ◽  
Kerstin Moog ◽  
Janine Zahlten ◽  
Andreas C. Hocke ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Activation ◽  
Rho Gtpase ◽  
Dominant Negative ◽  
Lung Epithelial Cells ◽  
Dominant Negative Mutant ◽  
Epithelial Cell Line ◽  
Phosphatidylinositol 3 Kinase ◽  
Lung Epithelial ◽  
Phosphatidylinositol 3

Streptococcus pneumoniae is the major pathogen of community-acquired pneumonia. The respiratory epithelium constitutes the first line of defense against invading lung pathogens, including pneumococci. We analyzed the involvement of Toll-like receptors (TLR) and Rho-GTPase signaling in the activation of human lung epithelial cells by pneumococci. S. pneumoniae induced release of interleukin-8 (IL-8) by human bronchial epithelial cell line BEAS-2B. Specific inhibition of Rac1 by Nsc23766 or a dominant-negative mutant of Rac1 strongly reduced cytokine release. In addition, pneumococci-related cell activation (IL-8 release, NF-κB-activation) depended on MyD88, phosphatidylinositol 3-kinase, and Cdc42 but not on RhoA. Pneumococci enhanced TLR1 and TLR2 mRNA expression in BEAS-2B cells, whereas TLR4 and TLR6 expression was constitutively high. TLR1 and 2 synergistically recognized pneumococci in cotransfection experiments. TLR4, TLR6, LPS-binding protein, and CD14 seem not to be involved in pneumococci-dependent cell activation. At the IL-8 gene promoter, recruitment of phosphorylated NF-κB subunit p65 was blocked by inhibition of Rac1, whereas binding of the phosphorylated activator protein-1 subunit c-Jun to the promoter was not diminished. In summary, these results suggest that S. pneumoniae activate human epithelial cells by TLR1/2 and a phosphatidylinositol 3-kinase- and Rac1-dependent NF-κB-recruitment to the IL-8 promoter.

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