Protein kinase D protects against oxidative stress-induced intestinal epithelial cell injury via Rho/ROK/PKC-δ pathway activation

Protein kinase D (PKD) is a novel protein serine kinase that has recently been implicated in diverse cellular functions, including apoptosis and cell proliferation. The purpose of our present study was 1) to define the activation of PKD in intestinal epithelial cells treated with H2O2, an agent that induces oxidative stress, and 2) to delineate the upstream signaling mechanisms mediating the activation of PKD. We found that the activation of PKD is induced by H2O2 in both a dose- and time-dependent fashion. PKD phosphorylation was attenuated by rottlerin, a selective PKC-δ inhibitor, and by small interfering RNA (siRNA) directed against PKC-δ, suggesting the regulation of PKD activity by upstream PKC-δ. Activation of PKD was also blocked by a Rho kinase (ROK)-specific inhibitor, Y-27632, as well as by C3, a Rho protein inhibitor, demonstrating that the Rho/ROK pathway also mediates PKD activity in intestinal cells. In addition, H2O2-induced PKC-δ phosphorylation was inhibited by C3 treatment, further suggesting that PKC-δ is downstream of Rho/ROK. Interestingly, H2O2-induced intestinal cell apoptosis was enhanced by PKD siRNA. Together, these results clearly demonstrate that oxidative stress induces PKD activation in intestinal epithelial cells and that this activation is regulated by upstream PKC-δ and Rho/ROK pathways. Importantly, our findings suggest that PKD activation protects intestinal epithelial cells from oxidative stress-induced apoptosis. These findings have potential clinical implications for intestinal injury associated with oxidative stress (e.g., necrotizing enterocolitis in infants).

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PKD in intestinal epithelial cells: rapid activation by phorbol esters, LPA, and angiotensin through PKC

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.4.c929 ◽

2001 ◽

Vol 280 (4) ◽

pp. C929-C942 ◽

Cited By ~ 67

Author(s):

Terence Chiu ◽

Enrique Rozengurt

Keyword(s):

Protein Kinase ◽

Epithelial Cells ◽

Phorbol Esters ◽

Intestinal Epithelial Cells ◽

Intestinal Cell ◽

Protein Kinase D ◽

Intestinal Epithelial ◽

Downstream Signaling ◽

Normal Rat ◽

Rapid Activation

Protein kinase C (PKC) is implicated in the regulation of multiple important functions in intestinal epithelial cells, but the downstream signaling targets of PKCs in these cells remain poorly characterized. Here we report that treatment of normal rat intestinal cell lines IEC-6 and IEC-18 with phorbol 12,13-dibutyrate (PDBu) led to a rapid and striking PKC-dependent activation of protein kinase D (PKD; also known as PKCμ). Unlike conventional and novel PKCs, PKD did not undergo downregulation in response to prolonged (24 h) exposure of IEC-6 or IEC-18 cells to PDBu. PKD was also rapidly activated in these cells by lysophosphatidic acid (LPA) or angiotensin in a concentration-dependent fashion via a PKC-dependent pathway. EC50 values were 0.1 μM and 2 nM for LPA and angiotensin II, respectively. LPA-induced PKD activation was prevented selectively by treatment with pertussis toxin. PKD activation was tightly associated with an increase in PKD autophosphorylation at serine 916. Our results identify PKD as a novel early point of convergence and integration of Gi and Gq signaling in intestinal epithelial cells.

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Etomidate Alleviates Ischemia-Anoxia Reperfusion Injury in Intestinal Epithelial Cells by Inhibiting the Activation of traf6-Regulated NF-KB Signaling

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2022.2990 ◽

2022 ◽

Vol 12 (5) ◽

pp. 1015-1021

Author(s):

Gen Lin ◽

Ruichun Long ◽

Xiaoqing Yang ◽

Songsong Mao ◽

Hongying Li

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Intestinal Epithelial Cells ◽

Ischemia Reperfusion ◽

Stress Factors ◽

Inflammatory Factors ◽

Intestinal Cell ◽

Intestinal Epithelial

Objective: The present study aimed to investigate the role of etomidate in intestinal cell ischemia and hypoxia-reperfusion injury and potential mechanisms. Method: In this study, we establish the intestinal epithelial cells ischemia-reperfusion model in vitro. CCK8 was used to detect cell viability and flow cytometry assay was used to detect apoptosis levels of treated OGD/R model cells. ELISA measured the expression level of oxidative stress factors and inflammatory factors. Furthermore, western blot assay was used to detect the expression the apoptosis-related factors and TNFR-associated factors in treated OGD/R model cells. Result: Etomidate does not affect the activity of intestinal epithelial cells, and can protect intestinal epithelial cells to reduce ischemiareperfusion injury, and the expression of inflammatory factors and oxidative stress in cells with mild intestinal epithelial ischemia-reperfusion injury. Etomidate alleviates apoptosis of intestinal epithelial ischemia-reperfusion injury cells. Etomidate inhibits the activation of traf6-mediated NF-κB signal during ischemia-anoxia reperfusion of intestinal epithelial cells. Conclusion: Taken together, our study demonstrated that etomidate attenuates inflammatory response and apoptosis in intestinal epithelial cells during ischemic hypoxia-reperfusion injury and inhibits activation of NF-κB signaling regulated by TRAF6.

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Oxidative stress enhances IL-8 and inhibits CCL20 production from intestinal epithelial cells in response to bacterial flagellin

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00089.2010 ◽

2010 ◽

Vol 299 (3) ◽

pp. G733-G741 ◽

Cited By ~ 20

Author(s):

Sabine M. Ivison ◽

Ce Wang ◽

Megan E. Himmel ◽

Jared Sheridan ◽

Jonathan Delano ◽

...

Keyword(s):

Oxidative Stress ◽

Protein Kinase ◽

Epithelial Cells ◽

Pathogenic Bacteria ◽

Intestinal Epithelial Cells ◽

Host Cells ◽

Dependent Manner ◽

Intestinal Epithelial ◽

Chemokine Production ◽

Molecular Signals

Intestinal epithelial cells act as innate immune sentinels, as the first cells that encounter diarrheal pathogens. They use pattern recognition molecules such as the Toll-like receptors (TLRs) to identify molecular signals found on microbes but not host cells or food components. TLRs cannot generally distinguish the molecular signals on pathogenic bacteria from those found in commensals, yet under healthy conditions epithelial immune responses are kept in check. We hypothesized that, in the setting of tissue damage or stress, intestinal epithelial cells would upregulate their responses to TLR ligands to reflect the greater need for immediate protection against pathogens. We treated Caco-2 cells with the TLR5 agonist flagellin in the presence or absence of H2O2 and measured chemokine production and intracellular signaling pathways. H2O2 increased flagellin-induced IL-8 (CXCL8) production in a dose-dependent manner. This was associated with synergistic phosphorylation of p38 MAP kinase and with prolonged I-κB degradation and NF-κB activation. The H2O2-mediated potentiation of IL-8 production required the activity of p38, tyrosine kinases, phospholipase Cγ, and intracellular calcium, but not protein kinase C or protein kinase D. H2O2 prolonged and augmented NF-κB activation by flagellin. In contrast to IL-8, CCL20 (MIP3α) production by flagellin was reduced by H2O2, and this effect was not calcium dependent. Oxidative stress biases intestinal epithelial responses to flagellin, leading to increased production of IL-8 and decreased production of CCL20. This suggests that epithelial cells are capable of sensing the extracellular environment and adjusting their antimicrobial responses accordingly.

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