PKC-ζ is required in EGF protection of microtubules and intestinal barrier integrity against oxidant injury

2002 ◽  
Vol 282 (5) ◽  
pp. G794-G808 ◽  
Author(s):  
A. Banan ◽  
J. Z. Fields ◽  
D. A. Talmage ◽  
L. Zhang ◽  
A. Keshavarzian

Using monolayers of human intestinal (Caco-2) cells, we showed that epidermal growth factor (EGF) protects intestinal barrier integrity against oxidant injury by protecting the microtubules and that protein kinase C (PKC) is required. Because atypical PKC-ζ isoform is abundant in wild-type (WT) Caco-2 cells, we hypothesized that PKC-ζ mediates, at least in part, EGF protection. Intestinal cells (Caco-2 or HT-29) were transfected to stably over- or underexpress PKC-ζ. These clones were preincubated with low or high doses of EGF or a PKC activator [1-oleoyl-2-acetyl- sn-glycerol (OAG)] before oxidant (0.5 mM H2O2). Relative to WT cells exposed to oxidant, only monolayers of transfected cells overexpressing PKC-ζ (2.9-fold) were protected against oxidant injury as indicated by increases in polymerized tubulin and decreases in monomeric tubulin, enhancement of architectural stability of the microtubule cytoskeleton, and increases in monolayer barrier integrity toward control levels (62% less leakiness). Overexpression-induced protection was OAG independent and even EGF independent, but EGF significantly potentiated PKC-ζ protection. Most overexpressed PKC-ζ (92%) resided in membrane and cytoskeletal fractions, indicating constitutive activation of PKC-ζ. Stably inhibiting PKC-ζ expression (95%) with antisense transfection substantially attenuated EGF protection as demonstrated by reduced tubulin assembly and increased microtubule disassembly, disruption of the microtubule cytoskeleton, and loss of monolayer barrier integrity. We conclude that 1) activation of PKC-ζ is necessary for EGF-induced protection, 2) PKC-ζ appears to be an endogenous stabilizer of the microtubule cytoskeleton and of intestinal barrier function against oxidative injury, and 3) we have identified a novel biological function (protection) among the atypical isoforms of PKC.

2001 ◽  
Vol 280 (5) ◽  
pp. G828-G843 ◽  
Author(s):  
A. Banan ◽  
J. Z. Fields ◽  
Y. Zhang ◽  
A. Keshavarzian

Using monolayers of human intestinal (Caco-2) cells, we showed that growth factors (GFs) protect microtubules and barrier integrity against oxidative injury. Studies in nongastrointestinal cell models suggest that protein kinase C (PKC) signaling is key in GF-induced effects and that cytosolic calcium concentration ([Ca2+]i) is essential in cell integrity. We hypothesized that GF protection involves activating PKC and maintaining normal [Ca2+]i. Monolayers were pretreated with epidermal growth factor (EGF) or PKC or Ca2+ modulators before exposure to oxidants (H2O2 or HOCl). Oxidants disrupted microtubules and barrier integrity, and EGF protected from this damage. EGF caused rapid distribution of PKC-α, PKC-βI, and PKC-ζ isoforms to cell membranes, enhancing PKC activity of membrane fractions while reducing PKC activity of cytosolic fractions. EGF enhanced 45Ca2+ efflux and prevented oxidant-induced (sustained) rises in [Ca2+]i. PKC inhibitors abolished and PKC activators mimicked EGF protection. Oxidant damage was mimicked by and potentiated by a Ca2+ ionophore (A-23187), exacerbated by high-Ca2+ media, and prevented by calcium removal or chelation or by Ca2+ channel antagonists. PKC activators mimicked EGF on both 45Ca2+ efflux and [Ca2+]i. Membrane Ca2+-ATPase pump inhibitors prevented protection by EGF or PKC activators. In conclusion, EGF protection of microtubules and the intestinal epithelial barrier requires activation of PKC signal transduction and normalization of [Ca2+]i.


2020 ◽  
Vol 117 (46) ◽  
pp. 28930-28938
Author(s):  
Toru Nakata ◽  
Elizabeth A. Creasey ◽  
Motohiko Kadoki ◽  
Helen Lin ◽  
Martin K. Selig ◽  
...  

Common genetic variants interact with environmental factors to impact risk of heritable diseases. A notable example of this is a single-nucleotide variant in the Solute Carrier Family 39 Member 8 (SLC39A8)geneencoding the missense variant A391T, which is associated with a variety of traits ranging from Parkinson’s disease and neuropsychiatric disease to cardiovascular and metabolic diseases and Crohn’s disease. The remarkable extent of pleiotropy exhibited bySLC39A8A391T raises key questions regarding how a single coding variant can contribute to this diversity of clinical outcomes and what is the mechanistic basis for this pleiotropy. Here, we generate a murine model for theSlc39a8A391T allele and demonstrate that these mice exhibit Mn deficiency in the colon associated with impaired intestinal barrier function and epithelial glycocalyx disruption. Consequently,Slc39a8A391T mice exhibit increased sensitivity to epithelial injury and pathological inflammation in the colon. Taken together, our results link a genetic variant with a dietary trace element to shed light on a tissue-specific mechanism of disease risk based on impaired intestinal barrier integrity.


2020 ◽  
Vol 11 (12) ◽  
pp. 10519-10533
Author(s):  
Liuying Zhu ◽  
Juan Li ◽  
Changhao Wei ◽  
Ting Luo ◽  
Zeyuan Deng ◽  
...  

A polysaccharide from Fagopyrum esculentum Moench bee pollen relieves antibiotic-induced microbiota dysbiosis to improve immune function and intestinal barrier integrity by increasing intestinal sIgA secretion and inhibiting inflammation.


2001 ◽  
Vol 281 (2) ◽  
pp. G412-G423 ◽  
Author(s):  
A. Banan ◽  
J. Z. Fields ◽  
Y. Zhang ◽  
A. Keshavarzian

Loss of intestinal barrier integrity is associated with oxidative inflammatory GI disorders including inflammatory bowel disease. Using monolayers of human intestinal epithelial (Caco-2) cells, we recently reported that epidermal growth factor (EGF) protects barrier integrity against oxidants by stabilizing the microtubule cytoskeleton, but the mechanism downstream of the EGF receptor (EGFR) is not established. We hypothesized that phospholipase C (PLC)-γ is required. Caco-2 monolayers were exposed to oxidant (H2O2) with or without pretreatment with EGF or specific inhibitors of EGFR tyrosine kinase (AG-1478, tyrphostin 25) or of PLC (L-108, U-73122). Other Caco-2 cells were stably transfected with a dominant negative fragment for PLC-γ (PLCz) to inhibit PLC-γ activation. Doses of EGF that enhanced PLC activity also protected monolayers against oxidant-induced tubulin disassembly, disruption of the microtubule cytoskeleton, and barrier leakiness as assessed by radioimmunoassay, quantitative Western blots, high-resolution laser confocal microscopy, and fluorometry, respectively. Pretreatment with either type of inhibitor abolished EGF protection. Transfected cells also lost EGF protection and showed reduced PLC-γ phosphorylation and activity. We conclude that EGF protection requires PLC-γ signaling and that PLC-γ may be a useful therapeutic target.


Gut Microbes ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 1946368
Author(s):  
Angélica Cruz-Lebrón ◽  
Ramona Johnson ◽  
Claire Mazahery ◽  
Zach Troyer ◽  
Samira Joussef-Piña ◽  
...  

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