Ischemia-induced cleavage of cadherins in NRK cells: evidence for a role of metalloproteinases.
Although ischemia has been shown to disrupt cell adhesion, the underlying molecular mechanism is unknown. In these studies, we adapted a model of ischemia-reperfusion to normal rat kidney (NRK) cells, examined disruption of the cadherin/catenin complex, and identified a role for matrix metalloproteinases (MMPs) in ischemia-induced cleavage of cadherins. In NRK cells, ischemia was induced by applying a thin layer of PBS solution supplemented with calcium and magnesium and a layer of mineral oil, which restricts exposure to oxygen. NRK cells exhibited extracellular 80-kDa and intracellular 40-kDa E-cadherin fragments after 4 h of ischemia, and at 6 h the expression of full-length E-cadherin decreased. While no fragments of N-cadherin, alpha-catenin, and gamma-catenin were observed at any time point, the detectable levels of these proteins decreased during ischemia. Ischemia was detected by an increase in pimonidazole adducts, as well as an increase in glucose transporter-1 protein expression. Ischemia did not decrease cell number, but there was a decrease in ATP levels. In addition, there was no evidence of cleaved caspase 3 or 9 during 6 h of ischemia. The MMP inhibitors GM-6001 and TAPI-O inhibited cleavage and/or loss of E- and N-cadherin protein expression. Tissue inhibitors of metalloproteinases (TIMP)-3 and to a lesser extent TIMP-2, but not TIMP-1, inhibit ischemic cleavage and/or loss of E- and N-cadherin. These results demonstrate that ischemia induces a selective metalloproteinase-dependent cleavage of E-cadherin and decrease in N-cadherin that are associated with a disruption of junctional contacts.
Covington
Marisa D
MD
Department of Pharmacology and Toxicology, College of Medicine, Texas A&M University System Health Science Center, College Station, 77843-1114, USA.
Bayless
Kayla J
KJ
Burghardt
Robert C
RC
Davis
George E
GE
Parrish
Alan R
AR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism*; D002449:Cell Aggregation; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D007511:Ischemia / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D012079:Renal Circulation / Q000502:physiology*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000502:physiology; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000502:physiology; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000502:physiology
D015820:Cadherins; D011480:Protease Inhibitors; D019715:Tissue Inhibitor of Metalloproteinase-1; D019717:Tissue Inhibitor of Metalloproteinase-3; D019716:Tissue Inhibitor of Metalloproteinase-2; D000255:Adenosine Triphosphate; D045726:Metalloproteases