Ischemia-induced cleavage of cadherins in NRK cells: Evidence for a role of metalloproteinases

Department of Pharmacology and Toxicology, College of Medicine, Texas A&M University System Health Science Center, College Station, 77843-1114, USA.
American journal of physiology. Renal physiology (Impact Factor: 3.25). 09/2005; 289(2):F280-8. DOI: 10.1152/ajprenal.00351.2004
Source: PubMed


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.

Download full-text


Available from: Alan R Parrish, Apr 07, 2015
  • Source
    • "Finally, alongside specific changes of cadherin expression mentioned earlier, MMPs and ADAMs may regulate the mesenchymalization and the level of cell–cell interaction of the CNC by degrading Cadherins as it has been proposed for trunk NC cells (Shoval et al., 2007). Indeed, MMP2 and ADAM13 can digest N- Cadherin and Cadherin-11, respectively (Covington et al., 2005, 2006; Hartland et al., 2009; McCusker et al., 2009). Despite MMPs and ADAMs molecules holding great potential as putative key players in CNC migration, we still have a lot to discover about their precise role. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The cephalic neural crest (NC) cells delaminate from the neuroepithelium in large numbers and undergo collective cell migration under the influence of multiple factors including positive and negative taxis, cell-cell interactions mediating cell sorting, cell cooperation, and Contact-Inhibition of Locomotion. The migration has to be tightly regulated to allow NC cells to reach precise locations in order to contribute to various craniofacial structures such as the skeletal and peripheral nervous systems. Several birth defects, syndromes, and malformations are due to improper cephalic NC (CNC) migration, and NC cell migration bears important similarities to cancer cell invasion and metastasis dissemination. Therefore, understanding how CNC cells interpret multiple inputs to achieve directional collective cell migration will shed light on pathological situations where cell migration is involved.
    genesis 04/2011; 49(4):164-76. DOI:10.1002/dvg.20700 · 2.02 Impact Factor
  • Source
    • "Normal rat kidney (NRK-52E) epithelial cells were cultured as described previously (Covington et al., 2005) in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal calf serum. For measurement of mitochondrial biogenesis, NRK52-E cells were treated with 10 ␮M DOI or vehicle for 24 h, stained with MitoTracker Red (200 nM) (Invitrogen) for 20 min, washed, and imaged by fluorescence microscopy. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mitochondrial dysfunction is both a cause and target of reactive oxygen species during ischemia-reperfusion, drug, and toxicant injury. After injury, renal proximal tubular cells (RPTC) recover mitochondrial function by increasing the expression of the master regulator of mitochondrial biogenesis, peroxisome-proliferator-activated-receptor-gamma-coactivator-1alpha (PGC-1alpha). The goal of this study was to determine whether 5-hydroxytryptamine (5-HT) receptor agonists increase mitochondrial biogenesis and accelerate the recovery of mitochondrial function. Reverse transcription-polymerase chain reaction analysis confirmed the presence of 5-HT2A, 5-HT2B, and 5-HT2C receptor mRNA in RPTC. The 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI; 3-10 microM) increased PGC-1alpha levels, expression of mitochondrial proteins ATP synthase beta and NADH dehydrogenase (ubiquinone) 1beta subcomplex 8 (NDUFB8), MitoTracker Red staining intensity, cellular respiration, and ATP levels through a 5-HT receptor and PGC-1alpha-dependent pathway. Similar effects were observed with the 5-HT2 agonist m-chlorophenylpiperazine and were blocked by the 5-HT2 antagonist 8-[3-(4-fluorophenoxy) propyl]-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one (AMI-193). In addition, DOI accelerated the recovery of mitochondrial function after oxidant-induced injury in RPTC. This is the first report to demonstrate 5-HT receptor-mediated mitochondrial biogenesis, and we suggest that 5-HT-agonists may be effective in the treatment of mitochondrial and cell injury.
    Journal of Pharmacology and Experimental Therapeutics 10/2009; 332(2):632-9. DOI:10.1124/jpet.109.159947 · 3.97 Impact Factor
  • Source
    • "In addition, MMPs are capable of cleaving the E-cadherin protein (Xian et al., 2005; Covington et al., 2005). We therefore examined if the reduction in E-cadherin abundance was due to cleavage by MMPs present in TIMP-3 null lungs. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs) throughout embryogenesis. We examined lungs from TIMP3 null mice and found decreased bronchiole branching, enhanced activity of MMPs and enhanced fibronectin degradation throughout lung development compared to controls. Activation of focal adhesion kinase (FAK) was also reduced from embryonic days 12.5 through 14.5 in TIMP3 null lungs. Treatment with a synthetic MMP inhibitor, GM6001, in utero enhanced the branching pattern in both wild type and null lungs accompanied by a restoration of fibronectin localization, signaling through FAK and epithelial cell proliferation in null lungs. Direct down-regulation of FAK abundance in WT lung organ culture by siRNA targeting resulted in reduced bronchiole branching, phenocopying the TIMP3 defect. We propose that enhanced MMP activity in the absence of TIMP3 interferes with focal ECM proteolysis, perturbing the intracellular signaling necessary for correct pattern formation of the bronchiole tree during bronchiole branching morphogenesis. Thus, TIMP3 can indirectly regulate epithelial cell proliferation via MMP inhibitory activity. While others have demonstrated this function for MMPs, and there is in vitro evidence that TIMP3 controls proliferation, to our knowledge this is the first evidence of TIMP3 regulating proliferation in vivo.
    Developmental Biology 11/2006; 298(2):540-54. DOI:10.1016/j.ydbio.2006.07.004 · 3.55 Impact Factor
Show more