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.