TY - JOUR
T1 - Stretch activation of Jun N-terminal kinase/stress-activated protein kinase in mesangial cells
AU - Ingram, Alistair J.
AU - James, Leighton
AU - Ly, Hao
AU - Thai, Kerri
AU - Scholey, James W.
N1 - Funding Information:
This work was supported by a grant from the Heart and Stroke Foundation of Canada and the Juvenile Diabetes Foundation/Medical Research Council of Canada to Dr. Scholey and from the Kidney Foundation of Canada to Dr. Ingram. Dr. Scholey was also supported by a Scholarship Award from the PMAC/MRC. Dr. James is supported by a research fellowship from the Kidney Foundation of Canada and Medical Research Council of Canada.
PY - 2000
Y1 - 2000
N2 - Background. Mesangial cells (MCs) grown on extracellular matrix (ECM)-coated plates and exposed to cyclic stretch/relaxation proliferate and produce ECM protein, suggesting that this may be a useful in vitro model for MC behavior in response to increased physical forces. The induction of c-fos in response to MC stretch has been shown. Stimuli that lead to c-fos induction pass through mitogen-activated protein (MAP) kinase pathways. We have seen early activation of jun N-terminal kinase/stress-activated protein kinase (SAPK/JNK) in MCs exposed to cyclic stretch. Accordingly, we studied SAPK/JNK activation in stretched MCs and the downstream consequences of this signaling. Methods. MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 2 to 60 minutes of cyclic strain (60 cycles per minute) by generation of vacuums of -10 to -27 kPa, inducing approximately 16 to 28% maximum elongation in the diameter of the surfaces. Control MCs were grown on coated rigid bottom plates. Protein levels (by Western blot) and activity assays for SAPK/JNK were performed under these conditions. We observed marked activation at -18 kPa and above and at two minutes, and then we studied activation mechanisms under these conditions. Nuclear protein binding to activator protein-1 (AP-1) consensus sequences was also examined. The role of calcium was studied with EGTA and BAPTA-AM to chelate extra- and intracellular calcium, respectively. Protein kinase C (PKC) was down-regulated by incubation with phorbol ester (PMA) for 24 hours prior to stretch. In unstretched MCs, A23187 was used as a calcium ionophore, and PKC was up-regulated with PMA application for 30 minutes to determine the effects on SAPK/JNK. Nuclear protein binding to AP-1 was also determined under these conditions. The effects of stretch, acute PMA, and A23187 on fibronectin mRNA levels were studied using reverse transcriptase-polymerase chain reaction (RT-PCR). Results. Cyclic strain/relaxation led to increased SAPK/JNK activity only at two minutes and -18 kPa and above. The activation of SAPK/JNK was dependent on intracellular calcium, with BAPTA-AM almost completely abrogating the response to stretch. EGTA was without effect. Down-regulation of PKC also led to a diminution of activity. In static cells, the calcium ionophore A23187 increased SAPK/JNK activity, and this was potentiated by acute PMA. Stretch, acute PMA, and A23187 all increased nuclear protein binding to AP-1 consensus sequences, mRNA levels for fibronectin were increased by stretch in MCs and by PMA and A23187 in static MCs. No change was observed in the amount of SAPK/JNK protein present in stretched MCs by Western blot. Conclusions. Stretch leads to early activation of SAPK/JNK in MCs. This is dependent on intracellular calcium and PKC and can be replicated by activation of these stimuli in static MCs. A downstream induction of nuclear protein binding to AP-1 consensus sequences was seen in a pattern that was completely concordant with the SAPK/JNK induction.
AB - Background. Mesangial cells (MCs) grown on extracellular matrix (ECM)-coated plates and exposed to cyclic stretch/relaxation proliferate and produce ECM protein, suggesting that this may be a useful in vitro model for MC behavior in response to increased physical forces. The induction of c-fos in response to MC stretch has been shown. Stimuli that lead to c-fos induction pass through mitogen-activated protein (MAP) kinase pathways. We have seen early activation of jun N-terminal kinase/stress-activated protein kinase (SAPK/JNK) in MCs exposed to cyclic stretch. Accordingly, we studied SAPK/JNK activation in stretched MCs and the downstream consequences of this signaling. Methods. MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 2 to 60 minutes of cyclic strain (60 cycles per minute) by generation of vacuums of -10 to -27 kPa, inducing approximately 16 to 28% maximum elongation in the diameter of the surfaces. Control MCs were grown on coated rigid bottom plates. Protein levels (by Western blot) and activity assays for SAPK/JNK were performed under these conditions. We observed marked activation at -18 kPa and above and at two minutes, and then we studied activation mechanisms under these conditions. Nuclear protein binding to activator protein-1 (AP-1) consensus sequences was also examined. The role of calcium was studied with EGTA and BAPTA-AM to chelate extra- and intracellular calcium, respectively. Protein kinase C (PKC) was down-regulated by incubation with phorbol ester (PMA) for 24 hours prior to stretch. In unstretched MCs, A23187 was used as a calcium ionophore, and PKC was up-regulated with PMA application for 30 minutes to determine the effects on SAPK/JNK. Nuclear protein binding to AP-1 was also determined under these conditions. The effects of stretch, acute PMA, and A23187 on fibronectin mRNA levels were studied using reverse transcriptase-polymerase chain reaction (RT-PCR). Results. Cyclic strain/relaxation led to increased SAPK/JNK activity only at two minutes and -18 kPa and above. The activation of SAPK/JNK was dependent on intracellular calcium, with BAPTA-AM almost completely abrogating the response to stretch. EGTA was without effect. Down-regulation of PKC also led to a diminution of activity. In static cells, the calcium ionophore A23187 increased SAPK/JNK activity, and this was potentiated by acute PMA. Stretch, acute PMA, and A23187 all increased nuclear protein binding to AP-1 consensus sequences, mRNA levels for fibronectin were increased by stretch in MCs and by PMA and A23187 in static MCs. No change was observed in the amount of SAPK/JNK protein present in stretched MCs by Western blot. Conclusions. Stretch leads to early activation of SAPK/JNK in MCs. This is dependent on intracellular calcium and PKC and can be replicated by activation of these stimuli in static MCs. A downstream induction of nuclear protein binding to AP-1 consensus sequences was seen in a pattern that was completely concordant with the SAPK/JNK induction.
KW - Calcium
KW - Cyclic stretch
KW - Extracellular matrix protein
KW - Mechanical strain
KW - Nuclear protein binding
KW - Protein kinase C
KW - Signaling
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U2 - 10.1046/j.1523-1755.2000.00305.x
DO - 10.1046/j.1523-1755.2000.00305.x
M3 - Article
C2 - 11012878
AN - SCOPUS:0033806613
SN - 0085-2538
VL - 58
SP - 1431
EP - 1439
JO - Kidney International
JF - Kidney International
IS - 4
ER -