Stretch activation of jun N-terminal kinase/stress-activated protein kinase in mesangial cells.
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.
Ingram
A J
AJ
Department of Medicine, McMaster University, Hamilton, and University of Toronto, Toronto, Ontario, Canada. ingrama@fhs.mcmaster.ca
James
L
L
Ly
H
H
Thai
K
K
Scholey
J W
JW
D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D016384:Consensus Sequence; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics; D015870:Gene Expression / Q000502:physiology; D005920:Glomerular Mesangium / Q000166:cytology* / Q000201:enzymology*; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*; D013314:Stress, Mechanical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism
D002273:Carcinogens; D002614:Chelating Agents; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate