Stretch activation of Jun N-terminal kinase/stress-activated protein kinase in mesangial cells

Background. Mesangial cells (MCs) grown on extracellular matrix (ECM)-coate d plates and exposed to cyclic stretch/ relaxation proliferate and produce ECM protein, suggesting that this may be a useful in vitro model for MC beh avior in response to increased physical forces. The induction of c-fos in r esponse 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/JN K activation in stretched MCs and the downstream consequences of this signa ling. Methods. MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexibl e-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 approxima tely 16 to 28% maximum elongation in the diameter of the surfaces. Control MCs were grown on coated rigid bottom plates. Protein levels (by Western bl ot) 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 prot ein binding to activator protein-1 (AP-1) consensus sequences was also exam ined. The role of calcium was studied with EGTA and BAPTA-AM to chelate ext ra- and intracellular calcium, respectively. Protein kinase C (PKC) was dow n-regulated by incubation with phorbol ester (PMA) for 24 hours prior to st retch. In unstretched MCs, A23187 was used as a calcium ionophore, and PKC was up-regulated with PMA application for 30 minutes to determine the effec ts on SAPK/JNK. Nuclear protein binding to AP-1 was also determined under t hese conditions. The effects of stretch, acute PMA, and A23187 on fibronect in mRNA levels were studied using reverse transcriptase-polymerase chain re action (RT-PCR). Results. Cyclic strain/relaxation led to increased SAPK/JNK activity only a t two minutes and -18 kPa and above. The activation of SAPK/JNK was depende nt 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 A23 187 increased SAPK/JNK activity, and this was potentiated by acute PMA. Str etch, acute PMA, and A23187 all increased nuclear protein binding to AP-1 c onsensus sequences. mRNA levels for fibronectin were increased by stretch i n MCs and by PMA and A23187 in static MCs. No change was observed in the am ount 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 activat ion of these stimuli in static MCs. A downstream induction of nuclear prote in binding to AP-1 consensus sequences was seen in a pattern that was compl etely concordant with the SAPK/JNK induction.