Heparin inhibits Ca2+/calmodulin-dependent kinase II activation and c-fos induction in mesangial cells

Abstract

Like vascular smooth-muscle cells, rat mesangial cells (RMCs) display an anti-mitogenic response to heparin. In particular, heparin partially suppresses the ability of quiescent RMCs to enter the cell cycle and induce c-fos expression. When the mitogenic stimulus is serum, phorbol ester or platelet-derived growth factor, this response appears to result from the ability of heparin to suppress activation of the extracellular-signal-regulated kinase family of mitogen-activated protein kinases. However, we have also shown that heparin suppresses c-fos expression in response to ionophores such as ionomycin, an event independent of mitogen-activated protein kinase [Miralem, Wang, Whiteside and Templeton (1996) J. Biol. Chem. 271, 17100-17106]. Here we identify this second heparin-sensitive pathway as involving Ca2+/calmodulin-dependent kinase (CaMK) II. Ionomycin (100 nM) caused a transient rise in intracellular Ca2+ concentration ([Ca2+]i) in quiescent RMCs to 386+/-55 nM, with an increase in CaMK II activity that peaked 30 s later. The accumulation of c-fos mRNA that ensued 30 min later was prevented when the increase in [Ca2+]i was prevented with the intracellular Ca2+ chelator, 1,2-bis-(2-aminophenyoxy)ethane-N,N,N',N'-tetra-acetic acid. The broad-specificity CaMK inhibitor, KT 5926, inhibited ionomycin-dependent c-fos induction at a concentration at which it was without effect on induction by serum or phorbol ester. The CaMK II-specific inhibitor, KN-93, likewise inhibited c-fos induction by ionomycin, but not by serum or phorbol ester. ML-7, an inhibitor of the CaMK-related myosin light-chain kinase (MLCK), was without effect. Heparin (1 microg/ml) suppressed ionomycin-dependent c-fos induction. It was without effect on [Ca2+]i, but inhibited the development of autonomous CaMK II activity. However, when heparin was added to the CaMK II assay solution in vitro, it was without effect on autonomous activity. Furthermore, heparin did not prevent full activation of CaMK II by the Ca2+-calmodulin complex in vitro. Heparin did not affect myosin light-chain phosphorylation or RMC contraction, processes mediated by MLCK. We conclude that ionomycin induces c-fos in RMCs through the CaMK II pathway, and that heparin prevents CaMK II activation by an indirect process mediated by other cell components. Heparin does not affect activation of the closely related CaMK, MLCK.

Involvement of Ca2+/Calmodulin-Dependent Protein Kinase II in Heparin-Stimulated Release of Hepatic Lipase Activity from Rat Hepatocytes

“…25) However, our results in rat hepatocytes suggested that the heparin-stimulated release of HTGL activity was associated with the activation of the intracellular Ca 2ϩ , calmodulin-and CaMK-IIsensitive process. Moreover, the CaMK-II activity in the hepatocytes incubated with heparin was seen to markedly increase.…”

Heparin Inhibits Phosphorylation and Autonomous Activity of Ca2+/Calmodulin-Dependent Protein Kinase II in Vascular Smooth Muscle Cells

“…A recent report by Miralem et al 22 has also shown that heparin inhibits CaM kinase II activity in mesangial cells, which like VSMC are sensitive to the antiproliferative effects of heparin. In the study reported here we provide significant insight into the molecular mechanism of action of heparin.…”

“…7,22 It is important to note that most of the studies establishing heparin as a pharmacological inhibitor of Ca 2ϩ channels have been performed using permeabilized cells. 19 we reasoned that CaM kinase II may be a target of heparin.…”

“…We show that heparin inhibits CaM kinase II phosphorylation induced by serum and Ca 2ϩ -mobilizing stimuli and consequently generation of Ca 2ϩ /calmodulin independent activity. This inhibitory effect of heparin on CaM kinase II activity cannot be due to "carryover" of heparin from the cell lysate to the kinase assay because: 1) our past studies have shown that the rinsing conditions used in these studies successfully removes almost all of the exogenously added heparin; 42 and 2) Miralem et al 22 have shown that addition of 1 g/ml heparin directly to the in vitro kinase assay mixture does not inhibit CaM kinase II activity. Further, the inhibition of phosphorylation is primarily achieved by the activation of protein phosphatase PP-2A.…”

Slow motility in hair cells of the frog amphibian papilla: Myosin light chain-mediated shape change

“…Since the selective MLCK inhibitor, ML-7 (Saitoh et al, 1987) is a very poor inhibitor of CaMK II (Miralem and Templeton, 1998), and the selective CaMK II & IV inhibitor, KN-62, has a very low affinity for MLCK (Tokumitsu et al, 1990;Davies et al, 2000), our results suggest that both MLCK and CaM kinases II and/or IV, might contribute to the iso-volumetric length change in RAPHCs. Thus, we sought to determine if these two Ca 2+ /CaM-dependent pathways act in parallel or series.…”