Andrea Huwiler

Stimulation by extracellular ATP and UTP of the mitogen‐activated protein kinase cascade and proliferation of rat renal mesangial cells

1 Extracellular ATP and UTP have been reported to activate a nucleotide receptor that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases C and D, respectively. Here we report that ATP and UTP potently stimulate mesangial cell proliferation. 2 Both nucleotides stimulate phosphorylation and activation of mitogen‐activated protein kinase and a biphasic phosphorylation of the up‐stream mitogen‐activated protein kinase kinase. 3 When added at 100 μm, ATPγS, UTP and ATP were the most potent activators of mitogen‐activated protein kinase. βγ‐imido‐ATP was somewhat less active and ADP and 2‐methylthio‐ATP caused a weak induction of enzyme activity. Activation of mitogen‐activated protein kinase by both ATP and UTP is dose‐dependently attenuated by the P2‐receptor antagonist, suramin. 4 The protein kinase C activator 12‐0‐tetradecanoylphorbol 13‐acetate, but not the biologically inactive 4α‐phorbol 12,13‐didecanoate, increased mitogen‐activated protein kinase activity in mesangial cells, suggesting that protein kinase C may mediate nucleotide‐induced stimulation of mitogen‐activated protein kinase. 5 Down‐regulation of protein kinase C ‐α and‐θ isoenzymes by 4 h or 8 h treatment with phorbol ester partially inhibited ATP‐ and UTP‐triggered mitogen‐activated protein kinase activation. Moreover, a 24 h treatment of mesangial cells with phorbol ester, a regimen that also causes depletion of protein kinase C‐∍ did not further reduce the level of mitogen‐activated protein kinase stimulation. 6 The specific protein kinase C inhibitor, CGP 41251, which displayed a selectivity for the Ca2+‐dependent isoenzymes, as compared to the Ca2+‐independent isoenzymes did not inhibit nucleotide‐stimulated mitogen‐activated protein kinase phosphorylation, thus implicating the involvement of a Ca2+‐independent protein kinase C isoform. 7 In summary, these results suggest that ATP and UTP trigger the activation of the mitogen‐activated protein kinase signalling cascade in mesangial cells and this may be responsible for the potent mitogenic activity of both nucleotides.

Cross-talk between secretory phospholipase A2 and cytosolic phospholipase A2 in rat renal mesangial cells

Incubation of rat glomerular mesangial cells with potent proinflammatory cytokines like interleukin 1beta, (IL- 1beta) triggers the expression of a non-pancreatic secretory phospholipase A2 (sPLA2) and increases the formation of prostaglandin E2. We show here that sPLA2 acts in an autocrine fashion on mesangial cells and induces a rapid activation of protein kinase C (PKC) isoenzymes delta and epsilon and of p42 mitogen-activated protein kinase (MAPK), two putative activators of cytosolic phospholipase A2 (cPLA2). sPLA2 also activates Raf-1 kinase in mesangial cells which integrates the signals coming from PKC for further processing along the MAPK cascade. Subsequently a phosphorylation and activation of cPLA2 is observed, thus arguing for a cross-talk between the two classes of PLA2. Pretreatment of cells with either the highly specific PKC inhibitor Ro-318220 or the highly specific MAPK kinase (MEK) inhibitor PD 98059 completely blocked the sPLA2-induced cPLA2 activation, indicating that both kinases are essential for the cross-talk between the two types of PLA2. The effect of sPLA2 is mimicked by lysophosphatidylcholine (LPC), a reaction product of sPLA2 activity. LPC stimulates PKC-epsilon, Raf-1 kinase and MAPK activation as well as cPLA2 activation with a subsequent increase in arachidonic acid release from mesangial cells. These data suggest that sPLA2 by cleaving membrane phospholipids and generating LPC and other lysophospholipids activates cPLA2 via the PKC/Raf-1/MAPK signalling pathway. Hence a network of interactions between different PLA2s is operative in mesangial cells and may contribute to the progression of glomerular inflammatory processes.

Nitric oxide stimulates stress-activated protein kinases in glomerular endothelial and mesangial cells

Exposure of rat glomerular mesangial cells and primary cultures of bovine glomerular endothelial cells to compounds releasing nitric oxide (NO), including MAHMANONOate, S‐nitrosoglutathione, and spermine‐NO, results in a time‐ and concentration‐dependent activation of stress‐activated protein kinases (SAPK) as measured by the phosphorylation of c‐Jun in a solid phase kinase assay. Dibutyryl cGMP had no effect on SAPK activity. Pretreatment of the cells with the tyrosine kinase inhibitor genistein strongly attenuated NO‐induced c‐Jun phosphorylation. Furthermore, N‐acetylcysteine markedly reduced the activation of SAPK in response to NO. These studies identify SAPK as a target for NO which may be critical for the NO‐induced apoptosis of glomerular mesangial and endothelial cells.

Transforming growth factor β2 stimulates acute and chronic activation of the mitogen-activated protein kinase cascade in rat renal mesangial cells

Exposure of rat glomerular mesangial cells to transforming growth factor β2 (TGFβ2) stimulates a biphasic mitogen‐activated protein kinase (MAP kinase) activation. A rapid increase in activity (maximal at 10 min) is followed by a second persistent level of activity which steadily increases over 24 h. The second peak of MAP kinase activity is markedly attenuated by the protein synthesis inhibitor cycloheximide and consequently is paralleled by a pronounced de‐novo synthesis of p42 and p44 MAP kinases as measured by immunoprecipitation of [35S]methionine‐labeled mesangial cells. In addition, an increased de‐novo synthesis of MAP kinase kinase (MEK), the upstream activator of MAP kinase, is observed in response to TGFβ2 stimulation. We propose that TGFβ‐induced activation and de‐novo synthesis of MAP kinases and MEK is important for the multifunctional actions of this cytokine in mesangial cells and its role in disease states characterized by excessive fibrosis.

Stimulation by extracellular ATP and UTP of the stress-activated protein kinase cascade in rat renal mesangial cells.

Extracellular adenosine 5′‐triphosphate (ATP) and uridine 5′‐triphosphate (UTP) have been shown to activate a nucleotide receptor (P2U receptor) in rat mesangial cells that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases C and D, respectively. This is followed by an increased activity of the mitogen‐activated protein kinase cascade and cell proliferation. Here we show that ATP and UTP potently stimulate the stress‐activated protein kinase pathway and phosphorylation of the transcription factor c‐Jun. Both nucleotides stimulated a rapid (within 5 min) and concentration‐dependent activation of stress‐activated protein kinases as measured by the phosphorylation of c‐Jun in a solid phase kinase assay. When added at 100 μm the rank order of potency of a series of nucleotide analogues for stimulation of c‐Jun phosphorylation was UTP>ATP=UDP=ATPγS>2‐methylthio‐ATP>βγ‐imido‐ATP= ADP>AMP=UMP=adenosine=uridine. Activation of stress‐activated protein kinase activity by ATP and UTP was dose‐dependently attenuated by suramin. Down‐regulation of protein kinase C‐α, ‐δ and ‐ε isoenzymes by 24 h treatment of the cells with 12‐O‐tetradecanoylphorbol 13‐acetate did not inhibit ATP‐ and UTP‐induced activation of c‐Jun phosphorylation. Furthermore, the specific protein kinase C inhibitors, CGP 41251 and Ro 31–8220, did not inhibit nucleotide‐stimulated c‐Jun phosphorylation, suggesting that protein kinase C is not involved in ATP‐ and UTP‐triggered stress‐activated protein kinase activation. Pretreatment of the cells with pertussis toxin or the tyrosine kinase inhibitor, genistein, strongly attenuated ATP‐ and UTP‐induced c‐Jun phosphorylation. Furthermore, N‐acetyl‐cysteine completely blocked the activation of stress‐activated protein kinase in response to extracellular nucleotide stimulation. In summary, these results suggest that ATP and UTP trigger the activation of the stress‐activated protein kinase module in mesangial cells by a pathway independent of protein kinase C but requiring a pertussis toxin – sensitive G‐protein and tyrosine kinase activation.

Interleukin‐1 stimulates de novo synthesis of mitogen‐activated protein kinase in glomerular mesangial cells

Interleukin‐1 (IL‐1) stimulates a lime‐ and concentration‐dependent mitogen‐activated protein (MAP) kinase activation in rat mesangial cells. A rapid increase in activity (maximal at 10 min) is followed by a second persistent level of activity which steadily increases over 24 h. The second peak of MAP kinase activity is paralleled by a marked de novo synthesis of p42 MAP kinase as measured by immunoprecipitation of [35S]methionine‐labelled mesangial cells and by a 60% increase in total p42 MAP kinase protein as detected by Western blot analysis. We propose that IL‐1 induced de novo synthesis of p42 MAP kinase is important for the multiplicity of long‐term actions of this cytokine in renal mesangial cells.

A role for protein kinase C-ϵ in angiotensin II stimulation of phospholipase D in rat renal mesangial cells

The role of Ca2+ and protein kinase C (PKC) in the regulation of phosphatidylcholine‐hydrolyzing phospholipase D (PLD) was investigated in angiotensin II‐stimulated mesangial cells. Elevation of cytosolic free Ca2+ by the calcium ionophore, A23187, or the Ca2+‐ATPase inhibitor, thapsigargin, slightly increased PLD‐stimulated phosphatidylethanol formation. However, chelation of cytosolic Ca2+ with high concentrations of quin 2 did not attenuate angiotensin II‐indueed phosphatidylethanol production, thus suggesting that Ca2+ is not crucially involved in agonist‐stimulated PLD activation. Stimulation of PKC by phorbol esters increased PLD activity in mesangial cells. Down‐regulation of PKC‐α and ‐δ isoenzymes by 8 h phorbol ester treatment still resulted in full PLD activation. In contrast, a 24 h treatment of mesangial cells with phorbol ester, a regimen that also causes depletion of PKC‐ϵ, abolished angiotensin II‐evoked phosphatidylethanol formation. In addition, the selective PKC inhibitor, calphostin C, attenuated hormone‐induced PLD activity. In summary, these data suggest that angiotensin II stimulation of phospholipase D appears to involve the PKC‐ϵ isoenzyme, activated by DAG derived from phosphoinositide hydrolysis.

Comparison of different tumour promoters and bryostatin 1 on protein kinase C activation and down-regulation in rat renal mesangial cells

The effects of a series of protein kinase C (PKC) activators with different spectra of biological activities and reportedly different patterns of PKC isoenzyme activation were examined in renal mesangial cells. Treatment of mesangial cells with the tumor promoters phorbol 12-myristate 13-acetate (PMA), debromoaplysiatoxin, dihydroteleocidin and thymeleatoxin, as well as with the marine natural product bryostatin 1, caused translocation and at least partial down-regulation of the PKC-alpha, -delta and -epsilon isoenzymes as assessed by immunoblot analysis. Bryostatin 1 mediates a faster depletion of PKC-alpha isoform than any of the other PKC activators. Thymeleatoxin, which has been reported to selectively activate PKC-alpha, -beta and -gamma, but not PKC-delta or -epsilon isoenzymes in vitro, turned out to exert the most potent effect on PKC-delta and -epsilon in mesangial cells and down-regulated these isotypes within 8-24 hr. None of the compounds tested affected cellular distribution or amount of PKC-zeta in mesangial cells. Thus, all of the PKC activators tested are able to translocate and down-regulate three of the four PKC isoenzymes present in mesangial cells although with different kinetics. All PKC activators stimulated a phospholipase A2-mediated arachidonic acid release, a phospholipase D-mediated phosphatidylcholine hydrolysis, a comparable small proliferative response and an inhibition of phospholipase C-mediated inositol trisphosphate generation. These results suggest: (i) that the PKC activators investigated in this study do not display any type of isotype-specificity that could be used to selectively activate or down-regulate PKC isoenzymes in intact cell-systems; (ii) that thymeleatoxin has a different isoenzyme selectivity in intact cells as compared to in vitro enzyme inhibition data; and (iii) PKC-zeta is resistant to all PKC activators investigated in this study.

Translocation of protein kinase C isoenzymes by elevated extracellular Ca2+ concentration in cells from a human giant cell tumor of bone

In this study we investigated the protein kinase C isoenzymes expressed by human osteoclast-like cells harvested from a giant cell tumor of bone (GCT23 cells), and by freshly isolated rat osteoclasts. Immunoblotting analysis revealed that the -alpha, -delta, and -epsilon, PKC isoforms, but not the -beta isoenzyme, are expressed by GCT23 cells. Immunofluorescence studies demonstrated that PKC-alpha, -delta, and -epsilon are homogeneously expressed by both mononuclear and multinucleated GCT23 cells, as well as by rat osteoclasts. Similar to authentic osteoclasts, GCT23 cells responded to an increase of extracellular Ca2+ concentration ([Ca2+]o) with a dose-dependent elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). An increase of [Ca2+]o stimulated the translocation of PKC-alpha from the cytosolic to the particulate fraction, suggesting the involvement of this isoenzyme in the signal transduction mechanism prompted by stimulation of the [Ca2+]o sensing. By contrast, PKC-delta was not altered by exposure to elevated [Ca2+]o, whereas PKC-epsilon underwent reciprocal translocation, disappearing from the insoluble fraction and increasing in the cytosol. The effects of PKC on GCT23 cell functions were investigated by treatment with phorbol 12-myristate, 13-acetate (PMA). We observed that activation of PKC by PMA failed to affect adhesion onto the substrate, but down-regulated the [Ca2+]o-induced [Ca2+]i increases. The latter effect was specific, since it was reversed by treatment with the PKC inhibitors staurosporine and chelerythrine.