Ca2+/Calmodulin-Dependent Protein Kinase II Stimulates c-fos Transcription and DNA Synthesis by a Src-Based Mechanism in Glomerular Mesangial Cells
Yuan Wang,
Rangnath Mishra and
Michael S. Simonson
Department of Medicine, Division of Nephrology, School of Medicine, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio.
Correspondence to Dr. Michael S. Simonson, Department of Medicine, Division of Nephrology, Biomedical Research Building, Rm. 427, Case Western Reserve University, 2065 Adelbert Road, Cleveland, OH 44106. Phone: 216-368-1251; Fax: 216-368-1249;
ABSTRACT. Mesangial cell growth factors elevate intracellularfree [Ca2+]i, but mechanisms linking [Ca2+]i to gene expressionand DNA synthesis are unclear. This study investigated the hypothesisthat Ca2+/calmodulin-dependent protein kinase II (CaMK II),which is activated by elevated [Ca2+]i, increases c-fos transcriptionand DNA synthesis via a Src-based mechanism. In cultured ratmesangial cells, dominant negative Src (SrcK-) blocked activationof the c-fos gene promoter by CaMK II 290, a constitutivelyactive form of CaMK II. Activation of the c-fos promoter byCaMK II 290 was also blocked by COOH-terminal Src kinase, whichphosphorylates and inactivates c-Src. A pharmacologic CaMK inhibitor,KN-93, did not block activation of the c-fos promoter by ectopicallyexpressed v-Src. Stimulation of c-Src by endothelin-1 requiredCaMK II activity, further supporting the notion that CaMK IIacts upstream of Src in a signaling cassette. Activation ofthe c-fos promoter by CaMKII290 and Src required the c-fos serumresponse element. Dominant negative SrcK- also blocked inductionof DNA synthesis in mesangial cells by CaMK II 290. Collectively,these results suggest that in mesangial cells Src protein tyrosinekinases act downstream of CaMKII in a signaling pathway in which[Ca2+]i induces the c-fos promoter and increases DNA synthesis.E-Mail: mss5@po.cwru.edu
Mesangial cells and the associated mesangial matrix are essentialfor normal structure and function of the glomerular capillaries(1–5). The mesangial phenotype is highly plastic, andcontrol of mesangial cell growth is critical for normal developmentof the glomerular tuft in metanephrogenesis and in the responseof adult kidneys to glomerular injury (6). In the adult kidney,mesangial cells are largely quiescent, with a renewal rate ofless than 1% (2–4). Glomerular injury can alter the phenotypeof mesangial cells, resulting in hypertrophy, hyperplasia, and/orexpansion of the mesangial matrix. This injury-dependent phenotypicswitch of mesangial cells contributes to glomerulosclerosisand is mediated in part by growth factors and inflammatory mediatorsthat evoke immediate early gene expression and DNA synthesis.The signal transduction pathways that control mesangial cellgrowth are complex but seem to share as an early event a rapidand transient increase in intracellular free [Ca2+]i (7,8).A variety of experiments in cultured mesangial cells and inexperimental models of glomerular injury with mesangial expansiondemonstrate that inhibiting the rise in intracellular free [Ca2+]iattenuates mesangial cell proliferation (2,4,9–11).
The molecular mechanisms linking [Ca2+]i to gene expressionand DNA synthesis in mesangial cells are unclear. In renal andnonrenal cells, recent evidence suggests a possible role inCa2+ signaling for non-receptor protein-tyrosine kinases ofthe Src family. Ca2+ influx through Ca2+-permeable ion channelsactivates c-Src tyrosine kinase activity in epithelial cells(12,13), in neurons (14), and in mesangial cells (15). The abilityof dominant negative c-Src to block c-fos immediate early geneinduction and neurite outgrowth by Ca2+ influx in neurons suggestsan important role for Src in cell signaling by Ca2+ (14). Inmesangial cells, Ca2+ influx and subsequent activation of Srcare necessary for c-fos immediate early gene induction by theendothelin-1 G protein-coupled receptor (15,16), which has beensuggested to stimulate mesangial cell growth in glomerular injury(17,18). These observations suggest c-Src as a candidate insignal transduction pathways that link elevation of cytosolicfree [Ca2+]i to c-fos induction in mesangial cells, but a functionalrole for c-Src in stimulating DNA synthesis by specific effectorsof Ca2+ signaling has not been demonstrated.
One of the primary mechanisms by which intracellular [Ca2+]iregulates signal transduction is through activation of the Ca2+/calmodulin-dependentprotein kinases (CaMK) (19). In this study, we asked whethera CaMK II signaling pathway activates c-fos transcription andmesangial cell growth by a Src-based mechanism. We report herethat dominant negative mutants of Src and the Src-inactivatingkinase Csk block activation of the c-fos promoter by CaMKII.Dominant negative Src mutants also inhibit mesangial cell DNAsynthesis stimulated by an active CaMKII290 mutant. We conclude,therefore, that CaMKII signals through Src and suggest thatthis pathway regulates genes that control cell growth in themesangium.
Cell Culture
Mesangial cell strains from male Sprague-Dawley rats were isolatedand characterized as previously reported (20). Cells were maintainedin RPMI 1640 medium supplemented with 17% fetal bovine serum,100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/mleach of insulin and transferrin, and 5 ng/ml of sodium seleniteat 37°C in 5% CO2 incubator. HeLa cells, A7r5 vascular smoothmuscle cells, and bovine pulmonary artery endothelial cellswere obtained from the American Type Culture Collection andwere cultured in DMEM supplemented with 10% fetal bovine serum,24 mM HEPES (pH 7.4), 100 U/ml penicillin, and 100 µg/mlstreptomycin.
Plasmids, Transient Transfections, and Reporter Gene Assays
The following plasmids were as described: p-356wt/fosLUC (16);point mutants (pm) of the c-fos promoter, pmxfosLUC (16,21);pSrcK-,K+ (22,23); pCMVCsk (24); pCMVCaMKII 290 (21); pMv-Src(25). Cells were transfected using the calcium phosphate methodas previously reported (16,26). Cells in 6-well plates (35 mm;2 x 105 cells/well) were transfected with 0.5 µg of thep-356wt/fosLUC, 0.1 µg pRSVGal internal control, and 2µg of pCMV or pCMVCaMKII290. When indicated, 2 µgpSrcK+/K- or pCMVCsk was also added. Total DNA was adjustedto 6 µg of DNA/well with pUC19. Total promoter strengthin control transfections was held equivalent by inclusion ofexpression plasmids lacking a cDNA insert. After incubatingwith precipitates for 16 to 18 h at 37°C, cells were thenwashed three times with DMEM and incubated with DMEM/0.5% FBSfor an additional 24 h before cell lysis (RLB Buffer, Promega).Luciferase activity was assessed as described previously (26),and relative light units were measured in a Berthold Lumat Luminometer(Wallac, Gaithersburg, MD) for two 10-s intervals. Transfectionefficiency was determined using a pRSVGal construct that directed galactosidase expression from the viral LTR. Galactosidaseactivity was measured using the Galacto-Light protocol as describedby the manufacturer (Tropix, Bedford, MA). All reporter geneassays were in the linear range. In experiments where KN-93(Calbiochem, San Diego, CA) was used to inhibit endogenous CaMKactivity, KN-93 at 10 µM was added 8 h after DNA additionand was present throughout the remaining transfection period.
Western Blotting of Epitope-Tagged CaMKII290
To determine if SrcK- repressed expression of CaMKII290, mesangialcells were transiently transfected with pCMV CaMK II 290 (containingan HA epitope tag (21)) and with increasing amounts of the plasmidexpressing dominant negative SrcK-. Forty-eight hours aftertransfection, cells in 100-mm dishes were washed once in DPBSand scraped into 1 ml of sample lysis buffer (0.5 M Tris-HCl[pH 6.8], 2.5 ml; 10% (wt/vol) SDS, 4.0 ml; glycerol, 2 ml;0.1% bromophenol blue, 0.5 ml; -mercaptoethanol, 0.5 ml; distilledwater, to 10 ml). The lysate was vortexed, boiled for 5 min,and aliquots were resolved on 8 to 16% SDS-PAGE gradient gelsand proteins transferred to 0.2-µm nitrocellulose filters.Transferred proteins were stained with Ponseau S, and the filterwas blocked in blocking buffer (1.0% BSA, 10 mM Tris [pH 7.5],100 mM NaCl, 0.1%Tween 20) with shaking overnight at 4°C.To detect the HA epitope-tagged CaMKII290 in transfected cells,the blots were incubated with a monoclonal anti-HA antibody(clone 12CA5; Boehringer Mannheim, Indianapolis, IN). Afterextensive washing, the appropriate peroxidase-labeled secondaryantibodies in blocking buffer (1:10,000) were added, and theproteins were detected by chemiluminescense (ECL; Amersham,Arlington Heights, IL). Typical exposure times were 30 to 60s. The same blot was reprobed with an antibody that recognizesrat -actin (Sigma, mAb A5316) to confirm equal protein loading.
Inhibition of Endothelin-1-Activated c-Src by a CaMKII Inhibitor
Serum-starved cells (24 h; 0.5% FBS) in 60-mm plates were stimulatedwith 100 nM endothelin-1 (ET-1), and the cells were preparedfor Western blotting as described above. To block CaMK II activity,cells were preincubated for 1 h with 20 uM of a myristolyatedautocamtide-2-inhibitory peptide (myr-AIP), a nonphosphorylatableanalog of autocamtide-2 that corresponds to the autophosphorylationsite of CaMK II (Calbiochem). This protocol with myr-AIP haspreviously been shown to inhibit CaMK II activation in culturedcells (27,28). The membranes were probed with 1:2000 of an affinity-purifiedpolyclonal phosphospecific antibody that recognizes the activeform (P-Tyr 416) of c-Src exactly as described by the manufacturer(Cell Signaling Technology, Beverly, MA). To confirm equal amountsof total c-Src, the blot was reprobed with mAb clone 327 (Calbiochem)as described by Lipsich et al. (29).
Measurements of DNA Synthesis in Transiently Transfected Cells
Mesangial cells in 6-well plates were transfected as describedabove, except that the amount of RSVGal was increased to 1 µgper well, and the fos luciferase reporter was omitted. Incorporationof 5-bromo-2-deoxyuridine (BrdU; Sigma, St. Louis, MO) intoDNA in transiently transfected cells (i.e., galactosidase–positivecells) was measured exactly as described previously (30). Briefly,transfection cells were labeled for 24 h with 20 µM BrdU;Sigma, St. Louis, MO). Monolayers were then washed twice withice-cold DPBS and fixed for 5 min on ice with 2.0% formaldehyde/0.2%glutaraldehyde in DPBS (Dulbecco phosphate-buffered saline).Cells expressing galactosidase were detected by histochemicalanalysis (30), and under these conditions 1 to 3% of mesangialcells were transfected (i.e., galactosidase–positive).BrdU incorporation into DNA was then identified by immunocytochemistryusing 6 µg/ml IgG of a specific monoclonal antibody (BoehringerMannheim, Indianapolis, IN) as described (30). Cells were visualizedunder brightfield microscopy (Nikon Diophot) with a neutralfilter. Red cells (i.e., expressing galactosidase–positive)with dark nuclei (BrdU-positive) and light nuclei (BrdU-negative)were counted, and percent inhibition of DNA synthesis was calculatedas follows:
(1)
where Nv = % BrdU-positive nuclei in cells transfected withvector only and Ne = % BrdU-positive nuclei in cells transfectedwith expression plasmid. Statistical significance was analyzedby the 2 test using InStat for Macintosh (GraphPad, SanDiego,CA).
A Dominant Negative Src Mutant (SrcK-) Blocks Activation of the c-fos Promoter by CaMK II
Our first approach was to ask if dominant negative mutants ofSrc would interfere with activation of the c-fos promoter byCaMK II. Kinase-inactive forms of Src (SrcK-) function as dominantnegative mutants and inhibit DNA synthesis stimulated by colonystimulating factor or platelet-derived growth factor in fibroblasts(23,31) and block stimulation of the c-fos promoter by the Gprotein-coupled ET-1 receptor in mesangial cells (16). We thereforetested whether dominant negative SrcK- (Lys 295 to Met) wouldblock stimulation of the c-fos promoter by a constitutivelyactive mutant of CaMK II, CaMK II 290 (Figure 1A). Mesangialcells were transiently transfected with a plasmid containinga genomic DNA fragment of the c-fos promoter (-356 to +109)that drives transcription of a luciferase reporter gene (Figure 1A)(16). Transfection with a plasmid vector expressing CaMKII 290 stimulated a 3.9-fold increase in c-fos promoter activity(Figure 1B). Cotransfection with a vector expressing SrcK- completelyblocked activation of the c-fos promoter by CaMK II 290 (Figure 1B).Inhibition by SrcK- was apparently specific as transfectionwith wild-type SrcK+ did not block CaMK II 290-stimulated c-fospromoter activity (Figure 1B). SrcK+ did not potentiate theeffect of CaMK II 290. These results suggest that CaMK II actsupstream of Src in a signaling cascade that activates the c-fospromoter.
Figure 1. SrcK- dominant negative mutant blocks Ca2+/calmodulin-dependent protein kinase II (CaMK II) signaling to c-fos. (A) Schematic diagram of plasmids used in transient transfections. p-356wt/fos LUC is a reporter construct containing -356 to +109 of the mouse c-fos promoter driving transcription of a luciferase reporter gene. SIE, sis-inducible element; SRE, serum response element; Ca/CRE, Ca2+-cAMP response element. pCMV CaMK II 290 and pSrcK- are expression plasmids encoding a truncated, constitutively activated form of CaMK II and a dominant negative Src mutant, respectively. (B) Glomerular mesangial cells were co-transfected with p-356wt/fos LUC and either pCMV or pCMV CaMK II 290 as described below. As indicated in the legend, plasmids encoding SrcK- or wild-type Src K+ were also added. Forty-eight hours after DNA addition, the cells were lysed and processed for luciferase and galactosidase expression. Data are mean ± SEM from four independent experiments in duplicate. ** P < 0.01 by t test.
One potential interpretation of the inhibitory effects of SrcK-is that SrcK- might block expression of CaMKII290. Therefore,in mesangial cells transfected with CaMKII290 and SrcK-, wemeasured CaMKII290 levels by Western blotting using the HA epitopetag fused to the NH2 terminus (Figure 1A). As shown in Figure 2,SrcK- did not inhibit CaMKII290 expression at any concentrationused in these experiments (lane 2 versus lanes 3 and 4 withSrcK-, Figure 2). Coexpression of SrcK+ (lanes 5 and 6, Figure 2)did not significantly alter CaMKII 290 levels, which suggeststhat the Src expression vector was without any apparent effecton CaMKII 290 expression under control of the CMV promoter/enhancer.We conclude that SrcK- does not inhibit expression of CaMKII290and that CaMKII does indeed act upstream of Src in the pathwayto c-fos.
Figure 2. CaMK II 290 expression is unaffected by SrcK-. Mesangial cells were transfected with pCMV (lane 1) or pCMV CaMK II 290 (lanes 2 to 6) as in Figure 1 but with co-transfection of SrcK- (2 µg, lane 3; 4 µg, lane 4) or SrcK+ (2 µg, lane 5; 4 µg, lane 6). Levels of HA epitope-tagged CaMKII290 (p33kDa) were then assessed by Western blotting. Results are representative of three independent experiments.
COOH-Terminal Src Kinase Also Inhibits Activation of the c-fos Promoter by CaMK II
An independent approach to inactivate c-Src is to express Csk,which phosphorylates the COOH-terminal tyrosine (Y527) in c-Srcand maintains the kinase in an inactive conformation (24,32).When a vector expressing Csk (Figure 3A) was transiently co-transfectedinto cells with the c-fos luciferase reporter, Csk inhibitedactivation of the c-fos promoter by CaMK II 290 (Figure 3B).As shown previously (16), Csk does not nonspecifically inhibitthe c-fos promoter because Csk does not block activation ofthe c-fos promoter by fetal bovine serum or a constitutivelyactive mutant of Raf-1 kinase (16). Also, Csk had no effecton c-fos luciferase activity in the absence of CaMK II 290,(i.e., pCMV; Figure 3B). Collectively, the results with SrcK-and Csk strongly suggest that CaMK II functions upstream ofSrc in a signaling pathway to the c-fos promoter.
Figure 3. Src-inactivating kinase Csk blocks induction of c-fos promoter by CaMK II 290. Mesangial cells were transfected with p-356wt/fos LUC and pCMV or pCMV CaMK II 290 as in Figure 1 but with co-transfection of a plasmid encoding full-length Csk (pCMVCsk) or the control vector lacking a cDNA insert (A). (B) Results (mean ± SEM) of three independent experiments in duplicate. ** P < 0.01.
Activation of Src by ET-1 Requires CaMK II
To determine whether CaMK II participates in Src activationin mesangial cells by physiologic ligands, we treated serum-starvedcells with 100 nM ET-1and measured Tyr-416 phospho-Src in thepresence and absence of myr-AIP to block CaMK II activity. ET-1increased phospho-Src levels at 10 min, and Src remained activatedat 60 min (Figure 4A). Ca2+ ionophore A23187, which increasesCaMK II activity, also increased phospho-Src levels but witha more rapid time course. Preincubation of cells with myr-AIPbefore addition of ET-1 greatly attenuated Src activation (Figure 4B);reprobing of the blot for total Src protein confirmed thepresence of unactivated Src. These results suggest that CaMKII is upstream of Src and participates in a pathway of Src activationby ET-1.
Figure 4. Blockade of CaMK II inhibits Src activation by endothelin-1 (ET-1). (A) Serum-starved cells were stimulated with 100 nM ET-1 or 10 µM Ca2+ ionophore A23187 and levels of phospho-Tyr-416 Src were measured by Western blotting with an activation state-specific antibody. The blot was reprobed with mAb against total Src to confirm equal protein loading (bottom panel). (B) Cells were preincubated with myr-AIP (20 µM for 1 h), a cell-permeable peptide that blocks CaMKII activation, before addition of ET-1 and measurement of phospho-Tyr-416 Src levels as in panel A. Identical results were observed in two independent experiments.
CaMK II Acts Upstream of Src in Diverse Cell Types
We next asked if the ability of CaMK II to function upstreamof Src was cell type–specific or was instead a commonfeature of diverse cell types. We conducted the same cotransfectionexperiments described above in Figure 1 with CaMK II 290 andSrcK- in A7r5 vascular smooth muscle cells, porcine aortic endothelialcells, and HeLa cells. SrcK-, but not SrcK+, blocked activationof the c-fos promoter by CaMK II 290 in A7r5 vascular smoothmuscle and endothelial cells (Figure 5). SrcK- also blockedactivation of the c-fos promoter by CaMK II 290 in HeLa cells,but SrcK+ potentiated activation by CaMK II 290 (Figure 5).It is not clear why SrcK+ potentiated stimulation by CaMK II290 in HeLa cells and not in the other cell types we studied,but this probably reflects cell type-specific differences incross-talk between CaMK II or Src. However, the key findingis that dominant negative SrcK- blocked activation of the c-fospromoter by CaMK II 290 in four distinct cell types and suggeststhat a CaMK II-Src signaling cassette is conserved.
Figure 5. Dominant negative SrcK- blocks activation of the c-fos promoter by CaMK II 290 in different cell types. Embryonic rat A7r5 vascular smooth muscle cells, porcine aortic endothelial cells, and human HeLa cells were transfected with p-356wt/fos LUC, pCMV CaMK II 290, and SrcK- or SrcK+ exactly as described in Figure 1. Data are mean ± SEM from three experiments in duplicate. Inhibition by SrcK- was significant (P < 0.01) in all cell types.
A Pharmacologic Antagonist of CaMK Fails to Block Activation of the c-fos Promoter by v-Src
Additional evidence placing CaMKII upstream of Src came fromexperiments in which a selective pharmacologic antagonist ofCaMK, KN-93 (33), did not block stimulation of the c-fos promoterby constitutively active v-Src (Figure 6). Eight hours afterco-transfecting mesangial cells with v-Src and the fos-luciferasereporter, KN-93 at 10 µM was added and maintained throughoutthe remainder of the transfection. KN-93 did not significantlyreduce activation of the c-fos promoter by v-Src and did notaffect basal promoter activity in cells transfected with theexpression vector alone (i.e., pM-MuLV, Figure 6). We have previouslyshown that 10 µM KN-93 blocks stimulation of the c-fospromoter by ET-1 (21), a finding that was confirmed here inconcurrent experiments to demonstrate that KN-93 was indeedeffective (Figure 6). The inability of a CaMK antagonist toblock c-fos promoter activation by Src is further evidence thatSrc functions downstream of CaMKII on a signaling pathway toc-fos.
Figure 6. A CaMK antagonist (KN-93) fails to block c-fos promoter activation by v-Src. Mesangial cells were co-transfected with p-356wt/fos LUC and p-v-Src (2 µg) or the parent expression vector pM-MuLV. Eight hours later, KN-93 (10 µM) was added and maintained throughout the transfection. In experiments to demonstrate that KN-93 was effective, mesangial cells transfected with p-356wt/fos LUC were treated with KN-93 followed by 100 nM endothelin-1 (ET-1). Results (mean ± SEM) of 4 independent experiments in duplicate. ** P < 0.01.
CaMKII/Src Signaling to c-fos Requires the c-fos Serum Response Element (SRE)
We next sought to determine which cis-element(s) of the c-fospromoter is targeted by CaMKII/Src signaling. Mesangial cellswere transfected with c-fos reporter constructs in which pointmutations have been introduced to inactivate specific cis-elementsas shown in Figure 7A. Inactivation of the SIE and FAP cis-elementshad no significant effect on activation of the c-fos promoterby either constitutively active CaMKII290 or by v-Src (pm6 andpm9, Figure 7A). Point mutations in the Ca/CRE, which respondsto Ca2+ and cAMP-based signaling pathways (34), slightly inhibitedthe response to CaMKII290 (i.e., 6.4-fold versus 4.3-fold withpm3) but did not alter the response to v-Src. In contrast, pointmutations in the SRE completely inhibited stimulation of thec-fos promoter by either CaMKII290 or v-Src (pm12, Figure 7A).Thus the SRE appears to be the critical cis-element requiredfor the CaMKII/Src signaling pathway. It is important to note,however, that full activation of the c-fos promoter by CaMKII290requires both the SRE and Ca/CRE cis-elements.
Figure 7. The c-fos SRE is necessary for activation of the c-fos promoter by CaMKII/Src signaling. (A) Mesangial cells were transfected with c-fos promoter LUC constructs with inactivating point mutations (pm) as indicated schematically at left. Cells were cotransfected with pCMV (open bars), pCMVCaMKII290 (solid bars), or pSrcK- (hatched bars). Data are means from three independent experiments; error bars are omitted for clarity but were never more than 20% of the mean. (B) Cells were transfected with a minimal promoter construct (p56LUC, 2 µg) or the same vector with a single copy of the c-fos SRE upstream of TATA (pSRELUC, 2 µg). Cotransfections were with pCMV alone (no addition), CaMKII290, or CaMKII290 plus dominant negative SrcK-. Data are mean ± SEM from four independent experiments.
To further investigate the role of the SRE in CaMKII/Src signaling,we transfected mesangial cells with a luciferase reporter constructin which transcription is controlled by a single copy of theSRE upstream of TATA, pSRELUC (26). Cotransfection of CaMKII290or CaMKII290 plus SrcK- failed to alter luciferase expressionfrom the parent vector, p56LUC (Figure 7B). However, in cellstransfected with pSRELUC, CaMKII290 stimulated a 4.7-fold increasein SRE cis-element activity that was markedly inhibited by SrcK-(i.e., 1.6-fold, Figure 7B). Taken together, these results suggestthat CaMKII/Src signaling targets the SRE of the c-fos promoter.
Dominant Negative Src K- Inhibits DNA Synthesis Stimulated by CamK II 290
To assess the biologic importance of Src in signaling by CaMKII, we asked whether Src is a downstream effector in pathwayswhereby CaMK II controls mesangial cell growth. To this end,we measured bromodeoxyuridine uptake into DNA in cells transientlytransfected with CaMK II 290. Transfected cells were identifiedby cotransfection with a lac Z gene and histochemical detectionof expressed galactosidase (Figure 8, A and B). BrdU was incorporatedinto DNA in 71% of cells transfected with CaMK II 290 (Figure 8, A and C)versus 22% of cells with the control vector (Figure 8C).Expression of Src K- attenuated the increase in DNA synthesisin cells expressing CaMK II 290 (Figure 8, B and C; 35% versus71%, P < 0.01 by 2 analysis). In contrast, SrcK+ had no effecton CaMK II 290-stimulated DNA synthesis. These results supportthe concept that Src acts downstream of CaMK II in a signalingcassette that controls gene expression and cell growth.
Figure 8. SrcK- inhibits DNA synthesis stimulated by CaMK II 290. Mesangial cells were transfected with pRSV lacZ, pCMV CaMK II 290, and either pSrcK- or pSrcK+. After 24 h, DNA was labeled by incorporation of BrdU for 24 h at 37°C. Transfected cells were then identified by histochemical detection of expressed galactosidase, and DNA synthesis was assessed by immunocytochemical detection of BrdU. Transfected cells with BrdU-positive nuclei (A) and BrdU-negative nuclei (B) were counted in three independent experiments, n > 300 cells for each condition taken from three independent experiments (C). Statistical significance was calculated by the 2 test.
Mesangial hypertrophy and hyperplasia are adaptive responsesto glomerular injury and are mediated by a variety of paracrineand autocrine growth factors that increase [Ca2+]i. Mesangialcell growth requires coupling of [Ca2+]i to transcription factorsthat activate and maintain hypertrophy and hyperplasia, butelucidation of the intracellular signals involved representsan important challenge. The results presented here demonstratea novel mechanisms whereby the Ca2+-dependent effector CaMKIIfunctions upstream of Src in a signaling pathway that activatesthe c-fos promoter and stimulates DNA synthesis in culturedmesangial cells.
Several lines of evidence suggest that CaMK II lies upstreamof Src in mesangial cells. We blocked CaMK II-stimulated c-fosinduction and DNA synthesis by expressing dominant negativeSrcK- and Csk to inactivate Src. SrcK- and Csk interfere withSrc activity by distinct molecular mechanisms, thereby providingan important internal control for possible nonspecific effectsof either protein. Transfection with the empty expression vectorlikewise did not reduce luciferase activity. That inactivationby SrcK- was specific was further supported by the finding thatoverexpression of wild-type SrcK+ did not alter activation ofthe c-fos promoter by CaMK II 290. SrcK- did not block expressionof CaMKII290 as verified by Western blotting of HA epitope-taggedCaMKII290 in mesangial cells transfected with SrcK-. Moreover,we have previously shown that SrcK- and Csk do not inhibit activationof the c-fos promoter by Raf-1, which functions downstream ofSrc and Ras (16). A pharmacologic CaMK inhibitor (KN-93) didnot significantly block activation of the c-fos promoter byv-Src, providing additional evidence that Src lies downstreamof CaMKII in this signaling pathway. We also demonstrated thatCaMKII acts upstream of Src in several different cell types.We had previously shown that Src activation by ET-1 requiresCa2+ influx and elevation of cytosolic free [Ca2+] (15), andthe present studies in mesangial cells demonstrated that Srcactivation by ET-1 was inhibited in the presence of myr-AIPto block CaMKII. Taken together, these results suggest thatCaMK II functions upstream of Src in a Ca2+-dependent signalingpathway.
An important caveat is that SrcK- and Csk might also inactivateclosely related Src PTK family members (23,24,31,32), so wecannot formally rule out participation of Src-related kinasesin CaMK II signaling. In addition, our experiments have notaddressed the question of how Ca2+ and CaMK II might activateSrc. We were unable to demonstrate a direct protein-proteininteraction between CaMK II and cSrc (Wang and Simonson, unpublishedresults). Src contains a consensus phosphorylation site forCaMKs (R-X-X-S/T) in the unique NH2 terminal sequence, raisingthe possibility of phosphorylation and control of Src activityby CaMK II. An indirect mechanism by which CaMK II activatesSrc might depend on some as yet unidentified cofactor or proteinas part of an heterooligomeric protein complex. Indeed, Zhaoet al. (12) propose that Ca2+ ionophore A23187 increases Srcactivity in keratinocytes by an indirect mechanism possiblyinvolving a Ca2+-stimulated tyrosine phosphatase activity orthe Ca2+-dependent association of three unidentified proteinswith c-Src. Another possibility is that CaMKII activates Ca2+channels and/or ion pumps (19), thereby elevating [Ca2+]i andstimulating Src PTK activity.
Our results with point mutants of the c-fos promoter suggestthat the SRE is required for CaMKII/Src signaling to the c-fosimmediate early gene. Point mutations in the SRE blocked activationof the c-fos promoter by CaMKII290 and Src. Mutation of theCa/CRE only modestly blocked the response to CaMKII 290 anddid not alter activation of the c-fos promoter by Src. The c-fosSRE is activated by the Ras/MAPK pathway, and it seems possiblethat CaMKII/Src signaling also activates the Ras/MAPK pathwayto c-fos. Indeed, Rosen et al. (35) have previously shown inPC12 cells that Ca2+ influx activates the Ras/MAPK pathway,and Rusanescu et al. (14) have further demonstrated that Srcis required for Ca2+ activation of Ras/MAPK. It is not entirelyclear how Ca2+ influx and Src activate Ras, but the mechanismsprobably involve Src-mediated phosphorylation of the Shc adapterprotein and subsequent recruitment of Grb2-Sos complexes (14).The new finding of the present experiments concerns the roleof CaMKII contributing to Src-based signaling to the c-fos SRE.
An important aspect of our study was to test a possible rolefor the CaMKII-Src signaling cassette in mesangial cell growth,which is a pathologic hallmark of many forms of glomerular disease.Previous experiments in cultured mesangial cells and in experimentalmodels of glomerular injury with mesangial growth demonstratethat inhibiting a rise in intracellular free [Ca2+]i attenuatesmesangial cell proliferation (2,4,9–11), but the molecularmechanisms and effectors of Ca2+ signaling that underlie theseresults remain unclear. We showed that ectopic expression ofCaMKII 290 stimulated DNA synthesis in quiescent mesangial cellsthat was effectively blocked by SrcK-. The control vector lackingSrcK- and the vector expressing SrcK+ had no effect on DNA synthesisdriven by CaMKII 290. These results are the first direct demonstrationin mesangial cells that activated CaMKII drives DNA synthesis,and the results also support a role for Src in this signal transductionpathway. Many mesangial cell growth factors stimulate a rapidand transient increase in intracellular free [Ca2+]I (7,8),and our experiments suggest that a CaMKII-Src signaling cassetteis one mechanism coupling the rise in [Ca2+]i to DNA synthesis.
Acknowledgments
We thank Sara Courtneidge, Hidesaburo Hanafusa, G. Stanley McKnight,and Michael G. Gilman for providing plasmids. We also thankWilliam Herman for excellent technical assistance, and GeorgeDubyak, Hsing-Jien Kung, and Mark Kester for helpful comments.Supported by grant DK-46939 from the National Institutes ofHealth.
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Received for publication May 30, 2002.
Accepted for publication October 2, 2002.
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Top
Abstract
Introduction
. Activation of the c-fos promoter by CaMK II 290 was also blocked by COOH-terminal Src kinase, which phosphorylates and inactivates c-Src. A pharmacologic CaMK inhibitor, KN-93, did not block activation of the c-fos promoter by ectopically expressed v-Src. Stimulation of c-Src by endothelin-1 required CaMK II activity, further supporting the notion that CaMK II acts upstream of Src in a signaling cassette. Activation of the c-fos promoter by CaMKII290 and Src required the c-fos serum response element. Dominant negative SrcK- also blocked induction of DNA synthesis in mesangial cells by CaMK II 290. Collectively, these results suggest that in mesangial cells Src protein tyrosine kinases act downstream of CaMKII in a signaling pathway in which [Ca2+]i induces the c-fos promoter and increases DNA synthesis. E-Mail: mss5@po.cwru.edu 
Gal internal control, and 2 µg of pCMV or pCMVCaMKII290. When indicated, 2 µg pSrcK+/K- or pCMVCsk was also added. Total DNA was adjusted to 6 µg of DNA/well with pUC19. Total promoter strength in control transfections was held equivalent by inclusion of expression plasmids lacking a cDNA insert. After incubating with precipitates for 16 to 18 h at 37°C, cells were then washed three times with DMEM and incubated with DMEM/0.5% FBS for an additional 24 h before cell lysis (RLB Buffer, Promega). Luciferase activity was assessed as described previously (26), and relative light units were measured in a Berthold Lumat Luminometer (Wallac, Gaithersburg, MD) for two 10-s intervals. Transfection efficiency was determined using a pRSV
2 test using InStat for Macintosh (GraphPad, SanDiego, CA). 
