Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada M5S 1A8
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ABSTRACT |
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Mesangial cell proliferation is an early event in several progressive renal diseases. When mesangial cells in culture are rendered quiescent by serum starvation and subsequently stimulated to proliferate, induction of c-fos is an early indicator of entry into the cell cycle. Several heparin-sensitive signals transduce these events. We have examined the potential roles of CaMK and PKA. Selective stimulation of CaMK with Ca2+ ionophores and of PKA with forskolin or dibutyryl cAMP both result in induction of c-fos mRNA. CaMK but not PKA signaling is suppressed by low concentrations of heparin. Cross talk between the pathways has been demonstrated in some cells, with evidence of CaMK phosphorylating cAMP response element binding protein (CREB) at an inhibitory site and PKA suppressing CaMK-dependent signaling. However, in the present study, both pathways phosphorylated CREB on Ser133 and induced c-fos in an additive manner. Serum, ionomycin, and forskolin all caused a rapid decline in cyclin D1 levels, but only serum effected a subsequent increase, indicative of cell cycle progression. We conclude that, in human mesangial cells, CaMK and PKA can both contribute to cell cycle entry, and, although induction of c-fos by CaMK requires active PKA, neither pathway antagonizes or synergizes c-fos induction by the other.
heparin; ionophores; second messengers; mitogenic response
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INTRODUCTION |
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MESANGIAL CELLS are smooth muscle-like cells of the renal glomerulus that adopt a proliferative myofibroblast phenotype in response to many different forms of injury (12, 25). In common with its effect on vascular smooth muscle cells, heparin has an antiproliferative effect on mesangial cells at a much lower concentration than with most other cell types (3). For instance, although much higher concentrations have been used in most studies aimed at elucidating mechanism, some smooth muscle cell lines respond to as little as 50 ng/ml heparin with a decrease in DNA synthesis after stimulation of quiescent cells with serum (29). This has led to consideration of the therapeutic potential of heparin in disorders that involve smooth muscle proliferation, e.g., in vascular restenosis after angioplasty, although successful regimens have not yet been achieved (14, 23). Nevertheless, the sensitivity to heparin may indicate a role of endogenous heparan sulfates in maintaining mesangial cell phenotype, and mesangial cell-derived heparan sulfate chains also suppress DNA synthesis in cultured mesangial cells (33).
We (17, 18, 34) previously used induction of the immediate-early response gene c-fos as an early indicator of entry of quiescent mesangial cells into the cell cycle after mitogenic stimulation. Heparin inhibits c-fos induction in response to serum and phorbol esters at least in part by inhibition of the Erk MAPK cascade but also through Erk-independent pathways (17, 18). In particular, induction of c-fos by Ca2+ signaling mediated through CaMK is heparin sensitive (17).
The c-fos promoter has been studied in detail. In addition to sis-inducible (32) and heat shock-responsive (11) elements further upstream, a serum response element (SRE) and a cAMP response element (CRE) are important for induction through the MAPK and CaMK pathways. Erk phosphorylates and activates the Ets-like transcription factor (Elk) that forms part of the transcription factor complex binding to SRE, whereas CaMK phosphorylates another factor in the same complex (19, 24). CRE binds the CRE binding protein (CREB), which is an important substrate of the cAMP-dependent protein kinase, PKA; PKA activates CREB by phosphorylation of Ser133 (6, 24). CREB is also a substrate for CaMK, which may activate it by phosphorylation of Ser133 or, alternatively, may inhibit CREB binding in some cell lines by phosphorylating it on Ser142 (16, 28). Thus PKA and CaMK might differentially activate c-fos transcription through CRE and SRE binding factors, respectively, or their signals might converge at CREB. Alternatively, CaMK might antagonize PKA signaling by inhibitory phosphorylation of CREB. Further cross talk between the pathways has been established in several cell lines with the demonstration that PKA effects inhibitory phosphorylation of CaMK IV (36).
The present study was undertaken to determine whether PKA-dependent signaling can potentially play a role in c-fos induction in human mesangial cells and, if so, whether it is heparin sensitive and/or has a role to play in modulating the involvement of CaMK.
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EXPERIMENTAL PROCEDURES |
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Materials. FBS and culture media were obtained from GIBCO BRL Life Technologies (Burlington, ON, Canada). NuSerum IV was from BD Bioscience (Mississauga, ON, Canada). Calmodulin, ionomycin, A-23187, kemptide, KT-5926, KT-5720, and H-89 were products of Calbiochem (La Jolla, CA). Forskolin and dibutyryl cAMP were obtained from Sigma (St. Louis, MO). Autocamtide-2 was from Bachem (Torrance, CA). Antibodies to CREB peptide 5-21 and specific for phospho-Ser133-CREB were obtained from Upstate (Lake Placid, NY). Anti-cyclin D1 was from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence (ECL) kits and Hybond-N nylon membrane were from Amersham Pharmacia Biotech (Baie d'Urfé, PQ, Canada). A cAMP assay kit was from Diagnostic Products (Los Angeles, CA), and TRIzol reagent was from Invitrogen/Life Technologies (Burlington, ON, Canada).
Cell culture. Human mesangial cells prepared from the uninvolved part of a kidney resected for renal cell carcinoma were described previously (35). In addition, human cells purchased from Clonetics (San Diego, CA) were used between passages 3 and 7 and showed similar responses to agonists and inhibitors with respect to c-fos induction. Cells were grown in RPMI 1640 medium with 10% FBS in a 5% CO2 environment at 37°C and passaged by trypsinization. For experiments, cells were passaged 1:3 by trypsinization and allowed to grow overnight before being made quiescent by growth for 48 h in 0.4% FBS. The resultant nearly confluent cultures were treated with agonists in serum-free medium at time zero. When inhibitors were used, they were added 30 min before agonists. Controls generally included cultures treated with serum-free medium alone and stimulation with 5% NuSerum.
Kinase assays.
Autonomous CaMK activity (referring to the calmodulin-independent
activity sustained by autophosphorylation) and total activity were
measured as previously described (17). Briefly, cells were lysed by several freeze-thaw cycles in 50 mM HEPES buffer containing 0.5% Nonidet P-40 with protease and phosphatase inhibitors and centrifuged (10 min at 17,000 g). For autonomous activity,
aliquots of the supernatant containing 10 µg of protein were
incubated at 30°C for 3 min in 10 vols of 50 mM HEPES (pH 7.5)
containing (in mM) 10 MgCl2, 0.1 ATP, 1 EGTA, and 0.01 autocamtide with 50 µCi/ml [-32P]ATP. For
total activity, EGTA was replaced with 3 mM CaCl2 and 1 µM calmodulin. Reactions were stopped by addition of 5%
trichloroacetic acid, and reaction mixtures were spotted on P81
phosphocellulose filters, washed with 75 mM
H3PO4, and counted by liquid scintillation. For
PKA assay, aliquots of the supernatant prepared as above and containing
5 µg of protein were incubated at 30°C for 30 min in 10 vols of 50 mM MOPS (pH 7.0) containing 0.25 mg/ml bovine serum albumin, 10 mM
MgCl2, 0.2 mM ATP, 1 mM EGTA, 0.2 mM kemptide, and 50 µCi/ml [
-32P]ATP in the presence or absence of 20 µM dibutyryl cAMP. Reaction mixtures were collected on P81 filters
and processed as above.
cAMP.
To measure cAMP levels, cells were lysed in 95% ethanol containing 20 mM HCl, scraped from the plate and held at 20°C for 24 h,
dried in a Speed-Vac apparatus, and dissolved in 150 µl of Tris-EDTA
buffer. Fifty microliters was used to measure total protein content and
one hundred microliters for cAMP determination according to
instructions supplied with the kit.
Western blotting. Total CREB and phosphorylated CREB were detected by Western blotting with antibodies to CREB peptide and specific for CREB phosphorylated on Ser133, respectively. After lysis in RIPA buffer, cell extracts were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with nonfat dry milk in phosphate-buffered saline and incubated overnight with 1 µg/ml antibody at 4°C and then at room temperature for 90 min with horseradish peroxidase-conjugated goat anti-rabbit IgG. The blot was washed in 0.05% Tween 20 and visualized with ECL according to instructions supplied with the ECL kit. Samples were processed for detection with anti-cyclin D1 in an identical manner.
Northern blotting. Total RNA was isolated with TRIzol reagent according to the manufacturer's instructions. Equal amounts of RNA (10-20 µg) were fractionated by 1.2% agarose-2.2 M formaldehyde gel electrophoresis and transferred to Hybond-N nylon membrane by overnight capillary blotting in 20× SSC (3.0 M NaCl, 0.3 M sodium citrate). Blots were hybridized with random primer radiolabeled c-fos probes in 10% dextran sulfate, 5× SSPE (0.98 M NaCl, 50 mM Na2HPO4, 50 mM NaH2PO4, 0.5 mM EDTA), 50% formamide, 5× Denhardt's solution, 250 µg/ml salmon sperm DNA, and 0.5% SDS at 42°C overnight. Levels of c-fos mRNA were quantitated by densitometry of the Northern blot autoradiograph and normalized to 18S rRNA after probing with labeled cDNA to rat 18S rRNA.
Statistical analysis. Values from multiple measurements are expressed as means ± SD. Single comparisons were performed with Student's t-test. Multiple comparisons were by one-way ANOVA with the Student-Newman-Keuls post hoc test. A P value of <0.05 is considered significant.
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RESULTS |
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Activation of PKA and CaMK pathways.
Ionomycin activates CaMK in human mesangial cells (Fig.
1), requiring ~10-fold higher
concentrations than observed in our previous work (17)
with rat mesangial cells. NuSerum had no effect on CaMK activity (data
not shown), indicating specificity for Ca2+ entry. Heparin
blocked this activation, consistent with our previous demonstration
(17) of heparin-sensitive CaMK activation in rat mesangial
cells.
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Induction of c-fos.
Stimulation of quiescent smooth muscle cells with serum leads to a
rapid transcriptional activation of the immediate-early response gene
c-fos (29). Forskolin caused an increase in
c-fos mRNA levels by 30 min after treatment of quiescent
cells that was sustained to at least 1 h (Fig.
3). The cell-permeant cAMP analog
dibutyryl cAMP caused a similar increase at 30 min that was transient,
probably because of lower stability of the reagent. Therefore,
subsequent experiments were carried out with forskolin. Two inhibitors
with high specificities for PKA, KT-5720 (10) and H-89
(4), were used to further implicate this pathway in c-fos induction. KT-5720 (inhibition constant = 56 nM
in vitro) was without effect at a concentration up to 100 nM (Fig.
4). However, it was reported that
the closely related inhibitor KT-5823 is inactive in vivo in mesangial
cells (2), perhaps because of poor uptake or metabolic
degradation. On the other hand, H-89 nearly completely blocked
forskolin-dependent c-fos induction.
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Phosphorylation of CREB.
Serum, ionomycin, and forskolin each caused an increase in
phosphorylation of CREB at Ser133 (Fig.
7). CREB phosphorylation in response to
forskolin was fully inhibited by H-89 and unaffected by KT-5926,
indicative of signaling predominantly through PKA. Neither inhibitor
decreased the level of phospho-CREB in response to ionomycin,
suggesting that Ca2+-dependent CREB phosphorylation is not
mediated through CaMK and does not depend on PKA.
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Cyclin D1 expression.
Implicit in the above-described studies is the idea that induction of
c-fos through CaMK or PKA pathways is an indication of early
entry of quiescent cells into the cell cycle. To assess further the
relevance of these events, we examined expression of the G1
marker cyclin D1 and progression to S phase with DNA synthesis. As in
our previous studies (33), serum stimulation caused a peak
of incorporation of [3H]thymidine into DNA after ~18 h,
indicative of progression to S phase. Not surprisingly, neither
ionomycin nor forskolin alone was able to stimulate progression to DNA
synthesis (data not shown). However, cyclin D1 was prominent in
quiescent cells and rapidly decreased on stimulation with serum,
ionomycin, or forskolin (Fig. 8). Whereas
levels rose with time in serum-stimulated cells, they remained low in
cells treated with ionomycin or forskolin, indicating failure to
progress beyond early G1.
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DISCUSSION |
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Mesangial cell proliferation is a hallmark of the early stages of a number of progressive renal diseases (12, 15, 25) and may indicate transition to a myofibroblast phenotype (5, 12). A large number of factors have been identified that stimulate mesangial cell proliferation characterized by entry into the cell cycle and induction of immediate-early response genes such as c-fos. These include cytokines, growth factors, and various injurious stimuli (26). Relatively few factors have been shown to suppress proliferation in the absence of toxicity, chief among them cAMP, nitric oxide, and heparin (26). This suggests that a number of pathways may be involved simultaneously in triggering a mitogenic response. The present study confirms that activation of CaMK in quiescent cultures of human mesangial cells is associated with induction of c-fos, as it is in rat mesangial cells (17). As in rat cells, the mechanism is sensitive to low concentrations of heparin. Heparin-sensitive Erk signaling is also involved in c-fos induction by serum in mesangial cells of both species (Refs. 18 and 34 and data not shown). The present study adds PKA to the list of kinase pathways that are involved in mesangial cell c-fos induction and thus are potentially factors in mesangial cell proliferation.
Unlike the CaMK and MAPK pathways, PKA activation is insensitive to heparin; heparin had no effect on the direct activation of the PKA pathway, either with the adenylyl cyclase activator forskolin or with serum-stimulated PKA activation. Nevertheless, we confirm that activation of PKA leads to induction of c-fos by observing phosphorylation of CREB on Ser133 and demonstrating inhibition of c-fos induction by forskolin in the presence of the PKA-specific inhibitor H-89. Because PKA is activated by serum and implicated in c-fos induction in cultured mesangial cells, it could be among the signaling pathways that contribute to the failure of heparin to completely block serum-stimulated DNA synthesis, as observed previously (33).
Independence of the PKA pathway from heparin is further indicated by the failure of heparin to influence either basal cAMP levels or the increased levels produced by either serum or forskolin. This in itself is noteworthy because factors that increase cAMP are associated with decreased mesangial cell proliferation in some studies (21, 26, 30). Increased cAMP inhibits activation of MAPK by endothelin and phorbol esters (8). Heparin induces capacitation of sperm by increasing cellular cAMP concentrations (31) and stimulates production of cGMP in endothelial cells (38). Therefore, because cAMP has antiproliferative effects in some circumstances, effects on cAMP could in principle account for heparin's antiproliferative effects. However, at concentrations that suppress c-fos, heparin is without effect on cAMP levels in human mesangial cells, and this seems to rule out a role for cAMP in the antiproliferative effects of heparin.
CREB can serve as a substrate for CaMK, but the significance of this is not always clear. CaMK actually inhibits CREB in several cell lines by phosphorylating it on an alternative site, Ser142 (16, 28). In this scenario, CaMK might serve to check the action of PKA. On the other hand, PKA engages in extensive cross talk with other signaling cascades (1). In smooth muscle cells it can function as a negative regulator of Erk (7) and antagonize cyclin-dependent kinases (1), and increased cAMP levels and PKA activity produce inhibitory phosphorylation of CaMK in several cell lines (36). However, the present results show that, in cultured mesangial cells, PKA acts independently of CaMK in inducing c-fos. On the other hand, induction by CaMK requires PKA activity. This requirement appears to be permissive, however. Activation of either CaMK or PKA with specific agonists appears neither to synergize nor inhibit induction of c-fos by the other. As would be expected if CaMK did not antagonize the activation of CREB by PKA, the CaMK inhibitor KT-5926 did not affect c-fos mRNA levels produced by forskolin. In fact, under conditions in which ionomycin significantly increased CaMK activity, the signal detected by anti-phospho-Ser133 antibody was significantly increased. The absence of an effect of heparin on PKA-dependent events provides further support for their independence from heparin-sensitive CaMK activation. The conclusion is that both CaMK and PKA stimulate early responses in human mesangial cells.
Surprisingly, then, the PKA inhibitor H-89 significantly decreased c-fos induction by ionophores. Both CaMK-dependent (ionomycin) and -independent (A-23187) pathways were affected, suggesting that PKA is required for Ca2+-dependent c-fos induction. Nuclear Ca2+ is required for CRE-dependent gene expression (9), and Ca2+ channel blockers decrease binding of CREB to CRE (27). We showed previously (37) that intracellular Ca2+ is necessary for mesangial cell induction of c-fos and subsequent incorporation of [3H]thymidine into DNA in response to a number of stimuli, including serum, ionomycin, platelet-derived growth factor, and endothelin. Although these observations point to important links between cAMP and Ca2+ second messenger signal transduction, the mechanism(s) underlying the effect of H-89 on ionophore-dependent c-fos induction remains unexplained. Likewise, it is unclear why the response of c-fos to A-23187 is unaffected by CaMK inhibition. However, A-23187 has multiple effects on Ca2+ entry and may provide Ca2+ pathways that signal independently of CaMK. The possibility also remains that A-23187 has a mechanism of action independent of effects on calcium, but the common effect of H-89 on the response of c-fos to both A-23187 and ionomycin makes this unlikely.
The induction of c-fos was examined here because it is requisite for cell cycle progression (22) and also because it is an early marker of exit from G0 into G1. Consistent with c-fos induction by selective activation of either Ca2+-dependent or PKA-activated signals, ionomycin and forskolin both cause a rapid decrease in cyclin D1 protein levels, as does serum. Cyclin D1 was previously shown to be present at significant levels in quiescent mesangial cells (13). On stimulation, it rapidly decreases and then rises progressively throughout G1 (20). Although not examined here, this increase may represent a rise in nuclear cyclin D1 (13). Both ionomycin and forskolin cause a decrease in cyclin D1 levels in quiescent cells, but they cause levels to remain low over the 4-h period examined. Thus, although activation of PKA and Ca2+ signaling pathways can both trigger early events of cell cycle entry, additional factors must be involved in progression.
In summary, CaMK and other Ca2+-dependent pathways can lead to cell cycle entry and induction of c-fos in human mesangial cells. These pathways do not antagonize PKA-mediated c-fos induction. Induction of c-fos by PKA is independent of, and additive with, the Ca2+-dependent pathways. CaMK-mediated induction of c-fos, but not that arising from the PKA pathway, is sensitive to heparin. An unexplained observation is that basal PKA activity is permissive for c-fos induction by Ca2+ ionophores.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Kidney Foundation of Canada.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. M. Templeton, Dept. of Laboratory Medicine and Pathobiology, Medical Sciences Bldg. Rm. 6302, Univ. of Toronto, 1 King's College Circle, Toronto, Canada M5S 1A8 (E-mail: doug.templeton{at}utoronto.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 2, 2002;10.1152/ajprenal.00074.2002
Received 22 February 2002; accepted in final form 21 June 2002.
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