The Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois
Submitted 18 March 2005 ; accepted in final form 5 April 2005
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ABSTRACT |
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signal transduction; heart failure; calcium; protein kinase C
Despite the potential negative impact of SERCA2 downregulation on cardiac structure and function, relatively few studies have addressed the mechanisms and signal transduction pathways responsible for these changes. Both transcriptional (1, 2, 17, 40, 48, 49, 53) and posttranscriptional (30, 31, 37, 39, 51) mechanisms may be involved, although the specific effectors responsible for SERCA2 downregulation are only now being identified. In a previous study from our laboratory (6), we showed that decreasing [Ca2+]i by blockade of voltage-gated, L-type Ca2+ channels substantially increased basal SERCA2 mRNA and protein levels in neonatal rat ventricular myocytes (NRVM), and stimulated sarcoplasmic reticulum Ca2+ uptake, resulting in a twofold increase in the rate of [Ca2+]i decline. In contrast, cyclic stretch of noncontracting NRVM substantially downregulated SERCA2 mRNA and protein levels via a mechanism independent of Ca2+ influx via voltage-gated, L-type Ca2+ channels (12). Furthermore, we and others (15, 16, 21, 36, 37) have shown that one or more isoenzymes of protein kinase C (PKC) are critical factors regulating SERCA2 gene expression in response to neurohormonal stimuli. For instance, Ho et al. (21) previously showed that phorbol myristate acetate (PMA), a potent activator of PKC-, -
, -
, and -
in cardiomyocytes, downregulated SERCA2 and prolonged the [Ca2+]i transient via activation of the Raf-MEK1-ERK1/2 pathway. Similarly, Andrews et al. (1) demonstrated that activation of the MKK6-p38MAPK signaling pathway also caused the transcriptional downregulation of SERCA2 promoter activity. However, the specific components linking Ca2+, cyclic stretch, and PKCs to the MAPK signaling pathways that regulate SERCA2 levels in cardiomyocytes remain unknown.
The Ca2+-dependent, nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (PYK2) is a member of the focal adhesion kinase (FAK) family of nonreceptor PTKs. PYK2 coordinates Ca2+-, integrin-, and PKC-dependent signaling pathways in some tissues (for review, see Ref. 3). PYK2 expression and/or phosphorylation are regulated by [Ca2+]i and PKCs in cardiomyocytes and vascular smooth muscle cells (7, 8, 41). As examined in various cell types, PYK2 acts as an important scaffolding protein, and transduces signals from G protein-coupled receptors to downstream MAPK signaling pathways depending on which signaling kinases and adapter proteins bind to the phosphorylated enzyme (10, 34). PYK2 has also been shown to link a variety of stressful stimuli, including Ca2+ overload, UV irradiation, and TNF- treatment to MAPK activation in several cell types (50). Recently, Hirotani et al. (20) demonstrated that PYK2 is an essential signaling component in endothelin- and phenylephrine-induced NRVM hypertrophy, perhaps acting via the Ca2+- and/or PKC-dependent activation of Rac1.
We (9) previously demonstrated that PYK2 expression and phosphorylation were significantly increased in adult rat ventricular myocytes in vivo during the transition from LVH to HF coincident with SERCA2 downregulation. Similarly, Melendez et al. (29) showed that PYK2 expression and phosphorylation were increased in a mouse model of dilated cardiomyopathy, but its exact role in these conditions has not been elucidated. In this study, we utilized replication-defective adenoviruses (Adv) encoding wild-type and mutant forms of PYK2 to interrogate their effects on cell growth, MAPK activation, and SERCA2 gene expression in NRVM. These data are presented to suggest a novel, and potentially important negative feedback mechanism regulating [Ca2+]i levels in cardiomyocytes.
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MATERIALS AND METHODS |
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Cell culture.
The animals were handled in accordance with the American Physiological Society "Guiding Principles in the Care and Use of Animals." All procedures involving the animals were approved by the Institutional Animal Care and Use Committee of Loyola University Medical Center. NRVM were isolated as previously described (42). NRVM were plated at a density of 400, 1,000, or 1,600 cells/mm2 onto collagen-coated 35, 60, or 100 mm dishes, or one-well Permanox chamberslides, and left undisturbed in a 5% CO2 incubator for 1824 h. Attached cells were maintained in DMEM/medium 199 (4:1) containing antibiotic/antimycotic solution. Under these serum-free culture conditions, NRVM displayed synchronous [Ca2+]i transients and beating activity (100150 beats/min) within 24 h after being plated.
Adenoviral constructs.
Replication-deficient Adv encoding WT and mutant forms of PYK2 were generated as previously described (27). Briefly, PYK2 cDNAs (kindly provided by J. T. Parsons, University of Virginia) were subcloned in frame into pEGFP-C1 vector (BD Biosciences-Clontech, Palo Alto, CA). The WTPYK2 contained the entire 1,009 amino acid coding sequence of rat PYK2 (43). The kinase-defective (KD) mutant consisted of a single amino acid substitution (K457A) within the central catalytic domain. The autophosphorylation site mutant consisted of a single amino acid substitution (Y402F), which is required for Src-family kinase binding. The green fluorescent protein (GFP)-tagged PYK2 inserts were then sequentially subcloned into pShuttle-CMV plasmid, and then pAdeno-X viral DNA (BD Biosciences-Clontech). Linearized pAdeno-X+GFP-PYK2 sequences were transfected into HEK-293 cells, and the resulting WT and mutant PYK2 Adv were amplified and purified by double CsCl gradient centrifugation. Replication-defective Adv encoding constitutively active (ca)MKK6 and caMKK7 were kindly provided by Dr. Yibin Wang (University of California at Los Angeles) (54, 55), whereas a replication-defective Adv encoding caMEK1 was kindly provided by Dr. Jeffrey Molkentin, University of Cincinnati (11). An Adv-expressing GFP (Adv-GFP) and an Adv-expressing nuclear-encoded (ne) -galactosidase (Adv-ne
gal) were used to control for nonspecific effects of Adv infection. The multiplicity of viral infection (MOI) was determined by dilution assay in human embryonic kidney (HEK)-293 cells grown in 96 well clusters. Cardiomyocytes were infected (60 min, 25°C with gentle agitation) with each Adv diluted in DMEM/medium 199. The medium was then replaced with virus-free DMEM/medium 199, and the cells cultured for an additional 8, 24, or 48 h.
Protein content and cell size.
For the analysis of NRVM protein content, cells were quantitatively scraped from dishes in 0.2 N perchloric acid (1 ml) and collected by centrifugation (10,000 g, 10 min). The precipitate was redissolved by incubation (60°C, 20 min) in 250 µl of 0.3 N KOH. Aliquots were used for analysis of total protein by the Lowry method with crystalline human serum albumin as standard, and for DNA using 33258 Hoechst dye and salmon sperm DNA as standard (46). For determination of cell surface area, cardiomyocytes were first loaded with 2'7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM [2 µM in a modified Krebs medium composed of (in mM) 135 NaCl, 5.9 KCl, 1.5 CaCl2, 1.2 MgCl2, 11.5 glucose, and 11.6 HEPES, pH 7.3, supplemented with 0.1% BSA and 0.2% Pluronic F-127 detergent] for 1 h, followed by 1-h incubation in BCECF-free Krebs buffer. Cells were viewed with a laser scanning confocal microscope (model LSM 510, Zeiss). Optical sections through the base of the cells (20 cells/field) were stored as digital images and analyzed with Image-1 software (Universal Imaging, West Chester, PA). A binary mask was created by setting the threshold brightness that distinguished the fluorescent cells from the black background. Cell area was determined as an exact count of the number of pixels that made up the object's binary mask multiplied by the area of a unit pixel (46).
Cell fixation and confocal microscopy. NRVM grown on chamberslides were infected with Adv-GFP or Adv-GFPWTPYK2 (10 MOI, 48 h). Cells were fixed, permeabilized, and stained with anti-paxillin mAb, as previously described (13, 14). Fluorescently labeled cells were viewed with a Zeiss LSM 510 confocal microscope.
Western blot analysis. NRVM were homogenized in lysis buffer (44). Equal amounts of extracted proteins (50 µg) were separated by SDS-PAGE and Western blot analysis on 7.5% or 10% polyacrylamide gels. Primary antibody binding was detected with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody, and visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL).
mRNA analysis. Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA). RNA was quantified by absorbance at 260 nm, and its integrity was determined by examining the 28S and 18S rRNA bands in ethidium bromide-stained agarose gels. SERCA2 and GAPDH mRNA levels were then analyzed with Northern blots or by real-time RT-PCR, as previously described (12, 36).
SERCA2 promoter activity.
NRVM plated at a density of 1,000 cells/mm2 were transiently transfected with a SERCA2 promoter plasmid construct pGL3-b-S2-HindIII/HincII (kindly provided by Dr. Warner Simonides, VU University Medical Center, Amsterdam) (53). The plasmid contained a fragment from the rat SERCA2 promoter (-3,263/+550 bp from the transcriptional start site) linked to the firefly luciferase reporter gene. A control plasmid, containing the Rous sarcoma virus-long terminal repeat (RSV-LTR), linked to a bacterial
gal reporter gene (kindly provided by Dr. Edward Kislauskis, University of Massachusetts Medical Center) was used in conjunction with the SERCA2 plasmid to control for transfection efficiency. Cells were transfected with 5 µg of the SERCA2 promoter plasmid along with 0.5 µg of control RSV-LTR
gal. The plasmids were added to 4:1 medium free of both serum, as well as antibiotic/antimycotic agents, in the presence of 10 µl/ml Lipofectamine 2000 reagent (Invitrogen Life Technologies, Carlsbad, CA). Cells were treated with the transfection cocktail for 6 h, and were then maintained in serum-free, 4:1 medium containing antibiotic/antimycotic agents for up to 72 h.
SERCA2 promoter activity was assessed using the protocol described in the Luciferase Assay System (Promega, Madison, WI). Briefly, 4:1 medium were removed and 300 µl of 1 x reporter lysis buffer were added to the cells. The cells were then quick-frozen in a methanol/dry ice bath and allowed to thaw on ice. Cells were scraped into prechilled 1.5-ml Eppendorf tubes, briefly vortexed, and then centrifuged at 12,000 g for 2 min at 4°C. Supernatants were transferred into a fresh set of tubes and maintained on ice. A manual luminometer (Lumat LB 9501, Berthold Systems, Aquilippa, PA) was used to measure luciferase activity. For each sample, 100 µl of luciferase assay reagent were added to 50 µl of cell lysate, and luminescence was measured over a 30-s period. Detection of RSV-LTR gal activity in cell lysates was performed using a
gal detection kit (catalog no. MO255, Marker Gene Technologies). Briefly, 20-µl aliquots of cell lysates were pipetted into individual wells in opaque 96-well plates. One hundred microliters of reaction buffer (100 mM NaPO4, 1 mM MgCl2, 10 mM
-mercaptoethanol, and 0.1% Triton X-100, pH 7.0) were added to each well. The fluorogenic substrate reagent 3-carboxyumbellifery
-D-galactopyranoside (50 µl, 5 mM) was added and samples were allowed to incubate (20 min) at room temperature. Stop buffer (500 mM glycine and 10 mM EDTA, pH 12.0; 100 µl) was then added and samples were allowed to incubate (10 min) at room temperature. Fluorescence values were measured on a Synergy HT Multi-Detection Plate Reader (Bio-Tek Instruments, Winooski, VT) using 390-nm excitation and 460-nm emission wavelengths. Promoter activity was reported as the ratio of luciferase luminescence values to
-galactosidase fluorescence values.
Data analysis. Results were expressed as means ± SE. Normality was assessed using the Kolmogorov-Smirnov test, and homogeneity of variance was assessed using Levene's test. Data from multiple groups were compared with one-way ANOVA, or one-way ANOVA on ranks, followed by Dunnett's test, or Student-Newman-Keuls test, where appropriate. Data from two groups were compared by paired t-test, or Wilcoxon's signed-rank test where appropriate. Differences among means were considered significant at P < 0.05. Data were analyzed using SigmaStat version 1.0 (Jandel Scientific, San Rafael, CA).
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RESULTS |
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GFPWTPYK2 expression alone does not cause NRVM hypertrophy. The effect of WTPYK2 overexpression on NRVM protein content and cell size were then examined. Total protein (µg/dish), DNA (µg/dish), and total protein/DNA ratio (Fig. 2A) were similar in uninfected, Adv-GFP-infected, and Adv-GFPWTPYK2-infected NRVM. Cell surface area (µm2/cell; Fig. 2B) was also unaffected. In contrast, the hypertrophic agent phorbol myristate acetate (PMA; 200 nM, 24 h) increased total protein/DNA ratio and cell size to a similar extent in each group. These data indicate that WTPYK2 overexpression alone was not sufficient to induce NRVM hypertrophy, but WTPYK2 overexpressing cells were still capable of undergoing hypertrophy in response to a hypertrophic stimulus.
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Inhibition of PYK2 increases basal SERCA2 mRNA levels.
We (8) previously demonstrated that the highly specific Src-family PTK inhibitor PP2 reduced basal, endothelin- and caPKC--induced PYK2 activation in NRVM. Therefore, we next examined whether inhibition of Src-family PTKs affected SERCA2 mRNA levels in these high-density, spontaneously contracting NRVM cultures. As seen in Fig. 4, PP2 (50 µM; 48 h) significantly increased SERCA2 mRNA levels 2.9-fold compared with untreated NRVM. However, PMA was still able to downregulate SERCA2 mRNA levels to 53 ± 11% of untreated NRVM, which is similar to the level of SERCA2 mRNA downregulation observed with WTPYK2 overexpression in the absence of PP2, or with PMA (Fig. 3).
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DISCUSSION |
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In previous studies (6, 12, 36, 37), we demonstrated that mechanical loading, [Ca2+]i, and PKCs are all important regulators of SERCA2 gene expression. Ca2+ influx via L-type Ca2+ channels substantially reduced SERCA2 mRNA and protein levels, and prolonged the time constant for relaxation of the [Ca2+]i transient compared with verapamil-treated NRVM (6). Our present results now provide a potential explanation for these findings, in that Ca2+ channel blockade markedly decreased PYK2 phosphorylation (8) and expression (7), thus eliminating a key component of a signaling pathway that may be important in regulating SERCA2 levels. Similarly, SERCA2 mRNA levels were increased by treating spontaneously contracting NRVM with the PKC inhibitors staurosporine and chelerythrine (37), and by overexpressing dominant-negative inhibitors of individual PKC isoenzymes (36), thereby inhibiting potential activators of a PYK2-dependent signal transduction pathway (8). It is also interesting to note that increasing Ca2+ influx/release activates PYK2 in many cell types, including NRVM (8, 29). Electrical stimulation of contraction also caused increased [Ca2+]i, and caused the membrane translocation of PKC- and PKC-
(47), thus providing additional links to the SERCA2 regulatory pathway.
PYK2 overexpression appeared to mediate SERCA2 downregulation via activation of the JNK and p38MAPK cascades. PYK2 has been identified as a potential upstream regulator of both SAPKs in NRVM (28), as well as in other cell types. For example, Tokiwa et al. (50) showed that WTPYK2 overexpression activated JNKs in PC12 cells, whereas a dominant-negative mutant of PYK2 interfered with UV light- or osmotic shock-induced activation of JNKs. Furthermore, Kodama et al. (24) demonstrated that ET-induced JNK activation in NRVM was mediated primarily via the PKC- and Ca2+-dependent activation of PYK2. PYK2 was found to associate with active Src, and the adapter proteins p130Cas and Crk, which are involved in SAPK activation in a variety of cells. In cardiac fibroblasts, angiotensin II-induced JNK activation was also dependent upon PYK2 via GTP-loading of Rac1 (33). A similar PYK2-dependent pathway was necessary for endothelin-induced activation of p38MAPK in human and rat mesangial cells (45). Finally, we (27) recently demonstrated in cultured chondrocytes that soluble fibronectin fragments activated a 5
1-integrin-dependent signaling pathway involving PKC-
, PYK2, JNKs, and p38MAPK, leading to stimulation of MMP-13 promoter activity and expression. Thus it seems likely that PYK2 activates the JNK and p38MAPK cascades via similar mechanisms in NRVM.
We found that specific functional domains within PYK2 were important in downregulating SERCA2 mRNA levels. We found that mutations at the putative autophosphorylation site and within the kinase domain decreased (but did not eliminate) the ability of PYK2 to elicit these effects. In fact, the kinase activity of the overexpressed transgene appeared to contribute relatively little to SERCA2 downregulation, as the GFPKDPYK2 mutant underwent efficient phosphorylation at Y402 despite a mutation that rendered the kinase catalytically inactive. Similar results were obtained by Lakkakorpi et al. (26), who found that in osteoclasts, PYK2 kinase activity was dispensable for efficient tyrosine phosphorylation at Y402 as well as at other sites. These data suggest that in some cell types, PYK2 can be phosphorylated at Y402 by another kinase, perhaps a member of the Src-family of PTKs. Of note, we recently demonstrated that basal and endothelin-induced phosphorylation of PYK2 at Y402 was inhibited by PP2, a relatively specific Src-family PTK inhibitor (8). It is also possible that the catalytically inactive, GFPKDPYK2 transgene underwent Y402 phosphorylation via bimolecular autophosphorylation involving the endogenous enzyme, as was recently demonstrated to occur in PC12 cells and Src/Yes/Fyn-deficient fibroblasts (35). In contrast to the kinase-deficient mutant, SERCA2 downregulation were nearly prevented by overexpression of PYK2 bearing the Y402F mutation. Phosphorylation of Y402 provides a docking site for Src-family PTKs to bind to and phosphorylate PYK2 at other sites, including Y579, Y580, and Y881. Lakkakorpi et al. (26) also showed that overexpressed Y402FPYK2 was only weakly phosphorylated at these additional sites, and therefore functioned as a dominant-negative inhibitor of PYK2-dependent cell spreading and bone resorption in osteoclasts. Furthermore, PP2 (which blocks PYK2 phosphorylation at Y402) substantially increased basal SERCA2 mRNA levels in NRVM (Fig. 4). Taken together, these results suggest that the PYK2/Src complex is both necessary and sufficient to generate downstream signals that are important in regulating SERCA2 gene expression in cardiomyocytes.
Our results differ somewhat from a recent study by Andrews et al. (1), which demonstrated that activation of the MKK6-p38MAPK pathway regulated cardiomyocyte [Ca2+]i via downregulation of SERCA2 gene transcription. They showed that overexpression of caMKK6 reduced SERCA2 promoter activity, and lowered steady-state levels of SERCA2 mRNA and protein resulting in prolongation of the [Ca2+]i transient. Although we found that p38MAPK activation alone was not sufficient to downregulate SERCA2 mRNA (Fig. 7), basal levels of JNK1/2 activation in their cells may have been relatively higher than in our experiments, therefore accounting for this difference. Nevertheless, our results indicating that PYK2 can mediate activation of the JNK and p38MAPK cascades support other studies, and confirm recently published work by Melendez et al. (28) indicating that PYK2 activates JNKs and p38MAPK in cardiomyocytes. Taken together, these studies provide a potential upstream link between integrin-, Ca2+-, and PKC-dependent pathways leading to SAPK activation and SERCA2 downregulation in cardiomyocytes.
PYK2 overexpression substantially reduced SERCA2 promoter activity, suggesting that PYK2-dependent signaling pathways reduced SERCA2 gene expression by decreasing the rate of SERCA2 transcription. PMA, a potent activator of both Ca2+-dependent (i.e., PKC- and PKC-
) and Ca2+-independent (i.e., PKC-
and PKC-
) PKC isoenzymes, also reduced SERCA2 promoter activity. Of note, PMA substantially activates all three MAPK cascades in NRVM, although their degree of activation by physiological stimuli is in part dependent on which PKC isoenzymes are differentially activated (19, 47, 52). However, PP2 did not prevent PMA-induced SERCA2 downregulation, suggesting that there is a PKC-dependent, but PYK2-independent pathway that also regulates SERCA2 promoter activity in NRVM. The most likely explanation for these data is that PMA activates a PKC-
/MEK1/ERK signaling cascade independently of PYK2 (19, 36, 46). However, additional studies using physiological agonists and highly specific inhibitors of the individual PKCs and MAPKs will be necessary to fully evaluate these complex signaling pathways.
A potential concern of our studies is that PYK2- and SAPK-mediated SERCA2 downregulation may be part of a more generalized change in mRNA expression in a compromised cardiomyocyte preparation. Indeed, prolonged PYK2 overexpression can trigger apoptosis in some cell culture systems (56), including NRVM (28), and both p38MAPK and JNKs have been implicated in the induction of cardiomyocyte apoptosis (5). We found that WTPYK2 overexpression had no significant effect on cardiomyocyte attachment, protein and DNA content, or the ability of the cells to respond to a hypertrophic stimulus, at least over the time course of these relatively brief cell culture experiments. Quantitative analysis of SERCA2 mRNA was consistently compared with that of another mRNA (GAPDH mRNA), and GAPDH mRNA levels did not systematically change relative to total RNA. Nevertheless, much of the data presented above provide only circumstantial evidence that PYK2 and SAPKs are important regulators of SERCA2 gene expression in NRVM. Although experiments utilizing PP2 suggest that PYK2 and Src-family PTKs are indeed involved, these inhibitors may be nonspecific. Furthermore, the majority of our studies utilize a "gain-of-function" approach to analyze potential, interacting signaling pathways. It is conceivable that overexpression of wild-type or constitutively active signaling kinases induces promiscuous, nonphysiological responses by causing phosphorylation reactions and complexation of signaling proteins that may not normally occur in response to the intricate, redundant signaling pathways operative in the intact heart. Overexpression of kinase-inactive, and phosphorylation-site mutant forms of PYK2, which function as dominant-negative inhibitors of PYK2 signaling in other cell types (4, 27, 50), is also problematic in NRVM because these molecules appear to still generate specific (or nonspecific) signals via their scaffolding function in the cell. Additional studies using highly specific, pharmacological inhibitors of PYK2 autophosphorylation, or "knockdown/knockout" strategies will be needed to fully define the role of this signaling kinase in the altered gene expression that accompanies LVH and HF.
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GRANTS |
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FOOTNOTES |
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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.
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