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Am J Physiol Heart Circ Physiol 279: H619-H629, 2000;
0363-6135/00 $5.00
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Vol. 279, Issue 2, H619-H629, August 2000

CD14-independent activation of cardiomyocyte signal transduction by bacterial endotoxin

Douglas B. Cowan1, Dimitrios N. Poutias1, Pedro J. Del Nido2, and Francis X. McGowan Jr1

Departments of 1 Anesthesia and 2 Cardiac Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the heart, lipopolysaccharide (LPS) induces the production of proinflammatory cytokines that cause myocardial dysfunction; however, the signaling pathways involved in cardiomyocyte responses are poorly understood. We studied LPS-induced signaling by treating cardiomyocyte cultures with 0.01-10 µg/ml LPS for 0-24 h in the presence or absence of 2.5% serum. Cytosolic and nuclear proteins were analyzed for expression and activation of protein kinases. Members of the extracellular signal-regulated kinase (ERK) and signal transducer and activators of transcription (STAT) protein families were uniformly expressed and specifically phosphorylated in response to LPS. Activation was biphasic; peaking at 5-10 min and 24 h after treatment. Inhibitor experiments provided evidence that ERK proteins may regulate STAT activity. Serum did not augment endotoxin-induced phosphorylation. Although cardiomyocytes expressed low levels of CD14 and LPS-binding protein, specific enzymatic removal of glycosyl phosphatidylinositol-linked receptors or incubation with an anti-CD14 antibody had no effect on kinase activation. Treatment of cells with an excess of detoxified LPS attenuated endotoxin-induced signaling. In addition, endotoxin stimulated specific binding of nuclear factors to AP-1, nuclear factor-kappa B (NF-kappa B), STAT1 (SIE, sis-inducible element), and STAT3 consensus-binding sequences. Finally, inhibition of ERK phosphorylation reduced, and NF-kappa B nuclear translocation prevented, tumor necrosis factor-alpha production. Our results indicate that LPS-induced activation of signal transduction in cardiomyocytes occurs by a CD14-independent mechanism.

lipopolysaccharide; kinase; receptor; phosphorylation; myocyte


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LIPOPOLYSACCHARIDE (LPS; endotoxin) is the major constituent from the outer membrane of gram-negative bacteria that evokes diverse and profound responses in mammalian cells. Although LPS is not believed to be intrinsically toxic, this glycolipid stimulates the release of reactive oxygen intermediates, arachidonic acid metabolites, and proinflammatory cytokines that contribute to the pathophysiology of sepsis and septic shock (6, 51, 54).

The cellular signaling events that lead to the LPS-induced production of inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha ), interleukin-1beta (IL-1beta ), and IL-6 have been well characterized in cells of reticuloendothelial origin. Several receptors are expressed on the surface of monocytes and macrophages that are involved in activating signal transduction pathways when bound by LPS ligand. Of these, CD14 has been the most intensely studied and is found in two distinct forms, as a glycosyl phosphatidylinositol (GPI)-linked membrane receptor on myeloid cells and as a serum protein lacking the GPI anchor (13, 39). The soluble form of CD14 can bind LPS and activate cells that otherwise do not express membrane-bound CD14 (e.g., endothelial, epithelial, and smooth muscle cells) (31, 40, 54).

The interaction of CD14 with physiologically relevant concentrations of LPS is facilitated by the plasma glycoprotein LPS-binding protein (LBP) (40, 53). The presence of serum can greatly enhance cellular activation due to LBP-mediated transfer of LPS to membrane-bound CD14. In addition, serum provides a source of LBP and soluble CD14. LBP also functions to neutralize the stimulatory effects of endotoxin by catalyzing the transfer of LPS into high-density lipoprotein particles (60). Although it is certain that LBP intensifies LPS-induced activation in myeloid and some nonmyeloid cells, it does not seem to be a component of the LPS and CD14 activating complex (54).

The question of how either the GPI-anchored receptor or the soluble form of CD14 communicates a ligand-specific signal to the cell interior is not completely understood. The idea that the LPS · CD14 complex is recognized by other transmembrane receptors that convey an activating signal through the plasma membrane has recently found support (13, 54). Two members of the Toll receptor family, TLR-2 and TLR-4, were demonstrated to mediate LPS-induced cellular signaling (37, 61). Whether either of these receptors functions in a cooperative manner with CD14 remains to be thoroughly investigated. However, there is preliminary support for CD14 acting synergistically with TLR-2 (24). Alternatively, membrane-bound CD14 may directly associate with heterotrimeric G proteins localized to membrane microdomains enriched with GPI-anchored receptors, cholesterol, sphingomyelin, and numerous signaling proteins (47). Although this represents an appealing means for LPS-induced signal transduction, some studies indicate that glycolipid-rich microdomains are irrelevant for LPS-mediated myeloid cell activation (38, 39). Another mechanism of CD14-dependent signal transmission is based on the finding that LPS can be directly transferred into phospholipid bilayers and internalized by vesicular transport (52, 60). This observation led to the hypothesis that LPS may signal, in part, by structurally mimicking ceramide (59). Interestingly, several investigators have shown that even though LPS may mediate or contribute to certain ceramide effects, there are important differences between the two stimuli (4, 29, 32). Moreover, LPS · CD14 internalization appears to be dissociated from activation in some instances (38) but not others (14, 45, 52). Taken together, these findings show that CD14-dependent mechanisms of LPS signal transduction are complex.

Several laboratories have found examples of CD14- and LBP-independent signaling in response to LPS stimulation (5, 30, 36). The involvement of Toll-like receptors, leukocyte beta 2-integrins, L-selectin, or other putative receptors have not been examined in these instances, yet CD14-independent mechanisms of endotoxin-induced signal transduction may prove significant in cells that are not directly in contact with blood (13, 37, 61).

Regardless of the mechanisms that transmit signals to the interior of a cell, many of the signaling pathways activated in response to LPS have been thoroughly examined in myeloid cells. These include the mitogen-activated protein kinase (MAPK) cascade (16, 57), the stress-activated protein kinase (SAPK) pathway (17, 50), members of the signal transducer and activators of transduction (STAT) protein family (27), p38 MAPK (18), and NF-kappa B (28). Outside these cell types, however, little is known about LPS-mediated activation of signal transduction.

In the heart, LPS leads to reduced contractility and beta -adrenergic responses, arrhythmia propagation, and cell death (8, 20, 62). Many of these phenomena can be tied to the production of interleukins and TNF-alpha , which, in turn, cause an increase in inducible nitric oxide synthase (iNOS) levels (23, 35, 48). In contrast, several other studies have implied that LPS-induced cardiac depression may involve nitric oxide (NO)-independent mechanisms (12, 22, 26, 41). Presently, the signal transduction pathways that underlie these events have not been examined, despite their obvious importance in the clinical management of sepsis and septic shock. This study examines the activation of early signaling events in the cardiomyocyte by endotoxin. To better understand the role of these pathways in LPS-stimulated proinflammatory cytokine production, we assessed their relationship to the stimulation of myocyte TNF-alpha release, one of the earliest cytokines produced in response to LPS. We conclude that a novel CD14-independent mechanism of endotoxin-mediated signal transmission appears to be required for the generation of early responses in cardiomyocytes.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and treatments. Two-day-old Wistar rat (Charles River) cardiomyocytes were isolated essentially as described earlier (46) using the Neonatal Cardiomyocyte Isolation System (Worthington). Contaminating cardiac fibroblasts were removed by preplating cell mixtures for 1.75 h in 75-mm2 culture flasks (Becton-Dickinson). Cardiomyocytes were initially plated in DMEM-F12 medium (Life Technologies) containing 10% fetal bovine serum (FBS), 10 µM cytosine beta -D-arabinofuranoside (Sigma), and 1% penicillin-streptomycin (Life Technologies) and allowed to attach for 24 h. Cells were then maintained in DMEM-F12 medium containing 10 µM cytosine beta -D-arabinofuranoside, 1% ITS (insulin, transferrin, and selenium solution; Collaborative Biomedical), 0.25 mg/ml fetuin (Sigma), 0.1× MEM vitamins (Life Technologies), 0.4× nonessential amino acids, 1% penicillin-streptomycin, 15 mM NaHCO3, and 0.5 µM CaCl2.

Rat astrocyte cultures were the gift of Dr. Joanne Chan (Dana Farber Cancer Institute). The RAW264.7 mouse macrophage cell line was purchased from American Type Culture Collection and plated in DMEM containing 10% FBS and 1% penicillin-streptomycin as specified by the supplier. All culture media preparations were tested for endotoxin contamination with the E-Toxate Limulus amebocyte lysate detection kit (Sigma).

Cell cultures were treated for various times (0, 5, 10, 30, 60 min and 24 h) with Salmonella typhosa LPS (Sigma) at a variety of concentrations (0.01, 0.1, 1.0, and 10.0 µg/ml). RAW264.7 cells were treated in the presence of 10% FBS unless noted otherwise, whereas cardiomyocytes were treated in both serum-free and serum-containing media as indicated in the text. Some cardiomyocytes were exposed to detoxified Salmonella typhimurium LPS (Sigma) at a 100-fold excess to S. typhosa LPS (0.1 µg/ml), whereas others were pretreated for 1 h with 0.5 U/ml phosphatidylinositol-specific phospholipase C (PI-PLC; Sigma) before being treated with 0.01 µg/ml LPS for 10 min. RAW264.7 cells were also treated with 0.5 U/ml PI-PLC and 100 ng/ml LPS in serum-free medium as described above. Activation of extracellular signal-regulated kinase (ERK) was prevented by treating cells with 50 µM PD-98059 (New England BioLabs). The concentration of the MAPK kinase (MEK) inhibitor PD-98059 was based on the IC50 values of 4 and 50 µM for MEK1 and MEK2, respectively. PD-98059 does not inhibit activation of other highly related dual-specificity kinases at concentrations <100 µM. Nuclear translocation of NF-kappa B was prevented with 5 µM SN-50 (Biomol).

For some experiments, cardiomyocyte and RAW264.7 cell cultures were pretreated for 1 h in serum-free medium with a 5 µg/ml concentration of purified anti-rat CD14 (ED9) monoclonal antibody (Serotec; see Immunoblot analyses) before being treated for 10 min with 100 ng/ml LPS plus anti-CD14 or antibody alone.

Immunoblot analyses. Proteins were isolated by rinsing the cells twice with ice-cold PBS (pH 7.4) and then lifting them from the plates with a rubber policeman. Cells were removed to 1.5-ml tubes and collected with a 10-s spin at 12,000 g. The cells were resuspended in a small volume of ice-cold lysis solution [150 mM NaCl, 20 mM Tris · HCl (pH 7.6), 1 mM EDTA, 0.5% sodium deoxycholate, 70 mM NaF, 1% Nonidet P-40, Complete protease inhibitor cocktail (Boehringer Mannheim), 200 µM sodium orthovanadate, and 2 µM phenylmethylsulfonyl fluoride] (9). After a 10-min incubation on ice with intermittent, brief vortexing, debris was pelleted in the microcentrifuge, and the supernatants were stored at -80°C. Protein concentrations were established using the bicinchoninic acid (BCA) protein determination kit (Pierce).

SDS-PAGE and transfer to nitrocellulose were performed as described earlier (2), and identical gels were stained with Coomassie brilliant blue R250 to confirm equal protein loading. Nitrocellulose membranes were rinsed in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 (TBS-T) and blocked in 5% nonfat milk/TBS-T for 1 h at 22°C on a rocking platform. Membranes were rinsed four times in TBS-T and then incubated overnight at 4°C on an orbital shaker with primary antibodies diluted in TBS-T containing 5% BSA (Sigma). The anti-ERK1 polyclonal antibody (K-23; Santa Cruz) was used at a concentration of 0.1 µg/ml to detect both ERK1 and ERK2, whereas the anti-phospho-MAPK polyclonal antibody (Promega) was diluted to 0.05 µg/ml to detect the dually phosphorylated (active) forms of ERK1 and ERK2. The anti-c-Jun NH2-terminal kinase 2 (JNK2) polyclonal antibody (FL; Santa Cruz), at a concentration of 0.1 µg/ml, and the anti-p38 MAPK polyclonal antibody (New England BioLabs), at 1:1,000 dilution, were used to detect total JNK2 and p38 proteins, respectively. We attempted to identify active forms of the latter two proteins with the anti-phospho-SAPK/JNK (New England BioLabs), p-JNK (Santa Cruz), and anti-ACTIVE JNK antibodies (Promega) as well as the anti-phospho-p38 MAPK (New England BioLabs) and anti-ACTIVE p38 antibodies (Promega) using the manufacturer's suggested dilutions. Total and active forms of STAT proteins were detected with the anti-STAT1 or anti-STAT3 antibodies and anti-phospho-STAT1 (Tyr-701) or anti-phospho-STAT3 (Tyr-705) antibodies (New England BioLabs) at the manufacturer's suggested dilutions. Nuclear (see below) and cytoplasmic extracts were examined for STAT3 (Ser-727) activation with a phospho-specific antibody (New England BioLabs) as outlined by the manufacturer. Two anti-CD14 monoclonal antibodies (ED2 and ED9; Serotec) were purified from ascites fluid using the E-Z Sep kit (Pharmacia) and were found to react equally well at a concentration of 1.0 µg/ml. Excess primary antibodies were washed from nitrocellulose membranes with three 10-min washes in TBS-T and incubated with appropriate horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies (Amersham) diluted 1:2,500 in 5% milk/TBS-T. After three 10-min washes in TBS-T, bound antibodies were detected with the enhanced chemiluminescence kit (Amersham) and exposed to Kodak X-Omat AR film.

In vitro kinase analysis. The p44/p42 MAPK Assay kit (New England BioLabs) was used to confirm activity of ERK1 and ERK2 as determined by immunoblotting with the phospho-specific MAPK antibody (Promega). The ability of immunoprecipitated active ERK1 and ERK2 to phosphorylate an Elk-1 recombinant fusion protein in vitro was determined by immunoblotting with an anti-phospho-(Ser-183)-Elk-1 antibody as described in the manufacturer's directions. A positive control (2 ng of active MAPK) was run along side cardiomyocyte samples that had been treated with LPS. Bound antibodies were detected with the Phototope-HRP Western Detection System (New England BioLabs).

RNA isolation and RT-PCR. Total RNA was isolated from cultured cells treated with LPS for 0, 5, 10, 30, and 60 min and 24 h essentially as described (7) and then digested with RQ1 RNase-free DNase (Promega) to remove any contaminating DNA. Spectrophotometric quantitation of purified RNA was confirmed visually by agarose-formaldehyde electrophoresis of RNA (2). RT-PCR was performed using the Access System (Promega) with primer pairs designed to the cDNA sequences of rat CD14 (15), rat LBP (49), and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Clontech). Purified total RNA (100 ng) from either cardiomyocytes treated with LPS for various times or untreated astrocytes was used for the reactions. The primer sets produced product sizes of 267 bp for CD14, 789 bp for LBP, and 452 bp for GAPDH. A linear range of amplification profile for each primer set was established on the basis of the following cycling parameters: reverse transcription step (1 cycle at 48°C for 45 min and 94°C for 2 min), PCR step (up to 40 cycles at 94°C for 30 s, 60°C for 45 s, and 68°C for 60 s), and extension step (1 cycle of 68°C for 7 min and 4°C soak). A midpoint of the linear amplification range was determined for CD14, LBP, and GAPDH RT-PCR products to be at cycles 33, 30, and 26, respectively. Negative controls included tubes that lacked RNA, AMV reverse transcriptase, or Tfl polymerase. The identity of each PCR product was confirmed by ligating the 267-, 452-, and 789-bp DNA fragments into the pCRII vector (Invitrogen) and sequencing these using standard molecular biology techniques (2). Equal volumes of amplified products were loaded in each lane of a 1% TAE (Tris, acetic acid, and EDTA)-buffered agarose gel and electrophoresced with the 1-kb DNA ladder (Life Technologies).

Electrophoretic mobility shift assays. Nuclear extracts were isolated from LPS-treated cardiomyocytes using the method of Andrews and Faller (1) and quantitated using the BCA protein determination kit (Pierce). Electrophoretic mobility shift assay reactions were carried out as described previously (10). Briefly, each 20-µl reaction contained 10 mM Tris · HCl (pH 7.6), 70 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA (pH 8.0), 25% glycerol, 0.2% Triton X-100, Complete protease inhibitor, 2.0 µg poly(dI-dC) · poly(dI-dC) (Pharmacia), 0.25 ng of labeled AP-1, NF-kappa B, (Promega), STAT1 (SIE, sis-inducible element), or STAT3 (Santa Cruz) consensus oligonucleotides, and, in some reactions, unlabeled oligonucleotides (either identical, mutated binding sites or unrelated sites of the same size). Probes were labeled with [gamma -32P]ATP and polynucleotide kinase using standard techniques (2) and purified with ProbeQuant G-50 columns (Pharmacia).

Measurement of TNF-alpha levels. Rat TNF-alpha levels were determined in cell culture medium samples using the Quantikine (R&D Systems) sandwich enzyme-linked immunosorbent assay (ELISA). Equal cardiomyocyte numbers were treated with 0.1 µg/ml LPS ± 50 µM PD-98059 and/or 5 µM SN-50. Culture medium was collected 4 h after treatment, and ELISAs were performed as described in the manufacturer's protocol.

Densitometric and statistical analyses. Densitometric determination of mean integrated areas under the curve from unsaturated autoradiograms of immunoblots was determined using Scion Image software (National Institutes of Health). Comparisons of densitometric data were made by analyzing the means ± SD with the Mann-Whitney test. Comparisons of TNF-alpha production as measured by a sandwich ELISA kit (R&D Systems) were made using an ANOVA followed by the Tukey-Kramer test.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Protein kinase activation in LPS-treated cardiomyocytes. Cardiomyocytes were cultured in either serum-free medium or in the presence of 2.5% FBS to determine the effect of serum components, such as soluble CD14 and LBP, on endotoxin-mediated signal transduction. The activation of various kinase proteins was studied using LPS concentrations ranging from 0.01 to 10 µg/ml. Initial immunoblotting experiments were conducted by using antibodies directed against either total or phosphorylated (active) forms of various pivotal protein kinases including ERK1, ERK2, p38, JNK1, JNK2, STAT1, and STAT3.

The dual phosphorylation of the 44- and 42-kDa ERK proteins was determined using a phospho-specific antibody. The phosphorylated proteins were compared with total ERK levels. ERK1 and 2 were phosphorylated at the same times (5 min, 10 min, and 24 h) regardless of the absence or presence of serum (Fig. 1, A and C, respectively). The background level of phosphorylated ERK1 and 2 detectable in the control cells was slightly higher in the serum-treated cultures (Fig. 1, A and C, lane 1). Densitometric analyses showed that serum treatment caused a significant (P = 0.02 vs. no serum) decrease in phosphorylation of these proteins at 24 h. Total ERK protein levels were not altered in response to LPS at the times studied in the cardiomyocyte cultures (Fig. 1, B and D); however, the phosphorylated form of ERK2 was detectable in these blots by its slightly slower migration in lanes 2, 3, and 6. Furthermore, similar times of activation and levels of phosphorylation of ERK1 and 2 were found for every LPS concentration studied (0.01, 0.1, 1, and 10 µg/ml) (results not shown). Figure 1 depicts a representative result from cells treated with 0.1 µg/ml LPS.


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  		Fig. 1.   Immunoblot analysis of phosphorylated and total extracellular signal-regulated kinase (ERK) protein in lipopolysaccharide (LPS)-treated cardiomyocytes. Cells were treated with 100 ng/ml endotoxin for 0, 5, 10, 30, and 60 min and 24 h before cell lysates were analyzed by immunoblotting with antibodies specific to active or total ERK proteins. Representative blots are from cells grown in serum-free defined medium (A and B) or in 2.5% fetal bovine serum (C and D). Essentially identical patterns of ERK activation (A and C) were found when cells were treated with LPS concentrations ranging from 0.01 to 10 µg/ml. Total protein levels remained the same at all times (B and D). Experiments were repeated at least 3 times; kD, kilodaltons.

Correlation of phosphorylation of ERK proteins with activity was accomplished using an in vitro kinase assay (Fig. 2). In this experiment, the ability of immunoprecipitated active ERK1 and 2 from endotoxin-treated, serum-free cell extracts to specifically phosphorylate the substrate transcription factor Elk-1 was determined. The Ser-383 phosphorylated form of Elk-1 was subsequently detected by immunoblot analysis. Often, two bands are seen in this assay when very high levels of phosphorylated Elk-1 are present. This is likely due to the incomplete denaturation of Elk-1, because this protein undergoes a change in conformation once it is phosphorylated. ERK activity was apparent in all of the lanes, with the highest levels at 10 min and 24 h after LPS treatment (Fig. 2, lanes 4 and 7). The basal level of ERK activity in untreated cells (lane 2) was approximately equivalent to that of the positive control (lane 1), which contained 20 ng of phospho-MAPK. Larger amounts of the positive control produced the characteristic double banding pattern (not shown). The results at 10 min and 24 h corroborate the findings from Fig. 1; however, the kinase assay showed no noticeable increase above background levels at 5 min.


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  		Fig. 2.   Mitogen-activated protein kinase (MAPK) activity in cardiomyocytes treated with 10 µg/ml LPS. Cells were treated for 0, 5, 10, 30, and 60 min and 24 h and then used to immunoprecipitate active MAPK and determine the specific phosphorylation of an Elk-1 fusion protein (lanes 2-7). The positive control (+) in lane 1 represents the level of Elk-1 phosphorylation that resulted from the immunoprecipitation of 20 ng of purified active ERK 2 protein. Elk-1 was detected with a phospho-specific antibody as described in METHODS. The kinase assay was repeated 3 times; a representative blot is shown.

Similar to ERK1 and 2, the STAT1 and STAT3 proteins were specifically tyrosine phosphorylated in a serum-independent fashion (Fig. 3 and not shown, respectively) at 5 min, 10 min, and 24 h after cardiomyocytes were treated with 10 µg/ml LPS. There were no differences in the total protein levels of either STAT at these times (Fig. 3, B and D). Untreated cells exhibited a small amount of STAT activation analogous to that observed for the ERK proteins (Fig. 3, A and C, lane 1). Interestingly, the STAT proteins appeared to have been phosphorylated to a greater extent at 5 min than at 10 min. Comparable results were found in cardiomyocytes treated with lower concentrations of LPS. Furthermore, inhibition of ERK activation through MEK1 inhibition with PD-98059 resulted in a substantial (P = 0.0008 vs. absence of MEK1 inhibitor) inhibition of supplemental phosphorylation of STAT3 at serine residue 727 (Fig. 4, top). The serine phosphorylated form of STAT3 was only detectable in the nuclear fraction from cardiomyocytes (Fig. 4, lanes 1-4), whereas total STAT3 was readily detectable in both the cytosol and nucleus (Fig. 4, bottom). PD-98059 had no effect on the tyrosine phosphorylation of either STAT1 or STAT3 (not shown).


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  		Fig. 3.   Immunoblot analysis of phosphorylated and total signal transducer and activators of transduction (STAT) proteins in LPS-treated cardiomyocytes. Cells were treated with 10 µg/ml endotoxin for 0, 5, 10, 30, and 60 min and 24 h before cell lysates were analyzed by immunoblotting with antibodies specific to active or total STAT1 (A and B) and STAT3 (C and D) proteins. Essentially identical patterns of STAT activation (A and C) were found when cells were treated with LPS concentrations ranging from 0.01 to 10 µg/ml. Total protein levels remained the same during the time course (B and D). All experiments were repeated at least 3 times.



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  		Fig. 4.   Immunoblot analysis of serine phosphorylated STAT3 protein in nuclear and cytosolic fractions from LPS-treated cardiomyocytes. Cells were treated with 1 µg/ml LPS for 5 min in the presence (+) or absence (-) of 50 µM PD-98059. Nuclear and cytosolic extracts were analyzed for Ser-727 phosphorylation of STAT3 (top) as well as total STAT3 protein levels (bottom). Results are representative of 3 independent experiments.

Total cellular JNK2 and p38 MAPK were detectable at moderate levels; however, there was no change at any of the times or as a consequence of incubation with serum. There was no measurable activation of the SAPK proteins, JNK1 or 2, or p38 MAPK as measured by phospho-specific antibodies obtained from several sources (results not shown).

Expression of CD14 and LBP in cardiomyocytes. CD14 was faintly detectable in cardiomyocyte lysates from cells treated with LPS for 0, 5, 10, 30, and 60 min and 24 h (Fig. 5, lanes 1-6). There were no apparent alterations in CD14 protein levels throughout these times. In addition, CD14 was more abundant in primary astrocytes and RAW264.7 cell extracts (Fig. 5, lanes 7 and 8, respectively), and results were the same whether CD14 was detected with the ED2 or ED9 primary antibody.


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  		Fig. 5.   Immunoblot analysis of CD14 protein in cardiomyocytes treated with LPS and in untreated astrocytes (ast) and RAW264.7 cells (raw). Mouse anti-rat CD14 antibodies (ED2 and ED9) were purified from ascites fluid before being used in immunoblotting experiments. Both primary antibodies reacted equally well with the separated protein samples (25 µg/lane) at the expected size of 55 kDa. A representative blot using the ED9 antibody is shown.

Verification of CD14 expression in cardiomyocytes was accomplished using RT-PCR (Fig. 6). In addition, the expression of LBP in these cells was established and compared with that of the housekeeping gene GAPDH. These results are not strictly semiquantitative; however, care was taken to assure that starting total RNA amounts were the same for each sample, and the cycling parameters were adjusted for each primer pair to ensure that PCR was stopped during the linear segment of the amplification profile. The RT-PCR products from LPS-treated cardiomyocytes are shown for the times indicated in Fig. 6 (lanes 2-7). Cardiomyocytes exhibited uniform low-level expression of both CD14 and LBP at the times studied. Untreated rat astrocyte mRNA was reverse transcribed and amplified as a positive control for CD14 (Fig. 6, lane 8) because it was known to be expressed in these cells (Fig. 5, lane 7) (46). Astrocyte RNA was negative for LBP (Fig. 6, lane 8). All reactions, with the exception of the water control (Fig. 6, lane 9), contained an equal amount of GAPDH amplification products.


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  		Fig. 6.   RT-PCR analysis of CD14, LPS-binding protein (LBP), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using RNA isolated from LPS-treated cardiomyocytes and untreated astrocytes. Lanes 1 and 10 contain molecular (M) size standards (GIBCO; 1-kb ladder), whereas lanes 2-9 contain samples. Cardiomyocytes were treated with 10 µg/ml LPS for 0, 5, 10, 30, and 60 min and 24 h (lanes 2-7). Cardiomyocyte and astrocyte (a, lane 8) reactions were carried out using 100 ng of total RNA. In lane 9, water was used as a negative (-) control.

Effect of PI-PLC and detoxified LPS treatment on kinase activity. Figure 7 shows the results of experiments that centered on the recognition of LPS and transmission of signal in cardiomyocytes. Figure 7A shows phosphorylated ERK, STAT1, and STAT3 proteins in LPS ± PI-PLC-treated cells, whereas Fig. 7B shows active ERK, STAT1, and STAT3 levels in cells treated with LPS ± detoxified LPS. Extracts were collected 10 min after LPS treatment. The total ERK and STAT protein levels did not change in response to these treatments (not shown). Figure 7A, lane 1, shows the basal level of ERK and STAT phosphorylation noted earlier (Figs. 1 and 3) in untreated cardiomyocytes. Figure 7A, lane 2, shows the activation of ERK and STAT kinases as a result of treatment with 0.01 µg/ml LPS for 10 min. In cardiomyocytes incubated with PI-PLC for 1 h before exposure to LPS, there was no difference in the level of phosphorylation (Fig. 7A, lane 3) compared with cells treated only with LPS (lane 2). When cells were treated with PI-PLC alone, active ERK and STAT levels were essentially at background (Fig. 7A, lane 4). In all instances, the culture medium was changed at the time of LPS treatment. This eliminated the possibility that soluble CD14, LBP, or other GPI-linked receptors were present in the culture medium at the time of LPS exposure. In addition, PI-PLC treatment was found to attenuate low-dose (0.01 µg/ml) LPS-mediated ERK activation in RAW264.7 cells (results not shown).


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  		Fig. 7.   Immunoblot analysis of active ERK and STAT protein in cardiomyocytes treated with LPS ± phosphatidylinositol-phospholipase C (PI-PLC) (A) or detoxified LPS (dLPS) (B). A: cells were pretreated with 0.5 U/ml PI-PLC (lanes 3 and 4) and then treated with 0.01 µg/ml LPS for 0 (lane 1) or 10 min (lanes 2 and 3). Protein lysates were isolated and used for immunoblotting with active ERK (top), STAT1 (middle), and STAT3 (bottom) antibodies. B: representative results from cells treated with 0.1 µg/ml LPS for 0 (lane 1) or 10 min (lanes 2 and 3) and 10 µg/ml dLPS (lanes 3 and 4). The experiment was repeated 3 times.

When a 100-fold molar excess of detoxified LPS was administered simultaneously with 0.1 µg/ml LPS, the phosphorylation of ERK and STAT proteins was greatly diminished compared with that in cells treated with LPS alone (Fig. 7B, lanes 2 and 3). Detoxified LPS (10 µg/ml) failed to induce kinase activity above control levels (Fig. 7B, lanes 1 and 4). Notice that the extent of ERK phosphorylation at 0.01 µg/ml (Fig. 7A, lane 2) was qualitatively the same as that at 0.1 µg/ml (Fig. 7B, lane 2).

The results presented in Fig. 8 demonstrate the activation of ERK1 and 2 in RAW264.7 cells in response to 100 ng/ml LPS treatment (A) and the LPS-induced phosphorylation of ERK proteins in both RAW264.7 cells and cardiomyocytes treated with PI-PLC and anti-CD14 antibody (B). All treatments were performed in serum-free medium. In the macrophage cell line, ERK protein phosphorylation was detected at high levels after 10 and 30 min of endotoxin exposure. Phosphorylation diminished at 60 min and was barely evident after 24 h of treatment (Fig. 8A, top, lanes 3-6). There was no detectable ERK activation at 0 or 5 min (Fig. 8A, top, lanes 1 and 2). Similar results were found at LPS concentrations ranging from 0.1 to 10 µg/ml. Therefore, activation of ERK1 and 2 in these cells differs from the phosphorylation profile seen in cardiomyocytes (Fig. 1). Comparable to levels in cardiomyocytes, total ERK protein levels did not significantly change at the times studied (Fig. 8A, bottom). Figure 8B shows that the effect of PI-PLC treatment and incubation with the ED9 anti-CD14 antibody was defined in both cell types after 10 min of 100 ng/ml LPS treatment. Figure 8B, top, shows that both PI-PLC and anti-CD14 antibody attenuated LPS-mediated MAPK signaling in macrophages (compare lane 2 with lanes 3 and 5, respectively). Figure 8B, bottom, demonstrates that neither PI-PLC nor anti-CD14 treatment reduced LPS-induced ERK protein activation in cardiomyocytes (lanes 2, 3, and 5). Both cell types showed little or no ERK phosphorylation in the absence of LPS (lanes 1, 4, and 6), regardless of the presence of PI-PLC (lane 4) or antibody (lane 6). Note that total protein and total ERK levels were the same for all lanes of Fig. 8B (not shown) and that the results shown in Fig. 8 are representative of three separate experiments.


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  		Fig. 8.   Immunoblot analysis of active and total ERK protein in RAW264.7 cells and cardiomyocytes treated with LPS ± PI-PLC or anti-CD14. A: RAW264.7 cells were treated with 100 ng/ml endotoxin for 0, 5, 10, 30, and 60 min and 24 h before cell lysates were analyzed by immunoblotting with antibodies specific to active (top) or total (bottom) ERK proteins. B: RAW264.7 cells (top) and cardiomyocytes (bottom) were also pretreated with PI-PLC or anti-CD14 and then treated with 100 ng/ml LPS ± PI-PLC or anti-CD14 for 10 min and analyzed for active ERK protein. The experiments in A were performed in the presence of serum, whereas experiments in B were conducted in serum-free medium. Total protein levels were the same in each lane, and the X-ray films shown are representative of 3 separate experiments.

Interaction of nuclear proteins with consensus DNA-binding sites. Nuclear proteins were isolated from cardiomyocyte cultures treated with 10 µg/ml LPS for 0, 5, 10, 30, and 60 min and 24 h and reacted with 32P-labeled DNA sequences corresponding to consensus binding sites for AP-1, NF-kappa B, SIE, and STAT3. Bound complexes were resolved using the electrophoretic mobility shift assay (Fig. 9). Adding unlabeled identical or nonidentical double-stranded (identical and nonidentical) competitor DNA at 50- and 500-fold molar excesses confirmed specificity of complex formation. Identical results were obtained from nuclear extracts derived from cardiomyocytes grown in serum-containing medium (not shown). LPS treatment stimulated specific binding of proteins to AP-1, NF-kappa B, SIE, and STAT3 sites. Aside from the 24-h treatment, the next greatest binding of protein to the AP-1 site was at 10 and 30 min, whereas the NF-kappa B site displayed increased binding from 5 min to 24 h. Nuclear proteins bound the STAT3 consensus binding site with increasing affinity from 5 to 60 min, whereas the SIE site had the greatest binding at 10 min.


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  		Fig. 9.   Electrophoretic mobility shift assays using consensus binding sites reacted with LPS-treated cardiomyocyte nuclear extracts. Cells were exposed to 10 µg/ml LPS for 0, 5, 10, 30, 60 min, and 24 h (lanes 1-6). Nuclear extract preparations from these cells were incubated with 32P-labeled consensus binding sites for AP-1, nuclear factor-kappa B (NFkappa B), STAT1 (SIE), and STAT3. Lanes 7 and 8 contain unlabeled identical (ic) and nonidentical competitor (nc) sequences to establish the specificity of DNA-protein interactions. After separation, the 6% gel was dried and exposed to film. Specifically bound DNA-protein complexes are indicated near the top of each gel, whereas free probe is shown near the bottom.

Effect of MAPK and NF-kappa B inhibitors on LPS-induced TNF-alpha production. A significant increase (P < 0.001) in TNF-alpha secretion was observed when cardiomyocytes were treated with 0.1 µg/ml LPS for 4 h compared with untreated cells (Fig. 10). The MEK1 inhibitor PD-98059 caused a significant reduction (P < 0.001) in LPS-induced TNF-alpha production, whereas SN-50, which prevents nuclear translocation of NF-kappa B, completely blocked TNF-alpha secretion (P < 0.001) in LPS-treated cells. The combination of both PD-98059 and SN-50 similarly prevented LPS-mediated TNF-alpha release in culture medium (P < 0.001).


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  		Fig. 10.   Tumor necrosis factor-alpha (TNF-alpha ) production in cardiomyocytes treated with LPS ± PD-98059 and/or SN-50. Cells were pretreated for 30 min with 50 µM PD-98059 and/or 5 µM SN-50. Four hours after treatment with 0.1 µg/ml LPS plus PD-98059 and/or SN-50 (as indicated), culture medium was analyzed using a sandwich ELISA kit for rat TNF-alpha . Results are presented as pg/ml TNF-alpha (means ± SD), where n = 4 and all experiments have an equal number of cells. An ANOVA followed by Tukey-Kramer analysis revealed a significant increase (**P < 0.001) in TNF-alpha secretion in cells treated with LPS alone (2nd bar) compared with untreated cardiomyocytes (1st bar). PD-98059 and SN-50, alone (3rd and 4th bars, respectively) or in combination (5th bar), significantly (*P < 0.001) reduced endotoxin-induced TNF-alpha production.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Inflammatory cytokines produced as a consequence of LPS exposure are implicated in heart failure and myocardial dysfunction (33, 56). Paradoxically, sublethal doses of endotoxin can provide delayed cardioprotective effects against ischemia-reperfusion injury (34). The mechanisms that control these diverse outcomes are not fully understood but likely result from the production of injurious mediators and the mobilization of cellular defenses, respectively. This study determined the early responses of cardiomyocytes to LPS exposure, because these events presumably account for both its beneficial and harmful effects.

In cardiomyocytes, members of the MAPK (ERK1 and 2) and Janus kinase (JAK)/STAT (STAT1 and STAT3) signaling pathways are activated following LPS exposure (Figs. 1-3). The p38 MAPK and SAPK (JNK1 and 2) cascades were not activated, contrary to what has been reported for other cell types. Because of the rapidity of activation, it is likely that phosphorylation of ERK and STAT proteins is the direct consequence of LPS treatment rather than a secondary effect. ERK and STAT phosphorylation occurred within 5-10 min following exposure to 0.01-10 µg/ml LPS. Serum had no stimulatory effect on kinase activity at any of these concentrations. The lack of serum-induced potentiation of endotoxin signal, particularly at the lower concentrations, is indicative of a CD14- and LBP-independent response (13, 39), despite low-level expression of both these proteins by cardiomyocytes (Figs. 5 and 6). Interestingly, serum consistently blunted ERK1 and 2 phosphorylation after 24 h of LPS treatment, whereas STAT activation was unaffected. The finding that LPS-induced TNF-alpha release is similarly reduced in adult cardiomyocytes in the presence of 10% FBS (8) supports our observation because TNF-alpha expression is, in part, controlled by the MAPK pathway (16). The latter assumption was tested by measuring the effect of MAPK inhibition on LPS-induced TNF-alpha secretion by cardiomyocyte cultures (Fig. 10). The MEK1 inhibitor PD-98059 was found to significantly (P < 0.001) (but not completely) reduce production of TNF-alpha . The lack of complete inhibition of TNF-alpha secretion by PD-98059 implies that the MAPK pathway is not solely responsible for LPS-mediated induction of this cytokine.

Our finding that cleavage of GPI-anchored receptors from the surface of cardiomyocytes did not prevent LPS-induced ERK or STAT activation (Figs. 7A and 8B) provides further evidence of CD14-independent signaling. Because cleaved receptors were washed from the plates before LPS treatment and the experiments were performed in serum-free medium (i.e., no endogenous or exogenous soluble CD14 or LBP), these results provide strong support for a CD14 receptor-independent mechanism of signal transduction in cardiomyocytes. Although endogenous CD14 and LBP could account for some late signaling events, as seen at 24 h, it is unlikely that these proteins contributed to early signaling. Furthermore, the activity of the PI-PLC used in these experiments was confirmed in LPS-treated RAW264.7 cells, in which ERK activation could be attenuated under identical conditions (Fig. 8B). Figure 8B further demonstrates that cardiomyocytes activate MAPK signaling in a CD14-independent fashion, unlike macrophages, because block of this GPI-anchored receptor with an anti-CD14 antibody before LPS treatment failed to decrease ERK phosphorylation. Figure 7B shows that a 100-fold excess of detoxified LPS can efficiently compete with LPS and inhibit kinase activation. This supports the notion that LPS-induced signaling in cardiomyocytes is receptor-mediated rather than relying on uncontrolled uptake through the plasma membrane. It also implies that the putative receptor recognizes detoxified LPS as a ligand, similar to the findings for CD14 (25). Competition by detoxified LPS can block LPS-stimulated TNF-alpha release from RAW264.7 cells but not from phagocytic Kupffer cells (30). Kupffer cells are similar to cardiomyocytes in that they express small quantities of CD14 (Figs. 5 and 6) and utilize a GPI-linked receptor-independent means of signal transmission (Fig. 7).

The activation of ERK proteins as measured using phospho-specific antibodies was confirmed with an in vitro kinase assay that measured the Ser-383 phosphorylation of a recombinant Elk-1 fusion protein by the ERK1 and 2 proteins (Fig. 2). Although the ability of ERK proteins to phosphorylate substrate was enhanced at 10 min and 24 h, there were no noticeable increases in Elk-1 phosphorylation at 5 min. This may be due to experimental differences in the speed of cell lysate preparation or inaccuracies encountered during the immunoprecipitation and wash steps of the in vitro kinase assay. Alternatively, there may be a short delay between the activation of ERK proteins and subsequent phosphorylation of Elk-1. Whether serum attenuates Elk-1 phosphorylation in a manner analogous to that of ERK phosphorylation remains to be investigated. Overall, ERK phosphorylation of Thr-202 and Tyr-204 residues, as detected by an anti-active MAPK antibody, correlate well with active ERK-mediated phosphorylation of the downstream target protein Elk-1.

The ternary complex transcription factor Elk-1 is a nuclear target of the ERK proteins known to induce c-fos and c-jun expression by binding to the serum response element and the 12-O-tetradecanoylphorbol-13-acetate response element, respectively (21). Because c-fos and c-jun are known members of the AP-1 sequence-specific transcriptional complex, the binding of nuclear proteins to a consensus AP-1 DNA sequence was examined in cardiomyocytes treated with LPS (Fig. 9). Specific DNA-protein complexes were found at moderate levels at 5, 10, and 30 min and at higher levels at 24 h following endotoxin exposure. The pattern of binding was not affected by the presence of FBS, providing more evidence that cardiomyocytes respond to LPS through a CD14-independent means. The induction of binding at the earlier time points is likely due to ERK and/or STAT activation; the more intense binding found at 24 h may result from additional convergent signals that amplify AP-1 complex formation. This amplification of signal is not the consequence of cell cycle effects because cardiomyocytes are nondividing cells.

Similarly to Elk-1, the STAT3 protein can stimulate AP-1 by binding to the c-fos promoter (42). STAT3 and AP-1 are critical for the activation of many downstream gene targets such as the acute phase reactants (19, 43). The rapid activation of STAT3 by LPS in cardiomyocytes may be indicative of a direct receptor tyrosine kinase-mediated activation, because JAK-induced activation is generally slower (20-30 min) (11). In contrast, LPS induction of STAT3 binding in rat liver required 75 min, implying that hepatocytes and cardiomyocytes respond to endotoxin through different signaling pathways (42).

The anti-active STAT3 antibody used in Fig. 3 recognizes tyrosine phosphorylated protein. Tyr-705 phosphorylation is obligatory for transcriptional activity of STAT3; however, this protein can also be serine phosphorylated. Ser-727 phosphorylation may supplement transcriptional activity, and this event has been proposed to be ERK dependent (11). Figure 4 shows that ERK proteins do serine phosphorylate STAT3 in the cardiomyocyte nucleus. Although the full significance of this event has yet to be determined, our preliminary data indicate that MEK1 inhibition increases nuclear factor binding to SIE and STAT3 consensus sites (not shown). This observation suggests that the ERK pathway reduces the expression of gene products downstream of STAT3. Intriguingly, ERK proteins appear to mediate the inhibition of STAT3 transcriptional activity induced by IL-6 (44).

NF-kappa B is another two-protein transcriptional complex composed from a family of related nuclear factors that are activated in response to LPS (58). Transcriptionally competent NF-kappa B is known to activate a variety of genes including many of the inflammatory cytokines (3, 51). Recent studies indicate that NF-kappa B-mediated IL-6 production requires both the ERK and p38 MAPK pathways to be activated (55). In cardiomyocytes, nuclear factors were induced to specifically bind the NF-kappa B consensus sequence following LPS stimulation (Fig. 9). The moderate binding found within the first hour of stimulation was greatly increased at 24 h. Similar to AP-1, the increase in NFkappa B binding after a day of endotoxin exposure is likely to result from a multiplicative effect due to convergence of a variety of signaling pathways. It is noteworthy that both AP-1 and NFkappa B are downstream of recently identified LPS-responsive Toll-like receptors (58). Remarkably, inhibition of NF-kappa B nuclear translocation completely blocked LPS-induced TNF-alpha secretion at 4 h (Fig. 10). This finding indicates that NF-kappa B (unlike MAPK) is absolutely required for TNF-alpha production by cardiomyocytes.

In conclusion, the stimulation of cardiomyocyte signal transduction by LPS through ERK and STAT pathways is independent of CD14. Definition of signal transmission events in these cells is critical for the development of receptor-specific therapies to prevent the initiation of deleterious mechanisms that result in contractile depression. Similarly, this knowledge may allow us to exploit any protective benefits of low-dose endotoxin treatment as a means of providing functional protection against ischemia-reperfusion injury in the heart.


    ACKNOWLEDGEMENTS

We thank Lina M. Garcia and Christina Cheng for technical and clerical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-52589 (to F. X. McGowan) and HL-46207 (to P. J. del Nido) as well as the Anesthesia Foundation of Children's Hospital.

Address for reprint requests and other correspondence: D. B. Cowan, Dept. of Anesthesia, Enders Rm. 1255, Children's Hospital, 300 Longwood Ave., Boston, MA 02115 (E-mail: cowan_d{at}a1.tch.harvard.edu).

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. §1734 solely to indicate this fact.

Received 14 September 1999; accepted in final form 26 January 2000.


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