PeproTech Inc.

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Regenhard, P.
Right arrow Articles by Phi-van, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Regenhard, P.
Right arrow Articles by Phi-van, L.
(Journal of Leukocyte Biology. 2001;69:651-658.)
© 2001 by Society for Leukocyte Biology

Involvement of PKA, PKC, and Ca2+ in LPS-activated expression of the chicken lysozyme gene

Petra Regenhard*, Ralph Goethe*,{dagger} and Loc Phi-van*

* Institut für Tierzucht und Tierverhalten Celle (FAL), Celle, Germany; and
{dagger} Institut für Mikrobiologie und Tierseuchen, Hannover, Germany

Correspondence: Dr. Loc Phi-van, Institut für Tierzucht und Tierverhalten, Dörnbergstr. 25-27, 29223 Celle, Germany. E-mail: loc.phi-van{at}fal.de


arrow
ABSTRACT
 
The lysozyme gene is activated in myelomonocytic HD11 cells in response to LPS. In this study, we described the involvement of LPS-activated signal transduction pathways in activation of the lysozyme gene. Pre-treatment of HD11 cells with H-89, H-7, TMB-8, or KN-93 resulted in inhibition of the LPS-enhanced lysozyme expression, suggesting that PKA, PKC, and Ca2+-dependent protein kinases participate in the LPS activation. CaMKII seems to be required for the processing of lysozyme transcripts. TPA and calcium ionophore A23187, when separately added to HD11 cells, stimulated the lysozyme expression effectively, and forskolin was ineffective. It is interesting that simultaneous treatment of cells with forskolin and calcium ionophore A23187 resulted in a potentiated increase in lysozyme mRNA expression, indicating a synergistic cooperation of PKA and Ca2+. This synergistic effect of PKA and Ca2+ was observed on the expression of a stably integrated CAT construct, controlled by the lysozyme promoter and the -6.1-kb enhancer containing binding sites for C/EBP and NF-{kappa}B/Rel. Therefore, we discussed the role of C/EBPß(NF-M), CREB, and NF-{kappa}B/Rel as possible targets for phosphorylation mediated by PKA, PKC, and Ca2+.

Key Words: myelomonocytes • inflammatory response • C/EBPß(NF-M) • CREB • NF-{kappa}B/Rel


arrow
INTRODUCTION
 
Lysozyme is one of the antibacterial proteins synthesized mainly in the chicken oviduct and in macrophages. In tubular gland cells of the oviduct, the expression of the lysozyme gene is regulated transcriptionally by steroid hormones [1 , 2 ]. During the differentiation of macrophages, the lysozyme gene is expressed at a low level in precursors and at a high level in mature macrophages and thus is a later marker gene for the myeloid lineage [3 4 5 ]. Inflammatory agents such as lipopolysaccharide (LPS) activate immunological and inflammatory responses, particularly in cells of immunological systems including B and T lymphocytes and macrophages [6 ]. Several studies demonstrated that treatment with LPS activated protein phosphorylation mediated by protein kinase C (PKC) [7 , 8 ], protein kinase A (PKA) [9 10 11 ], as well as protein tyrosine kinases (PTKs) [8 , 12 , 13 ]. Furthermore, LPS initiates hydrolysis of phosphatidylinositol-4,5-biphosphate (PIP2) to generate inositol triphosphate (IP3), which in turn leads to large elevations in intracellular levels of Ca2+ [14 , 15 ]. This second messenger can modulate the expression of target genes by activating Ca2+-dependent kinases, the most well-characterized of which are the Ca2+-calmodulin-dependent protein kinase II (CaMKII) isoenzymes, which are expressed in most tissues [16 17 18 ]. It has been shown that CCAAT/enhancer-binding protein (C/EBP)ß, a member of the bZip family of transcription factors, was phosphorylated by activation of CaMKII in response to increased intracellular calcium concentrations, and this phosphorylation at serine276 within the leucine zipper of C/EBPß appeared to confer calcium-regulated transcriptional activation of promoters containing binding sites for C/EPBß [19 ].

In macrophages, expression of several genes can be activated upon stimulation by LPS. We have shown previously that the lysozyme expression was elevated strongly in LPS-activated chicken myelomonocytic HD11 cells. This LPS activation was regulated at transcriptional and post-transcriptional levels [20 ]. The LPS-activated transcription was mediated by specific interaction of the myeloid-specific transcription factor C/EBPß(NF-M) with two C/EBPß-binding sites of the far-upstream -6.1-kb lysozyme enhancer [21 , 22 ]. Furthermore, treatment of HD11 cells with LPS increased the level of nuclear factor {kappa}B (NF-{kappa}B)p65/Rel-containing protein complexes binding to the NF-{kappa}B-binding site within the lysozyme promoter [4 ]. To determine the transduction pathways that mediate the LPS-activated lysozyme expression in this study, we investigated the effects of activators and inhibitors of protein kinases on the lysozyme expression in myelomonocytic HD11 cells. We found that pre-treatment of cells with H-7, H-89, TMB-8, and KN-93, but not with herbimycin A and Gö 6976, inhibited the LPS-activated lysozyme expression effectively, indicating involvement of PKC, PKA, Ca2+, and CaMKII in the LPS activation. Furthermore, in parallel with its stimulatory effect on the transcription, CaMKII seems to be required to process lysozyme transcripts. Treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or calcium ionophore A23187 increased the lysozyme expression. It is interesting that forskolin and calcium ionophore A23187, when added simultaneously, had a synergistic effect on the lysozyme expression, suggesting a synergism of transduction pathways mediated by PKA and Ca2+ involved in the LPS-triggered lysozyme expression. In this context, the role of C/EBPß(NF-M), CREB, and NF-{kappa}B/Rel as targets for protein kinases mediated by PKA, PKC, CaMKII, and Ca2+ is discussed.


arrow
MATERIALS AND METHODS
 
Cell culture
Myelomonocytic HD11 cells [23 ] were grown in Iscove’s modified Dulbecco’s medium (IMDM), supplemented with 8% fetal calf serum (FCS), 2% chicken serum, 100 U/ml penicillin, 100 µg/ml streptomycin at 37°C, and 5% CO2. For stimulation, cells were maintained in IMDM with 0.5% FCS for 48 h and then treated with 5 µM calcium ionophore A23187 (Sigma, Deisenhofen, Germany), 25 µM forskolin (ICN, Eschwege, Germany), 60 ng/ml TPA (Sigma), or 5 µg/ml LPS from Salmonella typhimurium (Sigma) for the indicated time periods. To inhibit protein phosphorylation by PKC, PKA, PTK, CaMKII, and Ca2+-dependent PKC and to inhibit release of Ca2+ from intracellular stores, cells were incubated first with H-7 and H-89 (both at ICN); herbimycin A (Sigma); and KN-93, Gö 6976, and TMB-8 (all at Calbiochem, Schwalbach, Germany) and then stimulated with LPS.

RNA preparation
For preparation of poly(A)+ RNA, 4 x 107 cells washed twice in phosphate-buffered saline (PBS) were lysed in 15 ml SSTE [0.1 M NaCl, 20 mM Tris-HCl (pH 7.5), 10 mM ethylenediaminetetraacetate (EDTA), and 0.5% sodium dodecyl sulfate (SDS)] and homogenized with a Janke & Kunkel Ultra-Turrax (Staufen, Germany). After a 30-min digestion with 300 µg/ml proteinase K at 37°C, the lysate was incubated with 100 mg oligo(dT) cellulose in the presence of 0.5 M NaCl at room temperature overnight. The oligo(dT) cellulose-bound poly(A)+ RNA was collected by centrifugation at 1600 rpm for 4 min and washed with four changes of 10 ml of a solution containing 10 mM Tris-HCl (pH 7.5), 0.3 M NaCl, 5 mM EDTA, and 0.1% SDS, and finally, poly(A)+ RNA was eluted with deionized water.

Northern analysis
Northern analysis was performed by a standard method [24 ]. Briefly, 4 µg poly(A)+ RNA denatured by 0.5 M glyoxal and 27% dimethyl sulfoxide (DMSO) were fractionated electrophoretically on 1.4% agarose gels containing 10 mM NaH2PO4 (pH 6.9) at 75 V for 2–3 h, capillary-transferred onto Hybond-N+ nylon membranes (Amersham Pharmacia Biotech, Braunschweig, Germany) [25 ], and immobilized by backing the blot at 80°C for 2 h. After a 4-h pre-hybridization in a solution containing 0.5 M Na2HPO4 (pH 7.2), 1 mM EDTA, and 7% SDS at 65°C, blots were hybridized to nick-translated plasmid DNA (3x106 cpm/ml) containing chicken lysozyme cDNA [26 ], lysozyme intron 1 and 2 [20 ], NF-M cDNA (22), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA [27 ] with 0.1 mg/ml yeast tRNA in the same solution at 65°C overnight. Hybridized blots were washed at 65°C once in 100 ml of 40 mM Na2HPO4 (pH 7.2), 1 mM EDTA, and 5% SDS and three times in 60 ml of 40 mM Na2HPO4 (pH 7.2), 1 mM EDTA, and 1% SDS for 15 min, followed by a short wash in 100 ml of 4 x standard saline citrate (SSC; 1xSSC: 150 mM NaCl and 15 mM sodium citrate, pH 7.0) at room temperature. After washing, blots were exposed to X-ray films with intensifying screens at -80°C. To quantify hybridization signals, autoradiograms were scanned by a scanning densitometer from Bio-Rad (München, Germany). Lysozyme RNA levels were normalized with respect to GAPDH on the same blots.

Stable transfection and chloramphenicol acetyltransferase (CAT) assay
HD11 cells were transfected with 20 µg pcEPCAT5 [21 ] and 2 µg ptkNeo [28 ] by the calcium phosphate co-precipitation method described previously [28 ]. The plasmid pcEPCAT5 carries the CAT reporter gene under transcriptional control by the chicken lysozyme promoter and the -6.1-kb lysozyme enhancer. The plasmid ptkNeo contains the Tn5 neomycin-resistance gene driven by the thymidine kinase promoter from Herpes simplex virus. Transfected cells were fed with fresh IMDM medium and incubated at 37°C for 24 h before 500 µg/ml G418 (Life Technologies, Karlsruhe, Germany) was added to select cells that had integrated DNA stably. G418-resistant clones were isolated 2–3 weeks later and grown to a density of 1 x 107 cells per 8.5-cm plate for stimulation with LPS, calcium ionophore A23187, and forskolin, as described above. After stimulation (24 h), cells washed twice in PBS were scraped by a rubber policeman in 1 ml TEN (40 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 150 mM NaCl) and pelleted by centrifugation in an Eppendorf centrifuge for 10 s. The pellet was suspended in 150 µL of 0.25 mM Tris-HCl (pH 7.5), and the suspension was sonified and cleared by centrifugation at 10,000 g for 10 min. Protein concentrations of cell extracts were determined by using a detergent- compatible protein assay kit from Bio-Rad, according to the instructions of the manufacturer. CAT assays were performed with 10–50 µg protein of each cell extract in the presence of 0.5 mM acetyl coenzyme A and 0.5 µCi [14C]chloramphenicol at 60 mCi/mmol (Amersham Buchler, Braunschweig, Germany), as described previously [28 ].

Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
Nuclear extracts from HD11 cells were prepared as described by Schreiber et al. [29 ]. EMSA was performed with radiolabeled oligonucleotides containing the C/EBPß(NF-M)-binding site TTTGGAAAT of the -6.1-kb lysozyme enhancer [21] or the NF-{kappa}B site of the lysozyme promoter [4], using 5 µg protein of nuclear extracts as described previously [22 ].


arrow
RESULTS
 
PKA and PKC, but not PTK, are essential for the LPS-activated lysozyme expression
To investigate transduction pathways that mediate the LPS-activated expression of the lysozyme gene, inhibitors of protein kinases were used. HD11 cells were incubated with LPS following treatment with H-7, H-89, or herbimycin A, and the levels of lysozyme RNA were determined by Northern analysis using the full-length lysozyme cDNA as probe. We have shown previously that LPS induced an accumulation of the lysozyme pre-mRNAs of 3.9, 2.1, and 0.8 kb [20 ]. Figure 1A 1B 1C shows the effects of these inhibitors on the lysozyme expression in LPS-activated cells. H-7 and H-89, inhibitors of PKC and PKA, respectively, were sufficient to inhibit the LPS-induced lysozyme expression completely at a concentration of 25 µM. This result indicates that PKC and PKA are essential for the LPS stimulation in HD11 cells. In contrast, herbimycin A, an inhibitor of PTK, failed to inhibit the LPS-activated lysozyme expression. Instead, treatment of cells with herbimycin A led to an increase in lysozyme RNA levels (2.1-fold).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 1. Effects of H-7, H-89, and herbimycin A on the LPS-induced lysozyme expression. Cells were pre-incubated with H-7 (A), H-89 (B), or herbimycin A (C) for 2 h before activation with 5 µg/ml LPS for 1 h (A and B) or 30 min (C). Poly(A)+ RNAs were isolated and analyzed sequentially by Northern blot hybridization to lysozyme and GAPDH cDNA. The arrow indicates the position of the mature, lysozyme mRNA.

CaMKII is required for the LPS-activated lysozyme transcription and splicing of lysozyme transcripts
Ca2+ can be mobilized from the endoplasmic reticulum by treatment of macrophages with LPS leading to increases in intracellular Ca2+ levels [14 , 15 ]. To confirm the role of Ca2+ in the LPS activation of the lysozyme expression, we used TMB-8 to block the release of Ca2+ from intracellular stores and thus to prevent Ca2+ increase in LPS-activated cells. Northern blot analysis on lysozyme mRNA from cells pre-treated with 25, 50, 100, and 200 µM TMB-8 and subsequently stimulated with LPS revealed that TMB-8 prevented the LPS-activated expression in a dose-dependent manner. The LPS activation was inhibited almost completely at the TMB-8 concentration of 200 µM (Fig. 2A ).



View larger version (43K):
[in this window]
[in a new window]
  		Figure 2. Inhibition of the LPS-induced lysozyme expression by TMB-8 and KN-93. HD11 cells pre-treated with TMB-8 (A), Gö 6976 (B), or KN-93 (C) for 2 h were activated with 5 µg/ml LPS for 1 h. Poly(A)+ RNAs were isolated and subjected to Northern blot analysis using lysozyme and GAPDH cDNA.

Next, we examined the involvement of Ca2+-dependent protein kinases in the LPS-activated lysozyme expression using a Ca2+-dependent PKC inhibitor (Gö 6976) and a CaMKII inhibitor (KN-93). Densitometric scanning of the hybridization signals revealed that Gö 6976 had no effect on the lysozyme expression, when the lysozyme RNA levels were normalized for GAPDH (Fig. 2B) . In contrast, when cells were pre-treated with 50 µM KN-93, followed by stimulation with LPS, the increased lysozyme expression was inhibited by ~50% (Fig. 2C) . It is interesting that Figure 2C shows further that in the presence of 50 µM KN-93, 8%, 87%, and 5% of lysozyme transcripts at 3.9, 2.1, and 0.8 kb, respectively, were present after 1 h of treatment with LPS, in contrast to 1%, 43%, and 56% of those obtained in LPS-stimulated cells without KN-93. Thus, the lysozyme transcripts in cells treated with KN-93 seem to be spliced more slowly than those in untreated cells. Therefore, the splicing of lysozyme transcripts in cells treated with and without KN-93 was pursued after inhibiting transcription by treatment with actinomycin D, an inhibitor of RNA polymerases. Before inhibition of transcription with actinomycin D, cells were pre-treated with or without KN-93 and then stimulated with LPS for 1 h. Figure 3 shows that after 30-, 60-, and 90-min inhibition of transcription by actinomycin D, 72%, 24%, and 22% of the lysozyme transcript at 2.1 kb still remained in cells treated with KN-93, compared with 6%, 0%, and 0% of that in untreated cells, suggesting an involvement of CaMKII in the processing of the lysozyme transcripts, particularly of the 2.1-kb transcript.



View larger version (60K):
[in this window]
[in a new window]
  		Figure 3. Effect of KN-93 on the processing of lysozyme transcripts. Cells pre-treated with 25 mM KN-93 for 2 h and then activated with 5 µg/ml LPS for 1 h were treated with 5 µg/ml actinomycin D (act D) for 30, 60, and 90 min. Poly(A)+ RNAs were isolated and analyzed by Northern blot hybridization using lysozyme and GAPDH cDNA.

To determine whether CaMKII is also involved in the LPS-activated transcription of the lysozyme gene, the effect of KN-93 on the transcription of a CAT gene driven by the lysozyme promoter and the -6.1-kb lysozyme enhancer (pcEPCAT5) was tested. Using a run-on transcription assay, we have shown previously that expression of this CAT construct, integrated stably in HD11 cells, was activated transcriptionally by LPS [20 ]. A G418-resistant HD11 cell line (pc5) containing the CAT construct was pre-treated with KN-93 and then activated with LPS. After 24 h of incubation, CAT activities were determined. As shown in Figure 4 , KN-93 was sufficient to suppress the LPS activation of CAT activity. Taken together, these data are consistent with the suggestion that CaMKII is required not only for the increased transcription of the lysozyme gene but also for the splicing/processing of its transcripts in LPS-activated cells.



View larger version (52K):
[in this window]
[in a new window]
  		Figure 4. KN-93 inhibits expression of a lysozyme-CAT construct integrated stably in an HD11 cell line (pc5). pc5 cells pre-treated with KN-93 for 2 h were incubated with or without 5 µg/ml LPS for 24 h before harvesting for preparation of cell extracts. CAT assays of cell extracts were performed as described in Materials and Methods. Acetylated chloramphenicol (Ac-Cm) was separated from unmodified chloramphenicol (Cm) by thin-layer chromatography on a silica plate.

Calcium ionophore A23187 and forskolin stimulate the lysozyme expression in myelomonocytic HD11 cells synergistically
To examine the lysozyme expression mediated by PKA, PKC, and Ca2+-dependent protein kinases, Northern analysis was performed with poly(A)+ RNA from HD11 cells treated with forskolin, TPA, and calcium ionophore A23187 using the full-length lysozyme cDNA as probe. As shown in Figure 5A , treatment of cells with forskolin induced the lysozyme expression weakly even after 12 h, and treatment with calcium ionophore A23187 resulted in moderate induction. It is interesting that forskolin and calcium ionophore A23187, when added simultaneously, induced significant accumulation of lysozyme RNA synergistically (Fig. 5A , lanes 4, 7, and 10). In a typical time-course experiment, at time zero, the levels of lysozyme RNA were quite low, and after the addition of forskolin and calcium ionophore, lysozyme RNA levels in HD11 cells remained unchanged for the first 4 h but were increased significantly following this lag phase and elevated continuously until at least 12 h of incubation (Fig. 5B) . In contrast to forskolin, TPA, a PKC activator, when separately added to HD11 cells, was able to stimulate the lysozyme expression significantly. When TPA and calcium ionophore A23187 were added simultaneously, additional stimulation of the expression was observed (Fig. 5C , lanes 3, 5, and 7). In contrast, no further stimulation was seen with TPA and forskolin (Fig. 5C , lanes 3, 9, and 11).



View larger version (50K):
[in this window]
[in a new window]
  		Figure 5. (A–C) Synergistic activation of the lysozyme expression by calcium ionophore A23187 and forskolin. Poly(A)+ RNAs, isolated from untreated HD11 cells (-) and from cells treated with calcium ionophore A23187, forskolin (FOR), and TPA, separately or simultaneously, were fractionated on 1.6% agarose gels and Southern-blotted onto nylon membranes. The blots were hybridized sequentially to lysozyme and GAPDH cDNA. Representative results shown in B are from one of three experiments.

Changes in processing of lysozyme transcripts in forskolin- and calcium ionophore A23187-treated HD11 cells
Forskolin and calcium ionophore A23187, like LPS, when added to HD11 cells, induced an accumulation of three larger lysozyme RNA species of 3.9, 2.1, and 0.8 kb. To analyze the processing pathway of lysozyme pre-mRNA in HD11 cells treated with forskolin and calcium ionophore A23187, lysozyme-specific intron sequences 1 and 2 were used as probes to be labeled and hybridized to poly(A)+ RNA on Northern blots. As shown in Figure 6 , RNA species at 3.9 and 2.1 kb were detected by intron 1, whereas the largest at 3.9 kb was detected by intron 2. Thus, these results are similar to our previous results obtained with RNA from LPS-activated HD11 cells [20 ]. Furthermore, the 0.8-kb RNA species was spliced completely but still larger than the mature lysozyme mRNA in control HD11 cells. Analysis of poly(A) tails by RNase H indicates an increase in poly(A) tail length in HD11 cells treated with forskolin and calcium ionophore A23187 (unpublished results).



View larger version (36K):
[in this window]
[in a new window]
  		Figure 6. Processing of the lysozyme primary transcript in calcium ionophore A23187 and forskolin-activated HD11 cells. Poly(A)+ RNAs isolated from untreated cells or from cells treated with calcium ionophore A23187 and forskolin for 8 and 10 h were fractionated on 1.6% agarose gels and Southern-blotted onto nylon membranes. The blots were hybridized to lysozyme cDNA or to intron sequences 1 and 2, followed by rehybridization to GAPDH.

Calcium ionophore A23187 and forskolin activate synergistically expression of a lysozyme-CAT construct integrated stably in HD11 cells
To determine whether forskolin and calcium ionophore A23187 activate the lysozyme expression at a transcriptional level, we measured the transcriptional and regulatory activity of the lysozyme promoter and the -6.1-kb enhancer in stably transfected HD11 cells treated with calcium ionophore A23187 and forskolin. G418-resistant cell lines containing a CAT gene controlled by these elements were established by co-transfection of HD11 cells with pcEPCAT5 (see above) and ptkNeo, a plasmid containing the selectable marker gene for neomycin phosphotransferase. Five of these lines were treated with forskolin and calcium ionophore A23187, and the effect of these drugs on the transcription of the CAT gene was investigated by determining CAT activities. Figure 7 shows five individual, G418-resistant cell lines expressing the integrated CAT gene. As expected, forskolin or calcium ionophore A23187, when separately added to these cells, induced the CAT activity weakly. In contrast, in four of five clones, simultaneous treatment of cells with forskolin and calcium ionophore A23187 resulted in a synergistic activation of CAT activity. Thus, this result suggests that the synergistic lysozyme activation by calcium ionophore A23187 and forskolin occurs at a transcriptional level.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 7. Synergistic effects of calcium ionophore A23187 and forskolin on expression of a lysozyme-CAT construct integrated stably in HD11 cells. Cells of five individual clones containing a CAT gene controlled by the lysozyme promoter and the -6.1-kb enhancer were treated with 5 µM calcium ionophore A23187, 25 µM forskolin, or with the two reagents simultaneously. Following an incubation of 24 h, cells were harvested for preparation of cell extracts. CAT assays of cell extracts were performed as described above. The relative CAT activities were mean values from three experiments. Standard deviations were <20%.

Forskolin and calcium ionophore A23187 induce binding activity of C/EBPß(NF-M) and NF-{kappa}B
The lysozyme promoter and the -6.1-kb lysozyme enhancer contain a NF-{kappa}B site and two C/EBPß-binding sites (D and E), respectively. These sites have been shown to mediate the LPS-activated lysozyme expression in HD11 cells [4 , 21 ]. Our data suggest that the transduction pathways mediated by PKA and Ca2+ contribute, at least in part, to the LPS activation of the lysozyme gene. To determine whether binding of C/EBPß(NF-M) to the -6.1-kb enhancer and NF-{kappa}B to the promoter may be regulated by these pathways, EMSA was performed with element D of the -6.1-kb lysozyme enhancer and NF-{kappa}B site of the promoter using nuclear extracts from HD11 cells treated with forskolin and calcium ionophore A23187 for 2 and 12 h. The C/EBPß(NF-M) binding to element D was stimulated significantly by forskolin (threefold and 8.2-fold) and calcium ionophore A23187 (2.3- and 5.6-fold) after 2 and 12 h of incubation, respectively (Fig. 8A ). Figure 8B shows that binding activity of NF-{kappa}B was not effected by forskolin but moderately induced by calcium ionophore A23187 after a 12-h incubation (~2.6-fold). Treatment of cells with both reagents, however, did not lead to further stimulation of the C/EBPß(NF-M) binding to element D (4.3- and 7.1-fold after 2 and 12 h, respectively) or NF-{kappa}B-binding activity (~2.8-fold after 12 h of stimulation).



View larger version (52K):
[in this window]
[in a new window]
  		Figure 8. Activation of (A) C/EBPß(NF-M)- and (B) NF-{kappa}B-binding activity by forskolin and calcium ionophore A23187. Nuclear extracts were prepared from untreated cells (A, lane 1), from cells treated with ethanol (A, lane 2, and B, lane 1), DMSO (A, lane 6, and B, lane 4), and with both reagents (A, lane 10, and B, lane 7) as controls or from cells treated with 25 µM forskolin (FOR), 5 µM calcium ionophore A23187 (A23187), or with the two reagents simultaneously. EMSA was performed with radiolabeled oligonucleotide D containing a C/EBPß-binding site [21 ] or lys{kappa}B containing the NF-{kappa}B site of the lysozyme promoter [4 ], using 5 µg protein of nuclear extracts in the presence or absence of unlabeled oligonucleotide D or lys{kappa}B (comp). The faster, migrating band, for which unlabeled lys{kappa}B does not compete, represents a nonspecific DNA protein complex.


arrow
DISCUSSION
 
Previously, we have shown that the lysozyme expression in chicken myelomonocytic HD11 cells activated by LPS is regulated by a multi-step process at transcriptional and post-transcriptional levels. This activation occurs very early and extends over 10 h after addition of LPS [20 ]. In this study, our work focused on the role of protein kinases and Ca2+ in the LPS response of the lysozyme expression. The data obtained from this study using various inhibitors of protein kinases and a calcium-release antagonist (TMB-8) indicate that PKA, PKC, and Ca2+ release from intracellular stores are involved in the LPS activation of the lysozyme expression. Although forskolin and calcium ionophore A23187, when added to the cells separately, induced the lysozyme expression weakly to moderately, inhibition of PKA by H-89 or blocking Ca2+ release by TMB-8, however, prevented the increased lysozyme expression by LPS completely. It is interesting that simultaneous treatment of cells with forskolin and calcium ionophore A23187 resulted in a potentiated induction of the lysozyme expression, thus confirming a synergistic cooperation between PKA and Ca2+ in the lysozyme gene activation. Because the synergistic activation by PKA and Ca2+ was observed after 2 h of treatment with forskolin and calcium ionophore A23187, PKA and Ca2+ seem to be required mainly for later activation by LPS. Our results also confirmed the involvement of PKC in the LPS activation of the lysozyme expression in HD11 cells. Activation of the lysozyme expression by TPA occurs late also. Thus, like PKA and Ca2+, PKC seems to be important for the later LPS activation. However, because the LPS-activated lysozyme expression is abolished completely by pre-treatment with H-7, H-89, and TMB-8, we believe that other essential factors in addition to PKA, PKC, and Ca2+ should be required for early stages of LPS activation. Taken together, our results demonstrate that PKA, PKC, and Ca2+ alone are necessary but not sufficient to mediate full induction of lysozyme expression by LPS, although their activators forskolin, TPA, and calcium ionophore A23187 are capable of stimulating the lysozyme expression in HD11 cells to some extent. It is surprising that protein tyrosine phosphorylation seems to be unnecessary for the LPS activation, because herbimycin A failed to inhibit the LPS-activated lysozyme expression, although PTK p53/56lyn is shown to be associated to the LPS receptor CD14, leading to activation of cytokine gene expression [30 ]. In contrast, it stimulated the lysozyme expression. Thus, our result seems to be consistent with the observation that herbimycin A is able to stimulate gene expression, e.g., expression of the 70-kD heat-stress protein [31 ] and the serotonin-reporter gene [32 ], and, furthermore, it can increase cytoplasmic calcium in rat osteoclasts [33 ].

Several transcription factors as target proteins for PKA, PKC, and Ca2+-dependent protein kinases are shown to be involved in the LPS activation, one of which should be the myeloid-specific transcription factor C/EBPß(NF-M) of the leucine zipper family. In HD11 cells, C/EBPß(NF-M), by interacting with the -6.1-kb enhancer, mediates the LPS-activated expression of the lysozyme gene [21 , 22 ]. C/EBPß has been shown to be regulated by phosphorylation mediated by PKA [34 ], PKC [35 ], CaMKII [19 ], and mitogen-activated protein (MAP) kinases [36 ]. Metz and Ziff [34 ] have demonstrated that C/EBPß, located mainly in the cytoplasm, requires phosphorylation to translocate to the nucleus of PC12 cells following forskolin treatment. Our results show that forskolin is capable of increasing the C/EBPß(NF-M)-binding activity to the -6.1-kb enhancer, although inhibition of PKA by H-89 has no effect on the C/EBPß(NF-M) mRNA expression (unpublished results). Because the C/EBPß(NF-M)-binding activity to the -6.1-kb enhancer has shown to be independent of phosphorylation [21 ], it is possible that this increase in binding activity may be a result of a translocation of C/EBPß(NF-M) from the cytoplasm into the nucleus that may be induced by PKA following treatment with forskolin. Several studies have demonstrated that the transactivation potential of C/EBPß for activation of gene expression can be enhanced by phosphorylation within the leucine zipper by calcium/calmodulin-dependent protein kinases [19 ], MAP kinases [36 ], and PKC-dependent protein kinases [35 ]. It is interesting that C/EBPß(NF-M) has been shown to be a repressed transcription factor with a concealed transactivation potential that can be de-repressed by phosphorylation [37 ]. Treatment of HD11 cells with LPS increases the level of C/EBPß(NF-M) protein complex to the -6.1-kb lysozyme enhancer, resulting in activation of the lysozyme expression. This increase was shown to be a result of enhanced transcription of the C/EBPß(NF-M) gene [22 ]. Our results indicate the requirement of PKC for the lysozyme mRNA expression. Also, PKC is shown to be essential for the C/EBPß(NF-M) mRNA expression (unpublished results). Therefore, these data suggest that de novo synthesis of C/EBPß(NF-M) mediates the PKC pathway required for the activation of the lysozyme expression.

It has been demonstrated that Ca2+ interacts synergistically with PKA to induce c-fos transcription via a convergent mechanism involving phosphorylation of the cAMP response element-binding protein (CREB) [38 ]. CREB, a Ca2+-regulated transcription factor, is a substrate for PKA [39 ] as well as for CaMKI and II [40 ]. Phosphorylation of serine133 stimulates the ability of CREB to activate gene expression [39 , 40 ]. Furthermore, phosphorylated CREB binds to the CREB-binding protein (CBP) and acts as a transcriptional co-activator of NF-{kappa}Bp65 [41 ]. The chicken lysozyme-promoter region contains no CRE but does contain a binding site for NF-{kappa}B/Rel. NF-{kappa}B/Rel-containing protein complexes binding to this element have been shown to be activated by LPS in HD11 cells [4 ]. Zhong et al. [42 ] have demonstrated that the binding of NF-{kappa}Bp65 to CREB via CBP is essential for NF-{kappa}B-enhanced transcriptional activity that is also dependent on phosphorylation of NF-{kappa}Bp65 induced by PKA. Also, Muroi and Suzuki [10 ] have shown the involvement of PKA in NF-{kappa}B/Rel activation in J774 cells. Thus, the synergistic effect of PKA and Ca2+ on the lysozyme expression in HD11 cells seems to be because of the involvement of PKA and Ca2+ in phosphorylation of C/EBPß(NF-M), CREB, and NF-{kappa}B/Rel.

PU.1, originally identified as the proto-oncogene Spi-1 [43 ], is shown to be implicated in LPS-inducible gene expression (for review, see [44 ]). Transciptional activity of PU.1 is enhanced by phosphorylation at serine148 following LPS stimulation [45 ]. By contrast, its binding activity is not stimulated in LPS-treated RAW264.7 [45 ] and HD11 cells [46 ]. The -2.7-kb lysozyme enhancer containing a PU.1-binding site in addition to a C/EBP site [46 , 47 ] is also able to confer LPS responsiveness in HD11 cells ([46 ] and unpublished results), suggesting an involvement of PU.1 in the LPS-activated lysozyme expression.

In summary, we have demonstrated that PKA, PKC, and Ca2+ regulate the LPS-activated lysozyme expression. It is tempting to speculate that C/EBPß(NF-M), CREB, NF-{kappa}B/Rel, and PU.1 may be the main targets for signal-transduction pathways mediated by PKA, PKC, and Ca2+ in LPS-activated myelomonocytic HD11 cells.


arrow
ACKNOWLEDGEMENTS
 
This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (L. P.). We thank S. Trampenau and K. Zimmermann for skillful technical assistance.

Received August 14, 2000; revised December 4, 2000; accepted December 5, 2000.


arrow
REFERENCES
 
    1
  1. Palmiter, R. D. (1972) Regulation of protein synthesis in chick oviduct J. Biol. Chem. 247,6450-6461[Abstract/Free Full Text]
    2. 2
  2. Hecht, A., Berkenstamm, A., Strömstedt, P-E., Gustafsson, J-A., Sippel, A. E. (1988) A progesterone responsive element maps to the far upstream steroid dependent DNase hypersensitive site of chicken lysozyme chromatin EMBO J 7,2063-2073[Medline]
    3. 3
  3. Hauser, H., Graf, T., Beug, H., Greiser-Wilke, I., Lindenmaier, W., Grez, M., Land, H., Giesecke, K., Schütz, G. (1981) Structure of the lysozyme gene and expression in the oviduct and macrophages Gallo, R. C. Graf, T. Mannweiler, K. Winkler, K. eds. Hematology and Blood Transfusion vol. 26,175-178 Springer Verlag Berlin.
    4. 4
  4. Phi-van, L. (1996) Transcriptional activation of the chicken lysozyme gene by NF-{kappa}Bp65(relA) and c-rel, but not by NF-{kappa}Bp50 Biochem. J. 313,39-44
    5. 5
  5. Cross, M., Mangelsdorf, I., Wedel, A., Renkawitz, R. (1988) Mouse lysozyme M gene: isolation, characterization, and expression studies Proc. Natl. Acad. Sci. USA 85,6232-6236[Abstract/Free Full Text]
    6. 6
  6. Hamilton, T. A., Adams, D. O. (1987) Molecular mechanism of signal transduction in macrophages Immunol. Today 8,151-158
    7. 7
  7. Novotney, M., Chang, Z. L., Uchiyama, H., Suzuki, T. (1991) Protein kinase C in tumoricidal activation of mouse macrophage cell lines Biochemistry 30,5597-5604[Medline]
    8. 8
  8. Shapira, L., Takashiba, S., Champagne, C., Amar, S., Van, D. T. (1994) Involvement of protein kinase C and protein tyrosine kinase in lipopolysaccharide-induced TNF-alpha and IL-1 beta production by human monocytes J. Immunol. 153,1818-1824[Abstract]
    9. 9
  9. Onodera, S. (1991) Elevation of intracellular cyclic adenosine 3', 5'-monophosphate levels in macrophages activated with lipopolysaccharide for tumor cell killing Chem. Pharm. Bull. (Tokyo) 39,991-993[Medline]
    10. 10
  10. Muroi, M., Suzuki, T. (1993) Role of protein kinase A in LPS-induced activation of NF-kappa B proteins of a mouse macrophage-like cell line, J774 Cell Signal 5,289-298[Medline]
    11. 11
  11. Fujihara, M., Muroi, M., Muroi, Y., Suzuki, T. (1993) Mechanism of lipopolysaccharide-triggered junB activation in a mouse macrophage-like cell line (J774) J. Biol. Chem. 268,14898-14905[Abstract/Free Full Text]
    12. 12
  12. Weinstein, S. L., June, C. H., DeFranco, A. L. (1993) Lipopolysaccharide-induced protein tyrosine phosphorylation in human macrophages is mediated by CD14 J. Immunol. 151,3829-3838[Abstract]
    13. 13
  13. Stefanova, I., Corcoran, M. L., Horak, E. M., Wahl, L. M., Bolen, J. B., Horak, I. D. (1993) Lipopolysaccharide induces activation of CD14-associated protein tyrosine kinase p53/56lyn J. Biol. Chem. 268,20725-20728[Abstract/Free Full Text]
    14. 14
  14. Prpic, V., Weiel, J. E., Somers, S. D., DiGuiseppi, J., Gonias, S. L., Pizzo, S. V., Hamilton, T. A., Herman, B., Adams, D. O. (1987) Effects of bacterial lipopolysaccharide on the hydrolysis of phosphatidylinositol-4,5-bisphosphate in murine peritoneal macrophages J. Immunol. 139,526-533[Abstract]
    15. 15
  15. Letari, O., Nicosia, S., Chiavaroli, C., Vacher, P., Schlegel, W. (1991) Activation by bacterial lipopolysaccharide causes changes in the cytosolic free calcium concentration in single peritoneal macrophages J. Immunol. 147,980-983[Abstract]
    16. 16
  16. Bennett, M. K., Erondu, N. E., Kennedy, M. B. (1983) Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain J. Biol. Chem. 258,12735-12744[Abstract/Free Full Text]
    17. 17
  17. Shenolikar, S., Lickteig, R., Hardie, D. G., Soderling, T. R., Hanley, R. M., Kelly, P. T. (1986) Calmodulin-dependent multifunctional protein kinase Evidence for isoenzyme forms in mammalian tissues. Eur. J. Biochem. 161,739-747
    18. 18
  18. Bulleit, R. F., Bennett, M. K., Molloy, S. S., Hurley, J. B., Kennedy, M. B. (1988) Conserved and variable regions in the subunits of brain type II Ca2+/calmodulin-dependent protein kinase Neuron 1,63-72[Medline]
    19. 19
  19. Wegner, M., Cao, Z., Rosenfeld, M. G. (1992) Calcium-regulated phosphorylation within the leucine zipper of C/EBPß Science 256,370-373[Abstract/Free Full Text]
    20. 20
  20. Goethe, R., Phi-van, L. (1998) Posttranscriptional lipopolysaccharide regulation of the lysozyme gene at processing of the primary transcript in myelomonocytic HD11 cells J. Immunol. 160,4970-4978[Abstract/Free Full Text]
    21. 21
  21. Goethe, R., Phi van, L. (1994) The far upstream chicken lysozyme enhancer at -6.1 kilobase, by interacting with NF-M, mediates lipopolysaccharide-induced expression of the chicken lysozyme gene in chicken myelomonocytic cells J. Biol. Chem. 269,31302-31309[Abstract/Free Full Text]
    22. 22
  22. Goethe, R., Phi-van, L. (1997) Evidence for an enhanced transcription-dependent de novo synthesis of C/EBPß in the LPS activation of the chicken lysozyme gene J. Leukoc. Biol. 61,367-374[Abstract]
    23. 23
  23. Beug, H., von Kirchbach, A., Döderlein, G., Conscience, J-F., Graf, T. (1979) Chicken hematopoietic cells transformed by seven strains of defective avian leukemia viruses display three distinct phenotypes of differentiation Cell 18,375-390[Medline]
    24. 24
  24. Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual 2nd ed. ,7.40-7.42 Cold Spring Harbor Laboratory Cold Spring Harbor, NY.
    25. 25
  25. Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis J. Mol. Biol. 98,503-517[Medline]
    26. 26
  26. Sippel, A. E., Land, H., Lindenmaier, W., Nguyen-huu, M. C., Wurtz, T., Timmis, K. N., Giesecke, K., Schütz, G. (1978) Cloning of chicken lysozyme structural gene sequences synthesized in vitro Nucleic Acids Res 5,3275-3294[Abstract/Free Full Text]
    27. 27
  27. Dugaiczyk, A., Haron, J. A., Stone, E. M., Dennison, O. E., Rothblum, K. N., Schwartz, R. (1983) Cloning and sequencing of a deoxyribonucleic acid copy of glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acid isolated from chicken muscle Biochemistry 22,1605-1613[Medline]
    28. 28
  28. Phi-van, L., von Kries, J. P., Ostertag, W., Strätling, W. H. (1990) The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes Mol. Cell. Biol. 10,2302-2307[Abstract/Free Full Text]
    29. 29
  29. Schreiber, E., Matthias, P., Müller, M. M., Schaffner, W. (1989) Rapid detection of octamer binding proteins with "mini-extracts", prepared from a small number of cells Nucleic Acids Res 17,6419[Free Full Text]
    30. 30
  30. Stefanova, I., Corcoran, M. L., Horak, E. M., Wahl, L. M., Bolen, J. B., Horak, I. D. (1993) Lipopolysaccharide induces activation of CD14-associated protein tyrosine kinase p53/56lyn J. Biol. Chem. 268,20725-20728
    31. 31
  31. Morris, S. D., Cumming, D. V. E., Latchman, D. S., Yellon, D. M. (1996) Specific induction of the 70-kD heat stress proteins by the tyrosine kinase inhibitor herbimycin-A protects rat neonatal cardiomyocytes J. Clin. Invest. 97,706-712[Medline]
    32. 32
  32. Prasad, P. D., Torres-Zamorano, V., Kekuda, R., Leibach, F. H., Ganapathy, V. (1997) Functional link between tyrosine phosphorylation and human serotonin transporter gene expression Eur. J. Pharmacol. 325,85-92[Medline]
    33. 33
  33. Kajiya, H., Okabe, K., Okamoto, F., Tsuzuki, T., Soeda, H. (2000) Protein tyrosine kinase inhibitors increase cytosolic calcium and inhibit actin organization as resorbing activity in rat osteoclasts J. Cell. Physiol. 183,83-90[Medline]
    34. 34
  34. Metz, R., Ziff, E. (1991) cAMP stimulates the C/EBP-related transcription factor rNFIL-6 to translocate to the nucleus and induce c-fos transcription Genes Dev 5,1754-1766[Abstract/Free Full Text]
    35. 35
  35. Trautwein, C., Caelles, C., van der Geer, P., Hunter, T., Karin, M., Chojkier, M. (1993) Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain Nature 364,544-547[Medline]
    36. 36
  36. Nakajima, T., Kinoshita, S., Sasagawa, T., Sasaki, K., Naruto, M., Kishomoto, T., Akira, S. (1993) Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6 Proc. Natl. Acad. Sci. USA 90,2207-2211[Abstract/Free Full Text]
    37. 37
  37. Kowenz-Leutz, E., Twamley, G., Ansieau, S., Leutz, A. (1994) Novel mechanism of C/EBPß(NF-M) transcriptional control: activation through derepression Genes Dev 8,2781-2791[Abstract/Free Full Text]
    38. 38
  38. Sheng, M., McFadden, G., Greenberg, M. E. (1990) Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB Neuron 4,571-582[Medline]
    39. 39
  39. Gonzalez, G. A., Montminy, M. R. (1989) Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133 Cell 59,675-680[Medline]
    40. 40
  40. Sheng, M., Thompson, M. A., Greenberg, M. E. (1991) CREB: a Ca2+-regulated transcription factor phosphorylated by calmodulin-dependent kinases Science 252,1427-1430[Abstract/Free Full Text]
    41. 41
  41. Gerritsen, M. E., Williams, A. J., Neish, A. S., Moore, S., Shi, Y., Collins, T. (1997) CREB-binding protein/p300 are transcriptional coactivators of p65 Proc. Natl. Acad. Sci. USA 94,2927-2932[Abstract/Free Full Text]
    42. 42
  42. Zhong, H., Voll, R. E., Ghosh, S. (1998) Phosphorylation of NF-kappaBp65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300 Mol. Cell 1,661-671[Medline]
    43. 43
  43. Moreau-Gachelin, F., Ray, D., Mattei, M. G., Tambourin, P., Tavitian, A. (1989) The putative oncogene Spi-1: murine chromosomal localization and transcriptional activation in murine acute erythroleukemias Oncogene 4,1449-1456[Medline]
    44. 44
  44. Sweet, M. J., Hume, D. A. (1996) Endotoxin signal transduction in macrophages J. Leukoc. Biol. 60,8-26[Abstract]
    45. 45
  45. Lodie, T. A., Reiner, M., Coniglio, S., Viglianti, G., Fenton, M. J. (1998) Both PU.1 and nuclear factor-{kappa}B mediate lipopolysaccharide-induced HIV-1 long terminal repeat transcription in macrophages J. Immunol. 161,268-276[Abstract/Free Full Text]
    46. 46
  46. Faust, N., Bonifer, C., Sippel, A. E. (1999) Differential activity of the -2.7 kb chicken lysozyme enhancer in macrophages of different ontogenic origins is regulated by C/EBP and PU.1 transcription factors DNA Cell Biol. 18,631-642[Medline]
    47. 47
  47. Ahne, B., Strätling, W. H. (1994) Characterization of a myeloid-specific enhancer of the chicken lysozyme gene. Major role for an Ets transcription factor-binding site J. Biol. Chem. 269,17794-17801[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. R. Mead, T. R. Hughes, S. A. Irvine, N. N. Singh, and D. P. Ramji
Interferon-gamma Stimulates the Expression of the Inducible cAMP Early Repressor in Macrophages through the Activation of Casein Kinase 2. A POTENTIALLY NOVEL PATHWAY FOR INTERFERON-gamma -MEDIATED INHIBITION OF GENE TRANSCRIPTION
J. Biol. Chem., May 9, 2003; 278(20): 17741 - 17751.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
L. Crepaldi, L. Silveri, F. Calzetti, C. Pinardi, and M. A. Cassatella
Molecular basis of the synergistic production of IL-1 receptor antagonist by human neutrophils stimulated with IL-4 and IL-10
Int. Immunol., October 1, 2002; 14(10): 1145 - 1153.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Masuda, Y. Yoshikai, K. Aiba, and T. Matsuguchi
Th2 Cytokine Production from Mast Cells Is Directly Induced by Lipopolysaccharide and Distinctly Regulated by c-Jun N-Terminal Kinase and p38 Pathways
J. Immunol., October 1, 2002; 169(7): 3801 - 3810.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. B. Fessler, K. C. Malcolm, M. W. Duncan, and G. S. Worthen
A Genomic and Proteomic Analysis of Activation of the Human Neutrophil by Lipopolysaccharide and Its Mediation by p38 Mitogen-activated Protein Kinase
J. Biol. Chem., August 23, 2002; 277(35): 31291 - 31302.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Zimmermann, K. Ahrens, S. Matthes, J.-M. Buerstedde, W. H. Stratling, and L. Phi-van
Targeted Disruption of the GAS41 Gene Encoding a Putative Transcription Factor Indicates That GAS41 Is Essential for Cell Viability
J. Biol. Chem., May 17, 2002; 277(21): 18626 - 18631.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Regenhard, P.
Right arrow Articles by Phi-van, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Regenhard, P.
Right arrow Articles by Phi-van, L.