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The Journal of Immunology, 1998, 160: 4970-4978.
Copyright © 1998 by The American Association of Immunologists

Posttranscriptional Lipopolysaccharide Regulation of the Lysozyme Gene at Processing of the Primary Transcript in Myelomonocytic HD11 Cells1

Ralph Goethe2 and Loc Phi-van3

Institut für Tierzucht und Tierverhalten (FAL), Celle, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lysozyme is increasingly expressed in macrophages in inflammatory response to bacterial LPS. In this study, we investigated the mechanisms that control expression of the lysozyme gene in myelomonocytic HD11 cells activated by LPS. Nuclear run-on transcription assays showed that LPS caused a 15-fold increase in the transcription rate of the lysozyme gene. However, Northern analyses with lysozyme cDNA and intron sequences revealed that the LPS-induced increase in nuclear lysozyme transcripts greatly exceeded the increase in transcription rate. Furthermore, nuclear lysozyme transcripts in untreated cells with a t1/2 of <10 min were more unstable than those accumulated in LPS-activated cells. We suggested, therefore, that the increased lysozyme expression following LPS treatment was largely due to a nuclear stabilization of the primary transcript. Interestingly, the increase in stability of the lysozyme primary transcript was accompanied by changes in nuclear processing including an increase in poly(A) tail length, which gradually shortened after entering the cytoplasm. The long lysozyme poly(A) tail, however, did not result in any increase in polysomal recruitment for translation or in stability of the cytoplasmic lysozyme mRNA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lysozyme is an antibacterial protein produced mainly in the chicken oviduct and in macrophages. In the tubular gland cells of the oviduct, the expression of lysozyme is dependent on steroid hormones (1). During the differentiation of macrophages, the lysozyme gene is continuously activated from a low level of expression in precursors such as myeloblasts to a high level of expression in mature macrophages. The lysozyme gene thus is a well-characterized marker for the myeloid lineage (2, 3, 4, 5). Bacterial endotoxins such as LPS, in the acute phase of bacterial infection, activate immunologic and inflammatory responses, particularly in cells of immunologic systems including B and T lymphocytes and macrophages (6). Macrophages, in response to LPS, increasingly express a variety of cytokines and antibacterial proteins including lysozyme (7, 8). We have previously shown that the LPS activation of the lysozyme gene in myelomonocytic cells was transcriptionally regulated by the myeloid-specific transcription factor C/EBPß4 in interaction with the far upstream -6.1-kb lysozyme enhancer (9, 10).

In the present study, we have employed the chicken myelomonocytic cell line HD11 (11) to investigate the mechanisms involved in the regulation of LPS-activated expression of the lysozyme gene. We show that the lysozyme gene in myelomonocytic HD11 cells was regulated at both the transcriptional and posttranscriptional levels in inflammatory response to LPS. Interestingly, the posttranscriptional regulation mechanism is a multistep process that involves changes in the transcript stability leading to a nuclear accumulation of unspliced and incompletely spliced transcripts, changes in poly(A) tail length, and subsequent cytoplasmic poly(A) shortening of the completely spliced lysozyme RNA transcripts. The altered processing of the lysozyme primary transcript involving the increase in poly(A) tail length may be associated with an enhanced nuclear lysozyme RNA stability.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant DNA plasmids

The plasmid pcEPCAT5 contains the CAT gene under transcriptional control of the lysozyme promoter and the -6.1-kb lysozyme enhancer (9). The plasmid ptkNeo contains the neomycin resistance gene controlled by the thymidine kinase promoter (-109/+51) from the herpes simplex virus (12). Plasmids pBSlysI1 and pBSlysI2 were constructed by insertion of a HindIII fragment containing lysozyme intron 1 sequence and an XbaI fragment containing intron 2 sequence into the HindIII site or XbaI site of the pBluescript SK+ (Stratagene, Heidelberg, Germany), respectively. These DNA fragments resulted from subcloned DNA probes of {lambda}lys30 (13).

Cell lines and cell culture

HD11 cells of an established chicken myelomonocytic cell line, transformed by the v-myc encoding retrovirus MC29 (11), were maintained in Iscove’s modified Dulbecco’s medium, supplemented with 8% FCS, 2% chicken serum, 100 U/ml penicillin, and 100 µg/ml streptomycin, at 37°C in humidified 95% air, 5% C02. The clone pc5 contains one copy of the CAT gene controlled by the lysozyme promoter and the -6.1-kb lysozyme enhancer (9). This clone was established from HD11 cells by stable calcium phosphate cotransfection performed as described previously (12) using 20 µg pcEPCAT5 and 2 µg ptkNeo followed by the selection for resistance to G418 (500 µg/ml). The copy number of the integrated CAT gene was determined by quantitative Southern blotting of genomic DNA cut with HindIII and SacI and hybridization to a 539-bp BamHI-HindIII fragment containing the -6.1-kb enhancer (14) as described previously (15).

CAT assay

pc5 cells were grown to a density of 5 x 106 cells per 8.5-cm plate. For stimulation with LPS, cells were maintained in Iscove’s modified Dulbecco’s medium with 0.5% FCS for 48 h before they were activated with 5 µg/ml LPS from Salmonella typhimurium (Sigma, Deisenhofen, Germany) for further 24 h. For extract preparation, cells were washed twice in PBS, scraped by a rubber policeman in TEN containing 40 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 150 mM NaCl, and pelleted in an Eppendorf centrifuge for 10 s. The cell pellet was suspended in 0.25 M Tris-HCl (pH 7.8), lysed by three cycles of freeze-thawing, and cleared by centrifugation at 10,000 x g for 10 min. Protein concentrations of cell extracts were determined by the method of Bradfort (Bio-Rad, München, Germany). CAT assays were performed with 50 µg protein of each cell extract as previously described (12).

RNA isolation

Poly(A)+ RNA preparation was performed according to the method described by Rahmdorf et al. (16). Briefly, 1 x 107 cells were washed twice with PBS, lysed in SSTE containing 10 mM NaCl, 0.5% (w/v) SDS, 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, and homogenized with a Janke & Kunkel Ultra-Turrax (Staufen, Germany). Following an incubation of the lysate with 300 µg/ml proteinase K for 30 min at 37°C, poly(A)+ RNA was prepared by binding to oligo(dT) cellulose (Boehringer, Mannheim, Germany) in the presence of 0.5 M NaCl. The bound poly(A)+ RNA was washed four times with a solution containing 10 mM Tris-HCl (pH 7.5), 0.3 M NaCl, 5 mM EDTA, and 0.1% (w/v) SDS, and then poly(A)+ RNA was eluated with deionized water.

To isolate nuclear and cytoplasmic poly(A)+ RNAs, cells were washed twice in PBS and lysed in a cell lysis buffer containing 10 mM Tris (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.5% (v/v) Nonidet P-40, 25 U/ml RNasin (Promega, Heidelberg, Germany). Following incubation at 4°C for 5 min, nuclei were separated from the cytoplasm by centrifugation of the cell lysate at 10,000 x g for 90 s, then washed twice with cell lysis buffer. Cytoplasmic and nuclear poly(A)+ RNAs were then isolated by binding with oligo(dT) cellulose as described above.

Total cellular RNA was isolated by using a RNeasy kit from Qiagen (Hilden, Germany). To eliminate traces of genomic DNA, RNA samples (50 µg) were incubated with 15 U of RNase-free DNase in the presence of 15 U of RNasin (Promega) in 100 µl of a buffer containing 10 mM Tris-HCl (pH 8.0), 50 mM KCl, and 1.5 mM MgCl2 at 37°C for 30 min. The samples were extracted twice with Roti-phenol (Roth, Karlsruhe, Germany) and precipitated with ethanol, and RNA pellets were dissolved in 100 µl of deionized water.

Northern analysis

For Northern analysis, RNAs were denatured by 0.5 M glyoxal and 27% (v/v) DMSO, electrophoretically fractionated through 1.4 to 1.8% agarose gels containing 10 mM NaH2PO4 (pH 6.9), Southern-transferred to nylon membranes (Appligene, Heidelberg, Germany) (17), and immobilized by baking at 80°C for 30 min. Plasmid DNA containing the chicken lysozyme cDNA (18), intron 1 (pBSlysI1), intron 2 (pBSlysI2), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (19) was labeled with [{alpha}-32P]dCTP (3000 Ci/mmol) (DuPont NEN, Bad Homburg, Germany) by using a nick translation kit from Life Technologies (Eggenstein, Germany). The blots were prehybridized, hybridized, washed according to the method described by Church and Gilbert (20), and exposed to x-ray films with intensifying screens at -80°C. To quantify hybridization signals, autoradiograms were scanned with an imaging densitometer from Bio-Rad. Lysozyme mRNA levels were normalized for GAPDH on the same blot.

Reverse transcription-polymerase chain reaction

RT-PCR of total RNA was performed by using a Titan One tube RT-PCR kit from Boehringer Mannheim. Briefly, the cDNA reaction was conducted with 0.5 or 1 µg of total RNA at 50°C for 30 min followed by a PCR with 43 cycles (each 1 min at 94°C and 1 min at 60°C) using the primer pair consisting of P1 (5'-ACGACACTGGCAACATGAGG-3') and P2 (5'-ATTCCAACATCACGCAGACC-3'). PCR products were fractionated on 2% agarose gels at 75 V for 3 h, stained with ethidium bromide, and quantified using an ethidium bromide gel documentation system from Bio-Rad.

Polyadenylation analysis with RNase H

To remove poly(A) tails, 4-µg poly(A)+ RNA samples were hybridized to 0.6 µg of oligo(dT)12–18 (Pharmacia, Freiburg, Germany), and digested with RNase H as described by Kleene et al. (21). The samples were extracted twice with Roti-Phenol (Roth) and precipitated with ethanol. RNA pellets were dissolved in 4 µl of deionized water and then subjected to Northern blotting. Blots were sequentially hybridized to nick-translated lysozyme cDNA and GAPDH cDNA as described above.

Run-on transcription assay

Nuclear run-on transcription assays were performed according to the procedure described by Greenberg and Ziff (22). Briefly, 1.5 x 107 cells were suspended in 1 ml of a Nonidet P40 lysis buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.5% (v/v) Nonidet P40 and incubated at 4°C for 5 min. Nuclei were harvested by centrifugation at 500 x g for 5 min at 4°C and then washed once with 2 ml of the Nonidet P40 lysis buffer. After centrifugation, nuclei were suspended in 100 µl of a storage buffer containing 50 mM Tris-HCl (pH 8.3), 5 mM MgCl2, 0.1 mM EDTA, and 40% (v/v) glycerol and frozen at -80°C. For run-on transcription, nuclei were thawed, immediately mixed with an equal volume of a run-on transcription buffer containing 10 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl, 0.5 mM each ATP, CTP, GTP, and 100 µCi [{alpha}-32P]UTP (800 Ci/mmol) (DuPont NEN), and incubated for 30 min at 30°C. RNA was isolated by treatment with 200 U of RNase-free DNase for 15 min at 30°C, followed by incubation with 800 µg/ml proteinase K for 45 min at 37°C, extraction with phenol-chloroform-isoamyl alcohol, and two cycles of precipitation, first with isopropanol and then with ethanol. For binding on nylon membranes, isolated DNA fragments containing the chicken lysozyme cDNA (18), the CAT gene (23), the c-rel cDNA (24), the GAPDH cDNA (19), or chicken genomic DNA were denatured with 0.2 N NaOH for 10 min at 37°C and slot-blotted in the presence of 0.125 x SSC onto nylon membranes (Appligene) using a Hybri-Slot Manifold (Life Technologies). For analysis of the synthesized RNAs, the baked filters were hybridized to the purified labeled RNAs according to the method developed by Church and Gilbert (20).

Polysome preparation

Polysome preparation was conducted as described previously (25) with some modifications. Briefly, 1 x 108 cells were washed twice with PBS, harvested, and lysed in 3 ml of a polysome buffer (PB) containing 20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 300 mM KCl, 4 µg/ml polyvinyl sulfate, 0.5% (v/v) Nonidet P40, 2 mM DTT, 25 U/ml RNasin (Promega), and 10 µg/ml cycloheximide. Following centrifugation of the homogenate at 12,000 x g for 15 min, the postmitochondrial supernatant was layered over a 6-ml cushion of 35% (w/v) sucrose in PB, and the polysomes were collected by centrifugation at 150,000 x g for 2 h at 4°C in a Beckman SW40 Ti rotor (Munich, Germany). Poly(A)+ RNAs from the polysome fraction and the postpolysomal supernatant were isolated and then subjected to Northern blotting as described above.

Immunoblotting

Cells were grown to a density of 107 cells per 8.5-cm plate and maintained in Iscove’s modified Dulbecco’s medium with 0.5% FCS for 48 h. Following washing twice with PBS, cells were incubated in 3 ml of the same medium with or without LPS for the indicated times. At various time points, cell culture medium was transferred into a new tube, and cells were washed twice with PBS, scraped in 0.8 ml TEN, harvested by centrifugation in an Eppendorf centrifuge for 15 s, and lysed in 250 µl of 0.25 M Tris-HCl (pH 7.8) by four freeze-thaw cycles. Cell lysates were cleared by centrifugation at 10,000 x g for 10 min.

For immunoblotting, 2 µl of each cell lysate and 10 µl of each cell culture medium were separated on 12% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes. Following blocking in NTT (150 mM NaCl, 30 mM Tris-HCl (pH 8.0), and 0.05% Tween 20) containing 0.5% gelatin for 30 min at room temperature, the blots were incubated with a rabbit anti-lysozyme antiserum at a 1:1000 dilution in NTT for 1 h at room temperature. After three 5-min washes in NTT, the blots were incubated with an alkaline phosphatase-conjugated anti-rabbit Ab (Dianova, Hamburg, Germany) diluted 1:5000 in NTT. Following washing twice in NTT, bound alkaline phosphatase was visualized by incubating the blots with 100 µg/ml nitro blue tetrazolium, 50 µg/ml 5-bromo-4-chloro-3indolylphosphate in 100 mM Tris-HCl (pH 9.5), 50 mM NaCl, and 5 mM MgCl2.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPS induction of an accumulation of lysozyme pre-mRNAs in myelomonocytic HD11 cells

Treatment with LPS caused increased expression of various genes in macrophages. Therefore, we examined the effect of LPS on expression of the lysozyme gene in myelomonocytic HD11 cells. Cells were cultured in the presence of LPS from S. typhimurium at 5 µg/ml for 10, 20, 30, and 60 min, and the levels of accumulated lysozyme poly(A)+ RNA were determined by Northern analysis using the full-length lysozyme cDNA (18). Figure 1Go shows that in contrast to control HD11 cells that expressed weakly the lysozyme gene, HD11 cells activated by LPS exhibited a marked increase in the level of lysozyme RNA transcripts (33- and 255-fold after 30 and 60 min of LPS treatment, respectively). In addition to the mature lysozyme mRNA, three lysozyme RNA transcripts were seen, the largest transcript at 3.9 kb appearing as early as 20 min after addition of LPS; the other two, at 2.1 and 0.8 kb, were subsequently observed within 30 and 60 min of LPS stimulation, respectively. In contrast, the mature lysozyme mRNA appeared to be constant during the incubation time periods. The results demonstrated that LPS stimulated an accumulation of lysozyme premRNAs in HD11 cells.



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  		FIGURE 1. LPS stimulates the accumulation of lysozyme pre-mRNAs in HD11 cells. Poly(A)+ RNAs isolated from cells activated with LPS for the indicated time periods were electrophoretically fractionated on a 1.4% agarose gel, Southern blotted, and sequentially hybridized to 32P-labeled lysozyme cDNA and GAPDH cDNA; the blot was autoradiographed for 3 days and 1 day, respectively. RNA sizes were estimated using RNA standards from Life Technologies. The long arrow indicates the position of the mature lysozyme mRNA.

 
LPS stimulates the transcription of the lysozyme gene

We have previously shown that LPS activated expression of a transiently transfected CAT gene controlled by the lysozyme promoter and the -6.1-kb lysozyme enhancer in HD11 cells (9). To investigate the transcriptional control of the lysozyme gene expression, the rate of its transcription in untreated and in LPS-activated HD11 cells and pc5 cells was determined in a run-on transcription assay. pc5 of the HD11 cell line harbors a stably integrated CAT gene controlled by the lysozyme promoter and the -6.1-kb lysozyme enhancer. Nuclei isolated from untreated cells and from cells activated with LPS for 30 min, 1 h, and 5 h were incubated for 30 min with [{alpha}-32P]UTP to elongate nascent RNA transcripts. The 32P-labeled RNA was isolated and then hybridized to immobilized DNA fragments specific to the lysozyme, c-rel, and GAPDH gene. The isolated 32P-labeled RNA from pc5 was hybridized to the CAT gene. Figure 2Go shows that the basal transcription of the lysozyme gene in untreated HD11 cells was low, but markedly increased ~15-fold after exposure to LPS for 30 min; this increase remained nearly constant during the indicated time periods. The c-rel gene was thought to be stimulated by LPS. Here, the rate of transcription determined under the same conditions was clearly increased (6-fold) after stimulation with LPS. The transcription of the GAPDH gene, on the other hand, was not influenced by LPS, consistent with the steady state levels of GAPDH mRNA in HD11 cells. pc5 exhibited a 17-fold increased CAT activity in response to LPS (Fig. 3Go), and this increase was clearly due to a 15-fold enhanced rate of transcription of the stably integrated CAT gene and thus consistent with our previous results obtained from transient transfection experiments (9). Taken together, these data show that the lysozyme gene was transcriptionally activated by LPS.



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  		FIGURE 2. LPS stimulates the rate of lysozyme transcription. Nuclei were isolated from untreated cells (-) or from cells activated with LPS for 30 min, 1 h, and 5 h and incubated in a run-on transcription assay with [{alpha}-32P]UTP for 30 min to elongate RNA transcripts as described in Materials and Methods. RNAs were isolated, and equal amounts of radiolaleled RNAs were hybridized to cDNA fragments specific to lysozyme, c-rel, GAPDH, and CAT and separated to chicken genomic DNA immobilized by slot blotting on nylon membranes.

 


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  		FIGURE 3. LPS stimulates expression of a stably integrated CAT gene controlled by the lysozyme promoter and the -6.1-kb lysozyme enhancer in pc5 cells. CAT assays were performed as described in Materials and Methods.

 
Evidence for an increase in lysozyme transcript stability in LPS-activated cells

Using this run-on transcription assay under the same conditions, we have previously shown that LPS induced an 3.5-fold increase in C/EBPß transcription rate that reflected the 3.2-fold increase in steady state level of C/EBPß mRNA with a t1/2 of 1 h (10). Thus, the run-on transcription used in our experiments reflected in vivo gene transcription. However, the 15-fold increased lysozyme gene transcription induced by LPS cannot fully account for the LPS-induced lysozyme gene expression. This induction exhibited a 33-fold and a 255-fold increase over the basal steady state level of lysozyme mRNA after 30 and 60 min of LPS treatment, respectively (Fig. 1Go). In fact, the LPS-induced increase in lysozyme transcripts exceeds the increase in transcription rate even more if the levels of LPS-induced pre-mRNAs are compared directly with those of newly synthesized lysozyme pre-mRNAs in untreated cells during the same time period. Since the lysozyme mRNA was stable with a t1/2 of ~9 h (see below), and the discrepancy between the LPS-induced increases in the lysozyme gene transcription rate and in the levels of lysozyme transcripts was already observed early within 60 min of LPS treatment, we suggested a nuclear co-/posttranscriptional process affecting the lysozyme transcript stability.

To evaluate this suggestion, we employed RT-PCR to estimate the half-life of nuclear lysozyme transcripts in untreated and in LPS-activated HD11 cells after inhibiting transcription with actinomycin D. First-strand lysozyme cDNA, synthesized by reverse transcription using primer P2, antisense to the 5' end of lysozyme intron 1, was derived only from lysozyme intron 1-containing RNA, but not from mRNA. PCR performed with this cDNA and two primers, P1, sense to the 5' end of exon 1, and P2, should yield a diagnostic 248-bp DNA fragment for nuclear lysozyme transcripts. In contrast to the Northern analysis data (Fig. 1Go), nuclear lysozyme transcripts were detected by RT-PCR not only in LPS-activated cells, but also in control untreated cells. The levels of transcripts in untreated cells, however, decreased rapidly in the presence of actinomycin D, in contrast to those detected in LPS-activated cells (Fig. 4Go). A densitometric scanning of the results revealed a t1/2 of <10 min for nuclear lysozyme transcripts in untreated cells and a t1/2 of ~30 min for those in LPS-activated cells. These results indicated that LPS alters the rate of degradation of lysozyme transcripts. Thus, not only the increase in lysozyme transcription, but also the nuclear stabilization of LPS-induced lysozyme primary transcript accounted for the large accumulation of lysozyme mRNA in LPS-activated cells.



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  		FIGURE 4. Analysis of nuclear lysozyme transcript stability. Untreated cells and cells activated with LPS for 30 min were treated with actinomycin D (Act. D) for 10, 20, 30, and 60 min. Total RNAs were isolated and treated with RNase-free DNase to eliminate genomic DNA. RT-PCR was performed with 1 µg total RNA from untreated cells or 0.5 µg RNA from LPS-activated cells. PCR products, resolved on a 2% agarose gel, were stained with ethidium bromide, and the intensity of the 248-bp band was quantified by densitometric scanning. DNA marker fragments were a 100-bp DNA ladder.

 
Delayed, sequential splicing of the lysozyme primary transcript in LPS-activated HD11 cells

The chicken lysozyme gene contains three intron sequences (1, 2, 3) of about 1270, 1810, and 79 bp, respectively (see Refs. 3, 13, and 26, and Fig. 6Go). To determine whether the three larger lysozyme transcripts at 3.9, 2.1, and 0.8 kb were unspliced or partially spliced lysozyme RNA, Northern blots of poly(A)+ RNAs from LPS-activated cells were hybridized to lysozyme cDNA and intron sequences of the lysozyme gene. As shown in Figures 5Go and 6, hybridization to intron 1 revealed two transcripts at 3.9 and 2.1 kb, whereas hybridization to intron 2 showed only the largest transcript at 3.9 kb. This 3.9-kb band should represent the primary transcript because it had a size expected from the published sequence of the lysozyme gene (13, 26). Fractionating poly(A)+ RNA from cells activated by LPS for 30 min on a 1.8% agarose gel revealed that the RNA band at 2.1 kb consisted of two transcripts (see Fig. 8Go, lanes 3 and 4). Because intron 3 contains only 79 bases, it is most likely that the upper band at 2.1 kb contained introns 1 and 3, whereas the lower band carried only intron 1. Interestingly, we never found transcripts containing both intron 2 and 3, suggesting that intron 1 cannot be spliced in the presence of intron 2. Thus, these results demonstrated a splicing pathway in which the intervening sequences, in contrast to most intron sequences, were sequentially and stepwise unusually slowly spliced from the primary transcript. The hybridization pattern presented in Figure 5Go shows clearly that intron 2 was the first and intron 1 was the last to be removed from the lysozyme primary transcript.



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  		FIGURE 6. Schematic representation of the splicing pathway of lysozyme primary transcript.

 


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  		FIGURE 5. The splicing pathway of the lysozyme primary transcript in LPS-activated HD11 cells. Poly(A)+ RNAs were isolated from cells activated with LPS for 0, 0.5, 2, and 6 h, fractionated on 1.4% agarose gels, and Southern blotted onto nylon membranes. The blots were hybridized to lysozyme cDNA, intron 1, and intron 2 and were subsequently stripped and rehybridized to GAPDH cDNA. The arrow indicates the position of the mature lysozyme mRNA.

 


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  		FIGURE 8. Analysis of poly(A) tails on lysozyme pre-mRNAs and mature mRNA. Poly(A)+ RNAs from untreated cells (-) or from cells activated with LPS for 30 min and subsequently treated with actinomycin D (Act. D) for the indicated time periods were subjected to RNase H digestion following hybridization to oligo(dT)12–18. The resulting deadenylated RNAs were analyzed by sequential Northern hybridization to lysozyme and GAPDH. The two arrows indicate two RNA bands after digestion with RNase H, and the long arrow indicates the position of the mature lysozyme mRNA.

 
LPS induces increases in poly(A) tail length of the lysozyme RNA transcripts

The hybridization pattern presented in Figures 1Go and 5Go shows that the LPS-induced lysozyme RNA transcript at 0.8 kb was completely spliced, but was still larger than the mature lysozyme mRNA from untreated HD11 cells, indicating a further step in the posttranscriptional processing of the LPS-induced lysozyme RNA transcripts. Therefore, this step was investigated after inhibiting transcription with actinomycin D, an inhibitor of RNA polymerases. Before the actinomycin D treatment, cells were activated with LPS for 30 min. As shown in Figure 7Go, A and B, the lysozyme 0.8-kb RNA transcript appeared first after 40 min of exposure to actinomycin D, and intron 1 was more slowly removed than intron 2 and 3. Interestingly, a continuous shortening of the 0.8-kb RNA transcript was observed throughout 15 h of actinomycin D addition. After 15 h, the size of the 0.8-kb RNA transcript was similar to that of the mature lysozyme mRNA in untreated cells (Fig. 7GoB).



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  		FIGURE 7. Northern blot analysis of the poly(A) tail shortening of LPS-induced lysozyme mRNA. HD11 cells were activated with LPS for 30 min and then treated with 5 µg/ml actinomycin D (Act. D) for the indicated time periods. Poly(A)+ RNA were isolated, and 4 µg were electrophoretically fractionated on 1.8% agarose gels and blotted onto nylon membranes. The blots were sequentially hybridized to lysozyme and GAPDH. The arrow indicates the position of the mature lysozyme mRNA.

 
Several studies have demonstrated posttranscriptional regulation involving changes in poly(A) tail length of completely spliced RNAs (27, 28, 29). To determine whether the difference in size of the LPS-induced 0.8-kb RNA transcript and the mature lysozyme mRNA may be due to a difference in their poly(A) tail length, we used RNase H and oligo(dT)12–18 to selectively digest the poly(A) tails. Poly(A)+ RNAs from untreated, from LPS-activated or from LPS- and actinomycin D-treated HD11 cells were hybridized to oligo(dT)12–18 and subsequently digested with RNase H, which specifically cleaves RNA in RNA:DNA hybrids. The deadenylated RNAs were analyzed by Northern blotting and hybridization to the lysozyme cDNA. As shown in Figure 8Go, when poly(A) tails were completely digested, the size difference between the LPS-induced 0.8-kb RNA transcript and the mature lysozyme mRNA was eliminated, indicating that their poly(A) tail lengths were quite different. These data confirmed that LPS induced increases in the poly(A) tail length of the lysozyme RNA transcripts and that the progressive shortening of LPS-induced lysozyme mRNA was due to a reduction in poly(A) tail length occurring in a 3'-to-5' direction. Figure 7GoB shows further that the poly(A) shortening of LPS-induced lysozyme mRNA occurred slowly throughout 15 h after actinomycin D addition. After this time period, the LPS-induced mRNA still contained a shortened but not completely removed poly(A) tail, which corresponded to the mature, uninduced lysozyme mRNA. Longer incubation with actinomycin D, up to 24 h, did not cause a decrease in poly(A) tail length (data not shown), indicating that the process of deadenylation could stop within 15 h of actinomycin D treatment. Interestingly, the poly(A) shortening was observed only from the LPS-induced mRNA, but never from the uninduced lysozyme mRNA during this time.

Posttranscriptional regulation of gene expression occurs frequently by changing the cytoplasmic message stability affecting steady state levels of specific mRNAs (31, 32). Furthermore, the increase in poly(A) tail length can influence the translation rate of specific mRNAs (33, 34, 35, 36). Therefore, we examined whether the increase in poly(A) tail length of lysozyme mRNA was accompanied by changes in the translational efficiency or in the message stability.

To examine the polysomal recruitment of lysozyme mRNA for translation, postmitochondrial supernatants from LPS-activated HD11 cells were fractionated by centrifugation through 35% sucrose gradients. Polysomal mRNA from the pellet and nontranslating mRNA from the supernatant were isolated and analyzed by Northern blotting. Northern blots probed to the lysozyme cDNA, presented in Figure 9Go, showed that all lysozyme mRNAs were associated with polysomes irrespective of their poly(A) tail length. Thus, the polysomal recruitment of lysozyme mRNA for translation did not change after exposure to LPS.



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  		FIGURE 9. Analysis of polysomal recruitment of lysozyme mRNA. HD11 cells were activated with LPS for 30 min or 6 h and then treated with or without actinomycin D (Act. D) for 3 and 15 h. Postmitochondrial supernatants were fractionated by 35% sucrose gradients into polysomal fractions (P) and postpolysomal supernatants (S). Poly(A)+ RNA was isolated from each fraction and analyzed by Northern hybridization to lysozyme cDNA.

 
Next, to determine whether the increase in poly(A) tail length of the lysozyme mRNA might be associated with the message stability, we measured the decay of lysozyme mRNA in the absence of transcription. HD11 cells were left untreated or activated with LPS and then incubated with 5 µg/ml of actinomycin D for 2 to 10 h, and the levels of lysozyme message were determined by Northern analysis. Unfortunately, a longer than 10-h incubation with actinomycin D at this concentration was very toxic to the cells (data not shown). Actinomycin D at a concentration of 5 µg/ml has been previously shown to be able to inhibit transcription in HD11 cells (10) (see also Fig. 7Go). Figure 10Go shows that the lysozyme mRNA was found to be stable in untreated cells and in LPS-activated cells; panel B shows a t1/2 of ~9 h for lysozyme mRNA in both untreated and LPS-activated HD11 cells.



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  		FIGURE 10. Analysis of cytoplasmic lysozyme mRNA stability. A, Untreated HD11 cells or LPS-activated cells were treated with actinomycin D (Act. D) for 2, 4, 6, 8, and 10 h. Total RNAs were isolated. 1/5 and 1/25 of RNA from untreated cells and LPS-activated cells, respectively, were analyzed by Northern hybridization to lysozyme cDNA. The blots were exposed to x-ray films for 6 days and for 12 h, respectively. Subsequently, the blots were stripped and rehybridized to GAPDH cDNA. Data shown are from a representative experiment. B, Stability of lysozyme mRNA in untreated cells ({square}) and LPS-activated cells ({blacksquare}) at various times after exposure to actinomycin D. Autoradiograms were analyzed by densitometric scanning. For each curve, the values at 2, 4, 6, 8, and 10 h were expressed as percentage of the value at time zero. Data shown are from two independent experiments.

 
Nuclear localization of the unspliced and incompletely spliced lysozyme RNA transcripts

Next, we determined the subcellular localization of the accumulated lysozyme pre-mRNAs induced by LPS. Cytoplasmic and nuclear poly(A)+ RNAs were fractionated from LPS-activated actinomycin D-treated HD11 cells; the subcellular distribution of the intron-containing lysozyme pre-mRNAs was analyzed by Northern blot hybridization to the lysozyme cDNA. A densitometric scanning of the autoradiogram presented in Figure 11Go revealed that most of the intron-containing lysozyme transcripts at 3.9 kb and 2.1 kb were, as expected, accumulated in the nucleus, whereas >90% of the 0.8-kb transcripts were located in the cytoplasm. This implied that the poly(A) tails of LPS-induced lysozyme mRNA began to shorten following translocation into the cytoplasm, while the change in poly(A) tail length occurred, on the other hand, in the nucleus. Rehybridizing the blot to GAPDH cDNA demonstrated the cytoplasmic localization of the mature GAPDH mRNA.



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  		FIGURE 11. Subcellular localization of lysozyme pre-mRNAs. HD11 cells were activated with LPS for 30 min and then treated with actinomycin D (Act. D) for the indicated time periods. Cytoplasmic and nuclear poly(A)+ RNAs were isolated and analyzed by Northern hybridization to lysozyme and GAPDH.

 
LPS stimulates lysozyme synthesis in myelomonocytic HD11 cells

To examine whether LPS-induced lysozyme mRNA is functional and translatable, we performed Western blot analysis for lysozyme. HD11 cells were left untreated or activated with LPS. After 1 to 9 h, cell culture media and cell lysates were subjected to Western blot analysis using an antiserum against chicken lysozyme. As shown in Figure 12Go, production and secretion of lysozyme were increased following stimulation with LPS. Since lysozyme was undetectable in control untreated cells, the maximal fold induction by LPS could not be determined. The increase in lysozyme production seemed to correlate temporally with the increase in lysozyme mRNA after LPS treatment.



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  		FIGURE 12. LPS stimulates lysozyme production. HD11 cells were treated with and without LPS for the indicated time periods. Following removal of the cell culture media, cells were disrupted, and cell lysates were isolated. Two microliters of each cell lysate (A) and 10 µl of each cell culture medium (B) were subjected to Western analysis using an antiserum against chicken lysozyme.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LPS has been shown to activate expression of various genes. The activation is regulated in many cases at the transcriptional level by various transcription factors such as nuclear factor {kappa}B (NF-{kappa}B) (37, 38, 39), activator protein 1 (AP-1) (40), or C/EBPß (9, 10, 41). Our previous study on the LPS activation of the lysozyme gene in myelomonocytic HD11 cells indicated a transcriptional control of the lysozyme gene regulated by LPS, which contributed to an accumulation of the lysozyme mRNA (9). In this detailed study, we show that both transcriptional and posttranscriptional mechanisms are involved in the regulation of the lysozyme gene in myelomonocytic cells in response to LPS. Analyses of nuclear and cytoplasmic RNAs demonstrated that the posttranscriptional regulation was a multistep process including a transient accumulation of the pre-mRNAs with an intranuclear elongation of poly(A) tail length and a subsequent cytoplasmic partial deadenylation of the completely spliced mRNA.

Primary transcripts of most eukaryotic genes have been shown to be spliced very quickly immediately after the transcription has been completed before leaving the nucleus to enter the cytoplasm. Recently, a number of studies have provided evidence that splicing can occur cotranscriptionally through binding of pre-mRNA splicing factors with the C-terminal domain of RNA polymerase II (for a review, see 42 . In contrast, splicing of the lysozyme primary transcript in LPS-activated HD11 cells was extremely slow, and the intron sequences 1, 2, and 3 have been shown to be completely removed at 3 h after transcription. Interestingly, the extremely slow splicing of the lysozyme gene primary transcript enabled us to employ Northern analysis using intron sequences to investigate its splicing pathway. In this pathway, the intron sequences carried in the primary transcript were sequentially spliced in the following order: intron 2 was the first and intron 1 was the last to be removed. Intron 2 and 3 were spliced out within 40 min, whereas intron 1 was completely removed after 3 h following the transcription. Furthermore, the splicing of intron 1 can occur after the splicing of intron 2 and 3 has been completed (Figs. 5Go and 6Go). Thus, the order of the splicing did not occur in a linear sequence from the 5' or 3' end of the primary transcript. As expected, all intron-containing lysozyme pre-mRNAs were retained in the nucleus. After the splicing had been completed, the lysozyme mRNA was quickly transported from the nucleus into the cytoplasm (Fig. 11Go), indicating that splicing complexes prevent the export of unspliced precursor RNAs from the nucleus. This direct competition between the processes of splicing complex formation and nuclear export has been reported by Legrain and Rosbash (43) and Chang and Sharp (44). In fact, mutated introns that were not recognized by the splicing machinery and not spliced were able to leave the nucleus (43, 44).

There is evidence indicating that a great part of eukaryotic primary transcripts is quickly degraded after transcription, whereas only a minor part can undergo the nuclear processing to become mature mRNAs before leaving the nucleus (45). Posttranscriptional regulation affecting the stability of unspliced transcripts has already been suggested (46, 47). In the present study, we investigated mechanisms involved in the accumulation of lysozyme pre-mRNAs in the nucleus following LPS treatment. Our data demonstrating that LPS activated transcription of the lysozyme gene also indicated that an intranuclear stabilization of the lysozyme primary transcript accounted, in part, for the nuclear accumulation of lysozyme transcripts, thus leading to a considerable increase in cytoplasmic lysozyme mRNA level. The increased stability of the nuclear transcripts, however, was clearly associated with LPS-activated transcription of the lysozyme gene and seemed to be caused by the altered processing of lysozyme pre-mRNAs, because simultaneous treatment with LPS and actinomycin D failed to activate accumulation of the lysozyme pre-mRNAs (data not shown). Interestingly, the splicing process of the lysozyme primary transcript occurred very slowly in LPS-activated cells. At present, we are not able to detect this splicing pattern in untreated cells, although transcription of the lysozyme gene in these cells was clearly detectable by a run-on transcription assay. Indeed, Northern hybridization to the lysozyme intron sequences 1 and 2 did not reveal any signal even after very long autoradiography. Thus, it is possible that the duration of the splicing process, but not the splicing itself, may be altered by LPS. Alternatively, the splicing process of the lysozyme primary transcript in untreated cells was the same in LPS-activated cells, but the lysozyme primary transcript could not be detected because of rapid degradation.

Interestingly, the increase in lysozyme transcript stability in LPS-activated cells seemed to be associated with an increase in the poly(A) tail length of the lysozyme transcript. This process appeared to occur in the nucleus, in contrast to many developmental systems of the mouse (36), Xenopus (33, 48), and Drosophila (49) in which preexisting short poly(A) tails of mRNAs were elongated in the cytoplasm.

Nuclear mechanisms increasing poly(A) tail length of nuclear RNAs have already been described (27, 29). It has been proposed that this process may be involved in the stability of cytoplasmic, mature mRNAs (29, 50, 51). Surprisingly, Ford et al. (52) have shown that poly(A) tails were able to stabilize RNA in an in vitro RNA stability system only with nuclear extracts. Our results show that the cytoplasmic message stability was similar in both untreated and LPS-activated HD11 cells. Although any correlation between an extended polyadenylation and a stabilization of primary transcripts has not been reported yet, we could not exclude the possibility that the increase in poly(A) tail length of the lysozyme transcript might result in an increased stability of the lysozyme pre-mRNAs, but not account for the stability of the cytoplasmic mature mRNA, because of the following two considerations. First, the mature lysozyme mRNA was a stable message with a t1/2 of ~9 h. Increasing its stability may result more likely in an extended half-life than in an elevated level of the lysozyme message and an increased lysozyme production of macrophages in early response to LPS or at the beginning of a bacterial infection. Second, the increased poly(A) tail length of lysozyme mRNA induced by LPS began to be gradually shortened immediately after entering the cytoplasm. It has already been reported that the rate of mRNA decay is determined by the rate of poly(A) tail removal (30). Like the stable ß-globin mRNA (30), the LPS-induced lysozyme mRNA underwent a slow, gradual poly(A) shortening for at least 15 h. It is possible that this process is a prerequisite to the lysozyme mRNA decay and a consequence of the poly(A) tail length increase induced by LPS. Perhaps the increased poly(A) tail has to shorten to reach normal size before the normal degradation pathway of the lysozyme mRNA can occur. Thus, the poly(A) tail shortening seems to be a control mechanism involved in the regulation of the lysozyme message stability.

Poly(A) tails have been shown to be involved in translational efficiency (53). Although the polysomal recruitment of the lysozyme mRNA was shown to be independent of the poly(A) tail length, the possibility that long poly(A) tails are nevertheless more suited for efficient translation than short poly(A) tails can not be excluded. Directly determining the translation rate of the lysozyme mRNA with different poly(A) tail lengths may better reveal differences in their translatability.

In a recent study, Huang and Carmichael (54) have provided evidence for a role of poly(A) tails in transport of RNA across the nuclear membrane. The nucleocytoplasmic transport is energy dependent and saturable and thus a carrier-mediated process (55). Perhaps this process can be facilitated by altered poly(A) tail length, particularly when a great amount of mRNA must be exported within a short time period, for example, following LPS treatment.

In summary, our study demonstrates a posttranscriptional multistep process of LPS-regulated lysozyme gene expression. The exact mechanism by which the stability of the primary transcript was increased and the role of each step remain to be investigated in a further study.


   Acknowledgments
 
We thank K. Zimmermann for skilful technical assistance, D. Wulf for RT-PCR, Prof. G. Mieskes (Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany) for the anti-lysozyme antiserum, and Prof. G. F. Gerlach (University of Hannover, Hannover, Germany) for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by grants to L.P. from the Deutsche Forschungsgemeinschaft. Back

2 Present address: Institut für Mikrobiologie und Tierseuchen, Bischofsholer Damm 15, 30173 Hannover, Germany. Back

3 Address correspondence and reprint requests to Dr. L. Phi-van, Institut für Tierzucht und Tierverhalten (FAL), Dörnbergstr. 25–27, 29223 Celle, Germany. E-mail address: Back

4 Abbreviations used in this paper: C/EBPß, CCAAT/enhancer binding protein ß; kb, kilobase(s); CAT, chloramphenicol acetyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TEN, 40 mM Tris-HCl (pH 7.5)/1 mM EDTA/150 mM NaCl. Back

Received for publication August 11, 1997. Accepted for publication January 26, 1998.


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