Regulation of C/EBPbeta mRNA expression and C/EBPbeta promoter activity by protein kinases A and C in a myelomonocytic cell line (HD11).

OBJECTIVE: The transcription factor CCAAT/enhancer- binding protein (C/EBP) beta is involved in inflammatory responses in immune cells, including myelomonocytic cells. In this study, signal transduction pathways regulating C/EBPbeta expression were investigated. METHODS: The expression of C/EBPbeta mRNA in cells treated with various activators and inhibitors of PKA and PKC was analyzed by Northern blot hybridization. C/EBPbeta promoter activity was investigated by transient transfection assays with C/EBPbeta promoter CAT constructs. RESULTS: Phorbol 12-myristate 13-acetate (PMA), forskolin and 3-isobutyl-1-methyl-xanthine (IBMX), an inhibitor of cAMP and cGMP phosphodiesterases, but not cGMP, when added to chicken myelomonocytic HD11 cells, markedly stimulated the C/EBPbeta mRNA expression. However, transfection experiments using HD11 cells showed that CAT constructs controlled by the 5' flanking sequence from -704 to +24 of chicken C/EBPbeta gene were activated by PMA, but not by forskolin. In contrast to forskolin, IBMX was able to activate the C/EBPbeta promoter CAT constructs. Further transient transfection experiments using other cell lines demonstrated that the chicken C/EBPbeta promoter was responsive to forskolin in mouse fibroblasts NIH3T3, but not in human hepatoma HepG2 cells. Increase in C/EBPbeta mRNA stability in HD11 cells was induced by forskolin and PMA. CONCLUSION: The results indicate that the C/EBPbeta gene is regulated transcriptionally as well as post-transcriptionally in response to forskolin and PMA, and the forskolin responsiveness of the C/EBPbeta promoter seems to depend on cellular cAMP turnover.

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

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 HD 11 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-kappaB/Rel. Therefore, we discussed the role of C/EBPbeta(NF-M), CREB, and NF-kappaB/Rel as possible targets for phosphorylation mediated by PKA, PKC, and Ca2+.

An origin of bidirectional DNA replication is located within a CpG island at the 3" end of the chicken lysozyme gene.

We previously identified a broad initiation zone of DNA replication at the chicken lysozyme gene locus. However, the existence of a highly preferred origin of bidirectional replication (OBR), often found in initiation zones, remained elusive. In order to re-examine this issue we used a competitive PCR assay to determine the abundance of closely spaced genomic segments in a 1 kb size fraction of nascent DNA. A sharp peak of nascent strand abundance occurred at the 3" end of the gene, where initiation events were 17 times more frequent than upstream of the gene. This primary initiation site, active in lysozyme expressing myelomonocytic HD11 cells and non-expressing hepatic DU249 cells, was found to reside within an unusually located CpG island. While most CpG islands are found at the 5" end of genes, the lysozyme gene island extends from the 3" end of the second intron and includes approximately 1.2 kb of 3" flanking DNA. As diagnosed by methylation-sensitive restriction enzymes, the island is largely non-methylated in HD11 cells, DU249 cells and inactive chicken erythrocytes. Furthermore, a DNase I hypersensitive site (HS) that is composed of two subsites separated by approximately 100 bp, was localised very close to the segment with the highest initiation activity. Our results suggest that the non-methylated CpG island and the HS provide an accessible chromatin structure for the lysozyme gene origin of replication.

An initiation zone of chromosomal DNA replication at the chicken lysozyme gene locus.

The chicken lysozyme gene domain is distinguished by a broad knowledge of how its expression is regulated. Here, we examined the in vivo replication of the lysozyme gene locus using polymerase chain reaction amplification and competitive polymerase chain reaction of size-fractionated, nascent DNA strands. We found that DNA replication initiates at multiple sites within a broad initiation zone spanning at least 20 kilobases, which includes most of the lysozyme gene domain. The 5' border of this zone is probably located downstream of the lysozyme 5' nuclear matrix attachment region. Preferred initiation occurs in a 3'-located subzone. The initiation zone at the lysozyme gene locus is also active in nonexpressing liver DU249 cells. Furthermore, examining the timing of DNA replication at the lysozyme gene locus revealed that the gene locus replicates early during S phase in both HD11 and DU249 cells, irrespective of its transcriptional activity.

Posttranscriptional lipopolysaccharide regulation of the lysozyme gene at processing of the primary transcript in myelomonocytic HD11 cells.

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 t(1/2) 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.

Evidence for an enhanced transcription-dependent de novo synthesis of C/EBPbeta in the LPS activation of the chicken lysozyme gene.

C/EBPbeta has been shown to mediate the lipopolysaccharide (LPS)-induced expression of the lysozyme gene through enhanced binding to the -6.1-kb lysozyme enhancer. In this study, we describe the LPS regulation of the C/EBPbeta gene in myelomonocytic HD11 cells. Northern analysis showed that the steady state level of C/EBPbeta mRNA increased in response to LPS. The half life of C/EBPbeta mRNA of about 1 h in HD11 cells was not affected by exposure to LPS. Nuclear run-on transcription experiments with isolated nuclei revealed that the rate of C/EBPbeta gene transcription was enhanced by LPS, demonstrating that the C/EBPbeta gene in HD11 cells was regulated at the transcriptional level in response to LPS. Furthermore, the LPS-induced binding activity of C/EBPbeta to the -6.1-kb lysozyme enhancer was dependent not only on protein synthesis, but also on transcription. Thus, these results suggested that the LPS-induced binding activity to the -6.1-kb lysozyme enhancer in HD11 cells was regulated by an enhanced transcription-dependent de novo synthesis of C/EBPbeta.

Dissection of the ability of the chicken lysozyme gene 5' matrix attachment region to stimulate transgene expression and to dampen position effects.

The chicken lysozyme gene domain is flanked by nuclear matrix attachment regions (MARs) on each side. We have previously shown that bilaterally flanking 5'MARs in stably transfected artificial genetic units enhance expression of a reporter transgene and dampen position effects of the chromatin structure at the site of integration. The 5' MAR was now dissected into smaller fragments that were monitored for effects on transgene expression in mouse 3T3 cells by a similar assay. Fragments, which contain 1.32 and 1.45 kb and represent the upstream and the downstream half, respectively, of the 5' MAR, retained the ability to stimulate transgene expression as well as the ability to reduce the variation in the level of expression. However, a 452 bp subfragment (H1-HaeII), which still exhibits specific binding to nuclear matrices and contains two high-affinity binding sites for the abundant nuclear matrix protein ARBP, lost both of those abilities. A dimerized 177 bp sequence from fragment H1-HaeII, which also binds selectively to nuclear matrices and includes a duplicated ARBP binding site, was also unable to stimulate reporter gene expression. Furthermore, a 0.65 kb subfragment containing an intrinsically bent sequence did not affect an elevated reporter gene expression and its dampening. Our results show that the ability of MAR fragments to bind to nuclear matrices is not sufficient to enhance and insulate transgene expression in stably transfected cells.

Transcriptional activation of the chicken lysozyme gene by NF-kappa Bp65 (RelA) and c-Rel, but not by NF-kappa Bp50.

The lysozyme gene is expressed at a low level in myeloblasts and is progressively activated to constitutively high expression in mature macrophages. The binding activity of the newly defined NF-kappa B/Rel family of transcription factors increases during the terminal differentiation of macrophages. In this study, I show that NF-kappa B/Rel-like proteins bind to the nuclear factor kappa B (kappa B)-like sequence of the lysozyme promoter. These binding activities were induced by treatment of HD11 cells with lipopolysaccharide. Immunomobility shift assays show that c-Rel is possibly a factor in the complexes that bind to the kappa B-like sequence lys kappa B. Binding activity to one of the protein complexes seems to be regulated by phosphorylation. In fact, overexpression of p65 and c-Rel stimulates expression of the chloramphenicol acetyltransferase gene controlled by the lysozyme promoter. Furthermore, co-transfection experiments reveal that the kappa B-like sequence within the lysozyme promoter mediates the transactivation by p65 and c-Rel. These results indicate that the p65 and c-Rel could be components of the protein complexes that bind to the kappa B-like sequence and this binding could contribute to the progressively activated expression of the lysozyme gene during the terminal differentiation of macrophages.

A non-curved chicken lysozyme 5' matrix attachment site is 3' followed by a strongly curved DNA sequence.

Matrix attachment regions (MARs) partition the genome into functional and structural loop-domains. Here, we determined the relative matrix affinity of cloned fragments of the chicken lysozyme 5' MAR. We show that this region contains a non-curved high-affinity binding site, which is 3' followed by a strongly curved DNA sequence that exhibits weak matrix binding. DNA curvature is not a physical property required for strong matrix binding. Possible biological functions of this sequence arrangement, particularly of the strongly curved DNA, are discussed.

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.

Matrix attachment regions (MARs) are DNA elements that dissect the genome into topologically separated domains by binding to a chromosomal skeleton. This study explored the putative influence of the MAR located 5' of the chicken lysozyme gene on expression of heterologous genes in heterologous cell systems. Expression of a construct with the chloramphenicol acetyltransferase (CAT) indicator gene controlled by the herpes simplex virus thymidine kinase promoter (TC) and a construct in which the same transcriptional unit is flanked by chicken lysozyme 5' MARs (MTCM) was assayed after stable transfection into rat fibroblasts. Median CAT activity per copy number in MTCM transfectants was elevated approximately 10-fold relative to that in TC transfectants. Total variation in normalized CAT activity decreased from more than 100-fold among TC transfectants to nearly 6-fold among MTCM transfectants. The steady-state level of transcripts and the relative rate of transcription were increased in MTCM transfectants, as shown by S1 nuclease and run-on transcription assays, respectively. The chicken lysozyme 5' MAR thus can confer elevated, less position-dependent expression on a heterologous promoter in cells of a different species by increasing the density of transcribing RNA polymerase molecules. MAR-mediated transcriptional enhancement suggests that MARs are important for gene expression and not just for DNA packaging.

Proacrosin and the differentiation of the spermatozoa.

Using the indirect immunofluorescence staining technique, the occurrence and localization of proacrosin, the zymogen form of acrosin, was studied during spermatogenesis in the bull, ram, boar and rabbit. Proacrosin staining was demonstrable for the first time in the early haploid spermatid and increased with the differentiation of the spermatid to spermatozoon. The spermatozoon is covered by a cap-like structure of uniform fluorescence corresponding to the acrosomal compartment of the male gamete. No fluorescence could be found in diploid spermatogenic cells, i.e., in spermatogonia and spermatocytes. An identical developmental pattern of proacrosin was observed with the indirect immunoperoxidase staining technique. However, with this staining technique a distinct distribution of proacrosin staining was observed in the acrosome of epididymal and ejaculated spermatozoa of the bull, ram, boar, rabbit and man. Proacrosin seems to be distributed in the acrosome in granules rather than in the homogeneous form, as was indicated by the results of indirect immunofluorescence staining.

Acrosin in the spermiohistogenesis of mammals.

Using the indirect immunofluorescence staining technique, the developmental pattern of acrosin during spermatogenesis of boar, ram, rabbit, mouse, rat, and Russian hamster (Phodopus sungorus) was studied. Specific antibodies against purified boar acrosin raised in rabbits cross-reacted with the acrosin of all species investigated thus suggesting that the antigenic determinants of the acrosin molecule cross-reacting with anti-boar acrosin antiserum have been highly conserved in mammalian evolution. During spermatogenesis acrosin was first demonstrable in haploid spermatids and increased in the course of the differentiation of the spermatids to spermatozoa. During the entire period of spermatid differentiation acrosin appeared in juxtaposition to the nucleus. In boar and ram the results obtained with the indirect immunofluorescence staining procedure were confirmed with the indirect immunoperoxidase staining method.

Proacrosin/acrosin activity during spermiohistogenesis of the bull.

Acrosin and its zymogen form, proacrosin, were extracted from early and late spermatids, from ejaculated and epididymal spermatozoa (caput, corpus, and cauda) of the bull. Activity of proacrosin/acrosin and the time course of proacrosin activation were studied. It turned out that proacrosin/acrosin activity is first demonstrable in haploid spermatids, increases during spermiohistogenesis in the testis, and remains nearly constant in epididymal and ejaculated spermatozoa.