Histones derived from replica-tion-independent mRNAs were originally suggested to Keywords C-terminal H4 peptides; extracellular function; histogranin; histone H4 mRNA variants; H4-v.1 C
Trang 1anti-inflammatory function of rat histone H4 mRNA
variants
Rene´ Poirier, Irma Lemaire and Simon Lemaire
Department of Cellular and Molecular Medicine, University of Ottawa, Canada
Histones are known to play a key role in the
pack-aging of DNA within eukaryotic cells The majority of
histone proteins or ‘core histones’ are produced during
the synthesis (S) phase of the cell cycle [1] Core
his-tone mRNAs do not end with a polyadenylated tail
but, instead, contain within their 3¢UTR a conserved
stem–loop sequence that is involved in their
matur-ation and function [2] There are also replicmatur-ation-
replication-independent histone variants that transcribe poly-adenylated mRNAs and whose translation products accumulate preferentially in nondividing, terminally differentiated tissues [3] In contrast with core histone mRNAs, histone mRNA variants can be expressed throughout all phases of the cell cycle in inducible and tissue-specific ways [3] Histones derived from replica-tion-independent mRNAs were originally suggested to
Keywords
C-terminal H4 peptides; extracellular
function; histogranin; histone H4 mRNA
variants; H4-v.1
Correspondence
S Lemaire, Department of Cellular and
Molecular Medicine, Faculty of Medicine,
University of Ottawa, 451 Smyth Road,
Ottawa, Ontario, Canada K1H-8M5
Fax: +1 613 562 5646
Tel: +1 613 562 5800 ext 8350
E-mail: slemaire@uottawa.ca
(Received 7 July 2006, accepted 1 August
2006)
doi:10.1111/j.1742-4658.2006.05444.x
Two histone H4 mRNA variants, H4-v.1 and histogranin mRNAs, were detected in the rat genome and measured in various tissues and isolated alveolar macrophages Medium to high levels of both mRNAs were present
in the liver, adrenal glands, thymus, bone marrow and alveolar
macrophag-es H4-v.1 cDNA contained an open reading frame that coded for unmodi-fied whole histone H4, whereas histogranin cDNA lacked the first ATG codon and contained an open reading frame that coded for modified (Thr89) H4-(84–102) The two genes displayed a sequence homologous (> 80%) to the open reading frame of core H4 somatic (H4s) and H4 ger-minal (H4g) and their variant nature was supported by the absence of histone consensus palindromic and purine-rich sequences in the proximal 3¢UTR, and the presence of a polyadenylation signal in the distal 3¢UTR and of specific upstream transcription factor-binding sites H4-v.1 and his-togranin transcripts, but not H4s transcript, were selectively induced by lipopolysaccharide and⁄ or interferon gamma in alveolar macrophages
In vitro transcription⁄ translation experiments with H4-v.1 and histogranin cDNA pCMV constructs produced peptides with the molecular mass (2 kDa) of the alternative histone H4 translation product which, like syn-thetic H4-(86–100) and [Thr89]H4-(86–100) or rat histogranin, inhibited lipopolysaccharide-induced prostaglandin E2 release from rat alveolar macrophages The synthetic peptides also inhibited the secretion of the CXC chemokine interleukin-8 (GRO⁄ CINC-1) in response to lipopolysac-charide The presence of H4-v.1 and histogranin mRNAs in tissues wherein immune reactions take place and the inhibitory effects of their translation products on prostaglandin E2 and interkeukin-8 secretion by activated alveolar macrophages suggest an anti-inflammatory function
Abbreviations
AM, alveolar macrophage; AP, amplification primer; BAL, bronchoalveolar lavage; EST, expressed sequence tag; GSP, gene-specific primer;
HN, histogranin; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; NF-jB, nuclear factor kappa B; OGP, osteogenic growth peptide; PGE2, prostaglandin E2; SP1, stimulating protein 1.
Trang 2constitute a replacement pool of histones for
nucleo-somal maintenance throughout the cell cycle [4]
Recently, their expression was shown to regulate
var-ious processes that comprise heterochromatin ectopic
spread [5–7], DNA transcription (H2A.Z) [8–10],
cen-tromere formation (CENP-A) [11–13], X chromosome
inactivation (macroH2A) [14,15] and DNA repair
(H2AX) [16–19] The observation that the translation
of histone mRNA variants follows the rules of typical
poly(A) track-containing mRNAs [20] suggests that
histone variants may also exert extranuclear functions
In this regard, histones were reported to modulate
pituitary hormone secretions [21–24], pathogenic
anti-body production [25–27], microbial [28,29] and
tumor-al [30] cell growth, osteogenesis [31,32], pain [33–36]
and macrophage proinflammatory functions [37]
Histogranin (HN), a slightly modified C-terminal
histone H4 peptide homologous to histone H4-(86–
100), was first isolated in our laboratory from bovine
adrenal medulla [38] The immunoreactive peptide was
detected in various rat tissues, including the pituitary,
adrenal glands, lungs, spleen, brain and plasma [39]
Synthetic HN was initially shown to block
N-methyl-d-aspartate-induced convulsions in mice [38] More
recently, HN and related peptides were also shown to
display in vivo nonopioid analgesic effects and in vitro
anti-inflammatory activity [33–40] Our initial search
to determine the structure of the HN gene was
unsuc-cessful but led to the discovery of the H4 mRNA
vari-ant H4-v.1 [41] H4-v.1 was first isolated and
sequenced from a bovine adrenal medullary cDNA
phage library [41] Bovine H4-v.1 was then shown to
be a polyadenylated mRNA coding for unmodified
whole histone H4 A similar mRNA variant was also
detected in the rat using a cDNA probe that
recog-nized part of the bovine H4-v.1 coding region,
although its sequence was not determined [42] A close
correlation was then observed between the level of
H4-v.1 in various rat tissues and alveolar macrophages
(AMs) and the amounts of the histone H4 C-terminal
peptides, osteogenic growth peptide (OGP) [31] and
H4-(86–100), but not whole histone H4 protein or core
H4 mRNA [42] This study suggested that the
extra-cellularly acting unmodified C-terminal histone H4
peptides OGP and H4-(86–100) may be translation
products of the alternative AUG start codon in
H4-v.1, but not core H4 mRNA On the other hand, the
modified nature of the C-terminal histone H4 peptide
HN indicated that its synthesis might depend upon the
expression of another H4 mRNA variant akin to some
other types of histone variant, such as the H3 mRNA
variants that produce modified histone H3 proteins
[43,44]
As no report has indicated the structure of rat H4-v.1 and HN mRNAs, we herein used rat genome data-bases to search for the H4 variant candidates as well
as specific molecular approaches and in vitro assays to assess their structure, expression and function We also verified whether the cell cycle regulatory region and site II element, known to regulate the expression of H4 genes [45–47] or other specific elements, were present upstream of the H4 mRNA variants The results con-firm the existence and illustrate the structures of H4-v.1 and HN genes, two polyadenylated histone H4 mRNA variants with characteristics of the replication-independent histone genes coding for unmodified whole histone H4 and a modified C-terminal histone H4 peptide, respectively The particular localization
of the two genes in rat tissues, the identification of upstream gene-specific regulatory elements and the
in vitro transcription⁄ translation experiments with gene-specific cDNA constructs indicate that the two genes are independently expressed and produce C-ter-minal H4 peptides with in vitro anti-inflammatory activity
Results
Gene BLASTsearch
A blast search of the rat TIGR database provided
an expressed sequence tag (EST) sequence (TC: 322388) that resembled that of the bovine H4-v.1 mRNA variant [41] Like bovine H4-v.1, this EST sequence contained an ORF that coded for unmodi-fied histone H4 and a 3¢UTR that ended with an AATAAA polyadenylation signal Conversely, the EST sequence was not complete at its 5¢ end, lack-ing the 5¢UTR and the first ATG initiation codon present in bovine H4-v.1 On the other hand, a tblastn search of NCBI for histone H4 in the rat genomic sequence provided another sequence (NW_047492.1|Rn17_2014:1861737–1862016) that did not code for whole histone H4, but a modified his-tone H4 C-terminal peptide, [Thr89]H4-(84–102) Since histone H4 is one of the most evolutionarily conserved proteins [48], it was assumed that if a gene with this modified H4 coding region was expressed, it could be the gene encoding the modified C-terminal H4 peptide HN [38], generating in this case [Thr89]H4-(84–102) as an immediate precursor of rat
HN Next, we sought to verify the expression of H4-v.1 and HN mRNA transcripts in total mRNA prep-arations from various rat tissues and isolated AMs, and determine the complete structures of rat H4-v.1 and HN mRNAs
Trang 3Localization of H4-v.1 and HN mRNAs
Because H4-v.1 mRNA and immunoreactive HN had
already been detected within various rat tissues using
probes and antibodies that recognized bovine H4-v.1
mRNA and HN, respectively [39,42], initial tests were
performed to assess the level of expression of rat
H4-v.1 and HN mRNAs in total RNA preparations of
various rat tissues by the use of real-time PCR with
gene-specific primers (GSPs) designed from the blast
information High levels of both HN and H4-v.1
mRNA transcripts were observed in the liver, adrenal
glands, thymus and bone marrow (Fig 1A) However,
the HN mRNA transcript was more widely
distri-buted, being also abundant in endocrine,
neuroendo-crine and central nervous system tissues such as the
pituitaries, the spinal cord and the brain Both
mRNAs were also detected in AMs, and their levels
were compared with that of core H4s (Fig 1B) HN
mRNA was markedly increased by incubation of AMs
in the presence of interferon gamma (IFN-c)
(8.97-fold), whereas H4-v.1 was significantly stimulated by
both lipopolysaccharide (LPS) (2.67-fold) and IFN-c
(3.48-fold) In contrast, the level of H4s mRNA,
although 4.7 and 6.2 times higher than those of control
H4-v.1 and HN mRNAs, respectively, was not
signifi-cantly affected by incubation of AMs with LPS or
IFN-c
Sequence determination of H4-v.1 and HN cDNAs
Determination of the length and sequences of H4-v.1
and HN mRNAs was accomplished by 3¢RACE and
5¢RACE experiments using a Marathon-ReadyTM rat
spleen cDNA library The 5¢RACE and 3¢RACE
ampli-cons of H4-v.1 and HN were designed to overlap one
another, resulting in complete cDNA structure
amplifi-cation The 5¢RACE and 3¢RACE amplicons were
inser-ted into the TOPO cloning vector (Invitrogen) and
sequenced Complete H4-v.1 and HN cDNAs
(Gen-Bank accession numbers: AY936209 and AY936210,
respectively) were compared with their respective
sequences within the rat genome H4-v.1 and HN cDNA
sequences were considered to be accurate if three
separ-ate sets of sequenced 5¢RACE and 3¢RACE amplicon
clones and the corresponding genome sequences in the
NCBI genome database could be matched The H4-v.1
cDNA transcript contained a short 5¢UTR (19 bp), an
ORF corresponding to unmodified whole histone H4, a
3147 bp missing intron, and a relatively long 3¢UTR
(965 bp) ending with a polyadenylation signal (AAT
AAA) and an auxiliary mRNA-processing
facilitator-like element (AAAGAT) (Fig 2A; AY936209) On the
other hand, the HN cDNA transcript contained a relatively long 5¢UTR (253 bp), a short ORF coding for MDVVYTLKRQGRTLYGFGG as an immediate
Fig 1 Relative abundance of H4-v.1 and HN mRNA transcripts in various rat tissues (A) and isolated AMs (B) (A) Total RNA was extracted from rat tissues (three pools of two animals) and the amounts of H4-v.1 and HN mRNAs were determined by real-time PCR with gene-specific primers as described in Experimental proce-dures The relative abundance of the cDNA amplicons was meas-ured in comparison with GAPDH, using the lung as a comparative tissue for calculation in the equation: mRNA ¼ 2 ) [(Ct mRNA test tissue ) Ct GAPDH test tissue) ) (Ct mRNA comparative tis-sue ) Ct GAPDH comparative tissue)] In (B), freshly isolated AMs (three preparations from two animals each) were incubated for
24 h in the absence or presence of LPS (1 lgÆmL)1) or IFN-c (100 UÆmL)1) prior to total RNA extraction and measurement of H4-v.1 and HN mRNA transcripts in comparison with core H4s Results represent the mean ± SEM of three duplicated sets of experi-ments Statistical significance was determined using one-way ana-lysis of variance followed by a Bonferonni comparison test (A)
*P 6 0.05 as compared with heart H4-v.1 cDNA amplicon;
§ P 6 0.05 as compared with heart HH cDNA amplicon; † P 6 0.05
as compared with H4-v.1 cDNA amplicon in the same tissue (B)
§ P 6 0.05 as compared with control H4-v.1 mRNA; *P 6 0.05 as compared with control HN mRNA.
Trang 4precursor of rat HN (VVYTLKRQGRTLYGF, the
portion of the peptide homologous to bovine HN [38])
and a relatively long 3¢UTR (273 bp) ending with a
non-canonical polyadenylation signal (TATAAA) and an
auxiliary mRNA-processing facilitator-like element
(AAAGAT) (Fig 2B; AY936209)
Comparisons of H4-v.1 and HN cDNAs with core
histone H4 cDNAs
A comparison of H4-v.1 and HN cDNAs with core
germinal (H4g) and somatic (H4s) histone H4 genes
showed 80–92% homology in a region corresponding
to the ORF of core histone H4 genes (Fig 3) The
nucleotide substitutions in H4-v.1 did not affect the
highly conserved amino acid structure of the whole
histone H4 protein or the alternative initiation
transla-tion product H4-(84–102) (Fig 2A) On the other
hand, the HN cDNA shared a high degree of
homol-ogy with the histone H4 coding region (Fig 3), but
lacked the first ATG codon necessary to translate the
whole histone H4 protein and contained a modified
codon (ACT coding for Thr instead of Ala) in the
alternative ORF sequence to code for [Thr89]H4-(84–
102) (Fig 2B)
Comparison of the structures of the proximal 3¢UTR of HN cDNA with those of H4g and H4s revealed a GC-rich stem–loop structure followed clo-sely by a purine-rich region similar to the histone con-sensus palindromic and purine-rich sequences of H4g and H4s (Table 1) However, the stem–loop structure found in HN was considered to be noncanonical, being distinct from that found in other histone genes [48]
No comparable stem–loop structure was found in the proximal 3¢UTR of H4-v.1 Interestingly, the 3¢UTR
of both H4-v.1 and HN cDNAs contained an ATTT repeat element (14 and 4 repeats, respectively; AY936209 and AY936210) that is known to play a post-transcriptional role in the synthesis of cytokines
in lymphoid cells [49] Finally, the distal 3¢UTR of H4-v.1 and HN cDNAs, but not H4g or H4s, con-tained a polyadenylation signal characteristic of his-tone cDNA variants (Table 1)
Comparison of upstream genome sequences of H4-v.1 and HN genes with those of H4s and H4g indicated a region similar to the site II cell cycle regula-tory domain of the replication-dependent histone H4 genes (Table 1) comprising a TATA box-like motif, a histone H4-specific GGTCCG element, and a motif homologous to the human histone H4 gene cell cycle
Fig 2 Schematic representation of H4-v.1
(A) and HN (B) genes in the rat genome.
Rat cDNA structures were determined by
combined 5¢RACE and 3¢RACE with a
Mara-thon-Ready rat spleen cDNA library as
des-cribed in Experimental procedures The
complete sequences of H4-v.1 and HN
cDNAs were submitted to the NIH GenBank
and have been given the accession
numbers AY936209 and AY936210,
respectively The ORFs of the H4-v.1 and
HN genes encode complete histone H4
protein and the H4 C-terminal peptide
MDVVYTLKRQGRTLYGFGG, respectively
(Fig 3) The 5¢UTR, ORF, 3¢UTR, stem–loop
sequence and polyadenylation signals are
located as indicated in the schemes Both
genes are preceded by the gene-specific
promoters as described in Table 1 and as
illustrated The H4-v.1 gene contains a
3.5 kb intron and does not contain the
canonical histone stem–loop sequence
30 nucleotides downstream of the ORF.
The HN gene does not contain an intron and
its stem–loop sequence downstream of the
ORF is distinct from that of histone H4
genes [48].
Trang 5control motif (5¢-CTTTCGGTTTT-3¢) [46] Other
potential transcriptional regulatory binding motifs close
to the site II regulatory domain of the H4-v.1 gene
included the mitogen-activated protein (MAP) kinase
transcription factor ELK-1 ()44taagacGGAActgcttt)28),
a MAP kinase substrate transcription factor involved
in cell growth [50] and the cyclic AMP response
elements CREB ()88tccgccTGACgctccctgttt)69) and
CREB-P1 ()152ttgctcttACATgaactgaaa)132), two
tran-scription factors involved in the regulation of metabolic
and neuronal activities [51] (Fig 2) Sequences
upstream of the HN gene comprised the Elk-1 motif
()33gtacacGGAAgttttag)17) [52], the GC box motif
()121aaatgaGGCGgagcaa)107), a specific stimulating
protein 1 (SP1)-binding site that can modulate the
action of the nuclear factor kappa B (NF-jB) DNA site [52] and the NF-jB motif ()181tgGGGAaaacccc
ag)167), a transcription factor involved in the matur-ation of immune cells and inflammmatur-ation processes [53] (Fig 2) None of these sequences found upstream of either the H4-v.1 gene or HN gene was observed upstream of the replication-dependent H4g (NCBI
#m27433) and H4s (NCBI #x13554) genes
Transcription⁄ translation in an in vitro wheat germ lysate system
The presence in H4-v.1 cDNA of both initial and alternative ATG codons should allow its translation into both whole histone H4 and the C-terminal peptide
Fig 3 Comparison between the structures
of the ORF of H4 somatic (H4s), H4 germi-nal (H4g) and H4-v.1 cDNAs and corres-ponding 5¢UTR and ORF in HN cDNA Analyses were done with the BLAST 2 sequences of NCBI Start and stop codons are indicated by bold letters H4s (accession number x13554) is 84% and 80% homolog-ous with H4-v.1 and HN, respectively, and 84% homologous with H4g (accession num-ber m27433) H4g is 92% and 88% homol-ogous with H4-v.1 and HN, respectively, whereas H4-v.1 is 89% homologous with
HN H4s, H4g and H4-v.1 cDNA sequences code for unmodified whole histone H4 HN cDNA does not contain the initial ATG codon found in H4s, H4g and H4-v.1 cDNAs, thus giving rise to a translation product (pro-HN) of 19 amino acids corres-ponding to the alternative translation prod-uct in the other genes with a modification at position 89 (T instead of A) The initial M (M 0 ) in the translated H4 protein is cleaved
to give rise to a protein of 102 amino acids [48].
Trang 6H4-(84–102) (Fig 3) [41] The presence in HN cDNA
of only the alternative H4 ATG codon should allow
its translation only into [Thr89]H4-(84–102) as an
immediate precursor of rat HN Experiments
per-formed using pCMVTnT vector constructs with the
in vitro wheat germ lysate-coupled transcription⁄
trans-lation system (TnT, Promega) indicated that the
H4-v.1 construct synthesizes two radiolabeled (Met35)
protein⁄ peptide products, one comigrating on SDS gel
electrophoresis with whole histone H4 (11.4 kDa), and
the other comigrating with synthetic H4-(84–102)
(approximately 2 kDa) (Fig 4A) On the other hand,
the HN construct produced only one radiolabeled
compound, which migrated within the expected
molecular mass range of [Thr89]H4-(84–102)
(approxi-mately 2 kDa) (Fig 4A) The empty pCMVTnT vector
did not produce any radiolabeled protein⁄ peptide
product, whereas the Promega luciferase control
plas-mid showed multiple radiolabeled products, the major
band corresponding to luciferase (60 kDa) (Fig 4A)
Inhibition of LPS-induced prostaglandin E2(PGE2)
release from cultured AMs
Prostaglandins are known to play an important role in
inflammation and pain Rat AMs stimulated with LPS
(1 lgÆmL)1), the archetypal bacterial antigen, produced
significant amounts of PGE2 (171.3 ± 23 pgÆmL)1
compared to 76 ± 8.9 pgÆmL)1for unstimulated cells)
As shown in Fig 4B, LPS-stimulated release of PGE2
from primary cultures of rat AMs was reduced to
49.8% and 46.3% of the control value in the presence
of 10)8m synthetic H4-(86–100) and [Thr89]H4-(86–
100) (rat HN), respectively Incubation of AMs in the presence of the transcription⁄ translation HN-pCMV-TnT and H4-v.1-pCMVHN-pCMV-TnT products (20 lL) also reduced LPS-evoked release of PGE2 to 54.5% and 49.4% of the control value, respectively (Fig 4B) In contrast, the transcription⁄ translation product (20 lL)
of the empty pCMVTnT plasmid had no significant effect on PGE2release
Inhibition of LPS-induced rat interleukin-8 (IL-8) (GRO/CINC-1)
The CXC chemokine IL-8 is a potent neutrophil chem-otactic and activating agent As IL-8 and its rat ana-log, GRO⁄ CINC-1, are reported to be produced by human and rat AMs [68], we next investigated whether the synthetic translation products of H4-v.1 and HN mRNAs also modulated the secretion of this inflam-matory cytokine Rat AMs incubated with LPS (1 lgÆmL)1) for 4 h released into the culture medium significant amounts of GRO⁄ CINC-1 (4023 ± 325 pgÆmL)1), whereas GRO⁄ CINC-1 was undetectable in culture supernatants of unstimulated AMs Incubation
of AMs with 10)8m H4-(86–100) and [Thr89]H4-(86– 100) significantly decreased LPS-induced GRO⁄
CINC-1 secretion (to 58.3% and 62.5% of the control value, respectively) (Fig 5)
AM survival following treatment with H4-v.1 and
HN products
To verify whether inhibition of LPS-induced PGE2 and GRO⁄ CINC-1 by H4-v.1 and HN gene products
Table 1 Analysis of 3¢UTR palindromic and purine-rich sequences a (A) and upstream histone H4-like site II regulatory domain b (B) in rat H4g (m27433), H4s (x13554), H4-v.1 (AY936209) and HN (AY936210) Sequence homologies were determined as indicated in Experimental pro-cedures.
A
CCACACCATCAGGCTGTGGATACATAGATAAGGCAACATGG95 TATAAA B
a Underlined sequences represent consensus stem–loop sequences for histone H4, the underlined bold letters indicate a noncanonical stem–loop structure, and bold italic letters indicate purine-rich sequences Superscript positive numbers indicate the position downstream of the stop codon TAAA repeat elements [49] are also present in H4-v.1 and HN 3¢UTRs b TATA-box sequences are indicated in bold letters, histone H4 subtype-specific GGTCCG elements are underlined and in bold letters, and interferon regulatory factor recognition motifs are underlined Superscript negative numbers indicate the position upstream of the cap site (*).cThe polyadenylation signals were preceded by
an auxiliary mRNA-processing facilitator-like element (AAAGAT).
Trang 7is related to possible cytotoxic effects on rat AMs, the
percentage of living cells was determined on the basis
of the cytoplasmic esterase conversion of calcein-AM
to the green fluorescent product calcein by living cells
Exposure to 10)8m H4-(86–100) or [Thr89]-H4-(86– 100) or to 20 lL of the transcription⁄ translation prod-ucts of either H4-v.1pCMVTnT, HNpCMVTnT or control pCMVTnT for up to 24 h had no effect on the percentage of viable green fluorescent cells, indicating
no loss of AM membrane integrity (Fig 6)
A
B
Fig 4 Electrophoretic gel separation of coupled transcription ⁄
trans-lation (TnT) products with H4-v.1 and HN cDNA constructs (A) and
inhibition of LPS-induced PGE2release (B) (A) Biosynthesis
experi-ments with Promega luciferase control DNA plasmid,
HN-pCMV-TnT, H4-v.1-pCMVTnT and emptied pCMVTnT plasmids were
performed, and the radioactive products were separated by gel
electrophoresis as described in Experimental procedures Arrows
show the molecular mass of [ 35 S]Met-labeled protein and peptide
products as determined by comparison with the electrophoretic
pattern of a Mark 12 molecular weight ladder (B) Biosynthetic
products (20 lL of reaction samples) obtained in parallel
experi-ments with unlabeled Met were incubated with primary cultures of
rat AMs as described in Experimental procedures, and their ability
to inhibit LPS-evoked PGE 2 secretion was compared with those
of synthetic H4-(86–100) and [Thr89]H4-(86–100) at 10)8M
*P 6 0.05 is considered significant as compared with control.
Fig 5 Inhibition of LPS-induced chemokine secretion (A) Rat AMs were stimulated for 4 h with LPS (1 lgÆmL)1) in the presence and absence of synthetic [Thr89] H4-(86–100)) and H4-(86–100) at
10)8M as described in Experimental procedures, and secretion of IL-8 (GRO ⁄ CINC-1) was measured in culture supernatants Results are means ± SEM of three experiments (*significantly different from control at P 6 0.05).
Fig 6 The percentage of live ⁄ dead cells following treatment of AMs with TnT products of H4-v.1 and HN cDNA constructs (20 lL reaction samples) and corresponding synthetic peptide products (10)8M ) was determined as described in Experimental procedures
by assessing the number of living cells, which take up calcein and convert it to F-calcein (green fluorescence), and dead cells, which take up ethidium bromide homodimer (red fluorescence).
Trang 8Each class of histones contains its gene variants Bovine
H4-v.1, the first reported example of a histone H4
mRNA variant in mammals [41], contains the
palin-dromic and purine-rich sequences typical of cell
cycle-dependent histone mRNAs with a 1.3 kb downstream
extension that terminates with a polyadenylated track
characteristically found in cell cycle-independent
his-tone mRNAs The present results indicate that rat
H4-v.1 cDNA differs somewhat from bovine H4-H4-v.1 cDNA
by the absence of the consensus palindromic and
pur-ine-rich sequences and the excision of an intron, two
characteristics of the Drosophila
replication-independ-ent histone H4 cDNA [54] Yet, like bovine H4-v.1 and
all other histone cDNA variants, rat H4-v.1 cDNA
contains a 3¢UTR extension that terminates with a
polyadenylation signal (AATAAA) On the other hand,
HN cDNA, the second polyadenylated histone
H4-rela-ted cDNA observed in rat, contains noncanonical
pal-indromic and purine-rich sequences in its proximal
3¢UTR and a noncanonical TATAAA polyadenylation
signal [55] in its distal 3¢UTR (Table 1; Fig 2)
Inter-estingly, whereas the presence or absence of intronic
and palindromic sequences varies among subtypes of
replication-independent histone mRNAs [56], the
pres-ence of a polyadenylation signal is typical of all
replica-tion-independent histone mRNAs [2], suggesting that
the expression of both H4-v.1 and HN genes may be
independent of the cell cycle
Even though the promoter regions of core histone
H4 genes are evolutionarily divergent among
verteb-rate species [47], they all contain an upstream region
named site II, consisting of the cell cycle control
ele-ment, H4 gene subtype eleele-ment, and TATA box
[45,46] The site II region is considered to play a key
role in the cell cycle dependency of expression [46,57]
On the other hand, histone gene variants are not
expected to be regulated by the same factors that
regulate the expression of core histone genes, because
variant transcripts accumulate preferentially in
nondi-viding and terminally differentiated cells [3] Analysis
of genomic sequences upstream of the H4-v.1 and
HN genes indicated that both sequences contain a
region similar to the histone H4 site II cell cycle
regu-latory domain (Table 1) [46] This finding suggests
that this region may not be the sole determinant for
cell cycle dependency of histone H4 gene expression
Further analyses of the upstream genome region
proximal of the H4-v.1 and HN genes indicated the
presence of specific transcription regulator-binding
sequences that were not present upstream of either
somatic or germinal core histone H4 genes (Fig 2)
Among these specific regulatory factor-binding motifs, the CREB and NF-jB sites may play an important role in the tissue-specific expression of H4-v.1 and
HN mRNAs, respectively In this regard, marked release of immunoreactive HN from perfused bovine adrenal glands has been observed when the glands are stimulated with carbamylcholine [39] As carbachol was shown to be a potent activator of NF-jB in iso-lated canine gastric parietal cells [58], we may hypo-thesize that the NF-jB-binding element located 167 nucleotides upstream of the rat HN cDNA cap site has a role to play in the production and release of
HN Interestingly, in contrast with granule prestored enkephalins and catecholamines, which were suc-cinctly and rapidly released after carbamylcholine sti-mulation, the release of HN started only 30 min after the beginning of the stimulation and lasted for more than 1 h, thus allowing sufficient time for transcrip-tion factor activatranscrip-tion, HN mRNA formatranscrip-tion and translation [39]
Post-transcriptional control of cell cycle-dependent histone mRNAs is monitored by the stem–loop struc-ture present within their 3¢UTRs [20] H4-v.1 mRNA does not contain a stem–loop sequence, whereas HN possesses a stem–loop sequence that differs from the histone stem–loop consensus sequence observed in H4g
or H4s (Table 1) However, as both the H4-v.1 and
HN genes are polyadenylated, their post-transcrip-tional maturation and processing are expected to be regulated like those of polyadenylated mRNAs [47] Interestingly, the 3¢UTRs in H4-v.1 and HN mRNAs also contain TAAA repeat elements (14 and 4 repeats, respectively) that are known to be involved in the post-transcriptional regulation of IL-2 in lymphoid cells [49] The presence of this repeat element in H4-v.1 and HN mRNAs may explain the particularly high abundance of these genes in lymphoid tissues (Fig 1) along with OGP and HN [39,42] In this regard, H4-v.1 and HN cDNA transcripts, as well as C-terminal H4 and HN related-peptides, were shown to be present
in nonreplicating and terminally differentiated rat AMs (Fig 1B) [42] Synthesis, storage, processing and release of C-terminal histone H4 and related peptides were suggested to follow the same route as cytokines [42], which are stored in microvesicles and processed and released via a lysosomal pathway [59] The distinct induction of the expression of the H4-v.1 and HN genes by the immunostimulants LPS and IFN-c in AMs suggests that the two genes may have distinct and⁄ or complementary functions in response to immu-nostimulants, whereas the noninduction of core H4s
by the same agents concurs with its known cell replica-tion dependency (Fig 1B)
Trang 9Examination of the ORF of rat H4-v.1, H4s and
H4g cDNAs revealed the presence of two ATG
initi-ation codons that allow their transliniti-ation into whole
histone H4 and the alternative C-terminal fragment
H4-(84–102) (Fig 3) Interestingly, the 5¢UTR of rat
H4.v-1 cDNA (19 nucleotides; Fig 2) was much
shor-ter than those of H4s and H4g cDNAs (> 100
nucleo-tides) The short 5¢UTR in rat H4-v.1 mRNA may
enhance leaky ribosomal scanning, as the first ATG
codon could be too close to the 5¢ end to be
recog-nized efficiently [60] Such a possibility is supported by
a previous observation indicating that LPS stimulation
of the expression of H4-v.1 mRNA in rat AMs is
accompanied by an increase in the cell contents of the
short peptides OGP and H4-(86–100), but not of
whole histone H4 or total H4 mRNA [42] The in vitro
biosynthesis experiments with the H4-v.1 cDNA
con-struct also indicate that at least part of the first ATG
codon may be skipped to produce the C-terminal
pep-tide H4-(84–102) (Fig 4A) The relatively high level of
production of complete histone H4 as compared with
H4-(84–102) by the H4-v.1 cDNA construct may be
due to the elongation of the 5¢UTR in the cDNA
con-struct by 5¢RACE Marathon adapter and 5¢ b-globin
leader sequences in the pCMVTnT vector On the
other hand, Bab et al [61] used a histone H4-CAT
reporter fused cDNA vector engineered to produce a
polyadenylated histone H4 mRNA The recombinant
construct produced different ratios of whole histone
H4-CAT and H4-(84–102)-CAT, depending upon the
cell type in which the vector was expressed Further
investigation is required to clarify whether, in the
in vivosituation, rat H4-v.1 synthesizes both whole
his-tone H4 and H4-(84–102) or mainly H4-(84–102), as
suggested by our previous experiments with
LPS-sti-mulated rat AMs [42]
Use of the coupled transcription⁄ translation system
with the HN pCMVTnT construct produced a single
radiolabeled compound with a molecular mass
corres-ponding to that of the expected translation product
[Thr89]H4-(84–102) The HN cDNA ORF has the
necessary translational start and stop codons to
pro-duce this modified H4 C-terminal peptide, but not the
first start codon necessary to produce total histone H4
(Figs 2B, 3) The ability of HN mRNA to translate a
small peptide allows the messenger to be considered as
a minigene Minigenes are well recognized for their
role in the regulation of gene expression [62–65]
With-out including the small interfering RNAs (siRNAs),
which affect gene expression but cannot be considered
as true genes, due to their lack of an ORF, there exist
at least two types of minigene that can affect
transcrip-tional or post-transcriptranscrip-tional gene expressions For
instance, Tenson et al [65] reported that the transla-tion of a minigene with an ORF coding for a peptide
of eight amino acids or fewer inhibits protein synthesis
by a phenomenon of ‘dropping off’ of the peptide from ribosomes under a form that is still attached to the tRNA corresponding to its C-terminal amino acid, thus creating a shortage of this tRNA for translation Other minigenes selectively inhibit the translation of the functional downstream cistron [63,64] As the bio-synthetic products of H4-v.1 and HN cDNA constructs display anti-inflammatory effects in isolated AMs com-parable to those of the synthetic H4 C-terminal pep-tides H4-(86–100) and [Thr89]H4-(86–100) (Figs 4B, 5), the question of whether the expression of these H4 mRNA variants can affect some transcriptional
or post-transcriptional gene regulatory mechanisms, in addition to producing the extracellularly acting anti-inflammatory peptides, remains to be investigated
In conclusion, a growing body of evidence indicates that various histones or histone-derived products act
in an extranuclear and⁄ or extracellular manner Such examples include the histones H2A and H2B, which display growth hormone- and prolactin-releasing activity [20–23], and the antimicrobial histone-H2A peptide buforin I, produced by the action of pepsin within gastric gland cells of the vertebrate stomach [28,29] In addition, the histone H4-derived peptides
HN and OGP have been shown to display antinoci-ceptive and osteogenic activities, respectively [32–37]; whereas synthetic C-terminal histone H4 peptides were reported to serve as potent epitopes for antigen-presenting cells in in vitro models of T-lymphocyte activation [25] In the present study, we further dem-onstrate that the C-terminal histone H4-related pep-tides transcribed from H4-v.1 and HN genes significantly inhibit the LPS-evoked release of PGE2
and IL-8 (GRO⁄ CINC-1), two potent
proinflammato-ry mediators produced by activated macrophages The particular interest in the effects of H4-(86–100) and [Thr89]H4-(86–100) (or rat HN) on AM GRO⁄
CINC-1 secretion derives from the knowledge that IL-8 pro-duction represents one of the primary responses of macrophages to inflammation, and that such an effect lasts as long as inflammation persists [69] IL-8 not only serves to attract inflammatory cells to a site of inflammation and keep them there, but also stimulates neutrophils to a higher activation state Its release from macrophages is evoked by LPS and cytokines such as IL-1, and its high plasma level is associated with various human inflammatory diseases [68,69] Therefore, the presence of the H4-v.1 and HN genes
in tissues (thymus, bone marrow) wherein immune reactions are known to take place and the potent
Trang 10inhibitory effects of their translation products on
macrophage proinflammatory functions suggest that
histone H4 mRNA variants may have an important
role in the physiology and⁄ or physiopathology of
inflammation
Experimental procedures
Computer-assisted analysis of genomic sequence
The National Center for Biotechnology Information
(NCBI; http://www.ncbi.nlm.nih.gov/BLAST/) and The
Institute for Genomic Research (TIgr; http://www.tigt.org/
tdb/tgi/) blast programs were used to gather information
regarding possible H4-related sequences carrying a
down-stream polyadenylation signal or coding for modified H4
proteins GSPs were made with the aid of the primer3:
www primer tool (http://biotools.umassmed.edu/bioapps/
primer3_http://www.cgi) Computer-assisted analysis of
potential upstream transcription factor-binding sites of the
H4-v.1 and HN genes was done using the matinspector
program (http://www.genomatix.de/cgi-bin/./eldorado/
main.pl) Comparison of upstream sequences with
homol-ogy to the site II cell cycle regulatory domain of vertebrate
H4 genes was done using the lalign program (http://
www2.igh.cnrs.fr/bin/lalign-guess.cgi) Analysis of potential
palindromic sequences within the 3¢UTRs of the mRNAs
was done with the aid of the mfold program (http://
biotools.idtdna.com/Analyzer/)
Real-time RT-PCR
Reverse transcription was performed on 250 ng of rat
(Sprague-Dawley) tissue total RNA preparations (RNeasy
Mini Kit for total RNA isolation; Qiagen Mississauga,
Canada), pretreated with amplification grade DNase 1
(Invitrogen, Burlington, Canada), using database-deduced
gene-specific H4-v.1 (5¢-ccagggttttgtttgtttttg-3¢), HN (5¢-ca
cagcctgatggtgtggattggtg-3¢) and GAPDH (5¢-aggtcaat
gaaggggtcgttg-3¢) antisense primers and 4 U of omniscript
reverse transcriptase (Qiagen) (where U is defined as
enzyme activity which incorporates 1 nmol TTP into
acid-insoluble products in 10 min at 37C with poly A template
RNA and oligo-dT12–18 primer) Real-time PCR was
per-formed using a standard QuantitectTMSYBRRGreen PCR
kit (Qiagen) protocol on an Applied Biosystems (Foster
City, CA) 7900HT Sequence Detection System PCR
amplifications (40 cycles) were performed using designed
rat H4-v.1 (sense, 5¢-ggcggctaagaaacaaagtg-3¢; antisense,
5¢-gaaaagttgggtggaagcaa-3¢) or rat HN (sense, 5¢-gccat
ggatgtggtctatact-3¢; antisense, 5¢-gccgaagccatagagagtg-3¢)
primers and the QuantiTect SYBR Green PCR Master Mix
(Qiagen) Validation was done with GAPDH using
rat-spe-cific GAPDH (sense, 5¢-aatggtgaaggtcggtgtgaac-3¢;
anti-sense, 5¢-aggtcaatgaaggggtcgttg-3¢) primers The relative quantification of mRNA transcripts was carried out by the comparative Ct (cycle threshold) method, the theoretical basis of which has previously been described in detail [66] Amplicons were cloned into the pCR 4-TOPO vector 2.0 using the TOPO TA cloning kit for sequencing (Invitro-gen), transformed, plated as outlined within Invitrogen’s TOPO TA kit manual, and sequenced with a DNA seq-uencer (AIB automatic seqseq-uencer; Biotechnology Research Institute, BMI Department, University of Ottawa)
5¢RACE and 3¢RACE of rat H4-v.1 and HN mRNAs
in a rat spleen cDNA library Full-length rat (Sprague-Dawley) H4-v.1 and HN cDNAs were obtained using 0.5 ng of a Marathon-ReadyTM spleen cDNA library (BD Biosciences Clontech, Paolo Alto, CA)
by two successive rounds of PCR with outside and nested gene-specific H4-v.1 and HN primers and the adapter sequence-specific amplification primers (APs) supplied with the Marathon-Ready spleen cDNA kit (AP1 and nested AP2 primers) The initial H4-v.1 5¢RACE and 3¢RACE reaction round used an H4-v.1 5¢-GSP (5¢-H4-v.1 GSP1: 5¢-tatagacatgcctgtagtatctgaacc-3¢) coupled with the adapter primer AP1 (5¢-ccatcctaatacgactcactatagggc-3¢), and an H4-v.1 3¢-GSP (3¢-H4-H4-v.1 GSP1: 5¢-ctacacggagcacgccaag-3¢) coupled with AP1 The initial HN 5¢RACE and 3¢RACE reaction round used an HN 5¢-GSP (5¢-HN GSP1: 5¢-aga ggtcctgagttcaattgct-3¢) coupled with AP1, and an HN 3¢-GSP (3¢-HN 3¢-GSP1: 5¢-ctaagcgcccaccgcaaagtcttg-3¢) coupled with AP1 First-round RACE PCR reactions included 2.5 U (where U is defined as enzyme activity which incor-porates 10 nmol dNTPs into acid-insoluble amplicon in
30 min at 72C) of pfuUltra Hotstart DNA polymerase (Stratagene, La Jolla, CA) and were performed in accord-ance with Stratagene’s PCR protocol, using 30 cycles of amplification Nested 5¢-H4-v.1 or 3¢-H4-v.1 RACE PCR reactions were conducted using a 1 : 100 dilution of the first PCR reactions with either the H4-v.1 5¢-nested GSP (5¢-H4-v.1 GSP2: 5¢-ccagggttttgtttgtttttg-3¢) coupled with AP2 (5¢-actcactatagggctcgagcggc-3¢), or the H4-v.1 3¢-nested GSP (3¢-H4-v.1 GSP2: 5¢-ccaagactaataaaataaacctgaagg-3¢) coupled with AP2 Nested 5¢ and 3¢ HN RACE reactions were conducted as above, with an HN 5¢-nested GSP
(5¢-HN GSP2: 5¢-tggcgcttgagagtatagacc-3¢) coupled with AP2, and an HN 3¢-nested GSP (3¢-HN GSP2: 5¢-ggatgtggtcta-tactctcaagc-3¢) coupled with AP2 The second-round nested RACE PCR reaction included 2.5 U of HotstarTaq DNA polymerase (Qiagen), and was performed in accordance with the manufacturer’s PCR protocol, with 25 cycles of amplification Amplicons were cloned into the pCR 4-TOPO vector 2.0 using the 4-TOPO TA cloning kit for sequencing (Invitrogen), transformed and plated as outlined
in Invitrogen’s TOPO TA kit manual, and sequenced