Download Rat maf related genes: specific expression in chondrocytes

Oncogene (1997) 14, 745 ± 750
 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
SHORT REPORT
Rat maf related genes: speci®c expression in chondrocytes, lens and spinal
cord
Masaharu Sakai1, Junko Imaki2, Kazuhiko Yoshida1, Akihiko Ogata1,
Yuko Matsushima-Hibiya1, Yoshinori Kuboki3, Makoto Nishizawa4 and Shinzo Nishi1
1
Department of Biochemistry, School of Medicine and 3Department of Biochemistry, School of Dentistry, Hokkaido University N15
W7, Kita-ku, Sapporo 060; 2Department of Anatomy, Nippon Medical College, Sendagi, Bunkyo-ku, Tokyo 113; 4Department of
Molecular Oncology, Kyoto University, Faculty of Medicine, Yoshida-Konoecho, Sakyo-ku, Kyoto 606, Japan
maf is a family of oncogenes originally identi®ed from
avian oncogenic retrovirus, AS42, encoding a nuclear
bZip transcription factor. We have isolated two maf
related cDNA clones, maf-1 and maf-2, from a rat liver
cDNA library. Comparison of the sequence homologies
of the proteins encoded by maf-1 and maf-2 with those of
c-maf and chicken mafB indicated that maf-1 and maf-2
are the rat homologues of mafB and c-maf, respectively.
Both genes are expressed at low levels in a wide variety
of rat tissues, including spleen, kidney, muscle and liver.
Immunohistochemical studies and in situ hybridization
analyses show that maf-1 and maf-2 are strongly
expressed in the late stages of chondrocyte development
in the femur epiphysis and the rib and limb cartilage of
15 day old (E15) embryo in rat. Cartilage cells, induced
by subcutaneous implantation of bone morphogenic
protein, also expressed maf-1 and maf-2. In situ
hybridization analyses of E15 embryos show that both
genes are expressed in the eye lens and the spinal cord as
well as the cartilage. However, the expression patterns of
maf-1 and maf-2 in lens and spinal cord are di€erent.
Keywords: maf; transcription factor; chondrocyte; lens;
spinal cord
The maf oncogene (v-maf) was initially identi®ed from
an avian oncogenic retrovirus, AS42, which induces
musculoaponeurotic ®brosarcoma in vivo and transforms chicken embryo ®broblast cells in vitro
(Nishizawa et al., 1989). The v-maf oncogene product
is a transcription factor containing a typical bZip DNA
binding domain. The binding consensus sequence of
the v-maf product (Maf recognition element, MARE, TGCTGACTCAGCA-) overlaps with the TPA responsive element (TRE), and cyclic AMP responsive
element (CRE) (Kataoka et al., 1994b). The maf
oncogene products can form homo- and hetero-dimers
with transcription factors having bZip structures, such
as Jun and Fos (Kataoka et al., 1994a,b, 1996;
Kerppola and Curran, 1994). Furthermore, several
maf-related genes have been identi®ed (i.e. mafB,
mafK, mafG, mafF and Nrl) (Fujiwara et al., 1993;
Kataoka et al., 1994a, 1995; Swaroop et al., 1992).
Interestingly, mafK, mafG and mafF encode small
proteins in which the N-terminal putative transactiva-
Correspondence: M Sakai
Received 23 May 1996; revised 7 October 1996; accepted 8 October
1996
tion domain is truncated. MafK forms a heterodimer
with NF-E2 p45, a transcription factor that regulates
the globin gene (Andrews et al., 1993; Igarashi et al.,
1994). Recently it was found that MafB interacts with
Ets-1, a transcription factor containing a helix ± turn ±
helix DNA binding domain, inhibits Ets-1 activity,
thus interrupting erythroid di€erentiation (Sieweke et
al., 1996). Maf-family transcription factors heterodimerize not only with bZip proteins, but also with
helix ± turn ± helix proteins. Thus, a wide variety of
Maf-heterodimers are formed, which in turn could
regulate a wide variety of target genes depending on
which heterodimer is formed. The mouse mafB gene
was identi®ed as the gene responsible for the
segmentation of hind brain during early development
by analyses of kreislar (Kr) mutant mice (Cordes and
Barsh, 1994). These ®ndings indicate that Maf
oncogene products play an important role in morphogenic processes and cellular di€erentiation.
Determination of maf expression should facilitate
identi®cation of target genes and would thus further
clarify maf and target gene functions. In this report, we
isolated two maf related cDNA clones, maf-1 and maf2, from a rat liver cDNA library and studied the
expression of these genes.
Approximately 16106 recombinant phages from a
rat liver cDNA library were screened with a v-maf
probe, and two clones, maf-1 and maf-2, were obtained
(nucleotide sequences of both cDNAs were submitted
to GenBank/EMBL/DDBJ DNA database, accession
numbers are U56241 and U56242 for maf-1 and maf-2,
respectively).
The predicted amino acid sequences of Maf-1 and
Maf-2 show great similarity with that of v-Maf. The
overall amino acid sequence homologies of Maf-1 and
Maf-2 with those of chicken MafB and c-Maf
(Kataoka et al., 1994a) are 84% and 81%, respectively; thus rat Maf-1 and Maf-2 appear to be the rat
homologues of MafB and c-Maf. The bZip structures
of all Mafs are conserved at more than 80% homology.
This suggests that these Maf-related proteins recognize
similar sequence elements. The DNA binding analyses
using E coli expressed proteins show that both Maf-1
and Maf-2 bind with almost equal anity to the
MARE consensus sequence. However, when subtle
variations of this sequence were used, Maf-1, Maf-2
and v-Maf showed signi®cant di€erences in binding
anity (manuscript in preparation).
Maf-1 is almost identical (98% homology) with the
Kr gene product, a mouse mafB homologue (Cordes
and Barsh, 1994).
Expression of Rat maf-related oncogenes
M Sakai et al
— Uterus
— Muscle
— Bone marrow
—
—
—
—
—
— Stomach
— Intestine
Brain
Heart
Lung
Spleen
Liver
—
— Kidney
M
maf1
maf2
➝
Figure 1 Transactivation of MARE-luciferase gene by Mafs.
MARE (-TGCTGACTCAGCA-) was ligated into the luciferase
expression vector, pGV-P (Nippon Gene, Toyama, Japan), and
co-transfected with indicated expression plasmids into HepG2
cells, a human hepatoma cell line. Transfection and determination
of luciferase activity were done as described (Sakai et al., 1995).
The v-maf and c-jun expression plasmids were previously
described (Sakai et al., 1995). Expression plasmids of maf-1 and
maf-2 were constructed by inserting the cDNA fragments
corresponding to amino acids 18-C-terminus and 19-C-terminus
for maf-1 and maf-2, respectively, into a human b-actin expression
vector (Gunning et al., 1987). Vec indicates expression vector
without insert
hybridization studies using 15 day old (E15) rat
embryos clearly show that maf-2 is strongly expressed
in the cartilage of ribs (Figure 3c) and limbs (Figure
3d). Almost identical results were obtained using the
maf-1 probe. These ®ndings suggest that Mafs are
closely associated with a chondrocyte di€erentiation
process not only in the adult epiphysis but also in
embryonic cartilage tissue. To con®rm the expression
of Mafs in chondrocytes, we used a BMP (bone
morphogenic protein) implantation system to induce
cartilage formation. BMP stimulates immature mesenchymal cells to create a process similar to
endochondral ossi®cation when they are subcuta-
➝
The transactivation functions of the proteins
encoded by maf-1 and maf-2 were con®rmed by a cotransfection assay using a fusion construct consisting of
a MARE and luciferase gene. Figure 1 shows both
maf-1 and maf-2 activate the gene containing MARE
with activities similar to those of v-maf. The activities
were much stronger than c-jun, although MARE
contained complete TRE (TPA responsive element, TGACTCAG-) sequence.
We investigated the tissue distribution of maf-1 and
maf-2 mRNAs by RNase protection analysis (Figure
2). maf-1 is expressed at low levels in a variety of
organs including spleen, muscle and liver. The tissue
distribution of maf-2 mRNA is similar to that of maf1, but not identical.
Immunohistochemical staining by speci®c antisera
against Maf-1 and Maf-2 did not detect any signi®cant
positive signals in liver, though mRNA is expressed
there (Figure 2). However, anti-Maf antibodies
strongly and speci®cally stained chondrocyte nuclei
located in the hypertrophic chondrocytes of the femur
epiphysis, and the immature proliferating chondrocytes
were stained very weakly (Figure 3a and b).
Hypertrophic chondrocytes are maturated non-dividing cells in chondrocyte di€erentiation. The antiserum
against Maf-1 yields almost the same results as
obtained with anti-Maf-2 antiserum. RNase protection
analyses detected mRNA of both mafs from femur
epiphysis but signals were weak (data not shown). This
is probably due to the fact that the expressed cells are
low in number in the dissected samples of femur
epiphysis as shown in Figure 3a and b. In situ
➝
746
GAPDH
Figure 2 Expression of maf-1 and maf-2 mRNA in rat tissues,
examined by RNase protection analysis. The RNAs from various
tissues were prepared using an RNA extraction kit (Nippon Gene
Co. Toyama, Japan). Nucleotides 829 ± 1037 and 1248 ± 1417 of
the maf-1 and maf-2 clones, respectively, were inserted into
pBluescript II vector (Stratagene, California, USA), and antisense RNA probes were prepared (approximately 16109 c.p.m./
mg). Ten micrograms of the total RNAs from indicated tissues
were used for RNase protection analysis as described previously
(Sakai et al., 1992). The same preparations of RNAs were
quantitated using an anti-sense probe for glyceraldehyde 3phosphate dehydrogenase (GAPDH). HpaII digested pUC18
DNA, terminally labeled by 32P, was used as a size marker
(M). Autoradiograms were exposed for 2 days and overnight for
mafs and GAPDH, respectively. This experiment was done three
times with nearly identical results
Expression of Rat maf-related oncogenes
M Sakai et al
747
Figure 3 Maf expression in chondrocytes. (a and b): The speci®c antibodies of Maf-1 and Maf-2 were prepared as follows:
Fragments of Maf-1 and Maf-2 proteins were produced in E coli as fusion proteins with maltose binding protein and glutathione Stransferase, respectively. maf-1 cDNA fragment corresponding to amino acids 49 ± 122 was inserted into pMal-c2 vector (New
England Biolab, USA) and a Maf-2 cDNA sequence encoding amino acids 64 ± 158 was inserted into pGEX-T2 vector (Pharmacia
LKB, Sweden). The fusion proteins were puri®ed according to the supplier's instructions, used to immunize rabbits, and antisera
were anity puri®ed by the corresponding antigens. Rats were sacri®ced by perfusion ®xation via the aorta with 4% freshly
prepared paraformaldehyde, and axial sections of knee joints were made. The sections were stained by anti-Maf-1 antibody using an
avidin-biotin-peroxidase complex method. (c and d): Expression of maf-2 mRNA in E15 rat ribs (c) and limbs (d) analysed by in situ
hybridization. The sections were dried and immersed in 1% Triton X-100 in 50 mM Tris/HCl, 25 mM EDTA (pH 8.0) and
acetylated with acetic anhydride. The sections were dehydrated and hybridized with the probes (16106 d.p.m./ml) at 55 ± 608C
overnight. Slides were rinsed in 46SSC, digested with RNase A (20 mg/ml) and washed sequentially in 26SSC, 16SSC, 0.56SSC,
then for 30 min in 0.16SSC at 608C. The sections were exposed to X-ray ®lm (Kodak X-omat) for 3 days, then dipped in NTB2
nuclear emulsion, (1 : 1 with water, Kodak), and exposed for 2 weeks. Radioactive RNA probes of maf-1 and maf-2 (nucleotides
195 ± 410 and 1248 ± 14170, respectively) were synthesized with [a-35S]UTP by T7 RNA polymerase. As a control for nonspeci®c
labeling, the adjoining series were treated with RNase A (20 mg/ml) for 30 min at 378C and then hybridized as above. In control
experiments, adjacent sections were treated with RNase before hybridization or sense-strand cRNA probes were used for
hybridization. Scale bar equals 480 mm for a, 100 mm for b, 50 mm for c and 250 mm for d
neously implanted with an insoluble carrier (Urist et
al., 1984). When a ®brous glass membrane is used as a
BMP carrier, cartilage formation is induced. Induction
of cartilage cells in this system is evidenced by type II
collagen synthesis (Kuboki et al., 1995). The ®brous
glass implants were removed after 2 weeks and
examined by Alcian blue staining and immunohistochemical staining using anti-Maf-1 antiserum. Alcian
blue stains proteoglycan is known to be speci®cally
produced by chondrocytes. Figure 4a and b show that
anti-Maf-1 positive cells correspond to the Alcian blue
stained cells. Both antisera, anti-Maf-1 and anti-Maf-2
gave apparently identical results. As in the epiphysis
chondrocytes (Figure 3a and b), the signals of bigger,
hypertrophic-like cells were stronger than those of
small, immature cells. RNase protection analyses in the
BMP implanted tissues were carried out (Figure 4c).
The implants, 1 and 2 weeks after implantation, were
removed and total RNAs were analysed by RNase
protection analyses together with RNAs from liver and
kidney as references. Figure 4c shows that maf-1 is
expressed strongly in both 1 and 2 week old implants.
maf-2 is also expressed, though not as strongly, and the
expression decreased at 2 weeks.
In situ hybridization studies of E15 rat embryos
indicate that both mafs are strongly expressed not only
in cartilage tissue but also in the eyes and spinal cord,
and have similar hybridization patterns. However,
when examined in detail, the expression locations
were di€erent in the eyes and spinal cord. The maf-1
and maf-2 genes were expressed in the lens but not in
the retina of the E15 rat eyes (Figure 5). Expression of
maf-1 was con®ned to the equator of the lens, while
maf-2 was distributed throughout the lens (Figure 5b
and c). Immunohistochemical staining of E16 rat lens
using speci®c antisera against Maf-2 protein strongly
and speci®cally stained nuclei in the outer equatorial
epithelium and lens ®bers (Figure 5d and e). The
Expression of Rat maf-related oncogenes
M Sakai et al
c
maf-1
➝
— 1W
— Kidney
—
— 2W
maf-2
➝
M
— Liver
BMP
Implanted
➝
748
GAPDH
Figure 4 Maf expression in BMP implanted tissues. BMP (0.3 mg) was absorbed to a piece of ®brous glass membrane GA-100
(106561 mm, Advantec Co. Tokyo, Japan) and was implanted subcutaneously in the back of rats (Wistar, male, 4 weeks old) as
described previously (Kuboki et al., 1995). After 1 or 2 weeks, the implants were removed and subjected to immunohistochemical
staining and RNA preparation. Two week old BMP implanted tissue was (a) stained with Alcian blue and hematoxylin, and (b) was
stained with anti-Maf-1 antiserum as described in the legend for Figure 3. (c) The total RNAs from implants were analysed by
RNase protection analysis using maf-1 (lower panel) and maf-2 (upper panel) anti-sense RNA probes as described in the legend for
Figure 2
developmental expression of maf-1 and maf-2 shows a
number of interesting correlations with the changing
pattern of cell proliferation and di€erentiation in the
developing rat lens. The most striking of these is the
observation that levels of both gene products rise in the
nuclei of the di€erentiating outer epithelial cells. It was
also interesting that maf-1 mRNA was con®ned to the
equator of the lens where the epithelial cells were just
di€erentiating to ®ber cells, in contrast to maf-2
mRNA which is detectable after the di€erentiation of
lens ®ber cells. These results suggest that both genes
play a role in the di€erentiation from epithelial cells to
®ber cells, whereas the maf-2 mRNA also participates
in the further di€erentiation of lens ®ber cells.
Figure 6 shows the mafs expression in spinal cord. It
is interesting that each gene is expressed in di€erent
regions of spinal cord. The maf-1 and maf-2 mRNA
positive cells in the spinal cord could be divided into
three groups according to a previous report about
somatostatin expression (Senba et al., 1982). The ®rst
two groups of cells are located in the dorsal part of the
dorsal horn and the ventral part of the dorsal horn,
and the third is located in the ventral horn. For E15, a
strong signal of maf-1 mRNA was observed in the
ventral part of the dorsal horn and the ventral horn of
the spinal cord. For maf-2, strong mRNA signals were
observed in the dorsal and ventral parts of the dorsal
horn and a much weaker signal was observed in the
Expression of Rat maf-related oncogenes
M Sakai et al
Figure 5 mafs expression in the lens. Sagittal sections of E15 rat
lens were (a) Nissl-stained, and in situ hybridization of mRNA for
(b) maf-1 and (c) maf-2 was performed. Immunoreactivity for
Maf-2 protein in the horizontal section of E16 rat lens (d) low
magni®cation and (e) high magni®cation. Bar indicates 250 mm
for a, b, c, d and 50 mm for e
ventral horn. Although details of cell lineages and
function of these regions in the spinal cord are not
known, the di€erent but related expression patterns of
maf-1 and maf-2 suggest there are slight functional
di€erences. This result shows that the initial differentiation and development of maf-1 and maf-2 mRNA
containing cells takes place during the prenatal period
of rats, before the establishment of normal synaptic
transmission.
Recently the expression pattern of Nrl, a member of
the maf-family transcription factor from mouse was
reported (Liu et al., 1996). Nrl is expressed mainly in
the retina of E14.5 embryo, but weak expression was
seen in the equatorial region of the lens where both
maf-1 and maf-2 are expressed. The maf-1 and maf-2
expression patterns are clearly di€erent from Nrl,
which is evenly expressed throughout the whole spinal
cord in E15.5. Both maf genes are expressed
predominantly in post-mitotic cells, suggesting that
the Mafs play a role in the ®nal di€erentiation process
in these cells. Nrl is also expressed in post-mitotic cells
of neurons (Liu et al., 1996). This gene family may play
a similar role though in di€erent cell types.
Study of the Kr mutant mouse indicates that mafB is
expressed in the rhombomere, and has an important
function in segmentation of the rhombomere in the
very early stages of development (Cordes and Barsh,
1994). This ®nding, together with our observations,
Figure 6 Expression of mafs mRNA in E15 rat spinal cord. Dark-®eld photographs of sagittal section in which mRNA was
detected by in situ hybridization (see Figure 3 legend) for (a) maf-1 and (b) maf-2. Autoradiographs of cross section of the spinal
cord for (c) maf-1 and (d) maf-2. Cresylviolet was used to stain the adjacent section in panel (e). Sense probes for maf-1 and maf-2
showed no hybridization (data not shown). Scale bar equals 150 mm
749
Expression of Rat maf-related oncogenes
M Sakai et al
750
strongly suggests that the Maf proteins not only have
an important role in hind brain morphogenesis in early
development, but also in the di€erentiation processes
of the cartilage, lenses and spinal cord. However, in Kr
mouse (i.e., a mutant strain of mafB), no abnormalities
of cartilage, lenses or spinal cord formation were
reported, except for a hyoid greater horn abnormality
(Frohman et al., 1993). It is possible that a functional
redundancy of MafB may be responsible for those
results as, in this study, both maf-1 and maf-2 were
shown to have similar, but not exact, expression
patterns.
In a preliminary study, Maf-1 could heterodimerized
with Fos family proteins (c-Fos, FosB, Fra1 and Fra2)
while Maf-2 could heterodimerized only c-Fos. These
heterodimers have signi®cantly di€erent DNA binding
speci®cities. It is quite possible that the many di€erent
heterodimers that can be formed are useful in enabling
a wide range of regulation of target genes with various
heterodimers having varying DNA binding speci®cities
and heterodimer protein components having varying
expression patterns. It was recently shown that lack of
the ATF-2 gene causes great abnormality in endochon-
dral ossi®cation at the epiphyseal plates (Reimold et
al., 1996). Since distribution of ATF-2 is similar to that
of Mafs, and ATF-2 can heterodimerize with Maf-1
and Maf-2 (manuscript in preparation), it is possible
that ATF-2/Maf(s) heterodimers function in chondrocyte di€erentiation.
Further analyses on the stage speci®c expression of
maf-1 and maf-2 in the cartilage, lens and spinal cord,
in conjunction with the identi®cation of speci®c genes
that are targets for transcriptional regulation by this
protein, should prove valuable in delineating the
molecular pathways underlying the regulation of
transcription during development in these tissues.
Acknowledgements
We thank Dr T Fujita, Tokyo Metropolitan Institute of
Gerontology, for kindly providing the rat liver cDNA
library and Dr John F Maune for critical reading of the
manuscript. This work was supported by grants from the
Ministry of Education, Science and Culture, Japan.
EMBL/Genbank accession No. for maf-1 and maf-2 are
maf-1: U56241 and maf-2: U56242.
References
Andrews NC, Kotkow KJ, Ney PA, Erdjument BH, Tempst
P and Orkin SH. (1993). Proc. Natl. Acad. Sci. USA, 90,
11488 ± 11492.
Cordes SP and Barsh GS. (1994). Cell, 79, 1025 ± 1034.
Frohman MA, Martin GR, Cordes SP, Halamek LP and
Barsh GS. (1993). Development, 117, 925 ± 936.
Fujiwara KT, Kataoka K and Nishizawa M. (1993).
Oncogene, 8, 2371 ± 2380.
Gunning P, Leavitt J, Muscat G, Ng SY and Kedes L. (1987).
Proc. Natl. Acad. Sci. USA, 84, 4831 ± 4835.
Igarashi K, Kataoka K, Itoh K, Hayashi N, Nishizawa M
and Yamamoto M. (1994). Nature, 367, 568 ± 572.
Kataoka K, Fujiwara KT, Noda M and Nishizawa M.
(1994a). Mol. Cell. Biol., 14, 7581 ± 7591.
Kataoka K, Noda M and Nishizawa M. (1994b). Mol. Cell.
Biol., 14, 700 ± 712.
Kataoka K, Igarashi K, Itoh K, Fujiwara KT, Noda M,
Yamamoto M and Nishizawa M. (1995). Mol. Cell. Biol.,
15, 2180 ± 2190.
Kataoka K, Noda M and Nishizawa M. (1996). Oncogene,
12, 53 ± 62.
Kerppola TK and Curran T. (1994). Oncogene, 9, 675 ±
684.
Kuboki Y, Saito T, Murata M, Takita H, Mizuno M, Inoue
M, Nagai N and Poole AR. (1995). Connective Tissue Res.,
32, 219 ± 226.
Liu Q, Ji X, Breitman ML, Hitchcock PF and Swaroop A.
(1996). Oncogene, 12, 207 ± 211.
Nishizawa M, Kataoka K, Goto N, Fujiwara KT and Kawai
S. (1989). Proc. Natl. Acad. Sci. USA, 86, 7711 ± 7715.
Reimold AM, Grusby MJ, Kosaras B, Fries JW, Mori R,
Maniwa S, Clauss IM, Collins T, Sidman RL, Glimcher
MJ and Glimcher LH. (1996). Nature, 379, 262 ± 265.
Sakai M, Matsushima HY, Nishizawa M and Nishi S. (1995).
Cancer Res., 55, 5370 ± 5376.
Sakai M, Muramatsu M and Nishi S. (1992). Biochem.
Biophys. Res. Commun., 187, 976 ± 983.
Senba E, Shiosaka S, Hara Y, Inagaki S, Sakanaka M,
Takatsuki K, Kawai Y and Tohyama M. (1982). J. Comp.
Neurol., 208, 54 ± 66.
Sieweke MH, Tekotte H, Frampton J and Graf T. (1996).
Cell, 85, 49 ± 60.
Swaroop A, Xu JZ, Pawar H, Jackson A, Skolnick C and
Agarwal N. (1992). Proc. Natl. Acad. Sci. USA, 89, 266 ±
270.
Urist MR, Huo YK, Brownell AG, Hohl WM, Buyske J,
Lietze A, Tempst P, Hunkapiller M and DeLange RJ.
(1984). Proc. Natl. Acad. Sci. USA, 81, 371 ± 375.