Download c/ebp beta is involved in the regulation of tissue specific expression

C/EBP BETA IS INVOLVED IN THE REGULATION OF TISSUE SPECIFIC EXPRESSION OF CARTILAGE-DERIVED
RETINOIC ACID-SENSITIVE PROTEIN
*OKAZAKI, K (A-NIH); *LI, J (A-NIH); +*SANDELL, L J (A-NIH)
+*Washington University School of Medicine at Barnes-Jewish Hospital, Department of Orthopaedic Surgery, St. Louis, MO. 314-454-7800, Fax: 314-454-5900,
[email protected]
Introduction
Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a secretory
protein primarily expressed in chondrocytes, but can be stimulated in
chondrosarcoma, melanoma and breast cancer1)2). CD-RAP is thought to affect
the metastatic activities of tumor cells by interfering with the attachment of
cells to the extracellular matrix3)4), but its physiological function in
chondrogenesis is unclear. During embryonic development, CD-RAP is
expressed from the beginning of chondrogenesis and the distribution of the
expression is restricted to cartilage with an only exception being the
mammary bud. We have previously generated transgenic mice harbouring
2251 bp or 2068 bp promoter of CD-RAP linked to β-galactosidase reporter
gene. The analysis of the transgene expression indicated that the 2251 bp
promoter induced the reporter gene expression in all cartilage, but the 2068 bp
promoter did not induce any expression5). This suggests that the 183 bp
fragment between –2251 to –2068 may contain important elements
responsible to cartilage specific expression. In this study, transcription factors
that bind to the 183 bp fragment were analyzed. We report here the
characterization and function of a binding protein, C/EBPβ, a member of the
CAAT/enhancer binding protein family of transcription factors, that binds to
the 183 bp fragment and acts as an repressor of CD-RAP expression in
undifferentiated C5.18 cells.
Methods
Identification of the factors which bind to the 183 bp fragment.
As the database analysis revealed a lot of potential elements around –2100, A
fragment from –2039 to –2119 was amplified by PCR, and used as a probe for
the electrophoretic mobility shift assay (EMSA). The nuclear extract was
isolated from C5.18, rat chondrogenic cell line, and various fragments within
the probe sequence were used as cold competitors. For supershift analysis,
various antibodies were incubated prior to adding the probe. The molecular
size of C/EBPβ in the nuclear extract was analysed by Western blot.
Transient transfection assay
Various promoters, 2251 and 2068, were subcloned into the luciferase reporter
plasmid pGL3-basic. Site-directed mutagenesis was performed within the
C/EBPβ binding site in the 2251-pGL3b. Each plasmid was co-transfected
with CMV-β-Gal internal control plasmid into C5.18 cells. The luciferase
activities were assayed and normalised to the β-galactosidase value.
tissue specific expression. Our previous study of transgenic mice showed that
2251 promoter is sufficient for the tissue specific expression in vivo, but 2068
(without the 183) is not. In preliminary studies, we have expressed a longer
CD-RAP promoter (3345 bp) that generates strong tissue-specific expression.
When the 183 bp fragment is removed from this construct, CD-RAP is
expressed widely in the animal. Consequently, the 183 bp fragment could be
absolutely critical for the chondrocyte-specific expression of CD-RAP. This
factor may also be important for other cartilage-specific gene expression.
C5.18 NE
Competitor
C/EBPβ antibody
+
-
+
cold
-
+
mu
-
+
-
+
+
Fig.1 EMSA for C/EBPβ binding site with C5.18 nuclear extract.
Competitor: cold; 100-fold excess cold probe, mu; C/EBP binding site-mutant
Results
The EMSA analysis revealed that C/EBPβ binds within the183 bp fragment,
from nucleotide –2074 to –2082 (Fig 1). Western blot analysis of the C5.18
nuclear extract showed that C/EBPβ was present as both the longer isoform of
38kDa (called LAP) and the truncated one of 20kDa (called LIP). Sitedirected mutagenesis at the C/EBP β binding site in the 2251 promoter
showed about 2-fold up regulation of luciferase activities compared to the
wild type 2251 promoter by transient transfection in C5.18 cells (Fig 2).
Discussion
The 183 bp CD-RAP promoter fragment contains indispensable elements for
tissue specific expression of CD-RAP. C/EBPβ binds to this DNA and
functions as a repressor. C/EBPβ is a member of CAAT/enhancer binding
protein family, which is a prototype family of basic leucine zipper
transcription factors. C/EBPβ can act as a repressor or activator depending on
the concentration of subunits LIP and LAP in the nucleus: that is, LAP forms
dimers with itself or other C/EBP that enhance gene expression while LIP
forms a heterodimer with LAP or other C/EBP and represses gene
expression6). C5.18 has features of undifferentiated mesenchymal cells and
has the potential to obtain chondrocytic phenotypes during extended culture
after confluence. The presence of both subunits of C/EBPβ in C5.18 cells is
consistent with the low level of CD-RAP expression, and the repressor
activity C/EBPβ. These data suggest that C/EBPβ may function to suppress
the CD-RAP expression in mesenchymal cells and contributes to its restricted
Fig 2. Relative luciferase activities of C5.18 cells transfected with luciferase
reporter vectors which contains 2251 promoter or the 2251 promoter
harbouring C/EBP binding site-mutant.
References
1.Dietz UH, Sandell LJ J. Biol. Chem. 271:3311-3316
2. Bosserhoff AK, Moser M, et al. J. Pathol. 187:446-454, 1999
3. Guba M, Bosserhoff AK, et al. Br. J. Cancer 83:1216-1222, 2000
4. Stoll R, Renner C, et al. EMBO J 20:340-349, 2001
5. Xie WF, Zhang X, Sandell LJ Matrix Biol. 19:501-509, 2000
6. Descombes P, Schibler U Cell 67:569-579, 1991
48th Annual Meeting of the Orthopaedic Research Society
Poster No: 0325