Generation of tartrate resistant acid phosphatase (TRAP) over

27 Pages·2009·0.84 MB·English

Generati o n o f t a r tra t e r e s ista n t a c id
phosphata s e o v er-express in g a n d k n ock-down
mod e ls f o r d i f ferentiati o n a n d p r oliferation
stud i e s o f m e senchym a l li n ea g e c e lls
Christ in a P a tlaka
Degr e e p r oje c t i n a p pli e d b i o technolo g y , M a st e r o f S c ien c e ( 2 y e ar s ) , 2 0 09
Examensarbe t e i t i l l ämp a d b i o tekn i k 3 0 h p t i l l m a sterexame n , 2 0 09
Biolo g y E d ucati o n C e nt r e , U p ps a l a U n iversi t y , a n d D i visi o n o f P a tholo g y , D e partme n t o f L a boratory
Medicin e , K a rolins k a In s titutet,Karolins k a U n ivers i t y H o spi t a l H u dding e , S t o ckholm
Superviso r s : P r ofess o r G ö r a n A n derss o n P h D a n d P e rn il l a L å n g P h D
Summary
Tartrate resistant acid phosphatase (TRAP) is a metalloenzyme, mostly expressed in
osteoclasts and macrophages. TRAP exists in a monomeric enzymatically inactive and a
cleaved enzymatically active isoform. In osteoclasts, TRAP has been suggested to participate
in bone resorption and to be secreted by osteoclasts. Recent data has additionally shown that
TRAP induces differentiation of pre-osteoblasts, while monomeric but not cleaved TRAP
induces proliferation and differentiation in adipocyte precursor cells which, just as
osteoblasts, are of mesenchymal stem cell origin. In light of these data, TRAP has been
suggested to function as a growth factor and that there are differential roles of the uncleaved
and cleaved isoform.
The aim of the project was the creation of TRAP-over-expressing and knock-down pre-
osteoblasts, pre-adipocytes and fibroblasts for future investigation of the role of TRAP-
expression during their differentiation and proliferation. Moreover, an attempt to generate a
vector carrying the mouse TRAP insert for future transfections was also made.
The generation of over-expressing and knock-down cells was done by stable transfection with
vectors carrying the full length coding sequence (CDS) for TRAP and shRNA constructs,
respectively. For the pre-adipocytes and fibroblasts, increase of the TRAP mRNA expression
and enzyme activity in the over-expressing cells and decrease on mRNA expression in the
knock-down cells was observed when compared to normal or mock-transfected cells.
However, for the over-expressing pre-osteoblasts, no difference on the mRNA level was
detected. For the knock down pre-osteoblasts, increase of TRAP mRNA expression was
observed which might be due to the insertion of siRNA. The cloning of mouseTRAP into the
vector was evaluated through restriction analysis that showed that further trials need to be
made for the generation of the construct.
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Introduction
Tartrate resistant acid phosphatase (TRAP, E.C.: 3.1.3.2) also known as purple acid
phosphatase (PAP) (38) or acid phosphatase type 5 (Acp 5) is a metalloenzyme that belongs
to the group of acid phosphatases. TRAP is mainly expressed in macrophages and osteoclasts
but also in some epithelial cells and neurons (14, 16, 21, 22). For that reason, the enzyme has
been extensively used as a marker for osteoclasts. Moreover, since osteoclasts are related to
bone resorption and it has been suggested that TRAP could be used as a marker for
pathological bone resorptive conditions (8, 14).
1.1 Gene Structure
Human TRAP is located on chromosome 19 (29) on locus 19p13.2 (24), while the mouse
TRAP gene was found to be located on chromosome 9. Mammalian TRAP genes have high
sequence similarity and gene structure (4).
The murine TRAP gene contains 5 exons and 4 introns and is regulated by three promoters
located upstream of alternative exon 1; 1A, 1B, 1C (figure 1) (39). It has been suggested that
TRAP promoters are differentially active in a tissue specific pattern, such as that 1B regulates
expression of TRAP in macrophages and epithelial cells in the liver and kidney, while 1C acts
in expression of TRAP in myeloid hematopoetic cells, e.g. macrophages and osteoblasts (33,
39).

Figure 1. Gene Structure of mouse TRAP. Alternative exon 1 (E1, A,B,C)

1.2 Structure of TRAP
Two structures of the TRAP enzyme are available up to date. Its structure is that of a β-
sandwich surrounded by α-helices. The residues participating mostly in the active site are
H195, H92, H223, D14 and Y55 which are also the residues that coordinate with the two iron
atoms (37). Comparison between the mammalian rat TRAP and other purple phosphatases
shows that the general structure of the enzymes is quite similar but differences occur at the
substrate binding pocket. (26)
As shown in figure 2, TRAP has been crystallized as both monomeric (37) and proteolytically
processed (26) forms. In figure 2a the form of TRAP containing the loop domain (Ser145-
Val161) is shown (Protein Data Bank I.D: 1qhw), while in figure 2b the cleaved form is
demonstrated (Protein Data Bank I.D: 1qfc).
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The cleavage takes place in the loop demonstrated by red in figure 2a. When the loop is
excised by proteases, the spectroscopic properties and kinetics of the active site are altered
due to the excision of Asp146 that interacts with a residue of the active site in the monomeric
form and affects the divalent metal ion (13).

Figure 2. TRAP structure. A. Uncleaved monomeric form of mammalian TRAP. The excised loop that is not
present on the cleaved form is colored red. B. Proteolytically cleaved form of mammalian TRAP (pdb 1a: 1qhw,
1b: 1qfc) (Pictures created using Swiss PDB viewer software)

1.3 Posttranslational modifications of TRAP
Mammalian TRAP has a molecular weight of approximately 33-35kDa (10), contains a redox
activedi-iron active site and is synthesized as a low activity proenzyme (monomeric or
isoform 5a) (9, 20) that targets against tyrosine and serine/threonine phosphorylated
substrates (27). Besides the monomeric form, TRAP has been found to exist in the cells in a
dimeric form (proteolytically cleaved also called isoform 5b), where two fragments of 15 and
21-23kDa are bound through a disulphide bond (32). As described for the structure of TRAP,
the second isoform occurs from the proteolytic cleavage of the isoform 5a in the loop region
(Ser145-Val161) that leaves the active site more exposed to the substrates (21, 27). In
osteoclasts, the activation of TRAP occurs when the exposed loop is excised by cysteine
proteinases (figure 3) (28). The dimeric active form has several substrates such as β-
umberlliferylphosphate and p-nitrophenolphosphate (30) (31)
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Figure 3. Proteolytic cleavage of TRAP. The loop region is in front of the active site. After the cleavage of the
loop by proteases the active site is accessible for substrates.

1.4 Biological Function and expression pattern of TRAP

In Osteoclasts and bone metabolism
As previously mentioned, TRAP is mainly expressed in osteoclasts where its presence has
been related to bone resorption. More specifically, it has been suggested to participate in the
intracellular degradation of collagen fragments (15). Additionally, it has also been suggested
that TRAP participates in the migration of osteoclast on the bone surface by
dephosphorylation of osteopontin (OPN) (11). Moreover monomeric TRAP has been shown
to be secreted from osteoclasts (35) and cleaved in the lacuna (19). Another study that
strengthens the involvement of TRAP in bone resorption comes from generated TRAP
deficient mice. These mice develop mild osteopetrosis with decreased bone turnover and late
mineralization, indicating that TRAP in osteoclasts serves as a regulator of normal bone
formation by controlling bone resorption and mineralization (17, 18)

In mesenchymal cells
Mesenchymal Stem Cells (MSC) were originally described by Friedenstein in 1966 and are
non-hematopoietic cells that have a high differentiation lineage potential and are derived from
the bone marrow (12). MSCs can also be found in several adult tissues such as adipose tissue,
periosteum, muscle, teeth, trabecular bone, etc. ( reviewed in (5)). One of the criteria of MSC
is their ability to differentiate into osteoblasts, chondrocytes and adipocytes (figure 4) (6).
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Figure 4. Potential Differentiation of MSCs

It has been shown that the cleaved isoform of TRAP can induce differentiation of osteoblasts.
More specifically, in osteoblasts TRAP seems to interact with receptor TRIP-I which when
bound to TRAP triggers a TGF-β differentiation pathway. In this way osteoblasts are rapidly
driven to differentiation (36).
Recent data has additionally shown that monomeric TRAP over-expressing mice develop
hyperplastic obesity. Monomeric TRAP, but not proteolytically cleaved TRAP, was found to
induce differentiation and proliferation of adipocyte precursor cells, suggesting that there is a
different role for this isoform. (23)

Uteroferrin in iron metabolism and proliferation
Moreover, uteroferrin a pig TRAP analog, has been found to act as a growth factor for
hematopoietic stem cells (2). Uteroferrin was also shown to participate in iron transfer from
mother to fetus on pigs. Uteroferrin acts by transferring its iron to fetal transferrin (3, 7).
Moreover, it has also been suggested that TRAP expression is regulated by iron (1).

1.5 Methodology

Short interference RNA and short hairpin RNA construct
Short interference RNA has been extensively used for the inhibition of expression of genes.
siRNA exists naturally in cells, and acts by targeting to complementary sequences on the
mRNA produced by the cells. When siRNA is bound to the mRNA the double stranded RNA
becomes a target for RDE-1 nuclease and is degraded (39)
Based on RNA interference technique short-hairpin RNA constructs can be produced. The
construct consist of a vector carrying a sequence that after transcription makes tight short-
hairpin turn with sequence complementary to the target gene (figure 5). The short hairpin is
recognized by the Dicer enzyme that digests it to siRNA. Since the short hairpin is included in
the vector, it is transferred to daughter cells, thus, providing a suitable vector for stable
inhibition of genes in cell lines.
5
vector
transcriptionof vector
shRNA
Dicer
siRNA

Figure 5. Mechanism of RNA interference by shRNA

1.6 Aim of study
The present study is based on the findings mentioned above concerning the ability of TRAP
to induce differentiation and proliferation in mesenchymal cells such as adipocyte and
osteoblast precursors.
The aim of the project was the generation of TRAP over-expressing and knock-down pre-
osteoblastic, pre-adipocytic and fibroblastic cell lines in order to further study the mechanism
by which TRAP induces proliferation and/or differentiation. The future goal is the use of the
generated cell lines to study the differentiation and proliferation pattern, in relation to TRAP
levels.
In an attempt to create those models, mouse pre-osteoblasts 3T3-E1, pre-adipocytes 3T3-L1
and fibroblasts NIH-3T3 were submitted to stable transfection with vectors carrying the
TRAP cDNA or shRNA for over-expression or knock-down respectively.
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Results
2.1 Generation of the pUIN-mouseTRAP construct
For the cloning, as described in the materials and methods section, the TRAP insert was
amplified using primers with overhangs and then ligated to the TOPO TA vector. After
restriction analysis using the EcoRI enzyme the clone that carried the insert was used for
further digestion with EcoRI. In figure 6a, the gel analysis of the TOPO TA vector after
digestion is shown. As it is shown after digestion, clones 1-5 contain the TRAP insert. After
the digestion, the insert with the EcoRI sites on both ends, was gel purified and ligated to the
pUIN vector, also digested with the EcoRI to create complementary ends.
In figure 6b, the restriction analysis of the clone generated after the ligation of the TRAP
insert to the pUIN vector is shown. The restriction was done using PstI restriction enzyme as
described in materials and methods. After restriction only one band was generated, indicating
that the insert is not present in the vector.

a. b.
Figures 6: Restriction analyses from cloning procedure. a. Analysis of the TOPO-TA vector after ligation
and transformation into TOP 10 competent cells. The restriction analysis was done using the EcoRI enzyme. On
lane 1 the 100bp DNA ladder was loaded. Lanes 2 and 8 are loaded with empty digested vector that serves as
control. Lanes 3 to 7 correspond to clones 1-5. b. Restriction analysis of pUIN vector, generated after ligation.
Lanes 2 and 3 correspond to the same sample (digested clone). The control used is shown on lane 1 and
corresponds to the uncut plasmid.

2.2 Construction of cell lines with a stable over-expression of TRAP
The stable transfection was performed in order to generate clones that carry the constructs of
interest through several generations of cells. The cells are intended to be used for 2-3 weeks
long differentiation experiments so there was a need for constant over-expression or knock
down of TRAP expression.
The expression of TRAP was measured at both the mRNA level as well as at the enzyme
activity level. In order to compare the TRAP expression, the cell lines transfected with the
constructs carrying the TRAP insert were compared to the mock transfected cell lines (cell
lines transfected with empty vector), using two different primer sets for TRAP. More
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specifically, one pair of primers was specific for the mouse TRAP construct while the second
pair was mouse and rat TRAP specific.
The two pairs of TRAP primers gave slightly different results, but in both cases, the mRNA
expression of TRAP seemed to be increased in the over-expressing pre-adipocytes (3T3-L1)
and fibroblasts (NIH-3T3) when compared to mock-transfected cells. Moreover, the TRAP
mRNA expression of untransfected 3T3-L1 cells was compared to the mRNA expression of
mock-transfected cells and was shown to be 3-7 times higher. However, for 3T3-E1 cells of
clone 4 there is no significant increase of TRAP (table I).
Apart from the mRNA level determination, the enzyme activity was measured and compared
for the 3T3-L1 and NIH-3T3 cell lines through the pNPP assay. For the 3T3-L1 cells the
enzyme activity of over-expressing cells was compared to that of untransfected cells, while
for NIH-3T3 cells, the enzyme activity of over-expressing was compared to mock transfected
cells (table I). For both over-expressing cell lines an increase in the enzyme activity was
observed. For the 3T3-L1 cells this is in accordance with the results of the TRAP mRNA
expression. However, the increase in the enzyme activity is lower than the increase in the
mRNA level. For 3T3-L1 cells, since the comparison is against the untransfected cells and not
mock transfected, the lower increase might relate to the difference of mRNA expression
between untransfected and mock-transfected cells, since the mock-transfected cells have
lower TRAP mRNA levels than untransfected cells. However, for the NIH-3T3 cell lines, the
same pattern was shown, even though only mock-transfected cells were used as a control.

Table I. Over expression of TRAP on mRNA and TRAP activity level after transfection and selection. Fold
difference of TRAP over-expressing cell lines. The fold difference was calculated comparing the TRAP over-
expressing cells to the mock transfected cells.
TRAPmRNA TRAP enzyme
activity
n=1
Cell Transfection Fold Fold SD qPCR repeats µU/µg Fold
line difference difference mTRAP/mrTRAP mTRAP/mrTRAP protein difference
mTRAP mrTRAP
primers primers
3T3- untransfected 7.4 3.28 4.6/2.6 4/3 0.01 1
L1
mock- 1 1 ND 1/1 ND ND
transfected
over- 33.42 73.51 8.9/- 2/1 0.07 4.32
expressing
NIH- mock- 1 1 ND 1/1 0.03 1
3T3 transfected
over- 39.39 73.51 ND 1/1 0.09 3.08
expressing
3T3- untransfected 1 1 0.42/- 2/1 ND ND
E1
clone
4
over- 1.14 1.17 0.34/- 2/1 ND ND
expressing
ND* not determined, n* experiment repeats

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2.3 Construction of TRAP knock-down cell lines.
Apart from the over-expressing cell lines, an attempt to create knockdown cell lines for TRAP
was made. The evaluation of the inhibition of TRAP expression was also done with qPCR
with the same method as for the over-expressing cell-lines. The cells used as reference in this
case were the ones transfected with the shRNA construct carrying scrambled RNA sequences
(negative control for knock down cell lines). The cell lines were transfected with one to four
different short hairpin constructs. For the 3T3-L1 and NIH-3T3 the cell lines transfected with
the shRNA B construct are presented. Moreover, the enzyme activity was also tested in the
3T3-L1 and NIH-3T3 knock-down cells and was compared to negative control cells. For the
generation of knock-down cell lines, the 3T3-E1 clone 14 was also tested (not included in the
over-expression of TRAP transfection experiments) (table II). The two clones of pre-
osteoblasts were used in separate experiments in order to verify if there are any differences
among them and in their reaction towards transfections.
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