1 Department of Medicine and Lady Davis Institute for Medical Research, Sir
Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, QC,
Canada H3T 1E2
2 Calcium Research Laboratory, Department of Medicine, McGill University Health
Centre and McGill University, Montreal, QC, Canada H3A 1A1
* Author for correspondence (e-mail: akarapli{at}ldi.jgh.mcgill.ca )
Accepted 23 April 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Indian hedgehog, Parathyroid hormone-related peptide, Chondrocytes, Protein kinase A
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Hedgehog (HH) proteins, a family of secreted morphogens, have been
implicated in a multitude of developmental processes
(Nusslein-Volhard et al.,
1980; Riddle et al.,
1993
; Roberts et al.,
1995
; Bitgood et al.,
1996
). All known HH members are proteolytically processed through
an autocatalytic mechanism to generate secreted peptides corresponding to the
N- and C-terminal domains of the native protein
(Porter et al., 1996a
;
Lee et al., 1994
;
Bumcrot et al., 1995
;
Valentini et al., 1997
). The
C-terminal domain is believed to possess the catalytic properties required for
HH cleavage, which occurs at a conserved Gly-Cys site. Cholesterol,
participating as a nucleophile in the autocatalytic process, attaches to the
C-terminal end of the nascent N-terminal domain and enhances its lipophilic
properties (Porter et al.,
1996a
; Porter et al.,
1996b
). The N-terminal domain is believed to possess all the known
biological activities of HH proteins and is highly conserved and
interchangeable amongst HH family members
(Vortkamp et al., 1996
). This
domain can bind its cognate receptor Patched (Ptc), a 12-transmembrane (TM)
protein that otherwise interacts with, and thereby inhibits, the 7-TM receptor
protein Smoothened (Smo) (Stone et al.,
1996
; Carpenter et al.,
1998
). The ligand-induced release of Smo from its interaction with
Ptc results in intracellular signal transduction.
Indian hedgehog (Ihh) has been shown to be a key regulator of chondrocyte
differentiation. In addition to its expression in kidney, gut and osteoblasts
(Valentini et al., 1997;
Bitgood and McMahon, 1995
), its
restricted expression by a discrete layer of chondrocytes in the early
hypertrophic zone of the epiphyseal cartilage has suggested a role for Ihh in
directing these cells to their final differentiated state
(Vortkamp et al., 1996
).
Retroviral-mediated overexpression of Ihh in chick limbs resulted in
inhibition of chondrocyte differentiation, as exhibited by reduced type X
collagen (Col10a1) expression
(Vortkamp et al., 1996
).
Subsequently, it was proposed that the effects of Ihh on chondrocyte
differentiation are indirect and occur via parathyroid hormone-related peptide
(PTHrP), a potent inhibitor of chondrocyte differentiation, expressed in the
resting zone cartilage (periarticular layer)
(Karaplis et al., 1994
).
Treatment of bone explants from wild-type mice with Sonic Hedgehog (Shh)
protein mimicked the ability of PTHrP to inhibit Col10a1 expression
(Vortkamp et al., 1996
;
Lanske et al., 1996
). These
findings, and the observation that HH protein did not affect Col10a1
expression in bone explants from PTHrP-null mice, led to the postulate that
PTHrP may act as a downstream mediator of Ihh action. The proposed model
suggests that Ihh acts, in a paracrine fashion on cells of the perichondrium,
to indirectly increase PTHrP expression in the periarticular cartilage; PTHrP
in turn, via activation of the PTHrP receptor (PTHR1), would then inhibit
differentiation in the growth plate.
However, several lines of evidence have indicated that Ihh may also have
direct effects on chondrocyte differentiation. Consistent with the general
observation that Ptc expression is upregulated in response to HH
signalling (Hooper and Scott,
1989; Phillips et al.,
1990
; Tabata and Kornberg,
1994
; Goodrich et al.,
1996
). Ptc transcripts have been reported in epiphysial
chondrocytes adjacent to the Ihh expression domain
(Vortkamp et al., 1998
).
Furthermore, similar patterns of expression were described for Smo
and Gli1, a member of the Gli family of transcription factors that
mediates gene expression in response to HH
(Vortkamp et al., 1998
;
Akiyama et al., 1999
).
Expression of these genes by growth plate chondrocytes suggests that these
cells may be directly responsive to Ihh.
Moreover, a number of in vitro studies have indicated that the N-terminal
domain of HH proteins can promote chondrogenesis. Thus, retrovirally
overexpressed Shh in limb bud micromass cultures resulted in induction of
cartilaginous nodules that were strongly positive for Col10a1
expression and alkaline phosphatase (ALP) activity in the absence of PTHrP
upregulation (Stott and Chuong,
1997). Additionally, recombinant N-terminal-Ihh induced
Col10a1 and Ptc expression in ATDC5 embryonic carcinoma
cells undergoing chondrocytic differentiation, suggesting that this domain of
Ihh harbors the ability to induce hypertrophy
(Akiyama et al., 1999
).
In this study, we have used the rat CFK-2 chondrocytic cell line to investigate the role of Ihh and its interplay with the PTHrP signalling pathway in chondrocyte differentiation. We present evidence indicating that Ihh or its N-terminal domain harbors the capacity to induce chondrogenic differentiation, an effect mimicked by recombinant N-terminal HH protein. We also show that PTHrP, through a protein kinase A (PKA)-dependent mechanism, inhibits Ihh-mediated differentiation and that Ihh in turn impedes PKA stimulation by PTHrP.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A PCR-based method for site directed mutagenesis (QuikChange, Strategene) was used to generate the W160G mutation where the following primers altered nucleotide 661 of Ihh from a T to a G nucleotide (resulting in a tryptophan to glycine substitution): 5'-CTCTGTCATGAACCAGGGGCCCGGTGTG-3' as the sense primer and 5'-CAGTTTCACACCGGGCCCCTGGTTCATGACAGAG-3' as the antisense primer.
The cDNA encoding the constitutively active PTHR1 (PTHR1 H223R) was generously provided by H. Juppner (Massachusetts General Hospital and Harvard Medical School, Boston, MA). This was subsequently subcloned into the pcDNA3.1/Zeo mammalian expression vector (Invitrogen). The cDNA encoding for full-length rat PTHrP was also subcloned into pcDNA3.1/Zeo.
Cell culture and transfections
CFK-2 cells were maintained in Dulbecco's Modified Eagle Medium (Gibco BRL)
supplemented with 10% fetal bovine serum (Wisent). To induce differentiation,
cells were grown to confluence in 10% FBS after which serum was gradually
reduced by 2% decrements every 2 days, and cells were maintained at 2% FBS
thereafter until 16 days of post confluent growth. For some experiments medium
was supplemented with 1x10-7 M forskolin,
1x10-8 M human PTHrP (1-34) or varying concentrations of
recombinant N-Shh (Curis, Cambridge, MA). N-Shh was modified by an addition of
a hydrophobic eight-carbon chain (octyl group) to the N-terminal cysteine and
this is reported by the suplier to increase the biological activity of the
native peptide by up to 10-fold. N-Shh protein was suspended in `Octyl' buffer
(PBS pH 7.2, 50 µM DTT conjugated to N-octylmaleimide, 350 µM free DTT
and 0.5% DMSO) and this buffer was also used as vehicle control, where
indicated.
Generation of stably transfected CFK-2 cell populations were performed by electroporation. Cells were grown on 10 cm dishes and upon reaching 60% confluence were washed once in PBS and trypsinized. Cells were then suspended in HBS (20 mM Hepes pH 7.4, 0.14 M NaCl, 5 mM KCl, 2.5 mM MgSO4, 25 mM glucose, 1 mM CaCl2) at a density of 5x105 cells/ml, supplemented with plasmid DNA (2 µg), and electroporated at 240V/500µF. Following 48 hours of recovery, cells were subjected to selection by the addition of G418 at a final concentration of 500 µg/ml. Selection proceeded for 10 days and subsequently cells were maintained in G418 (500 µg/ml). Coexpression of PTHrP or PTHR1 H223R with Ihh was achieved by subjecting stable populations of Ihh-transfected CFK-2 cells to a secondary transfection with PTHrP-pcDNA3.1/Zeo or PTHR1 H223R-pcDNA3.1/Zeo. These were selected by growth in media containing 300 µg/ml of zeocin and 500 µg/ml of G418.
Transient transfection of COS-1 cells was performed by the calcium phosphate precipitation method. Briefly, DNA (2 µg/7x105 cells) was added to a solution of 2.5 M CaCl2 and precipitated by the addition of HeBs solution (0.28 M NaCl, 0.05 M Hepes pH 7.05, 1.5 mM Na2HPO4). Precipitated DNA was layered onto cells and incubated for an 8 hour period. Cells were then washed five times in PBS and medium replaced for a recovery period of 48 hours. Alternatively, cells were transfected using FugeneTM 6 (Roche) according to the manufacturer's specifications.
Northern blot analysis
Total RNA was obtained from cell monolayers by guanidium isothiocyanate
(GTC)/CsCl extraction. Briefly, cells were washed once with PBS and
homogenized with 0.5 ml of GTC (4 M guanidium isothiocyanate, 0.1 M Tris-HCl
pH 7.5, 1% ß-mercaptoethanol) and passaged through a 25-gauge needle.
Homogenates were layered on top of a 5.7 M CsCl/0.01 M EDTA cushion and
ultracentrifuged overnight at 120,000 g in a SW40 rotor.
Pellets were washed with 70% ethanol and suspended in DEPC-treated water.
Alternatively, RNA was extracted by Trizol, as specified by the manufacturer
(Gibco BRL).
Typically, aliquots (20 µg) of total RNA were size fractionated on a 1.5% agarose/formaldehyde gel and transferred overnight onto a supported nitrocellulose membrane using 20x SSC buffer (3 M NaCl, 0.003 M Na citrate, pH 7.0). Hybridization of membranes to 32P-labeled probes was performed in a buffer containing 40% formamide, 10% dextran SO4, 4x SSC, and 1x Denhardt's blocking solution with 0.1 mg/ml salmon sperm DNA. Membranes were washed once in 2x SSC/0.1% SDS at room temperature and once in 0.1x SSC/0.1% SDS for 15 minutes at 58°C before exposing to film.
All probes were radiolabeled by the random priming method (Roche). Probes corresponding to the N and C terminus of Ihh were generated by PCR using the primers described above. A 390 bp probe verified by DNA sequencing corresponding to Ptc cDNA was generated by RT-PCR from total RNA extracted from CFK-2 cells. The oligonucleotides used to obtain this fragment were 5'-GGACTTCCAGGATGCCATTTGACAGTG-3' as the sense primer and 5'-GCCGTTGAGGTAGAAAGGGAACTG-3' as the antisense primer and were based on the mouse Ptc cDNA sequence. The cDNA probe for rat Pthr1 was generously provided by H. Juppner (Endocrine Unit, Massachusetts General Hospital, Boston, MA); the cDNA probe for rat Col2a1 was a gift from Y. Yamada (National Institute of Dental Research, Bathesda, MD); the probe for mouse Col10a1 was kindly provided by K. Lee (Massachusetts General Hospital, Boston, MA). Northern blots were assessed quantitatively by videodensitometric analysis (Scion Image).
Western blotting
For western blot analysis of proteins, transiently transfected COS-1 cells
were lysed 48 hours following transfection by scraping monolayers into 300
µl of ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1
mM sodium orthovanadate, 10 mM sodium glycerophosphate, 50 mM NaF, 1% (v/v)
Triton X-100, 0.1% ß-mercaptoethanol, 10 mM PMSF, 5 µg/ml aprotinin, 5
µg/ml leupeptin). Upon one freeze-thaw cycle at -70°C, debris was
removed by centrifugation of lysates for 10 minutes at 12,000
g/4°C. Approximately 25 µl of cleared lysates were
analyzed by SDS-PAGE. For preparation of protein from conditioned media,
medium was centrifuged briefly to remove cellular debris, and proteins were
precipitated by the addition of five volumes of acetone and incubation on ice
for 1 hour. Centrifugation at 10,000 g for 15 minutes
generated a pellet that was suspended in 100 µl of lysis buffer of which 20
µl aliquots were used for analysis.
Protein samples were loaded onto a 12% SDS-polyacrylamide gel and
transferred onto a nitrocellulose membrane. Membranes were blocked in TBST
buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween 20) and 5% milk for 1
hour, and then incubated with primary antibodies that were diluted in
TBST/0.5% milk [1:300 for -N-Ihh (Santa Cruz, CA), 1:500 for
-myc] for 1 hour. Secondary antibodies [horseradish
peroxidase-conjugated anti-goat IgG or anti-mouse IgG (Santa Cruz and Sigma,
respectively), 1:2000 with TBST/0.5% milk] were incubated with membranes for 1
hour and bands were visualized with the BM chemiluminescence blotting
substrate (Roche), according to manufacturer's instructions.
ALP activity assay
Cell monolayers were washed with PBS, lysed in 300 µl ALP lysis buffer
(0.15 M Tris pH 9.0, 0.1 mM ZnCl2, 0.1 mM MgCl2), and
subjected to one freeze-thaw cycle at -70°C. Lysates were cleared by a 10
minute centrifugation at 10000 g and 50 µl aliquots were
analyzed spectrophotometrically at 410 nm with ALP assay solution (7.5 mM
p-nitrophenyl phosphate (Sigma reagent 104), 1.5 M Tris pH 9.0, 1 mM
ZnCl2, 1 mM MgCl2). Protein concentrations were
determined by the method of Lowry using the Bio-Rad DC protein assay kit
(Bio-Rad).
PKA assay
CFK-2 cells were grown to 3 days post-confluence in the presence of 10%
FBS, 500 µg/ml G418, 50 µg/ml ascorbate and 10 mM
ß-glycerophosphate. Following serum starvation for 24 hours, cells were
then stimulated for 20 minutes in the presence of 10-8 M PTHrP 1-34
and 300 µM isobutylmethylxanhine (IBMX) or with IBMX alone. Cells were
rinsed with PBS, placed on ice and lysed in PKA assay buffer (25 mM Tris-HCl,
0.5 mM EDTA, 0.5 mM EGTA, 10 mM ß-mercaptoethanol, 0.5% Triton X-100, 1
µg/ml leupeptin, 1 µg/ml aprotinin, 0.5 mM PMSF). Measurement of PKA
activity in cell lysates was performed using a PKA assay kit (Upstate
Biotech.). Briefly, reactions were performed in the presence of 5 µl of
lysate, 83 µM kemptide substrate, 0.33 µM PKC inhibitor peptide, 3.33
µM CaMK inhibitor (R24571) and 83.3 µM ATP/16.67 µCi
[-32P]ATP. To demonstrate specificity for phosphorylation by
PKA, reactions were also performed in the presence of both kemptide substrate
and 1 µM of PKA inhibitor peptide. After an incubation of 10 minutes at
30°C, 15 µl samples were blotted onto phosphocellulose P81 paper,
washed four times in 0.75% phosphoric acid, once in acetone, and radioactivity
was measured in a scintillation counter. Values were normalized to protein
concentrations of the lysates and to specific radioactivity (cpm/pmol) of the
reaction mix.
Statistics
Statistical analysis of results was performed using one-way analysis of
variance (ANOVA). Sample values were determined to be significantly different
between groups when P values were in the order of P0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Next, an N-Ihh variant harboring a single base pair substitution that
alters a tryptophan to a glycine residue at position 160 of the Ihh peptide
was constructed (N-Ihh W 160G; Fig.
4A). This mutation was first described in human Shh in patients
afflicted with the autosomal dominant form of holoprosencephaly
(Roessler et al., 1996). It
was therefore deduced that this residue, conserved in all mammalian HH
proteins, may be critical for the protein's bioactivity and that its
alteration results in loss of function. Expression of N-Ihh W160G in
transfected COS-1 cells was comparable with that of its wild-type counterpart,
as observed from western blot analysis, indicative of appropriate translation
and maintenance of immunoreactivity to
-Ihh-N
(Fig. 1C).
|
Ihh and N-Ihh induce Ptc expression in CFK-2 cells
We then used an in vitro system, the CFK-2 chondrocytic cell line, to
examine the actions of Ihh on chondrocyte biology. CFK-2 cells have been shown
previously to undergo progressive differentiation manifested by expression of
chondrocytic markers such as type II collagen (Col2a1), type X
collagen (Col10a1), link protein, and Pthr1
(Bernier et al., 1990;
Bernier and Goltzman, 1993
;
Henderson et al., 1996
;
Wang et al., 2001
). To examine
the effects of Ihh on CFK-2 differentiation, we generated stably transfected
populations of these cells expressing Ihh, N-Ihh or N-Ihh W160G
(Fig. 1A). As control, CFK-2
cell populations transfected with pcDNA3 vector were also generated. Stably
transfected CFK-2 cells were then subjected to a 16-day postconfluent culture
period with gradual serum withdrawal (as described in Materials and Methods)
during which samples of total cellular RNA were obtained intermittently.
To verify the status of HH-signalling activity, the expression of
Ptc-receptor was examined in stably transfected CFK-2 cells that
underwent postconfluent culture (Fig.
2A). Since Ptc has been described as a downstream target
gene of HH-signalling, whose levels are presumably dependent on the amount of
HH ligand present (Hooper and Scott,
1989; Phillips et al.,
1990
; Tabata and Kornberg,
1994
; Goodrich et al.,
1996
), we also examined the expression levels of the transgene in
each cell population using a probe encoding the N-terminal domain of Ihh.
Whereas pcDNA3-transfected cells expressed low constitutive levels of
Ptc, Ihh- and N-Ihh-transfectants demonstrated a robust elevation in
Ptc expression levels (Fig.
2A-C). In comparison to pcDNA3-transfected cells, N-Ihh W160G was
capable of inducing Ptc expression, albeit to a lesser degree than
its wild-type counterpart, suggesting that the W160G mutation only partially
impedes the activity of Ihh (Fig.
2B). In addition, we found that the relative Ptc
expression (Ptc/transgene/GAPDH) was higher in
Ihh-transfected cells than in N-Ihh-transfectants, suggesting that the native
molecule may harbour more potent biological activity
(Fig. 2C).
|
Since the N-terminal domains of HH proteins have been shown to be
interchangeable with respect to biological action
(Vortkamp et al., 1996), we
further verified our findings by incubating naive CFK-2 cells with increasing
concentrations of recombinant N-terminal Shh peptide (N-Shh) in the culture
medium. Induced Ptc expression was initially detected in response to
N-Shh concentrations as low as 10-10 M and reached maximal levels
at 10-8 M of the peptide (Fig.
2D). These results verified that N-Shh, similarly to Ihh, induces
Ptc expression in CFK-2 cells and that this is dependent on ligand
concentration.
Ihh and N-Ihh induce chondrogenic differentiation in CFK-2 cells
Since Ihh expression has been localized in situ predominantly to
pre-hypertrophic and hypertrophic growth plate chondrocytes, we assessed
whether CFK-2 cells could differentiate under the influence of Ihh
overexpression or in response to exogenous N-Shh. Differentiation of CFK-2
cells was assessed initially by measuring changes in ALP activity over a
16-day postconfluent culture period (Fig.
3A, left). Whereas control pcDNA3-transfected cells displayed low
levels of ALP activity, Ihh- and to a lesser extent N-Ihh-expressing cells
displayed increasing levels of ALP over time. In a separate experiment, ALP
induction was also compared in N-Ihh and its mutant variant, N-Ihh W160G
(Fig. 3A, right). Whereas N-Ihh
induced ALP activity in CFK-2 cells subjected to an 8-day postconfluent
period, N-Ihh W160G failed to do so. Similarly, Ihh and N-Ihh, but not N-Ihh
W160G, induced the expression of the chondrogenic marker Col2a1,
whose levels increased during postconfluent culture
(Fig. 3B). We then examined
Col10a1 expression as a definitive marker of the hypertrophic stage
of differentiation. Whereas Col10a1 expression was detected at low
levels in control pcDNA3 transfectants, Ihh and N-Ihh induced Col10a1
to a much greater extent (Fig.
3B). Surprisingly, cells transfected with N-Ihh W160G exhibited
similar Col10a1 levels to those observed in Ihh or N-Ihh
transfectants. This suggests that N-Ihh W160G may be capable of transmitting
sufficient levels of HH-signalling to selectively induce certain markers of
differentiation (Col10a1) but not others (ALP, Col2a1)
(Fig. 3B).
|
To examine the effects of recombinant N-Shh on chondrocytic differentiation, CFK-2 cells were subjected to increasing concentrations of this peptide over a postconfluent culture period of 8 days (Fig. 3C). Dose-dependent increases in ALP activity and Col2a1 and Col10a1 expression were observed, that were maximal in response to 10-8 M N-Shh.
Taken together, these observations suggest that Ihh and N-Ihh can induce CFK-2 cells to attain chondrogenic properties exemplified by elevated ALP activity, Col2a1 and Col10a1 expression, and that this effect is mimicked by recombinant N-Shh.
PTHrP antagonizes HH-mediated chondrogenic differentiation through a
PKA-dependent pathway
In bone cells, PTHrP mediates most of its biological actions through
activation of its cognate G-protein-coupled receptor, PTHR1, leading to
stimulation of adenylate cyclase and consequent PKA activation
(Shigeno et al., 1988;
Capehart and Biddulph, 1991
).
PKA has been widely described as an inhibitor of HH-signalling in multiple
systems (Hammerschmidt et al.,
1996
; Chen et al.,
1999
; Wang et al.,
1999
). We therefore examined the effects of induced PKA activity
on HH-mediated differentiation in CFK-2 cells. Naive CFK-2 cells were
subjected to treatment with N-Shh (5x10-9 M) in the presence
or absence of PTHrP (1x10-8 M) or forskolin (10-6
M), a potent activator of adenylate cyclase, and their differentiation state
was assessed after an 8-day culture period. Whereas N-Shh treatment promoted
high ALP activity in CFK-2 cells, this was strongly impeded by co-treatment of
the cells with PTHrP (Fig. 4A).
Similarly, PTHrP led to severe dampening of HH-induced Col2a1 and
Col10a1 mRNA expression (Fig.
4B). In contrast, PTHrP had no effect on N-Shh-induced
Ptc expression. Moreover, all the antagonistic effects exerted by
PTHrP on HH-action were also mimicked by forskolin suggesting that this
phenomenon may be attributed to PKA activation
(Fig. 4B).
To test this hypothesis further, we generated stably transfected CFK-2 cell populations expressing Ihh alone or in combination with PTHrP or PTHR1 (H223R), a mutant variant of PTHR1 known to selectively and constitutively activate the PKA pathway (Schipani and Juppner, 1995). Upon examination of these cells during postconfluent growth, it was observed that Ihh induced ALP enzymatic activity, but that this was strongly perturbed by co-expressing Ihh in conjunction with PTHrP or PTHR1 (H223R) (Fig. 4C). These findings, showing that PTHrP action and constitutive activation of adenylate cyclase through PTHR1 similarly abrogate Ihh function, implicate PKA as the effector of PTHrP-mediated antagonism of HH-signalling.
Ihh dampens PTHrP responsiveness of CFK-2 cells
In the growth plate, PTHR1 expression is observed in proliferating
chondrocytes but is strongest in the prehypertrophic layer adjacent to and
overlapping with Ihh-expressing cells
(Valentini et al., 1997). One
possibility for modulation of HH action by PTHrP may therefore be through
differential PTHR1 expression. To examine this possibility, northern blot
analysis for Pthr1 mRNA expression was performed in CFK-2 cells
treated with PTHrP (10-8 M) or N-Shh (5x10-9 M)
for 7 days during postconfluent growth and compared with cells that were
treated with vehicle alone. In contrast to vehicle-treated cells, N-Shh
treatment resulted in strong upregulation of Pthr1 mRNA expression,
whereas PTHrP treatment alone had no effect
(Fig. 5A, left). Furthermore,
upregulation of Pthr1 expression was antagonized by concomitant
treatment of cells with PTHrP or forskolin, indicative of an HH-specific
effect that can be antagonized by PTHrP-mediated activation of PKA
(Fig. 5A, right).
|
To examine whether increased Pthr1 mRNA expression also results in amplified PTHrP responsiveness, we measured PKA activity directly in CFK-2 cells following transient treatment with PTHrP (1-34). As HH-mediated chondrocytic differentiation can be modulated by PKA activation, measuring its activity serves as an assessment of the cells' responsiveness to HH-inhibitory signals. Examination of PKA activity in pcDNA3-transfected CFK-2 cells that underwent 2 days of post confluent growth and were then treated with PTHrP (IBMX + PTHrP) showed a nearly tenfold increase in activity over cells treated with IBMX alone (Fig. 5B, left). In contrast, an approximately 50% reduction in PTHrP-mediated PKA activity was consistently apparent in cells transfected with either Ihh or N-Ihh. As control, both wild-type and transfected cells showed similar PKA responses to forskolin (data not shown). Furthermore, western blot analysis for the catalytic subunit isoforms of PKA showed no significant difference between treatment groups (Fig. 5B, right), suggesting that reduced PTHrP stimulation of PKA likely did not arise from differences in PKA protein expression levels. To further confirm these results, naive CFK-2 cells were treated with vehicle or increasing concentrations of N-Shh for 7 days after which PKA activity was measured upon transient treatment with PTHrP. In agreement with the previous experiment, HH-treated cells displayed a significantly dampened response to PTHrP that was apparent even at the lowest concentration of N-Shh (Fig. 5C). This implies that HH may negatively regulate signalling through PTHR1, ultimately leading to impeded PKA activity.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our initial monitoring of HH action in CFK-2 cells was performed by
examination of Ptc mRNA expression, a transcriptional target of
HH-signalling (Goodrich et al.,
1996). Preceding the activation of chondrogenic markers, a robust
upregulation of Ptc-receptor expression was observed in both
Ihh/N-Ihh transfected cells, indicative of strong HH-responsiveness in CFK-2
cells. Consistently, recombinant N-Shh also induced Ptc expression in
a dose-dependent fashion. Whereas Ihh/N-Ihh transfectants showed little
variation in Ptc expression over the period of culture, it was noted
that Ihh was a stronger inducer of Ptc than N-Ihh when transgene
expression levels were considered (Fig.
2). This difference may be attributed to the cholesterol moiety
present on the N-terminal peptide generated from the wild-type form of Ihh,
inherently absent from its recombinant truncated counterpart. Indeed, other
observations have indicated that lipid-modified forms of N-terminal Shh have
increased potency in ALP activation in C3H10T1/2 cells, despite unaltered
receptor binding capacity (Pepinsky et
al., 1998
). The existence of sterol sensing domains (SSD) in both
the hedgehog receptor Ptc and in the hedgehog-releasing
protein, Dispatched, would also suggest an important biological role
for cholesterol modification of the N-terminal domain
(Burke et al., 1999
).
The inherent capacity of the N-terminal domain of Ihh to mediate
chondrogenic differentiation in CFK-2 cells is further confirmed by the
ability of a specific missense mutation introduced in this protein domain to
partially abrogate this function. The W160G mutation has been described in an
individual case of holoprosencephaly ascribing a presumptive loss of function
to Shh, although this finding was not confirmed in vitro
(Roessler et al., 1996). The
corresponding residue in mouse Shh, Trp117, was shown to localize
adjacent to the first
-helix of the peptide
(Hall et al., 1995
). Since at
least one residue (Asp115) residing within this
-helix was shown to be
involved in Ptc binding and activation of HH-signalling
(Pepinsky et al., 2000
), it is
likely that Trp117/Trp160 may also be crucial for this function. Here, we
report that the W160G mutation reduces the capacity of N-terminal Ihh to
stimulate Ptc expression, indicative of a partial loss in
HH-signalling activity. That N-Ihh W160G is unable to induce ALP activity and
Col2a1 expression, but yet capable of Col10a1 induction,
further corroborates the observation that this protein may act as a partial
agonist of HH-signalling. Moreover, in comparison to other mutations mapping
to the N-terminal of Shh, mutations at Trp117 are associated with a milder
form of holoprosencephaly and this may be attributed to the partial signalling
capacity of this variant (Roessler et al.,
1996
).
In this study we show that HH-signalling can promote chondrogenic
differentiation. In contrast, previous in vivo studies have indicated that Ihh
promotes chondrocyte proliferation, in part through a PTHrP-independent
mechanism, while mediating PTHrP-dependent actions that result in delay of
chondrocytic hypertrophy (Karp et al.,
2000). That Ihh mediates its hypertrophic inhibitory actions
through transcriptional activation of PTHrP at the periarticular layer was
suggested from observations of chick limbs retrovirally overexpressing Ihh or
murine bone explants treated with recombinant N-Shh
(Vortkamp et al., 1996
;
Lanske et al., 1996
). However,
further studies have demonstrated that signalling by TGF-ß, but not BMPs,
can also elicit PTHrP activation in the periarticular cartilage, suggesting
that Ihh may act indirectly through a relay mechanism
(Serra et al., 1999
;
Minina et al., 2001
). From
these studies, and others (St-Jacques et
al., 1999
), it was concluded that the propensity to activate PTHrP
was indispensable for achieving HH-mediated inhibition of hypertrophy and that
ablation of PTHrP completely abrogated this effect. In apparent contrast to
these studies, we demonstrate that HH-signalling activates, rather than
inhibits, chondrogenic differentiation in CFK-2 cells. Thus, activation of
Col10a1 by HH is indicative of the potential that this peptide has in
driving CFK-2 cells toward a progressive state of hypertrophic
differentiation. This observation is consistent with other in vitro reports
describing the propensity of Shh or Ihh to induce hypertrophic marker
expression in micromass cultures and in embryonic carcinoma cells
(Akiyama et al., 1999
;
Stott and Chuong, 1997
). It is
also consistent with the initial delay in chondrocyte maturation and
Col10a1 expression described in Ihh-null mice
(St-Jacques et al., 1999
).
This phenomenon was initially attributed to a perturbation of chondrocytic
proliferation (Karp et al.,
2000
); however later studies have indicated that ablation of
HH-signalling in growth plate chondrocytes selectively interfered with their
proliferative capacity but did not affect their differentiation programme
(Long et al., 2001
).
Alternatively, a delay in Col10a1 expression can indicate that Ihh
may have direct inductive influences on chondrocytic differentiation. Ihh may
play a temporal role in promoting early chondrocyte differentiation, analogous
to the role Shh has in promoting somitic chondrogenesis
(Murtaugh et al., 1999
). The
fact that Ihh-null mice eventually display an increase in
hypertrophic chondrocytes indicates that factors other than Ihh are required
for this process. These factors may include members of the bone morphogenetic
protein (BMP) family, as these were shown to induce chondrocytic hypertrophy
(Enomoto-Iwamoto et al., 1998
;
Grimsrud et al., 1999
;
Terkeltaub et al., 1998
). The
fact that it is possible to observe the intrinsic HH differentiating capacity
in CFK-2 cells in vitro may be due to the fact that PTHrP, which is normally
induced through the negative regulatory response observed in vivo, is not
stimulated by Ihh in this system. However, addition of exogenous PTHrP
mimicked this in vivo effect.
PTHrP-induced inhibition of chondrogenic differentiation mediated directly
through activation of PTHR1 is well described
(Schipani et al., 1997;
Chung et al., 1998
). Here, we
demonstrate for the first time that PTHrP action impedes HH-mediated
differentiation in CFK-2 cells. Thus, treatment of CFK-2 cells with N-Shh in
the presence of PTHrP or forskolin resulted in complete abrogation of ALP
activity and Col2a1 and Col10a1 expression, suggesting that
this effect was mediated through a PKA-dependent pathway. In further
agreement, Ihh-mediated ALP activity was prevented by overexpressing PTHR1
H223R, a variant of PTHR1 described in patients with Jansen-type metaphyseal
chondrodysplasia, and known to selectively and constitutively activate the PKA
pathway (Schipani and Juppner, 1995). The in vivo observation that
overexpression of PTHR1 H223R1 in the growth plate leads to a delay in
chondrocytic hypertrophy is also consistent with our results
(Schipani et al., 1997
). PKA
has been widely described as a negative regulator of the HH-signalling pathway
and appears to exert its function through direct phosphorylation of specific
consensus sites present in Gli family members and their Drosophila
homologue, Ci (Hammerschmidt et al.,
1996
; Chen et al.,
1999
; Wang et al.,
1999
). Thus, an attractive scenario emerges where PTHrP signalling
modulates, via PKA, one or more of the Gli factors
(Fig. 6). The fact that
Ptc induction by HH was refractory to PTHrP or forskolin treatment
indicates the inhibition is rather selective. Such selectivity may reflect
divergent functions of different Gli factors that are context-dependent
(Ruiz i. Altaba, 1999
). Thus,
phosphorylation of Gli3, but not Gli1, by PKA results in its proteolysis and
formation of an alternate repressor form that downregulates transcription of
certain HH target genes (Chen et al.,
1999
; Chen et al.,
1998
; Wang et al.,
2000
). We have observed the expression of Gli2 and Gli3 expression
in CFK-2 cells (data not shown) and speculate that complex regulation of HH
target genes may be regulated by relative levels of repressor and activator
forms of these proteins.
|
PTHR1 has previously been shown to mediate the inhibitory effects of PTHrP
on differentiation (Chung et al.,
1998; Chung et al.,
2001
), a process involving activation of the cAMP but not the
phospholipase C-dependent pathway (Guo et
al., 2001
). Interestingly, our data demonstrates an interaction
between the HH and PTHrP signalling pathways at the level of PTHR1 regulation.
This is evidenced by HH-dependent transcriptional upregulation of
Pthr1 mRNA, an effect that is antagonized by PTHrP or forskolin.
Paradoxically, HH also renders cells less responsive to PTHrP and this is
indicative of a functional inhibition of PTHR1 by HH action. These finding
have several implications that could converge with in vivo observations.
First, the fact that Pthr1 mRNA expression appears to be highest in
the prehypertrophic cells that lie adjacent to, and overlap with, cells
expressing Ihh may suggest the possibility of Pthr1 being a
transcriptional target of HH-signalling
(St-Jacques et al., 1999
).
Second, Pthr1 expression in proliferating chondrocytes is associated
with enhanced responsiveness to PTHrP resulting in suppression of the
hypertrophic markers ALP and Col10a1, a response that is diminished
in hypertrophic chondrocytes (Iwamoto et
al., 1994
). Thus, signalling by Ihh may be required for inhibition
of signalling via PTHR1, and consequently of PTHrP action, in order to allow
prehypertrophic chondrocytes to proceed to their final differentiated state.
However, HH signalling does not appear to completely inhibit PTHrP action, as
cells treated with HH peptide remained responsive to transient and long-term
PTHrP treatment. This suggests rather, that signalling by HH and PTHrP have
interactive feedback mechanisms that allow for the appropriate pace of
differentiation to occur (Fig.
6).
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akiyama, H., Shigeno, C., Iyama, K., Ito, H., Hiraki, Y., Konishi, J. and Nakamura, T. (1999). Indian hedgehog in the late-phase differentiation in mouse chondrogenic EC cells, ATDC5: upregulation of type X collagen and osteoprotegerin ligand mRNAs. Biochem. Biophys. Res. Commun. 257,814 -820.[Medline]
Bernier, S. M. and Goltzman, D. (1993). Regulation of expression of the chondrocytic phenotype in a skeletal cell line (CFK2) in vitro. J. Bone Miner. Res. 8, 475-484.[Medline]
Bernier, S. M., Desjardins, J., Sullivan, A. K. and Goltzman, D. (1990). Establishment of an osseous cell line from fetal rat calvaria using an immunocytolytic method of cell selection: characterization of the cell line and of derived clones. J Cell Physiol. 145,274 -285.[Medline]
Bitgood, M. J. and McMahon, A. P. (1995). Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev. Biol. 172,126 -138.[Medline]
Bitgood, M. J., Shen, L. and McMahon, A. P. (1996). Sertoli cell signalling by Desert hedgehog regulates the male germline. Curr. Biol. 6, 298-304.[Medline]
Bumcrot, D. A., Takada, R. and McMahon, A. P. (1995). Proteolytic processing yields two secreted forms of sonic hedgehog. Mol. Cell Biol. 15,2294 -2303.[Abstract]
Burke, R., Nellen, D., Bellotto, M., Hafen, E., Senti, K. A., Dickson, B. J. and Basler, K. (1999). Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signalling cells. Cell 99,803 -815.[Medline]
Capehart, A. A. and Biddulph, D. M. (1991). Development of PTH-responsive adenylate cyclase activity during chondrogenesis in cultured mesenchyme from chick limb buds. Calcif. Tissue Int. 48,400 -406.[Medline]
Carpenter, D., Stone, D. M., Brush, J., Ryan, A., Armanini, M.,
Frantz, G., Rosenthal, A. and de Sauvage, F. J. (1998).
Characterization of two patched receptors for the vertebrate hedgehog protein
family. Proc. Natl. Acad. Sci. USA
95,13630
-13634.
Chen, Y., Gallaher, N., Goodman, R. H. and Smolik, S. M.
(1998). Protein kinase A directly regulates the activity and
proteolysis of cubitus interruptus. Proc. Natl. Acad. Sci.
USA 95,2349
-2354.
Chen, Y., Cardinaux, J. R., Goodman, R. H. and Smolik, S. M.
(1999). Mutants of cubitus interruptus that are independent of
PKA regulation are independent of hedgehog signalling.
Development 126,3607
-3616.
Chung, U. I., Lanske, B., Lee, K., Li, E. and Kronenberg, H.
(1998). The parathyroid hormone/parathyroid hormone-related
peptide receptor coordinates endochondral bone development by directly
controlling chondrocyte differentiation. Proc. Natl. Acad. Sci.
USA 95,13030
-13035.
Chung, U. I., Schipani, E., McMahon, A. P. and Kronenberg, H.
M. (2001). Indian hedgehog couples chondrogenesis to
osteogenesis in endochondral bone development. J. Clin.
Invest. 107,295
-304.
Enomoto-Iwamoto, M., Iwamoto, M., Mukudai, Y., Kawakami, Y.,
Nohno, T., Higuchi, Y., Takemoto, S., Ohuchi, H., Noji, S. and Kurisu, K.
(1998). Bone morphogenetic protein signalling is required for
maintenance of differentiated phenotype, control of proliferation, and
hypertrophy in chondrocytes. J. Cell Biol.
140,409
-418.
Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A. and Scott, M. P. (1996). Conservation of the hedgehog/patched signalling pathway from flies to mice: induction of a mouse patched gene by Hedgehog. Genes Dev. 10,301 -312.[Abstract]
Grimsrud, C. D., Romano, P. R., D'Souza, M., Puzas, J. E., Reynolds, P. R., Rosier, R. N. and O'Keefe, R. J. (1999). BMP-6 is an autocrine stimulator of chondrocyte differentiation. J. Bone Miner. Res. 14,475 -482.[Medline]
Guo, J., Lanske, B., Liu, B. Y., Divieti, P., Kronenberg, H. M.
and Bringhurst, F. R. (2001). Signal-selectivity of
parathyroid hormone (PTH)/PTH-related peptide receptor-mediated regulation of
differentiation in conditionally immortalized growth-plate chondrocytes.
Endocrinology 142,1260
-1268.
Hall, T. M., Porter, J. A., Beachy, P. A. and Leahy, D. J. (1995). A potential catalytic site revealed by the 1.7-A crystal structure of the amino-terminal signalling domain of Sonic hedgehog. Nature 378,212 -216.[Medline]
Hammerschmidt, M., Bitgood, M. J. and McMahon, A. P. (1996). Protein kinase A is a common negative regulator of Hedgehog signalling in the vertebrate embryo. Genes Dev. 10,647 -658.[Abstract]
Henderson, J. E., He, B., Goltzman, D. and Karaplis, A. C. (1996). Constitutive expression of parathyroid hormone-related peptide (PTHrP) stimulates growth and inhibits differentiation of CFK2 chondrocytes. J. Cell Physiol. 169, 33-41.[Medline]
Hooper, J. E. and Scott, M. P. (1989). The Drosophila patched gene encodes a putative membrane protein required for segmental patterning. Cell 59,751 -765.[Medline]
Iwamoto, M., Jikko, A., Murakami, H., Shimazu, A., Nakashima,
K., Iwamoto, M., Takigawa, M., Baba, H., Suzuki, F. and Kato, Y.
(1994). Changes in parathyroid hormone receptors during
chondrocyte cytodifferentiation. J. Biol. Chem.
269,17245
-17251.
Karaplis, A. C., Luz, A., Glowacki, J., Bronson, R. T., Tybolewicz, V. L. J., Kronenberg, H. M. and Mulligan, R. C. (1994). Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev. 8, 277-289.[Abstract]
Karp, S. J., Schipani, E., St-Jacques, B., Hunzelman, J.,
Kronenberg, H. and McMahon, A. P. (2000). Indian hedgehog
coordinates endochondral bone growth and morphogenesis via parathyroid hormone
related-protein-dependent and independent pathways.
Development 127,543
-548.
Lanske, B., Karaplis, A. C., Lee, K., Luz, A., Vortkamp, A., Pirro, A., Karperien, M., Defize, L. H. K., Ho, C., Mulligan, R. C. et al. (1996). PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273,663 -666.[Abstract]
Lee, J. J., Ekker, S. C., von Kessler, D. P., Porter, J. A., Sun, B. I. and Beachy, P. A. (1994). Autoproteolysis in hedgehog protein biogenesis. Science 266,1528 -1537.[Medline]
Long, F., Zhang, X. M., Karp, S., Yang, Y. and McMahon, A.
P. (2001). Genetic manipulation of hedegehog signaling in the
endochondral skeleton reveals a direct role in the regulation of chondrocyte
proliferation. Development
128,5099
-5108.
Minina, E., Wenzel, H. M., Kreschel, C., Karp, S., Gaffield, W.,
McMahon, A. P. and Vortkamp, A. (2001). BMP and Ihh/PTHrP
signaling interact to coordinate chondrocyte proliferation and
differentiation. Development
128,4523
-4534.
Murtaugh, L. C., Chyung, J. H. and Lassar, A. B.
(1999). Sonic hedgehog promotes somitic chondrogenesis by
altering the cellular response to BMP signalling. Genes
Dev. 13,225
-237.
Nusslein-Volhard, C. K. and Wieschaus, E. (1980). Mutations affecting number and polarity in Drosophila. Nature 287,795 -801.[Medline]
Pepinsky, R. B., Zeng, C., Wen, D., Rayhorn, P., Baker, D. P.,
Williams, K. P., Bixler, S. A., Ambrose, C. M., Garber, E. A., Miatkowski, K.
et al. (1998). Identification of a palmitic acid-modified
form of human Sonic hedgehog. J. Biol. Chem.
273,14037
-14045.
Pepinsky, R. B., Rayhorn, P., Day, E. S., Dergay, A., Williams,
K. P., Galdes, A., Taylor, F. R., Boriack-Sjodin, P. A. and Garber, E. A.
(2000). Mapping sonic hedgehog-receptor interactions by steric
interference. J. Biol. Chem.
275,10995
-11001.
Phillips, R. G., Roberts, I. J., Ingham, P. W. and Whittle, J. R. (1990). The Drosophila segment polarity gene patched is involved in a position-signalling mechanism in imaginal discs. Development 110,105 -114.[Abstract]
Porter, J. A., Ekker, S. C., Park, W. J., von Kessler, D. P., Young, K. E., Chen, C. H., Ma, Y., Woods, A. S., Cotter, R. J., Koonin, E. V. and Beachy, P. A. (1996a). Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86,21 -34.[Medline]
Porter, J. A., Young, K. E. and Beachy, P. A.
(1996b). Cholesterol modification of hedgehog signalling proteins
in animal development. Science
274,255
-259.
Riddle, R. D., Johnson, R. L., Laufer, E. and Tabin, C. (1993). Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75,1401 -1416.[Medline]
Roberts, D. J., Johnson, R. L., Burke, A. C., Nelson, C. E.,
Morgan, B. A. and Tabin, C. (1995). Sonic hedgehog is an
endodermal signal inducing Bmp-4 and Hox genes during induction and
regionalization of the chick hindgut. Development
121,3163
-3174.
Roessler, E., Belloni, E., Gaudenz, K., Jay, P., Berta, P., Scherer, S. W., Tsui, L. C. and Muenke, M. (1996). Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. Nat. Genet. 14,357 -360.[Medline]
Ruiz i. Altaba, A. (1999). Gli proteins encode
context-dependent positive and negative functions: implications for
development and disease. Development
126,3205
-3216.
Serra, R., Karaplis, A. C. and Sohn, P. (1999).
Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects
of transforming growth factor beta (TGF-ß) on endochondral bone
formation. J. Cell Biol.
145,783
-794.
Schipani, E., Kruse, K. and Juppner, H. (1995). A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 268,98 -100.[Medline]
Schipani, E., Lanske, B., Hunzelman, J., Luz, A., Kovacs, C. S.,
Lee, K., Pirro, A., Kronenberg, H. M. and Juppner, H. (1997).
Targeted expression of constitutively active receptors for parathyroid hormone
and parathyroid hormone-related peptide delays endochondral bone formation and
rescues mice that lack parathyroid hormone-related peptide. Proc.
Natl. Acad. Sci. USA 94,13689
-13694.
Shigeno, C., Yamamoto, I., Kitamura, N., Noda, T., Lee, K.,
Sone, T., Shiomi, K., Ohtaka, A., Fujii, N. and Yajima, H.
(1988). Interaction of human parathyroid hormone-related peptide
with parathyroid hormone receptors in clonal rat osteosarcoma cells.
J. Biol. Chem. 263,18369
-18377.
St-Jacques, B., Hammerschmidt, M. and McMahon, A. P.
(1999). Indian hedgehog signalling regulates proliferation and
differentiation of chondrocytes and is essential for bone formation.
Genes Dev. 13,2072
-2086.
Stone, D. M., Hynes, M., Armanini, M., Swanson, T. A., Gu, Q., Johnson, R. L., Scott, M. P., Pennica, D., Goddard, A., Phillips, H. et al. (1996). The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature 384,129 -134.[Medline]
Stott, N. S. and Chuong, C. M. (1997). Dual
action of sonic hedgehog on chondrocyte hypertrophy: retrovirus mediated
ectopic sonic hedgehog expression in limb bud micromass culture induces novel
cartilage nodules that are positive for alkaline phosphatase and type X
collagen. J. Cell Sci.
110,2691
-2701.
Tabata, T. and Kornberg, T. B. (1994). Hedgehog is a signalling protein with a key role in patterning Drosophila imaginal discs. Cell 76,89 -102.[Medline]
Terkeltaub, R. A., Johnson, K., Rohnow, D., Goomer, R., Burton, D. and Deftos, L. J. (1998). Bone morphogenetic proteins and bFGF exert opposing regulatory effects on PTHrP expression and inorganic pyrophosphate elaboration in immortalized murine endochondral hypertrophic chondrocytes (MCT cells). J. Bone Miner. Res. 13,931 -941.[Medline]
Valentini, R. P., Brookhiser, W. T., Park, J., Yang, T., Briggs,
J., Dressler, G. and Holzman, L. B. (1997).
Post-translational processing and renal expression of mouse Indian hedgehog.
J. Biol. Chem. 272,8466
-8473.
Vortkamp, A., Lee, K., Lanske, B., Segre, G. V., Kronenberg, H. M. and Tabin, C. J. (1996). Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273,613 -622.[Abstract]
Vortkamp, A., Pathi, S., Peretti, G. M., Caruso, E. M., Zaleske, D. J. and Tabin, C. J. (1998). Recapitulation of signals regulating embryonic bone formation during postnatal growth and in fracture repair. Mech. Dev. 71,65 -76.[Medline]
Wang, B., Fallon, J. F. and Beachy, P. A. (2000). Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100,423 -434.[Medline]
Wang, D., Canaff, L., Davidson, D., Corluka, A., Liu, H., Hendy,
G. N. and Henderson, J. E. (2001). Alterations in the sensing
and transport of phosphate and calcium by differentiating chondrocytes.
J. Biol. Chem. 276,33995
-34005.
Wang, G., Wang, B. and Jiang, J. (1999).
Protein kinase A antagonizes Hedgehog signalling by regulating both the
activator and repressor forms of Cubitus interruptus. Genes
Dev. 13,2828
-2837.