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Endocrine Research Unit (W.C., C.T., R.B., D.S.) and the Departments of Dermatology (L.K.) and Medicine, Veterans Affairs Medical Center, University of California, San Francisco, California 94121; and the Department of Radiobiology, University of Utah School of Medicine (S.M.), Salt Lake City, Utah 84112; Department of Medicine (G.S.), Brockton/West Roxbury Veterans Affairs Medical Center, West Roxbury, Massachusetts, and Harvard Medical School, Boston, Massachusetts
Address all correspondence and requests for reprints to: Dr. Dolores M. Shoback, 111N, Endocrine Research Unit, 4150 Clement Street, San Francisco, California 94121. E-mail: dolores{at}itsa.ucsf.edu
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2
mM Ca2+). RNA levels for aggrecan and type II
collagen decreased with increasing [Ca2+]o
(ID50, 2.0 and 4.1 mM Ca2+,
respectively). RNA levels for type X collagen and alkaline phosphatase
were also reduced by high [Ca2+]o with
ID50 values of approximately 2.9 and 1.6 mM
Ca2+, respectively. These responses were rapid, in that
increasing [Ca2+]o from 1.0 to more than 6
mM suppressed aggrecan RNA levels by about 50%, and
lowering [Ca2+]o from 2.9 to 1.0
mM increased aggrecan RNA levels by about 300% within
4 h. As Ca2+ receptors (CaRs) mediate extracellular
Ca2+ sensing in parathyroid and kidney, we assessed the
expression of CaRs in these cells. C5.18 cells stained positively for
CaR protein with an anti-CaR antiserum and for CaR RNA by in
situ hybridization. An approximately 150-kDa protein was
detected by immunoblotting with anti-CaR antiserum. CaR antisense
oligonucleotides suppressed the expression of CaR protein and enhanced
RNA levels of aggrecan in C5.18 cells. These data support the idea that
CaRs are expressed in this cell system and may be involved in
regulating chondrogenic gene expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many studies have shown that changes in extracellular [Ca2+] ([Ca2+]o) act as a primary signal to regulate crucial cell functions (9). In the parathyroid, high [Ca2+]o activates phospholipase C and intracellular Ca2+ mobilization (10, 11, 12), events that ultimately lead to the suppression of PTH secretion (13). In thyroid parafollicular cells, high [Ca2+]o increases intracellular [Ca2+] and calcitonin release (14). In keratinocytes, increases in [Ca2+]o accelerate cell differentiation (15). Substantive evidence indicates that membrane Ca2+ receptors (CaRs) detect changes in [Ca2+]o and couple them to downstream actions in all of these systems (16). These receptors belong to the G protein-coupled receptor superfamily and couple to the activation of phospholipase C (17, 18), mobilization of intracellular Ca2+, inhibition of cAMP formation (17), and cation influx (10).
Previous studies support a regulatory role of [Ca2+]o in the expression of chondrogenic genes. Investigators have found that increasing the medium Ca2+ concentration enhanced type II and X collagen production in cultures of tibio-tarsus chondrocytes isolated from chick embryos (19). In the eggshell-less chicken model, Ca2+ deficiency induced chondrogenesis in the calvaria, a site at which intramembranous bone formation usually occurred (4, 5). Additional studies in this model identified a population of calvarial cells that preferentially differentiated into cartilage when maintained at low [Ca2+]o (4, 20). These studies suggested that chondrogenic cells have the ability to detect and respond to different [Ca2+]o and that these responses may differ according to the specific anatomical site of origin of the cells.
In the present study, we examined whether chondrogenic gene expression is modulated by changes in [Ca2+]o in a nontransformed clonal chondrogenic cell line, RCJ3.1C5.18, abbreviated C5.18 hereafter. These cells, derived from fetal rat calvaria, express several chondrogenic markers and form cartilage nodules in culture (21, 22). Changes in [Ca2+]o modulate the expression of chondrogenic genes in these cells, which express both protein and transcripts homologous to known CaRs. We further found that CaR antisense expression altered aggrecan RNA levels in these cells. Taken together, these studies lend support to the idea that CaRs are present in chondrogenic cells and may be important in regulating cartilage-specific gene expression.
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Materials and Methods |
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1(II) complementary DNA
(cDNA) probes were provided by Dr. Yoshi Yamada (NIH, Bethesda, MD).
The rat alkaline phosphatase (ALP) cDNA probe was provided by Dr.
Gideon Rodan (Merck, Sharp, and Dohme Research Laboratories, West Point, PA). The 18S and rat cyclophilin (CP)
cDNA probes were obtained from Dr. Mei-Jhy Su (Maxim Biotech, Inc.,
South San Francisco, CA) and Dr. J. Gregor Sutcliffe (Scripps Research
Institute, La Jolla, CA), respectively. Chamber slides were purchased
from Becton Dickinson and Co. (Franklin Lakes, NJ). FCS
was purchased from Intergen (Purchase, NY), and culture
media were obtained from the Cell Culture Facility of the University of
California-San Francisco. Digoxigenin (DIG) and RNA polymerase were
obtained from Boehringer Mannheim (Indianapolis, IN).
Biotinylated tyramide reagent and streptavidin peroxidase were
purchased from DAKO Corp. (Carpinteria, CA). All other
reagents were obtained from Sigma Chemical Co. (St. Louis,
MO), previously noted suppliers (23), or as specified.
Cell culture
The chondrogenic cell line C5.18 was provided by Dr. Jane E.
Aubin (University of Toronto, Toronto, Canada) and maintained in
standard medium (SM) (21, 22) containing 15% FCS and dexamethasone
(DEX; 10-7 M) and studied within 25 passages.
Cells were usually replated before reaching confluence using 0.05%
(wt/vol) trypsin and 0.5 M EDTA1 to dissociate
cell monolayers. For experiments, cells were initially plated at a
density of 1.6 x 104 cells/cm2 in 96-well
plates for alcian green staining, 10- or 15-cm culture dishes for RNA
extraction, or chamber slides for morphological studies. Cells
typically reached confluence 48 h after seeding and then the
medium was switched to a supplemented medium [SM plus ß-glycerol
phosphate (10 mM), and ascorbic acid (50 µg/ml)] to
enhance the formation of cartilage nodules (24). When the
Ca2+ concentration in the medium was varied, appropriate
volumes of 1 M CaCl2 were added to a
calcium-free supplemented medium. Ionized Ca2+ was
determined at room temperature using a Nova CRT 8 analyzer (Nova
Biomedical, Waltham, MA), and values are reported in Table 1
. All [Ca2+]o
values reported in this paper reflect the ionized Ca2+
concentration.
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Oligonucleotide transfection
The antisense oligonucleotide sequence was from the region
flanking the putative translation start site of the rat brain CaR (25).
C5.18 cells were transfected with CaR sense
(5'-GAGAAGGCAACGCTATGGCATCG-3') or antisense
(5'-CGATGCCATAGCGTTGCCTTCTC-3') phosphorothioate oligonucleotides
(Cruachem, Inc., Dulles, VA). Cells that were 50–70% confluent were
transfected with oligonucleotides (100 nM) using
Lipofectamine reagent (8 µg/ml) according to the manufacturer’s
instructions (Life Technologies, Gaithersburg, MD). Five
hours after transfection, media were replaced with supplemented media
containing 1.8 mM Ca2+. After 48- to 72-h
incubation, membrane protein and total RNA were purified for Western
and Northern analyses, respectively.
Northern blotting
Total RNA was extracted from cultured C5.18 cells using RNA
Stat-60 (Tel-Test, Inc., Friendswood, TX). RNA (25
µg/sample) was electrophoresed on agarose gels and transferred to
Hybond-N+ nylon membranes (Amersham Corp.,
Arlington Heights, IL). cDNA probes were labeled with
[-32P]deoxy-CTP (Amersham Corp.) using the Prime-It II
DNA labeling kit (Stratagene, La Jolla, CA). Membranes
were hybridized with labeled probes and washed sequentially with 2, 1,
and 0.1 x SSC (standard saline citrate) at 65 C for 15 min.
Thereafter, each membrane was hybridized with CP and/or 18S cDNA probes
after stripping the membranes. Hybridization signals were detected on
film and quantified densitometrically using NIH image 1.5.2 software
(NIH) on a microcomputer. In all cases, the ratio of signals from the
RNA of interest compared with either CP or 18S RNA was determined. The
ratio of signals at 0.4 mM Ca2+ is defined as
100% in each experiment unless otherwise stated. The data presented
are representative of at least two independent experiments unless
otherwise specified.
Immunocytochemistry
For immunocytochemistry, C5.18 cells were cultured in chamber
slides, fixed for 30 min, and then stained. Fixed cells were treated
with H2O2 (0.6%, vol/vol) in methanol (80%,
vol/vol) to reduce endogenous peroxidase activity and blocked with PBS
containing nonfat dry milk (1%, wt/vol), fish skin gelatin (0.3%,
wt/vol), goat serum (3%, vol/vol), and Tween-20 (0.01%, vol/vol).
Cells were then incubated with anti-CaR antiserum 21825A (500
nM) (17). This antiserum was raised in a rabbit against a
peptide epitope (amino acids 215–236) in the extracellular domain of
the bovine parathyroid CaR, which is completely conserved in rat and
human CaRs. To assess specificity, sections were treated with either
antiserum preincubated with excess peptide (1000-fold) or nonimmune
rabbit serum. After several washes, cells were incubated with
peroxidase-conjugated goat antirabbit IgG (1:100) at room temperature
for 60 min, followed by diaminobenzidine (DAB) staining using SigmaFast
DAB tablets and counterstaining with aqueous hematoxylin.
In situ hybridization
C5.18 cell monolayers were fixed, embedded in paraffin, and
sectioned. Sections were hybridized to a DIG-labeled sense or antisense
RNA probes (26) prepared from the human keratinocyte CaR cDNA template
(nucleotides 2442–2746) (15). Probes were diluted in hybridization
solution [2 x SSC, 12.5 x Denhardt’s solution, formamide
(50%), SDS (0.5%), salmon sperm DNA (0.25 µg/ml), sodium
pyrophosphate (0.5%), and Tris-HCl (10 mM, pH 7.4)] and
then applied to sections, which were incubated overnight at 42 C.
Sections were washed and incubated first with ribonuclease A (20
µg/ml) and then with a series of solutions of increasing stringency.
Sections were exposed for 1 h to anti-DIG-horseradish peroxidase
antibody (1:500) in buffer [BSA (2%), fish skin gelatin (0.5%), NaCl
(500 mM), Tween-20 (0.1%), and Tris-HCl (10
mM, pH 7.6)], then incubated with biotinylated tyramide
reagent for 15 min, followed by streptavidin-peroxidase for 15 min.
Signals were visualized with DAB chromogen substrate for 5 min.
Sections were counterstained with methyl green.
Immunoblotting
Crude membrane protein fractions were prepared from C5.18 cells
cultured in supplemented medium for 10 days and human embryonic kidney
293 (HEK-293) cells expressing wild-type bovine parathyroid CaR (17).
Immunoblotting was performed with affinity-purified rabbit anti-CaR
peptide antiserum (21825A; 50 nM) as previously described
(17) after electrophoresis on 6% SDS-PAGE gels and transfer to
nitrocellulose membranes. Immunoreactivity was developed by incubating
blots with peroxidase-conjugated goat antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA; 1:4000), ECL assay kits were
used for signal detection (Amersham Corp.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Nodules containing alcian
green-stainable material generally appeared 2 days postconfluence in
the supplemented medium (Fig. 1b). The sizes of the nodules increased
with time in culture, as did the amount of alcian green staining within
the nodules (Fig. 1c).
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). RNA transcripts for
the 1-subunit of type II collagen
[1-(II)] were detectable in day 1 cultures and
increased with the duration of culture (Fig. 2b). RNA encoding the
1-subunit of type X collagen [1-(X)],
however, could barely be detected in the first 3 days of culture, but
its level profoundly increased thereafter (Fig. 2c). Levels of ALP RNA
did not change significantly throughout the culture period (Fig. 2d).
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and data not shown). At a [Ca2+]o of 4
mM or more, staining was reduced to about 40% of maximum
at 0.4 mM Ca2+.
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. In
C5.18 cells that were initially grown at 2.9 mM
Ca2+ for 7 days, the subsequent reduction of
[Ca2+]o to less than 2.9 mM for 7
days produced a concentration-dependent increase in alcian green
staining (Fig. 4). On the other hand, if these same cultures, grown at
2.9 mM Ca2+ for 7 days, were switched to a
[Ca2+]o more than 2.9 mM for 7
days, this further decreased alcian green staining compared with the
levels seen in cultures continuously exposed to 2.9 mM
Ca2+ for 14 days (Fig. 41). The inhibitory effects of
culturing cells for 7 days at more than 6 mM
Ca2+ were also reversible (Fig. 4). Thus, high
[Ca2+]o did not appear to have nonspecific
toxic effects on matrix production by these cells.
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2 mM
Ca2+). Suppression was evident when RNA levels were
normalized to either CP or 18S RNA levels (Fig. 5a and Table 2). The latter two RNA levels were not
affected by changing [Ca2+]o (Fig. 5a and
data not shown). RNA levels for 1(II), an early
cartilage marker, were also decreased by high
[Ca2+]o in a dose-dependent manner (Fig. 5b
and Table 2). The higher ID50 of about 4.1 mM
indicated that this marker was less sensitive to
[Ca2+]o. Suppression was maximal at more than
6 mM Ca2+, to less than 10% of the levels at
0.4 mM Ca2+.
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1(X)
and ALP. These genes are considered markers of the chondrocyte
phenotype that appear later in differentiation (8). Total RNA levels
for 1(X) and ALP were reduced by increases in
[Ca2+]o with ID50 values of about
2.9 and 1.6 mM, respectively (Fig. 5c
and Table 2). The
densitometric ratio of 1(X) RNA and CP RNA fell at
[Ca2+]o of more than 5.2 mM to
less than 10% of the maximum ratio at 0.4 mM
Ca2+ (see Table 2). In contrast, ALP RNA levels were
maximally suppressed by about 50% when
[Ca2+]o was increased to more than 6
mM (Fig. 5d and Table 2).
As these results were obtained after 12 days in culture, we next
determined whether responses to different
[Ca2+]o could occur more rapidly. In these
studies, postconfluent cells were grown at 1.0 mM
Ca2+ for 7 days to confluence, and then
[Ca2+]o was increased to more than 6
mM for between 0.25–72 h. We found that high
[Ca2+]o suppressed aggrecan RNA levels by
50% within 4 h and by more than 90% within 72 h, compared
with control levels at 1.0 mM Ca2+ (Fig. 6a). In the reverse experiment, when
cells grown at 2.9 mM Ca2+ for 7 days were
switched to 1.0 mM Ca2+ for 0.25–72 h,
aggrecan RNA levels rose 3-fold within 4 h and about 8-fold in
72 h compared with control levels at 2.9 mM
Ca2+ (Fig. 6b).
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). Staining intensity was slightly
increased in areas of nodule formation, which are localized by alcian
green staining (Fig. 7a). There were no qualitative differences in CaR
staining at any time points examined during culture (1–12 days) either
with or without DEX (data not shown). Similarly, there were no
significant changes in CaR protein expression by Western blotting
during this time in culture (data not shown). At higher magnification,
CaR staining localized to the cell membrane and the perinuclear regions
(Fig. 7b). This pattern is comparable to that of HEK-293 cells stably
transfected with bovine parathyroid CaR (data not shown).
Immunoreactivity appeared to be specific, as staining was absent when
cells were treated with antiserum preincubated with the immunizing
peptide or with nonimmune antisera (data not shown).
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). Hybridization with a CaR sense probe was
negative (Fig. 7d), confirming the specificity of the signal for CaR
transcripts.
To assess the molecular weight of bands reacting with anti-CaR
antiserum, we blotted crude membrane fractions from C5.18 cells and
HEK-293 cells stably expressing the bovine parathyroid CaR (17). Our
antisera detected three major protein bands of about 140, about 160,
and more than 205 kDa in membranes prepared from these HEK-293 cells
(17) (see Fig. 7e). In C5.18 cells, there was a predominant band with a
molecular mass of approximately 150 kDa and two less intense but
specific higher molecular mass bands (>205 kDa; Fig. 7e). We also
consistently observed two fainter bands of 180–190 kDa molecular mass
in these cells. This immunoreactivity was judged to be specific, as it
was absent when preimmune sera was substituted for anti-CaR antisera
(data not shown) and was abolished by preincubation of anti-CaR
antisera with excess peptide (Fig. 7f).
To determine whether there was a link between expression of CaRs and
chondrogenic gene expression, we transfected CaR antisense
oligonucleotides into C5.18 cells and examined aggrecan RNA levels.
Transfection of cells with antisense oligonucleotides reduced CaR
protein expression by 46.3 ± 5.5% compared with that of cells
transfected with sense oligonucleotides (P < 0.05;
n = 3; Fig. 8, a and c). Steady
state aggrecan RNA levels, however, increased by 77.2 ± 18.1%
compared with levels in sense DNA-transfected control cells
(P < 0.02; n = 3; Fig. 8, b and c). These
findings suggested that interruption of CaR expression correlated with
changes in aggrecan RNA expression.
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Discussion |
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Ca2+ is critical at several points in cartilage development. In early stages of chondrogenesis, mesenchymal cells interact and form condensed aggregates from which chondrocytes are derived (3). In vitro studies using limb-bud cells show that cell-cell interactions in the condensation and aggregation steps are modulated by extracellular Ca2+ (3). After condensation and aggregation, chondrocytes progress through stages of proliferation, maturation, and hypertrophy. To study the effects of [Ca2+]o on steps beyond cell aggregation, we used postconfluent C5.18 cells. At 1–3 days postconfluence, C5.18 cells resemble maturing chondrocytes in that they express aggrecan and type II collagen. At 6 days postconfluence, these cells express type X collagen, which is a feature of hypertrophic chondrocytes (8). Our results show that changes in [Ca2+]o modulate levels of matrix protein synthesis in both early and late postconfluent cultures, suggesting that [Ca2+]o may be able to regulate chondrogenic function in cells beyond aggregation.
The [Ca2+]o we observed to have significant effects on chondrogenic gene expression in C5.18 cells were within the 1.0–4.0 mM range. Although some of these [Ca2+]o appear high compared with serum ionized [Ca2+] measured in vivo, these [Ca2+]o are well within the range over which Ca2+-sensing receptors are active (16). It is also important to note that [Ca2+] in the cartilage microenvironment in vivo may be higher than that in serum due to the active mobilization of Ca2+ from serum to the matrix (8).
Our studies showed that increases in [Ca2+]o suppressed the expression of chondrogenic genes with varying ID50 values and maximal responses. The RNA levels for aggrecan and type II and type X collagen were profoundly affected, with more than 90% suppression by high [Ca2+]o. Only about 50% of ALP expression, however, was sensitive to changes in [Ca2+]o. The ID50 values for extracellular Ca2+-induced suppression of steady state messenger RNA (mRNA) levels of ALP, aggrecan, type X collagen, and type II collagen were about 1.6, 2.0, 2.9, and 4.1 mM Ca2+, respectively. This range of sensitivities to Ca2+ could reflect differences in the sensitivity of regulatory events beyond membrane CaRs that are triggered in response to a change in [Ca2+]o and that ultimately lead to changes in gene transcription rates and RNA stability. Although we found that steady state RNA levels decrease in response to high [Ca2+]o for all of these genes, different mechanisms could readily be involved depending on the gene in question.
It is well known that cells possess many signaling pathways that are responsive to changes in [Ca2+]o. In parathyroid cells, for example, high [Ca2+]o increases InsP production, elevates [Ca2+]i, and suppresses cAMP accumulation. Different levels of ligand (i.e. Ca2+) are required for activating these pathways. Changes in [Ca2+]i are detectable at 1 mM Ca2+, increases in InsPs production typically require at least 2 mM Ca2+, and cAMP levels are decreased by 1.5 mM Ca2+ or more (10). In terms of cell function, PTH secretion is rapidly suppressed (within minutes) by [Ca2+]o greater than 1.0 mM (10, 13, 29). In contrast, changes in prepro-PTH mRNA levels require more than 12 h, and higher [Ca2+]o (2 or 3 mM) are needed to achieve significant suppression (30). These differences in the sensitivity of important physiological responses in the parathyroid to Ca2+ could be directly relevant to our findings in C5.18 cells. Levels of receptor expression could also explain the greater ID50 values in C5.18 cells compared with parathyroid cells. Finally, in addition to extracellular Ca2+, there are many other regulators that impact on steady state mRNA levels for the genes we studied. Clearly, changes in a given mRNA level reflects an integration of all the regulatory factors that control expression of that gene.
The effects of high [Ca2+]o on the parameters we studied are likely to be specific. Cartilage nodule formation was assessed by staining with alcian green, which interacts with matrix proteoglycans. Alcian green was concentrated specifically in the nodules, and the intensity of staining correlated with both the number and size of the nodules. It is unlikely that differences in staining intensity at different [Ca2+]o were due to nonspecific effects of Ca2+ during the procedure. After fixation, cells were extensively washed with PBS, water, and acetic acid solutions to remove any residual Ca2+ before the addition of alcian green, which was followed by further washing with acetic acid and water. We confirmed that this procedure removed Ca2+ deposits in the cultures by alizarin red staining (data not shown). Therefore, the reduction in alcian green staining in these cultures was probably due to the suppression of matrix synthesis by high [Ca2+]o and not to nonspecific effects of Ca2+ in the medium. In addition, the effects of changing [Ca2+]o on aggrecan RNA levels were promptly reversible, also arguing against nonspecific or toxic effects of high Ca2+.
How do our studies relate to previous work on the role of Ca2+ in chondrogenic gene expression? The observation that high [Ca2+]o suppresses RNA levels of chondrogenic genes in C5.18 cells differs from findings in other systems. Bonen and Schmid (19) reported that raising [Ca2+]o increased type II and X collagen production in cultures of tibio-tarsus chondrocytes isolated from chick embryos. Reginato et al. (6) showed that Ca2+ deficiency, due to the absence of the eggshell, decreased type X collagen synthesis, cell hypertrophy, and mineralization in the embryonic sternal cartilage (6). These defects were corrected by supplementation of the embryo cultures with Ca2+ (6).
There are several explanations for differences between C5.18 cells and other cartilage-based systems. 1) The model systems are different. Many previous studies used embryonic chondrocytes (3), which include cells at all points in differentiation, including condensation and aggregation. Ca2+ stimulates condensation and aggregation (3). C5.18 cells are thought to represent a later stage in differentiation at which Ca2+ could have different effects. Bonen and Schmid (19) used primary chondrocytes, which are heterogeneous populations. Reginato et al. (6) studied whole chick embryos. C5.18 cells are a clonal chondrogenic cell line, highly synchronized in their stage of differentiation (21). 2) The cells of origin for the different models could be relevant. C5.18 cells were derived from fetal calvaria, a site of intramembranous bone formation. Tibio-tarsus and sternal cartilage, in contrast, are sites where endochondral bone forms. Differences in the effects of [Ca2+]o may be due to phenotypic features of the cells of origin. This idea is supported by the observation that Ca2+ deficiency in eggshell-less chick embryo reduced type X collagen synthesis, hypertrophy, and mineralization in sternal cartilage but enhanced cartilage nodule formation in calvaria (4). This latter result is similar to our findings in C5.18 cells. Despite differing effects of [Ca2+]o among these model systems, these in vitro studies are in agreement that [Ca2+]o affects chondrocyte function.
Findings from immunoblotting, immunocytochemistry, and in situ hybridization support the idea that a CaR, similar to those already identified in parathyroid (16), kidney (31), and brain (25), is expressed in C5.18 cells. The sizes of the protein bands in C5.18 cell membranes detected by the anti-CaR antiserum differ modestly from those in parathyroid (16), kidney (31), and CaR-expressing HEK-293 cells (17, 18). Different posttranslational processing (e.g. glycosylation) could account for these differences (32).
Our studies with CaR-specific antisense oligonucleotides suggest that changes in CaR expression impact on aggrecan expression. When CaR protein levels were reduced by about 50%, aggrecan RNA levels increased by 70–80%. The cor- relation between CaR expression and aggrecan RNA levels supports a potential causal link between these parameters. Further studies are required to test this hypothesis, uncover the underlying mechanism, and determine its physiological importance in chondrogenesis.
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Footnotes |
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Received May 20, 1998.
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 T. Yamaguchi, N. Chattopadhyay, O. Kifor, C. Ye, P. M. Vassilev, J. L. Sanders, and E. M. Brown Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line Am J Physiol Cell Physiol, February 1, 2001; 280(2): C382 - C393. [Abstract] [Full Text] [PDF]
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 E. M. Brown and R. J. MacLeod Extracellular Calcium Sensing and Extracellular Calcium Signaling Physiol Rev, January 1, 2001; 81(1): 239 - 297. [Abstract] [Full Text] [PDF]
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W. Chang, C. Tu, T.-H. Chen, L. Komuves, Y. Oda, S. A. Pratt, S. Miller, and D. Shoback Expression and Signal Transduction of Calcium-Sensing Receptors in Cartilage and Bone Endocrinology, December 1, 1999; 140(12): 5883 - 5893. [Abstract] [Full Text]
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D. Wang, L. Canaff, D. Davidson, A. Corluka, H. Liu, G. N. Hendy, and J. E. Henderson Alterations in the Sensing and Transport of Phosphate and Calcium by Differentiating Chondrocytes J. Biol. Chem., August 31, 2001; 276(36): 33995 - 34005. [Abstract] [Full Text] [PDF]
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C.-L. Tu, W. Chang, and D. D. Bikle The Extracellular Calcium-sensing Receptor Is Required for Calcium-induced Differentiation in Human Keratinocytes J. Biol. Chem., October 26, 2001; 276(44): 41079 - 41085. [Abstract] [Full Text] [PDF]
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W. Chang, T.-H. Chen, S. Pratt, and D. Shoback Amino Acids in the Second and Third Intracellular Loops of the Parathyroid Ca2+-sensing Receptor Mediate Efficient Coupling to Phospholipase C J. Biol. Chem., June 23, 2000; 275(26): 19955 - 19963. [Abstract] [Full Text] [PDF]
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) or >6
mM Ca2+ (
) for 7 days. Media were then
changed to those containing different [Ca2+]o
as shown (0.4 to >6 mM) for 7 more days. On day 14,
cultures were stained with alcian green, and stain was eluted and
quantified. Symbols * and + depict results from cells that were
continuously grown in 1 mM or more than 6 mM
Ca2+, respectively, for 14 days.
) or the percent reduction (
).
These results represent data from three transfections.