Articles in PresS. Am J Physiol Cell Physiol (October 15, 2014). doi:10.1152/ajpcell.00037.2014
1
Caveolin-1 Regulates P2X7Receptor Signaling in Osteoblasts
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Vimal Gangadharan1, Anja Nohe1, Jeffrey Caplan1,2,
3
Kirk Czymmek1,2, Randall L. Duncan1, 2
4 5
Author Affiliation: 1Department of Biological Sciences, University of Delaware, Newark, DE 19716
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2
Bioimaging Center, Delaware Biotechnology Institute, Newark, DE 19711
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Corresponding Author: Randall Duncan, Ph.D. Department of Biological Sciences 118 Wolf Hall University of Delaware Newark, DE 19716, USA Tel: 302.831.4296 Fax: 302.831.2281 Email: [email protected]
Running Head: CAV1 regulates P2X7R signaling in Osteoblasts
22 23 24 25 26 27
Copyright © 2014 by the American Physiological Society.
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Abstract
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The synthesis of new bone in response to a novel applied mechanical load requires a
30
complex series of cellular signaling events in osteoblasts and osteocytes. The activation
31
of the purinergic receptor, P2X7R, is central to this mechanotransduction signaling
32
cascade. Recently, the P2X7R have been found to be associated with caveolae, a
33
subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1),
34
the primary protein constituent of caveolae in osteoblasts, results in increased bone
35
mass, leading us to hypothesize that the P2X7R is scaffolded to caveolae in
36
osteoblasts. Thus, upon activation of the P2X7R, we postulate that caveolae are
37
endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation
38
of MC3T3-E1 pre-osteoblasts showed that CAV1 was translocated to the denser
39
cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X7R
40
agonist, BzATP, induced endocytosis of CAV1, which was inhibited when MC3T3-E1
41
cells were pretreated with the specific P2X7R antagonist, A-839977. The P2X7R co-
42
fractionated with CAV1 but, using Superresolution Structured Illumination Microscopy
43
(SR-SIM), we found that only a sub-population of the P2X7R existed in these lipid
44
microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced
45
the intracellular Ca2+ response to BzATP, suggesting that caveolae regulate P2X7R
46
signaling. This proposed mechanism is supported by increased mineralization observed
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in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that
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caveolae regulate P2X7R signaling upon activation by undergoing endocytosis and
49
potentially carrying with it other signaling proteins, hence controlling the spatio-temporal
50
signaling of P2X7R in osteoblasts.
2
51
Keywords: Caveolin-1, Purinergic signaling, P2X7R, Endocytosis, Osteoblasts
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3
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Introduction
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Purinergic signaling plays a critical role in regulating skeletal mechanotransduction, an
55
intricate process through which physical stimulus is converted into a biochemical
56
response (7, 23). We, and others, have shown that ATP, and other nucleotides, are
57
released in response to fluid shear stress in vitro and activate purinergic (P2) receptors
58
on the membrane of the osteoblasts (13, 20, 29, 38). Although there are many subtypes
59
of P2 receptors present in osteoblasts (4), knockout of the P2X7R, a ligand gated ion
60
channel, resulted in an osteopenic phenotype (22), suggesting that this receptor is
61
important in skeletal maintenance. Further in vivo loading experiments show that load-
62
induced bone formation is significantly abrogated when the P2X7R is genetically
63
deleted, demonstrating the importance of this receptor in mechanotransduction (23).
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Several characteristics distinguish the P2X7R from the other P2X receptors. This
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receptor is 10-30 times more specific for 2’,3’-(benzoyl-4-benzoyl)-ATP (BzATP) than
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ATP (28) and activation of the receptor by BzATP induces dynamic membrane blebbing
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(39). P2X7R activation has been associated with the opening of a large pore on the
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plasma membrane that allows the entry of molecules up to 900 Da in size (28, 46),
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although recent reports suggests that this pore is actually a pannexin (19). Though the
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prolonged activation of this receptor has been known to trigger apoptosis in certain cell
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types, we have shown there is no caspase-3 activation observed in osteoblasts when
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stimulated with BzATP (23). Stimulation of the P2X7R with BzATP in cells of the
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osteoblastic lineage results in the expression of osteogenic markers such as osterix and
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osteocalcin (31) and the release of PGE2 (23), all of which can eventually lead to
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osteogenesis. Signaling via the P2X7R is also known to activate PLD and PLA2, leading 4
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to the synthesis of LPA that, in turn, has been attributed to membrane blebbing (31).
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P2X7R activation also induces ERK phosphorylation in a PKC-dependent manner in
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osteoblasts (26). The receptor has a significant role in inflammatory responses, wherein
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it was shown the receptor activation induces the release of inflammatory cytokines from
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macrophages and osteoclasts (42). Overall this ligand gated ion channel has a major
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role in several intracellular processes in a cell type specific manner.
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Recent reports suggest that a sub-population of the P2X7R exists in caveolae, 60-100
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nm invaginations of the plasma membrane (30, 49), in cells of the submandibular
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glands, lung alveolae and macrophages (3, 12, 16). These plasma membrane
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structures are widely expressed in adipocytes, fibroblasts, endothelial cells and
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osteoblasts (34, 43, 44) and are formed from a subset of components of lipid rafts, such
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as sphingolipids and cholesterol, and are stabilized by 22 kD integral proteins called
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caveolins (34).There are three isoforms of caveolin, CAV1, CAV2 and CAV3. CAV1 and
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CAV2 are found in non-muscle cells, whereas CAV3 is the primarily found in skeletal
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and some smooth muscle cells (47). The removal of CAV1 and CAV3 prevents the
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formation of caveolae in these cells, but there was no apparent changes when CAV2
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was suppressed (5, 11). Numerous studies have demonstrated an important role for
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caveolae in cell signaling and scaffolding of signaling molecules in several cell types
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(24, 27, 35). Besides anchoring second messengers, caveolae can also attenuate
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signaling by endocytosis therefore regulating the presence of the receptors along with
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other signaling proteins on the plasma membrane (17, 36).
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The importance of CAV1 and caveolae in regulation of the skeleton is emphasized by a
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study that demonstrate the knockout of CAV1 in mice increases trabecular and cortical 5
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bone which appears to result from increased osteoblast function (40). Because both
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P2X7R and CAV1 are important in skeletal maintenance and the response of bone to
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mechanical loading, we hypothesized that the P2X7R is present in caveolae of
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osteoblasts and further postulated that disruption of caveolae will alter P2X7R mediated
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signaling.
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Materials and Methods
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Cell culture: MC3T3-E1 cells, a murine preosteoblast-like cell line (ATCC), were grown
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in α-Modified Eagles Medium (α-MEM) that was supplemented with 10% FBS (Gemini),
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100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained in a humidified
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incubator in a 5% CO2/95% air mixture and subcultured every 72 hrs. MC3T3-E1 cells
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were serum starved for 12-16 hrs prior to all experiments. All cell culture media and
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antibiotics were purchased from Sigma-Aldrich, St Louis, MO.
111
Reagents and antibodies: Adenosine 5’ Triphosphate disodium salt (ATP); 2’, (3’)-O-
112
(4-Benzoylbenzoyl) adenosine 5’ triphosphate triethylammonium salt (BzATP) and
113
puromycin dihydrochloride (Puromycin) were purchased from Sigma-Aldrich. ATP (250
114
µM; from a stock of 5 mM in PBS) and BzATP (150 µM; from a stock of 6 mM in dH2O)
115
were used for activation of P2X7R. A-839977 (500 nM; from stock of 500 µM in DMSO),
116
a potent P2X7R antagonist was purchased from Tocris Bioscience. Rabbit polyclonal
117
anti-CAV1 and mouse monoclonal anti-CAV1 were obtained from BD Biosciences (San
118
Jose, CA). Rabbit polyclonal anti-P2X7R and FITC conjugated rabbit anti-P2X7R were
119
purchased from Alomone Labs (Jerusalem, Israel). Anti-GAPDH antibody obtained from
120
Abcam (Cambridge, MA) was used as loading control in Western blots. The caveolar
6
121
marker, Alexa Fluor®-555 Cholera Toxin B (CTX-B) and AF-488 donkey anti-rabbit IgG
122
were purchased from Life Technologies (Carlsbad, CA). siRNA against CAV1 was
123
obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and the Sure Silencing
124
plasmid vectors coding shRNA against CAV1 with Puromycin resistance (KM03639P)
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were purchased from SABiosciences (Frederick, MD).
126
Detergent free isolation of caveolin enriched membrane fractions: Sucrose
127
gradient fractionation was used to isolate caveolin enriched membrane fractions as
128
described by Song, et al. with some modifications (45). Briefly, MC3T3-E1 cells were
129
grown to 80-90% confluence in 150 mm Petri dishes and washed with cold PBS. In the
130
experiments with agonist treatment, the cells were grown in reduced serum (0.2%) for
131
14-16 hrs prior to treatment. The cells were scraped into 2 ml of ice cold 500 mM
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sodium carbonate containing protease inhibitors (Calbiochem, San Deigo, CA). The cell
133
lysate was then homogenized by sonication, centrifuged at 9000xg for 10 min, and the
134
supernatant was collected. Protein concentration was measured using BCA assay and
135
equal concentrations were subjected to discontinuous sucrose gradient fractionation.
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A 45% sucrose-lysate solution was made by adding 90% sucrose in MBS (25 mM MES,
137
150 mM NaCl, pH 6.5) to the lysate. This mixture was then placed at the bottom of an
138
ultracentrifuge tube and a discontinuous gradient was prepared on top of this mixture by
139
layering with 4 ml of 35% and 5% sucrose in MBS containing 250 mM sodium
140
carbonate. The gradient was subjected to ultracentrifugation for 18 hrs at 39,000 rpm at
141
4°C with a SW41-TI rotor (Beckman Coulter). A light scattering band was typically
142
observed at the 5%-35% interface where most of the caveolae was enriched. Twelve 1
143
ml fractions were collected from the top of the gradient and the proteins present in each 7
144
fraction were precipitated using TCA-Acetone. The precipitates were then resuspended
145
in sample buffer and equal volumes of the fractions were subjected to Western blotting
146
for the respective proteins.
147
Immunocytochemistry: Cells were seeded at a density of 2.5x103/cm2 onto glass
148
cover slips coated with rat tail type I collagen (100 µg/ml in 0.02 N acetic acid, BD,
149
Franklin Lakes, NJ) and grown to 80-90% confluency. Experimentally, cells were treated
150
with 250 µM ATP or 150 µM BzATP. In one group, cells were pretreated with 500 nM of
151
A-839977 for 30 min prior to the addition of 150 µM BzATP. After 10 min of stimulation,
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cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA)
153
containing 0.1% Triton X-100. Fixed cells were washed with PBS and blocked with
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blocking buffer containing 3% BSA and 10% donkey serum for 2 hrs. Rabbit anti-
155
caveolin-1 antibody (0.5 µg) was then added and the samples were incubated overnight
156
at 4°C. Following incubation, the samples were washed with blocking buffer, and then
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the secondary antibody, AF-488 donkey anti-rabbit IgG, was added. The cells were then
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mounted on microscopy slides with Prolong Gold (Life Technologies) and visualized
159
using the Zeiss LSM 780 confocal microscope with a EC Plan Neofluar 40x/1.3 Oil
160
objective lens. Quantification was carried out by manually counting the number of cells
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with strong membrane staining pattern for CAV1 within the field of view. A minimum of
162
30 cells were counted for each treatment and the percentage of cells with plasma
163
membrane CAV1 was calculated. Each experiment was repeated three times with
164
different cell passages. The line profile of CAV1 staining illustrating the intensity of
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labeling across the cell was created using the ImageJ software.
8
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Triton X-100 extraction: MC3T3-E1 cells grown on 100mm Petri dishes were washed
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three times with ice cold PBS and lysed in 500 µl of cold Triton X-100 lysis buffer (1%
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Triton X-100, 50 mM Tris, 150 mM NaCl, pH 6.5) containing protease inhibitors. The
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cells were scraped off the plate and proteins were extracted for 30 min at 4°C. The
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lysate was then centrifuged at 9000xg for 30 min and supernatant was collected as the
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soluble fraction (S). The pellet containing the detergent-resistant caveolae was
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resuspended in 500 µl RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5%
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sodium deoxycholate, 1% Triton X-100 and protease inhibitors) and sonicated to obtain
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the insoluble fraction (IS). Equal volumes of both fractions were subjected to Western
175
blotting and probed for CAV1 and the P2X7 receptor.
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siRNA strategy for transient knockdown of CAV1: Small interfering RNA was used
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to transiently knockdown CAV1 in MC3T3-E1 cells as per manufacturer’s instructions. In
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brief, MC3T3-E1 cells were seeded at a cell density of 2.5x103/cm2 onto rat tail type-1
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collagen-coated (100 µg/ml; BD) glass bottom dishes (MatTek Corporation, Ashland,
180
MA) and grown to 50-60% prior to transfection. For transfection, the culture medium
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was removed and the cells washed with PBS, followed by addition of 800 µl Opti MEM
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(Life Technologies). siRNA against CAV1 (0.5 µM; sc-29942) and 6 µl of Oligofectamine
183
(Life Technologies) were diluted in 100 µl of OptiMEM, respectively. The reagents were
184
then gently mixed and incubated for 20 min at room temperature before being added to
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each dish. After 5-7 hrs of incubation with the siRNA, fresh culture media containing
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10% FBS was added to the cells. Experiments were conducted after 72 hrs post
187
transfection since Western blotting demonstrated maximum suppression was achieved
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after 72 hrs. Scrambled siRNA (sc-37007) and PBS were used as controls. 9
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Generation of stable CAV1 knockdown (CAV1 KD) cells: To generate a clonal line of
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MC3T3-E1 cells with stable knockdown of CAV1, SureSilencing shRNA plasmid with
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Puromycin resistance was used. Five sets of plasmids were provided; of which four
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were specifically targeted against CAV1 mRNA and the fifth plasmid was a non-specific
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scrambled plasmid that served as a control. Following the manufacturers protocol, each
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of the plasmids was amplified after transformation into the competent DH5-alpha cells.
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The plasmids were isolated using QIAGEN Mini Prep and concentration was measured.
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Prior to transfection with the shRNA encoding plasmids, MC3T3-E1 cells (2.5x103/cm2)
197
were grown on a 12 well plate for 24 hrs. 6 µl of the transfection reagent, Lipofectin (Life
198
Technologies) was diluted in 100 µl of OptiMEM and mixed with 100 µl of OptiMEM
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containing 1.2 µg of the plasmid and incubated for 20 min at room temperature.
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Meanwhile the growth medium on the cells was replaced with 800 µl of fresh OptiMEM.
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After incubation, the transfection mixture was added to the MC3T3-E1 cells and
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incubated for 5-7 hrs at 37°C. The transfection reagents were then removed and fresh
203
culture media containing 10% FBS was added. After 72 hrs post transfection, the
204
culture media was supplemented with 5 µg/ml of Puromycin, the minimum inhibitory
205
concentration based on the dose response for MC3T3-E1 cells. Cells that did not take
206
up the plasmid failed to develop a resistance to the antibiotic and died. Within a week,
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colonies of MC3T3-E1 cells with Puromycin resistance were beginning to develop.
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These colonies were isolated for approximately 8 weeks and were continuously
209
screened for antibiotic resistance. A control group of untransfected MC3T3-E1 cells
210
were also screened to ensure the antibiotic concentration remained effective to
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completely kill the non-transfected cells. Four different clones were isolated of which 10
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one was the scrambled control and the extent of CAV1 knockdown was measured using
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immunoblotting. GAPDH was used as the loading control.
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Western Blotting: To determine the protein levels of CAV1 following knockdown the
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siRNA or shRNA treatment, MC3T3-E1 cells were lysed using RIPA buffer and equal
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amounts of protein were subjected to SDS-PAGE. To detect CAV1, P2X7R and GAPDH
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in the sucrose fractionation studies and in the Triton-X100 extraction experiments, equal
218
volumes of the samples were loaded onto 4-12% gradient NuPAGE gels (Life
219
Technologies). After electrophoresis, the proteins were transferred to nitrocellulose
220
membrane and blocked ON with 5% milk in TBS/T (TBS containing 0.1% Tween-20).
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The blots were probed with antibodies against CAV1, P2X7R and GAPDH using the
222
respective antibodies.
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Calcium imaging: Intracellular Ca2+ levels [Ca2+]i were measured in MC3T3-E1 cells
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using a fluorescent Ca2+ chelator, Fluo-4 and imaged using Zeiss 510 confocal
225
microscope with a Zeiss 10X C-Apochromat water immersion lens (0.45 numerical
226
aperture). As previously described, MC3T3-E1 cells were grown on rat tail type I
227
collagen-coated glass bottom dishes until cells reached 70% - 80% confluency. Cells
228
were then loaded with the calcium indicator, 10 µM Fluo-4 (Life Technologies) for 20
229
min at 37ºC. The cells were then washed with HBSS to remove excess dye and allowed
230
to recover for 15 min before observation. A 488nm excitation laser was used to visualize
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[Ca2+]i and the changes in intensity were measured as a time lapse. Images were
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collected over a period of 1 min prior to the addition of BzATP to establish the baseline
233
and continued for 4 min after the addition of BzATP. The data was analyzed using the
234
Zeiss ZEN Physiology software module (version 2011) and the change in intensity over 11
235
baseline was calculated to determine the increase in intracellular calcium over baseline.
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The calcium response to BzATP was measured in control and CAV1 suppressed
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MC3T3-E1 cells. The experiment was repeated 3 times from different cell passages,
238
with a minimum of 15 cells under each condition.
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Co-localization of P2X7R and cholera toxin B subunit: AF 555-Cholera toxin B (CTX-
240
B; 5 μg/ml) subunit was added to MC3T3-E1 cells grown on coverslips and incubated
241
on ice for 20 min. Subsequently, the cells were washed with PBS and fixed in 4%
242
paraformaldehyde for 20 min at 4ºC. FITC conjugated rabbit anti-P2X7R antibody was
243
used to target the extracellular domain of the receptor after blocking the fixed cells with
244
3% BSA. Prolong Gold was used to mount the immunostained cells on a slide and
245
imaged using the Zeiss ELYRA PS.1 superresolution microscope with the
246
PlanApochromat 63X oil immersion objective (1.4 numerical aperture). Structural
247
illumination data was acquired with 5 rotations and 3 phases to obtain improved
248
resolution and quantification. The excitation lasers used were 488 nm and 561 nm. To
249
minimize artifacts, channels were aligned with an affine channel alignment from a multi-
250
color bead calibration, auto noise filtered, and displayed using a baseline cut for
251
accurate representation for colocalization analysis. Mander’s correlation coefficient was
252
measured by setting an appropriate threshold for each channel using the Zen 2011
253
software. The values describe the total amount of P2X7R colocalized with the CTX-B
254
over the entire CTX-B bound region (8). Mander’s correlation coefficient were measured
255
for 12 randomly selected individual cells over two different experiments and averaged.
256
Mineralization of CAV1 KD cells with BzATP treatment: CAV1 KD cells were seeded
257
in a 12 well plate at 2.5x103/cm2 cell density and grown to confluency after which, the 12
258
cell culture media supplemented with 50 μg/ml of ascorbic acid and 10 mM of β-
259
glycerophosphate (BGP). The cells were treated with the P2X7R agonist, BzATP (150
260
μM) and after 24 hrs fresh media was added. The same process was carried out for 21
261
days with BzATP addition on alternative days. The control cells with the scrambled
262
shRNA were also subjected to the same treatment. The extent of mineralization was
263
then measured either by using von Kossa staining or Alizarin red staining.
264
von Kossa Staining: After 21 days in culture, the cells were washed with cold PBS and
265
fixed in 4% paraformaldehyde for 20 min at 4ºC. 5% aqueous silver nitrate was added to
266
the cells after rinsing with distilled water. The samples were then exposed to UV light for
267
30 min; then the excess silver nitrate was washed off with H2O. The regions of mineral
268
deposits were seen as dark nodules on the plate. Digital images of the wells were taken
269
and the extent of mineralization was quantified using ImageJ software. The intensity of
270
the control untreated cells was set as the threshold and comparisons were made with
271
the suppression of CAV1 and with BzATP treatment in CAV1 KD cells. The data was
272
normalized to the extent of mineralization in the control untreated cells and expressed
273
as fold change.
274
Alizarin Red Staining: After 21 days in culture the cells were washed gently with PBS
275
and fixed in 100% ethanol for 20 min, then air dried. Alizarin red stain (Lifeline Cell
276
Technology, Fredrick MD) was added to the samples and washed away with running
277
water after 15 min. Images were taken randomly encompassing different regions of the
278
well using the Nikon SMZ 1500 light microscope and quantified using ImageJ
13
279
Statistical Analysis: Densitometric analysis was carried out to quantify the extent of
280
CAV1 translocation from the buoyant fractions to the denser fractions using ImageJ
281
software. The samples were all normalized to the protein concentration of the lysate.
282
Mean peak [Ca2+]i response was expressed as a percentage increase in intensity over
283
baseline intensity levels. Experiments were repeated at least three times with cells from
284
3 different cell passages and the data are presented as mean ± SE. Significance of all
285
experiments were considered by One way analysis of variance (ANOVA) and the
286
significance of multiple comparisons was made using the Tukey-Kramer post hoc test.
287
Results:
288
P2X7R activation induces CAV1 endocytosis: To determine if purinergic signaling
289
can stimulate caveolar endocytosis, sucrose gradient fractionation was performed on
290
MC3T3-E1 cells treated with 250 µM ATP. Due to the high concentration of cholesterol
291
and sphingolipids in the caveolae, CAV1 is found in the less dense fractions of the
292
gradient when subjected to ultracentrifugation. However when CAV1 translocates from
293
the membrane microdomains, it loses the buoyant characteristic associated with the
294
lipid microdomains and is found in the denser fractions with other cytosolic components
295
(21). Fig 1A shows that in control conditions, CAV1 was mostly found in the 3-6
296
fractions of the gradient, equivalent to the 5%-35% gradient interface. However, when
297
the cells were stimulated with 250 µM of ATP for 10 min at 37ºC, most of the CAV1 is
298
seen in the denser fractions (9-12 fractions). Normalized densitometric analysis of 3
299
separate experiments shows a significant reduction in the CAV1 present in lipid
300
microdomain fractions in Fig 1B.
14
301
To test our hypothesis that CAV1 is endocytosed due to the activation of P2X7R,
302
MC3T3-E1 cells were treated with ATP, BzATP or BzATP after pretreatment with the
303
specific P2X7R inhibitor, A-839977, for 10 min. Changes in CAV1 localization was
304
determined using immunocytochemistry. Under control conditions, CAV1 was localized
305
to the plasma membrane as indicated by the arrows (Fig. 1C, i), however, when the
306
cells were treated with 250 µM ATP (Fig 1C, ii) or 150 µM BzATP (Fig 1C, iii), the
307
characteristic membrane CAV1 staining was significantly reduced. Treatment with
308
P2X7R agonists resulted in a diffuse and cytosolic staining. Finally, this change in CAV1
309
localization to purinergic stimulation was significantly attenuated when the cells were
310
pretreated with 500 nM A-839977 (Fig 1C, iv), which blocks BzATP induced [Ca2+]i
311
influx (18). Quantification the images shows, under control conditions, approximately
312
60% of cells showed the characteristic CAV1 staining on the plasma membrane and it
313
was significantly reduced with either ATP or BzATP treatment (p
1
Caveolin-1 Regulates P2X7Receptor Signaling in Osteoblasts
2
Vimal Gangadharan1, Anja Nohe1, Jeffrey Caplan1,2,
3
Kirk Czymmek1,2, Randall L. Duncan1, 2
4 5
Author Affiliation: 1Department of Biological Sciences, University of Delaware, Newark, DE 19716
6
2
Bioimaging Center, Delaware Biotechnology Institute, Newark, DE 19711
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Corresponding Author: Randall Duncan, Ph.D. Department of Biological Sciences 118 Wolf Hall University of Delaware Newark, DE 19716, USA Tel: 302.831.4296 Fax: 302.831.2281 Email: [email protected]
Running Head: CAV1 regulates P2X7R signaling in Osteoblasts
22 23 24 25 26 27
Copyright © 2014 by the American Physiological Society.
28
Abstract
29
The synthesis of new bone in response to a novel applied mechanical load requires a
30
complex series of cellular signaling events in osteoblasts and osteocytes. The activation
31
of the purinergic receptor, P2X7R, is central to this mechanotransduction signaling
32
cascade. Recently, the P2X7R have been found to be associated with caveolae, a
33
subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1),
34
the primary protein constituent of caveolae in osteoblasts, results in increased bone
35
mass, leading us to hypothesize that the P2X7R is scaffolded to caveolae in
36
osteoblasts. Thus, upon activation of the P2X7R, we postulate that caveolae are
37
endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation
38
of MC3T3-E1 pre-osteoblasts showed that CAV1 was translocated to the denser
39
cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X7R
40
agonist, BzATP, induced endocytosis of CAV1, which was inhibited when MC3T3-E1
41
cells were pretreated with the specific P2X7R antagonist, A-839977. The P2X7R co-
42
fractionated with CAV1 but, using Superresolution Structured Illumination Microscopy
43
(SR-SIM), we found that only a sub-population of the P2X7R existed in these lipid
44
microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced
45
the intracellular Ca2+ response to BzATP, suggesting that caveolae regulate P2X7R
46
signaling. This proposed mechanism is supported by increased mineralization observed
47
in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that
48
caveolae regulate P2X7R signaling upon activation by undergoing endocytosis and
49
potentially carrying with it other signaling proteins, hence controlling the spatio-temporal
50
signaling of P2X7R in osteoblasts.
2
51
Keywords: Caveolin-1, Purinergic signaling, P2X7R, Endocytosis, Osteoblasts
52
3
53
Introduction
54
Purinergic signaling plays a critical role in regulating skeletal mechanotransduction, an
55
intricate process through which physical stimulus is converted into a biochemical
56
response (7, 23). We, and others, have shown that ATP, and other nucleotides, are
57
released in response to fluid shear stress in vitro and activate purinergic (P2) receptors
58
on the membrane of the osteoblasts (13, 20, 29, 38). Although there are many subtypes
59
of P2 receptors present in osteoblasts (4), knockout of the P2X7R, a ligand gated ion
60
channel, resulted in an osteopenic phenotype (22), suggesting that this receptor is
61
important in skeletal maintenance. Further in vivo loading experiments show that load-
62
induced bone formation is significantly abrogated when the P2X7R is genetically
63
deleted, demonstrating the importance of this receptor in mechanotransduction (23).
64
Several characteristics distinguish the P2X7R from the other P2X receptors. This
65
receptor is 10-30 times more specific for 2’,3’-(benzoyl-4-benzoyl)-ATP (BzATP) than
66
ATP (28) and activation of the receptor by BzATP induces dynamic membrane blebbing
67
(39). P2X7R activation has been associated with the opening of a large pore on the
68
plasma membrane that allows the entry of molecules up to 900 Da in size (28, 46),
69
although recent reports suggests that this pore is actually a pannexin (19). Though the
70
prolonged activation of this receptor has been known to trigger apoptosis in certain cell
71
types, we have shown there is no caspase-3 activation observed in osteoblasts when
72
stimulated with BzATP (23). Stimulation of the P2X7R with BzATP in cells of the
73
osteoblastic lineage results in the expression of osteogenic markers such as osterix and
74
osteocalcin (31) and the release of PGE2 (23), all of which can eventually lead to
75
osteogenesis. Signaling via the P2X7R is also known to activate PLD and PLA2, leading 4
76
to the synthesis of LPA that, in turn, has been attributed to membrane blebbing (31).
77
P2X7R activation also induces ERK phosphorylation in a PKC-dependent manner in
78
osteoblasts (26). The receptor has a significant role in inflammatory responses, wherein
79
it was shown the receptor activation induces the release of inflammatory cytokines from
80
macrophages and osteoclasts (42). Overall this ligand gated ion channel has a major
81
role in several intracellular processes in a cell type specific manner.
82
Recent reports suggest that a sub-population of the P2X7R exists in caveolae, 60-100
83
nm invaginations of the plasma membrane (30, 49), in cells of the submandibular
84
glands, lung alveolae and macrophages (3, 12, 16). These plasma membrane
85
structures are widely expressed in adipocytes, fibroblasts, endothelial cells and
86
osteoblasts (34, 43, 44) and are formed from a subset of components of lipid rafts, such
87
as sphingolipids and cholesterol, and are stabilized by 22 kD integral proteins called
88
caveolins (34).There are three isoforms of caveolin, CAV1, CAV2 and CAV3. CAV1 and
89
CAV2 are found in non-muscle cells, whereas CAV3 is the primarily found in skeletal
90
and some smooth muscle cells (47). The removal of CAV1 and CAV3 prevents the
91
formation of caveolae in these cells, but there was no apparent changes when CAV2
92
was suppressed (5, 11). Numerous studies have demonstrated an important role for
93
caveolae in cell signaling and scaffolding of signaling molecules in several cell types
94
(24, 27, 35). Besides anchoring second messengers, caveolae can also attenuate
95
signaling by endocytosis therefore regulating the presence of the receptors along with
96
other signaling proteins on the plasma membrane (17, 36).
97
The importance of CAV1 and caveolae in regulation of the skeleton is emphasized by a
98
study that demonstrate the knockout of CAV1 in mice increases trabecular and cortical 5
99
bone which appears to result from increased osteoblast function (40). Because both
100
P2X7R and CAV1 are important in skeletal maintenance and the response of bone to
101
mechanical loading, we hypothesized that the P2X7R is present in caveolae of
102
osteoblasts and further postulated that disruption of caveolae will alter P2X7R mediated
103
signaling.
104
Materials and Methods
105
Cell culture: MC3T3-E1 cells, a murine preosteoblast-like cell line (ATCC), were grown
106
in α-Modified Eagles Medium (α-MEM) that was supplemented with 10% FBS (Gemini),
107
100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained in a humidified
108
incubator in a 5% CO2/95% air mixture and subcultured every 72 hrs. MC3T3-E1 cells
109
were serum starved for 12-16 hrs prior to all experiments. All cell culture media and
110
antibiotics were purchased from Sigma-Aldrich, St Louis, MO.
111
Reagents and antibodies: Adenosine 5’ Triphosphate disodium salt (ATP); 2’, (3’)-O-
112
(4-Benzoylbenzoyl) adenosine 5’ triphosphate triethylammonium salt (BzATP) and
113
puromycin dihydrochloride (Puromycin) were purchased from Sigma-Aldrich. ATP (250
114
µM; from a stock of 5 mM in PBS) and BzATP (150 µM; from a stock of 6 mM in dH2O)
115
were used for activation of P2X7R. A-839977 (500 nM; from stock of 500 µM in DMSO),
116
a potent P2X7R antagonist was purchased from Tocris Bioscience. Rabbit polyclonal
117
anti-CAV1 and mouse monoclonal anti-CAV1 were obtained from BD Biosciences (San
118
Jose, CA). Rabbit polyclonal anti-P2X7R and FITC conjugated rabbit anti-P2X7R were
119
purchased from Alomone Labs (Jerusalem, Israel). Anti-GAPDH antibody obtained from
120
Abcam (Cambridge, MA) was used as loading control in Western blots. The caveolar
6
121
marker, Alexa Fluor®-555 Cholera Toxin B (CTX-B) and AF-488 donkey anti-rabbit IgG
122
were purchased from Life Technologies (Carlsbad, CA). siRNA against CAV1 was
123
obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and the Sure Silencing
124
plasmid vectors coding shRNA against CAV1 with Puromycin resistance (KM03639P)
125
were purchased from SABiosciences (Frederick, MD).
126
Detergent free isolation of caveolin enriched membrane fractions: Sucrose
127
gradient fractionation was used to isolate caveolin enriched membrane fractions as
128
described by Song, et al. with some modifications (45). Briefly, MC3T3-E1 cells were
129
grown to 80-90% confluence in 150 mm Petri dishes and washed with cold PBS. In the
130
experiments with agonist treatment, the cells were grown in reduced serum (0.2%) for
131
14-16 hrs prior to treatment. The cells were scraped into 2 ml of ice cold 500 mM
132
sodium carbonate containing protease inhibitors (Calbiochem, San Deigo, CA). The cell
133
lysate was then homogenized by sonication, centrifuged at 9000xg for 10 min, and the
134
supernatant was collected. Protein concentration was measured using BCA assay and
135
equal concentrations were subjected to discontinuous sucrose gradient fractionation.
136
A 45% sucrose-lysate solution was made by adding 90% sucrose in MBS (25 mM MES,
137
150 mM NaCl, pH 6.5) to the lysate. This mixture was then placed at the bottom of an
138
ultracentrifuge tube and a discontinuous gradient was prepared on top of this mixture by
139
layering with 4 ml of 35% and 5% sucrose in MBS containing 250 mM sodium
140
carbonate. The gradient was subjected to ultracentrifugation for 18 hrs at 39,000 rpm at
141
4°C with a SW41-TI rotor (Beckman Coulter). A light scattering band was typically
142
observed at the 5%-35% interface where most of the caveolae was enriched. Twelve 1
143
ml fractions were collected from the top of the gradient and the proteins present in each 7
144
fraction were precipitated using TCA-Acetone. The precipitates were then resuspended
145
in sample buffer and equal volumes of the fractions were subjected to Western blotting
146
for the respective proteins.
147
Immunocytochemistry: Cells were seeded at a density of 2.5x103/cm2 onto glass
148
cover slips coated with rat tail type I collagen (100 µg/ml in 0.02 N acetic acid, BD,
149
Franklin Lakes, NJ) and grown to 80-90% confluency. Experimentally, cells were treated
150
with 250 µM ATP or 150 µM BzATP. In one group, cells were pretreated with 500 nM of
151
A-839977 for 30 min prior to the addition of 150 µM BzATP. After 10 min of stimulation,
152
cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA)
153
containing 0.1% Triton X-100. Fixed cells were washed with PBS and blocked with
154
blocking buffer containing 3% BSA and 10% donkey serum for 2 hrs. Rabbit anti-
155
caveolin-1 antibody (0.5 µg) was then added and the samples were incubated overnight
156
at 4°C. Following incubation, the samples were washed with blocking buffer, and then
157
the secondary antibody, AF-488 donkey anti-rabbit IgG, was added. The cells were then
158
mounted on microscopy slides with Prolong Gold (Life Technologies) and visualized
159
using the Zeiss LSM 780 confocal microscope with a EC Plan Neofluar 40x/1.3 Oil
160
objective lens. Quantification was carried out by manually counting the number of cells
161
with strong membrane staining pattern for CAV1 within the field of view. A minimum of
162
30 cells were counted for each treatment and the percentage of cells with plasma
163
membrane CAV1 was calculated. Each experiment was repeated three times with
164
different cell passages. The line profile of CAV1 staining illustrating the intensity of
165
labeling across the cell was created using the ImageJ software.
8
166
Triton X-100 extraction: MC3T3-E1 cells grown on 100mm Petri dishes were washed
167
three times with ice cold PBS and lysed in 500 µl of cold Triton X-100 lysis buffer (1%
168
Triton X-100, 50 mM Tris, 150 mM NaCl, pH 6.5) containing protease inhibitors. The
169
cells were scraped off the plate and proteins were extracted for 30 min at 4°C. The
170
lysate was then centrifuged at 9000xg for 30 min and supernatant was collected as the
171
soluble fraction (S). The pellet containing the detergent-resistant caveolae was
172
resuspended in 500 µl RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5%
173
sodium deoxycholate, 1% Triton X-100 and protease inhibitors) and sonicated to obtain
174
the insoluble fraction (IS). Equal volumes of both fractions were subjected to Western
175
blotting and probed for CAV1 and the P2X7 receptor.
176
siRNA strategy for transient knockdown of CAV1: Small interfering RNA was used
177
to transiently knockdown CAV1 in MC3T3-E1 cells as per manufacturer’s instructions. In
178
brief, MC3T3-E1 cells were seeded at a cell density of 2.5x103/cm2 onto rat tail type-1
179
collagen-coated (100 µg/ml; BD) glass bottom dishes (MatTek Corporation, Ashland,
180
MA) and grown to 50-60% prior to transfection. For transfection, the culture medium
181
was removed and the cells washed with PBS, followed by addition of 800 µl Opti MEM
182
(Life Technologies). siRNA against CAV1 (0.5 µM; sc-29942) and 6 µl of Oligofectamine
183
(Life Technologies) were diluted in 100 µl of OptiMEM, respectively. The reagents were
184
then gently mixed and incubated for 20 min at room temperature before being added to
185
each dish. After 5-7 hrs of incubation with the siRNA, fresh culture media containing
186
10% FBS was added to the cells. Experiments were conducted after 72 hrs post
187
transfection since Western blotting demonstrated maximum suppression was achieved
188
after 72 hrs. Scrambled siRNA (sc-37007) and PBS were used as controls. 9
189
Generation of stable CAV1 knockdown (CAV1 KD) cells: To generate a clonal line of
190
MC3T3-E1 cells with stable knockdown of CAV1, SureSilencing shRNA plasmid with
191
Puromycin resistance was used. Five sets of plasmids were provided; of which four
192
were specifically targeted against CAV1 mRNA and the fifth plasmid was a non-specific
193
scrambled plasmid that served as a control. Following the manufacturers protocol, each
194
of the plasmids was amplified after transformation into the competent DH5-alpha cells.
195
The plasmids were isolated using QIAGEN Mini Prep and concentration was measured.
196
Prior to transfection with the shRNA encoding plasmids, MC3T3-E1 cells (2.5x103/cm2)
197
were grown on a 12 well plate for 24 hrs. 6 µl of the transfection reagent, Lipofectin (Life
198
Technologies) was diluted in 100 µl of OptiMEM and mixed with 100 µl of OptiMEM
199
containing 1.2 µg of the plasmid and incubated for 20 min at room temperature.
200
Meanwhile the growth medium on the cells was replaced with 800 µl of fresh OptiMEM.
201
After incubation, the transfection mixture was added to the MC3T3-E1 cells and
202
incubated for 5-7 hrs at 37°C. The transfection reagents were then removed and fresh
203
culture media containing 10% FBS was added. After 72 hrs post transfection, the
204
culture media was supplemented with 5 µg/ml of Puromycin, the minimum inhibitory
205
concentration based on the dose response for MC3T3-E1 cells. Cells that did not take
206
up the plasmid failed to develop a resistance to the antibiotic and died. Within a week,
207
colonies of MC3T3-E1 cells with Puromycin resistance were beginning to develop.
208
These colonies were isolated for approximately 8 weeks and were continuously
209
screened for antibiotic resistance. A control group of untransfected MC3T3-E1 cells
210
were also screened to ensure the antibiotic concentration remained effective to
211
completely kill the non-transfected cells. Four different clones were isolated of which 10
212
one was the scrambled control and the extent of CAV1 knockdown was measured using
213
immunoblotting. GAPDH was used as the loading control.
214
Western Blotting: To determine the protein levels of CAV1 following knockdown the
215
siRNA or shRNA treatment, MC3T3-E1 cells were lysed using RIPA buffer and equal
216
amounts of protein were subjected to SDS-PAGE. To detect CAV1, P2X7R and GAPDH
217
in the sucrose fractionation studies and in the Triton-X100 extraction experiments, equal
218
volumes of the samples were loaded onto 4-12% gradient NuPAGE gels (Life
219
Technologies). After electrophoresis, the proteins were transferred to nitrocellulose
220
membrane and blocked ON with 5% milk in TBS/T (TBS containing 0.1% Tween-20).
221
The blots were probed with antibodies against CAV1, P2X7R and GAPDH using the
222
respective antibodies.
223
Calcium imaging: Intracellular Ca2+ levels [Ca2+]i were measured in MC3T3-E1 cells
224
using a fluorescent Ca2+ chelator, Fluo-4 and imaged using Zeiss 510 confocal
225
microscope with a Zeiss 10X C-Apochromat water immersion lens (0.45 numerical
226
aperture). As previously described, MC3T3-E1 cells were grown on rat tail type I
227
collagen-coated glass bottom dishes until cells reached 70% - 80% confluency. Cells
228
were then loaded with the calcium indicator, 10 µM Fluo-4 (Life Technologies) for 20
229
min at 37ºC. The cells were then washed with HBSS to remove excess dye and allowed
230
to recover for 15 min before observation. A 488nm excitation laser was used to visualize
231
[Ca2+]i and the changes in intensity were measured as a time lapse. Images were
232
collected over a period of 1 min prior to the addition of BzATP to establish the baseline
233
and continued for 4 min after the addition of BzATP. The data was analyzed using the
234
Zeiss ZEN Physiology software module (version 2011) and the change in intensity over 11
235
baseline was calculated to determine the increase in intracellular calcium over baseline.
236
The calcium response to BzATP was measured in control and CAV1 suppressed
237
MC3T3-E1 cells. The experiment was repeated 3 times from different cell passages,
238
with a minimum of 15 cells under each condition.
239
Co-localization of P2X7R and cholera toxin B subunit: AF 555-Cholera toxin B (CTX-
240
B; 5 μg/ml) subunit was added to MC3T3-E1 cells grown on coverslips and incubated
241
on ice for 20 min. Subsequently, the cells were washed with PBS and fixed in 4%
242
paraformaldehyde for 20 min at 4ºC. FITC conjugated rabbit anti-P2X7R antibody was
243
used to target the extracellular domain of the receptor after blocking the fixed cells with
244
3% BSA. Prolong Gold was used to mount the immunostained cells on a slide and
245
imaged using the Zeiss ELYRA PS.1 superresolution microscope with the
246
PlanApochromat 63X oil immersion objective (1.4 numerical aperture). Structural
247
illumination data was acquired with 5 rotations and 3 phases to obtain improved
248
resolution and quantification. The excitation lasers used were 488 nm and 561 nm. To
249
minimize artifacts, channels were aligned with an affine channel alignment from a multi-
250
color bead calibration, auto noise filtered, and displayed using a baseline cut for
251
accurate representation for colocalization analysis. Mander’s correlation coefficient was
252
measured by setting an appropriate threshold for each channel using the Zen 2011
253
software. The values describe the total amount of P2X7R colocalized with the CTX-B
254
over the entire CTX-B bound region (8). Mander’s correlation coefficient were measured
255
for 12 randomly selected individual cells over two different experiments and averaged.
256
Mineralization of CAV1 KD cells with BzATP treatment: CAV1 KD cells were seeded
257
in a 12 well plate at 2.5x103/cm2 cell density and grown to confluency after which, the 12
258
cell culture media supplemented with 50 μg/ml of ascorbic acid and 10 mM of β-
259
glycerophosphate (BGP). The cells were treated with the P2X7R agonist, BzATP (150
260
μM) and after 24 hrs fresh media was added. The same process was carried out for 21
261
days with BzATP addition on alternative days. The control cells with the scrambled
262
shRNA were also subjected to the same treatment. The extent of mineralization was
263
then measured either by using von Kossa staining or Alizarin red staining.
264
von Kossa Staining: After 21 days in culture, the cells were washed with cold PBS and
265
fixed in 4% paraformaldehyde for 20 min at 4ºC. 5% aqueous silver nitrate was added to
266
the cells after rinsing with distilled water. The samples were then exposed to UV light for
267
30 min; then the excess silver nitrate was washed off with H2O. The regions of mineral
268
deposits were seen as dark nodules on the plate. Digital images of the wells were taken
269
and the extent of mineralization was quantified using ImageJ software. The intensity of
270
the control untreated cells was set as the threshold and comparisons were made with
271
the suppression of CAV1 and with BzATP treatment in CAV1 KD cells. The data was
272
normalized to the extent of mineralization in the control untreated cells and expressed
273
as fold change.
274
Alizarin Red Staining: After 21 days in culture the cells were washed gently with PBS
275
and fixed in 100% ethanol for 20 min, then air dried. Alizarin red stain (Lifeline Cell
276
Technology, Fredrick MD) was added to the samples and washed away with running
277
water after 15 min. Images were taken randomly encompassing different regions of the
278
well using the Nikon SMZ 1500 light microscope and quantified using ImageJ
13
279
Statistical Analysis: Densitometric analysis was carried out to quantify the extent of
280
CAV1 translocation from the buoyant fractions to the denser fractions using ImageJ
281
software. The samples were all normalized to the protein concentration of the lysate.
282
Mean peak [Ca2+]i response was expressed as a percentage increase in intensity over
283
baseline intensity levels. Experiments were repeated at least three times with cells from
284
3 different cell passages and the data are presented as mean ± SE. Significance of all
285
experiments were considered by One way analysis of variance (ANOVA) and the
286
significance of multiple comparisons was made using the Tukey-Kramer post hoc test.
287
Results:
288
P2X7R activation induces CAV1 endocytosis: To determine if purinergic signaling
289
can stimulate caveolar endocytosis, sucrose gradient fractionation was performed on
290
MC3T3-E1 cells treated with 250 µM ATP. Due to the high concentration of cholesterol
291
and sphingolipids in the caveolae, CAV1 is found in the less dense fractions of the
292
gradient when subjected to ultracentrifugation. However when CAV1 translocates from
293
the membrane microdomains, it loses the buoyant characteristic associated with the
294
lipid microdomains and is found in the denser fractions with other cytosolic components
295
(21). Fig 1A shows that in control conditions, CAV1 was mostly found in the 3-6
296
fractions of the gradient, equivalent to the 5%-35% gradient interface. However, when
297
the cells were stimulated with 250 µM of ATP for 10 min at 37ºC, most of the CAV1 is
298
seen in the denser fractions (9-12 fractions). Normalized densitometric analysis of 3
299
separate experiments shows a significant reduction in the CAV1 present in lipid
300
microdomain fractions in Fig 1B.
14
301
To test our hypothesis that CAV1 is endocytosed due to the activation of P2X7R,
302
MC3T3-E1 cells were treated with ATP, BzATP or BzATP after pretreatment with the
303
specific P2X7R inhibitor, A-839977, for 10 min. Changes in CAV1 localization was
304
determined using immunocytochemistry. Under control conditions, CAV1 was localized
305
to the plasma membrane as indicated by the arrows (Fig. 1C, i), however, when the
306
cells were treated with 250 µM ATP (Fig 1C, ii) or 150 µM BzATP (Fig 1C, iii), the
307
characteristic membrane CAV1 staining was significantly reduced. Treatment with
308
P2X7R agonists resulted in a diffuse and cytosolic staining. Finally, this change in CAV1
309
localization to purinergic stimulation was significantly attenuated when the cells were
310
pretreated with 500 nM A-839977 (Fig 1C, iv), which blocks BzATP induced [Ca2+]i
311
influx (18). Quantification the images shows, under control conditions, approximately
312
60% of cells showed the characteristic CAV1 staining on the plasma membrane and it
313
was significantly reduced with either ATP or BzATP treatment (p
