Age-related changes in gap junctional intercellular communication in osteoblastic cells.
ABSTRACT Aging demonstrates deleterious effects upon the skeleton which can predispose an individual to osteoporosis and related fractures. Despite the well-documented evidence that aging decreases bone formation, there remains little understanding whereby cellular aging alters skeletal homeostasis. We, and others, have previously demonstrated that gap junctions-membrane-spanning channels that allow direct cell-to-cell conductance of small signaling molecules-are critically involved in osteoblast differentiation and skeletal homeostasis. We examined whether the capacity of rat osteoblastic cells to form gap junctions and respond to known modulators of gap junction intercellular communication (GJIC) was dependent on the age of the animal from which they were isolated. We observed no effect of age upon osteoblastic Cx43 mRNA, protein or GJIC. We also examined age-related changes in PTH-stimulated GJIC. PTH demonstrated age-dependent effects upon GJIC: Osteoblastic cells from young rats increased GJIC in response to PTH, whereas there was no change in GJIC in response to PTH in osteoblastic cells from mature or old rats. PTH-stimulated GJIC occurred independently of changes in Cx43 mRNA or protein expression. Cholera toxin significantly increased GJIC in osteoblastic cells from young rats compared to those from mature and old rats. These data demonstrate an age-related impairment in the capacity of osteoblastic cells to generate functional gap junctions in response to PTH, and suggest that an age-related defect in G protein-coupled adenylate cyclase activity at least partially contributes to decreased PTH-stimulated GJIC. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1979-1984, 2012.
- SourceAvailable from: Eric C Beyer[show abstract] [hide abstract]
ABSTRACT: We have examined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromal cells (BMC) and trabecular bone osteoblasts (HOB), and in the human osteogenic sarcoma cell line, SaOS-2. Both HOB and BMC cells were functionally coupled, since microinjection of Lucifer yellow resulted in dye transfer to neighboring cells, with averages of 3.4 +/- 2.8 (n = 131) and 8.1 +/- 9.3 (n = 51) coupled cells per injection, respectively. In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4 +/- 1.8 coupled cells per injection, n = 100). Dye diffusion was inhibited by octanol (3.8 mM), an inhibitor of gap junctional communication. All of the osteoblastic cells expressed mRNA for connexin43 and connexin45, but not for connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similar quantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced significantly less Cx43 protein than either HOB or BMC, as assessed by immunofluorescence and immunoprecipitation. Conversely, more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and transformed human osteoblastic cells correlates with the level of expression of Cx43, which appears to mediate intercellular communication in these cells. Gap junctional communication may serve as a means by which osteoblasts can work in synchrony and propagate locally generated signals throughout the skeletal tissue.Journal of Clinical Investigation 06/1993; 91(5):1888-96. · 12.81 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Gap junctions consist of arrays of intercellular channels composed of integral membrane proteins called connexin in vertebrates. Gap junction channels regulate the passage of ions and biological molecules between adjacent cells and, therefore, are critically important in many biological activities, including development, differentiation, neural activity, and immune response. Mutations in connexin genes are associated with several human diseases, such as neurodegenerative disease, skin disease, deafness, and developmental abnormalities. The activity of gap junction channels is regulated by the membrane voltage, intracellular microenvironment, interaction with other proteins, and phosphorylation. Each connexin channel has its own property for conductance and molecular permeability. A number of studies have tried to reveal the molecular architecture of the channel pore that should confer the connexin-specific permeability/selectivity properties and molecular basis for the gating and regulation. In this review, we give an overview of structural studies and describe the structural and functional relationship of gap junction channels.Cellular and Molecular Life Sciences CMLS 10/2010; 68(7):1115-29. · 5.62 Impact Factor
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ABSTRACT: Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106-01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter-luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2. 8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106-01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.Molecular Biology of the Cell 09/1998; 9(8):2249-58. · 4.60 Impact Factor
Age-Related Changes in Gap Junctional Intercellular Communication in
Damian C. Genetos,1Zhiyi Zhou,2Zhongyong Li,2Henry J. Donahue2
1Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California-Davis, Davis, California,
2Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, The Pennsylvania State University College of Medicine,
Received 7 March 2012; accepted 22 May 2012
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22172
fractures. Despite the well-documented evidence that aging decreases bone formation, there remains little understanding whereby
cellular aging alters skeletal homeostasis. We, and others, have previously demonstrated that gap junctions—membrane-spanning
channels that allow direct cell-to-cell conductance of small signaling molecules—are critically involved in osteoblast differentiation and
skeletal homeostasis. We examined whether the capacity of rat osteoblastic cells to form gap junctions and respond to known modula-
tors of gap junction intercellular communication (GJIC) was dependent on the age of the animal from which they were isolated. We
observed no effect of age upon osteoblastic Cx43 mRNA, protein or GJIC. We also examined age-related changes in PTH-stimulated
GJIC. PTH demonstrated age-dependent effects upon GJIC: Osteoblastic cells from young rats increased GJIC in response to PTH,
whereas there was no change in GJIC in response to PTH in osteoblastic cells from mature or old rats. PTH-stimulated GJIC occurred
independently of changes in Cx43 mRNA or protein expression. Cholera toxin significantly increased GJIC in osteoblastic cells
from young rats compared to those from mature and old rats. These data demonstrate an age-related impairment in the capacity of
osteoblastic cells to generate functional gap junctions in response to PTH, and suggest that an age-related defect in G protein-coupled
adenylate cyclase activity at least partially contributes to decreased PTH-stimulated GJIC.
Published by Wiley Periodicals, Inc. J Orthop Res
Aging demonstrates deleterious effects upon the skeleton which can predispose an individual to osteoporosis and related
? 2012 Orthopaedic Research Society.
osteoblast; aging; connexion 43; gap junction; parathyroid hormone
Gap junctions are membrane-spanning channels that
facilitate intercellular communication by allowing pas-
sive flux of intracellular signaling molecules, such as
Ca2þ, cyclic AMP (cAMP), and ATP, to pass from the
cytosol of one cell directly into the cytosol of another.1
Gap junction channels are composed of protein subu-
nits termed connexins (Cx), at least 20 of which have
been identified in mammalian species.2Hexameriza-
tion of Cx generates a connexon, which can function
unapposed in the plasmalemma (Cx hemichannel), or
can bind to another connexon in a neighboring cell to
generate a functional gap junction. In adult bone, the
predominant gap junction protein expressed is con-
nexin 43 (Cx43) but Cx45 and 46 are also expressed,3
although Cx46 is retained in monomeric form in the
golgi network and thus does not present itself within
The role of gap junctions and gap junction intercel-
lular communication (GJIC) in skeletal metabolism
and homeostasis is well-established. Several studies
have presented compelling evidence that GJIC is criti-
cal for osteoblastic cell differentiation into mature
bone-forming cells both in vitro and in vivo.5–9Addi-
tionally, previous studies demonstrate that GJIC con-
tributes to the responsiveness of osteoblastic cells in
vitro to diverse anabolic signals including parathyroid
hormone,10electromagnetic fields,11and fluid shear
stress.12–15Gap junction channels also allow osteo-
cytes to communicate mechanical signals they detect
to osteoblastic cells.16The critical importance of con-
nexons, especially Cx43, are noted in murine models
and in human disease.17In mice, targeted Cx43 muta-
genesis elicits neonatal lethality because of severe car-
diovascular malformation18; nonetheless, these mice
demonstrate hypomineralization of craniofacial bones,
as well as delayed ossification of appendicular and axi-
al skeleton.6Similar results are observed in mice with
osteoblast and osteocyte-specific deletion of Cx43.19
These mice also display altered bone adaptation to
mechanical load.20In humans, mis-sense mutations
in Cx43 produce the rare genetic disease oculodento-
digital dysplasia (ODDD; OMIM 164200),21involving,
(reviewed in).22Taken together these studies indicate
that GJIC involving Cx43 significantly contributes to
Bone formation rates decrease with increasing
age,23,24suggesting that decreased osteoblastic activity
may contribute to age-related osteopenia.25Osteoblast
activity is determined to a large extent by the capacity
of osteoprogenitors and osteoblasts to adapt to changes
in their extracellular environment. The ability of oste-
oblastic cells to adapt to hormonal or biophysical sig-
nals appears to decrease with advanced age.26–28Since
GJIC contributes to bone cell responsiveness to extra-
cellular signals, we postulated that GJIC may change
as a function of age. Furthermore, we have previously
reported an age-related decrease in PTH-stimulated
cAMP accumulation in osteoblastic cells27and since
Grant sponsor: National Institute of Arthritis and Musculoskele-
tal and Skin Diseases; Grant number: R03AR057547;
Grant sponsor: National Institute on Aging; Grant number:
Correspondence to: Henry J. Donahue (T: 717-531-4819; F: 717-
531-0349; E-mail: firstname.lastname@example.org)
? 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2012
dependent on cAMP,27,29,30we also postulated that
PTH-stimulated GJIC decreases as a function of osteo-
We examined GJIC in primary cultures of rat osteo-
blastic cells isolated from young (4-month-old), mature
(12-month-old), and old (24–28-month-old) rats. In
order to examine the mechanisms underlying any age-
related changes we found in GJIC, we also examined
PTH-stimulated Cx43 mRNA and protein expression,
and PTH and cholera toxin (CTX)-stimulated GJIC
in ROB. We observed that PTH stimulated GJIC in
young ROBs, but not mature or old ROBs, and this
occurred independently of PTH-stimulated changes in
Cx43 mRNA or protein expression. In agreement with
our previous work upon age-related changes in cAMP
induction, we observed impaired CTX-stimulated GJIC
in osteoblastic cells from mature and old rats com-
pared to those from young rats, suggesting that aging
may alter the mechanisms whereby GJIC communica-
tion is regulated.
Rat parathyroid hormone fragment 1–34 (rPTH[1–34]) was
purchased from Bachem. RNeasy RNA isolation kits were
purchased from Qiagen. Reagents for real time RT-PCR
were purchased from Applied Biosystems, Foster City, CA.
Calcein-AM and 1,10-dioctadecyl-3,3,30,30-tetramethylindocar-
bocyanine perchlorate (DiI) were purchased from Molecular
Probes, Grand Island, NY. Dulbecco’s modified Eagle’s media
(DMEM), and penicillin/streptomycin were purchased from
Gibco, Grand Island, NY. Fetal bovine serum (FBS) was
purchased from Hyclone Laboratories, Rockford, IL. CTX
was purchased from Calbiochem, Billerica, MA. All other
reagents were purchased from Fisher Scientific (St. Louis,
MO) and were of tissue culture grade.
Primary Culture of Rat Osteoblastic Cells
Rat osteoblastic cells were isolated from the tibiae and femo-
rae of 4-, 12-, and 24–28-month-old male Fisher 344 rats as
previously described.27,31Briefly, sub-periosteal osteoblastic
cells were isolated by sequential collagenase digestion at
378C. Second digestion cells were collected by centrifugation
and placed into T-25 tissue culture flasks and cultured in
DMEM supplemented with 20% FBS, 100 IU/ml penicillin
and 100 mg/ml streptomycin. Cells were allowed to reach con-
fluence and then subcultured into culture dishes or glass
slides for each experiment. We have previously demonstrated
that cells isolated in this manner express phenotypic charac-
teristics of osteoblastic cells.31
Assessment of GJIC
GJIC between osteoblastic cells was assessed by a double
fluorescent labeling technique.32‘‘Acceptor’’ cells were plated
at a density of 4 ? 104cells/cm2on 25 mm glass and were
allowed to reach 90% confluence, over a period of 24–48 h.
Simultaneously, ‘‘donor’’ cells were plated at a density of
4 ? 104cells/cm2in 35 mm tissue culture dishes and main-
tained at 378C over the same time period. To measure GJIC,
donor cells were washed twice with phosphate-buffered
saline (PBS), incubated with 10 mM calcein-AM, 10 mM DiI,
1% pluronic acid, and 20 mg/mL BSA in Hank’s Balanced
Salt Solution, and incubated for 20 min at 378C. Following
incubation, donor cells were washed with PBS, collected via
trypsin, and 500 donor cells were added to confluent mono-
layers of unlabeled acceptor cells and incubated for 20 min at
378C. Thereafter, coverslips were removed, washed in PBS
and inverted onto microscope slides; GJIC was evaluated
using a Nikon epifluorescence microscope (Nikon EFD-3,
Optical Apparatus Co., Ardmore, PA) and visualized to locate
the calcein and DiI loaded cells, respectively. Calcein-AM,
because of its small size (MW 994.87), can be transferred
to neighboring cells when functional (open) gap junctions are
established between donor and acceptor cells. The fluorescent
dye DiI intercalates within cell membranes and does not
transfer to neighboring cells via GJIC and is used to identify
Both donor cells and acceptor cells were exposed for
20 min at 378C to vehicle control, 10?10–10?6M rPTH [1–34]
or 10?10–10?6M CTX. Each vehicle or drug was also present
during the formation of GJIC between donor and acceptor
cells. For each condition, the number of calcein-positive ac-
ceptor cells coupled to each of 40 DiI positive donor cells was
recorded. The average number of acceptor cells per donor cell
was then calculated. The number of donor cells counted was
40 as it was the maximum number of coupled donor cells
under any condition.
Western Blotting and Quantitative Real Time RT-PCR
Osteoblastic cells were plated at 5,000/cm2in 10 cm2tissue
culture dishes and cultured to 80% confluence (3–4 days).
The cells were then exposed for 2 h at 378C to rPTH [1–34]
(10?10–10?6M) or CTX (10?10–10?7M) or vehicle control, af-
ter which whole cell protein lysate or RNA was isolated.
Western immunoblotting and qtPCR was subsequently per-
formed as described previously.33
Basal Gja1 and Cx43 protein expression and basal GJIC
(Fig. 1a–c) are reported as mean ? SEM. The remaining
data are presented as fold-change in coupled cells compared
to age-matched, vehicle control in order to minimize inter-an-
imal variability and are presented as mean ? SEM. Data
were analyzed by ANOVA followed by Tukey or Dunnet’s
test. p < 0.05 was considered statistically significant. For
CTX studies, osteoblastic cells from one young animals
showed no responsiveness to CTX, and were deemed suitable
for exclusion from analysis by Q-test.34
Basal Cx43 Expression and GJIC Is Not Altered as a
Function of Age of Animal From Which Osteoblastic
Cells Were Isolated
We first examined the influence of donor animal age
on Cx43 mRNA, protein, and GJIC expression in oste-
oblastic cells isolated from young, mature, and old
rats. We found no significant influence of donor animal
age on basal Gja1 (Cx43 mRNA; Fig. 1a) or protein
(Fig. 1b). Similarly, we observed no age-related change
basal GJIC in osteoblastic cells (Fig. 1c).
Age of Donor Animal Influences PTH-Stimulated
GJIC in Osteoblastic Cells
Osteoblastic cells isolated from young rats demonstrat-
ed a statistically significant increase in the number of
coupled cells in response to PTH treatment (Fig. 2a).
GENETOS ET AL.
JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2012
Exposure to PTH (10?11–10?8M) revealed a trend for
increased GJIC in osteoblastic cells isolated from
young rats, which reached statistical significance
at 10?9and 10?8M PTH relative to vehicle controls.
In contrast, PTH at any dose used had no effect upon
GJIC in osteoblastic cells isolated from mature or old
rats (Fig. 2a). We found no effect of PTH upon Cx43
mRNA (Fig. 2b) or protein (Fig. 2c), indicating that
PTH increases GJIC via post-translational mecha-
nisms in osteoblastic cells from young rats.
Increasing cAMP AccumulationDoes Not StimulateGJICin
Osteoblastic Cells Isolated From Older Animals
We have previously demonstrated an age-related im-
pairment in cAMP synthesis in response to CTX.27
CTX exerted a dose-dependent increase in GJIC in os-
teoblastic cells from young animals, but had no effect
upon osteoblastic cells from mature or old rats (Fig. 3).
Gap junctions are critical regulators of cell–cell com-
munication and are involved in diverse developmental
demonstrate no differences in Cx43 expression or GJIC. Osteo-
blastic cells were isolated as described in Materials and Methods
Section and plated. (a) Basal Gja1 mRNA expression relative to
18s rRNA was examined by qPCR, and is not altered as a func-
tion of age of donor animal. (b) Basal Cx43 protein expression
relative to GAPDH was examined by western immunoblotting,
and also demonstrated no age-related differences. (c) Formation
of GJIC was assessed by parachute assay, and demonstrated no
age-related differences. n ¼ 3–5 per age group.
Osteoblastic cells from young, mature, and old rats
of de novo Gja1 or Cx43 expression. Osteoblastic cells were
isolated and GJIC was evaluated as described in Materials
and Methods. (a) Recombinant PTH (1–34) dose-dependently
increased the degree of GJIC in osteoblastic cells isolated
from young, but not mature or old, rats. (b) Recombinant PTH
(1–34; 10?11–10?7M) had no effect on Gja1 mRNA expression in
osteoblastic cells from young, mature, or old rats. (c) Recombi-
nant PTH (1–34; 10?11–10?7M) had no effect on Cx43 protein
expression in osteoblastic cells from young, mature, or old rats. a
indicates p < 0.05 compared to osteoblastic cells from mature
rats; b indicates p < 0.05 versus osteoblastic cells from old rats;
c indicates p < 0.05 young treated versus control. n ¼ 2–4
animals per age group per treatment.
PTH-induced GJIC is age-related and independent
AGING AND GAP JUNCTIONS IN OSTEOBLASTS
JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2012
and homeostatic function. Within cardiac muscle, they
rapidly propagate action potentials generated in the
sinoatrial (SA) node to enable the myocardium to gen-
erate synchronous myocardial contraction.35Within
the vasculature, GJIC between the endothelium and
the underlying smooth muscle contributes to endothe-
lium-dependent dilation.36Since the discovery of gap
junctions within the skeleton,37tremendous effort has
been exerted to understand the role and importance of
Cx, GJIC, and hemichannels in skeletal development.
Ample data have demonstrated a role for gap junctions
and Cx, particularly Cx43, in osteoblast differentiation
and adult bone homeostasis. Chemical inhibition of
GJIC promotes transdifferentiation of osteoblasts into
adipocytes9and attenuates osteoprogenitor differenti-
ation in vitro.7,38Similar findings are reported in
vivo.6What has been studied less extensively, howev-
er, is the influence of aging upon Cx43 expression and
GJIC in osteoblastic cells. Within, we examined Cx43
expression in osteoblastic cells derived from young,
mature, and old rats, and further examined the capaci-
ty for GJIC in response to hormonal and chemical
We observed no influence of donor animal age
upon Gja1 or Cx43 protein expression, nor was basal
GJIC significantly different among these age groups
(Fig. 1a–c). In contrast, age-related decreases in Cx43
expression have been observed in the SA node,39endo-
thelium,40fibroblasts,41dental pulp,42and the blad-
der.43While we observed no significant difference in
basal Cx43 expression or GJIC among the groups, we
did find that PTH was able to stimulate GJIC only
in osteoblastic cells from young animals. (Fig. 2a).
PTH stimulates GJIC by increasing Cx43 mRNA and
protein expression, although this occurs over a longer
time-scale (hours rather than minutes). We examined
whether there was an age-related impairment of Cx43
mRNA or protein in response to PTH. While there was
an age-related impairment in rapid activation of GJIC
by PTH (likely independent of de novo protein synthe-
sis; Fig. 2a), no cells from any population demonstrat-
ed changes in Cx43 (Gja1) mRNA (Fig. 2b) or protein
(Fig. 2c) after 2 h of PTH treatment.
Increasing cAMP positively correlates with in-
creased GJIC, and it is through cAMP signaling that
PTH acutely stimulates GJIC.44We have previously
demonstrated that osteoblastic cells from mature and
old rats are less capable of generating cAMP in re-
sponse to PTH or CTX compared to osteoblastic cells
from young rats.27Therefore, we examined whether
an age-related inability to generate cAMP in response
to hormonal or chemical signals also prevents forma-
tion of gap junctions and GJIC in mature and aged
mice. PTH dose-dependently increased GJIC in osteo-
blastic cells from young, but not mature or old, rats.
These populations of cells have similar levels of PTH/
PTHrP receptor mRNA and numbers of PTH receptors
per cell,27indicating that age-related defects in PTH
signaling, but not receptor number or binding affinity,
are a likely cause. Since PTH-stimulated cAMP accu-
mulation decreases as a function of age27and cAMP
mediates PTH stimulation of GJIC,45the age-related
decrease in PTH-stimulate GJIC reported could result
from defective cAMP accumulation. CTX, which ADP-
ribosylates Gasto synthesize cAMP, mimicked the ca-
pacity of PTH to promote GJIC in young, but, as was
the case with PTH, did not increase GJIC in osteoblas-
tic cells from mature and old rats. This suggests that
an age-related defect in Gasprotein coupled adenylate
cyclase activity at least partially contributes to de-
creased PTH-stimulated GJIC.
Insummary, our results
cells isolated from mature (12-month-old) and old
(24–28-month-old) rats display decreased PTH- and
CTX-stimulated GJIC. The mechanism underlying this
decreased responsiveness to PTH involves uncoupling
of Gs. protein to adenylate cyclase. Given the critical
role GJIC plays in osteoblastic differentiation and
bone adaptation to mechanical load, this age-related
defect in PTH regulation of cAMP may contribute to
age-related bone loss.
This work was supported by Award Number R03AR057547
from the National Institute of Arthritis and Musculoskeletal
and Skin Diseases (D.C.G.) and Award Number AG013087
from the National Institute on Aging (H.J.D.).
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