(Received for publication, January 19, 1996; and in revised form, March 7, 1996)
From the
To evaluate whether intercellular coupling via connexin43 gap junction channels modulates hormonal responsiveness of cells in contact, we have created osteoblastic cell lines deficient in connexin43. Osteoblastic ROS 17/2.8 cells were transfected with a plasmid containing an antisense cDNA construct to rat connexin43. Control transfection did not alter cell-to-cell coupling nor connexin43 mRNA or protein expression relative to nontransfected ROS 17/2.8 cells. In contrast, stable transfection with an antisense connexin43 cDNA resulted in two clones, RCx4 and RCx16, which displayed significant decreases in connexin43 mRNA and protein expression and were dramatically deficient in cell-to-cell coupling. Phenotypically, all transfectants retained osteoblastic characteristics. However, cells rendered connexin43-deficient through antisense transfection displayed a dramatic attenuation in the cAMP response to parathyroid hormone. Alterations in hormonal responses were not due to changes in parathyroid hormone receptor number or binding kinetics nor to alterations in adenylyl cyclase activity. These results indicate that gap junctions may be required for mediating hormonal signals. Furthermore, these experiments support a regulatory role for connexin43-mediated intercellular communication in the modulation of hormonal responses within elaborately networked bone cells.
Although evidence of a critical role for gap junctional
intercellular communication in tissue development and morphogenesis is
emerging(1, 2, 3, 4) , the role of
cell-to-cell coupling in fully developed tissue is less clear. Recent
investigations, however, suggest that gap junctional coupling
contributes to the coordinated responses of cellular networks to
extracellular signals. For instance, pharmacological inhibitors of
intercellular coupling have been shown to inhibit adrenocorticotropic
hormone-induced steroidogenesis in adrenal cells(5) ,
bombesin-stimulated cytosolic calcium oscillations in pancreatic
acini(6) , the secretory effect of thyrotrophic releasing
hormone on pituitary cells(7) , and alpha adrenergic receptor agonist-stimulated contractions in smooth
muscle cells(8) . However, these studies are difficult to
interpret because the agents used to inhibit gap junctional coupling,
in most cases lipophilic long chain alcohols, may have many nonspecific
effects on cells(9) . More precise approaches, which directly
and specifically inhibit intercellular coupling in a particular cell
type, are required to better understand gap junctional influences on
cellular responses to extracellular signals.
To more directly and
specifically assess the role of intercellular communication on the
ability of cell ensembles to respond to extracellular signals, we
developed two clonal osteoblastic cell lines with markedly reduced dye
coupling and expression of connexin43 (Cx43). ()There are
several reasons why osteoblastic cells present a useful paradigm to
examine the role of intercellular communication in cellular responses
to extracellular signals. Osteoblasts communicate with one another via
gap junctions both in organ culture (10) and in
vitro(11, 12, 13, 14, 15, 16) ,
and cell-to-cell communication is believed to be critical for the
coordinated cell behavior necessary in bone tissue development (17, 18, 19, 20) . Additionally, the
particular gap junction proteins expressed by osteoblasts have been
characterized(11, 12, 13, 14, 15, 16) ,
and osteotropic hormones, such as PTH, have been shown to regulate cell
coupling in osteoblastic cells(16, 21, 22) .
Finally, there is considerable evidence for a cell density-dependent
and possibly intercellular coupling-dependent effect of extracellular
signals on osteoblastic cell
networks(23, 24, 25) . Therefore, to examine
the hypothesis that cell-to-cell coupling is critical for the
coordinated response of cellular networks to extracellular signals, we
rendered an osteoblastic cell line, ROS, Cx43-deficient. ROS were
chosen because they express phenotypic characteristics typical of well
differentiated osteoblasts (26, 27) and demonstrate
abundant gap junctions(14, 16) .
Cx43 deficiency was accomplished through stable transfection of a plasmid DNA containing cDNA antisense to the mRNA of rat Cx43, the predominant connexin isoform in bone(11, 12, 13, 14, 15, 16, 28) . Antisense transfectants were found to be functionally uncoupled relative to controls and maintained their osteoblastic phenotype. However, cells deficient in functional coupling displayed a dramatic decrease in response to PTH.
For immunoblotting, gels were transferred onto nitrocellulose, blocked, and blotted as described previously(38) . All incubations and washes were performed at room temperature. Incubations with primary antibody to Cx43 and horseradish peroxidase-conjugated secondary antibody required dilutions of 1:1000. Substrates for immunodetection were added according to the manufacturer's instructions (Amersham Corp.), and blots were exposed for various times to Kodak X-Omat AR film.
Figure 1:
Quantification of dye coupling in
osteoblastic cells. Cells were plated at 50,000 cells/cm.
Individual cells were loaded with 0.1 mM carboxyfluorescein at
room temperature and visualized after 5 min under both phase contrast (A and C) and fluorescent illumination (B and D). Panels A and B demonstrate well
coupled ROS; panels C and D show inhibition of dye
coupling in antisense transfectant RCx16. The arrows indicate
injected cells. These results are typical of an individual dye loading
in which 5-10 different cells were routinely examined per dish
and over 25 dishes were used.
Figure 2: Gap junctional coupling in osteoblastic cells. Functional coupling was reduced more than 9-fold in confluent cultures of antisense transfectants RCx4 (1.2 ± 1.1) and RCx16 (1.5 ± 1.3) and in subconfluent ROS (ROS-s; 2.4 ± 1.4) and subconfluent bG (bG-s; 2.1 ± 1.6) as compared with confluent ROS or bG (15.5 ± 2.4 and 13.9 ± 1.9, respectively). Each bar represents the mean (±S.E.) number of adjacent cells that take up dye within 5 min. *, significantly different from either ROS or bG (p < 0.01; n = 25-50 cells).
Gap junction deficiency in both RCx4 and RCx16 was verified by the addition of dbcAMP at 10 µM, a concentration found to enhance functional coupling by as much as 50% in ROS (Fig. 3). These results demonstrate dbcAMP increased coupling in both ROS and bG by 49.8 and 48.6%, respectively. Functional coupling increased within 10 min in both ROS and bG and peaked in ROS by 30 min and in bG by 40 min. On the other hand, dye coupling was unaffected by dbcAMP in RCx16 and RCx4 (6.7 and 0.92% increase over basal, respectively).
Figure 3:
Effect of dbcAMP on gap junctional
coupling in osteoblastic cells. Cells plated at 50,000 cells/cm were exposed to either 10 µM dbcAMP or vehicle
control. Functional coupling was quantified by dye injections with 0.1
mM carboxyfluorescein at room temperature. The number of
neighboring cells with dye was recorded every 5 min for 80 min. The
addition of dbcAMP increased coupling in ROS (thick solid
line) and bG (thick dashed lines) by 50% within 20 or 30
min, respectively. The inability of antisense transfectants RCx4 (thin solid line) and RCx16 (thin dashed line) to
significantly up-regulate functional coupling in response to dbcAMP
demonstrates that Cx43 expression had been successfully reduced and
that neither recruitable membrane nor cytoplasmic Cx43 protein reserves
were sufficiently available to enable maximal dye coupling. Each data
point represents the mean (±S.E.) number of cells coupled (n = 3). *, significantly different from basal values within
each group (p < 0.01).
To determine if uncoupling with antisense transfection was reflected in Cx43 mRNA levels, Northern blot analysis was performed on transfected and nontransfected ROS. Fig. 4A demonstrates that both ROS (lane 1) and control transfectants, bG (lane 2), expressed abundant Cx43 mRNA of approximately 3.0 kb in size. Cx43-deficient clones, RCx4 (lane 3) and RCx16 (lane 4), displayed virtual elimination of Cx43 mRNA expression. Normalization of mRNA abundance to GAPDH, a gene with transcription levels in ROS that remained stable independent of plating density, revealed that mRNA levels in RCx16 and RCx4 were reduced by 78 and 92%, respectively, relative to ROS (Fig. 4B).
Figure 4: Connexin43 and GAPDH mRNA expression in osteoblastic cells. 20 µg of total RNA was subjected to Northern blot analysis using either a 890-base pair rat Cx43 cDNA radiolabeled probe (A) or a 1.4-kb rat GAPDH radiolabeled probe (B). RCx4 clones displayed a lack of Cx43 mRNA (A, lane 3), whereas RCx16 displayed a decreased expression (A, lane 4) relative to ROS (A, lane 1) and bG (A, lane 2). All cells examined displayed equivalent mRNA expression of the housekeeping gene, GAPDH (B). These data are typical of three similar experiments.
Western blot analysis of transfected and nontransfected cells revealed a pattern of Cx43 protein expression similar to that observed for mRNA expression and functional coupling experiments (Fig. 5). ROS (lane 1) and bG (lane 2) showed the predominant presence of a 43-kDa band consistent with the nonphosphorylated form of Cx43 (Cx43-NP) from rat heart(3) . ROS and bG also expressed two bands (Cx43-P1 and Cx43-P2) at reduced levels relative to Cx43-NP, also consistent with the expression of two phosphorylated forms of Cx43(3) . In both antisense transfected clones, RCx4 and RCx16, a greater than 86% reduction in expression for all forms of Cx43 protein was observed using the polyclonal antibody to rat Cx43.
Figure 5: Western blot analysis of connexin43 protein in osteoblastic cells. Equivalent amounts of total cell extract enriched for membrane proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed using a polyclonal antibody to rat Cx43. Lack of immunoreactivity for nonphosphorylated Cx43 (Cx43-NP) and all phosphorylated forms (Cx43-P1 and Cx43-P2) was observed for RCx4 (lane 3). RCx16 demonstrated greatly reduced immunoreacitivity for all Cx43 forms, whereas ROS (lane 1) and bG (lane 2) showed abundant immunoreactivity for all forms of Cx43 protein. These data are typical of three similar experiments.
Indirect immunostaining of ROS (Fig. 6A) revealed punctate distribution of Cx43 immunoreactivity particularly abundant at the interface of adjoining cells. Numerous immunoreactive sites within the cytoplasm, located primarily along the perinuclear border, were also apparent. Although this pattern of staining was observed in bG, there was no Cx43 immunoreactivity at the cell membrane interface or within the cytoplasm in either RCx4 (data not shown) or RCx16 (Fig. 6B).
Figure 6:
Photomicrographs of Cx43 immunoreactivity
in osteoblastic cells. Cells plated at 50,000 cells/cm and
grown for 48 h were fixed, permeabilized, and incubated with a
polyclonal antibody to rat Cx43. A fluorescein-conjugated secondary
antibody was used for visualization under a broadband ultraviolet
light. ROS cells (A) exhibit a punctate staining pattern that
represents gap junctions composed of Cx43 located primarily in the
plasma membrane between adjacent cells. Protein reserves of Cx43 are
those spots located perinuclearly. RC
16 (B) show no
immunoreactivity at the cell surface and little within the cytoplasm.
These experiments were repeated four to seven times for each cell line
examined.
Osteoblastic characteristics include expression of type I collagen, osteocalcin, and PTH/PTHrP receptor mRNA; therefore, Northern blot analysis was performed to identify any loss of these osteoblastic phenotypic markers associated with transfection. RCx4, RCx16, and bG as well as ROS demonstrated a uniform expression for the 3.0-kb type I collagen transcript, the 0.6-kb osteocalcin transcript, and the 2.3-kb PTH/PTHrP receptor transcript as assessed by quantification of total cellular RNA normalized to GAPDH (data not shown). Another characteristic of the osteoblast phenotype is the presence of alkaline phosphatase activity, a marker for osteoblastic differentiation that is associated with mineralization(42) . We found alkaline phosphatase abundance was similar in ROS, bG, RCx4, and RCx16 (data not shown).
Figure 7:
PTH-stimulated cAMP response in
osteoblastic cells. Intracellular cAMP responses were measured by
radioimmunoassay after 15-min exposures at 37 °C to concentrations
of rPTH(1-34) from 10 to 10
M. In A, cells were plated at 50,000
cells/cm
and grown for 60 h. PTH stimulated cAMP
accumulation in a concentration-dependent manner in both ROS (thick
solid line) and bG (thick dashed line). However,
PTH-stimulated cAMP accumulation in RCx4 (thin solid line) and
RCx16 (thin dashed line) was dramatically attenuated. The peak
cAMP response to 10
M rPTH(1-34) in
RCx4 and RCx16 was only 26.2 and 21.9% of that of ROS, respectively. In B, the intracellular cAMP response of ROS (thin solid
line) and bG (thin dashed line) plated at 5-10,000
cells/cm
and grown for 48 h are shown. An 80% reduction in
functional coupling in these low density cultures resulted in the
attenuation of PTH-stimulated cAMP by 50 and 55% in ROS and bG,
respectively. Confluent cultures of ROS (thick solid line) and
bG (thick dashed line) are shown for comparison and are the
same values as those presented in A. The average basal cAMP
response from all experiments was 8.1 ± 1.7 pmol/million cells.
All data represent means ± S.E. of three to six experiments
performed in duplicate. *, significantly different from either ROS or
bG (p < 0.05).
To determine
whether decreased hormonal responsiveness in antisense transfectants
could be due to reductions in PTH binding or receptor availability,
radiolabeled binding studies were performed on cells at equivalent
densities. Radioiodonated parathyroid hormone related protein,
PTHrP(1-36), a ligand with binding kinetics for the PTH/PTHrP
receptor equivalent to PTH(41) , was found to specifically bind
to the receptor protein in all cells analyzed (Table 1). These
data confirm that bG, RCx4, and RCx16 contain a receptor protein that
binds PTH analogs. From nonlinear regression of competition binding
curves for PTHrP, the EC and the number of specific
binding sites were obtained. Both bG and RCx16 bound PTHrP with
affinities equivalent to ROS, whereas RCx4 was found to exhibit an
increased affinity for the receptor. Scatchard transformation, used to
estimate receptor densities from cells in late log phase growth,
revealed that ROS, bG, and RCx16 expressed similar numbers of PTH/PTHrP
surface receptors per cell. Cx43-deficient RCx4, with a 2.5-fold higher
receptor binding affinity, were found to express fewer PTH/PTHrP
receptors per cell. To determine whether the adenylyl cyclase in
transfected cells was fully functional and to verify a uniform response
of the enzyme in the absence of intercellular connectivity, cells were
exposed to 10
, 10
, and
10
M forskolin, a direct activator of
adenylyl cyclase. Forskolin-stimulated cAMP accumulation was similar in
all cells examined (Fig. 8).
Figure 8:
Forskolin-stimulated cAMP accumulation in
osteoblastic cells. Cells plated at 50,000 cells/cm were
exposed to 10
, 10
, or
10
M forskolin for 15 min. Intracellular
cAMP was quantified by radioimmunoassay. Forskolin stimulated cAMP
accumulation in a concentration-dependent manner in ROS (thick
solid line), bG (thick dashed line), RCx4 (thin solid
line), and RCx16 (thin dashed line). There were no
significant differences in cAMP responses among the cell lines
examined. The average basal cAMP response from all experiments was 7.9
± 0.8 pmol/million cells. The data represent means ± S.E.
of four experiments performed in duplicate.
We also examined hormonal
responsiveness in subconfluent ROS and bG. Subconfluent cells displayed
less cell-to-cell contact and, consequently, up to 80% reduction in
cell coupling (Fig. 2). Additionally, PTH-stimulated cAMP
accumulation in subconfluent cells was significantly (p <
0.05) reduced relative to confluent cells (Fig. 7B). In
subconfluent ROS and bG, exposure to 10M PTH resulted in an approximately 6-fold increase in cAMP over
basal, whereas in confluent cells this exposure resulted in a 13-fold
increase in cAMP accumulation. The diminished intracellular cAMP values
found in subconfluent ROS and bG, cells that were poorly coupled, were
similar to those exhibited by Cx43-deficient antisense transfectants.
In order to define a physiological function for cell-to-cell communication in ensembles of bone cells, we examined hormonal responsiveness in uncoupled transfectants that maintained phenotypic characteristics typical of osteoblastic cells. PTH was chosen because it is a potent regulator of bone metabolism exerting a direct effect on osteoblastic behavior(42) . In osteoblastic cells such as ROS, PTH, via binding to its receptor, activates multiple second messenger systems, including the cAMP-protein kinase A cascade(43) . Indeed, we found that PTH stimulated cAMP accumulation in a concentration-dependent manner in ROS, as has been previously demonstrated(39, 41) . However, in antisense transfectants RCx4 and RCx16, cells exhibiting decreased Cx43 and, consequently, decreased coupling, PTH-stimulated cAMP accumulation was dramatically attenuated. Additionally, we found that subconfluent ROS and bG, cells that display decreased coupling, similarly demonstrated decreased cAMP responses to PTH. However, the cAMP response to PTH was greater in subconfluent ROS and bG than in antisense transfectants, probably because coupling was greater in subconfluent ROS and bG relative to antisense transfectants. Taken together, these data indicate that a decrease in cell-to-cell coupling contributes to decreased hormonal responsiveness in ROS.
A possible explanation for this decreased hormonal responsiveness in RCx4 and RCx16 could be an alteration in PTH/PTHrP receptor gene expression, protein abundance or binding kinetics possibly as a result of plasmid transfection per se. However, we found PTH/PTHrP receptor gene expression remained similar in all cell lines examined. Although radio-ligand binding studies revealed that one Cx43-deficient clone, RCx4, expressed fewer PTH/PTHrP receptors on the cell surface, RCx4 cells also demonstrated a higher (2.5-fold) binding affinity for the PTH analog. Thus, RCx4 cells may, by clonal selection, have been enriched for a higher affinity receptor, which could compensate for decreased receptor number. Furthermore, had the cells been adequately intercellularly connected, it is likely that the number of receptors present in RCx4 (approximately 11,300) would have been sufficient for maximal stimulation of cAMP by PTH(44, 45, 46) . In any case, we also observed diminished PTH-stimulated cAMP accumulation in RCx16, another Cx43-deficient clone that did not demonstrate any reduction in the number of cell surface PTH/PTHrP receptors or alteration in receptor binding affinity. Thus, in these experiments, attenuation of PTH-stimulated cAMP accumulation in antisense transfectants cannot be explained by diminished PTH receptor expression, availability, or binding kinetics.
Another explanation
for our results could be that antisense transfection resulted in a
dysfunctional adenylyl cyclase or some other component of the cAMP
generating mechanism. If this were the case, one would expect
transfected cells to display an attenuated cAMP response to forskolin,
a compound that directly activates the catalytic subunit of adenylyl
cyclase. Yet, forskolin-stimulated cAMP accumulation was similar in
ROS, bG, RCx4, and RCx16. This suggests that a defect in the mechanism
by which cAMP is generated does not contribute to reduced PTH
responsiveness in antisense transfectants. However, associated with the
activation of adenylyl cyclase is the translocation of at least one
G-protein(32) , and it is possible that transfection resulted
in alterations in G-protein function. If increased G-protein coupled
receptor binding energy (such as that observed with PTHrP binding in
RCx4) were associated with a conformation that activated G-proteins, it
would predict a lower ED value for adenylyl cyclase
response. Yet, we observed that upon direct activation of adenylyl
cyclase, neither the ED
nor the maximal cAMP response
differed between RCx4 and ROS, suggesting the transducing G-proteins
were not disturbed with transfection or that the affinity of ligand
binding to these receptors is dissociable from receptor activation
properties.
Without any apparent defect in the generation of cAMP in
Cx43-deficient RCx4 and RCx16, our studies suggest that gap junctions
may act as conduits for optimal intracellular responsiveness to
hormones such as PTH. Perhaps, as suggested by Christ et
al.(8) , gap junctions act to amplify the effects of local
receptor activation by permitting the spread of second messengers to
adjacent cells that are not directly activated by the agonist. Thus, a
mechanism may exist whereby agonist-stimulated increases in a second
messenger, such as cytosolic Ca in responsive cells
could be communicated via gap junctions to nonresponsive cells wherein
the activity of other second messenger systems, such as adenylyl
cyclase, are potentiated (43, 47, 48, 49) . More importantly,
such a mechanism may not be unique to bone
cells(5, 6, 7, 8, 50) .
Amplification of local receptor activity would be particularly
important when there are variabilities in hormone receptor expression
and/or in gap junctions between interconnected cells. With such
variabilites, gap junctional coupling would regulate hormone-stimulated
cellular behavior to various degrees in different cellular systems.
Munari-Silem and co-workers (5) found that the
adrenocorticotropic hormone-stimulated cAMP response in uncoupled
adrenal cells was not altered even at submaximal hormone
concentrations. Our findings suggest an attenuated cAMP response to PTH
in Cx43-deficient cells at all hormone concentrations examined. These
differences could be explained by a uniform or homogeneous
adrenocorticotropic hormone-stimulated cAMP response within individual
bovine adrenal cells, whereas our results suggest a more heterogenous
cAMP response to PTH in individual ROS cells. In fact, Civitelli et
al. (51) have shown that among individual osteoblasts, the
response to PTH is quite heterogenous such that only 30% of
osteoblastic cells would respond to maximal doses of 10M PTH on an individual basis. Hence, in a population of
cells exhibiting a heterogenous response to a hormone on an individual
basis, gap junctions could provide a mechanism by which the net
ensemble response would be greater than the sum of the individual
responding cells.
This role of gap junctions and their regional
specificity in more mature osteoblasts may explain certain
abnormalities or loss of cellular signaling observed in the aging
skeleton. Indeed, in preliminary experiments, ()we find
functional coupling is significantly reduced in osteoblastic cells
isolated from 28-month-old (aged) rats relative to those isolated from
4-month-old (young) rats. The decreased cAMP response to PTH we (52) and others (53, 54, 55) have
reported in osteoblastic cells isolated from aged rats is strikingly
similar to the decreased cAMP response to PTH observed in antisense
transfected Cx43-deficient cells. If the preliminary results are
confirmed, they could have important implications regarding the
interplay between age-related changes in gap junctional coupling and
intracellular signaling.
In summary, using an antisense transfection strategy, we have provided the first direct evidence that in ROS 17/2.8 cells, a well differentiated osteoblastic cell line, intercellular coupling is predominately mediated via gap junctions composed of Cx43. By the selective elimination of a specific connexin, we have been able to demonstrate that decreasing the abundance of a single gap junction protein can alter the responsiveness of a cellular ensemble to a hormonal signal. More importantly, our data reveal that cell-to-cell communication may be a critical component in the pathway by which more mature cellular networks coordinate their responsiveness to extracellular signals.