(Received for publication, July 13, 1994; and in revised form, October 12, 1994)
From the
Ciliary neurotrophic factor (CNTF) is a cytokine whose actions
are largely restricted to the nervous system because of the predominant
neuronal distribution of its receptor, CNTFR. In this study, we
sought to define the binding characteristics of CNTF to cultured
sympathetic neurons and cell lines of neuronal origin. We report that
I-CNTF binds to cultured sympathetic neurons, MAH, PC12,
and EW-1 cells via high and low affinity receptors that can be
distinguished on the basis of their dissociation constants (K
10
M and K
10
M). Competition experiments showed that the IC
for rat and human CNTF were, respectively, 65 pM and 5
nM for sympathetic neurons and 75 pM and 1.2 nM for EW-1 cells. Interestingly, leukemia inhibitory factor (LIF)
did not compete for CNTF binding even at 100 nM concentration.
The binding of
I-CNTF to sympathetic neurons involved all
three components of the CNTF receptor complex, namely CNTFR
, LIFR,
and gp130, as shown by cross-linking experiments. CNTF and LIF
treatments down-regulated CNTF binding to sympathetic neurons and EW-1
cells, suggesting that heterologous ligands can regulate CNTF receptor
levels, which may in turn modulate the efficacy of CNTF in vitro and in vivo.
Ciliary neurotrophic factor (CNTF) ()was initially
identified, purified, and molecularly cloned based on its ability to
support the survival of parasympathetic neurons of the chick ciliary
ganglion (Adler et al., 1979; Lin et al., 1989;
Stöckli et al., 1989). Subsequent studies
have revealed that CNTF can also enhance the survival of sensory
neurons (Skaper and Varon, 1986), motor neurons (Sendtner et
al., 1990; Arakawa et al., 1990; Oppenheim et
al., 1991), cerebellar neurons (Lärkfors et al., 1994), and hippocampal neurons (Ip et al.,
1991). CNTF inhibits proliferation and enhances cholinergic properties
of neuronal precusors from the sympathetic ganglion (Ernsberger et
al., 1989) and stimulates cholinergic differentiation of mature
sympathetic neurons (Saadat et al., 1989). In addition,
effects of CNTF on developing oligodendrocytes (Louis et al.,
1993), denervated (Helgren et al., 1994) and intact (Gurney et al., 1992; Forger et al., 1993) skeletal muscles
have also been documented.
Although CNTF does not contain a signal sequence which is typically associated with secreted proteins, Curtis et al. (1994) have demonstrated that CNTF can be retrogradely transported by adult sensory neurons from the periphery, suggesting a means by which CNTF gains access to neurons. Moreover, both sensory and motor neurons show greatly increased transport of CNTF following peripheral nerve lesion. Taken together, these observations suggest a possible role for CNTF in motor and sensory neurons after injury. Indeed, potent effects of CNTF toward motor neurons have been demonstrated in vitro (Arakawa et al., 1990; Wong et al., 1993) and confirmed in vivo using an axotomized-facial nerve paradigm (Sendtner et al., 1990) or mouse mutants that exhibit neuromuscular deficits (Sendtner et al., 1992; Mitsumoto et al., 1994). These studies suggested therapeutic potential of CNTF in musculomotor diseases and have led to human clinical trials for amyotrophic lateral sclerosis (Barinaga, 1994).
Since the cloning of the CNTF-binding protein
(hereon CNTFR) the signal transduction pathway that mediates the
effects of CNTF has been studied extensively. CNTFR
shows greatest
homology to the
component of the IL-6 receptor (Davis et
al., 1990). Recent findings using MAH cells and other neuronal
cell lines have demonstrated that CNTF, leukemia inhibitory factor
(LIF), IL-6, and oncostatin M share common signaling pathways which
involve gp130, the signal transducing component for the IL-6 receptor
(Ip et al., 1992); and all except IL-6 also share another
receptor component LIFR (for review, see Ip and Yancopoulos, 1992).
This family of cytokines has recently been shown to utilize the Jak/Tyk
family of cytoplasmic tyrosine kinases (Stahl et al., 1994).
gp130 was first identified as a signal transducer for the IL-6 receptor
and found to confer high affinity binding to the low affinity IL-6
receptor-
(Taga et al., 1989; Hibi et al.,
1990). LIFR, on the other hand, was originally cloned as a LIF-binding
protein (Gearing et al., 1991). It has been suggested that
gp130 and LIFR together form a functional LIF receptor complex (Gearing et al., 1992; Ip et al., 1992; Davis et al.,
1993a) and that addition of soluble CNTFR
to this complex is
sufficient to convert a functional LIF receptor into a functional CNTF
receptor (Ip et al., 1992; Stahl et al., 1993; Davis et al., 1993b). Whereas the broad distribution of both LIFR
and gp130 accounts for the very widespread actions of LIF in neural and
non-neural tissues, the limited distribution of CNTFR
(predominantly to the nervous system) is consistent with the much more
restricted actions of CNTF (Ip et al., 1993).
While the
therapeutic potential of CNTF is becoming apparent and its signal
transduction pathway is being unraveled, the binding characteristics of
CNTF to its receptor, the first step of the signal transduction
cascade, need to be elucidated. Given the predominant neuronal
distribution of CNTFR, this study aimed to examine the binding and
pharmacological characteristics of
I-CNTF to post-mitotic
(sympathetic) neurons as well as cell lines of neuronal origin.
For cross-linking experiments, SCG
neurons were incubated with 500 pMI-CNTF for 2
h at room temperature. The receptor(s) was cross-linked to the ligand
by the homobifunctional cross-linking agent DSS (150 µM,
for 30 min, room temperature), followed by a wash with 50 mM Tris, 1 mM EDTA (pH = 7) to inactivate DSS.
Protein lysates were boiled in sample buffer; equivalents of
6000-8000 neurons were loaded per lane and subjected to
SDS-polyacrylamide gel electrophoresis on 7.5% acrylamide gels. The
gels were dried and exposed to film. Trophic factors tested for their
ability to compete with radiolabeled CNTF included rat CNTF, human
CNTF, CNTF-myc (Squinto et al., 1990), NGF, brain-derived
neurotrophic factor (BDNF), neurotrophin-3 (NT-3), all at
concentrations of 100 nM.
In this study, cultured rat sympathetic neurons were used,
for two reasons, as a model to study CNTF receptor binding. First, it
is well established that homogeneous population of SCG neurons can be
obtained by tissue culture techniques; thus specific binding reflects
only I-CNTF binding to sympathetic neurons and not to
nonneuronal cells. Second, there is a well-defined physiological
response of SCG neurons to CNTF, i.e. the stimulation of
cholinergic phenotype and suppression of adrenergic phenotype (Saadat et al., 1989). We compared the binding of
I-CNTF
to SCG neurons with that of other adrenergic cells, namely, PC12 cells
and MAH cells. EW-1 was also examined as an example of a responsive,
but non-adrenergic cell type.
Figure 1:
Time course of I-CNTF binding in SCG neurons. SCG cultures were
incubated with 100 pM
I-CNTF and binding was
brought to a steady state (120 min; open circles) before the
addition of 100 nM unlabeled rat CNTF to all wells (closed
circles). At the times indicated, the neurons in wells were washed
and solubilized for counting of radioactivity. Values shown here are
from a representative experiment, with points representing the mean of
triplicate determinations.
Figure 2:
Saturation binding of I-CNTF (insets) and Scatchard analysis in SCG neurons (A),
EW-1 (B), PC12 (C), and MAH (D) cells.
Cultures were incubated with 15 concentrations of
I-CNTF
(20 pM-10 nM). The specific binding (closed
circles), defined as the difference between total (open
squares) and nonspecific (open circles) binding, was
saturable in all cell types examined. Values shown here are from a
representative experiment, with points representing the mean of
triplicate determinations.
In SCG neurons, the biphasic Scatchard plot
approached linearity at both extremes of I-CNTF
concentrations (Fig. 2A), suggesting the presence of
two classes of receptors. Indeed, Scatchard analysis revealed two
dissociation constants with values of 1.1 pM and 2.4
nM, and the number of low affinity binding sites is 87-fold
higher than that of the high affinity sites (approximately 6.5
10
/neuron and 7.5
10
/neuron,
respectively). As a result, the K
derived from
kinetic studies (950 pM) is much closer to the low affinity
binding constant. Thus, the K
derived from our
kinetic studies is an approximation and mostly for the purpose of
comparison; the K
derived from Scatchard analysis
is more accurate.
Scatchard plots of I-CNTF binding to
EW-1, PC12, and MAH cells also revealed two classes of receptors (Fig. 2, B-D). The high affinity receptors in these
cell lines were 6-9-fold lower in affinity than those of SCG
neurons (8.6 pM for EW-1, 9.3 pM for PC12, and 5.7
pM for MAH). With the exception of MAH cells (K
= 412 pM), the
dissociation constants of the low affinity receptor were similar in all
cells, 2.4, 1, and 3.4 nM in SCG neurons, EW-1 cells and PC12
cells, respectively (summarized in Table 1). Primary sympathetic
neurons, by far, contained the greatest number of both high and low
affinity receptor/cell than any of the cell lines examined, most likely
a result of their extensive neuritic network. The ratio of high to low
affinity sites is similar in SCG neurons and MAH cells (1:87 and 1:69,
respectively), but lower in PC12 and, particularly in EW-1 cells (1:20
and 1:5, respectively).
Figure 3:
Pharmacological characterization of I-CNTF binding in SCG neurons (A) and EW-1 cells (B). Cultures were incubated with 200 pM
I-CNTF in the presence of 15 concentrations of
unlabeled rat CNTF (open circles), human CNTF (closed
circles), LIF (open squares), IL-6 (closed
squares), or BDNF (triangles). Values shown here are from
a representative experiment, with points representing the mean of
triplicate determinations.
Figure 4:
Cross-linking of I-CNTF to
SCG neurons. SCG cultures were incubated with 500 pM
I-CNTF in the presence of rat CNTF, human CNTF,
CNTF-myc, NGF, NT-3, or BDNF (all at 100 nM). The receptor was
cross-linked to the ligand by DSS (150 µM), solubilized,
and the cell lysates fractionated by SDS gel electrophoresis. Each lane
was loaded with pooled lysate of three culture wells of approximately
equal number of SCG neurons.
Figure 5:
Regulation of I-CNTF
binding. SCG neurons (A) and EW-1 cell (B) were
pretreated with CNTF, LIF, or IL-6 for 24 and 72 h, respectively, prior
to
I-CNTF binding assay. Values shown here are from a
representative experiment, with points representing the mean of
triplicate determinations ± S.E.
In parallel with the discovery and complete characterization
of a number of neurotrophic factors has been the molecular
characterization of their receptors (for review, see Davis and
Yancopoulos, 1993; Ip and Yancopoulos, 1993). The biological effects
and signal transduction pathways for one of these factors, CNTF, have
been extensively studied, and binding properties of CNTF to its
receptor have been reported in transformed hematopoietic cell lines
(Gearing et al., 1994). Considering the predominant neuronal
distribution of CNTFR, knowledge of CNTF binding characteristics
in neurons is important for understanding the physiological role of
CNTF in vivo. In this study, we sought to define the binding
characteristics of
I-CNTF to primary sympathetic neurons
and to cell lines of neuronal origin. We report that
I-CNTF binds to cultured SCG neurons, EW-1, MAH, and PC12
cells via two classes (high and low affinity) of receptors which can be
distinguished on the basis of their dissociation constants (K
10
M and K
10
M). A hallmark of neurotrophic factors is their potency
in producing biological effects; the high affinity binding constant of
CNTF is in good agreement with the observed potency of its biological
effects on chick ciliary ganglion neurons (EC
=
2 pM; Masiakowski et al., 1991) and rat
sympathetic neurons (EC
=
20 pM;
Sadaat et al., 1989). It has been shown that the three CNTF
receptor components (CNTFR
, LIFR, and gp130) are initially
unassociated on the cell surface and are brought together in a
step-wise fashion upon CNTF binding (Stahl and Yancopoulos 1993). The
non-signal transducing binding of CNTF to CNTFR
is most likely low
affinity, whereas high affinity binding probably ensues when the
tripartite receptor complex is formed. Interestingly, PC12 cells, which
lack LIFR, (
)confer both high and low affinity binding,
suggesting that CNTF
CNTFR
complex binds to gp130 with high
affinity with or without further heterodimerizing with LIFR. This is in
contrast to the findings in transformed hematopoietic cells where LIFR,
but not gp130, was shown to be critical for generating high affinity
CNTF binding (Gearing et al., 1994).
CNTF and LIF have been
shown to elicit identical functional responses in SCG neurons
(Kotzbauer et al., 1994), and they both induce tis11 and c-fos gene expression and the phosphorylation of LIFR
and gp130 in MAH and EW-1 cells (Ip et al., 1993; Stahl et
al., 1993). Similarly, retrograde transport of I-CNTF in lesioned sciatic nerve was inhibited by excess
unlabeled CNTF as well as LIF (Curtis et al., 1994). In the
present study, however, we found that the 2 cytokines have different
binding characteristics. LIF, even at 100 nM concentration,
did not compete for
I-CNTF binding in SCG neurons. While
CNTF and LIF share common signal transduction components (LIFR and
gp130), CNTFR
is unique for CNTF. Thus, although LIF does not bind
to CNTFR
, it can deplete LIFR and gp130 and, in turn, should block
high affinity CNTF binding. However, because the concentration of
I-CNTF used in our competition experiments (100-300
pM) labeled both high and low affinity binding sites, and
since low affinity sites were in great excess, changes in high affinity
binding would most likely be obscured.
In addition to the classical
physiologic responses that neurotrophic factors are known to evoke,
they also regulate and modulate the functional properties of their own
receptors. They may increase or decrease the number of their receptors
or change the efficiency of binding to their receptors. For instance,
intrathecal infusion of NGF to dorsal root ganglia resulted in an
up-regulation of low affinity NGF receptor mRNA levels (Verge et
al., 1992). Our findings showed that CNTF binding to SCG neurons
was down-regulated by prior treatment with either CNTF or LIF. It is
most likely that these decreases in specific binding were due to
down-regulation of receptors, and not simply competition by any
residual CNTF left over from treatment because similar down-regulation
was also evident after LIF treatment. As LIF and CNTF share common
receptor signaling subunits, gp130 and LIFR, all downstream signals
produced by these two ligands are identical. However, CNTF is the only
cytokine that binds to CNTFR (for review, see Stahl and
Yancopoulos, 1993). LIF does not bind to nor require
CNTFR
for signal transduction, but was able to down-regulate CNTF
binding in sympathetic neurons; this strongly suggests that such
down-regulation is a specific event and not due to competition by any
residual CNTF left over from treatment. Moreover, results from kinetic
studies have shown that CNTF dissociates from its receptor to an almost
base-line level within 20 min following a cold chase (Fig. 1)
and within 40 min even without cold chase (data not shown). Since the
cultures were washed for 60 min prior to binding, it is unlikely that
there was sufficient CNTF left behind to alter the binding results.
Taken together, the data represented in Fig. 5were not the
result of receptor saturation, but rather, specific down-regulation of
CNTF binding after LIF or CNTF treatment.
Unlike the competition
experiments, where the ability of LIF to compete for CNTFR sites
was specifically measured, the regulation experiments might reflect the
binding of LIF to any of the three components in the CNTFR complex. In
this case, LIF binds to LIFR which then heterodimerizes with gp130 and
initiates signal transduction; upon activation of their specific
receptor components, the signal transduction pathways of CNTF and LIF
converge downstream in the neuron. It is possible that SCG neurons
cannot distinguish whether the signal was elicited by CNTF or LIF;
thus, although LIF does not compete for CNTF binding, it can indirectly
down-regulate CNTFR
. This may have functional consequences in
vivo under pathological and pharmacological conditions. If LIF is
present in the vicinity where CNTF is ``released'' or
administered in vivo, it may cause an indirect down-regulation
of CNTF receptors and, in turn, may alter the efficacy of CNTF. Thus,
binding characteristics of CNTF and regulation of its receptor complex
provide crucial information to the use of CNTF as a therapeutic agent.