(Received for publication, May 2, 1995; and in revised form, August 14, 1995)
From the
Trk receptors are a family of genes implicated in the survival,
differentiation, and growth of certain neurons and tumors of the
nervous system. A better understanding of the regulation of Trk
receptors is relevant for developmental and oncological studies. Human
neuroblastoma (NB) cell lines constitutively express low levels of TrkA
mRNA, while TrkB mRNA is not readily detectable. Differentiation of NB
cells is accompanied by a differential modulation of Trk expression in
human NB cells. Nanomolar concentrations of RA induce a stable increase
of TrkB mRNA. A transient induction of TrkA mRNA levels requires
micromolar concentrations of RA. Induction of both TrkA and TrkB mRNA
does not require new protein synthesis. However, RA-induced TrkB mRNA
expression is transcriptionally regulated, while the transient
RA-induced increase of TrkA mRNA is a consequence of extended mRNA
stability. Interferon (IFN-
) selectively increases TrkA mRNA
without affecting TrkB mRNA levels. Similar to RA, IFN-
does not
modify the transcriptional rate of TrkA mRNA, but rather increases TrkA
mRNA stability. Thus, RA and IFN-
differentially regulate TrkA or
TrkB expression in the same cell type by predominantly transcriptional
(TrkB) or post transcriptional (TrkA) mechanisms. Such experiments
indicate the complexity of Trk mRNA regulation and also indicate
compounds that may affect neurotrophin responsiveness in developing
neural cells.
The Trk family of tyrosine kinase receptors plays a crucial role
in neuronal survival, differentiation, function, and target organ
innervation during development (Snider et al., 1994). Although
originally described as an oncogene (Martin-Zanca et al.,
1986), the Trk gene product has been recently identified as the high
affinity receptor for nerve growth factor (NGF) (Kaplan et al., 1991a; Klein et al., 1991a). The Trk gene
family includes at least three members (TrkA, TrkB, and TrkC) that are
structurally and functionally related (Barbacid et al., 1991).
Their interactions with neurotrophins are complex; NGF interacts with
TrkA, BDNF binds TrkB, NT-3 binds TrkC and (to a lesser extent) TrkA
and TrkB, while NT4/5 binds TrkB and (to a limited extent) TrkA (Kaplan et al., 1991b; Soppet et al., 1991; Klein et
al., 1991a, 1991b; Squinto et al., 1991; Lambelle et
al., 1991; Glass et al., 1991; Berkemeier et
al., 1991; Ip et al., 1992; Tsoulfas et al.,
1993).
Despite the significance of the Trks to the development of the nervous system, little is known about the mechanisms by which these genes are regulated. In situ hybridization studies show that the expression of Trk mRNAs increase during embryonal life yet decreases in most tissues after birth (Klein et al., 1990b; Martin-Zanca et al., 1990; Masana et al., 1993). The observation that the survival dependence of trigeminal neurons switches from BDNF to NT3 or NGF during development implies that specific neural populations may also change their pattern of Trk receptors expression (Buchman et al., 1993; Verdi et al., 1994). Such studies indicate that a complex set of environmental signals must be coordinated to regulate Trk expression. Therefore a critical issue toward understanding the development of neuronal cells is to determine the mechanisms that control Trk expression.
Neurotrophins regulate
the expression of their specific receptors, as well as other Trk family
members. NGF induces TrkB mRNA in astrocytes (Kumar et al.,
1993) and TrkA mRNA in the basal forebrain and in PC12 cells (Holtzman et al., 1992). NT-3 induces TrkA in proliferating neuroblasts
(Verdi et al., 1994). Increased intracellular cAMP levels
induce TrkB in astrocytes (Kumar et al., 1993) and TrkA in the
immortalized sympathoadrenal MAH cell line (Birren et al.,
1992). In retinal cells, the steady-state levels of TrkB and TrkC mRNAs
increase upon exposure to light and decrease in the dark (Okazawa et al., 1994). IFN- recently has been shown to be able to
increase TrkA mRNA levels in NB cell lines (Shikata et al.,
1994). Members of the nuclear hormone receptor family also modulate
expression of Trk receptors. Estrogen increases TrkA mRNA expression in
adult sensory neurons (Sohrabji et al., 1994), while a thyroid
hormone receptor transfected into the mouse N2 cell line increases TrkB
and decreases TrkA and TrkC mRNA levels (Pastor et al., 1994).
RA modulates expression of TrkA receptors in immature chick sympathetic
neurons (Rodriguez-Tebar et al., 1991). Furthermore, RA
induces neurite extension in human NB cells expressing BDNF via
induction of TrkB mRNA and p145
expression (Kaplan et al., 1993).
The variety of compounds capable of altering
Trk expression indicates that diverse signal transduction pathways
regulate Trk gene expression. Retinoids play a key role during normal
morphogenesis of the developing chick limb (Wagner et al.,
1990) and are being viewed with increasing importance in the nervous
system since retinoid deprivation during embryogenesis causes central
nervous system damage in the chick (Krishnamurthy et al.,
1963). Sites of RA synthesis have been noted in the spinal cord
(McCaffery et al., 1994a), in the basal forebrain (McCaffery et al., 1994b), and in the retina (McCaffery et al.,
1993), and RA increases neuron survival and axon outgrowth in spinal
cord cultures (McCaffery et al., 1994b). IFNs are well known
to be a key element of the host defense against viruses and tumors.
IFNs regulate the expression of several genes at either the
transcriptional or post-transcriptional levels, such as the 2-5A
synthetase that induce cell resistance to viruses, and interleukins
that control growth and differentiation of many types of cells (Vilcek et al., 1994). Although IFN- is typically produced by T
lymphocytes and natural killer cells, sensory neurons have been shown
to produce an interferon-like molecule called N-IFN-
(Olsson et al., 1994). N-IFN-
has similar activities to
lymphocyte derived IFN-
, in that it can control myoblast
proliferation, as well as regulate induction of major
histocompatibility complex antigens in muscle and macrophage cell
cultures (Olsson et al., 1994).
In this report we use human
NB cells as a model system to study the molecular mechanisms regulating
Trk genes expression in an in vitro differentiation model
(Kaplan et al., 1993). We examine TrkA and TrkB gene
regulation in both the presence and absence of RA or IFN- and
describe distinct mechanisms of regulation of these genes.
Figure 1: Northern blot analysis of the kinetic of TrkA and TrkB mRNA in RA-treated KCNR cells. Twenty-five µg of total RNA were loaded in each lane and probed with TrkA cDNA, and filters were stripped and rehybridized with TrkB and GAPDH cDNA probes. Results were normalized by densitometric scanning according to GAPDH signals and background hybridization in the lane corresponding to the day 6 control was excluded in analysis. Northern blot analysis revealed a 3.2-kb TrkA mRNA and 9.4- and 8.4-kb TrkB mRNA species. TrkB mRNA species are similar in size to the previously reported rat TrkB mRNA encoding full-length TrkB (9.0 kb) and the truncated form of TrkB lacking the catalytic domain (7.4 kb) (Middlemas et al., 1991) (Kaplan et al., 1993). These results are supported by experiments done by Nakagawara, in which only the 9.4-kb mRNA species intensely hybridize with a probe for the intracellular domain of TrkB (Nakagawara et al., 1995). A, KCNR cells were treated with 5 µM RA, or control solvent for 3, 6, and 9 days. B, KCNR cells were treated with 5 µM RA for 3, 6, and 12 h. C, KCNR cells were treated with increasing concentrations of RA or control solvent as indicated.
During development, gradients of RA exist in tissues that have been postulated to influence morphogenesis (Hunter et al., 1991). Previously, we determined that nanomolar concentrations of RA induced TrkB mRNA, yet TrkA levels were relatively unaffected at 8 days (Kaplan et al., 1993). Since the increase in TrkA levels occurred within hours of RA treatment, KCNR cells were treated for 24 h with different concentrations of RA. Increases in TrkA expression were detected only in cells treated with higher RA concentrations (5 µM RA) (Fig. 1C). The apparent increase in TrkA mRNA in picomolar RA-treated cells was due to overloading of this lane, as indicated by densitometric analysis of TrkA mRNA levels normalized to GAPDH mRNA levels. In contrast, TrkB mRNA expression was induced by nanomolar RA (Fig. 1C). These results indicate that micromolar concentrations of RA are necessary to induce TrkA mRNA, whereas nanomolar concentrations are required to induce TrkB mRNA.
Figure 2:
Regulation of TrkA and TrkB mRNA
expression in RA-treated NB cells. A, KCNR cells were treated
for 2 days with 5 µM RA or control solvent. Ongoing
transcription was inhibited for the stated times with actinomycin D.
Cells were harvested, and 25 µg of total RNA utilized in Northern
blot analysis. Blots were hybridized with TrkA cDNA, stripped and
rehybridized with TrkB and GAPDH cDNA probe. Signals were normalized by
densitometric analysis to 28 S. B, half-life of the TrkA mRNA
at different time was calculated normalizing values obtained by
densitometric analysis of the TrkA signal to the 28 S intensity. Open circles, untreated cells; closed circles, 5
µM RA-treated cells. C, nuclei were isolated from
cells treated with 5 µM RA or control solvent. One µg
of TrkA, TrkB and GAPDH insert/lane was immobilized using a slot blot
apparatus. Typically, 3 10
cpm of nuclear run on
products were hybridized to each blot for 48 h at 42 °C, filters
were washed at 0.1
SSC, 0.1% SDS at 65 °C. For KCNR cells
we show TrkA and TrkB transcripts from two different experiments;
signals were normalized by densitometric analysis to their GAPDH. D, KCNR cells were treated with 5 µM RA, control
solvent, and cycloheximide to inhibit new protein synthesis. Cells were
harvested at the indicated time, and 25 µg of total RNA was
utilized for Northern blot analysis. Blots were hybridized with TrkA
cDNA, stripped, and rehybridized with TrkB and GAPDH
cDNA.
The early induction of Trk mRNA by RA suggests that RA may also directly regulate Trk gene transcription. To evaluate this hypothesis, nuclei were isolated from two different NB cell lines, KCNR and NGP. Previous studies indicated that RA induces Trk mRNA in NGP (Lucarelli et al., 1994). Both cell lines were treated for 2 days with 5 µM RA and gene transcription assessed by nuclear run-on assays. TrkA mRNA transcription was not significantly altered in nuclei from RA-treated cells, compared to nuclei from control cells in either cell line (Fig. 2C). However, TrkB mRNA transcription was increased 2- and 27-fold in nuclei from RA-treated KCNR and NGP cells compared to controls, respectively. These data indicate that in both the KCNR and NGP cell lines, RA stimulates TrkB but not TrkA gene transcription.
To verify whether RA-induced Trk gene expression requires de novo protein synthesis, KCNR cells were treated with RA or control solvent in the presence or absence of cycloheximide (CHX, 5 µg/ml), which inhibits 99% of protein synthesis (Thiele et al., 1988). RA-induced TrkA or TrkB mRNA expression occurred in the presence of CHX, indicating that de novo protein synthesis was not required to mediate RA induction of TrkA or TrkB mRNA (Fig. 2D). In the presence of CHX, TrkA and TrkB mRNA levels increased above those detected in the absence of CHX. This indicates that basal steady-state Trk mRNA levels may be affected by short-lived proteins.
Figure 3:
Northern blot analysis of TrkA and TrkB
mRNA expression in KCNR cells treated with IFN- and IFN-
. A, KCNR cells were treated with 5 µM RA, 1000
units/ml IFN-
, 1000 units/ml IFN-
, and combinations thereof.
Cells were harvested after 3 days and 25 µg of total RNA utilized
for Northern blot analysis. The filter was probed with TrkA cDNA and
reprobed with TrkB and GAPDH cDNA. B, KCNR cells were treated
for the indicated time with 1000 units/ml IFN-
or control solvent,
harvested, and 25 µg of the total RNA utilized for Northern blot
analysis. Blots were hybridized with TrkA cDNA probe, stripped, and
reprobed with GAPDH cDNA. C, levels of TrkA mRNA expression
were calculate normalizing TrkA mRNA values obtained by densitometric
analysis to GAPDH.
Figure 4:
Regulation of TrkA and TrkB mRNA
expression in KCNR cells. A, KCNR cells were treated with 1000
units/ml IFN- or control solvent. After 3 days cells were exposed
to 5 µg/ml actinomycin D for the indicated time, harvested, and 25
µg of total RNA were utilized for Northern blot analysis. The
filter was hybridized with TrkA cDNA, stripped, and reprobed with GAPDH
cDNA. B, half-life of TrkA mRNA at different times was
calculated normalizing values obtained by densitometric analysis of the
TrkA signal to the signal obtained for GAPDH. Open circles,
untreated cells; closed circles, 5 µM RA-treated
cells. C, nuclei were isolated from cells treated for 3 days
with 1000 units/ml IFN-
or control solvent. TrkA transcription was
assessed; hybridizing filters were 1 µg of TrkA, TrkB, and GAPDH
insert immobilized/lane. A total of 3
10
cpm of
nuclear run-on product were hybridized to each blot. D, KCNR
cells were treated with 1000 units/ml IFN-
, and/or 5 µg/ml
cycloheximide to block new protein synthesis. Cells were harvested
after 24 h, and 25 µg of total RNA were utilized for Northern blot
analysis. Blots were hybridized with TrkA cDNA, stripped, and reprobed
with GAPDH cDNA.
To evaluate whether
IFN- could influence TrkA mRNA stability, cells were treated for 3
days with IFN-
or control solvent and incubated for 2 and 4 h in
the presence of 5 µg/ml actinomycin D to inhibit new mRNA
synthesis. In control cells the half-life of TrkA mRNA was
approximately 3 h (Fig. 4B). In contrast, TrkA mRNA in
IFN-
-treated cells was stable for a period longer than 4 h (Fig. 4A). The estimated half-life of TrkA mRNA in
control cells, calculated in Fig. 2B and 4B,
is consistently and clearly shorter than the half-life of TrkA mRNA in
RA- and IFN-
-treated cells. These results suggest that the
increase in the mRNA steady-state levels is primarily mediated by an
increase in the stability of TrkA mRNA.
Figure 5:
TrkA autophosphorylation in KCNR cells
treated with IFN-. Cells were treated with 1000 units/ml IFN-
or control solvent for 3 days and then stimulated with NGF or control
solvent for 5 min. Full-length or truncated Trk were precipitated with
an anti-pan Trk antibody. Receptor autophosphorylation was analyzed by
Western blot analysis with an anti-Tyr(P)
antibody.
This study details the mechanisms of Trk gene regulation in
NB cells and shows that the changes in the steady-state levels of TrkA
and TrkB mRNA are mediated via distinct mechanisms of gene regulation.
Although Trk genes play a key role in the development and function of
the nervous system, there has been little analysis of the molecular
mechanisms by which these genes are regulated. Our studies indicate
that TrkA mRNA is constitutively expressed in NB cells and RA induces a
transient increase in the steady-state mRNA levels that is primarily
mediated by an increase in mRNA stability. In contrast, the basal level
of TrkB mRNA is typically below the levels detected by Northern blot
analysis. However, RA induces a sustained increase in TrkB mRNA that is
accompanied by an increase in TrkB mRNA transcription. Furthermore,
IFN- increases TrkA, but not TrkB mRNA levels. TrkA mRNA induction
by either IFN-
or RA is principally mediated by increasing TrkA
mRNA stability.
A direct role of RA on TrkB gene transcription is supported by experiments showing that RA induces an increase in TrkB mRNA transcription and by studies indicating that the increase in steady-state TrkB mRNA levels occurs within a few hours of treatment and in the absence of de novo protein synthesis. In KCNR cells, there is only a 2-fold increase in TrkB mRNA transcription at 3 days, while TrkB mRNA steady-state levels are increased 12-fold increase over controls at similar time. Although we were not able to evaluate whether the stability of TrkB mRNA was increased by RA, the difference in transcriptional increase and the steady-state levels would indicate that RA may also enhance TrkB mRNA stability. In contrast to the regulation of TrkB, increases in TrkA mRNA transcription were not detected in two RA-treated NB cell lines. RA enhancement of TrkA mRNA stability may be the major determinant for the increase in the steady-state TrkA mRNA levels. However, similar to the regulation of TrkB, TrkA mRNA levels increase after a few hours of RA treatment in the absence of de novo protein synthesis. Thus, it is possible that RA mediates an increase in TrkA transcription, although it is below the level of sensitivity of nuclear run-on assays. Treatment of NB cells with CHX results in increases in TrkA and TrkB mRNA steady state. This result suggests that short lived proteins negatively regulate TrkA and TrkB mRNA and may affect either mRNA stability, or regulate mRNA transcription.
Distinct concentrations
of RA regulate Trk gene expression; 10M RA
is required to induce TrkA mRNA, whereas 1000-fold less
(10
M RA) induces TrkB mRNA. The
observation that concentrations of RA in the embryonic retina that
express high levels of TrkB (Jelsma et al., 1993) are
approximately 500 nM (McCaffery et al., 1993)
provides physiologic support to our finding that nanomolar
concentrations of RA induce TrkB mRNA in vitro. Retinoid
signal transduction is mediated by two types of nuclear receptors,
retinoic acid receptor and retinoic X receptor, that bind specific
sequences (RARE) in the promoter of genes such as the RAR-
(de The et al., 1990) and homeobox genes (Wang et al., 1985).
Experiments on the human Hox B homeobox gene have shown that the
interactions between RA and the retinoid receptors are complex and
concentration-related, since RA concentrations ranging from 100 nM to 100 pM activate different gene clusters (Simeone et al., 1990). Extension of these studies to RA-regulated Trk
gene expression suggests that expression of Trk genes are selectively
dependent on RA concentration. Although the presence of RARE in the
promoter region of TrkA and TrkB is still not known, our results would
support the hypothesis that a RARE exists in the TrkB promoter,
although we cannot exclude the possibility that a RARE also exists in
the TrkA promoter. However, it is also possible that the increase in
TrkA stimulated by RA is not a direct effect, but rather a consequence
of the ability of micromolar, but not nanomolar, concentrations of RA
to arrest NB cell growth (Matsumoto et al., 1995). Increases
in NGF responsiveness have been noted in another NB cell model in which
proliferation was arrested by aphidicolin, a drug that inhibits DNA
polymerase (LoPresti et al., 1992), as well as in
aphidicolin-treated proliferating neuroblast from embryonic sympathetic
ganglia (Verdi et al., 1994). Delineation of the molecular
mechanism by which RA may affect transcription of Trk genes awaits
characterization of the promoters of these two genes.
The present
study provides the first analysis of the mechanism of regulation of
TrkA by IFN in neuronal cells. IFN- has been shown to regulate the
expression of many genes in the immune system at either the
transcriptional or post-transcriptional level. Our evidence indicates
that, similar to RA, IFN-
regulates TrkA mRNA primarily by a
post-transcriptional mechanism. Determination of TrkA mRNA stability
indicates that TrkA mRNA decays at a slower rate in IFN-
-treated
cells compared to control. Furthermore, we do not detect any changes in
TrkA transcription in nuclear run-on experiments. These data are
similar to studies in which IFN-
has been shown to
post-transcriptionally regulate an increase in Interleukin-8 mRNA in
human monocytes (Bosco et al., 1994), and a decrease in
c-fos mRNA in macrophages (Radzioch et al., 1991). A
role for IFN-
in the nervous system development has not been
identified; however, IFN-
-mediated induction of TrkA may be an
important step for enhancement of neural cell survival during periods
of immune cell activation.
Differential expression of TrkA and TrkB
is well documented in the nervous system, and the ability to switch
receptor phenotype is inferred from studies documenting changes in
neuronal responses to selective neurotrophins (Birren et al.,
1993; DiCicco-Bloom et al., 1993; Davies et al.,
1994). NB tumors are derived from cells in the embryonal neural crest
destined to be sympathetic ganglia or chromaffin cells. NB tumors have
been shown to express different patterns of Trk receptors as well
(Nakagawara et al., 1993; Nakagawara et al., 1994).
NB tumors in patients with a good prognosis express relatively high
levels of TrkA (Nakagawara et al., 1992), while many of those
who have a poor prognosis have tumors that express BDNF and TrkB mRNA
(Nakagawara et al., 1994). It is possible that NB tumors arise
from cells at different stages of differentiation or from distinct
neural crest cell lineages. Aside from being a prognostic marker,
differential Trk expression may contribute to the variable prognosis of
patients with NB tumors. Recently, we have found that activation of the
BDNF-TrkB signal transduction pathway stimulates NB cell invasion, a
property of metastatic NB cells, while activation of the NGF-TrkA
signal transduction pathway may inhibit cell invasion (Matsumoto et
al., 1995). When a cell line derived from a poor prognosis patient
produces high levels of TrkA by gene transfection, treatment with NGF
arrests cell growth (Matsushima et al., 1993). These studies
indicate that the biology of a tumor from a poor prognosis patient may
be altered by high levels of TrkA expression. Although we find that
IFN- stimulates a 2-fold increase in TrkA autophosphorylation in
NGF stimulated cells, it is not clear whether this increase is
sufficient to alter cell growth and differentiation. Current studies
are aimed at defining factors that can induce a high level of TrkA
expression in NB cells, by either increasing TrkA transcription or mRNA
stability.