(Received for publication, October 18, 1994; and in revised form, January 17, 1995)
From the
The gene regulatory functions of the human IL-2 receptor (IL-2R)
were reconstituted in transiently transfected hepatoma cells. The
combination of IL-2R and -
mediated a strong stimulation via
the cytokine response element of the
-acid
glycoprotein gene and the hematopoietin receptor response element, but
none via the IL-6 response element or the sis-inducible
element. IL-2R
enhanced 10-fold the sensitivity of the
IL-2R
complex to respond to IL-2 or IL-15, but did not
modify the specificity or the magnitude of maximal gene regulation. A
homodimerizing chimeric receptor G-CSFR-IL-2R
could mimic the
IL-2R action. The IL-2R-mediated gene regulation was similar to that
seen with receptors for IL-4 and IL-7, but differed from that for IL-6
type cytokines, thrombopoietin, erythropoietin, and growth hormone. The
activation of STAT proteins by the IL-2R was assessed in transfected
L-cells and COS-1 cells. Although IL-2R subunits were highly expressed
in these cells, no STAT protein activation was detectable. Transient
overexpression of JAK3 was unable to change the signaling specificity
of the hematopoietin receptors in rat hepatoma, L-, and COS cells, but
established a prominent activation of the IL-6 response elements by the
IL-2R and IL-4R in HepG2 cells. The data support the model that the
IL-2R and related hematopoietin receptors produce at least two separate
signals which control gene expression.
Hematopoietin receptors are members of a gene family
characterized by common structural motifs in their extracellular and,
in some cases, also in their intracellular
domains(1, 2) . Several groups of receptors within
this family have been identified based on the shared use of signaling
subunits. The groups include those depending on the IL-2R (
)(receptors for IL-2, -4, -7, -9, -13, and
-15)(3, 4, 5, 6) , the IL-3R
(receptors for IL-3, -5, and GM-CSF)(2) , and gp130 (receptors
for IL-6, IL-11, LIF, oncostatin M, and CNTF)(7, 8) .
Homodimeric hematopoietin receptors include those for
G-CSF(9, 10) , EPO(11) ,
prolactin(12) , growth hormone(13) , and probably
thrombopoietin(14, 15) .
Each hematopoietin receptor has been associated with control of proliferation of hematopoietic cells. A modulating effect on transcription of early growth response genes, such as c-fos, c-jun, junB, and c-myc, has been demonstrated for several of these receptors(10, 16, 17, 18, 19, 20) . However, the function of hematopoietin receptors, whose expression has been maintained during the course of cell differentiation, appears to involve the transcriptional control of differentiated genes such as neuropeptide genes by LIF and CNTF in neuronal cells(21, 22) , genes for myeloperoxidase, elastase, and G-CSFR by G-CSF in granulocytes(18, 23, 24, 25) , cytokine genes by IL-2, IL-15, and IL-12 in NK cells(24, 25, 26) , or acute phase plasma protein genes by IL-6-type cytokines in hepatic cells(27) . A major unanswered question is whether the regulation of proliferation and of differentiated gene expression by a given hematopoietin receptor is mediated by identical or distinct signal-transducing mechanisms.
The intracellular initiation of signal transduction by
ligand-occupied hematopoietin receptors involves an immediate
phosphorylation of the receptor cytoplasmic domain concomitant with the
activation of receptor-associated protein tyrosine kinases. These
protein tyrosine kinases, depending upon the cell type, include members
of the Janus kinase family (JAK/Tyk) (28, 29) and src-related protein tyrosine
kinases(30, 31, 32, 33) . The change
in the phosphorylation state of the receptor promotes the binding of
the STAT (signal transducer and activation of transcription) proteins
to the receptor(28, 29) . One of the consequences of
protein tyrosine kinase action is the phosphorylation of the
receptor-recruited STAT protein(s) that lead to the STAT protein
dimerization and activation of DNA binding activity (34) . STAT
protein complexes binding to the sis-inducible element (SIE)
of the c-fos gene, termed SIF, for example, has been
recognized to be a common target of growth factors and hematopoietin
receptor signals(35) . Components of the DNA-bound complexes
include dimers of STAT-1, primarily activated by IFN(36) ,
STAT-3 (or APFR) (34, 37, 38) activated by
IL-6-type cytokine, STAT-5 activated by prolactin(39) , and
IL-4 STAT (or STAT-6) activated by IL-4(40, 41) .
Structure/function analyses of hematopoietin receptor subunits
suggest that distinct subregions of the cytoplasmic domains of the
signaling receptor subunits control specific cell responses, such as
the regulation of early growth response genes by IL-2R (20, 42) and IL-3R
(as part of the
GM-CSFR)(17) , and differentiated genes in monocytic,
neuroblastoma, and hepatic cells by gp130, LIFR, and G-CSFR (7, 9, 10, 43) . To assess the
complexity of intracellular signaling pathways which are activated by
various hematopoietin receptor types and which control expression of
differentiated genes, we developed tissue culture systems in which
receptor-specific gene regulatory functions were
reconstituted(44, 45) . By applying this experimental
approach, we have established a sensitive assay system for defining
common signaling mechanisms utilized by seemingly related receptor
structures. In this study, we used rat and human hepatoma, L-, and COS
cells for probing the interaction of IL-2R and representative members
of the other hematopoietin receptor groups with the intracellular
signal transduction machinery.
For analyzing receptor expression and SIF activation, L- and COS-1 cells in 15-cm diameter culture dishes were transfected with 30 µg of receptor expression vector in 3 ml. The transfected cultures were subdivided and, after a 24-h recovery, maintained for an additional 16 h in serum-free medium. Cytokine treatments were then carried out for 15 min (except where indicated).
To identify the
expression of the chimeric G-CSFR protein, transfected cells were
washed three times with phosphate-buffered saline, scraped off the
dish, and collected by centrifugation. Cells were solubilized in lysis
buffer (10 cells per 100 µl) containing 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5 mM Na
VO
, 1 mM phenylmethylsulfonyl
fluoride, 1 mM EDTA, 10 µg of aprotinin, and 10 µg of
leupeptin. After 20 min on ice, the extracts were centrifuged for 10
min at 25,000
g. The supernatants were pretreated with
20 µl of Protein G-Sepharose beads (Pharmacia Biotech Inc.) for 1 h
and then reacted for 16 h with 5 µl of rabbit anti-human G-CSFR
serum(33) . The immune complexes were collected on Protein
G-Sepharose beads and eluted by washing four times with lysis buffer.
After solubilization by boiling in SDS sample buffer, proteins were
separated on a 6% SDS-polyacrylamide gel and electroblotted onto
Immobilon membrane (Millipore). The membrane was incubated for 6 h with
sheep anti-human G-CSFR. This sheep antiserum was generated in
collaboration with Greystone Therapeutics, Inc. by intranodal injection
of maltose binding protein fused to the extracellular domain of human
G-CSFR (residues 48 to 316). The membrane was then treated with rabbit
anti-goat immunoglobulin followed by alkaline phosphatase-conjugated
goat anti-rabbit immunoglobulin (Bio-Rad). The Western blot was
developed with a 5-bromo-4-chloro-3-indolyl phosphate p-toluidine/p-nitro blue tetrazolium chloride
reaction (Bio-Rad).
Figure 1:
Reconstitution of IL-2R function in
H-35 cells. Expression vectors for the IL-2R subunits (indicated at the top) were transfected together with pAGP(3
DRE)-GRE-CAT into H-35 cells. Four subcultures of each transfection
were treated with medium alone (Control) or medium containing
either IL-2, IL-15, or IL-6. CAT activity was quantitated and expressed
relative to the control in each experimental series (number above the autoradiogram).
The three
IL-2R subunits individually did not reconstitute an IL-2 response.
Although the combination of IL-2R and -
or IL-2R
and
-
has been described to reconstitute IL-2 binding
activity(52) , no regulation of the reporter gene was achieved.
The combination of IL-2R
and -
, however, produced an
20-fold stimulation of CAT activity. Inclusion of IL-2R
did
not detectably improve the magnitude of regulation. In several
independent experiments, we observed that the maximal IL-2 response was
consistently one-half of the IL-6 response (see Fig. 1and Fig. 5below) and that there was no discernible difference in the
response to IL-2 and IL-15 (Fig. 1).
Figure 5:
IL-2 regulation of various cytokine
response elements. The combinations of IL-2R, -
, and -
were co-transfected with the CAT reporter gene constructs indicated at
the bottom into H-35 cells. The -fold stimulation of the CAT
activity by IL-2, IL-6, and IL-1
was determined. Mean and S.D. of
three separate experiments are shown.
The regulation via the
AGP gene elements is not specific to hematopoietin receptors and is
also accomplished by various other cytokines and hormones such as IL-1,
tumor necrosis factor, insulin, and hepatocyte growth
factor(72, 73) . Therefore, we developed an
alternative response element that was specific to hematopoietin
receptor signals and that could be used to compare the activities of
different hematopoietin receptors. Oligonucleotides representing
modified IL-6RE/APFR (60, 74) sequences were
synthesized, multimerized, incorporated into CAT reporter gene
constructs, and tested for regulation by co-expressed hematopoietin
receptors. We selected one construct that was highly responsive to the
signal of hematopoietin receptors which contained at least an
equivalent of the box 1 motif (e.g. G-CSFR(27)). The sequence was termed ``hematopoietin receptor response
element'' (HRRE).
Cells co-transfected with IL-2R subunits and HRRE-CAT construct yielded a 300-fold stimulation of CAT gene expression by IL-2 that was similar to that achieved by IL-6 (Fig. 2A). The regulation of HRRE differed from that of AGP-CytRE by not being responsive to either IL-1 or insulin. The magnitude of IL-2 stimulation of HRRE was somewhat variable from experiment to experiment, probably due to the relatively low basal activity of the HRRE-CAT construct in untreated cells. Nevertheless, the IL-2R activity appeared to be similar to that of the functionally related IL-7R and other members of the hematopoietin receptor family such as GHR, EPOR, LIFR, and G-CSFR (Fig. 2B). An exception was the relatively low regulation mediated by IL-4R. Furthermore, GHR was unique among the transfected hematopoietin receptors in that it consistently produced an elevated basal expression of the CAT reporter gene suggesting a ligand-independent signaling reaction. Taken together, the results indicate that IL-2R is capable of exerting a prominent cytokine- and hematopoietin-receptor signal in the heterologous hepatoma cells which are comparable to those elicited by other members of the hematopoietin receptor family including the resident IL-6R.
Figure 2:
Regulation of pHRRE-CAT gene by
hematopoietin receptors in H-35 cells. In two separate experimental
series (A and B), H-35 cells were transfected with
pHRRE-CAT and the mixture of expression vectors for IL-2R, -
,
and -
(A) or with pHRRE-CAT and the receptor expression
vectors indicated at the top in B. Subcultures of
each transfected cell culture were treated with the cytokines listed at
the bottom. CAT activity was determined in 10-fold serially
diluted extracts and calculated relative to the control culture in each
experimental group (values given above the autoradiographic
image).
Figure 3:
A,
dose response of IL-2R action. H-35 cells were transfected with pAGP(4
CytRE)-SV-CAT (top panel) or pHRRE-CAT (bottom
panel) together with the expression vectors for the indicated
IL-2R subunits. Cultures were divided into seven subcultures which were
then treated with the IL-2 concentrations listed on the abscissa. The change in CAT activity relative to the control
culture (0 nM IL-2) was calculated. B, effect of
anti-IL-2R
on IL-2R function. H-35 cells were transfected with
expression vectors for IL-2R
, -
, and -
and pHRRE-CAT.
The cultures were divided into two sets of 10 subcultures. One set was
treated with serially diluted IL-15 (5 duplicates) and the other with
IL-2. In each set, one of each duplicate received 1 µg/ml
nonspecific immunoglobulin, and the other 1 µg/ml anti-TAC (anti-IL-2R
). After 24 h, the increase of CAT activity
relative to the control cultures was
determined.
To assess whether the cytoplasmic domains of IL-2R and
-
initiated signaling independently of each other, we transfected
chimeric receptors consisting of the extracellular domain of G-CSFR and
the transmembrane and intracellular domain of either IL-2R
or
-
. The chimeric receptors were predicted to function as
ligand-induced homodimers. Like the bona fide IL-2R ( Fig. 1and 2A), the G-CSFR-IL-2R
chimera proved to
be as active in regulating AGP-CAT (Fig. 4A) and
HRRE-CAT (Fig. 4B). G-CSFR-IL2R
was ineffective on
AGP-CAT (Fig. 4A) and HRRE-CAT (data not shown) and did
not significantly enhance the magnitude of stimulation achieved by
G-CSFR-IL-2R
(Fig. 4A and data not shown).
Figure 4: Activity of the G-CSFR-IL-2R chimeras. The receptor expression vectors, combined with the CAT gene constructs listed at the top in A or HRRE-CAT in B, were transfected into H-35 cells. Subcultures were treated with the cytokines listed at the bottom, and the CAT activity was quantitated relative to the control cultures.
The gene element specificity of the signals derived from the
IL-2R or G-CSFR-IL-2R was not restricted to H-35 cells.
Transfection of the same reporter gene constructs and receptor
expression vectors into human HepG2 cells yielded essentially the same
pattern of regulation except that the numerical values for the
magnitudes of cytokine responses were not as high as in H-35 cells
(data not shown; see also Fig. 11below).
Figure 11:
Action of JAK3. A, effect of
JAK3 on receptor action in HepG2 cells. HepG2 cells were transfected
with a mixture containing the plasmid DNAs indicated at the top. The reporter gene was pHP(5 IL-6RE)-CAT. The
subcultures were treated with the cytokines as indicated at the bottom. In each experimental set, the CAT activities were
normalized to the internal major urinary protein marker and expressed
relative to untreated control culture that did not receive JAK3. B, dose response of JAK3. Two sets each of HepG2 and H-35
cells were transfected with a DNA mixture containing increasing amounts
of pCD-JAK3 and constant amounts of expression vector for IL-2R
,
IL-2R
, IL-4R, and IL-7R or EPOR alone. The reporter construct for
all was pHP(5
IL-6RE)-CAT. Subcultures of each set were treated
with IL-2, IL-4, IL-7, EPO, or IL-6, and the -fold stimulation of CAT
activity was taken as an indicator for the action of the appropriate
receptor listed at the left.
The salient features of the
receptor assay developed for L-cells are shown in Fig. 6. In
this example, we used progressively truncated G-CSFR to demonstrate (a) the relevance of three box motifs in the cytoplasmic
domain for signaling and (b) the relationship of receptor
expression, SIF activation, and CAT gene regulation. All transfected
plasmids were expressed as detected by analysis of mRNA (Northern blot)
and protein (Western blot). The box 3 motif containing G-CSFR 130 and
96 yielded a prominent activation of SIF-A with a minor one of SIF-B.
In contrast, G-CSFR(56) , lacking box 3 motif, was only
minimally effective. The transfected receptor activated the same SIF
forms as the endogenous IL-6R and IL-11R, and these were clearly
different from SIF-C activated by IFN (Fig. 6). Although
the relative electrophoretic mobilities of the SIF complex of L-cells
were indistinguishable from those of IL-6-treated H-35 cells, the
composition of SIF activities was not identical. In H-35 cells, the
immediate response to all IL-6-type cytokine receptors was a strong
stimulation of all three SIF forms with only SIF-A being maintained
during subsequent hours.
The ability of G-CSFR forms to
activate SIFs appeared to correlate with the ability to regulate the
IL-6RE-CAT construct (Fig. 6, bottom panel).
Figure 6:
Functional analysis of G-CSFR in L-cells.
Six cultures of L-cells in 15-cm dishes were transfected with 3 ml of a
solution of DNA-DEAE-dextran mixture consisting of 10 µg of pHP(5
IL-6RE) CAT and 30 µg of expression vector for G-CSFR forms
listed at the top. After 16 h, each cell culture was released
by trypsin and divided into one culture in a 15-cm dish (for Northern
and Western blot and GMSA) and 2 cultures in 3.5-cm dishes (for CAT
activity). Twenty-four h later, the cells in the 15-cm dishes were
changed to serum-free medium, and, after 16 h, cells were treated for
15 min with serum-free medium containing G-CSF. The cells were scraped
off the dish, and one-third of the cell suspension was removed for RNA
extraction, one-third for immunoprecipitation and Western blot
analysis, and the remainder for GMSA. Aliquots of 20 µg of total
cellular RNA were analyzed by Northern blot hybridization to
P-labeled cDNA encoding the extracellular domain of
G-CSFR. Equal loading of RNA is demonstrated by the ethidium bromide
staining of the 18 S rRNA band (top panels). The cells for
Western blot analysis were lysed, and the supernatant fraction after
centrifugation was subjected to immunoprecipitation with rabbit
anti-human G-CSFR. The precipitates were separated and processed for
Western blotting. For GMSA, 5 µl of whole cell extract was reacted
with
P-labeled SIE. Nuclear extract of IL-6-treated H-35
cells served as a standard (Std). For comparison, the
activation of SIF in control L-cells treated with serum-free medium
alone (control) or containing IL-6, IL-11, or IFN-
is illustrated.
The autoradiogram of SIF complex as formed by extract of
G-CSFR-transfected cells was exposed for 3 days. The autoradiogram of
the complex by the H-35 standard and untreated L-cells was exposed for
6 h. The two cultures in 3.5-cm dishes were treated for 24 h with or
without G-CSF, and the CAT activity was determined. The relative change
in CAT activity is indicated above the
autoradiogram.
IL-2
did not increase SIF activity in nontransfected L-cells (Fig. 7A). Although transfection of either the
combination of IL-2R, -
, and -
or G-CSFR-IL-2R
reconstituted an IL-2 regulation of co-transfected HRRE-CAT construct (Fig. 7B, bottom panel), no IL-2-inducible
protein binding to either SIE or HRRE was detectable (Fig. 7B, middle panel). The expression of the
transfected receptor was demonstrated in the case of G-CSFR-IL-2R
(Fig. 7B, top panel).
Figure 7:
Action of IL-2R in L-cells. A,
response of untransfected L-cells. Cells in 10-cm dishes were treated
for 15 min with medium alone or containing IL-6, LIF, or IL-2. Whole
cell extracts were subjected to GMSA. Positions of SIF-A, -B, and -C
were compared to the standard consisting of nuclear protein extracted
from IL-6-treated H-35 cells. The autoradiogram was exposed for 16 h. B, two separate cultures of L-cells in 15-cm dishes were
transfected with 3 ml of DNA-DEAE-dextran solution containing pHRRE-CAT
(10 µg) and either a mixture of IL-2R, -
, -
(10
µg each) or G-CSFR-IL-2R
(30 µg). The cultures were
divided into two 10-cm dishes and two 3.5-cm dishes. The 10-cm dishes
were treated for 15 min with either medium alone or IL-2 or G-CSF.
Whole cell extracts were prepared. One-fourth of the cells transfected
with G-CSFR was removed prior to extraction and subjected to Western
blot analysis. Duplicate aliquots of 5 µl of cell extract were used
for GMSA using either
P-labeled SIE or HRRE as probe.
Extract of IL-6-treated H-35 cells was included at the left as
standard. The autoradiogram was exposed for 5 days. Cells in 3.5-cm
dishes were treated for 24 h as indicated at the bottom, and
the CAT activity was determined. Relative change is listed above the
autoradiogram.
The failure to detect SIF activation by IL-2R in L-cells may be attributed to either one or both of the following: (a) insufficient expression of the receptor subunits or (b) lack of signal transducing components (i.e. JAK3 and its target STAT proteins) that are utilized by IL-2R to produce SIF activity in lymphocytes(58, 75, 76, 77) .
To
assess the influence of receptor levels, we transfected the IL-2R forms
into COS-1 cells which yielded high expression of the plasmids as
determined by mRNA (Fig. 8A) and protein analysis (Fig. 8B). COS-1 cells that received either the
combination of IL-2R, -
, and -
(Fig. 9A, lanes 3 and 4) or G-CSFR-IL-2R
(Fig. 9B) failed to show any receptor-inducible SIF
activity. Similarly negative was the transfected G-CSFR-IL-2R
(Fig. 9B, lane 13). COS-1 cells were able to
utilize transfected receptor forms to activate SIF as demonstrated by
the example of G-CSFR-gp130 (Fig. 9A, lanes
7-11), G-CSFR-LIFR (Fig. 9B, lane
20), or G-CSFR (lane 21). The response elicited by these
receptors was compared to the response derived from the endogenous IL-6 (lane 12) or LIF receptor (lane 5). Surprisingly,
hematopoietin receptors, such as G-CSFR-MPL (lane 14), EPOR (lane 17), and GHR (lane 19), all of which, like
IL-2R, were unable to stimulate expression of IL-6RE-CAT constructs,
did nevertheless activate SIF. These data suggest that receptor action
on gene expression (HRRE or IL-6RE) in either hepatoma or L-cells does
not strictly correlate with the ability of the same receptor to connect
with the SIF activation pathway as measured in L-cells and COS cells.
Figure 8: Expression of transfected receptors in COS-1 cells. COS-1 cells in 15-cm dishes were transfected with expression vector for the indicated receptors. A, total cell RNA was extracted, and 20-µg aliquots were analyzed by Northern blot hybridization. Autoradiograms were exposed for 24 h. B, cells were lysed, and the chimeric receptors were immunoprecipitated with rabbit anti-human G-CSFR. The extract from nontransfected COS-1 cells served as a control. The precipitates were subjected to electrophoresis on a 6% SDS gel. The proteins were analyzed by Western blotting using sheep anti-human G-CSFR. The receptors migrated with an apparent molecular size of 120 to 100,000.
Figure 9: Activation of SIF by transfected receptors. COS-1 cells were transfected in two separate experiments (A and B), with the expression vectors for the receptors indicated at the bottom. Subcultures were treated for 15 min with the listed cytokines or suramin (in A, lanes 7-10, the G-CSFR concentration in ng/ml). Whole cell extracts were prepared and analyzed by GMSA using SIE as a probe. The SIF pattern of H-35 cells treated with IL-6 served as standard (Std). The autoradiograms were exposed for 3 days.
To assess the potential of IL-2R to regulate SIF activity, we subjected IL-2-deprived CTLL-2 cell cultures to an IL-2 restimulation (Fig. 10A). A transient activation of a SIF pattern was detected (lane 6) that consisted of two binding complexes co-migrating with SIF-A and SIF-B of either IL-6-treated H-35 cells (lane 1) or L-cells (lane 3).
Figure 10:
A,
stimulation of SIF in CTLL-2 cells by IL-2. A culture of CTLL-2 cells
was maintained 16 h in RPMI containing 0.5% fetal calf serum without
IL-2. The cells (75 10
) were centrifuged, and the
pellet was resuspended in 15 ml of serum-free minimal essential medium
and divided into 3 equal aliquots. To two cultures, IL-2 was added,
and, after 15 min at 37 °C, the control (lane 5) and one
IL-2-treated culture (lane 6) were extracted. The second
IL-2-treated culture (lane 7) was extracted after 2 h. Whole
cell extracts were subjected to GMSA using SIE as probe. The standard
was a nuclear extract of IL-6-treated H-35 cells (lane 1),
and, for comparison, whole cell extracts of control L-cells (lane
2) or L-cells treated for 15 min with IL-6 (lane 3) or
IFN-
(lane 4) were included. The autoradiogram was
exposed for 24 h. B, expression of JAK3 mRNA. A polyadenylated
RNA fraction (10 µg) from CTLL-2, L-, and H-35 cells was subjected
to Northern blot analysis using
P-labeled mouse JAK3 cDNA
as probe. The autoradiogram is shown in the upper panel. The
mRNA band and the position of the rRNA markers are indicated. The lower panel shows the ethidium bromide staining pattern of the
gel-separated RNAs.
The fact that SIF activities are induced in CTLL-2 cells by IL-2 suggests that these cells do have a signaling pathway involving STAT proteins and that this pathway does not exist in L-cells and COS-1 cells and probably also in hepatoma cells (see Fig. 12below). JAK3 and/or its target STAT proteins appeared as a mostly likely constituent of such a lymphoid cell-restricted SIF regulatory pathway(58, 75, 76) . We determined JAK3 mRNA in L-, H-35, and CTLL-2 cells by Northern blot analysis (Fig. 10B) and, as expected, observed a prominent JAK3 mRNA signal in CTLL-2 cells. However, both L-cells and H-35 cells also revealed a hybridizing band, albeit at a much lower relative level. The result in Fig. 10suggested that an insufficient amount of JAK3 relative to the overexpressed IL-2R subunits might be one reason for the lack of STAT activation in L-cells and COS-1 cells. To correct this presumed unfavorable ratio, we prepared an expression vector for JAK3, co-transfected that along with IL-2R subunits into each of the test cells used in this study, and determined its influence on the receptor action, i.e. on gene regulation and SIF activation.
Figure 12: Reconstitution of SIF activation by JAK3. L-, COS-1, and HepG2 cells were similarly transfected with the plasmid DNAs containing the expression vector indicated at the top. Subcultures in 6-cm diameter Petri dishes (L- and COS-1 cells) or 6-well cluster plates (HepG2 cells) were treated for 15 min with the cytokines listed above the panels. Whole cell extracts were subjected to GMSA using SIE as probe. For comparison of the electrophoretic pattern of IL-2- and IL-4-inducible SIFs, normal human NK cells were included in the analysis. The autoradiograms for the lanes containing the transfected cell extracts were exposed for 3 days. The standards consisting of extracts from cytokine-treated, but not transfected, cells were exposed for 6 h. The lanes with control and IL-4-treated NK cell extract were exposed for 3 days, and the lane of IL-2-treated cells for 8 h. The position of the SIF-A, -B, and -C (defined by the pattern of the IL-6 treatment) is indicated at the left. The IL-2- and IL-4-induced bands are marked by open arrowheads at the right.
When we
included JAK3 into the H-35 cell assays as shown in Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5, we
did not observe an appreciable change in qualitative pattern of CAT
gene regulation by any of transfected or endogenous hematopoietin
receptors. In particular, we could not detect any activation of IL-6RE
CAT constructs by either IL-2R, IL-4R, or IL-7R (data not shown; see
also Fig. 11B). However, JAK3 enhanced approximately 2-
to 10-fold the basal and maximally 2-fold the cytokine-stimulated
expression of the HRRE and CytRE CAT constructs in those cells that
received IL-2 and -
, or the combination of IL-2R
with
either IL-4R or IL-7R (data not shown). The action of the chimeric
receptors G-CSFR-IL-2R
and G-CSFR-IL-2R
(individually or in
combination), as well as of G-CSFR-MPL, IL-6-type cytokine receptors,
G-CSFR, EPOR, and GHR was unaffected by JAK3.
A different result was
obtained when the effects of overexpressed JAK3 on gene regulation was
determined in HepG2 cells (Fig. 11). Similar to the findings in
H-35 cells, JAK3 was ineffective in modulating the action of IL-6-type
cytokine receptors, G-CSFR-MPL, EPOR, and GHR. In combination with the
functional receptors that included the IL-2R subunit, JAK3
elevated signaling toward HRRE and CytRE. Surprising, however, was that
in the presence of JAK3 both the IL-2R
complex and
IL-4R, but not IL-7R, gained the ability to activate CAT gene
constructs containing the IL-6RE (Fig. 11A) or SIE
(data not shown). The magnitude of stimulation was in the range of that
mediated by IL-6R. Although JAK3 did not elicit an IL-6 signal with
either G-CSFR-IL-2R
or G-CSFR-IL-2R
, with both chimeric
receptors combined, JAK3 again mediated a prominent IL-6RE regulation (Fig. 11A).
Dose-response analyses indicated that maximal IL-6RE regulatory action of both IL-2R and IL-4R was achieved with a ratio of 0.2 µg of JAK3 expression vector versus 1 µg of receptor expression vector (Fig. 11B). Higher relative amounts of JAK3 expression vector in the transfection mixture did not enhance or diminish receptor action. The comparison also revealed that the JAK3-dependent signaling was more effective with IL-4R than IL-2R (Fig. 11B) and that the relative activity of the two receptors was reversed and then compared to the HRRE signal (see Fig. 2B). The finding that JAK3 established an IL-6-type response by IL-2R and IL-4R in HepG2 cells suggested that an additional productive signal pathway via JAK3 has been reconstituted.
To identify whether the action of JAK3 was also necessary for the activation of STAT proteins in L-cells and COS-1 cells, a combination of expression vectors for JAK3, IL-2R, and IL-4R was transfected into these cells, and the cytokine effect on SIF activities was measured. Neither receptor reconstituted a SIF activity in L-cells (Fig. 12). By contrast, in COS-1 cells, we observed an IL-4-, but not an IL-2-mediated SIF stimulation. The response to the two cytokines was, however, not detectably modified by the co-transfected JAK3. In both cell types, we established by mRNA analysis that the JAK3 expression vector was highly active (data not shown). From these results, we concluded that in L-cells and COS-1 cells the failure of reconstituting an IL-2R response on STAT protein was probably not due to lack of JAK3 but due to the absence of JAK3-regulated STAT proteins.
Based on the observations that prominent IL-6-type gene regulation correlated with SIF activation (Fig. 6) and that JAK3 introduced this type of gene regulation through IL-2R and IL-4R in HepG2 cells (Fig. 11), we expected that JAK3 will also reconstitute SIF activation in HepG2 cells. Therefore, we applied the transient transfection protocol established for receptor analysis in L-cells to HepG2 cells. Although based on mRNA analysis the relative level of receptor expression per transfected cell culture was approximately 15 times lower in HepG2 cells than L-cells (data not shown), we could nevertheless reconstitute a JAK3-sensitive STAT protein activation by both the IL-2R and IL-4R (Fig. 12). The induced SIF complex co-migrated with SIF-B and -C and yielded an electrophoretic pattern comparable to that activated by the same cytokines in human NK-cells. Taken together (Fig. 13), we conclude that: (a) HepG2 cells do express the STAT proteins that are substrates of JAK3; (b) these STAT proteins may serve as mediators of the IL-6 signal; (c) the specificity of gene regulation by hematopoietin receptors is controlled by the receptor subunit complex, receptor-associated kinase(s), STAT proteins, and gene elements; and (d) reconstitution of the receptor signaling pathways in heterologous cell systems permit dissection of the complexity and specificity of the signal transduction pathways leading to the control of gene expression.
Figure 13: A summary of cell responses activated by hematopoietin receptors. The diagram lists the receptor forms with the subunit combinations believed to be necessary for signaling action. The position of the box 1, 2, and 3 motifs (LIFR contains 2 copies of the box 3 motif but no functional box 1) and the two IL-4 STAT binding sites in IL-4R are marked by bars. The IL-6R and LIFR represent endogenous receptors, the others are those transfected in this study. Below the diagrams, the qualitative responses elicited by the receptors are listed. The regulation of specific gene elements by the receptors has been determined in hepatoma cells. The activation of STAT proteins, which are detectable as SIFs, is identified in L- and COS-1 cells (the specific forms of STAT proteins involved in the SIF complexes have not yet been determined). The ability of the receptor to activate the JAK3-specific pathway is defined in HepG2 cells. Wherever we could not detect a positive action (less than 10% of maximal response), no entry has been made.
The various types of hematopoietin receptors are often expressed in cells of widely different phenotypes; their unique cellular contexts thus preclude a direct comparison of receptor action. Recognizing the structural relationship among hematopoietin receptors, some common signaling mechanisms have been suspected(2, 28, 29) . We reasoned that the regulation of differentiated genes in one given cell type will provide defining features of receptor signals which are more readily assessed than the proliferative cell response activated by the same receptors in hematopoietic cells.
Our study has demonstrated that transient
expression of IL-2R and -
subunits in hepatic cells
reconstitutes IL-2- and IL-15-specific gene activation (Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5); a
process that is, in part, shared among other members of the
hematopoietin receptor family (Fig. 13). The similarity in the
cell response, e.g. HRRE regulation, suggests that each of the
hematopoietin receptors, regardless of its cellular origin, might
utilize the same signal transduction mechanism in the test cells and
that this mechanism might also be operative in other, nonhepatic cell
types. The reconstitution of IL-2R signaling function in the
heterologous cell systems permitted us to define the contribution of
lymphoid-specific JAK3 and its substrate STAT proteins in regulating
expression of differentiated genes (Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10Fig. 11Fig. 12).
The fact that we gained a JAK3-dependent regulatory action on IL-6RE,
while maintaining essentially unchanged the HRRE and CytRE regulation,
strongly suggests that two separate IL-2R signals exist. One signal
pathway is independent of JAK3, is targeted to HRRE and CytRE, and is
not detectably correlated with SIF activation; the other signal
requires JAK3 and appropriate substrate STAT proteins and leads to gene
activation via IL-6RE.
Our results present new aspects of hematopoietin receptor function; that is, the control of differentiated genes. Since the characterization of the receptors occurred in a heterologous cell system, the question arises as to the physiologic relevance of the potential receptor function in the homologous cell system, i.e. IL-2R action in lymphoid cells. The fact that the proliferation signal produced by IL-2R could, in part, be correlated with the control of immediate early growth response genes(16, 20, 42) suggests that gene regulatory mechanisms in lymphoid cells exist and that these may involve pathways analogous to those uncovered in transfected hepatic cells. An IL-2R signaling mechanism similar to that in hepatic cells seems even more likely to be found in differentiated lymphocytes, such as in NK cells in which the monocyte-derived IL-15 does not exert a proliferative response but stimulates the expression of several cytokine genes(26) .
The ease of gene transfection and the prominence of specific gene regulation in hepatic cells offer the great advantage of transient genetic manipulation and facilitate dissection of signaling pathways linking the receptor with the regulation of specific gene elements. Clearly, hepatic or fibroblastic cells cannot fully substitute for lymphoid cells because of their cell type-specific differences in make-up of signal transducing molecules and in the set of target genes controlled by the signals. However, these differences render our assay system even more attractive for studying hematopoietin receptors because the specific function of the cell-type restricted signaling factor(s) can be defined by complementation. The broadening of the IL-2R signal specificity by JAK3 in HepG2 cells is a case in point ( Fig. 11and Fig. 12).
One shortcoming of hepatoma cells is that the isolation of stably transfected lines is difficult and unpredictable and, therefore, makes these cells, unlike lymphoid cells(69, 70) , unsuitable for analyzing the proliferative response, if any, elicited by the introduced receptor. To define the overlap of proliferation and gene control with respect to the signaling pathways, we will have to rely on comparison of data derived from multiple systems.
The fact that transfected IL-2R and other nonhepatic receptors mediated transcriptional activation of specific gene constructs ( Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5and 10; (45) ) indicated an effective interaction of these receptors with the signal transduction mechanism in the heterologous system. Although the responses appeared receptor-specific, the results do not unequivocally prove that the action of the introduced receptor subunits was fully accountable for the observed regulation, because hepatoma cells, like most other cells, possess a set of hematopoietin receptor subunits, some of which may potentially contribute to or interfere with the transfected receptor action.
We showed (Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5)
that the combination of human IL-2R and -
is necessary to
elicit a HRRE signal in our cell system. The same subunit requirements
were noted for proliferative response and control of early response
genes in pre-B-cells(68, 69, 70) . The
importance of the cytoplasmic domain of the two subunits for the
hepatic action was inferred from experiments involving IL-2R subunits
with truncated cytoplasmic domains (Fig. 1) and G-CSFR-IL-2R
chimeras (Fig. 2). The cytoplasmic domain of IL-2R
in the
artificial context of the G-CSFR-mediated dimer performs essentially
the same signaling event as the
complex. A related
observation was made by Nelson et al.(70) who
demonstrated that similar proliferative signals were generated by the
normal
complex and the chimeric construct
c-kit-IL-2R
in BAF cells. Interestingly, this
c-kit-IL-2R
-restricted regulation was cell type-specific
because it was not reproducible in CTLL-2 cells. The combined data
suggest that the
subunit acts as a critical determinant in
initiating signal transduction both in hematopoietic and hepatic cells.
The functional relevance of specific regions in the
subunit
cytoplasmic domain has been documented by the identification of the
structural elements which are critical for signaling in hematopoietic
cells and fibroblasts(16, 68, 78) . However,
every IL-2R reconstitution experiment using wild-type subunits
indicated that the IL-2R
subunit is also essential for signaling.
Furthermore, the same obligatory function of the IL-2R
was
observed in reconstituting active IL-4R and IL-7R (Fig. 2; (5) and (6) ).
Although the IL-2R
dimer appears to be a functional entity (Fig. 4; (70) ),
it remains unclear whether the IL-2R
subunit, as an artificial
homodimer, has only limited signaling specificity (restricted to HRRE)
or whether it has redundant IL-2R
function. The complementation
experiment (Fig. 11) suggests, however, that the IL-2R
cytoplasmic domain is required for JAK3-dependent signal and is not
dispensable by IL-2R
dimerization.
The IL-2R signaling activity to HRRE, as well as the much lower activity of IL-4R, could not be correlated with the activation of STAT proteins as defined by binding to SIE (Fig. 7, Fig. 9, and Fig. 12). Since CTLL-2 cells (Fig. 10A) and NK cells (Fig. 12) showed an IL-2-responsive STAT activation, we concluded that SIF activation was not involved in signaling to HRRE and that the STAT-inducing factors operating in lymphocytes are not present in our test cells. JAK3 was the logical candidate factor for the cell type-restricted activity of the IL-2R action on SIF(58, 76, 77, 78, 79, 80) . The lymphoid-specific STAT activation may not be solely determined by JAK3 expression (JAK3 mRNA was detectable in L-cells and H-35 cells; Fig. 10A), but also by JAK3-specific substrates, i.e. STAT proteins. Attempts to reconstitute the missing SIF activation and IL-6RE regulation in our cell systems by overexpressing JAK3 indicated that the combination of JAK3 and JAK3-activated STAT proteins contributes to the functional specificity. The results from H-35 cells (Fig. 11B) and L- and COS-1 cells (Fig. 12) suggest that these cells, even when supplemented with JAK3, are unable to signal since the appropriate STAT proteins are missing. However, HepG2 cells seem to contain STAT proteins that serve as substrates for JAK3, but appear to be deficient in JAK3, explaining the prominent complementation of IL-2R action by JAK3 ( Fig. 11and Fig. 12).
We have not identified the STAT
proteins that constitute the IL-2R-inducible SIF seen in Fig. 10A and Fig. 12. Since the IL-4R receptor
proved to be the most prominent activator of IL-6RE in the presence of
JAK3, we assume that the recently described IL-4 STAT (41) is
participating in this regulation. The presence of that factor in the
hepatic cells is not surprising, since Hou et al.(41) detected by mRNA analysis high level expression of
IL-4 STAT in the liver. The IL-4R contains two box 3-related sequences
that serve as binding sites for IL-4 STAT (41; indicated in Fig. 13). Because the IL-2R enables signaling of the IL-4R
by JAK3, one possible explanation which is in agreement with the
analysis of receptor subunit-associated kinases (75, 76, 77) is that the IL-2R
subunit
provides JAK3 kinase to the receptor signaling reaction leading to the
phosphorylation of IL-4 STAT or any other STAT protein bound to IL-4R (40, 41) . If so, the same IL-2R
function is
expected to be a part of the signaling by IL-2R (
complex) and IL-7R. The contribution of IL-2R
appears not only to
include presentation of STAT proteins, but also the interaction with
JAK1 by its membrane proximal region(58, 79) . It
remains to be shown whether the JAK1-specific pathway that is
considered to be general to hematopoietin receptors (28, 29) is mediating the HRRE signal via a yet to be
detected STAT protein.
The working model of IL-2R providing
JAK3 and the IL-2R
, the STAT proteins, and probably JAK1 to the
signaling reaction also seems to apply to the heterodimeric form of the
G-CSFR-IL-2R
(Fig. 13). Indeed, this
experimental system will allow us to define the precise cytoplasmic
domains in each chimeric subunit that provides the respective function
for the JAK3-dependent STAT activation leading to IL-6RE induction in
HepG2 cells.
This study focuses on the ability of hematopoietin receptors to control gene expression. The comparison of the specificity by which these receptors control the proliferative response in nonhepatic cells reveals common traits in signaling. Like the regulation of CytRE in hepatoma cells(45) , box 1 and 2 motifs are required in the signaling subunit for generating the proliferative signal by IL-2R(16, 69, 70) , GM-CSFR(17, 81) , EPOR(19, 55, 82, 83) , PRLR(84) , gp130(7, 80, 85, 86) , and G-CSF(9, 10, 18) . Analysis of the GHR indicated, however, that a proliferative signal was already achieved with just the box 1 motif present(87, 88) . In several of these cases, JAK2 has been implied as being a potential mediator of the proliferative signal. It remains to be shown which of the signaling pathways characterized in the present studies (Fig. 13) is also part of the growth regulation and activation of an immediate gene growth response. A larger challenge will be to identify the molecular mechanisms that determine the specificity by which the hematopoietin receptors accomplish their tasks in the different cell types.