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.

EXPERIMENTAL PROCEDURES

Cells and Cell Treatments

Receptor and JAK3 Expression Vectors

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CAT Reporter Gene Constructs

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Transfection

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).

Gel Mobility Shift Assay (GMSA)

Analysis of Receptor Expression in L- and COS-1 Cells

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 NaVO, 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).

RESULTS

IL-2R Regulates Transcription in H-35 Cells

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).

Relative Contribution of IL-2R Subunits to Signaling

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.

The Cytoplasmic Domain of IL-2R Is Sufficient for Regulation

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 IL-2R Signals Do Not Act on IL-6RE or SIE

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.

IL-2R Does Not Activate SIF in Transfected Cells

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.

JAK3 Reconstitutes an IL-6 Signal and SIF Activation by IL-2R in HepG2 Cells

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.

DISCUSSION

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.

FOOTNOTES

*

This work was supported by National Institutes of Health Grant CA26122, American Chemical Society Grant DHP-111B, and Grant 2709 of the Women's and Children's Health Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Permanent address: Dept. of Ophthalmology, School of Medicine, Tohoku University, Sendai 980, Japan.

¶

To whom correspondence and reprint requests should be addressed. Tel.: 716-845-4587; Fax: 716-845-8389.

()

The abbreviations used are: IL-, interleukin; AGP, -acid glycoprotein; APP, acute phase protein; CAT, chloramphenicol acetyltransferase; DRE, distal regulatory element; EPO, erythropoietin; G-CSF, granulocyte-colony stimulatory factor; GH, growth hormone; GMSA, gel mobility shift assay; GRE, glucocorticoid response element; HP, haptoglobin; HRRE, hematopoietin receptor response element; CytRE, cytokine response element; JAK, Janus kinase; LIF, leukemia inhibitory factor; IFN, interferon; -R, receptor; PRL, prolactin; SIE, sis-inducible element; SIF, sis-inducible factor; STAT, signal transducer and activator of transcription.

()

Ziegler, S. F., Morella, K. K., Anderson, D., Kumaki, N., Leonard, W. J., Cosman, D., and Baumann, H.(1995) Eur. J. Immunol., in press.

()

C.-F. Lai, S. Immenschuh, D. P. Gearing, S. F. Ziegler, and H. Baumann, submitted for publication.

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