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J. Biol. Chem., Vol. 277, Issue 49, 47517-47523, December 6, 2002

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Interleukins 19, 20, and 24 Signal through Two Distinct Receptor Complexes

DIFFERENCES IN RECEPTOR-LIGAND INTERACTIONS MEDIATE UNIQUE BIOLOGICAL FUNCTIONS^*

Julia Parrish-Novak, Wenfeng Xu, Ty Brender^§, Lena Yao^¶, Crystal Jones^§, Jim West, Cameron Brandt, Laura Jelinek^¶, Karen Madden, Patricia A. McKernan^§, Donald C. Foster, Stephen Jaspers^§, and Yasmin A. Chandrasekher^§

From the Departments of  Cytokine and Receptor Biology, ^§ In Vitro Biology, and ^¶ Genetics, ZymoGenetics, Inc., Seattle, Washington 98102

Received for publication, May 24, 2002, and in revised form, September 12, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytokines that signal through Class II receptors form a distinct family that includes the interferons and interleukin 10 (IL-10). Recent identification of several IL-10 homologs has defined a cytokine subfamily that includes AK155, IL-19, IL-20, IL-22, and IL-24. Within this subfamily, IL-19, IL-20, and IL-24 exhibit substantial sharing of receptor complexes; all three are capable of signaling through IL-20RA/IL-20RB, and IL-20 and IL-24 both can also use IL-22R/IL-20RB. However, the biological effects of these three cytokines appear quite distinct: immune activity with IL-19, skin biology with IL-20, and tumor apoptosis with IL-24. To more fully elucidate their interactions with the receptor complexes, we have performed a series of in vitro assays. Reporter, proliferation, and direct STAT activation assays using cell lines expressing transfected receptors revealed differences between the receptor complexes. IL-19 and IL-24 also exhibited growth inhibition on a cell line endogenously expressing all three receptor subunits, an effect that was seen at cytokine levels two orders of magnitude above those required for STAT activation or proliferation. These results demonstrate that, although this subclass exhibits receptor complex redundancy, there are differences in ligand/receptor interactions and in signal transduction that may lead to specificity and a distinct biology for each cytokine.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interleukin 10 (IL-10)¹ and the related cytokines IL-20 (1), IL-19 (2), IL-24 (mda-7) (3), IL-22 (4, 5), and AK155 (6) form a distinct subfamily of ligands that bind and signal through Class II cytokine receptors. The IL-10 receptor (for review, see Ref. 7) consists of two subunits, a private alpha subunit (IL-10RA) and a beta subunit (IL-10RB), previously known as CRF2-4, that is also part of the IL-22 receptor complex (4). IL-10 modulates gene expression in responsive cell types through activation of the Jak/STAT signal transduction pathway (for reviews, see Refs. 8-11), in particular activating STATs 1, 3, and 5 (7, 12).

Recently, extensive cross-reactivity of the IL-10 subfamily ligands IL-20, IL-19, and IL-24 with two different receptor complexes consisting of IL-20RA/IL-20RB and also IL-22R/IL-20RB was observed (13, 14).² Although activation of a single receptor complex by multiple ligands is not unusual (e.g. the interferon / system), sharing of single receptor subunits between distinct ligand-specific complexes is more common. The sharing of identical receptor complexes within the IL-10 ligand subfamily raises the question of signal specificity. Each of the three ligands we have studied, IL-20, IL-19, and IL-24, appears to have unique biological activity. IL-20 has shown in vitro activity on keratinocytes, and the IL-20 transgenic phenotype has led to further investigation of IL-20 in skin biology (1). IL-19 has been reported to directly affect immune cells (2). Mice transgenic for IL-19, with the same promoters and similar expression levels as with IL-20, have no overt skin phenotype.³ IL-24 appears to function as a proapoptotic cytokine in a variety of tumors (15-18). These observations suggest distinct physiological roles for each ligand despite the sharing of receptor complexes. We therefore designed a series of experiments to better elucidate the interactions between these three ligands and their receptor complexes.

Here we show that all three receptor subunits are expressed in similar cell types in lung tissue, suggesting that at least in some instances a given cell type may respond to a given ligand through both receptor complexes simultaneously. To more fully study the interactions of each ligand with each receptor complex, we have performed both proliferation and reporter assays in two cell types stably transfected with each complex. The results of these assays indicate that each receptor complex responds to its respective ligands in a quantitatively similar fashion. However, growth inhibition experiments on the ovarian carcinoma cell line NIH:OVCAR-3, which endogenously expresses all three receptor subunits, suggest both that receptor recognition alone cannot adequately explain differences in ligand activity and that alternative signaling pathways may be involved. Another series of experiments to further study the activation of individual STAT proteins showed that in transfected cells STAT3 is activated at low (physiological) ligand concentrations, whereas STAT1 activation is seen at much higher levels.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RT-PCR Analysis on Human Tissues-- RT-PCR was performed on a human Rapid-Scan gene expression panel (Origene Technologies, Inc.) using primers 5'-ccccagacacggtctacagcat-3' and 5'-gggtcaggccgaagaactcatat-3' to amplify a 440-bp fragment of human IL22R. PCR conditions are 94 °C for 2 min, followed by 35 cycles of 94 °C for 15 s, 72 °C for 90 s, then a final extension step of 72 °C for 2 min. (See www.origene.com/ge_rs_dlink.html for a description of Rapid-Scan panel construction and control message amplification.)

In Situ Hybridization-- Hybridizations were carried out as described previously for IL-20 receptor subunits (1). For IL22R, two independent probes were designed and corresponded to nt 1910-2783 and nt 208-1081 with respect to sequence AF286095. BLASTn searches using our databases, which include all known class II cytokine receptors, confirmed that the probes were specific. A human alpha actin probe was used as a positive control for the tissue samples, which corresponds to nt 603-1328 of sequence NM_001613. A sense probe was used as a negative control. PCR products containing the working sequence of the T7 RNA polymerase promoter were used as templates for synthesis of digoxigenin-labeled antisense RNA probes (Riboprobe in vitro Transcription System, Promega). Hybridization was carried out at 60 °C with 50% formamide/2× SSC. The signals were amplified with two to three rounds of tyramide signal amplification (TSA in situ indirect kit, PerkinElmer Life Sciences) and visualized with Vector Red substrate kit (Vector Laboratories). Slides were counterstained with hematoxylin. Four different samples of normal human lung tissue were used with each probe. All tissues were tested with positive control probes and confirmed to be suitable for in situ hybridization analysis.

Luciferase Assay-- Luciferase reporter assays were performed as described previously (19) using BHK570 cells stably transfected with IL-20RA and IL-20RB or with IL-22R and IL-20RB and utilizing the STAT-driven luciferase reporter cassette. The STAT elements included in this construct are STATs 1, 3, 4, 5, and 6. Cells were switched to serum-free medium overnight prior to treatment with serial dilutions of IL-19, IL-20, and IL-24 in the presence or absence of IL-20RA/IL-20RB-soluble receptor. Cells were lysed and luciferase reporter activity was determined in triplicate for each data point using a Berthold MicroLumat Plus luminometer.

BaF3 Proliferation Assays-- The BaF3 cells were stably transfected with full-length Class II receptor subunits alone or in combinations and treated with IL-20, IL-19, and IL-24. For end point proliferation assays, cells were cultured at 5000 cells/well with variable cytokine concentrations for 72 h at 37 °C. Alamar Blue (Accumed) was added to the cells, and plates were read 24 h later on a fmax plate reader (Molecular Devices, Sunnyvale, CA) using the Softmax Pro program, 544-nm excitation, and 590-nm emission. For kinetic proliferation assays, cells were cultured at 5000 cells/well with 60 pM human IL-20, IL-19, or IL-24 in 96-well flat-bottomed plates at 37 °C. Each well was pulsed with 250 nCi of [³H]thymidine 6 h prior to harvest. Plates were harvested and counted at 24, 48, 72, and 96 h of culture.

Growth Inhibition Assay-- The human ovarian carcinoma cell line NIH:OVCAR-3 (20) was obtained from ATCC and cultured in Invitrogen RPMI 1640 medium with L-glutamine supplemented with 20% fetal bovine serum, 1% sodium pyruvate, 20 mM Hepes, and 10 µg/ml insulin. For growth inhibition assays, cells were plated in culture media at 5000 cells/well in 96-well flat-bottomed tissue culture-treated plates (Corning Costar) and allowed to adhere for 24 h in a 37 °C, 5% CO₂ incubator. Cells were treated in triplicate with each dose of each cytokine for 48 h. Relative live cell counts were determined using MTT Cell Titer 96 Nonradioactive Proliferation assay (Promega) according to the manufacturer's specifications. Percent inhibition was defined as the average of 100  100 ×[(A₅₇₂  A₆₅₀ for unknown)/(A₅₇₂  A₆₅₀ for growth media control)].

STAT Translocation Assay-- BHK570 cells stably expressing each receptor complex were plated in flat-bottomed 96-well plates at 2000 cells per well. The next day, cells were refed with serum-free medium and starved for 5-16 h. Cytokines were serially diluted into serum-free medium then added to cells to achieve final cytokine concentrations of 0.15-20 nM. Plates were incubated at 37 °C for 45 min (STAT1) or 20 min (STAT3). Immediately following incubation, plates were washed, fixed, and stained using protocols provided with STAT1 and STAT3 HitKit reagent kits (Cellomics, Inc., Pittsburgh, PA). Plates were analyzed using the ArrayScan II instrument running the nuclear translocation protocol (Cellomics, Inc.). A minimum of 100 cells per well was analyzed. Data were subjected to one-tailed unpaired t test with Welch's correction using Prism (GraphPad) software.

Protein Expression-- Untagged recombinant human IL-20 was produced in baculovirus. C-terminally FLAG-tagged IL-19 and IL-24 were expressed in BHK570 cells. IL-20RA- and IL-20RB-soluble receptors were expressed as homodimeric IgG fusion proteins in BHK570 cells. For construction of heterodimeric IL-20RA/IL-20RB, the extracellular domains of IL-20RA and IL-20RB were fused to human IgG with a Gly-Ser (×4) spacer between. In addition, the IL-20RA carried a C-terminal EE affinity tag (GEYMPME), and the IL-20RB carried a C-terminal His₆ tag. The receptor subunits were co-expressed in BHK-570 cells, and the IgG fusions were isolated from the culture media by protein-A chromatography. The heterodimers were purified away from the homodimers by immobilized metal chromatography utilizing an imidazole step gradient elution.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Receptor Subunits-- Recent studies in our laboratory and others have shown redundant interactions between the IL-10 homologs IL-19, IL-20, and IL-24 and their newly identified receptor complexes IL-20RA/IL-20RB and IL-22R/IL-20RB (1, 13, 14). To better understand the functional relevance of the receptor redundancy found in this IL- 20 subfamily, we performed a variety of experiments. Expression analysis using RT-PCR showed that IL20RA is the most widely expressed of the three receptor subunits. Previous RT-PCR analysis using Origene panels showed that both IL20RA and IL20RB mRNAs are highly expressed in skin and testis, and are also expressed in a variety of other tissues, including the lung and ovary (1). Because IL-22R is a shared alpha subunit, we evaluated an identical Origene panel for the expression of IL22R mRNA (Fig. 1). Table I provides a summary of our previous data directly compared with the new data shown in Fig. 1. IL22R is expressed in a few tissues that lack IL20RB expression, notably pancreas, small intestine, and fetal liver. With the exception of peripheral blood lymphocytes, IL20RB was only expressed in tissues that also showed IL20RA expression; most of these lacked IL22R expression. Co-expression of IL22R and IL20RB was only observed in tissues that also expressed IL20RA. Of these tissues, skin and lung exhibited robust expression of all three receptors.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   RT-PCR analysis of IL22R. PCR was performed on a Rapid-Scan human tissue panel (Origene) according to the manufacturer's instructions. Shown is the gel representing the 100× (100 pg each cDNA) series.

Because IL20RA, IL20RB, and IL22R are all expressed in the lung, we performed in situ hybridization on lung sections to evaluate whether the same cell types express all three receptors. The results show that epithelial cells as well as immune infiltrates exhibit positive staining for all three receptor subunits (Fig. 2). Taken together, the RT-PCR and in situ hybridization analyses show that the cellular/tissue content can be similar for IL20RA, IL20RB, and IL22R.

View larger version (67K): [in this window] [in a new window]   Fig. 2.   In situ hybridization analysis of IL20RA, IL20RB, and IL22R subunits in human lung. Probes were detected using tyramide signal amplification signal amplification with Vector Red substrate, producing red positive signals. Probes used were IL20RA (a), IL20RB (b), and IL22R (c). Alpha actin-positive controls are shown in d at low magnification to show positive staining in smooth muscle fibers and in e at high magnification. A negative control probe is shown in f. Bars indicate 10 µm except panel d, where the bar indicates 100 µm.

STAT Reporter Activation-- To evaluate functional interactions between the IL-20 ligand subfamily and the two receptor complexes, we utilized cells with stably transfected receptors and those determined to express the receptor subunits endogenously and assayed a variety of endpoints. In the first of these experiments, BHK570 cells were stably transfected with IL-20RA/IL-20RB, IL-22R/IL-20RB, or individual receptor subunits alone and treated with increasing amounts of IL-19, IL-20, and IL-24. These cells were also stably transfected with a reporter construct consisting of the firefly luciferase gene driven by promoter/enhancer sequences comprised of tandem STAT elements. In all cases where individual receptor subunits were transfected alone, there was no detectable luciferase production (data not shown). As described earlier (1), there was a dose-dependent increase in reporter luciferase activity in response to IL-20 treatment of BHK cells stably transfected with IL-20RA/IL-20RB (Fig. 3A). IL-19 and IL-24 were equipotent to IL-20 in this assay, with a half-maximal response between 20 and 40 pM. To confirm the specificity of activation, all three ligands were tested in the luciferase assay in the presence or absence of a heterodimeric IL-20RA/IL-20RB-soluble receptor (SR). A 50-fold excess of the SR resulted in nearly complete inhibition of IL-20 effects. Concurrent soluble receptor treatment resulted in a similar inhibition of IL-19- and IL-24-stimulated luciferase activity.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Luciferase reporter assays using BHK cell lines stably expressing (A) IL-20RA/IL-20RB or (B) IL-22R/IL-20RB. The solid symbols and lines represent cytokine alone; open symbols and dashed lines represent cytokine with a constant 30 nM soluble heterodimeric IL-20RA/IL-20RB. Samples were compared with negative controls using a one-tailed t test; significance is indicated as: *, p < 0.05; **, p < 0.01; and ***, p < 0.001. For panel A, significance is indicated for (top to bottom) IL-20, IL-19, and IL-24 at each point. For panel B, significance is indicated for (top to bottom) IL-20 and IL-24 at each point.

A similar set of experiments was performed using the same parental cell line stably transfected with IL-22R/IL-20RB and treated with increasing amounts of IL-19, IL-20, and IL-24 (Fig. 3B). IL-19 had no effect on this cell line. IL-20 and IL-24 were equipotent in stimulating luciferase output in this cell line, with half-maximal stimulation occurring around 60 pM. IL-20 and IL-24 activities were again blocked with SR treatment.

We next wanted to determine which soluble receptors were capable of blocking ligand activity. As described above, IL-20RA/IL-20RB heterodimeric soluble receptor blocked luciferase activity stimulated by IL-20, IL-19, and IL-24 on BHK cells bearing either combination of receptors. In similar luciferase assays, neither IL-20RA nor IL-22R was capable of blocking the activity of any of the three ligands (data not shown). In contrast, IL-20RB-soluble receptor alone did block the activity of IL-19 and IL-24, at >1000-fold excess for IL-19 and >100-fold excess for IL-24 (data not shown). Note that this soluble receptor is much less effective than the heterodimeric IL-20RA/IL-20RB, which fully blocked ligand activity at 50-fold excess (see above). Soluble IL-20RB had no effect on the activity of IL-20 at any concentration (data not shown). An additional binding assay (21), using soluble receptors to detect ligands transiently expressed in COS-7 cells, further confirmed the specific interaction of IL-20RB with both IL-19 and IL-24 (data not shown).

Proliferation Assays-- We next evaluated proliferation of receptor-transfected BaF3 cells using Alamar Blue as an end point live cell number readout (Fig. 4). IL-20, IL-19, and IL-24 all stimulated proliferation of BaF3 cells stably transfected with both IL-20RA and IL-20RB (Fig. 4A). All three ligands showed equipotent activity on this cell line, with a half-maximal response occurring between 60 and 75 pM. BaF3 cells were also stably transfected with IL-22R and IL-20RB alone or in combination and treated with these ligands. Both IL-20 and IL-24 stimulate proliferation through IL-22R/IL-20RB (Fig. 4B), with a half-maximal response detected at ~6 pM: 10-fold lower than that observed with BaF3 cells expressing IL-20RA/IL-20RB.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Proliferation assays using BaF3 cell lines stably expressing (A) IL-20RA/IL-20RB or (B) IL-22R/IL-20RB. Cells were cultured for a total of 96 h in the presence of the indicated concentration of ligand. The fluorescent readout is proportional to the number of live cells in the culture well. Values plotted represent mean (±S.D.) for quadruplicate cultures. Statistical significance is indicated as in Fig. 3.

Given the differences in ligand potency on BaF3 cells expressing each receptor complex, we next evaluated the kinetics of the growth response in these cells using [³H]thymidine incorporation assays. Fig. 5A shows that all three ligands stimulated a similar and continuous growth of BaF3 cells expressing IL-20RA/IL-20RB over a 72-h period. IL-20 and IL-24, but not IL-19, also stimulated the growth of BaF3 cells transfected with IL-22R/IL-20RB (Fig. 5B). The rate of growth stimulated by each ligand was similar for the same receptor complex, however, there were obvious differences in growth kinetics between the two cell lines. Cells transfected with IL-22R/IL-20RB proliferated much more rapidly, surpassing the maximal level of growth seen with the other complex within about 40 h. The decline in growth rate seen at 96 h is due to saturation of the culture. At its maximum, the growth rate of these cells is about double that of the cells expressing IL-20RA/IL-20RB.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Tritiated thymidine incorporation assays using BaF3 cell lines stably expressing (A) IL-20RA/IL-20RB or (B) IL-22R/IL-20RB. Cells were cultured for the times indicated in the presence of 60 pM of each ligand. Wells were pulsed 6 h prior to harvest. Values plotted represent mean (±S.D.) for quadruplicate cultures. In panel B, the plot representing IL-19 overlays the plot representing control (untreated) cells; in every other case the treated cells were highly significantly different (p < 0.001) from control.

Growth Inhibition Assays-- To perform assays on a cell type endogenously expressing all three receptors, we performed RT-PCR analysis on a variety of cell lines and identified one, NIH:OVCAR-3 (20), which expresses IL20RA, IL20RB, and IL22R (data not shown). Because IL-24 is known to inhibit growth of a variety of tumor cell lines (3, 15), we chose to measure ligand-induced growth inhibition in these cells. IL-19 and IL-24, but not IL-20, treatment resulted in a dose-dependent growth-inhibitory effect (Fig. 6), with half-maximal responses at about 30 nM: 500- to 5000-fold greater than those measured for proliferation or reporter activation in the receptor-transfected BaF3 and BHK cell lines. This result was confirmed in three independent assays. Concurrent STAT reporter activation experiments with the NIH:OVCAR-3 cells revealed that treatment with IL-19, IL-20, or IL-24 did not result in activation of the STAT pathway (data not shown). Similar results were obtained when a nuclear translocation assay was performed.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   NIH:OVCAR-3 cells are growth-inhibited by IL-24 and IL-19. Cells were treated for 48 h in the presence of the indicated concentration of each cytokine, or retinoic acid (RA) as a positive control. Bars represent mean (±S.D.) percent inhibition from triplicate cultures. Data were compared with untreated cultures using one-tailed t test; *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

STAT Translocation Assays-- To determine whether STAT recruitment changes with increasing ligand doses, we directly analyzed STAT protein nuclear translocation using the BHK cell lines stably expressing each receptor complex. These assays measure activation of individual signaling elements by measuring their localization in the nuclear versus cytoplasmic compartment in resting or stimulated cells (22). For our assays, STAT1- and STAT3-specific detection reagents were used. In BHK cells bearing the IL-22R/IL-20RB receptor complex, both IL-20 and IL-24 robustly translocate STAT1 and STAT3 (Fig. 7, A and B). The STAT3 translocation reaches the half-maximal level at 1-5 pM cytokine, whereas the STAT1 translocation is half-maximal at 150-800 pM and has dropped to background levels at ligand concentrations below 30 pM.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   STAT translocation assays using BHK cells expressing IL-22R/IL-20RB. Each data point represents the mean difference between nuclear and cytoplasmic localization of STAT1 (A) or STAT3 (B) for each treatment; at least 100 individual cells were analyzed for each point. Statistical significance markers and error bars were omitted for clarity; due to the large number of events measured, differences as small as 10% are highly significant.

We previously reported nuclear translocation of STAT3 but not STAT1 in response to IL-20 (1); similarly, both IL-19 and IL-24 translocate STAT3 in BHK cells bearing IL-20RA/IL-20RB (data not shown). In this set of experiments, we did observe some STAT1 recruitment at high IL-20 doses. Similar results were obtained with IL-19 or IL-24, with half-maximal STAT1 translocation at about 400 pM (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We show that functional differences exist in the IL-20 subfamily in which IL-20, IL-19, and IL-24 exhibit sharing of receptor complexes. Although STAT reporter activation and preferential activation of STAT3 versus STAT1 are similar between complexes, differences become apparent when ligand-induced proliferation is compared between receptor complexes. An ovarian carcinoma cell line endogenously expressing all three receptor subunits also responded differentially to the ligands; IL-19 and IL-24 were growth inhibitory, whereas IL-20 was not.

Our expression data show that, in most tissues expressing the common subunit IL20RB, the only other subunit expressed is IL20RA. Thus in the majority of cases the cytokines in this subfamily will be expected to signal through IL-20RA/IL-20RB. IL22R is found in a few tissues lacking IL20RB expression; these include adult and fetal liver, colon, small intestine, and pancreas. In these tissues its role as part of the IL-22 receptor is expected to predominate, consistent with reports of IL-22 proinflammatory activity on hepatocytes (5) as well as recent data showing IL-22 activity on pancreatic acinar cells (23). Interestingly, we did not find any tissue or cell type in which IL22R was expressed with IL20RB in the absence of IL20RA; rather, tissues co-expressing IL22R with IL20RB also expressed IL20RA. We detected IL20RA and IL22R messages in lung immune infiltrates, which consist largely of specialized macrophages (24, 25). These messages are detectable neither in peripheral blood leukocytes (Fig. 1 and Table I) nor in resting or activated peripheral immune subsets (26). Thus they appear to be up-regulated in immune cells only under certain maturation or stimulation conditions. Taken together, these data suggest that, although the primary receptor complex in most cell types is IL-20RA/IL-20RB, in some cell types expressing all three receptor subunits the net signal transduced by a given ligand would depend upon a more complicated set of interactions between that ligand and the two signaling complexes.

We studied the types of signals transduced by each receptor complex separately in transfected BHK and BaF3 cells. Although the EC₅₀ values for STAT reporter activation in BHK cells were very similar for all ligand/receptor combinations, there were substantial differences between receptor complexes in their ability to promote proliferation of BaF3 cells. Both IL-20 and IL-24 exhibit 10-fold lower EC₅₀ values for proliferation of BaF3 cells bearing IL-22R/IL-20RB as compared with cells bearing IL-20RA/IL-20RB (summarized in Fig. 8). Although it could be argued that this result may be due to higher ligand affinity for IL-22R/IL-20RB or to higher expression levels of that receptor complex on the assay cell line, it should be noted that the EC₅₀ values for both proliferation and reporter activation are 25- to 200-fold below the measured affinities of the ligands for receptors, where these values are known (Fig. 8). This suggests that these signaling events require only fractional receptor occupancy, and are thus reasonably insensitive to small differences in affinity or receptor number. A more likely explanation is that the signal transduced by IL-22R/IL-20RB differs from that of IL-20RA/IL-20RB in a way that favors proliferation.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Summary of the activities of each ligand on each receptor complex. Affinity values are from (a) our preliminary estimate (data not shown); (b) Ref. 1; (c) Ref. 14.

To further study the differences in signal transduction, we examined nuclear translocation of individual STAT proteins in transfected BHK cells. IL-20 and IL-24 appear equivalent in function when acting through IL-22R/IL-20RB, with robust activation of STAT3 showing EC₅₀ values of 1-5 pM. The EC₅₀ value for STAT1 activation is substantially higher, in the 150-800 pM range. When the STAT translocation assays were done on BHK cells bearing IL-20RA/IL-20RB, all three ligands activated STAT3 equivalently. Activation of STAT1 through this complex was slightly more robust with IL-19 and IL-24 as compared with IL-20, but the EC₅₀ values for both activities were consistent with those observed for cells bearing IL-22R/IL-20RB. Taken together our STAT activation data show that at low (below 100 pM) ligand concentrations, STAT3 activation is favored over STAT1 activation through either receptor complex.

STAT3 is involved with signal transduction through a wide variety of receptors, and, not surprisingly, ablation of the STAT3 gene results in embryonic lethality (for review, see Refs. 10 and 27). As a result, the critical actions of STAT3 are studied in tissue-specific knockouts. Because the three receptor subunits we studied are all expressed in skin, we were particularly interested in the recent report of keratinocyte-specific ablation of STAT3 (28). Keratinocytes from these mice failed to migrate in response to epidermal growth factor, transforming growth factor , hepatocyte growth factor, or IL-6, and this defect resulted in impaired wound healing and abnormal hair cycles. The keratinocytes showed normal proliferative responses, leading the authors to conclude that the proliferative response was due to activation of one or more STAT3-independent pathways, whereas the migratory response was STAT3-dependent.

We have shown that IL-19, IL-20, and IL-24 activate STAT3 through either receptor complex. We previously reported that IL-20 also activates STAT3 in the human keratinocyte cell line HaCaT and have shown marked synergy of IL-20 with epidermal growth factor, IL-1, or tumor necrosis factor  in STAT-luciferase reporter assays (1). Our IL-20 transgenic phenotype and our observation of high receptor expression in psoriatic skin (1) would appear to favor a role of IL-20 in proliferation of keratinocytes. We did not observe changes in keratinocyte migration in IL-20 transgenic mice, but it is possible that such a defect might have become apparent had the mice survived beyond the neonatal period. Interestingly, transgenic mice overexpressing IL-19 have no apparent skin phenotype,³ and we are unaware of any changes in skin caused by administration of IL-24 (although IL-24 is up-regulated in wound healing (29)). One hypothesis is that the hyperproliferation of keratinocytes seen in IL-20 transgenics is due to activation of a STAT3-independent pathway unique to IL-20. Future work with mice lacking each of the individual receptor subunits will prove helpful in determining the relative importance of each ligand in skin structure and remodeling.

Growth inhibition assays on NIH:OVCAR-3 cells revealed a functional divergence among the three ligands. IL-19 and IL-24 at doses above 600 pM inhibited the growth of this cell line, whereas IL-20 had no effect. To our knowledge, this is the first report of cytostatic effects of IL-19. The growth inhibitory effect that we detected on OVCAR cells did not appear to be cytotoxicity, because the cells grew normally following removal of the cytokines. The cytostatic effect does not seem to be a general response to treatment with these ligands, because none of the ligands at any concentration affects the growth rate of BHK cells transfected with either receptor complex (data not shown). Several groups have tried to dissect the putative pathways involved in the growth inhibitory activity of IL-24 using adenoviral delivery of this protein. The early work described this activity of IL-24 as growth-suppressing (3). Su et al. (15) showed nucleosomal DNA degradation in human breast cancer cells infected with adenoviral IL-24. These previous studies were done using adenoviral delivery of IL-24, whereas our experiments utilized purified proteins. Future experiments will need to better differentiate which effects of IL-24 are specific only to adenoviral delivery of this protein.

Because the growth inhibitory effect does not correlate with the receptor complex specificities of the ligands, it may be that the growth inhibition associated with IL-19 and IL-24 results from utilization of another signaling pathway. This pathway could involve signaling through the double-stranded RNA-dependent protein kinase, as has recently been observed in lung cancer cell lines (30). Use of an alternative pathway is supported by the failure of any of the ligands to activate the STAT/luciferase reporter or nuclear translocation assay in this cell line. Another possibility is that aberrant receptor complexes form at saturating doses of cytokine. Because IL-19 and IL-24 both bind to IL-20RB alone, a quality that IL-20 lacks, they could induce homodimerization of IL-20RB. They could also induce the formation of heterodimers between IL-20RB and an unknown additional receptor subunit. A third possibility is that IL-20 has a lower affinity for the receptor complexes than do IL-19 and IL-24, such that saturation kinetics are reached at the concentrations tested for the latter two but not for IL-20. An example from the interferon / system supports this hypothesis. A low affinity ligand, interferon , has antiviral properties similar to those of interferon  but is at least 30-fold less toxic. Thus in this system toxicity is associated with saturation binding and is related to the K_d value, whereas maximal antiviral activities are induced with only fractional receptor occupancy (31). A similar mechanism could be responsible for the disparate cytostatic effects of IL-19 and IL-24 as compared with IL-20 despite their similar activities in every other type of assay.

We have demonstrated substantial functional differences between the two receptor complexes in the IL-20 subfamily, reflected in proliferation assays. The growth inhibition induced by IL-19 and IL-24 in the OVCAR cells revealed a novel difference between ligands that is not explained by receptor specificity. Future work will be needed to dissect this ligand/receptor system further to better explain the biological differences seen in this family.

	ACKNOWLEDGEMENTS

We thank all of our colleagues at ZymoGenetics, Inc. for support, contributions to the work, and comments on the manuscript. In particular we recognize the contributions of Donna Prunkard, Rolf Kuestner, Jeff Ellsworth, Karyn Carlson, Stacy Schlutsmeyer, Harold Storey, Craig Ostrander, Carl Birks, Michael Fitzpatrick, Claire Noriega, Nancy Jenkins, Jon Berry, Nels Hamacher, Mark Rixon, and Tom Bukowski.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of In Vitro Biology, ZymoGenetics, Inc., 1201 Eastlake Ave. East, Seattle, WA 98102. Tel.: 206-442-6600; Fax: 206-442-6608; E-mail: chandray@zgi.com.

Published, JBC Papers in Press, September 25, 2002, DOI 10.1074/jbc.M205114200

² J. Parrish-Novak, W. Xu, T. Brender, L. Yao, C. Jones, J. West, C. Brandt, L. Jelinek, K. Madden, P. A. McKernan, D. C. Foster, S. Jaspers, and Y. A. Chandrasekher, unpublished data.

³ Transgenic group, ZymoGenetics, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: IL-10, interleukin 10; STAT, signal transducers and activators of transcription; RT, reverse transcription; wt, wild type; SR, soluble receptor; BHK, baby hamster kidney cells.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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