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Keywords:

  • IL-33;
  • knockout;
  • promoter;
  • ST2

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Conflict of interest
  8. References

IL-33 signals through ST2, which is expressed either as a full-length signaling receptor or a truncated soluble receptor that can suppress IL-33 activity. Previous data suggest that soluble ST2 mRNA in fibroblasts is coupled to a serum-inducible proximal promoter, while full-length ST2 expression in immune cells is directed from a distal promoter. In order to better understand the function of the alternative promoters and how they ultimately affect the regulation of IL-33, we generated a mouse in which the ST2 proximal promoter is deleted. Promoter deletion had no impact on ST2 expression in mast cells or their ability to respond to IL-33. In contrast, it resulted in a complete loss of both soluble and full-length ST2 mRNA in fibroblasts, which corresponded with both an inability to secrete soluble ST2 and a defect in IL-33 responsiveness. Importantly, in spite of the fibroblast defect, soluble ST2 concentrations were not reduced in the serum of naïve or allergen-exposed knockout mice. In summary, we found that ST2 promoter usage is largely cell-type dependent but does not dictate splicing. Moreover, the proximal promoter is not a major driver of circulating soluble ST2 under the conditions tested.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Conflict of interest
  8. References

il-33 is a tissue-derived cytokine that enhances Th2- and allergy-associated inflammation by activating a membrane-spanning receptor known as ST2 (or ST2L). ST2L encompasses a ligand-binding domain combined with an intracellular TIR domain required for signaling. In addition, a soluble form of the receptor (sST2) is encoded by a transcript variant that lacks the exons for the transmembrane and cytoplasmic domains. sST2 binds to IL-33 but is unable to transmit a signal thereby acting as a decoy molecule that regulates inflammation by neutralizing IL-33 in solution [1]. Regulation of sST2 expression is therefore related to regulation of IL-33 activity.

The sST2 transcript was identified over 20 years ago as a gene induced in either mouse [2] or rat [3] fibroblasts in response to oncogenes, serum, and other mitogenic stimuli. Optimal sST2 induction in fibroblasts requires a TPA-responsive enhancer element upstream of the promoter [4]. In comparison, the ST2L transcript represents an alternatively spliced mRNA [5] expressed predominantly in mast cells and other hematopoietic cell lineages. Mast cells and Th2 cells employ a more distal promoter, which contains Th2-associated GATA elements and lies 10 kb upstream of the promoter described in fibroblasts [6, 7].

Several studies have addressed the link between the unique ST2 promoters and generation of either ST2L or sST2. A study with rat cells suggested that expression of the two ST2 variants is largely governed by transcriptional regulation, with sST2 linked to the proximal promoter in fibroblasts and ST2L linked to the distal promoter in hematopoetic cells [8]. However, another group found ST2L expression in mouse mast cells to be dependent on the distal promoter and ST2 expression in fibroblasts (mostly sST2, but also ST2L) linked to the proximal promoter, suggesting that promoter usage was cell type but not transcript specific [6]. Collectively, these findings suggest that ST2 promoter usage is mostly cell-type specific and that transcription from the proximal promoter in fibroblasts is a potential source of sST2 in vivo.

Soluble ST2 protein is present in serum at up to ng/mL concentrations and is often elevated in inflammatory, infectious, or other disease situations [9-11]. Circulating sST2 concentration is also considered a potentially useful biomarker for predicting outcomes in patients with cardiovascular disease [12]. A number of stimuli induce sST2 gene expression, such as LPS, allergens [1], and cytokines [13]. Besides fibroblasts, sST2 is also expressed by endothelial, epithelial, and activated immune cells however it is difficult to ascertain the precise cellular source of circulating sST2 in vivo [14].

In order to better understand the regulation of ST2 mRNA transcription and splicing, we specifically deleted the mouse ST2 proximal promoter and associated enhancer element. We hypothesized that fibroblasts and possibly other abundant tissue cell types are major sources of sST2 protein in vivo and that deletion of the proximal promoter would result in less circulating sST2 and thus disruption of normal IL-33 regulation. Instead, we found that although loss of the proximal promoter abolished fibroblast-specific ST2 expression, it had no obvious impact on the amount of circulating sST2.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Conflict of interest
  8. References

Generation of mice

Figure 1A is a map of the mouse ST2 locus illustrating the location of the two promoters, the intron-exon organization and the targeting strategy to generate the proximal promoter and enhancer knockout. Figure 1B illustrates the alternative splicing whereby exons 9–11 are either included in the final spliced ST2L mRNA, or not included thereby leading to incorporation of an alternative stop codon and the generation of sST2. We selectively deleted the ST2 proximal promoter (with noncoding exon 1b) and its associated enhancer element. The resulting locus contains in their place a single loxP site, yet still retains the distal promoter and all coding exons. Homozygous knockout mice bred normally and nearly all animals lacked overt developmental or pathological manifestations. However, interestingly, two homozygous knockout mice spontaneously developed what appeared to be subcutaneous tumors on their neck and trunk and a third animal was found moribund due to unknown causes (not shown). Possibly relevant to these observations are previous findings that sST2 is correlated with progression of breast cancer [15] and that sST2 may modulate tumor cell activity in vitro [16].

image

Figure 1. ST2 locus and effect of promoter deletion on ST2 expression in splenocytes or mast cells. (A) Mouse ST2 locus and the strategy used to delete the proximal promoter and enhancer region. * = stop codon; LA = Long arm; SA = short arm. Locations of primers used for RT-PCR analysis are indicated. (B) Forms of ST2 protein that arise due to alternate splicing. (C) Products of isoform-specific RT-PCR assays of cDNA from splenocytes (from two different mice) or BMMCs (from three different mice). (D) Flow cytometric analysis of c-kit and ST2 expression in BMMCs from wild type and KO mice. IgG control is with wild-type cells. (E) IL-6 production from BMMCs isolated from the indicated mice and following 24-h stimulation in vitro with 10 ng/mL of IL-33. Data are plotted as mean ± 95% Confidence interval (CI) of triplicates and are representative of two experiments.

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Expression of ST2 in promoter knockout splenocytes and mast cells

Based on previous findings, we predicted that proximal promoter deletion would not disrupt expression of ST2L in immune cells. We performed a PCR designed to specifically amplify sST2 or ST2L cDNAs, as indicated in Fig. 1A, and found that as expected ST2L mRNA was expressed similarly in both wild type and knockout splenocytes (Fig. 1C). Little to no expression of sST2 was detected in splenocytes. Therefore, consistent with previous data, we found splenocytes express predominantly the ST2L isoform and deletion of the proximal promoter did not abolish ST2L expression.

We also found that deletion of the proximal promoter had minimal effects on the expression of ST2 in bone marrow-derived mast cells (BMMCs) (Fig. 1C). BMMCs express both sST2 and ST2L transcripts and neither isoform was affected by promoter deletion. Also, BMMCs from knockout mice developed normally in vitro (based on c-kit expression) and expressed equivalent amounts of ST2L on the cell surface compared with wild-type BMMCs (Fig. 1D). Moreover, knockout BMMCs responded to IL-33 by secreting equivalent amounts of IL-6 as compared with wild-type BMMCs (Fig. 1E). These observations indicate that expression of ST2L in mast cells is driven predominantly by the distal promoter.

Expression of ST2 in fibroblasts and differential promoter usage

We next examined the effect of proximal promoter deletion on ST2 expression in fibroblasts. First, we quantitated total ST2 expression using a qPCR assay that measures both ST2L and sST2. ST2 expression was abolished in promoter deficient fibroblasts compared with the high amounts of total ST2 expression seen in wild-type fibroblasts (Fig. 2A). In contrast, BMMCs from both wild type and knockout mice expressed similar amounts of ST2, consistent with the results shown in Fig. 1. We treated fibroblasts with either PMA or PDGF, which have previously been shown to increase sST2 expression [4], however these agents induced minimal sST2 expression in the promoter-deficient fibroblasts compared with wild-type cells. These results imply that the large majority of ST2 expression in fibroblasts, even following activation, is dependent on the proximal promoter and enhancer element.

image

Figure 2. Expression of sST2 and ST2L and promoter usage. (A) qPCR analysis of ST2 mRNA in tail fibroblasts (from wild-type and KO mice) and BMMCs from two different mice. Fibroblasts were stimulated for 4 h. Expression relative to the control gene HPRT. "LOD" = limit of detection using threshold of 40 cycles. Data representative of two experiments. (B) RT-PCR products obtained using combinations of 5′ primers specific for the two different promoters and 3′ primers specific for sST2 or ST2L using cDNA from cells as indicated. Fibroblasts were stimulated for 4 h. Data representative of two experiments. (C) qPCR measurement of fibroblast-expressed genes in wild-type or KO cells (each bar representing one sample). Fold induction is shown for wild-type cells (no treatment versus IL-33 normalized to HRPT expression). Data representative of two experiments. (D) sST2 protein in supernatants from tail fibroblasts treated with 10% FBS or 100 ng/mL PMA for 0, 2.5, 6, and 24 h. Data representative of two experiments, with one sample/mouse in each experiment, and are expressed as% maximum. (E) CXCL1 concentrations in supernatants of fibroblast cultures treated for 24 h with 5 ng/mL of IL-33 or a combination of IL-17 (10 ng/mL) and TNF-α (0.5 ng/mL). Data representative of two experiments.

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Next, a series of PCR assays were performed to measure sST2 or ST2L transcripts initiated from either the distal or proximal promoter (primer locations indicated in Fig. 1A). The majority of ST2 expression in BMMCs was linked to exon 1a of the distal promoter (both sST2 and ST2L); however, some ST2L expression was associated with the proximal promoter (Fig. 2B). In contrast, both sST2 and ST2L expression in fibroblasts were linked to the proximal promoter, either in untreated cells or following activation with serum, PMA, PDGF, or a combination of IL-17 and TNF. This was true for both primary tail-derived fibroblasts and 3T3 fibroblasts. No fibroblast expression was associated with the distal promoter, even though very low amounts of sST2 transcript could be detected in stimulated knockout fibroblasts samples (Fig. 2A and other data not shown), suggesting there may be additional sites of ST2 RNA initiation.

Interestingly, wild-type fibroblasts expressed both sST2 and ST2L (Fig. 2B). In order to determine if fibroblasts were responsive to IL-33, we measured the gene expression of a panel of inflammatory mediators following IL-33 treatment. As shown in Fig. 2C, IL-33 stimulation for 4 h resulted in induced expression of a selective set of chemokines and cytokines in wild type, but not promoter knockout tail fibroblasts (induction of CXCL1, CXCL10, and CCL2, but not CCL27, TGF-β1, or IL-18). This observation is consistent with another report describing IL-33 activity on fibroblasts [17] and, moreover, suggests that fibroblasts are a potential source of the neutrophil-attracting chemokine CXCL1, which is induced by IL-33 in vivo [18].

We next measured the production of sST2 protein from fibroblasts. Wild-type tail fibroblasts and 3T3 fibroblasts both secreted sST2 protein in response to stimulation with either serum, PMA or IL-33 (Fig. 2D and data not shown). In contrast, knockout fibroblasts produced no sST2 protein under any of the stimulation conditions tested. The proximal promoter is thus essential for sST2 protein secretion from fibroblasts. Knockout fibroblasts were able to produce the chemokine CXCL1 in response to IL-17+TNF treatment, indicating that these cells remain functionally responsive to stimulation and capable of secreting other factors (Fig. 2E). CXCL1 secretion could also be induced from wild-type fibroblasts by treatment with IL-33; however, promoter-deficient fibroblasts were completely nonresponsive, consistent with their lack of ST2L expression.

Production of sST2 protein in vivo

Our findings up to this point indicated that the proximal promoter and enhancer element are crucial for sST2 and ST2L expression by fibroblasts. Next, in order to determine to what extent fibroblasts contribute to sST2 production in vivo, we measured the

concentration of circulating sST2 in mice. As shown in Fig. 3A, serum contained roughly 5–7 ng/mL of sST2 protein regardless of whether it was collected from wild-type or knockout naïve animals, suggesting the proximal promoter is dispensable for steady-state sST2. Concentrations of sST2 have been shown to be increased in mice following challenge with an allergen [1] and we found that intranasal exposure of wild-type mice with house dust mite allergen (HDM) led to a dose-dependent increase in circulating sST2 after 48 h (Fig. 3B). Importantly, following a 10μg HDM exposure, sST2 was increased equivalently in wild type and promoter knockout mice (Fig. 3C), indicating that the proximal promoter is not required for the increase in sST2 in response to allergen challenge. Taken together, these findings imply that the proximal promoter and enhancer element are not crucial for the steady state or allergen-induced production of circulating sST2 protein.

image

Figure 3. Circulating sST2 concentrations in wild-type and knockout mice. (A) Concentrations of sST2 in the sera of wild-type and KO mice (n = 3). Data plotted as mean ± SEM with each symbol representing data from an individual mouse and are representative of two experiments. (B and C) Concentrations of sST2 in the sera of C57BL/six mice following intranasal administration of D. Farinae (HDM) allergen for 2 weeks ((B), titration of HDM, (n = 5/group), (C), mice treated with saline (n = 3) or 10 μg of HDM (n = 7)). Blood was collected 48 h following the final HDM administration. Data are plotted as mean ± 95% CI and are representative of two experiments; p-value was determined by unpaired t-test.

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Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Conflict of interest
  8. References

We conducted a novel genetic evaluation of the ST2 locus in mice by examining the effect of specifically deleting the proximal promoter and its associated enhancer element. Consistent with early work [6], we found that the two ST2 promoters are used preferentially in different cell types but that promoter usage is not linked to the generation of alternate ST2 transcripts. In mast cells the majority of both sST2 and ST2L expression was linked to the distal promoter, whereas in fibroblasts nearly all of the expression was directed by the proximal promoter. Although the specific mechanisms regulating promoter usage and splicing are not well understood, the general pattern of ST2 regulation appears to be conserved between rodents and humans. The intron-exon organization is preserved in humans and mice and GATA elements are associated with the distal promoters in both species. Moreover, like in the mouse, human hematopoetic cells predominantly use the distal promoter for expression of both sST2 and ST2L, while human fibroblasts almost exclusively use the serum-responsive proximal promoter [19, 20].

Ultimately, we are interested in improving our understanding of ST2 expression and the role both ST2L and sST2 play in IL-33 biology. We hypothesized that because the proximal promoter is associated with sST2 mRNA expression from abundant tissue cells such as fibroblasts, deletion of this promoter would significantly diminish sST2 protein production. This is not what we observed. In contrast, the absence of the proximal promoter did not decrease circulating sST2 concentrations, either in naïve or allergen-challenged mice. Although the cellular source of sST2 in the blood is still not known, these findings suggest fibroblasts are not a major source under the conditions tested. It remains possible, however, that fibroblasts and/or the proximal promoter and enhancer are important for sST2 induction in other physiological settings; this is something future studies with these mice may help reveal.

In the course of these experiments we also found that fibroblasts use the proximal promoter to express ST2L and are functionally responsive to IL-33, as demonstrated by the gene induction of the neutrophil-attracting CXCL1 and other chemokines. Examination of these mice in models of fibrosis could therefore be informative due to the central role of fibroblasts and recent evidence implicating IL-33 in fibrotic disease [21]. Finally, we hypothesize that there are other nonimmune cell types that require the proximal promoter for ST2L expression and that these mice may thus be useful for examining tissue-specific IL-33 responses in vivo.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Conflict of interest
  8. References

Mice

A targeting vector was constructed to delete a region in the ST2 locus beginning 4490 bp upstream of the +1 initiation site (ACGTGGGT) in exon 1b and ending at the 3′ end of exon 1b (83 bp downstream from the +1 site), as illustrated in Fig. 1A. The targeting construct was electroporated into 129×C57Bl/6 F1 hybrid ES cells and clones were then transfected with a CRE recombinase-expressing plasmid to delete the Neo cassette prior to injecting for germline transmission in C57Bl/6 mice using standard conditions.

Cell isolation and in vitro stimulation

For splenocytes, spleens were minced and single cell suspensions were collected through a nylon mesh. RBCs were lysed and cells were cultured for 3 h in RPMI with 10% FBS prior to RNA isolation. For mast cells, bone marrow cells were cultured in Iscove's Modified Dulbecco's Medium supplemented with 10% FBS, IL-3 (5 ng/mL, Amgen), and SCF (100 ng/mL, Amgen) at approximately 2–5 × 105 cells/mL. Every 3–4 days nonadherent cells were transferred to new flasks. Flow cytometry was performed after 5 weeks using antibodies to ST2 (MD Bioproducts, clone DJ8) and c-kit (CD117, BD Pharmingen, clone 2B8). BMMCs were cultured overnight at 105 cells/well with or without IL-33 (Amgen) and IL-6 was measured in the supernatant by ELISA (R&D Systems).

For fibroblasts, deboned tails from 12-week-old euthanized mice were minced in HBSS followed by digestion in a 1:1 solution of collagenase (Type XI-S Sigma in HBSS; 2000 U/mL) at 37°C for 30 min, and then 0.05% trypsin at 37°C for 20 min, followed by quenching (DMEM + 15% heat-inactivated calf serum). Cells were cultured in 10 cm plates for 5–7 days. For stimulations for RT-PCR and CXCL1 secretion assays, fibroblasts (or 3T3 cells) were seeded at 100,000 cells/mL in 24-well plates. After 24 h in low serum (0.5%) cells were stimulated with 10% FBS, 100 ng/mL PMA, 10 ng/mL PDGF, 10 ng/mL IL-17 + 0.5 ng/mL TNF-α, or 5 ng/mL IL-33 for 4 or 24 h. For the sST2 secretion assays fibroblasts were stimulated with PMA or 10% FBS as above for 2.5, 6, or 24 h.

PCR analysis

Total RNA was extracted from cells and cDNA was synthesized. The primers for PCR for promoter-independent expression included: ST2.E7: 5′-GATGTCCTGTGGCAGATTAACA-3′ and ST2.sol: 5′-TGGAAGACAGAAACATTCTGGA-3′ for soluble ST2 and ST2.E7 and ST2.FL: 5′-AGCAACCTCAATCCAGAACACT-3′ for full-length ST2. For the promoter-dependent analysis the isoform-specific primers ST2.sol and ST2.FL were used in combination with the promoter-specific primers ST2.proximal: 5′-GTAGCCTCACGGCTCTGAGC-3′ and ST2.distal: 5′-GATGGCTAGGACCTCTGGC-3′. Real-time PCR was conducted using custom Taqman Low Density Arrays (Applied Biosystems) and quantification was determined using the comparative Ct method.

HDM challenge

C57BL/6 (wild type) mice (9–11 weeks of age) received intranasal challenge with 50 μL of a saline solution containing designated amount of Dermatophagoides farinae HDM (Greer Labs, Lenoir, NC) on days 1, 3, 6, 8, 10, and 13. Serum was collected 48 h after the last challenge.

Serum analyte analysis

Blood was collected via the axillary artery and stored in serum separator tubes (BD, Franklin Lakes, NJ). Soluble ST2 and CXCL1 were measured using ELISA assays (R&D Systems).

Statistical analysis

Prism (GraphPad Software) was used for all statistical analyses, as described in the figure legends.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Conflict of interest
  8. References
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Abbreviations
BMMC

bone marrow-derived mast cell

HDM

house dust mite allergen