A novel distal enhancer mediates cytokine induction of mouse RANKl gene expression

Abstract

Chronic inflammatory states are associated with increased bone loss. This increase is often linked to an elevation in receptor activator of nuclear factor-kappaB ligand (RANKL), a TNFalpha-like factor essential to osteoclast formation. In this study, we document the ability of IL-6 in combination with IL-6 soluble receptor (IL-6/IL-6sR) and oncostatin M to induce Rankl expression in stromal cells via signal transducer and activator of transcription 3 (STAT3). We used chromatin immunoprecipitation-tiled DNA microarray analysis to determine sites of action of STAT3 at the Rankl locus and to assess the consequences of binding on histone H4 acetylation and RNA polymerase II recruitment. Both IL-6/IL-6 soluble receptor and oncostatin M stimulated STAT3 binding upstream of the Rankl transcriptional start site. Although previously identified enhancers bound STAT3, a more distal enhancer termed mRLD6 was a particular focus of STAT3 binding. When fused to a heterologous promoter, this enhancer was highly active, containing two functionally active STAT response elements. Importantly, small interfering RNA knockdown of Stat3 mRNA and protein, but not that of Stat1 or Stat5a, was effective in limiting Rankl mRNA up-regulation. Interestingly, although RNA polymerase II and histone H4 acetylation marked many of the enhancers under basal conditions, the levels of both were strongly increased after cytokine treatment, particularly at mRLD6. Finally, mRLD6 was also a target for forskolin-induced cellular response element-binding protein (CREB) recruitment, which potentiated cytokine activity. Our studies provide new insight into mechanisms by which glycoprotein 130 activating cytokines induce RANKL expression.

Figure 1

Induction of Rankl mRNA levels in ST2 cells is enhanced by OSM or IL-6/IL-6sR. A, Induction of Rankl transcript levels by OSM in vitro. ST2 cells were treated with OSM concentrations ranging from 0.5–20 ng/ml for 6 h. B, Induction of Rankl transcript levels by IL-6/IL-6sR in vitro. ST2 cells were treated with 50 ng/ml IL-6 or 100 ng/ml IL-6sR or cotreated with 100 ng/ml IL-6sR and increasing concentrations of IL-6 ranging from 5–50 ng/ml for 6 h. Total RNA was isolated, reverse transcribed, and analyzed by quantitative PCR using primers specific to Rankl and β-actin. Rankl transcript levels were normalized to β-actin. Each value represents the average of three independent experiments ± sem compared with control samples using a one-way nonparametric ANOVA analysis with single variance followed by a Tukey multiple-comparison post-test. *, P < 0.05.

Figure 2

ChIP-chip analysis reveals localization of pSTAT3 to the Rankl gene locus in response to the gp130 cytokines OSM and IL-6/IL-6sR. ST2 cells were treated with 20 ng/ml OSM or 50 ng/ml IL-6 and 100 ng/ml IL-6sR (IL-6/R) for 6 h and then subjected to ChIP analysis using antibodies to pSTAT3-Tyr705 or IgG. Immunoprecipitated DNA was amplified by ligation-mediated PCR, labeled with Cy3 or Cy5, and cohybridized to custom DNA microarrays as described in Materials and Methods. The upper panel depicts the Rankl gene locus. Nucleotide positions (Mb) are shown on chromosome 14 (chr14; February 2006 assembly), whereas the Rankl gene and the positions of the distal enhancer regions are indicated below the chromosomal position and designated by descending gray bands. A, Interaction of pSTAT3 with the Rankl gene locus under basal conditions. The data track represents the log₂ ratios of fluorescence obtained from a vehicle-treated (Veh) sample precipitated with antibodies to either pSTAT3 or IgG (pSTAT3_veh vs. IgG) (basal pSTAT3). B, Interaction of pSTAT3 with the Rankl gene locus after treatment with OSM. The data tracks represent the log₂ ratios of fluorescence obtained from an OSM-treated sample precipitated with antibodies to either pSTAT3 or IgG (pSTAT3_OSM vs. IgG) (total pSTAT3) or samples treated with either vehicle or OSM and precipitated with an antibody to pSTAT3 (pSTAT3_OSM vs. pSTAT3_veh) (net inducible pSTAT3). C, Interaction of pSTAT3 with the Rankl gene locus in response to IL-6/IL-6sR. Data tracks represent the log₂ ratios of fluorescence as in B with IL-6/IL-6sR as the inducer. All peaks highlighted in red represent statistically significant peaks [false discovery rate (FDR), P < 0.05].

Figure 3

ChIP-chip analysis reveals the presence and recruitment of RNA pol II at the Rankl gene locus in response to the gp130-activating cytokines OSM and IL-6. ST2 cells were treated with 20 ng/ml OSM or 50 ng/ml IL-6 and 100 ng/ml IL-6sR (IL-6/R) for 6 h and then subjected to ChIP analysis using antibodies to RNA pol II or IgG. Samples were prepared as described in Materials and Methods. The upper panel depicts the Rankl gene locus as in Fig. 2. A, Interaction of RNA pol II with the Rankl gene locus under basal conditions. The data track represents the log₂ ratios of fluorescence obtained from a vehicle-treated (Veh) sample precipitated with antibodies to either RNA pol II or IgG (RNA pol II_veh vs. IgG) (basal RNA pol II). B, Interaction of RNA pol II with the Rankl gene locus after treatment with OSM. The data tracks represent the log₂ ratios of fluorescence obtained from an OSM-treated sample precipitated with antibodies to either RNA pol II or IgG (RNA pol II_OSM vs. IgG) (total RNA pol II) or samples treated with either vehicle or OSM and precipitated with an antibody to RNA pol II (RNA pol II_OSM vs. RNA pol II_veh) (net inducible RNA pol II). C, Interaction of RNA pol II with the Rankl gene locus in response to IL-6/IL-6sR. Data tracks represent the log₂ ratios of fluorescence as in B with IL-6/IL-6sR as the inducer. All regions highlighted in red represent statistically significant peaks [false discovery rate (FDR), P < 0.05].

Figure 4

ChIP-chip analysis reveals basal and inducible levels of H4ac at the Rankl gene locus in response to the gp130-activating cytokines OSM and IL-6/IL-6sR. ST2 cells were treated with 20 ng/ml OSM or 50 ng/ml IL-6 and 100 ng/ml IL-6sR (IL-6/R) for 6 h and then subjected to ChIP analysis using antibodies to H4ac or IgG. A, Levels of H4ac with the Rankl gene locus under basal conditions. The data track represents the log₂ ratios of fluorescence obtained from a vehicle-treated sample precipitated with antibodies to either H4ac or IgG (H4ac_veh vs. IgG) (basal H4ac). B, the Rankl gene locus after treatment with OSM. The data tracks represent the log₂ ratios of fluorescence obtained from an OSM-treated sample precipitated with antibodies to either H4ac or IgG (H4ac_OSM vs. IgG) (total H4ac) or samples treated with either vehicle or OSM and precipitated with an antibody to H4ac (H4ac_OSM vs. H4ac_veh) (net inducible H4ac). C, Interaction of H4ac with the Rankl gene locus in response to IL-6/IL-6sR. Data tracks represent the log₂ ratios of fluorescence as in B with IL-6 as the inducer. All regions highlighted in red represent statistically significant peaks [false discovery rate (FDR), P < 0.05].

Figure 5

The mRLD6 enhancer region together with the D5a region are transcriptionally active in response to OSM. ST2 cells were transfected with pTK-luc control or the indicated Rankl enhancer region reporter constructs (250 ng) and pCH110-βgal (50 ng) in triplicate. Cells were treated for 20 h with 0.5–20 ng/ml OSM and harvested, and both luciferase activity and β-galactosidase activity were measured. Luciferase activity was normalized to β-galactosidase activity. Each value represents the average of three independent experiments ± sem compared with control samples using a one-way nonparametric ANOVA analysis with single variance followed by a Tukey multiple-comparison post-test. *, P < 0.05.

Figure 6

The gp130 cytokine regulation of the mRLD6 enhancer region is mediated by synergistic STAT-binding elements. A, Conserved regions of mRLD6. University of California, Santa Cruz Genome Browser (http://genome.uscs.edu)-derived conservation plot of the Rankl mRLD6 enhancer region (February 2006 assembly) shows location and size of the cloned mRLD6 fragments. B, Transcriptional activity of mRLD6 subfragments. The pTK-luc vector, pTK-mRLD6, pTK-mRLD6 proximal, and pTK-mRLD6 distal reporter constructs (250 ng) were transfected into ST2 cells with pCH110-βgal (50 ng). Cells were treated for 20 h with 20 ng/ml OSM or 50 ng/ml IL-6 and 100 ng/ml IL-6sR, and luciferase activity was assessed and normalized as described in Materials and Methods. C, Conservation of STAT response elements and identification of mutations. Conservation comparison of putative STAT-binding elements located in the mRLD6 region of several species is depicted using the TFSearch algorithm (36). The mutations shown were introduced into the pTK-luc mRLD6 distal enhancer region using site-directed mutagenesis. D and E, STAT3-mediated cytokine activity is abrogated upon mutation of the STAT-binding elements. The pTK-luc vector, pTKmRLD6 distal, pTKmRLD6 distal mut1, pTKmRLD6 distal mut2, or pTKmRLD6 distal mut1/2 reporter constructs (250 ng) were transfected into ST2 cells with pCH110-βgal (50 ng). Cells were treated with 20 ng/ml OSM (D), 50 ng/ml IL-6, or 100 ng/ml IL-6sR or cotreated with 100 ng/ml IL-6sR with IL-6 concentrations ranging from 5–50 ng/ml (E). Cells were harvested after 20 h, and both luciferase activity and β-galactosidase activity were measured. Luciferase activity was normalized to β-galactosidase activity. Each value represents the average of three independent experiments ± sem compared with control samples using a one-way nonparametric ANOVA analysis with single variance followed by a Tukey multiple-comparison post-test. *, P < 0.05. chr14, Chromosome 14.

Figure 6

The gp130 cytokine regulation of the mRLD6 enhancer region is mediated by synergistic STAT-binding elements. A, Conserved regions of mRLD6. University of California, Santa Cruz Genome Browser (http://genome.uscs.edu)-derived conservation plot of the Rankl mRLD6 enhancer region (February 2006 assembly) shows location and size of the cloned mRLD6 fragments. B, Transcriptional activity of mRLD6 subfragments. The pTK-luc vector, pTK-mRLD6, pTK-mRLD6 proximal, and pTK-mRLD6 distal reporter constructs (250 ng) were transfected into ST2 cells with pCH110-βgal (50 ng). Cells were treated for 20 h with 20 ng/ml OSM or 50 ng/ml IL-6 and 100 ng/ml IL-6sR, and luciferase activity was assessed and normalized as described in Materials and Methods. C, Conservation of STAT response elements and identification of mutations. Conservation comparison of putative STAT-binding elements located in the mRLD6 region of several species is depicted using the TFSearch algorithm (36). The mutations shown were introduced into the pTK-luc mRLD6 distal enhancer region using site-directed mutagenesis. D and E, STAT3-mediated cytokine activity is abrogated upon mutation of the STAT-binding elements. The pTK-luc vector, pTKmRLD6 distal, pTKmRLD6 distal mut1, pTKmRLD6 distal mut2, or pTKmRLD6 distal mut1/2 reporter constructs (250 ng) were transfected into ST2 cells with pCH110-βgal (50 ng). Cells were treated with 20 ng/ml OSM (D), 50 ng/ml IL-6, or 100 ng/ml IL-6sR or cotreated with 100 ng/ml IL-6sR with IL-6 concentrations ranging from 5–50 ng/ml (E). Cells were harvested after 20 h, and both luciferase activity and β-galactosidase activity were measured. Luciferase activity was normalized to β-galactosidase activity. Each value represents the average of three independent experiments ± sem compared with control samples using a one-way nonparametric ANOVA analysis with single variance followed by a Tukey multiple-comparison post-test. *, P < 0.05. chr14, Chromosome 14.

Figure 7

STAT3 knockdown eliminates OSM-induced Rankl expression via the mRLD6 enhancer region. A, Stat3 knockdown eliminates OSM-induced Rankl transcription. ST2 cells were transfected with 40 nm siRNA as indicated, incubated for 48 h, and then treated with 20 ng/ml OSM for an additional 6 h. RNA was isolated, reverse transcribed, and analyzed by quantitative PCR for Rankl expression. Levels were normalized to β-actin. B, Stat3 knockdown eliminates OSM-induced pTK-luc mRLD6 reporter activity. The pTK-luc mRLD6 distal reporter construct (250 ng) and pCH110-βgal (50 ng) were transfected into ST2 cells in the presence of cyclophilin B (CycloB), Stat1, Stat3, or Stat5a siRNA (40 nm). After a 48-h preincubation, cells were treated overnight with 20 ng/ml OSM or 50 ng/ml IL-6 and 100 ng/ml IL-6sR. Cells were harvested, and both luciferase activity and β-galactosidase activity were measured. Luciferase activity was normalized to β-galactosidase activity. Each value represents the average of three independent experiments ± sem compared with cyclophilin B control samples using a nonparametric t test. *, P < 0.05.

Figure 8

CREB is recruited to the mRLD6 enhancer region and potentiates cytokine-induced reporter activity. A, ChIP-chip analysis reveals the localization of CREB to the Rankl gene locus in response to forskolin. ST2 cells were treated with forskolin (Fsk) (10⁻⁶ m) for 6 h and then subject to ChIP analysis using antibodies to CREB or IgG. Samples were prepared as described in Materials and Methods. The upper panel depicts the Rankl gene locus as in Fig. 2. Data tracks represent the log₂ ratios of fluorescence obtained from 1) a vehicle-treated sample precipitated with antibodies to either CREB or IgG (CREB_veh vs. IgG) (basal CREB), 2) a forskolin-treated sample precipitated with antibodies to either CREB or IgG (CREB_OSM vs. IgG) (total CREB), and 3) forskolin-treated and vehicle-treated samples precipitated with antibody to CREB (CREB_OSM vs. CREB_veh) (net inducible CREB). All regions highlighted in red represent statistically significant peaks [false discovery rate (FDR), P < 0.05]. B, Forskolin and OSM cotreatment potentiates activity of the mRLD6 enhancer. The pTK-luc vector, pTK-luc mRLD6, pTK-luc mRLD6 distal, pTK-luc mRLD6 mut1, or pTK-luc mRLD6 mut2 reporter constructs (250 ng) and pCH110-βgal (50 ng) were transfected into ST2 cells and the cells treated with 20 ng/ml OSM, Fsk (10⁻⁶m), or both for 20 h. Cells were then harvested, and both luciferase activity and β-galactosidase activity were measured. Luciferase activity was normalized to β-galactosidase activity. Each value represents the average of three independent experiments ± SEM compared with control samples using a nonparametric T test. *, P < 0.05.