Oncostatin M-Induced and Constitutive Activation of the JAK2/STAT5/CIS Pathway Suppresses CCL1, but Not CCL7 and CCL8, Chemokine Expression

Christoph Hintzen, Claude Haan, Jan P. Tuckermann, Peter C. Heinrich and Heike M. Hermanns
J Immunol November 15, 2008, 181 (10) 7341-7349; DOI: https://doi.org/10.4049/jimmunol.181.10.7341

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    OSM-induced expression of CCL1, CCL7, and CCL8. A, The mRNA levels of CCL1, CCL7, and CCL8 in HDFs treated with 20 ng/ml OSM are determined by RNase protection assays and one representative of three independent experiments is shown. B, RNase protection assay results were quantified by phosphoimager, the relative mRNA levels were normalized to those of GAPDH and expressed as fold induction relative to unstimulated cells. The values shown are mean ± SD (n = 3). ∗, p < 0.05 vs control. C, Protein amounts of CCL1 and CCL8 in culture supernatants of OSM-treated HDFs (20 ng/ml) were determined by ELISA. Data are expressed as mean ± SD (n = 4). ∗, p < 0.05 vs control. D, Protein amounts of CCL1 and CCL8 in culture supernatants of OSM- (20 ng/ml), IL-1β- (10 ng/ml) or TNF-α- (10 ng/ml) treated HDFs (1 and 24 h) were determined by ELISA. Data are expressed as mean ± SD (n = 3). ∗, p < 0.05 vs control.

  • FIGURE 2.
    FIGURE 2.

    OSM-induced CCL1 and CCL8 expression mediates migration of primary monocytes and T lymphocytes. A and B, Culture supernatants of OSM-treated HDFs (20 ng/ml, 3 h) were used to analyze the potential of secreted chemokines to induce migration in a modified Boyden chamber assay as described in Materials and Methods. The numbers of migrated monocytes or T lymphocytes were determined and compared with controls (untreated DMEM). Values shown are mean ± SD (n = 3). ∗, p < 0.05 vs control; d, p < 0.05 vs Ab control.

  • FIGURE 3.
    FIGURE 3.

    OSM-induced CCL1 and CCL8 expression is dependent on MAPK signaling. A, HDFs were pretreated with 10 μM SB202190, 10 μM U0126, or 10 μM SP600125 for 30 min, exposed to 20 ng/ml OSM for 3 h and secreted protein amounts of CCL1 or CCL8 were measured by ELISA from culture supernatants. Data are expressed as mean ± SD (n = 5). ∗, p < 0.05 vs control. B, HDFs were stimulated with 20 ng/ml OSM for 30 min. Subsequently, cells were washed and cultivated for additional 20 min in OSM-free medium containing actinomycin D (4 μM) to block transcription. Afterward, 10 μM SB202190 or 10 μM U0126 were added to the medium before the cells were finally stimulated a second time with OSM for the indicated time periods. CCL1 mRNA and GAPDH mRNA levels were analyzed by RT-PCR. One representative of three independent experiments is shown. C, HDFs were transfected with TTP siRNA as described in Materials and Methods. Forty-eight hours after transfection, cells were pretreated with 10 μM SB202190 for 20 min and subsequently stimulated with 20 ng/ml OSM for 3 h. The amounts of CCL1 or CCL8 in culture supernatants were measured by ELISA. Data are expressed as mean ± SD (n = 3). ∗, p < 0.05 vs control. D, HDFs were transfected with control, c-Jun, or c-Fos siRNA, respectively. The amounts of CCL1 or CCL8 in culture supernatants were measured by ELISA. Data are expressed as mean ± SD (n = 4). ∗, p < 0.05 vs control. To analyze knock-down efficiency whole cellular lysates were subjected to Western blot analysis using specific Abs against c-Fos and c-Jun. Tubulin expression served as loading control.

  • FIGURE 4.
    FIGURE 4.

    CCL1 and TCA-3 expression are negatively regulated by STAT5 activation. A, Western blot detection of whole cellular extracts of siRNA transfected HDFs, treated with OSM (20 ng/ml, 3 h), using specific antisera against phospho-STAT1, -STAT3, or -STAT5. Subsequently the blots were stripped and reprobed with antisera recognizing proteins irrespectively of their activation. B, Amounts of CCL1 or CCL8 in culture supernatants of transfected HDFs were measured by ELISA. Data are expressed as mean ± SD (n = 5). ∗, p < 0.05 vs control. C, MEF cells were treated with 20 ng/ml murine OSM for the indicated time periods. The mRNA levels of TCA-3 were determined by RNase protection assays and a representative experiment of three independent experiments is shown. RNase protection assay results were quantified and the relative levels of TCA-3 following OSM stimulation were normalized to those of GAPDH and compared with unstimulated cells. The values shown are mean ± SD (n = 3). ∗, p < 0.05 vs control. D, ELISA to detect protein amounts of TCA-3 in MEF cells treated with murine OSM (20 ng/ml) for the indicated time periods.

  • FIGURE 5.
    FIGURE 5.

    The STAT5-dependent negative regulation of CCL1 expression is mediated by CIS. A, Western blot detection of whole cellular extracts of MEFs, treated with OSM (20 ng/ml for the indicated time periods), using Abs specific for phosphorylated STAT1, STAT3, STAT5, ERK1/2, and p38. The blots were stripped and reprobed with antisera recognizing the proteins irrespectively of their activation. B, RT-PCR to detect SOCS1, SOCS3, and CIS mRNA levels in MEFs stimulated with 20 ng/ml murine OSM for 1 h. One representative of three independent experiments is shown. C, HDFs were transfected with CIS siRNA and then stimulated with 20 ng/ml human OSM for 3 h. The protein amounts of CCL1 or CCL8 in culture supernatants were measured by ELISA. Data are expressed as mean ± SD (n = 4). ∗, p < 0.05 vs control. D, MEF cells were transiently transfected with expression vectors for muCIS or control vector and stimulated with mOSM as indicated. The protein amounts of TCA-3 in culture supernatants were measured by ELISA. Data are expressed as mean ± SD (n = 3). ∗, p < 0.05 vs MOCK-transfected control.

  • FIGURE 6.
    FIGURE 6.

    The JAK2 V617F mutation leads to CCL1 suppression. A, HELs were left untreated or incubated with 1 μM JAK inhibitor-1 (JI-1) or the solvent DMSO for 12 h. Lysates were prepared and subjected to Western blot analysis using Abs specific for phosphorylated JAK2, STAT5, and Erk1/2. The blots were stripped and reprobed with antisera recognizing the proteins irrespectively of their activation. B, HELs were preincubated with 1 μM JAK inhibitor-1 (JI-1) for 12 h and then stimulated with 10 ng/ml IL-1β or 20 ng/ml OSM for 1 h. RNA was isolated and RT-PCR used to detect CIS mRNA. C, Primary monocytes and HEL cells were stimulated with 10 ng/ml IL-1β for 3 h. The protein amounts of CCL1 in culture supernatants were measured by ELISA. Data are expressed as mean ± SD (n = 4). ∗, p < 0.05 vs control. D, HELs were preincubated with 1 μM JI-1 for 12 h and then stimulated 10 ng/ml IL-1β or 20 ng/ml OSM for 3 h. The protein amounts of CCL1 in culture supernatants were measured by ELISA. Data are expressed as mean ± SD (n = 3). ∗, p < 0.05 vs control. E, HELs were transfected with control or CIS siRNA and stimulated with 10 ng/ml IL-1β or 20 ng/ml OSM for 3 h. The protein amounts of CCL1 in culture supernatants were measured by ELISA. Data are expressed as mean ± SD (n = 3). ∗, p < 0.05 vs control. To analyze knock-down efficiency RT-PCR was used to detect CIS mRNA levels.

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