Figure 4.
Figure 4. Human sIL-1ra is encoded in a direct PPARβ/δ target gene in fibroblasts. (A) Expression of sIL-1ra and icIL-1ra mRNA (left) and protein (right) in OTC keratinocytes (KCTRL and KPPARβ/δ) and fibroblasts (FCRTL and FPPARβ/δ). sIL-1ra and icIL-1ra mRNA were analyzed by qPCR and normalized to ribosomal protein P0. The sIL-1ra level was determined by ELISA from medium of KCTRL/FCTRL and KCTRL/FPPARβ/δ OTCs. The icIL-1ra levels were measured by ELISA from cell lysates. (B) PPRE1 and PPRE3 of the human sIL-1ra gene are functional. Transactivation assay in fibroblasts cotransfected with a luciferase (luc) promoter driven by the human sIL-1ra promoter and pEF1–β-galactosidase as control of transfection efficiency. Relative positions of the three putative PPREs (PPRE 1–3) and their mutants (mPPRE 1–3) are represented in closed and open ovals, respectively. Cells were treated with either 500 nM GW501516 (GW) and/or 10 ng/ml IL-1β for 24 h. Luciferase activity was measured, and normalized reporter activity is shown as fold induction as compared with untreated fibroblasts. (C) EMSA of human sIL-1ra PPRE1 and PPRE3. Radiolabeled PPRE1 (left) and PPRE3 (middle) were incubated either with RXRα, PPARβ/δ, or both. NSC denotes nonspecific competitor, the nonfunctional MEd DR1 element in the malic enzyme promoter. SC denotes nonradiolabeled consensus PPRE (conPPRE). As positive control, conPPRE was used. Mutated consensus PPRE is denoted by mconPPRE. PPARβ/δ did not bind to PPRE2 and mutated PPRE probes (mconPPRE, mPPRE1, mPPRE2, and mPPRE3; right). (D) PPARβ/δ binds to PPRE1 and PPRE3 of the human sIL-1ra gene in fibroblasts. ChIP assays were conducted using preimmune IgG or antibodies against PPARβ/δ (AB) in FCTRL (WT) and FPPARβ/δ (kd) fibroblasts extracted from two independent OTCs (OTC1 and OTC2). The regions spanning PPRE1 and PPRE2 of the sIL-1ra gene were amplified using appropriate primers (Table S1). A control region between PPRE1 and PPRE2 served as negative control. *, P < 0.05; **, P < 0.01. Data are mean ± SEM, n = 4.

Human sIL-1ra is encoded in a direct PPARβ/δ target gene in fibroblasts. (A) Expression of sIL-1ra and icIL-1ra mRNA (left) and protein (right) in OTC keratinocytes (KCTRL and KPPARβ/δ) and fibroblasts (FCRTL and FPPARβ/δ). sIL-1ra and icIL-1ra mRNA were analyzed by qPCR and normalized to ribosomal protein P0. The sIL-1ra level was determined by ELISA from medium of KCTRL/FCTRL and KCTRL/FPPARβ/δ OTCs. The icIL-1ra levels were measured by ELISA from cell lysates. (B) PPRE1 and PPRE3 of the human sIL-1ra gene are functional. Transactivation assay in fibroblasts cotransfected with a luciferase (luc) promoter driven by the human sIL-1ra promoter and pEF1–β-galactosidase as control of transfection efficiency. Relative positions of the three putative PPREs (PPRE 1–3) and their mutants (mPPRE 1–3) are represented in closed and open ovals, respectively. Cells were treated with either 500 nM GW501516 (GW) and/or 10 ng/ml IL-1β for 24 h. Luciferase activity was measured, and normalized reporter activity is shown as fold induction as compared with untreated fibroblasts. (C) EMSA of human sIL-1ra PPRE1 and PPRE3. Radiolabeled PPRE1 (left) and PPRE3 (middle) were incubated either with RXRα, PPARβ/δ, or both. NSC denotes nonspecific competitor, the nonfunctional MEd DR1 element in the malic enzyme promoter. SC denotes nonradiolabeled consensus PPRE (conPPRE). As positive control, conPPRE was used. Mutated consensus PPRE is denoted by mconPPRE. PPARβ/δ did not bind to PPRE2 and mutated PPRE probes (mconPPRE, mPPRE1, mPPRE2, and mPPRE3; right). (D) PPARβ/δ binds to PPRE1 and PPRE3 of the human sIL-1ra gene in fibroblasts. ChIP assays were conducted using preimmune IgG or antibodies against PPARβ/δ (AB) in FCTRL (WT) and FPPARβ/δ (kd) fibroblasts extracted from two independent OTCs (OTC1 and OTC2). The regions spanning PPRE1 and PPRE2 of the sIL-1ra gene were amplified using appropriate primers (Table S1). A control region between PPRE1 and PPRE2 served as negative control. *, P < 0.05; **, P < 0.01. Data are mean ± SEM, n = 4.

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