Journal: Endocrinology

Article Title: Leptin enhances human beta-defensin-2 production in human keratinocytes.

doi: 10.1210/en.2008-0343

Figure Lengend Snippet: FIG. 1. mRNA expression of the leptin receptor Ob-Rb in keratino- cytes (KC) incubated for 12 h with or without IL-1 (1 ng/ml). The cDNA of human peripheral blood mononuclear cells (PBMC) was used as the positive control for Ob-Rb. The results shown are representa- tive of four separate experiments.

Article Snippet: Recombinant human leptin, human leptin receptor/Fc chimera, which is composed of the leptin receptor extracellular domain fused to human IgG1 Fc, and control human IgG1 Fc were purchased from R&D Systems (Minneapolis, MN). c-Jun N-terminal kinase (JNK) inhibitor SP600125 was purchased from Biomol (Plymouth Meeting, MA). p38 MAPK inhibitor SB202190, MAPK/ERK kinase (MEK) inhibitor PD98059, and Janus kinase (JAK) 2 inhibitor AG490 were from Calbiochem (La Jolla, CA).

Techniques: Expressing, Incubation, Positive Control

Journal: Endocrinology

Article Title: Leptin enhances human beta-defensin-2 production in human keratinocytes.

doi: 10.1210/en.2008-0343

Figure Lengend Snippet: FIG. 2. Stimulation of IL-1-induced hBD-2 secretion by leptin (A and B) and mRNA expression (C). A, Keratinocytes were incubated with either the medium alone or with IL-1 (1 ng/ml) in the presence of the indicated concentrations of leptin. At 48 h, the amount of hBD-2 secreted was analyzed. The data in A are presented as the mean SEM of triplicate cultures and represent the data from four experiments. *, P 0.05 vs. the controls; †, P 0.05 vs. IL-1 alone. B and C, Keratinocytes were preincubated with 1 g/ml of the leptin receptor/ IgG1 Fc chimera (LR) or control human IgG1 Fc (Fc) for 30 min and were then incubated with 1 ng/ml IL-1 in the presence or absence of 10 ng/ml leptin. At 48 h, hBD-2 secretion was analyzed (B), whereas at 12 h, the hBD-2 mRNA levels were analyzed by real-time RT-PCR (C). The data in B and C are presented as the mean SEM (n 4). *, P 0.05 vs. the controls; †, P 0.05 vs. IL-1 alone; ‡, P 0.05 vs. IL-1 plus leptin. In C, the hBD-2 mRNA levels were normalized to the mRNA levels of GAPDH; these are represented as fold induction relative to control keratinocytes treated with medium alone.

Article Snippet: Recombinant human leptin, human leptin receptor/Fc chimera, which is composed of the leptin receptor extracellular domain fused to human IgG1 Fc, and control human IgG1 Fc were purchased from R&D Systems (Minneapolis, MN). c-Jun N-terminal kinase (JNK) inhibitor SP600125 was purchased from Biomol (Plymouth Meeting, MA). p38 MAPK inhibitor SB202190, MAPK/ERK kinase (MEK) inhibitor PD98059, and Janus kinase (JAK) 2 inhibitor AG490 were from Calbiochem (La Jolla, CA).

Techniques: Expressing, Incubation, Control, Quantitative RT-PCR

Journal: Endocrinology

Article Title: Leptin enhances human beta-defensin-2 production in human keratinocytes.

doi: 10.1210/en.2008-0343

Figure Lengend Snippet: FIG. 3. The effects of antisense oligonucleotides on hBD-2 secretion (A) and mRNA expression (B). Keratinocytes were transfected with antisense oligonucleotides (ASO) against transcription fac- tors or control scrambled oligonucleotides (Con) (0.2 M each) and were treated with IL-1 (1 ng/ ml) in the presence or absence of leptin (10 ng/ml). At 48 h, the amount of hBD-2 secreted was ana- lyzed (A), whereas at 12 h, the mRNA levels were analyzed by real-time RT-PCR (B). *, P 0.05 vs. the controls; †, P 0.05 vs. IL-1; ‡, P 0.05 vs. IL-1 plus leptin. The data are presented as the mean SEM (n 4). In B, the hBD-2 mRNA levels were normalized to the mRNA levels of GAPDH; these are presented as fold induction.

Article Snippet: Recombinant human leptin, human leptin receptor/Fc chimera, which is composed of the leptin receptor extracellular domain fused to human IgG1 Fc, and control human IgG1 Fc were purchased from R&D Systems (Minneapolis, MN). c-Jun N-terminal kinase (JNK) inhibitor SP600125 was purchased from Biomol (Plymouth Meeting, MA). p38 MAPK inhibitor SB202190, MAPK/ERK kinase (MEK) inhibitor PD98059, and Janus kinase (JAK) 2 inhibitor AG490 were from Calbiochem (La Jolla, CA).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR

Journal: Endocrinology

Article Title: Leptin enhances human beta-defensin-2 production in human keratinocytes.

doi: 10.1210/en.2008-0343

Figure Lengend Snippet: FIG. 4. Stimulation of the basal and IL-1-induced transcriptional activities of NF-B (A), STAT1 (B), and STAT3 (C) by leptin. Kera- tinocytes transfected with pRL-tk and pNF-B-luc, pGAS-luc, or pSTAT3-luc were incubated with IL-1 (1 ng/ml) in the presence or absence of the indicated concentrations of leptin. At 18 h, the tran- scriptional activities were analyzed. *, P 0.05 vs. the controls with the medium alone. The data are represented as the mean SEM (n 4).

Article Snippet: Recombinant human leptin, human leptin receptor/Fc chimera, which is composed of the leptin receptor extracellular domain fused to human IgG1 Fc, and control human IgG1 Fc were purchased from R&D Systems (Minneapolis, MN). c-Jun N-terminal kinase (JNK) inhibitor SP600125 was purchased from Biomol (Plymouth Meeting, MA). p38 MAPK inhibitor SB202190, MAPK/ERK kinase (MEK) inhibitor PD98059, and Janus kinase (JAK) 2 inhibitor AG490 were from Calbiochem (La Jolla, CA).

Techniques: Transfection, Incubation

Journal: Endocrinology

Article Title: Leptin enhances human beta-defensin-2 production in human keratinocytes.

doi: 10.1210/en.2008-0343

Figure Lengend Snippet: FIG. 5. The effects of signal inhibitors on IL-1- and/or leptin-induced hBD-2 secretion (A) and mRNA expression (B). Keratinocytes were preincubated for 30 min with 25 M AG490 (AG), 10 M PD98059 (PD), 1 M SB202190 (SB), or 10 M SP600125 (SP), and were then incubated with 1 ng/ml IL-1 in the pres- ence or absence of 10 ng/ml leptin. At 48 h, hBD-2 secretion was analyzed (A), whereas at 12 h, hBD-2 mRNA levels were analyzed by real-time RT-PCR (B). The data represent the mean SEM (n 4). In B, hBD-2 mRNA levels were nor- malized to the mRNA levels of GAPDH and are shown as a fold induction. *, P 0.05 vs. the controls; †, P 0.05 vs. IL-1 alone; ‡, P 0.05 vs. IL-1 plus leptin.

Article Snippet: Recombinant human leptin, human leptin receptor/Fc chimera, which is composed of the leptin receptor extracellular domain fused to human IgG1 Fc, and control human IgG1 Fc were purchased from R&D Systems (Minneapolis, MN). c-Jun N-terminal kinase (JNK) inhibitor SP600125 was purchased from Biomol (Plymouth Meeting, MA). p38 MAPK inhibitor SB202190, MAPK/ERK kinase (MEK) inhibitor PD98059, and Janus kinase (JAK) 2 inhibitor AG490 were from Calbiochem (La Jolla, CA).

Techniques: Expressing, Incubation, Quantitative RT-PCR

Journal: Endocrinology

Article Title: Leptin enhances human beta-defensin-2 production in human keratinocytes.

doi: 10.1210/en.2008-0343

Figure Lengend Snippet: FIG. 7. IL-1 and/or leptin-induced phosphorylation of IB (A), ty- rosine- or serine-phosphorylation of STAT1 (B) and STAT3 (C). Ker- atinocytes were preincubated for 30 min with 25 M AG490 (AG) or 1 M SB202190 (SB), and were then treated with IL-1 (1 ng/ml) for 5 min in the presence or absence of leptin (10 ng/ml) to analyze the phosphorylation by Western blotting. The results represent the data from four separate experiments.

Article Snippet: Recombinant human leptin, human leptin receptor/Fc chimera, which is composed of the leptin receptor extracellular domain fused to human IgG1 Fc, and control human IgG1 Fc were purchased from R&D Systems (Minneapolis, MN). c-Jun N-terminal kinase (JNK) inhibitor SP600125 was purchased from Biomol (Plymouth Meeting, MA). p38 MAPK inhibitor SB202190, MAPK/ERK kinase (MEK) inhibitor PD98059, and Janus kinase (JAK) 2 inhibitor AG490 were from Calbiochem (La Jolla, CA).

Techniques: Phospho-proteomics, Western Blot

Journal: Endocrinology

Article Title: Leptin enhances human beta-defensin-2 production in human keratinocytes.

doi: 10.1210/en.2008-0343

Figure Lengend Snippet: FIG. 6. The effects of signal inhibitors on the activities of NF-B (A), STAT1 (B), and STAT3 (C). Keratinocytes transfected with pRL-tk and pNF-B-luc, pGAS-luc, or pSTAT3-luc were preincubated for 30 min with 25 M AG490 (AG) or 1 M SB202190 (SB), and were then incubated with 1 ng/ml IL-1 in the presence or absence of 10 ng/ml leptin. At 18 h, transcriptional activities were analyzed. *, P 0.05 vs. the controls; †, P 0.05 vs. IL-1 alone; ‡, P 0.05 vs. leptin alone; §, P 0.05 vs. IL-1 plus leptin. The data represent the mean SEM (n 4).

Article Snippet: Recombinant human leptin, human leptin receptor/Fc chimera, which is composed of the leptin receptor extracellular domain fused to human IgG1 Fc, and control human IgG1 Fc were purchased from R&D Systems (Minneapolis, MN). c-Jun N-terminal kinase (JNK) inhibitor SP600125 was purchased from Biomol (Plymouth Meeting, MA). p38 MAPK inhibitor SB202190, MAPK/ERK kinase (MEK) inhibitor PD98059, and Janus kinase (JAK) 2 inhibitor AG490 were from Calbiochem (La Jolla, CA).

Techniques: Transfection, Incubation

Journal: Endocrinology

Article Title: Leptin enhances human beta-defensin-2 production in human keratinocytes.

doi: 10.1210/en.2008-0343

Figure Lengend Snippet: FIG. 8. IL-1 and/or leptin-induced phosphorylation of JAK2 (A) and p38 MAPK (B). Keratinocytes were preincubated for 30 min with 25 M AG490 (AG) or 1 M SB202190 (SB), and were then treated for 5 min with IL-1 (1 ng/ml) in the presence or absence of leptin (10 ng/ml) to analyze the phosphorylation by Western blotting. The results rep- resent the data from four separate experiments.

Article Snippet: Recombinant human leptin, human leptin receptor/Fc chimera, which is composed of the leptin receptor extracellular domain fused to human IgG1 Fc, and control human IgG1 Fc were purchased from R&D Systems (Minneapolis, MN). c-Jun N-terminal kinase (JNK) inhibitor SP600125 was purchased from Biomol (Plymouth Meeting, MA). p38 MAPK inhibitor SB202190, MAPK/ERK kinase (MEK) inhibitor PD98059, and Janus kinase (JAK) 2 inhibitor AG490 were from Calbiochem (La Jolla, CA).

Techniques: Phospho-proteomics, Western Blot

Journal: Endocrinology

Article Title: Leptin enhances human beta-defensin-2 production in human keratinocytes.

doi: 10.1210/en.2008-0343

Figure Lengend Snippet: FIG. 9. A schematic model for the pathways in IL-1 plus leptin- induced hBD-2 expression. Black and white arrows indicate IL-1- induced and leptin-induced pathways, respectively. Ser-P, Serine phosphorylation; Tyr-P, tyrosine phosphorylation.

Article Snippet: Recombinant human leptin, human leptin receptor/Fc chimera, which is composed of the leptin receptor extracellular domain fused to human IgG1 Fc, and control human IgG1 Fc were purchased from R&D Systems (Minneapolis, MN). c-Jun N-terminal kinase (JNK) inhibitor SP600125 was purchased from Biomol (Plymouth Meeting, MA). p38 MAPK inhibitor SB202190, MAPK/ERK kinase (MEK) inhibitor PD98059, and Janus kinase (JAK) 2 inhibitor AG490 were from Calbiochem (La Jolla, CA).

Techniques: Expressing, Phospho-proteomics

Journal: European journal of biochemistry

Article Title: Differential regulation of fatty acid amide hydrolase promoter in human immune cells and neuronal cells by leptin and progesterone.

doi: 10.1111/j.1432-1033.2004.04427.x

Figure Lengend Snippet: Fig. 1. Effect of leptin and progesterone on FAAH activity and expression. (A) Effect of leptin on the activity of FAAH in human U937 and CHP100 cells and on the protein content and the mRNA of FAAH in U937 cells. These cells were incubated for 24 h with leptin alone, or with 10 nM ( 150 ngÆmL)1) leptin in the presence of 3 lgÆmL)1 soluble leptin receptor (sLR) (100% activity in U937 cells is 350 ± 30 pmolÆmin)1Æmg protein)1; protein content, 0.280 ± 0.040 A405 and mRNA level, 11 000 ± 1300 c.p.m; 100% activity in CHP100 cells, 25 ± 3 pmolÆmin)1Æmg pro- tein)1). (B) Effect of progesterone on the same samples as in (A). Cells were incubated with progesterone alone, or with 1 lM progesterone in the presence of 10 lM RU486. In both panels: *P < 0.05 vs. control, **P < 0.01 vs. control and #P < 0.01 vs. 10 nM leptin or vs. 1 lM progesterone, respectively. In both pan- els, vertical bars represent SD values. (C) Up- per panel, Western blot analysis of U937 cells, treated with 10 nM leptin or 1 lM progesterone and reacted with specific anti-FAAH (top) or anti-actin (bottom) Igs. The positions of FAAH and actin are indicated to the right. Lower panel, RT-PCR analysis of cDNA of the same samples as in (C, upper panel). The expected sizes of the amplicons (199 base pairs for FAAH and 258 base pairs for 18S rRNA) are indicated to the right. (D) Same as in (C) on CHP100 cells. Data shown in (C,D) are rep- resentative of three independent experiments.

Article Snippet: Anti-human insulin-like growth factor I receptor (anti-IGF-IR) and human leptin receptor/Fc chimeras (soluble LR, sLR) were purchased from R &D Systems (Minneapolis, MN, USA).

Techniques: Activity Assay, Expressing, Incubation, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: European journal of biochemistry

Article Title: Differential regulation of fatty acid amide hydrolase promoter in human immune cells and neuronal cells by leptin and progesterone.

doi: 10.1111/j.1432-1033.2004.04427.x

Figure Lengend Snippet: Fig. 2. Characterization of leptin receptor (LR) and progesterone receptor (PR). (A) Saturation curves of the binding of 125I-labeled leptin to human U937 or CHP100 cells, alone or in the presence of 100 nM cold leptin (data of the cold leptin experiment with CHP100 cells were superimposable to those of U937 cells, and were omitted for the sake of clarity). Western blot analysis of cell extracts (20 lg per lane), reacted with anti-LR (B) or anti-PR (C) Igs. Molecular mass markers and the position of LR and PR are indicated to the right. Data shown in (B,C) are representative of three independent experiments. In (A), vertical bars represent SD values.

Article Snippet: Anti-human insulin-like growth factor I receptor (anti-IGF-IR) and human leptin receptor/Fc chimeras (soluble LR, sLR) were purchased from R &D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Labeling, Western Blot

Journal: European journal of biochemistry

Article Title: Differential regulation of fatty acid amide hydrolase promoter in human immune cells and neuronal cells by leptin and progesterone.

doi: 10.1111/j.1432-1033.2004.04427.x

Figure Lengend Snippet: Fig. 3. Activation of downstream signals by leptin (L) or progesterone (P). Human U937 or CHP100 cells were treated for 24 h with 10 nM leptin (A) or 1 lM progesterone (B), then lysates (50 lg of protein) were immunoblotted with the specific antibody against the inactive (total) or active (phosphorylated, phospho) form of STAT3 (A), or against Ikaros iso- forms Ik1, Ik2 and Ik3 (B). The positions of (phospho) proteins are indicated to the right. These data are representative of three inde- pendent experiments. Quantitative analysis by ELISA of the same samples (25 lg of protein) shown in (A,B) is reported in (C,D), where *P < 0.01 vs. control (CTR), and vertical bars represent SD values.

Article Snippet: Anti-human insulin-like growth factor I receptor (anti-IGF-IR) and human leptin receptor/Fc chimeras (soluble LR, sLR) were purchased from R &D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: European journal of biochemistry

Article Title: Differential regulation of fatty acid amide hydrolase promoter in human immune cells and neuronal cells by leptin and progesterone.

doi: 10.1111/j.1432-1033.2004.04427.x

Figure Lengend Snippet: Fig. 4. Analysis of FAAH promoter by transient expression. (A) Proximal upstream region of human FAAH gene. Left and right handed arrows denote SP1 sites in the – and + strands, respectively. Pentagonal box: Ikaros (Ik) site; oval box: CRE-like site. (B) 5¢ Flanking regions of the human FAAH gene were cloned in the PstI/XbaI sites of pCAT3-basic vector, wt: )107 to +1 upstream region containing the two SP1 sites flanking the Ikaros site and the CRE-like site; mutL: same as wt but with the mutated CRE-like sequence; mutIk: same as wt but with mutated Ikaros site; mutated sites are in white. Human U937 cells and CHP100 cells were transfected with the aforementioned constructs and left untreated or treated with leptin or progesterone. Transfection efficiency was monitored by the use of thymidine kinase b-galactosidase construct. The values of CAT activity were normalized to b-gal activity and to the protein content and are expressed as percentage with respect to the activity of the empty vector, pCAT3 basic, which was set to 100%. *P < 0.01 vs. control, #P < 0.01 vs. leptin or progesterone alone, and horizontal bars represent SD values.

Article Snippet: Anti-human insulin-like growth factor I receptor (anti-IGF-IR) and human leptin receptor/Fc chimeras (soluble LR, sLR) were purchased from R &D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Clone Assay, Plasmid Preparation, Sequencing, Transfection, Construct, Activity Assay, Control

Journal: European journal of biochemistry

Article Title: Differential regulation of fatty acid amide hydrolase promoter in human immune cells and neuronal cells by leptin and progesterone.

doi: 10.1111/j.1432-1033.2004.04427.x

Figure Lengend Snippet: Fig. 5. Analysis of FAAH promoter by electrophoretic mobility-shift assay (EMSA). EMSA experiments were performed with 3 lg of U937 or CHP100 nuclear extracts, prepared before (–) and after (+) sti- mulation with 10 nM leptin (A) or 1 lM progesterone (B,C). (A) Complex formation (indicated by an arrow) was performed with oligonucleotides as 32P-labeled probes for mutated (mutL) or wild-type (wt) CRE-like site. (B) Complex formation (indicated by an arrow) in U937 cells, performed with 32P-labeled oligonucleotide corresponding to the Ikaros site. Lane O represents 32P-labeled oligonucleotide alone, which contained the Ikaros site; competitor wild type (wt) was a cold oligonucleotide containing the Ikaros site derived from vasoactive intestinal peptide receptor-1 promoter; competitor mutant (mut) was a cold oligonucleotide containing the mutated Ikaros site used in transfection experiments. (C) Same as in (B) with CHP100 cells.

Article Snippet: Anti-human insulin-like growth factor I receptor (anti-IGF-IR) and human leptin receptor/Fc chimeras (soluble LR, sLR) were purchased from R &D Systems (Minneapolis, MN, USA).

Techniques: Electrophoretic Mobility Shift Assay, Labeling, Derivative Assay, Mutagenesis, Transfection