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Endocrinology, doi:10.1210/en.2008-0343
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Endocrinology Vol. 149, No. 10 5189-5198
Copyright © 2008 by The Endocrine Society

Leptin Enhances Human β-Defensin-2 Production in Human Keratinocytes

Naoko Kanda and Shinichi Watanabe

Department of Dermatology, Teikyo University School of Medicine, Tokyo 173-8605, Japan

Address all correspondence and requests for reprints to: Dr. Naoko Kanda, Department of Dermatology, Teikyo University School of Medicine, 11-1 Kaga-2 Itabashi-Ku, Tokyo 173-8605, Japan. E-mail: nmk{at}med.teikyo-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin, an adipocyte-derived cytokine/hormone, modulates innate and adaptive immunity. Human β-defensin-2 (hBD-2) produced by epidermal keratinocytes promotes cutaneous antimicrobial defense, inflammation, and wound repair. We examined the in vitro effects of leptin on hBD-2 production in human keratinocytes. hBD-2 secretion and mRNA expression were analyzed by ELISA and RT-PCR, respectively. Although leptin alone was ineffective, it enhanced IL-1β-induced hBD-2 secretion and mRNA expression in keratinocytes. IL-1β- and IL-1β plus leptin-induced hBD-2 production both were suppressed by antisense oligonucleotides against nuclear factor-{kappa}B (NF-{kappa}B) p50 and p65; the latter was also suppressed by antisense signal transducer and activator of transcription (STAT)1 and STAT3. IL-1β enhanced the transcriptional activity of NF-{kappa}B, whereas leptin enhanced STAT1 and STAT3 activity. The p38 MAPK inhibitor SB202190 suppressed IL-1β- and IL-1β plus leptin-induced hBD-2 production, IL-1β-induced NF-{kappa}B activity, and leptin-induced STAT1 and STAT3 activity; contrastingly, the Janus kinase (JAK) 2 inhibitor AG490 suppressed IL-1β plus leptin-induced hBD-2 production and leptin-induced STAT1 and STAT3 activity. IL-1β induced serine phosphorylation of inhibitory {kappa}B{alpha}, STAT1, and STAT3. Leptin induced tyrosine and serine phosphorylation of STAT1 and STAT3, both of which were suppressed by AG490, and serine phosphorylation was also suppressed by SB202190. IL-1β or leptin individually induced threonine/tyrosine phosphorylation of p38 MAPK, whereas only leptin induced tyrosine phosphorylation of JAK2, suggesting that leptin may enhance hBD-2 production in keratinocytes by activating STAT1 and STAT3 via JAK2 and p38 MAPK in cooperation with NF-{kappa}B, which is activated by IL-1β. Leptin may promote cutaneous antimicrobial defense, inflammation, and wound repair via hBD-2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN IS A 16-kDa protein that is mainly secreted from adipocytes; it is in circulation and also leaks into the tissues (1, 2, 3). Leptin is also produced by skeletal muscle, the placenta, or the stomach. Leptin acts as a metabolic hormone; it acts on the hypothalamus to suppress food intake and increase energy expenditure (1, 2, 3). Leptin also belongs to a cytokine superfamily binding to type-1 receptors; six different splice variants of the leptin receptor gene db (Ob-Ra through Ob-Rf) have been identified, and all receptors share the same extracellular ligand-binding domain but differ in the intracellular portion and presumably in the ability to transduce signals (1, 2, 3). The full-length form of the leptin receptor gene (Ob-Rb) is known to be predominantly responsible for active signal transduction (3). Leptin enhances the production of reactive oxygen species and chemotaxis of neutrophils, enhances the cytotoxicity of natural killer cells, induces the production of proinflammatory cytokines such as IL-1, IL-6, or TNF-{alpha} in monocyte/macrophages (1, 2), and mediates the differentiation of Th1 cells (1, 2). Leptin also promotes antimicrobial host defense (4, 5, 6); leptin-deficient mice show increased susceptibility to pneumonitis caused by Klebsiella pneumoniae or Streptococcus pneumoniae, and exogenous leptin improves bacterial clearance by activating the phagocytotic activity of macrophages or neutrophils (4, 5, 6). It has recently been proved that leptin may function in the skin; human follicular papilla cells produce leptin and express Ob-Rb (7). Ob-Rb is also detected on human or murine epidermal keratinocytes, and exogenous leptin enhances the proliferation of these cells in vitro (8). Thus, it is plausible that leptin may also contribute to cutaneous antimicrobial defense systems, although that has not been precisely examined thus far.

Human β-defensin-2 (hBD-2), an antimicrobial peptide, is produced by epidermal keratinocytes, and its production is enhanced in cutaneous infection or inflammatory diseases such as psoriasis vulgaris (9, 10). hBD-2 kills gram-negative bacteria and protects the skin from infection due to these bacteria (11). In addition, hBD-2 potentiates skin inflammation; by binding to the CC chemokine receptor 6, it induces: 1) chemotaxis of memory T cells or immature dendritic cells (12); and 2) the production of IL-6, CC chemokine ligand 20, CXC chemokine ligand 10, and CC chemokine ligand 5 in keratinocytes (10). hBD-2 also promotes wound repair by increasing the proliferation of keratinocytes or angiogenesis (10). Because blood leptin levels increase during infection or inflammation (2, 4, 5), leptin, which leaks into tissues from the circulation and/or is produced in the skin, may act on epidermal keratinocytes and modulate their hBD-2 production in the skin lesions associated with infection or inflammatory diseases.

In this study we investigated the in vitro effects of leptin on hBD-2 production in human keratinocytes. We found that in these cells, leptin enhances hBD-2 production synergistically with IL-1β.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
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).

Culture of keratinocytes
Human neonatal foreskin keratinocytes were purchased from Clonetics (Walkersville, MD). The keratinocytes were cultured in serum-free keratinocyte growth medium containing keratinocyte basal medium (KBM) supplemented with 0.5 µg/ml hydrocortisone, 5 ng/ml epidermal growth factor, 5 µg/ml insulin, and 0.5% bovine pituitary extract. The cells from the third passage were used. Each of the following experiments was performed four times using the same lot of keratinocytes.

hBD-2 secretion
Keratinocytes (5 x 104 cells per well) were seeded in triplicate in 0.4 ml keratinocyte growth medium onto 24-well plates, adhered overnight, and washed. Cell differentiation was induced by incubation for 48 h with KBM containing a high concentration of calcium (1.25 mM) because hBD-2 production is enhanced in differentiated keratinocytes (13). The cells were incubated with 1 ng/ml IL-1β and/or the indicated concentrations of leptin in high-calcium KBM for 48 h. The hBD-2 concentrations in the culture supernatants were measured by ELISA (Peprotech, Rocky Hill, NJ). In some experiments, keratinocytes were preincubated for 30 min with the indicated concentrations of signal inhibitors before the addition of IL-1β or leptin. An IL-1β concentration of 1 ng/ml was adopted in this study because at this concentration, the viability of the keratinocytes was not affected (>95% of keratinocytes were viable), and this concentration was close to the IL-1β concentrations in the supernatants from lipopolysaccharide-stimulated human macrophages (14) or tissue homogenates from a murine hyperplasia skin model of psoriasis (15). Thus, IL-1β may exist at this concentration in skin wounds, infections, or inflammatory diseases. The concentrations of the signal inhibitors were selected on the basis of published data from our own and other laboratories (15, 16, 17, 18).

RT-PCR
The keratinocytes were incubated for 12 h as described previously. Total cellular RNA was extracted and reverse transcribed to produce cDNA. The cDNAs were analyzed for the mRNA expression of the functional leptin receptor Ob-Rb by conventional semiquantitative PCR as described previously (18). As positive controls for Ob-Rb mRNA expression, cDNA of normal human peripheral blood mononuclear cells was obtained from MTC multiple tissue cDNA panels (Clontech, San Jose, CA) and was used for PCR. PCR was performed as follows: one cycle of denaturation at 95 C for 3 min; 25 cycles of denaturation at 95 C for 30 sec, annealing at 58 C for 30 sec, and extension at 72 C for 30 sec; and a final extension at 72 C for 3 min. PCR products were analyzed by electrophoresis, and densitometric analysis was performed using the ATTO lane analyzer version 3 (ATTO Corp., Osaka, Japan).

Real-time quantitative PCR for hBD-2 mRNA was performed in a fluorescence temperature cycler (LightCycler; Roche Diagnostics GmbH, Mannheim, Germany), using specific forward and reverse primers as described (19). A SYBR Green I system was used in the reaction: 20 µl of the PCR mixture containing 2 µl cDNA, 3 mM MgCl2, 0.5 µM of each primer, and 2 µl of the reagent from light cycler-FastStart DNA Master SYBR Green I mix (Roche Diagnostics GmbH). Initial denaturation at 95 C for 10 min was followed by 45 cycles, each cycle consisting of denaturation at 95 C for 15 sec, annealing at 62 C for 5 sec, and elongation at 72 C for 15 sec. Cycle-to-cycle fluorescence emission readings were monitored and analyzed using LightCycler software (Roche Diagnostics GmbH) according to the manufacturer’s instruction. The hBD-2 mRNA expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and was shown as a fold induction relative to control keratinocytes treated with medium alone.

Plasmid and transfection
pNF-{kappa}B-luc, pGAS-luc, and pSTAT3-luc (Clontech) contain four copies of nuclear factor-{kappa}B (NF-{kappa}B) elements, two copies of signal transducer and activator of transcription (STAT)1-binding sequences, and four copies of STAT3-binding sequences, respectively, in front of the TATA box upstream of the firefly luciferase reporter. Transient transfection was performed using Fugene 6 (Roche, Indianapolis, IN) as described previously (20, 21, 22). The keratinocytes were plated in 35-mm dishes and were grown up to approximately 60% confluence. pNF-{kappa}B-luc, pGAS-luc, or pSTAT3-luc, and the herpes simplex virus thymidine kinase promoter-linked renilla luciferase vector (pRL-tk) mixed with Fugene 6 were added to the keratinocytes. After 6 h, the cells were washed and incubated for 48 h in high-calcium KBM, and were then treated with 1 ng/ml IL-1β and/or the indicated concentrations of leptin. After 18 h, the firefly and renilla luciferase activities of the cell extracts were quantified using the dual luciferase assay system (Promega Corp., Madison, WI). The transcriptional activities of NF-{kappa}B, STAT1, and STAT3 were expressed as ratios of the firefly/renilla luciferase activity.

Western blotting
Western blotting was performed as described (21). Briefly, keratinocytes were incubated for 5 min with 1 ng/ml IL-1β and/or the indicated concentrations of leptin, and lysed in a lysis buffer, and the resultant lysates were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were then blocked, probed with primary antibodies [anti-inhibitory {kappa}B (I{kappa}B) {alpha}, anti-JAK2, anti-p38 MAPK, anti-phospho-I{kappa}B{alpha}, anti-phospho-JAK2, anti-Thr/Tyr-phospho-p38 MAPK from Santa Cruz Biotechnology, Santa Cruz, CA; anti-STAT1, anti-STAT3, anti-phospho-STAT1 (Tyr-701), anti-phospho-STAT3 (Tyr-705), anti-phospho-STAT1 (Ser-727), anti-phospho-STAT3 (Ser-727), from Millipore Corp., Billerica, MA], and then with appropriate secondary antibodies conjugated to HRP (Santa Cruz Biotechnology), and visualized with ECL detection system (Amersham Biosciences, Arlington Heights, IL) according to the manufacturer’s instructions.

Treatment with antisense oligonucleotides
Antisense oligonucleotides against NF-{kappa}B p50, NF-{kappa}B p65, STAT1, STAT3, and control scrambled oligonucleotides were synthesized by phosphoroamidite chemistry, as described previously (20, 21, 22, 23, 24). The oligonucleotides are: p50 (5'-TGG ATC TTC TGC CAT TCT-3'); p65 (5'-GGG GAA CAG TTC GTC CAT GGC-3'); STAT-1 (5'-TAC CAC TGA GAC ATC CTG-3'); STAT3 (5'-GCT CCA GCA TCT GCT GCT TC-3'); and scrambled control oligonucleotide (5'-CAG CGC TGA CAA CAG TTT CAT-3'). The keratinocytes were transfected for 6 h with the indicated oligonucleotides (0.2 µM each) premixed with Fugene 6 in keratinocyte growth medium. The cells were treated with high-calcium KBM for 48 h, and were then incubated with 1 ng/ml IL-1β and/or the indicated concentrations of leptin.

Statistical analyses
One-way ANOVA with Dunnet’s multiple comparisons test was used, and the results are shown in Fig. 2AGo. One-way ANOVA with Tukey-Kramer multiple comparisons test was used, and the results are presented in Figs. 2Go, B and C, and 3. One-way ANOVA with Dunnet’s multiple comparisons test was used, and the results are shown in Fig. 4Go. One-way ANOVA with Tukey-Kramer multiple comparisons test was used, and the results are presented in Figs. 5Go and 6Go. A P value of less than 0.05 was considered statistically significant.


Figure 2
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  		FIG. 2. Stimulation of IL-1β-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-1β (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; {dagger}, P < 0.05 vs. IL-1β alone. B and C, Keratinocytes were preincubated with 1 µg/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-1β 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; {dagger}, P < 0.05 vs. IL-1β alone; {ddagger}, P < 0.05 vs. IL-1β 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.

 

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

 

Figure 5
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  		FIG. 5. The effects of signal inhibitors on IL-1β- and/or leptin-induced hBD-2 secretion (A) and mRNA expression (B). Keratinocytes were preincubated for 30 min with 25 µM AG490 (AG), 10 µM PD98059 (PD), 1 µM SB202190 (SB), or 10 µM SP600125 (SP), and were then incubated with 1 ng/ml IL-1β in the presence 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 normalized to the mRNA levels of GAPDH and are shown as a fold induction. *, P < 0.05 vs. the controls; {dagger}, P < 0.05 vs. IL-1β alone; {ddagger}, P < 0.05 vs. IL-1β plus leptin.

 

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

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expressions of the leptin receptor Ob-Rb in human keratinocytes
Human keratinocytes expressed the mRNA of the functional leptin receptor Ob-Rb (Fig. 1Go). Treatment with IL-1β did not alter the Ob-Rb mRNA level in the keratinocytes at 12 (Fig. 1Go), 24, or 48 h (data not shown).


Figure 1
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  		FIG. 1. mRNA expression of the leptin receptor Ob-Rb in keratinocytes (KC) incubated for 12 h with or without IL-1β (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 representative of four separate experiments.

 
Leptin enhances IL-1β-induced hBD-2 production in keratinocytes
We examined the effects of leptin on basal and IL-1β-induced hBD-2 production in the keratinocytes. Although leptin did not alter the basal hBD-2 secretion, it increased IL-1β-induced hBD-2 secretion in a dose-dependent manner (Fig. 2AGo). The stimulatory effect of leptin peaked at 10 ng/ml, which increased the hBD-2 secretion to 4.13-fold of that induced by IL-1β alone. Leptin-induced enhancement in hBD-2 secretion was suppressed by the soluble chimeric leptin receptor fused to human IgG1 Fc, but not by the control human IgG1 Fc (Fig. 2BGo), indicating that the enhancement may be specifically mediated by the leptin receptor. The interferon-{gamma} or IL-17 concentrations in the supernatants of the IL-1β- and/or leptin-stimulated keratinocytes were less than the detectable levels, as examined by ELISA (<8 pg/ml or <15 pg/ml, respectively). The leptin concentrations in the supernatants of IL-1β-stimulated or nonstimulated keratinocytes were less than detectable level as examined by ELISA (<7.8 pg/ml), and leptin was not detected in the cell lysates of these cells by Western blotting (supplemental Fig. S1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Thus, cultured keratinocytes did not produce detectable amounts of leptin, and the stimulatory effects on hBD-2 production appeared to be predominantly dependent on exogenous leptin. In parallel with protein secretion, leptin enhanced the IL-1β-induced hBD-2 mRNA expression at 12 h without altering the basal hBD-2 mRNA levels as examined by real-time RT-PCR, and the enhancement was specifically blocked by the chimeric leptin receptor (Fig. 2CGo). This result was observed even at 6 and 18 h (data not shown).

NF-{kappa}B may be involved in hBD-2 production induced by IL-1β or IL-1β plus leptin, and STAT1 and STAT3 may be involved only in hBD-2 production induced by IL-1β plus leptin
The hBD-2 promoter contains NF-{kappa}B-, STAT1-, or STAT3-binding sequences, and these factors may transactivate the hBD-2 gene (25). Using antisense oligonucleotides against these factors, we thus examined their involvement in leptin- and/or IL-1β-induced hBD-2 production. The antisense oligonucleotides at the indicated concentration did not affect the viability of keratinocytes (>94% of the keratinocytes were viable) and selectively reduced the protein levels of the respective transcription factors (supplemental Fig. S2). The antisense oligonucleotides against p50 and p65 remarkably reduced hBD-2 secretion (Fig. 3AGo) and mRNA expression (Fig. 3BGo) induced by IL-1β alone or by IL-1β plus leptin. These results suggest that NF-{kappa}B may be required for hBD-2 production by IL-1β or IL-1β plus leptin. Antisense STAT1 and STAT3 reduced the IL-1β plus leptin-induced hBD-2 secretion (Fig. 3AGo) and mRNA expression (Fig. 3BGo); however, these proteins did not alter hBD-2 production induced by IL-1β alone. These results suggest that STAT1 and STAT3 may be involved in the stimulatory effects of leptin on IL-1β-induced hBD-2 production.


Figure 3
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  		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 factors or control scrambled oligonucleotides (Con) (0.2 µM each) and were treated with IL-1β (1 ng/ml) in the presence or absence of leptin (10 ng/ml). At 48 h, the amount of hBD-2 secreted was analyzed (A), whereas at 12 h, the mRNA levels were analyzed by real-time RT-PCR (B). *, P < 0.05 vs. the controls; {dagger}, P < 0.05 vs. IL-1β; {ddagger}, P < 0.05 vs. IL-1β 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.

 
Leptin enhances STAT1 and STAT3 activities without altering basal or IL-1β-induced NF-{kappa}B activity
We then examined whether leptin alone or together with IL-1β can alter the transcriptional activities of NF-{kappa}B, STAT1, and STAT3 in the keratinocytes. When administered alone, leptin did not alter NF-{kappa}B activity (Fig. 4AGo); however, it enhanced STAT1 and STAT3 activity in a dose-dependent manner (Fig. 4Go, B and C); 10 ng/ml leptin brought about a 2.27-fold and 2.02-fold increase in the basal STAT1 and STAT3 activities, respectively. IL-1β alone increased the NF-{kappa}B activity to 2.15-fold that of the control (Fig. 4AGo); however, it did not alter the basal or leptin-induced STAT1 (Fig. 4BGo) and STAT3 activities (Fig. 4CGo). Leptin did not further enhance IL-1β-induced NF-{kappa}B activity (Fig. 4AGo).

These results indicate that IL-1β may induce hBD-2 production by activating NF-{kappa}B, and leptin may further enhance IL-1β-induced hBD-2 production by activating STAT1 and STAT3.

p38 MAPK may be involved in hBD-2 production induced by IL-1β or IL-1β plus leptin, whereas JAK2 may be involved only in that by IL-1β plus leptin
It is reported that leptin activates STAT1 or STAT3 via the stimulation of Ob-Rb-associated JAK2 or activates MEK/ERK, p38 MAPK, or JNK signaling pathways according to the cell type (2, 3). Thus, we examined the involvement of these signals in leptin-induced stimulation of hBD-2 production that occurred synergistically with IL-1β by using specific inhibitors of these signals. We confirmed that PD98059, SB202190, SP600125, and AG490 at the indicated doses specifically suppressed the activation of respective signals, ERK, p38 MAPK, JNK, and JAK2 in human keratinocytes (supplemental Fig. S3). hBD-2 secretion (Fig. 5AGo) and mRNA expression (Fig. 5BGo) induced by IL-1β alone were reduced by the p38 MAPK inhibitor SB202190 but not by AG490, PD98059, or SP600125, i.e. the inhibitors of JAK2, MEK, and JNK, respectively. These results indicate the involvement of p38 MAPK but not of JAK2, MEK/ERK, or JNK in the stimulation of hBD-2 production by IL-1β alone. On the other hand, leptin plus IL-1β-induced hBD-2 secretion (Fig. 5AGo) and mRNA expression (Fig. 5BGo) were suppressed by AG490, and also by SB202190 but not by PD98059 or SP600125, indicating the involvement of JAK2 in addition to p38 MAPK in leptin plus IL-1β-induced hBD-2 production. Similar results were obtained when another MEK inhibitor, i.e. U0126, or p38 MAPK inhibitor, i.e. SB203580, was used (data not shown).

IL-1β-induced activation of NF-{kappa}B requires p38 MAPK, whereas leptin-induced activation of STAT1 and STAT3 requires p38 MAPK and JAK2
We then examined whether JAK2 or p38 MAPK may be involved in IL-1β and/or leptin-induced stimulation of NF-{kappa}B, STAT1, or STAT3 activities. IL-1β-induced stimulation of NF-{kappa}B activity was reduced by SB202190 but not by AG490 (Fig. 6AGo). Similar results were obtained with NF-{kappa}B activities induced by IL-1β plus leptin. Leptin-induced stimulation of STAT1 or STAT3 activity was suppressed by AG490 and SB202190 (Fig. 6Go, B and C). Similar results were obtained with STAT1 or STAT3 activity induced by leptin plus IL-1β. These results suggest that p38 MAPK and JAK2 may be involved in leptin-induced activation of STAT1 and STAT3, and p38 MAPK may also be involved in IL-1β-induced activation of NF-{kappa}B.

p38 MAPK may be involved in leptin-induced serine phosphorylation of STAT1 and STAT3, whereas JAK2 may be involved in leptin-induced tyrosine and serine phosphorylation of STAT1 and STAT3
NF-{kappa}B p50/p65 is normally sequestered in the cytoplasm by its interaction with I{kappa}B, and upon stimulation, I{kappa}B is phosphorylated, ubiquitinated, and degraded, leading to the release of active NF-{kappa}B (26). Thus, we examined the phosphorylation of I{kappa}B in IL-1β- and/or leptin-stimulated keratinocytes. IL-1β alone induced Ser32 phosphorylation of I{kappa}B{alpha} (Fig. 7AGo, first panel). The IL-1β-induced phosphorylation of I{kappa}B{alpha} was also confirmed by a slower migrating form of I{kappa}B{alpha} (Fig. 7AGo, second panel). The phosphorylation of I{kappa}B{alpha} was not blocked by SB202190 or AG490. These results suggest that p38 MAPK may not be involved in I{kappa}B{alpha} phosphorylation but in another step(s) leading to the activation of NF-{kappa}B by IL-1β. Leptin did not enhance the phosphorylation of I{kappa}B{alpha} in the presence or absence of IL-1β (Fig. 7AGo, first and second panels).


Figure 7
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  		FIG. 7. IL-1β and/or leptin-induced phosphorylation of I{kappa}B{alpha} (A), tyrosine- or serine-phosphorylation of STAT1 (B) and STAT3 (C). Keratinocytes were preincubated for 30 min with 25 µM AG490 (AG) or 1 µM SB202190 (SB), and were then treated with IL-1β (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.

 
Phosphorylation of STAT1 and STAT3 at the Tyr701 or Tyr705 residues, respectively, induces their dimerization, nuclear translocation, and DNA binding (27). Phosphorylation of STAT1 and STAT3 at Ser727 is essential to maximize their transactivation potential. Thus, we examined the tyrosine or serine phosphorylation of STAT1 and STAT3 in IL-1β- and/or leptin-stimulated keratinocytes. As shown in first panels of Fig. 7Go, B and C, leptin alone induced Tyr701 phosphorylation of STAT1 and Tyr705 phosphorylation of STAT3, and tyrosine phosphorylation was suppressed by AG490 but not by SB202190, indicating that JAK2 may be required for tyrosine phosphorylation of STAT1 and STAT3 by leptin. IL-1β did not enhance tyrosine phosphorylation of STAT1 or STAT3 in the presence or absence of leptin (Fig. 7Go, B and C, first panels, respectively). Leptin alone induced serine phosphorylation of STAT1 and STAT3 (Fig. 7Go, B and C, second panels, respectively), both of which were suppressed by SB202190 and AG490, indicating the requirement of p38 MAPK and JAK2 for serine phosphorylation by leptin. IL-1β alone slightly increased the serine phosphorylation levels of STAT1 and STAT3, and the phosphorylation was not blocked by SB202190 or AG490 (Fig. 7Go, B and C, second panels), indicating the serine phosphorylation without involvement of p38 MAPK or JAK2. IL-1β plus leptin yielded additive effects on the serine phosphorylation levels of STAT1 and STAT3 (Fig. 7Go, B and C, second panels).

These results totally suggest that p38 MAPK may be involved in serine phosphorylation of STAT1 and STAT3 by leptin but not in serine phosphorylation by IL-1β; in contrast, JAK2 may be involved in both tyrosine and serine phosphorylation of STAT1 and STAT3 by leptin.

Leptin and IL-1β individually activate p38 MAPK, and leptin simultaneously activates JAK2
We examined whether leptin and/or IL-1β may activate JAK2 or p38 MAPK in the keratinocytes. The activation of JAK2 induces its autophosphorylation at the tyrosine residues (3), whereas p38 MAPK is activated by dual phosphorylation at the threonine and tyrosine residues (28). Leptin alone induced autophosphorylation of JAK2, which was suppressed by AG490 but not by SB202190 (Fig. 8AGo). IL-1β did not enhance autophosphorylation of JAK2 in the presence or absence of leptin. Leptin alone or IL-1β alone enhanced phosphorylation of p38 MAPK, both of which were suppressed by SB202190 but not by AG490 (Fig. 8BGo). IL-1β plus leptin additively potentiated p38 MAPK phosphorylation, which were suppressed by SB202190 but not by AG490. These results suggest that leptin and IL-1β individually activate p38 MAPK and that leptin simultaneously activates JAK2. Leptin-induced activation of JAK2 and p38 MAPK may independently occur in keratinocytes.


Figure 8
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  		FIG. 8. IL-1β and/or leptin-induced phosphorylation of JAK2 (A) and p38 MAPK (B). Keratinocytes were preincubated for 30 min with 25 µM AG490 (AG) or 1 µM SB202190 (SB), and were then treated for 5 min with IL-1β (1 ng/ml) 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In keratinocytes, IL-1β alone induced hBD-2 production via the activation of NF-{kappa}B, which was partially dependent on p38 MAPK. Leptin potentiated the IL-1β-induced hBD-2 production by activating STAT1 and STAT3 via JAK2 and p38 MAPK (Fig. 9Go); JAK2-induced tyrosine-phosphorylation and p38 MAPK-induced serine-phosphorylation of STAT1/3 (Fig. 7Go) may potentiate their transcriptional activity. Leptin alone neither activated NF-{kappa}B nor induced hBD-2 expression. Possibly hBD-2 gene expression may predominantly depend on NF-{kappa}B (29), and the activated STAT1 and/or STAT3 by themselves may not drive its expression without the activation of NF-{kappa}B. Possibly, compared with STAT1/3, the interaction of NF-{kappa}B with transcriptional coactivators and/or general transcriptional machinery on hBD-2 promoter may be more potent, and such interaction may more effectively induce histone acetylation on this promoter. However, STAT1 and STAT3 activated by leptin might cooperate with NF-{kappa}B activated by IL-1β on the hBD-2 gene and may potentiate its expression (Fig. 9Go). The possible cooperation between NF-{kappa}B and STAT1/3 on the hBD-2 gene may be mediated by the summing up of the interaction between individual transcription factors and basal transcriptional components such as TATA-binding proteins (30). Alternatively, this transcriptional synergy on the hBD-2 gene may be mediated by the direct interaction between NF-{kappa}B and STAT1/3, which may stabilize their DNA binding and, thus, create a highly stable multiprotein complex (31). It is also reported that NF-{kappa}B and STATs cooperatively promote the expression of other genes, such as CXCL9 or interferon regulatory factor-1 (32, 33).


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

 
Leptin activated JAK2 and p38 MAPK in keratinocytes, and each signal appeared to occur independently because the inhibitor of one signal did not suppress the activation of the other (Fig. 8Go). When leptin binds to the Ob-Rb receptor, the receptor-associated JAK2 is activated, and the receptor is phosphorylated on the tyrosine residues, which recruit STAT1 and STAT3 through the src homology domain 2 (3). The recruited STAT1 and STAT3 may further undergo tyrosine phosphorylation by the activated JAK2, which may induce their dimerization, nuclear translocation, and DNA binding (34, 35). Leptin-Ob-Rb binding may also activate p38 MAPK, and the activated p38 MAPK or its downstream kinase may bring about serine phosphorylation of STAT1 and STAT3, which may potentiate their transcriptional capacity. It is reported that a small guanosine triphosphatase, namely RhoA/Rho-associated kinase, may be the upstream mediator of leptin-induced activation of p38 MAPK; leptin-Ob-Rb binding induces activation and translocation of RhoA to the membrane caveolae, and the activated RhoA further stimulates Rho-associated kinase, which induces the activation of p38 MAPK in rat cardiomyocytes (36). We preliminarily found that leptin-induced activation of p38 MAPK in human keratinocytes was suppressed by Rho-associated kinase inhibitor Y27632 and that leptin activated RhoA in the same cells (supplemental Fig. S4). Thus, in human keratinocytes, RhoA may be involved in the activation of p38 MAPK by leptin. Although the JAK2 inhibitor AG490 did not suppress p38 MAPK activation by leptin (Fig. 8BGo), it suppressed serine phosphorylation of STAT1 and STAT3 by leptin (Fig. 7Go, B and C). These results indicate that JAK2 may contribute to serine phosphorylation of STAT1 and STAT3 by a mechanism independent of p38 MAPK (Fig. 9Go). Several such mechanisms can be hypothesized. First, STAT1 and STAT3 may have to dock on the Ob-Rb to undergo serine phosphorylation, and tyrosine phosphorylation of Ob-Rb by JAK2 may be necessary for creating such docking sites. Second, the other serine kinase(s), which is dependent on JAK2 but independent of p38 MAPK, may phosphorylate STAT1 or STAT3 in the leptin-stimulated keratinocytes; Akt or protein kinase C {delta} is involved in serine phosphorylation of STAT1 and STAT3 (37, 38). It has been reported that JAK2 activated by leptin stimulates: 1) phosphatidylinositol 3-kinase, upstream of Akt and/or protein kinase C {delta}, in Ob-Rb-transfected HEK293 cells (39); or 2) phospholipase C{gamma}2 upstream of protein kinase C in platelets (40). Alternatively, STAT1 or STAT3 may have to be dimeric to be recognized by p38 MAPK or its downstream kinase, or STAT1 or STAT3 may have to translocate to the nucleus to move on to p38 MAPK or its downstream kinase; this is because p38 MAPK is known to be imported into the nuclei in leptin-stimulated rat cardiomyocytes (36). These possibilities should be examined by further studies.

IL-1β activated p38 MAPK, which was involved in the activation of NF-{kappa}B (Fig. 6AGo). However, p38 MAPK activity was not required for the phosphorylation of I{kappa}B{alpha} (Fig. 7AGo). By binding to the IL-1 receptor, IL-1β induces the association between the IL-1 receptor-associated kinase and the IL-1 receptor, and the subsequent recruitment of the adaptor protein TNF receptor-associated factor 6, which further activates the NF-{kappa}B-inducing kinase and the downstream I{kappa}B kinase, leading to phosphorylation of I{kappa}B{alpha} (41). Possibly, p38 MAPK activated by IL-1β may stimulate the different steps for NF-{kappa}B activation (Fig. 9Go); p38 MAPK may be involved in phosphorylation and activation of the TATA-binding protein, which is a general transcriptional apparatus for NF-{kappa}B-mediated transcription (42). Alternatively, mitogen- and stress-activated protein kinase-1 located downstream of p38 MAPK may phosphorylate NF-{kappa}B p65 at Ser276 and increase its transactivation capacity (43).

IL-1β induced serine phosphorylation of STAT1 and STAT3 independently of tyrosine phosphorylation (Fig. 7Go, B and C). The serine phosphorylation of STAT1 and STAT3 by IL-1β did not involve p38 MAPK (Fig. 7Go, B and C), which was different from serine phosphorylation of STAT1 and STAT3 by leptin. It is reported that IL-1β may promote the interaction between the IL-1 receptor-associated kinase and STAT1 or STAT3, and recruits thus far unknown serine kinase(s) that phosphorylates STAT1 or STAT3 (44). IL-1β alone did not promote the transcriptional activity of STAT1 or STAT3 (Fig. 4Go, B and C). These results indicate that STAT1 or STAT3 that undergo serine phosphorylation without tyrosine phosphorylation may be unable to translocate to the nucleus, bind DNA, or transactivate the hBD-2 gene. IL-1β also additively enhanced serine phosphorylation of STAT1 and STAT3 together with leptin (Fig. 7Go, B and C); however, it did not further enhance their transactivation capacities (Fig. 4Go, B and C). Possibly, the serine phosphorylation-mediated activation of STAT1 or STAT3 may be saturated by leptin alone.

Although IL-1β activated p38 MAPK (Fig. 8BGo), it did not enhance STAT1 or STAT3 activity in the presence or absence of leptin (Fig. 4Go, B and C). Besides, leptin did not enhance NF-{kappa}B activity in the presence or absence of IL-1β (Fig. 4AGo); however, it activated p38 MAPK (Fig. 8BGo). Together, p38 MAPK activity may lead to the activation of different transcription factors according to the stimulus; IL-1β contributes to the activation of NF-{kappa}B and leptin, to the activation of STAT1/3. The outcome may depend on the intracellular localization, amounts, or activities of p38 MAPK, NF-{kappa}B p50/p65, STAT1, and STAT3. Thus, the downstream events from p38 MAPK differ between leptin and IL-1β, however, if both stimuli were added simultaneously, p38 MAPK activity was additively enhanced, and individual downstream events may converge onto the hBD-2 gene and cooperatively induce its expression (Fig. 9Go).

Leptin alone did not induce I{kappa}B phosphorylation (Fig. 7AGo) or activation of NF-{kappa}B (Fig. 4AGo) in human keratinocytes. Differently from these results in keratinocytes, JAK2-activating stimuli up-regulated NF-{kappa}B by indirect activation of I{kappa}B kinase via phosphatydilinositol-3 kinase in human leukemia K562 cells (45), and leptin activated NF-{kappa}B in human HT-29 colon cancer cells (46). Possibly, whether leptin activates NF-{kappa}B may differ according to the cell type used, and may depend on the expression levels of Ob-Rb and/or its status of coupling to the NF-{kappa}B-stimulating signals.

Our present study revealed that leptin acted on epidermal keratinocytes and induced hBD-2 production in these cells: its novel function in cutaneous immunity. In cutaneous infections, inflammatory diseases like psoriasis, or wounds, hBD-2 production in epidermal keratinocytes is enhanced (9, 13). In these conditions, systemic or local leptin levels are increased, and IL-1β, a proinflammatory cytokine, may coexist (4, 6). Thus, our present results suggest that leptin may be a candidate stimulus for hBD-2 production in such situations. The hBD-2 released from keratinocytes by leptin plus IL-1β may chemoattract CC chemokine receptor 6-positive cells, such as immature dendritic cells or memory T cells (12). Besides, when keratinocytes are exposed to microbes such as gram-negative bacteria, the cells may deposit the surface hBD-2 onto the surface-bound microbes immediately after its release, thereby killing the microbes (47). The released hBD-2 may in turn act on keratinocytes (10), and induce their proliferation and production of proinflammatory cytokines/chemokines in an autocrine/paracrine manner.

It has been suggested that leptin plays a key role in autoimmune diseases (1), and our results support that the probable involvement of leptin in the pathogenesis of psoriasis, a cutaneous autoimmune disease. Leptin may induce the infiltration of immature dendritic cells or memory T cells and hyperproliferation of keratinocytes in psoriatic lesions via induction of hBD-2. Systemic leptin levels positively correlate with the total body fat mass (48), and obesity is reported to be linked with a higher risk of psoriasis (49, 50). Thus, adipocyte-derived leptin may mediate this linkage by inducing hBD-2. A soluble chimeric leptin receptor may act as a therapeutic agent for psoriatic patients having increased leptin levels.

The optimal in vitro concentration of leptin for hBD-2 induction (10 ng/ml) is nearly equal to its in vivo serum concentrations [15.74 ± 9.12 ng/ml in healthy adults (51)], though leptin levels in the skin may be lower. However, serum leptin levels and leptin levels at infected sites decline during fasting and are also decreased in malnourished hosts with chronic illnesses, such as cancer or chronic obstructive lung diseases (52, 53); moreover, such hosts are more susceptible to infection and delayed wound repair (6, 8). Thus, reduced leptin levels may be responsible for compromised antimicrobial defense or delayed wound healing in malnourished hosts (6). In such hosts, leptin administration may prove to be effective therapeutically because it augments antimicrobial defense and wound repair. Our results support that leptin may play a key role in cutaneous antimicrobial defense, inflammation, and wound repair. Leptin may also modulate the expression of other proinflammatory, antimicrobial, or wound-healing molecules such as LL-37 or hBD-3 in keratinocytes. The other novel effects of leptin on keratinocytes are currently under investigation.


   Acknowledgments
 
We thank Ms. Hiroko Sato for the maintenance of the keratinocytes.


   Footnotes
 
This work is supported in part by the grant from the Japan Society for the Promotion of Science (18244120).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 12, 2008

Abbreviations: GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; hBD-2, human β-defensin-2; I{kappa}B, inhibitory {kappa}B; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; KBM, keratinocyte basal medium; MEK, MAPK/ERK kinase; NF-{kappa}B, nuclear factor-{kappa}B; pRL-tk, herpes simplex virus thymidine kinase promoter-linked renilla luciferase vector; STAT, signal transducer and activator of transcription.

Received March 11, 2008.

Accepted for publication May 30, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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