QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.091645v1 315/2/796    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kanda, N. Articles by Watanabe, S. Search for Related Content PubMed PubMed Citation Articles by Kanda, N. Articles by Watanabe, S. Pubmed/NCBI databases Compound via MeSH Substance via MeSH

INFLAMMATION AND IMMUNOPHARMACOLOGY

Prostaglandin E₂ Enhances Neurotrophin-4 Production via EP3 Receptor in Human Keratinocytes

Department of Dermatology, Teikyo University, School of Medicine, Tokyo, Japan

Received for publication June 28, 2005
Accepted August 2, 2005.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Atopic dermatitis is characterized by increased skin innervation. The expression of neurotrophin-4 is enhanced in the epidermal keratinocytes of lesions with atopic dermatitis and may be related to hyperinnervation in these lesions. Prostaglandin E₂ (PGE₂) levels are increased in lesions with atopic dermatitis; thus, PGE₂ may be involved in the development of this disease. We examined the in vitro effects of PGE₂ on neurotrophin-4 production in human keratinocytes. PGE₂ and EP1/EP3 agonist sulprostone increased neurotrophin-4 secretion and mRNA levels without altering its mRNA stability. Antisense Sp1 oligodeoxynucleotide and Sp1 inhibitor mithramycin A suppressed PGE₂ and sulprostone-induced neurotrophin-4 expression, indicating the requirement for Sp1 for expression. PGE₂ or sulprostone markedly enhanced the phosphorylation, DNA binding, and transcriptional activity of Sp1 and modestly increased Sp1 mRNA and protein levels. PGE₂ or sulprostone induced the membrane translocation of protein kinase C and the phosphorylation of extracellular signal-regulated kinase (ERK). PGE₂-induced increases in neurotrophin-4 expression, Sp1 transcriptional and DNA-binding activity, Sp1 mRNA and protein levels, and ERK phosphorylation were suppressed by antisense EP3 oligodeoxynucleotide, inhibitors of phosphatidylinositol-specific phospholipase C, conventional protein kinase C, and mitogen-activated protein kinase/ERK kinase 1 (MEK1). These results suggest that PGE₂ enhances neurotrophin-4 production by activating Sp1 via the EP3/phosphatidylinositol-specific phospholipase C/protein kinase C/MEK1/ERK pathway. PGE₂ may promote innervation in skin lesions with atopic dermatitis via the induction of neurotrophin-4.

A lipid mediator, prostaglandin E₂ (PGE₂), is produced by a variety of cells in the skin, such as mast cells, macrophages, dendritic cells, and keratinocytes (Kanda et al., 2004). PGE₂ may play either a pro- or an anti-inflammatory role in the skin that is dependent on the target cell type or activation status; the intradermal injection of PGE₂ induces local vasodilation and wheals (Greaves and Camp, 1988). On the other hand, PGE₂ enhances the production of immunosuppressive type 2 cytokines, such as interleukin-4 or interleukin-10, and thus suppresses the induction of delayed-type hypersensitivity in the skin (Shreedhar et al., 1998). PGE₂ levels are elevated in atopic skin lesions (Reilly et al., 2000). Thus, it is anticipated that PGE₂ regulates NT-4 production by keratinocytes in lesions with atopic dermatitis. However, the effects of PGE₂ on NT-4 production have not been precisely examined.

Prostaglandin E₂ binds four different G-protein-coupled receptors, EP1 to EP4, on the cell surface (Negishi et al., 1995). Keratinocytes express all four receptors with much higher levels of EP2 and EP3 than those of EP1 and EP4 (Kanda et al., 2004). The activation of EP1 or EP3 on keratinocytes induces intracellular Ca²⁺ signal via the activation of phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the activation of EP2 or EP4 generates a cyclic AMP signal (Kanda et al., 2004). Because the activation of PI-PLC generates diacylglycerol that stimulates protein kinase C (PKC) (Negishi et al., 1990), PGE₂ stimulates PKC via EP1 or EP3 in rat growth zone chondrocytes (Sylvia et al., 2001) or bovine adrenal chromaffin cells (Negishi et al., 1990). PGE₂ also activates ERK that is dependent on or independent of PKC via EP1 or EP3 in human lung cancer A549 cells (Yano et al., 2002) and rat mesangial cells (Suganami et al., 2001).

In this study, we investigated the in vitro effects of PGE₂ on NT-4 production by human keratinocytes. We found that PGE₂ has a stimulatory effect and further analyzed the mechanism of the effects.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Reagents. PGE₂, prostaglandin E₁ alcohol (1-OH PGE₁), and sulprostone were purchased from Cayman Chemical (Ann Arbor, MI). H-89, cycloheximide, mithramycin A, curcumin, and actinomycin D were purchased from Sigma Chemical Co. (St Louis, MO). U73122 [GenBank] , PD98059, Gö6976, and rottlerin were obtained from Calbiochem (La Jolla, CA).

Culture of Keratinocytes. Human neonatal foreskin keratinocytes were cultured in a serum-free KGM medium (Cambrex Bio Science Walkersville, Walkersville, MD) consisting of 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. Cells in the third passage were used.

NT-4 Secretion. Keratinocytes (5 x 10⁴/well) were seeded in triplicate into 24-well plates in 0.4 ml of KGM, adhered overnight, washed, and incubated with KBM including 1 µM indomethacin for 24 h. The cells were washed and treated with the indicated concentrations of PGE₂, sulprostone, or 1-OH PGE₁ in KBM, including indomethacin, for the indicated periods. The culture supernatants were assayed for NT-4 by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). In some experiments, the keratinocytes were preincubated with signal inhibitors for 30 min before the addition of PGE₂, sulprostone, or 1-OH PGE₁.

Reverse Transcription-Polymerase Chain Reaction. The keratinocytes were incubated as above for the indicated periods, and then the total cellular RNA was extracted and reverse-transcribed to produce cDNA (Kanda and Watanabe, 2003). The cDNA was thermocycled for polymerase chain reaction (PCR) as described previously (Kanda and Watanabe, 2003; Pang et al., 2003). The primers for amplification and the sizes of the respective PCR products were as follows: NT-4, 5'-GCTGTGGACTTGCGTGG-3' and 5'-GCACATAGGACTG-3' for 209 bp; Sp1, 5'-ACAGGTGAGCTTGACCTCAC-3' and 5'-GTTGGTTTGCACCTGGTATG-3' for 370 bp; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GCAGGGGGGAGCCAAAAGGG-3' and 5'-TGCCAGCCCCAGCGTCAAAG-3' for 566 bp (Küst et al., 2002; Kanda and Watanabe, 2003; Pang et al., 2003). PCR was performed by one denaturing cycle at 95°C for 3 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, and a final extension at 72°C for 3 min. PCR products were analyzed by electrophoresis, and densitometric analysis was performed using ATTO lane analyzer version 3 (ATTO Corporation, Osaka, Japan). NT-4 or Sp1 mRNA levels were normalized to those of GAPDH.

mRNA Stability Analysis. The keratinocytes were treated with PGE₂, sulprostone, or 1-OH PGE₁ for 8 h. RNA synthesis was blocked by actinomycin D (5 µg/ml), and RNA was isolated 0, 1, 2, 4, 8, 16, and 32 h later. Reverse transcription (RT)-PCR was performed as described above, and the decay of mRNA was determined from the band density ratios of NT-4/GAPDH as described previously (Kanda et al., 2004).

Plasmid and Transfection. Translucent Sp1 reporter vector (Luc Sp1) containing Sp1 enhancer elements (5'-ATTCGATCGGGGCGGGGCGAGATTAGATTCGATCGGGGCGGGGCGAG-3', consensus sequence underlined) in front of TATA box upstream of firefly luciferase reporter was purchased from Panomics (Redwood City, CA). Transient transfection was performed with FuGENE 6 (Roche Diagnostics, Indianapolis, IN) as described previously (Kanda et al., 2004). The keratinocytes were plated in 35-mm dishes and grown to approximately 60% confluence. Luc Sp1 (1 µg) and 0.2 µg of herpes simplex virus thymidine kinase promoter-linked Renilla luciferase vector (phRL-TK) (Promega, Madison, WI) mixed with 3 µl of FuGENE 6 was added to the keratinocytes. After 24 h, the cells were washed and incubated in KBM containing indomethacin for 24 h and then treated with PGE₂, sulprostone, or 1-OH PGE₁. After 24 h, the firefly and Renilla luciferase activities of the cell extracts were quantified using the dual luciferase assay system (Promega). Sp1 transcriptional activity was expressed as a ratio of firefly:Renilla luciferase activity.

Electrophoretic Mobility Shift Assay. Electrophoretic mobility shift assay (EMSA) was performed using an EMSA kit (Panomics) by incubating a biotin-labeled probe with a nuclear extract. The probe used was double-stranded oligonucleotide with consensus Sp1 sequence (5'-ATTCGATCGGGGCGGGGCGAGC-3'). The keratinocytes were pretreated with signal inhibitors for 30 min and then treated with PGE₂, sulprostone, or 1-OH PGE₁ for 2 h. Nuclear extracts were obtained from the keratinocytes using a nuclear extraction kit (Panomics) according to the manufacturer's instructions. For the gel shift assays, 5 µg of nuclear extracts were incubated with 10 ng of the labeled probe at room temperature for 30 min. Protein-DNA complexes were separated by 6% polyacrylamide gel and electrically transferred to a Biodyne B membrane (Pall Corporation, East Hills, NY) for chemiluminescence band detection. In the antibody supershift experiments, nuclear extracts were preincubated with rabbit polyclonal anti-Sp1 or Sp3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min before the addition of the probe.

Treatment with Antisense Oligodeoxynucleotide. Antisense oligonucleotides against Sp1, Sp3, EP1, or EP3 proteins were synthesized as phosphorothioate-modified oligonucleotides and were high-pressure liquid chromatography-purified as described previously (Hata et al., 1998; Su et al., 2004). The oligonucleotides were Sp1 (5'-ATATTAGGCATCACTCCAGG-3'), Sp3 (5'-AGTAGCAGCACTTGGAATCTGGACT-3'), EP1 (5'-GCAAGGGCTCATGTCAGG-3'), EP3 (5'-GTCTCCTTCATGTTGGC-3'), and control-scrambled (5'-AGTACCAGGACTTCGAATGTGCACT-3'). The keratinocytes were transfected with a final volume of 0.2 µM of the indicated oligonucleotides premixed with FuGENE 6 in KGM for 24 h. The medium was aspirated, and the cells were cultured with KBM including indomethacin (1 µM) for 24 h and then treated with PGE₂, sulprostone, or 1-OH PGE₁. In some experiments, these antisense oligonucleotides were transfected together with Luc Sp1 and phRL-TK.

Western Blotting. The phosphorylation of ERK was analyzed by Western blotting. The keratinocytes were pretreated with signal inhibitors for 30 min and then treated with PGE₂, sulprostone, or 1-OH PGE₁ for 10 min. The cells were lysed, and equal amounts of whole-cell lysates (20 µg/lane) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked and incubated with anti-phospho-ERK1/2 or anti-ERK1/2 antibody (Santa Cruz Biotechnology) followed by peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA). The blots were developed using an enhanced chemiluminescence kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK), and band intensity was measured by densitometric analysis.

The amount and phosphorylation status of Sp1 protein were also determined by Western blotting. The keratinocytes were treated with PGE₂, sulprostone, or 1-OH PGE₁ for 2 h. The nuclear extracts were separated and electrophoretically resolved and then transferred to membranes. The membranes were incubated with anti-Sp1 or anti-Oct-1 antibody (Santa Cruz Biotechnology) and then with secondary antibodies and developed as described above.

The subcellular localization of PKC isoforms was examined as described previously (Kanda and Watanabe, 2004). Nuclei and debris were removed from the whole-cell lysates by centrifugation (500g, 5 min), and this postnuclear fraction was centrifuged (10,500g, 90 min). The supernatant was saved as the cytosolic fraction. The pellet was homogenized in the same buffer, except that it contained 0.1% Triton X-100. The samples were mixed continuously for 1 h at 4°C and then centrifuged as described above. This supernatant was saved as the membrane fraction. Twenty micrograms of proteins of the cytosolic or membrane fractions were subjected to SDS-polyacrylamide gel electrophoresis and transferred as above, and the blots were incubated with anti-PKC, I, II, , , , , or antibodies (Santa Cruz Biotechnology) followed by secondary antibodies and developed as above. The antisense oligonucleotide-induced reduction in the respective protein levels was examined by Western blotting using the whole-cell lysates and anti-EP1 and EP3 antibodies (Cayman Chemical), anti-Sp1, Sp3, or GAPDH antibody (Santa Cruz Biotechnology), and secondary antibodies as described above.

Measurement of PGE₂ Release. The keratinocytes were incubated with KBM in the presence or absence of 1 µM indomethacin for 48 h. The supernatant PGE₂ amount was measured by enzyme-linked immunosorbent assay.

Statistical Analyses. Statistical evaluation of the results was performed by one-way analysis of variance using Dunnet's multiple comparison test for Fig. 1, A and B, or by one-way analysis of variance using Scheffe's multiple comparison test for Figs. 2B, 3B, 4A, 7A, and 7F. The results were considered significant at a value of P < 0.05.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Dose- and time-dependent enhancement of NT-4 secretion by PGE2 or sulprostone. A, the keratinocytes were pretreated with or without cycloheximide (CHX; 10 µg/ml) and then incubated with the indicated doses of PGE2, sulprostone, or 1-OH PGE1 for 48 h. The culture supernatants were assayed for NT-4. B, the cells were incubated with PGE2, sulprostone, or 1-OH PGE (each 10-6 M) for the indicated periods. The values are mean ± S.D. of triplicate cultures and representative of four separate experiments. *, P < 0.05, significantly different from the control values.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Effects of antisense EP3 on PGE2 or sulprostone-induced increases in NT-4 secretion and steady-state NT-4 mRNA levels. A, the keratinocytes were transfected with antisense oligonucleotide (AS) against EP1, EP3, or control-scrambled oligonucleotide (Con) (each 0.2 µM). At 24 h, the whole-cell lysates were analyzed for EP1, EP3, or GAPDH expression by Western blotting. The results are representative of four separate experiments. B, keratinocytes transfected with or without antisense oligonucleotides were incubated with PGE2 or sulprostone (each 10-6 M). At 48 h, the culture supernatants were analyzed for NT-4 secretion. The data are mean ± S.E.M. of four separate experiments. *, P < 0.05, significantly different from the control values; , P < 0.05, significantly different from the values with PGE2 alone; , P < 0.05, significantly different from the values with sulprostone alone. C, keratinocytes transfected with or without antisense oligonucleotides were incubated with PGE2, sulprostone (Sul), or 1-OH PGE1 (1-OH) (each 10-6 M). At 8 h, RNA was isolated and RT-PCR was performed. NT-4 mRNA levels were normalized to those of GAPDH and are shown as a -fold induction with control cultures equal to 1. The results in C are representative of four separate experiments.

View larger version (42K): [in this window] [in a new window]   Fig. 3. Involvement of Sp1 in PGE2-induced increases in NT-4 mRNA (A) or protein secretion (B). The keratinocytes were transfected with antisense oligonucleotide (AS) against Sp1, Sp3, or control-scrambled (Con) oligonucleotide (each 0.2 µM) or pretreated with mithramycin A (Mith; 10-7 M) or curcumin (Cur; 10-5 M) and then treated with PGE (10-6 M). A, RT-PCR was performed at 8 h, and NT-4 mRNA levels were normalized to those of GAPDH and are shown as a fold induction. The results are representative of four separate experiments. B, the culture supernatants were analyzed for NT-4 secretion at 48 h. The data are mean ± S.E.M. of four separate experiments. *, P < 0.05, significantly different from the control values; , P < 0.05, significantly different from the values with PGE2 alone.

View larger version (48K): [in this window] [in a new window]   Fig. 4. Enhancement of Sp1 transcriptional activity (A) and DNA binding (B) induced by PGE2 or sulprostone. A, the keratinocytes were transfected with Luc Sp1 and phRL-TK with or without antisense oligonucleotide (AS) against EP1, EP3, Sp1, Sp3, or control-scrambled oligonucleotide (Con) (each 0.2 µM). The cells were preincubated with or without mithramycin A (Mith; 10-7 M) and then incubated with PGE2, sulprostone (Sul) or 1-OH PGE1 (1-OH) (each 10-6 M). After 24 h, relative luciferase activity was analyzed. The data are mean ± S.E.M. of four separate experiments. *, P < 0.05, significantly different from the control values; , P < 0.05, significantly different from the values with PGE2 alone. B, the keratinocytes were transfected with antisense oligonucleotides or preincubated with mithramycin A and incubated with PGE2, sulprostone, or 1-OH PGE1 as above. After 2 h, nuclear extracts were obtained and EMSA was performed using an Sp1 probe. In the supershift assays, the nuclear extracts were incubated with anti-Sp1 or Sp3 antibody (Abs) for 30 min before the addition of the probe. The arrows indicate supershifted complexes. The results are representative of four separate experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 7. Enhancement of ERK1/2 phosphorylation by PGE2 or sulprostone. The keratinocytes were transfected with antisense oligonucleotide (AS) against EP1, EP3, or control-scrambled oligonucleotide (Con) (each 0.2 µM) or pre-incubated with U73122 [GenBank] (U; 10-6 M), Gö6976 (Gö; 10-8 M), rottlerin (Rott; 10-6 M), PD98059 (PD; 10-5 M), or H-89 (10-7 M) and then incubated with PGE2, sulprostone (Sul), or 1-OH PGE (1-OH) (each 10-6 M) for 10 min. The whole-cell lysates were blotted with anti-phospho-ERK antibody (A) or anti-ERK (B) antibody. Phosphorylated ERK (p-ERK) or total ERK levels are corrected to those in the control (set as 1.0). The results shown are representative of four separate experiments.

	Results

Top Abstract Materials and Methods Results Discussion References

The keratinocytes constitutively secreted small amounts of NT-4 for 48 h (mean ± S.E.M. 172.0 ± 22.4 pg/10⁶ cells, n = 4), and the secretion was dose-dependently increased by PGE₂ or EP1/EP3 agonist sulprostone, whereas the secretion volume was not altered by EP2/EP4 agonist 1-OH PGE₁ (Fig. 1A), indicating that EP1 or EP3 is involved in PGE₂-induced NT-4 secretion. PGE₂ and sulprostone (10^-6 M) increased NT-4 secretion 9.9- and 10.3-fold compared with the controls, respectively. PGE₂ or sulprostone-induced increases in NT-4 secretion were abolished by cycloheximide (Fig. 1A), indicating the requirement of de novo protein synthesis. A significant increase in NT-4 secretion compared with the controls was observed beginning 8 h after PGE₂ or sulprostone stimulation, increasing and continuing up to 48 h (Fig. 1B).

To identify the PGE₂ receptor isoforms involved in NT-4 secretion, we studied whether antisense oligodeoxynucleotide against EP1 or EP3 suppresses PGE₂ or sulprostone-induced NT-4 secretion. Antisense oligonucleotide against EP1 or EP3 selectively suppressed the expression of the respective receptor protein (Fig. 2A). Antisense EP3 suppressed PGE₂ or sulprostone-induced NT-4 secretion, but antisense EP1 or control-scrambled oligonucleotide did not (Fig. 2B), indicating that EP3 but not EP1 is involved in PGE₂ or sulprostone-induced NT-4 secretion. We next examined whether PGE₂ or sulprostone increases NT-4 mRNA levels in the keratinocytes. At 8 h of incubation, PGE₂ and sulprostone increased NT-4 mRNA levels 6.9- and 7.6-fold compared with the controls, respectively, whereas the levels were not altered by 1-OH PGE₁ (Fig. 2C). Antisense EP3 blocked PGE₂, but antisense EP1 did not block PGE₂ or sulprostone-induced increases in NT-4 mRNA level, which paralleled the results for NT-4 secretion (Fig. 2B). These results suggest that PGE₂ or sulprostone increases NT-4 production to pretranslational level via EP3.

We next examined whether PGE₂ or sulprostone increased the stability of NT-4 mRNA. The estimated half-life of NT-4 mRNA was mean ± S.E.M. (n = 4) 8.3 ± 0.9, 8.0 ± 0.9, 8.2 ± 0.8, and 8.2 ± 0.9 h in the control, PGE₂, sulprostone, and 1-OH PGE (each 10^-6 M treated keratinocytes), respectively, and there were no significant differences in the comparison of either of the two groups (P > 0.05 by one-way analysis of variance using Scheffe's multiple comparison test). Thus, PGE₂ or sulprostone did not increase the stability of NT-4 mRNA, indicating that these agents enhance NT-4 production at the transcriptional level.

Involvement of Sp1 in PGE₂ or Sulprostone-Induced NT-4 Expression. Although human NT-4 promoter has not yet been completely identified, rat NT-4 transcription can be controlled by two promoters, a proximal promoter containing several Sp1-binding sites and a distal promoter containing two activator protein-1 (AP-1) sites (Salin et al., 1997). Thus, we analyzed the involvement of transcription factors Sp1 or AP-1 in PGE₂-induced NT-4 expression using specific inhibitors and antisense oligonucleotides. Treatment with antisense oligonucleotide against Sp1 or Sp3 selectively reduced the levels of phosphorylated (105 kDa) and nonphosphorylated (90 kDa) Sp1 and levels of large (100 kDa) and small (60 kDa) Sp3 isoforms, respectively (data not shown). Mithramycin A (an inhibitor of Sp1 binding to its GC-rich recognition sequence) or antisense Sp1 suppressed PGE₂-induced NT-4 mRNA expression (Fig. 3A) and protein secretion (Fig. 3B), whereas AP-1 inhibitor curcumin, control-scrambled oligonucleotide, or antisense Sp3 did not. Similar results were obtained when sulprostone was used instead of PGE₂ (data not shown). These results suggest that Sp1 is responsible, whereas AP-1 may be dispensable for PGE₂ or sulprostone-induced NT-4 expression. We then analyzed whether PGE₂ or sulprostone enhances the transcriptional activity or DNA binding of Sp1.

Effects of PGE₂ or Sulprostone on the Transcriptional Activity, DNA Binding, Phosphorylation, and mRNA or Protein Levels of Sp1. PGE₂ or sulprostone enhances the transcriptional activity and DNA binding of Sp1. The keratinocytes were transiently transfected with luciferase vector linked to GC-rich Sp1 binding sequences in front of TATA box (Luc Sp1). PGE₂ or sulprostone increased Sp1-dependent transcriptional activity, whereas 1-OH PGE₁ did not (Fig. 4A). Mithramycin A or antisense Sp1 suppressed PGE₂-induced increases in Sp1 transcriptional activity, whereas antisense Sp3 or control oligonucleotide did not, supporting the existence of Sp1-specific activity. PGE₂ (Fig. 4A) or sulprostone-induced increases (data not shown) in Sp1 activity were suppressed by antisense EP3 but not by antisense EP1. These results suggest that PGE₂ or sulprostone increases Sp1-dependent transcriptional activity via EP3. EMSA was then performed using nuclear extracts and an oligonucleotide probe containing consensus GC-rich Sp1 binding sequences. At 2 h of incubation, PGE₂ (Fig. 4B, lane 2) or sulprostone (lane 3) increased the amount of DNA-protein complex with the Sp1 probe, whereas 1-OH PGE₁ did not alter the amount (lane 4). PGE₂ (Fig. 4B) or sulprostone-induced increases (data not shown) in the complex were suppressed by antisense EP3 (lane 7) but not by antisense EP1 (lane 6). The DNA-protein complex induced by PGE₂ was completely abolished by mithramycin A (lane 8), and anti-Sp1 antibody supershifted the complexes (lanes 9 and 11) but anti-Sp3 antibody did not (lanes 10 and 12), indicating the presence of Sp1 in the complexes. These results suggest that PGE₂ or sulprostone enhances Sp1 binding to DNA via EP3.

We then investigated whether PGE₂ or sulprostone-induced Sp1 binding and transcriptional activity was due to increased Sp1 protein expression and/or post-translational modification. PGE₂ or sulprostone markedly increased the ratio between phosphorylated and nonphosphorylated Sp1 19.0- or 24.5-fold, respectively, compared with the controls (Fig. 5A), whereas 1-OH-PGE₁ was much less potent at a 1.8-fold increase. PGE₂ (Fig. 5A) or sulprostone-induced Sp1 phosphorylation (data not shown) was suppressed by antisense EP3 but not by antisense EP1. The total protein level of Sp1 in the nuclear extract was slightly increased by 84 and 55% after PGE₂ and sulprostone treatment, respectively (Fig. 5A). In parallel to protein levels, PGE₂ and sulprostone modestly increased Sp1 mRNA levels by 222 and 169%, respectively (Fig. 5B). The PGE₂-induced increases in Sp1 protein (Fig. 5A) and mRNA levels (Fig. 5B) were suppressed by antisense EP3 but not by antisense EP1. These results suggest that PGE₂ or sulprostone markedly enhances Sp1 phosphorylation and modestly increases mRNA and protein levels via EP3, and these effects may lead to the enhancement of Sp1 binding and transcriptional activity.

View larger version (58K): [in this window] [in a new window]   Fig. 5. Enhancement of Sp1 phosphorylation and increases in Sp1 protein or mRNA levels induced by PGE2 or sulprostone. The keratinocytes were transfected with antisense oligonucleotide (AS) against Sp1, Sp3, or control-scrambled oligonucleotide (Con) (each 0.2 µM) and then incubated with PGE2, sulprostone (Sul), or 1-OH PGE1 (1-OH) (each 10-6 M) for 2 h. A, the nuclear extracts were blotted with anti-Sp1 or Oct-1 antibody. The band intensity ratio of phosphorylated Sp1 (pSp1)/non-phosphorylated Sp1 was corrected to that in the control (set as 1.0). The band intensity ratio of total Sp1 (phosphorylated and nonphosphorylated Sp1)/Oct-1 was corrected to that in the control. B, RNA was subjected to RT-PCR, and Sp1 mRNA levels were normalized to those of GAPDH and are shown as a -fold induction. The results shown are representative of four separate experiments.

Involvement of the PI-PLC-PKC-MEK1-ERK Pathway in PGE₂-Induced Sp1 Activation and NT-4 Production.

To know the signaling pathway(s) involved in PGE₂ or the sulprostone-induced activation of Sp1, we examined whether specific signaling enzyme inhibitors suppress the effects of these agents. PGE₂-induced increases in Sp1 transcriptional activity (Fig. 6A), DNA binding (Fig. 6B), phosphorylation levels (Fig. 6C), and in protein (Fig. 6C) or mRNA levels (Fig. 6D) were completely suppressed by PI-PLC inhibitor U73122 [GenBank] , conventional PKC inhibitor Gö6976, and PD98059, which suppresses ERK by inhibiting MEK1 to phosphorylate ERK1/2. On the other hand, novel PKC inhibitor rottlerin or PKA inhibitor H-89 did not block the effects of PGE₂ on Sp1 levels and activities. Similar results were obtained when sulprostone was used instead of PGE₂ (data not shown). These results suggest that PI-PLC, conventional PKC, and MEK1 are responsible for PGE₂ or the sulprostone-induced activation of Sp1.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Suppression of PGE2-induced Sp1 activation or NT-4 expression by inhibitors of PI-PLC, conventional PKC, and MEK1. A, the keratinocytes were transfected with Luc Sp1 and phRL-TK and were preincubated with U73122 [GenBank] (U; 10-6 M), Gö6976 (Gö; 10-8 M), rottlerin (Rott; 10-6 M), PD98059 (PD; 10-5 M), or H-89 (10-7 M) and then incubated with PGE2 (10-6 M). After 24 h, luciferase activity was analyzed. B, C, D, E, and F, keratinocytes without transfection were preincubated with inhibitors and then incubated with PGE2 as above. At 2 h, the nuclear extracts were subjected to EMSA using an Sp1 probe (B) or blotted with anti-Sp1 or Oct-1 antibody (C). The band intensity ratio of phosphorylated Sp1 (pSp1)/nonphosphorylated Sp1 was corrected to that in the control (set as 1.0). The band intensity ratio of total Sp1 (phosphorylated and nonphosphorylated Sp1)/Oct-1 was corrected to that in the control. At 2 (D) or 8 h (E), RNA was subjected to RT-PCR, and Sp1 (D) or NT-4 (E) mRNA levels were normalized to those of GAPDH and are shown as a -fold induction. At 48 h, the culture supernatants were analyzed for NT-4 secretion (F). The results in B, C, D, and E are representative of four separate experiments. The data in A and F are mean ± S.E.M. of four separate experiments. *, P < 0.05, significantly different from the control values; , P < 0.05, significantly different from values with PGE2 alone.

Because PGE₂-induced NT-4 expression seemed to be mediated by Sp1, we analyzed whether signal inhibitors suppressing the PGE₂-induced activation of Sp1 similarly suppressed PGE₂-induced NT-4 expression. In parallel to the results for Sp1, PGE₂-induced increases in NT-4 mRNA levels (Fig. 6E) and protein secretion (Fig. 6F) were completely suppressed by U73122 [GenBank] , Gö6976, and PD98059, whereas these were not altered by rottlerin or H-89. Similar results were obtained when sulprostone was used instead of PGE₂ (data not shown). These results suggest that PI-PLC, conventional PKC, and MEK1 are responsible for PGE₂ or the sulprostone-induced expression of NT-4 as well as the activation of Sp1 by these agents.

MEK1 activates ERK via dual phosphorylation on threonine and tyrosine residues (Kanda and Watanabe, 2004). Thus, we examined the phosphorylation status of ERK after treatment with PGE₂ or sulprostone. At 10 min, PGE₂ or sulprostone enhanced the dual phosphorylation of ERK1 (44 kDa) and ERK2 (42 kDa), whereas 1-OH PGE₁ did not (Fig. 7). The total ERK1/2 levels were not altered by these agents. PGE₂ (Fig. 7) or sulprostone-induced ERK1/2 phosphorylation (data not shown) was suppressed by antisense EP3, U73122 [GenBank] , Gö6976, and PD98059 while not suppressed by antisense EP1, rottlerin, or H-89. The results suggest that PGE₂-induced ERK activation is mediated via EP3 and requires PI-PLC, conventional PKC, and MEK1 activities. Human keratinocytes express conventional PKC, novel PKC, , and , and atypical PKC (Reynolds et al., 1994). The translocation of PKC from cytosol to membrane has been commonly accepted as an index of PKC activation (Reynolds et al., 1994). Thus, we examined whether conventional PKC is translocated from cytosol to membrane by PGE₂ or sulprostone. PGE₂ or sulprostone induced the membrane translocation of PKC from cytosol (Fig. 8). The membrane translocation of PKC by PGE₂ was suppressed by U73122 [GenBank] and antisense EP3 but not by antisense EP1 (Fig. 8). Other conventional PKC isoforms (PKCI, II, or ) were not detected in the cytosol or membrane fractions from the keratinocytes (data not shown). The membrane translocation of PKC, , , and was not induced by PGE₂ or sulprostone (data not shown). The results suggest that PKC is selectively activated by PGE₂ or sulprostone via EP3 and PI-PLC.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Membrane translocation of PKC induced by PGE2 or sulprostone. The keratinocytes were transfected with antisense oligonucleotide (AS) against EP1 and EP3 (each 0.2 µM) or preincubated with U73122 [GenBank] (U; 10-6 M) and then incubated with PGE2, sulprostone (Sul), or 1-OH PGE1 (1-OH) (each 10-6 M) for 5 min. Cytosolic and membrane fractions were isolated from the whole-cell lysates and were immunoblotted with anti-PKC antibody. PKC protein levels are shown as a -fold induction. The results are representative of four separate experiments.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, PGE₂ enhanced NT-4 production in keratinocytes via EP3 receptor. There have been no reports on the function of EP3 in neurotrophin production. Our findings thus suggest a novel key role of EP3 in NT-4 induction. EP3 may mediate NT-4 production in other cell types than keratinocytes and may play a neurotrophic role in tissues outside the skin. The stimulation of EP3 may also potentiate the production of other neurotrophins such as nerve growth factor or NT-3. We should further examine whether the activation of EP3 induces the production of these neurotrophins in human keratinocytes.

The EP3-mediated transcription of NT-4 was dependent on the activity of Sp1. Human NT-4 promoter has not been completely characterized; however, it may have several Sp1-binding sites because antisense Sp1 suppressed NT-4 expression. Further studies should identify the Sp1 elements responsible for human NT-4 transcription.

The EP3-mediated the transcriptional activity and DNA binding of Sp1 (Fig. 4, A and B) positively correlated with its phosphorylation level (Fig. 5A), indicating that phosphorylation may potentiate the transcriptional activity of Sp1. The phosphorylation of Sp1 may dissociate transcriptional repressor(s) from Sp1, such as Sp1-I and p74, which inhibits the DNA binding and transcriptional activity of Sp1, respectively (Chen et al., 1994; Murata et al., 1994), or from Sp1-bound promoter, such as histone deacetylase 1 (Choi et al., 2002). Alternatively, the phosphorylation of Sp1 may recruit transcriptional coactivators such as p300 or enhance its interaction with basal transcriptional machinery such as dTAF_II110 (Gill et al., 1994).

The stimulation of EP3 by PGE₂ activated ERK (Fig. 7), and this kinase was essential for the phosphorylation and transcriptional activity of Sp1 (Fig. 6). It has also been reported that the activation of ERK leads to the enhancement of the phosphorylation and DNA binding of Sp1 in human gastric adenocarcinoma cells (Merchant et al., 1999). Because Sp1 contains six putative ERK phosphorylation sites (Merchant et al., 1999), ERK may directly phosphorylate and activate Sp1 in PGE₂-stimulated keratinocytes. Alternatively, kinase(s) downstream of ERK may phosphorylate Sp1 in these cells. Further studies should identify direct Sp1 kinase induced by PGE₂ and phosphorylation sites on Sp1. In parallel to the enhanced phosphorylation of Sp1 (Fig. 5A), the protein and mRNA levels of Sp1 were increased by PGE₂ (Fig. 5, A and B), although the magnitude of the increased expression (approximately 2- or 3-fold compared with the controls) was much lower compared with that of phosphorylation (approximately 20-fold). It is possible that the increased expression of Sp1 may just result from the enhanced phosphorylation of Sp1, because Sp1 promoter itself contains several Sp1-binding elements and is positively regulated by its own gene product, Sp1 protein (Nicolas et al., 2001).

The activation of ERK via EP3 depended on the activity of PI-PLC and conventional PKC in the keratinocytes (Fig. 7). EP3 is linked to PI-PLC, and PI-PLC generates diacylglycerol, which activates conventional and novel PKCs (Negishi et al., 1990). The stimulation of EP3 by PGE₂ led to the activation of conventional PKC dependently on PI-PLC in human keratinocytes (Fig. 8). PKC was also activated by PGE₂ via PI-PLC in rat osteoblasts in vitro (Tang et al., 2005). On the other hand, in the medullary thick ascending limb of rat kidney, novel PKC was activated by PGE₂ in vitro only in the presence of arginine vasopressin, whereas PKC was not (Aristimuno and Good, 1997). PGE₂ in vivo activated novel PKC in rat colonic mucosa (Conte et al., 2004). Thus, PKC isoforms activated by PGE₂ may vary according to cell type, species, or experimental conditions and may reflect the relative expression, activity, or intracellular compartmentalization of individual PKC isoforms. PKC phosphorylates and activates c-Raf (Kolch et al., 1993), and the activated c-Raf may further phosphorylate and activate MEK1 that catalyzes the phosphorylation of ERK. Thus, the activation of EP3 may trigger the signaling cascade of PI-PLC-PKC-c-Raf-MEK1-ERK, resulting in the activation of Sp1. EP1 is also linked to the PI-PLC-PKC pathway in rat growth-zone chondrocytes (Sylvia et al., 2001) or linked to the activation of ERK in rat kidney mesangial cells (Suganami et al., 2001). However, antisense EP1 did not suppress the PGE₂-induced activation of PKC (Fig. 8) or ERK (Fig. 7) in human keratinocytes, indicating EP1-independent effects. This is possibly because the expression level and/or PGE₂ affinity of EP1 are much lower than those of EP3 in human keratinocytes (Kanda et al., 2004).

EP2/EP4 agonist 1-OH PGE₁ slightly increased the phosphorylation, mRNA, or protein levels of Sp1 (Fig. 5, A and B); however, it did not significantly increase the DNA binding and transcriptional activity of Sp1 (Fig. 4, A and B) or expression of NT-4 (Figs. 1 and 2). EP2 or EP4 is linked to PKA (Kanda et al., 2004), and Sp1 can be phosphorylated and activated by PKA (Black et al., 2001). Thus, PKA activated by 1-OH PGE₁ via EP2 or EP4 may phosphorylate Sp1. However, the phosphorylation level may be less than the threshold for driving the Sp1-dependent transcription of NT-4. The kinase(s) activating Sp1 may vary according to cell type or stimuli and reflect the relative activities of the signaling molecules in the target cells.

Our present findings indicate that PGE₂ in vivo promotes skin innervation via the induction of NT-4, especially in lesions with atopic dermatitis associated with elevated PGE₂ levels. The level of PGE₂ released from keratinocytes in vitro (4.21 pmol/10⁶ cells, corresponding to approximately 0.52 nM) was less than the threshold (10 nM) for NT-4 induction (Fig. 1A) and may not enhance NT-4 production per se. However, in skin lesions with atopic dermatitis, more than the threshold levels of PGE₂ might be released from neighboring cells such as macrophages or mast cells infiltrating the lesions (Imayama et al., 1995; Kiekens et al., 2001). In atopic skin lesions, the amounts of proinflammatory cytokines, tumor necrosis factor-, interleukin-1, or interferon- may be increased and this may promote PGE₂ release from the neighboring cells by inducing the expression of cyclooxygenase-2 (Takayama et al., 2002). Thus, in skin lesions with atopic dermatitis, PGE₂ from cells neighboring keratinocytes may act on EP3 on keratinocytes and promote the synthesis and secretion of NT-4. The secreted NT-4 may support the survival and sprouting of nerve fibers, especially sensory C-fibers, and thus sustain a pruritic sensation. In addition, the secreted NT-4 may modulate skin inflammation by regulating the survival or activity of macrophages, mast cells, eosinophils, or T cells containing trkB or p75NTR. PGE₂ in skin lesions with atopic dermatitis thus may contribute to cross-talk between the immune and nervous systems by the induction of NT-4. We should further examine whether the topical addition of PGE₂ enhances NT-4 expression in the keratinocytes of skin lesions with atopic dermatitis and whether antisense oligonucleotide against EP3 blocks the effects of PGE₂. EP3 may be a novel therapeutic target for atopic dermatitis.

	Acknowledgements

 
We thank Hiroko Sato for the maintenance of the keratinocytes.

	Footnotes

 
This work was partly supported by aid from the KANAE Medical Research Foundation.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.091645.

ABBREVIATIONS: PGE₂, prostaglandin E₂; NT-4, neurotrophin-4; p75NTR, p75 neurotrophin receptor; AP-1, activator protein-1; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; MEK, mitogen-activated protein kinase/ERK kinase; 1-OH PGE₁, prostaglandin E₁ alcohol; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; PD98059, 2'-amino-3'-methoxyflavone; H-89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide; U73122 [GenBank] , 1-[6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)-hexyl]-1H-pyrrole-2,5-dione; PI-PLC, phosphatidylinositol-specific phospholipase C; KBM, keratinocyte basal medium; RT, reverse transcription; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; PKA, protein kinase A; KGM, keratinocyte growth medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; phRL-TK, promoter-linked Renilla luciferase vector; Luc, luciferase.

Address correspondence 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

	References

Top Abstract Materials and Methods Results Discussion References

 

Aristimuno PC and Good DW (1997) PKC isoforms in rat medullary thick ascending limb: selective activation of the -isoform by PGE₂. Am J Physiol 272: F624-F631.
Barbacid M (1994) The Trk family of neurotrophin receptors. J Neurobiol 25: 1386-1403.[CrossRef][Medline]
Black AR, Black JD, and Azizikhan-Clifford J (2001) Sp1 and Krüppel-like factor family of transcription factors in cell growth regulation and cancer. J Cell Physiol 188: 143-160.[CrossRef][Medline]
Carter BD, Kaltschmidt C, Kaltschmidt B, Offenhauser N, Bohm-Matthaei R, Baeuerle PA, and Barde YA (1996) Selective activation of NF-kappa B by nerve growth factor through the neurotrophin receptor p75. Science (Wash DC) 272: 542-545.[Abstract]
Chen LI, Nishinaka T, Kwan K, Kitabayashi I, Yokoyama K, Fu YH, Grunwald S, and Chiu R (1994) The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator. Mol Cell Biol 14: 4380-4389.[Abstract/Free Full Text]
Choi HS, Lee JH, Park G, and Lee YI (2002) Trichostatin A, a histone deacetylase inhibitor, activates the IGFBP-3 promoter by up-regulating Sp1 activity in hepatoma cells; alteration of the Sp1/Sp3/HDAC1 multiprotein complex. Biochem Biophys Res Commun 296: 1005-1012.[CrossRef][Medline]
Conte M, Soper B, Chang Q, and Tepperman B (2004) The role of PKC and PKC in the neonatal rat colon in response to hypoxia challenge. Pediatr Res 55: 27-33.[CrossRef][Medline]
Gill G, Pascal E, Tseng ZH, and Tjian R (1994) A glutamine-rich hydrophobic patch in transcription factor Sp1 contacts the dTAFII110 component of the Drosophila TFIID complex and mediates transcriptional activation. Proc Natl Acad Sci USA 91: 192-196.[Abstract/Free Full Text]
Greaves MW and Camp RDR (1988) Prostaglandins, leukotrienes, phospholipase, platelet activating factor and cytokines: an integrated approach to inflammation of human skin. Arch Dermatol Res 280: S33-S41.
Grewe M, Vogelsang K, Ruzicka T, Stege H, and Krutmann J (2000) Neurotrophin-4 production by human epidermal keratinocytes: increased expression in atopic dermatitis. J Investig Dermatol 114: 1108-1112.[CrossRef][Medline]
Hata Y, Duh E, Zhang K, Robinson GS, and Aiello LP (1998) Transcription factors Sp1 and Sp3 alter vascular endothelial growth factor receptor expression through a novel recognition sequence. J Biol Chem 273: 19294-19303.[Abstract/Free Full Text]
Imayama S, Shibata Y, and Hori Y (1995) Epidermal mast cells in atopic dermatitis. Lancet 346: 1559.[CrossRef][Medline]
Kanda N, Mitsui H, and Watanabe S (2004) Prostaglandin E₂ suppresses CCL27 production through EP2 and EP3 receptors in human keratinocytes. J Allergy Clin Immunol 114: 1403-1409.[CrossRef][Medline]
Kanda N and Watanabe S (2002) Substance P enhances the production of interferon-induced protein of 10 kDa by human keratinocytes in synergy with interferon-. J Investig Dermatol 119: 1290-1297.[CrossRef][Medline]
Kanda N and Watanabe S (2003) 17-Estradiol inhibits oxidative stress-induced apoptosis in keratinocytes by promoting bcl-2 expression. J Investig Dermatol 121: 1500-1509.[CrossRef][Medline]
Kanda N and Watanabe S (2004) Histamine enhances the production of nerve growth factor in human keratinocytes. J Investig Dermatol 121: 570-577.[CrossRef]
Kiekens BCM, Thepen T, Oosting AJ, Bihari IC, van de Winkel JG, BruijnzeelKoomen CA, and Knol EF (2001) Heterogeneity within tissue-specific macrophage and dendritic cell populations during cutaneous inflammation in atopic dermatitis. Br J Dermatol 145: 957-965.[CrossRef][Medline]
Kolch W, Heidecker G, Kochs G, Hummel R, Vahidi H, Mischak H, Finkenzeller G, Marme D, and Rapp UR (1993) Protein kinase C activates RAF-1 by direct phosphorylation. Nature (Lond) 364: 249-252.[CrossRef][Medline]
Küst BM, Copray JC, Brouwer N, Troost D, and Boddeke HW (2002) Elevated levels of neurotrophins in human biceps brachii tissue of amyotrophic lateral sclerosis. Exp Neurol 177: 419-427.[CrossRef][Medline]
Merchant JL, Du M, and Todisco A (1999) Sp1 phosphorylation by Erk 2 stimulates DNA binding. Biochem Biophys Res Commun 254: 454-461.[CrossRef][Medline]
Murata Y, Kim HG, Rogers KT, Udvadia AJ, and Horowitz JM (1994) Negative regulation of Sp1 trans-activation is correlated with the binding of cellular proteins to the amino terminus of the Sp1 trans-activation domain. J Biol Chem 269: 20674-20681.[Abstract/Free Full Text]
Nassenstein C, Braun A, Erpenbeck VJ, Lommatzsch M, Schmidt S, Krug N, Luttmann W, Renz H, and Virchow JC Jr (2003) The neurotrophins nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4 are survival and activation factors for eosinophils in patients with allergic bronchial asthma. J Exp Med 198: 455-467.[Abstract/Free Full Text]
Negishi M, Ito S, and Hayaishi O (1990) Involvement of protein kinase C in prostaglandin E₂-induced catecholamine release from cultured bovine adrenal chromaffin cells. J Biol Chem 265: 6182-6188.[Abstract/Free Full Text]
Negishi M, Sugimoto Y, and Ichikawa A (1995) Prostaglandin E receptors. J Lipid Mediat Cell Signal 12: 379-391.[CrossRef][Medline]
Nicolas M, Noe V, Jensen KB, and Ciudad CJ (2001) Cloning and characterization of the 5'-flanking region of the human transcription factor Sp1 gene. J Biol Chem 276: 22126-22132.[Abstract/Free Full Text]
Pang JX, Cheng XY, Xu W, and Wu SG (2003) Antisense Sp1 oligodeoxynucleotide decreases telomerase activity by inhibiting hTERT mRNA expression in Jurkat T cells. Acta Pharmacol Sin 24: 91-96.[Medline]
Reilly DM, Parslew R, Sharpe GR, Powell S, and Green MR (2000) Inflammatory mediators in normal, sensitive and diseased skin types. Acta Derm Venereol 80: 171-174.[CrossRef][Medline]
Reynolds NJ, Baldassare JJ, Henderson PA, Shuler JL, Ballas LM, Burns DJ, Moomaw CR, and Fisher GJ (1994) Translocation and down-regulation of protein kinase C isoenzymes- and - by phorbol ester and bryostatin-1 in human keratinocytes and fibroblasts. J Investig Dermatol 103: 364-369.[CrossRef][Medline]
Segal RA and Greenberg ME (1996) Intracellular signaling pathways activated by neurotrophic factors. Annu Rev Neurosci 19: 463-489.[Medline]
Salin T, Timmusk T, Lendahl U, and Metsis M (1997) Structural and functional characterization of the rat neurotrophin-4 gene. Mol Cell Neurosci 9: 264-275.[CrossRef][Medline]
Shreedhar V, Giese T, Sung VW, and Ulrich SE (1998) A cytokine cascade including prostaglandin E₂, IL-4 and IL-10 is responsible for UV-induced systemic immune suppression. J Immunol 160: 3783-3789.[Abstract/Free Full Text]
Su JL, Shih JY, Yen ML, Jeng YM, Chang CC, Hsieh CY, Wei LH, Yang PC, and Kuo ML (2004) Cyclooxygenase-2 induces EP1- and HER-2/Neu-dependent vascular endothelial growth factor-C up-regulation: a novel mechanism of lymphangiogenesis in lung adenocarcinoma. Cancer Res 64: 554-564.[Abstract/Free Full Text]
Suganami T, Tanaka I, Mukoyama M, Kotani M, Muro S, Mori K, Goto M, Ishibashi R, Kasahara M, Yahata K, et al. (2001) Altered growth response to prostaglandin E₂ and its receptor signaling in mesangial cells from stroke-prone spontaneously hypertensive rats. J Hypertens 19: 1095-1103.[CrossRef][Medline]
Sugiura H, Omoto M, Hirota Y, Danno K, and Uehara M (1997) Density and fine structure of peripheral nerves in various skin lesions of atopic dermatitis. Arch Dermatol Res 289: 125-131.[CrossRef][Medline]
Sylvia VL, Del Toro F Jr, Hardin RR, Dean DD, Boyan BD, and Schwartz Z (2001) Characterization of PGE₂ receptors (EP) and their role as mediators of 1,25- (OH)₂D₃ effects on growth zone chondrocytes. J Steroid Biochem Mol Biol 78: 261-274.[CrossRef][Medline]
Takayama K, Garcia-Cardena G, Sukhova GK, Comander J, Gimbrone MA, and Libby P (2002) Prostaglandin E₂ suppresses chemokine production in human macrophages through the EP4 receptor. J Biol Chem 277: 44147-44154.[Abstract/Free Full Text]
Tang C-H, Yang R-S, and Fu W-M (2005) Prostaglandin E₂ stimulates fibronectin expression through EP₁ receptor, phospholipase C, protein kinase C and c-Src pathway in primary cultured rat osteoblasts. J Biol Chem 280: 22907-22916.[Abstract/Free Full Text]
Yano T, Zissel G, Muller-Qernheim J, Jae Shin S, Satoh H, and Ichikawa T (2002) Prostaglandin E₂ reinforces the activation of Ras signal pathway in lung adenocarcinoma cells via EP3. FEBS Lett 518: 154-158.[CrossRef][Medline]

This article has been cited by other articles:

				I. M. Macias-Perez, R. Zent, M. Carmosino, M. D. Breyer, R. M. Breyer, and A. Pozzi Mouse EP3 {alpha}, {beta}, and {gamma} Receptor Variants Reduce Tumor Cell Proliferation and Tumorigenesis in Vivo J. Biol. Chem., May 2, 2008; 283(18): 12538 - 12545. [Abstract] [Full Text] [PDF]

				M. F. Riera, M. N. Galardo, E. H. Pellizzari, S. B. Meroni, and S. B. Cigorraga Participation of phosphatidyl inositol 3-kinase/protein kinase B and ERK1/2 pathways in interleukin-1{beta} stimulation of lactate production in Sertoli cells Reproduction, April 1, 2007; 133(4): 763 - 773. [Abstract] [Full Text] [PDF]

				D. Roosterman, T. Goerge, S. W. Schneider, N. W. Bunnett, and M. Steinhoff Neuronal control of skin function: the skin as a neuroimmunoendocrine organ. Physiol Rev, October 1, 2006; 86(4): 1309 - 1379. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.091645v1 315/2/796    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kanda, N. Articles by Watanabe, S. Search for Related Content PubMed PubMed Citation Articles by Kanda, N. Articles by Watanabe, S. Pubmed/NCBI databases Compound via MeSH Substance via MeSH