LZTS3/TAGLN Suppresses Cancer Progression in Human Colorectal Adenocarcinoma Through Regulating Cell Proliferation, Migration, and Actin Cytoskeleton.

BACKGROUND: Numerous studies have confirmed that the leucine zipper tumor suppressor (LZTS) gene family plays a vital role in modulating transcription and cell cycle control, especially in colorectal cancer. This study aimed to evaluate the potential of leucine zipper tumor suppressor family member 3 (LZTS3) as a marker for COAD. METHODS: Bioinformatics, immunohistochemistry, and Western blotting were applied to assess the expression of LZTS3 in tissues. Gene overexpression or silencing was used to examine the biological roles of LZTS3 and validated using an in vivo nude mouse-human tumor model. RESULTS: The results obtained in this study indicate that LZTS3 is highly expressed in COAD. RTCA, Transwell, actin stain, and in vitro transfection experiments confirmed that LZTS3 expression inhibits tumor cell proliferation and cell migration. The results obtained in the nude mouse-human tumor model are consistent with those obtained in vitro. In particular, LZTS3 may exert biological effects by targeting the NOTCH signaling pathway. Furthermore, TAGLN was demonstrated to be a downstream target of LZTS3. CONCLUSION: This is the first study to demonstrate the important role of LZTS3 in the proliferation and migration of COAD and to shed light on the molecular mechanism underlying the tumor-suppressing role of LZTS3.

Genome-wide Analysis of Histone H3 Lysine 27 Trimethylation Profiles in Sciatic Nerve of Chronic Constriction Injury Rats.

The histone H3 lysine 27 trimethylation (H3K27me3) is one of the most important chromatin modifications, which is associated with injury-activated gene expression in Schwann cells (SCs). However, the alteration of genome-wide H3K27me3 enrichments in the development of neuropathic pain is still unknown. Here, we applied the chromatin immunoprecipitation sequencing (ChIP-seq) approach to identify the alteration of differential enrichments of H3K27me3 in chronic constriction injury (CCI) sciatic nerve of rats and potential molecular mechanisms underlying the development of neuropathic pain. Our results indicated that CCI increased the numbers of SCs displaying H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) and H3K27me3 in the sciatic nerve. ChIP-seq data showed that CCI significantly changed H3K27me3 enrichments on gene promoters in the sciatic nerve. Bioinformatics analyses exhibited that genes gaining H3K27me3 were mostly associated with regulation of cell proliferation, response to stress and oxidation-reduction process. Genes losing this mark were enriched in neuronal generation, and MAPK, cAMP as well as ERBB signaling pathways. Importantly, IL1A, CCL2, NOS2, S100A8, BDNF, GDNF, ERBB3 and C3 were identified as key genes in neuropathic pain. CCI led to significant upregulation of key genes in the sciatic nerve. EZH2 inhibitor reversed CCI-induced increases of H3K27me3 and key gene protein levels, which were accompanied by relieved mechanical allodynia and thermal hyperalgesia in CCI rats. These results indicate that genes with differential enrichments of H3K27me3 in SCs function in various cellular processes and pathways, and many are linked to neuropathic pain after peripheral nerve injury.

Role of the nuclear receptor subfamily 4a in mast cells in the development of irritable bowel syndrome.

The activation of mast cells (MCs) and mediator release are closely related to the pathophysiology of irritable bowel syndrome (IBS). However, the exact underlying mechanisms are still not completely understood. The nuclear receptor subfamily 4a (Nr4a) is a family of orphan nuclear receptors implicated in regulating MC activation, degranulation, cytokine/chemokine synthesis and release. Acute and chronic stress trigger hypothalamic-pituitaryadrenal axis (HPA) activation to induce the release of corticotropin-releasing hormone (CRH), resulting in MC activation and induction of the Nr4a family. Our newest data showed that Nr4a members were specially over-expressed in colonic MCs of the chronic water-avoidance stress (WAS)-induced visceral hyperalgesia mice, suggesting that Nr4a members might be involved in the pathophysiology of visceral hypersensitivity. In this review, we highlight the present knowledge on roles of Nr4a members in the activation of MCs and the pathophysiology of IBS, and discuss signaling pathways that modulate the activation of Nr4a family members. We propose that a better understanding of Nr4a members and their modulators may facilitate the development of more selective and effective therapies to treat IBS patients.

Calcitonin gene-related peptide induces the histone H3 lysine 9 acetylation in astrocytes associated with neuroinflammation in rats with neuropathic pain.

AIMS: Calcitonin gene-related peptide (CGRP) as a regulator of astrocyte activation may facilitate spinal nociceptive processing. Histone H3 lysine 9 acetylation (H3K9ac) is considered an important regulator of cytokine and chemokine gene expression after peripheral nerve injury. In this study, we explored the relationship between CGRP and H3K9ac in the activation of astrocytes, and elucidated the underlying mechanisms in the pathogenesis of chronic neuropathic pain. METHODS: Astroglial cells (C6) were treated with CGRP and differentially enrichments of H3K9ac on gene promoters were examined using ChIP-seq. A chronic constriction injury (CCI) rat model was used to evaluate the role of CGRP on astrocyte activation and H3K9ac signaling in CCI-induced neuropathic pain. Specific inhibitors were employed to delineate the involved signaling. RESULTS: Intrathecal injection of CGRP and CCI increased the number of astrocytes displaying H3K9ac in the spinal dorsal horn of rats. Treatment of CGRP was able to up-regulate H3K9ac and glial fibrillary acidic protein (GFAP) expression in astroglial cells. ChIP-seq data indicated that CGRP significantly altered H3K9ac enrichments on gene promoters in astroglial cells following CGRP treatment, including 151 gaining H3K9ac and 111 losing this mark, which mostly enriched in proliferation, autophagy, and macrophage chemotaxis processes. qRT-PCR verified expressions of representative candidate genes (ATG12, ATG4C, CX3CR1, MMP28, MTMR14, HMOX1, RET) and RTCA verified astrocyte proliferation. Additionally, CGRP treatment increased the expression of H3K9ac, CX3CR1, and IL-1ß in the spinal dorsal horn. CGRP antagonist and HAT inhibitor attenuated mechanical and thermal hyperalgesia in CCI rats. Such analgesic effects were concurrently associated with the reduced levels of H3K9ac, CX3CR1, and IL-1ß in the spinal dorsal horn of CCI rats. CONCLUSION: Our findings highly indicate that CGRP is associated with the development of neuropathic pain through astrocytes-mediated neuroinflammatory responses via H3K9ac in spinal dorsa horn following nerve injury. This study found that CGRP act on their astrocytic receptors and lead to H3K9 acetylation (H3K9ac), which are mainly associated with proliferation-, autophagy-, and inflammation-related gene expression. The number of astrocytes with H3K9ac expression is increased after nerve injury. Inhibition of CGRP attenuates the development of neuropathic pain, which was accompanied by the suppression of H3K9ac, CX3CR1, and IL-1ß expression in CCI rats.

Effect of Interleukin-1ß on Gene Expression Signatures in Schwann Cells Associated with Neuropathic Pain.

Interleukin-1ß (IL-1ß) plays a critical role in the development of neuropathic pain through activation of Schwann cells (SCs) after nerve injury. Here, we applied an RNA sequencing (RNA-seq) approach to identify the effect of IL-1ß on gene signatures of a rat SC line (RSC96) and the potential molecular mechanisms underlying the development of neuropathic pain. RNA-seq data demonstrated a total of 57 significantly differentially expressed genes (DEGs) with 35 up-regulated and 22 down-regulated between SCs treated with IL-1ß, and control SCs without treatment. Bioinformatics analysis showed that key upregulated DEGs included those associated with immune and inflammation-related processes, neurotrophin production and SC proliferation. Five proteins encoded by key upregulated DEGs (Ceacam1, Hap1, Irs3, Lgi4 and Mif) were further verified by Western blot. Consistent with the RNA-Seq results, the expression of key genes was confirmed in SCs by immunofluorescence of the chronic constriction injury (CCI) sciatic nerve in rats. Furthermore, we demonstrated that treatment with IL-1ß resulted in an increase in p38/ERK phosphorylation, and activators of p38/ERK enhanced the effect of IL-1ß on the expression some of the key genes, whereas p38/ERK inhibitors reversed these effects. In conclusion, the present study highlights key genes involved in the development of neuropathic pain through activation of SCs after nerve injury. Identification of these genes and subsequent evidence of their mediation by IL-1ß treatment promote our understanding of molecular mechanisms of nerve injury induced neuropathic pain, and highlight potential molecular targets for the treatment of neuropathic pain.

Calcitonin gene-related peptide regulates spinal microglial activation through the histone H3 lysine 27 trimethylation via enhancer of zeste homolog-2 in rats with neuropathic pain.

BACKGROUND: Calcitonin gene-related peptide (CGRP) as a mediator of microglial activation at the transcriptional level may facilitate nociceptive signaling. Trimethylation of H3 lysine 27 (H3K27me3) by enhancer of zeste homolog 2 (EZH2) is an epigenetic mark that regulates inflammatory-related gene expression after peripheral nerve injury. In this study, we explored the relationship between CGRP and H3K27me3 in microglial activation after nerve injury, and elucidated the underlying mechanisms in the pathogenesis of chronic neuropathic pain. METHODS: Microglial cells (BV2) were treated with CGRP and differentially enrichments of H3K27me3 on gene promoters were examined using ChIP-seq. A chronic constriction injury (CCI) rat model was used to evaluate the role of CGRP on microglial activation and EZH2/H3K27me3 signaling in CCI-induced neuropathic pain. RESULTS: Overexpressions of EZH2 and H3K27me3 were confirmed in spinal microglia of CCI rats by immunofluorescence. CGRP treatment induced the increased of H3K27me3 expression in the spinal dorsal horn and cultured microglial cells (BV2) through EZH2. ChIP-seq data indicated that CGRP significantly altered H3K27me3 enrichments on gene promoters in microglia following CGRP treatment, including 173 gaining H3K27me3 and 75 losing this mark, which mostly enriched in regulation of cell growth, phagosome, and inflammation. qRT-PCR verified expressions of representative candidate genes (TRAF3IP2, BCL2L11, ITGAM, DAB2, NLRP12, WNT3, ADAM10) and real-time cell analysis (RTCA) verified microglial proliferation. Additionally, CGRP treatment and CCI increased expressions of ITGAM, ADAM10, MCP-1, and CX3CR1, key mediators of microglial activation in spinal dorsal horn and cultured microglial cells. Such increased effects induced by CCI were suppressed by CGRP antagonist and EZH2 inhibitor, which were concurrently associated with the attenuated mechanical and thermal hyperalgesia in CCI rats. CONCLUSION: Our findings highly indicate that CGRP is implicated in the genesis of neuropathic pain through regulating microglial activation via EZH2-mediated H3K27me3 in the spinal dorsal horn.

Altered gene expression signatures by calcitonin gene-related peptide promoted mast cell activity in the colon of stress-induced visceral hyperalgesia mice.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is possibly involved in recruitment of mucosal mast cells (MCs) in the gut that may be associated with the development of irritable bowel syndrome (IBS), but the role of CGRP on the activation of MCs is still unknown. METHODS: Using RNA sequencing (RNA-seq), we examined differentially expressed genes (DEGs) in mouse MCs following CGRP treatment. The expression of key genes in colonic MCs and their relationship with CGRP-containing fibers were examined by immunofluorescence in chronic water-avoidance stress (WAS)-induced visceral hyperalgesia mice. KEY RESULTS: A total of 29 DEGs were found significantly changed with 28 upregulated and 1 downregulated following treatment of MCs with CGRP. Bioinformatics analysis showed that key higher DEGs included those associated with response to corticotropin-releasing hormone (CRH), regulation of transcription, MC activation, and proliferation. These processes are enriched for genes associated with stress-induced MC activation in IBS. Western blot verified changes in representative DEGs (Nr4a3, Crem, Gpr35, FosB, Sphlk1) and real-time cell analysis (RTCA) verified the MC proliferation. The vast majority of colonic MCs nearly CGRP-containing fibers in WAS mice overexpressed only Nr4a3 with little to no FosB, Gpr35, Sphlk1, or Crem expression. Nr4a3 knockdown may attenuate the promotion effect of CGRP on MC viability. CONCLUSIONS & INFERENCES: Our results suggest that CGRP is a critical regulator of key expressed genes in MC activation. Nr4a3 as a novel regulator of MC function may have an effect on stress-induced visceral hyperalgesia, and this may represent the novel target for drug development.

ChIP-seq Profiling Identifies Histone Deacetylase 2 Targeting Genes Involved in Immune and Inflammatory Regulation Induced by Calcitonin Gene-Related Peptide in Microglial Cells.

Calcitonin gene-related peptide (CGRP) is a mediator of microglial activation at the transcriptional level. The involvement of the epigenetic mechanism in this process is largely undefined. Histone deacetylase (HDAC)1/2 are considered important epigenetic regulators of gene expression in activated microglia. In this study, we examined the effect of CGRP on HDAC2-mediated gene transcription in microglial cells through the chromatin immunoprecipitation sequencing (ChIP-seq) method. Immunofluorescence analysis showed that mouse microglial cells (BV2) expressed CGRP receptor components. Treatment of microglia with CGRP increased HDAC2 protein expression. ChIP-seq data indicated that CGRP remarkably altered promoter enrichments of HDAC2 in microglial cells. We identified 1271 gene promoters, whose HDAC2 enrichments are significantly altered in microglia after CGRP treatment, including 1181 upregulating genes and 90 downregulating genes. Bioinformatics analyses showed that HDAC2-enriched genes were mainly associated with immune- and inflammation-related pathways, such as nitric oxide synthase (NOS) biosynthetic process, retinoic acid-inducible gene- (RIG-) like receptor signaling pathway, and nuclear factor kappa B (NF-κB) signaling pathway. The expression of these key pathways (NOS, RIG-I, and NF-κB) were further verified by Western blot. Taken together, our findings suggest that genes with differential HDAC2 enrichments induced by CGRP function in diverse cellular pathways and many are involved in immune and inflammatory responses.

Quantitative Proteomics Identify the Possible Tumor Suppressive Role of Protease-Activated Receptor-4 in Esophageal Squamous Cell Carcinoma Cells.

Exposure to carcinogens of tobacco smoke may result in methylation of protease-activated receptors-4 (PAR4) gene and further induces the loss of PAR4 expression, which is considered to be involved in carcinogenesis of esophageal squamous cell carcinoma (ESCC). Here we employed a TMT-based quantitative proteomic approach to identify PAR4-regulated changes of proteomic profiles in ESCC cells and to identify potentially therapeutic value. A total of 33 proteins were found significantly changed with 15 up-regulated and 18 down-regulated in PAR4-activating peptide (PAR4-AP) treated ESCC cells compared with controls. Bioinformatics analysis showed that key higher expressed proteins included those associated with apoptosis and tumor suppressor (e.g. CASP9), and lower expressed proteins included those associated with anti-apoptosis, autophagy and promoting cell proliferation (e.g. CHMP1B, PURA, PARG and HIST1H2AH). Western blot verified changes in five representative proteins including CASP9, CHMP1B, PURA, PARG and HIST1H2AH. Immunohistochemistry analysis showed that CHMP1B, PURA, PARG and HIST1H2AH expression in ESCC tissues were significantly higher than those in adjacent nontumorous tissues. Our findings will be helpful in further investigations into the functions and molecular mechanisms of PAR4 in ESCC.

Activation of PAR4 Upregulates p16 through Inhibition of DNMT1 and HDAC2 Expression via MAPK Signals in Esophageal Squamous Cell Carcinoma Cells.

A previous study showed that a downexpression of protease-activated receptor 4 (PAR4) is associated with the development of esophageal squamous cell carcinoma (ESCC). In this study, we explored the relationship between PAR4 activation and the expression of p16, and elucidated the underlying mechanisms in PAR4 inducing the tumor suppressor role in ESCC. ESCC cell lines (EC109 and TE-1) were treated with PAR4-activating peptide (PAR4-AP). Immunohistochemistry for DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) was performed in 26 cases of ESCC tissues. We found that DNMT1 and HDAC2 immunoreactivities in ESCC were significantly higher than those in adjacent noncancerous tissues. PAR4 activation could suppress DNMT1 and HDAC2, as well as increase p16 expressions, whereas silencing PAR4 dramatically increased HDAC2 and DNMT1, as well as reduced p16 expressions. Importantly, the chromatin immunoprecipitation-PCR (ChIP-PCR) data indicated that treatment of ESCC cells with PAR4-AP remarkably suppressed DNMT1 and HDAC2 enrichments on the p16 promoter. Furthermore, we demonstrated that activation of PAR4 resulted in an increase of p38/ERK phosphorylation and activators for p38/ERK enhanced the effect of PAR4 activation on HDAC2, DNMT1, and p16 expressions, whereas p38/ERK inhibitors reversed these effects. Moreover, we found that activation of PAR4 in ESCC cells significantly inhibited cell proliferation and induced apoptosis. These findings suggest that PAR4 plays a potential tumor suppressor role in ESCC cells and represents a potential therapeutic target of this disease.

Analyses of gene expression profiles in the rat dorsal horn of the spinal cord using RNA sequencing in chronic constriction injury rats.

BACKGROUND: Neuropathic pain is caused by damage to the nervous system, resulting in aberrant pain, which is associated with gene expression changes in the sensory pathway. However, the molecular mechanisms are not fully understood. METHODS: Wistar rats were employed for the establishment of the chronic constriction injury (CCI) models. Using the Illumina HiSeq 4000 platform, we examined differentially expressed genes (DEGs) in the rat dorsal horn by RNA sequencing (RNA-seq) between CCI and control groups. Then, enrichment analyses were performed for these DEGs using Gene Ontology (GO) function, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Hierarchical Cluster, and protein-protein interaction (PPI) network. RESULTS: A total of 63 DEGs were found significantly changed with 56 upregulated (e.g., Cxcl13, C1qc, Fcgr3a) and 7 downregulated (e.g., Dusp1) at 14 days after CCI. Quantitative reverse-transcribed PCR (qRT-PCR) verified changes in 13 randomly selected DEGs. GO and KEGG biological pathway analyses showed that the upregulated DEGs were mostly enriched in immune response-related biological processes, as well as 14 immune- and inflammation-related pathways. The downregulated DEGs were enriched in inactivation of mitogen-activated protein kinase (MAPK) activity. PPI network analysis showed that Cd68, C1qc, C1qa, Laptm5, and Fcgr3a were crucial nodes with high connectivity degrees. Most of these genes which have previously been linked to immune and inflammation-related pathways have not been reported in neuropathic pain (e.g., Laptm5, Fcgr3a). CONCLUSIONS: Our results revealed that immune and defense pathways may contribute to the generation of neuropathic pain after CCI. These mRNAs may represent new therapeutic targets for the treatment of neuropathic pain.

Downregulation of iNOS, IL-1ß, and P2X7 Expression in Mast Cells via Activation of PAR4 Contributes to the Inhibition of Visceral Hyperalgesia in Rats.

Protease-activated receptor 4 (PAR4) is implicated in the inhibition of visceral hyperalgesia. In the present study, the effects of PAR4 activation on visceral hypersensitivity and expression of inflammatory mediators, including interleukin-1ß (IL-1ß), P2RX7 purinergic receptor (P2X7), inducible nitric oxide synthase (iNOS), and tryptase, in mast cells (MCs) were investigated via in vivo and in vitro studies. The numbers of tryptase-positive MCs with extensive PAR4, P2X7, and iNOS expression were increased in the colons of visceral hyperalgesia rats compared with controls. Intracolonic administration of PAR4-activating peptide (PAR4-AP) significantly attenuated the visceral hypersensitivity to colorectal distention and reduced the iNOS, IL-1ß, P2X7, and tryptase protein and mRNA levels in the colonic mucosa. Treatment of rat bone marrow MCs (BMMCs) with PAR4-AP also reduced the iNOS, IL-1ß, P2X7, and tryptase protein and mRNA levels. ERK1/2 and p38 activators (t-butylhydroquinone, tBHQ, and U-46619) reversed the suppressive effect of PAR4 activation on IL-1ß and iNOS expression, whereas ERK1/2 and p38 inhibitors (PD98059 and SB203580) reversed the suppressive effect of PAR4 activation on P2X7 and tryptase expression. Our results indicate that the downregulation of inflammatory mediators, including iNOS, IL-1ß, P2X7, and tryptase, in MCs that are mediated by PAR4 activation could inhibit visceral hyperalgesia via the mitogen-activated protein kinase (MAPK) signal pathway.

Hepatitis B virus X protein influences enrichment profiles of H3K9me3 on promoter regions in human hepatoma cell lines.

We previously showed that hepatitis B virus (HBV) X protein (HBx) could promote the trimethylation of histone H3 lysine 9 (H3K9me3) to repress tumor suppressor genes in hepatocellular carcinoma (HCC). In this work, we analyze 23,148 human promoters using ChIP-chip to determine the effects of HBx on H3K9me3 enrichments in hepatoma cells with transfection of HBx-expressing plasmid. Immunohistochemistry for HBx and H3K9me3 was performed in 21 cases of HBV-associated HCC tissues. We identified that H3K9me3 immunoreactivity was significantly correlated with HBx staining in HCC tissues. ChIP-chip data indicated that HBx remarkably altered promoter enrichments of H3K9me3 in hepatoma cells. We identified 25 gene promoters, whose H3K9me3 enrichments are significantly altered in hepatoma cells transfected HBx-expressing plasmid, including 19 gaining H3K9m3, and six losing this mark. Most of these genes have not been previously reported in HCC, and BTBD17, MIR6089, ZNF205-AS1 and ZP1 have not previously been linked to cancer; only two genes (DAB2IP and ZNF185) have been reported in HCC. Genomic analyses suggested that genes with the differential H3K9me3 enrichments function in diverse cellular pathways and many are involved in cancer development and progression.

Protease-activated receptor 4 activation increases the expression of calcitonin gene-related peptide mRNA and protein in dorsal root ganglion neurons.

Accumulating evidence demonstrates that nociceptor activation evokes a rapid change in mRNA and protein levels of calcitonin gene-related peptide (CGRP) in dorsal root ganglion (DRG) neurons. Although the colocalization of CGRP and protease-activated receptor-4 (PAR4), a potent modulator of pain processing and inflammation, was detected in DRG neurons, the role of PAR4 activation in the expression of CGRP has not been investigated. In the present study, the expression of CGRP and activation (phosphorylation) of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in rat DRG neurons were measured by immunofluorescence, real-time PCR, and Western blotting after AYPGKF-NH2 (selective PAR4-activating peptide; PAR4-AP) intraplantar injection or treatment of cultured DRG neurons. The expression of CGRP in cultured DRG neurons was also assessed after treatment with AYPGKF-NH2 with preaddition of PD98059 (an inhibitor for ERK1/2 pathway). Results showed that PAR4-AP intraplantar injection or treatment of cultured DRG neurons evoked significant increases in DRG cells displaying CGRP immunoreactivity and cytoplasmic and nuclear staining for phospho-ERK1/2 (p-ERK1/2). Percentages of total DRG neurons expressing both CGRP and PAR4 or p-ERK1/2 also increased significantly at 2 hr after PAR4-AP treatment. Real-time PCR and Western blotting showed that PAR4-AP treatment significantly increased expression of CGRP mRNA and protein levels in DRG neurons. The PAR4 activation-evoked CGRP expression both at mRNA and at protein levels was significantly inhibited after p-ERK1/2 was inhibited by PD98059. These results provide evidence that activation of PAR4 upregulates the expression of CGRP mRNA and protein levels in DRG neurons via the p-ERK1/2 signal pathway.

Interleukin-1ß increased the expression of protease-activated receptor 4 mRNA and protein in dorsal root ganglion neurons.

Protease-activated receptor-4 (PAR4) is localized in primary sensory neurons and is believed to implicate in the modulation of nociceptive mechanisms. The pro-inflammatory cytokine interleukin-1ß (IL-1ß) is involved in the generation of hyperalgesia in pathological states such as neuropathy and inflammation. Previous studies have shown that IL-1ß enhances the expression of PAR4 in many cell types but the effect of this cytokine on primary sensory neuron PAR4 expression is less clear. In the present study, we evaluated in rat dorsal root ganglion (DRG) neurons the influence of IL-1ß on PAR4 mRNA and protein levels after IL-1ß intraplantar injection into the hind-paw or treatment of cultured DRG neurons. The expression of PAR4 in cultured DRG neurons was also assessed after treatment with IL-1ß with pre-addition of phorbol-12-myristate 13-acetate (PMA, a PKC activator) or chelerythrine chloride (a PKC inhibitor). We found that IL-1ß intraplantar injection into the hind-paw or long-term exposure of cultured DRG neurons to IL-1ß significantly increased the proportion of DRG neurons expressing PAR4 immunoreactivity. Real-time PCR and western blotting showed that IL-1ß treatment also significantly elevated PAR4 mRNA and protein levels in DRG neurons. This IL-1ß effect was enhanced in DRG neurons when DRG cultures were pre-treatment with the PMA. But pre-incubation with chelerythrine chloride strongly inhibited the IL-1ß-induced increase of PAR4 mRNA and protein levels. These results demonstrate that the expression of PAR4 mRNA and protein induced by IL-1ß is PKC signaling pathway dependent.