mRNA-based vaccines and therapeutics: an in-depth survey of current and upcoming clinical applications.

mRNA-based drugs have tremendous potential as clinical treatments, however, a major challenge in realizing this drug class will promise to develop methods for safely delivering the bioactive agents with high efficiency and without activating the immune system. With regard to mRNA vaccines, researchers have modified the mRNA structure to enhance its stability and promote systemic tolerance of antigenic presentation in non-inflammatory contexts. Still, delivery of naked modified mRNAs is inefficient and results in low levels of antigen protein production. As such, lipid nanoparticles have been utilized to improve delivery and protect the mRNA cargo from extracellular degradation. This advance was a major milestone in the development of mRNA vaccines and dispelled skepticism about the potential of this technology to yield clinically approved medicines. Following the resounding success of mRNA vaccines for COVID-19, many other mRNA-based drugs have been proposed for the treatment of a variety of diseases. This review begins with a discussion of mRNA modifications and delivery vehicles, as well as the factors that influence administration routes. Then, we summarize the potential applications of mRNA-based drugs and discuss further key points pertaining to preclinical and clinical development of mRNA drugs targeting a wide range of diseases. Finally, we discuss the latest market trends and future applications of mRNA-based drugs.

Epithelial cell adhesion molecule (EpCAM) regulates HGFR signaling to promote colon cancer progression and metastasis.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is known to highly expression and promotes cancer progression in many cancer types, including colorectal cancer. While metastasis is one of the main causes of cancer treatment failure, the involvement of EpCAM signaling in metastatic processes is unclear. We propose the potential crosstalk of EpCAM signaling with the HGFR signaling in order to govern metastatic activity in colorectal cancer. METHODS: Immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET) was conducted to explore the extracellular domain of EpCAM (EpEX) and HGFR interaction. Western blotting was taken to determine the expression of proteins in colorectal cancer (CRC) cell lines. The functions of EpEX in CRC were investigated by proliferation, migration, and invasion analysis. The combined therapy was validated via a tail vein injection method for the metastasis and orthotopic colon cancer models. RESULTS: This study demonstrates that the EpEX binds to HGFR and induces downstream signaling in colon cancer cells. Moreover, EpEX and HGF cooperatively mediate HGFR signaling. Furthermore, EpEX enhances the epithelial-to-mesenchymal transition and metastatic potential of colon cancer cells by activating ERK and FAK-AKT signaling pathways, and it further stabilizes active ß-catenin and Snail proteins by decreasing GSK3ß activity. Finally, we show that the combined treatment of an anti-EpCAM neutralizing antibody (EpAb2-6) and an HGFR inhibitor (crizotinib) significantly inhibits tumor progression and prolongs survival in metastatic and orthotopic animal models of colon cancer. CONCLUSION: Our findings illuminate the molecular mechanisms underlying EpCAM signaling promotion of colon cancer metastasis, further suggesting that the combination of EpAb2-6 and crizotinib may be an effective strategy for treating cancer patients with high EpCAM expression.

2,6-Difluorobenzamide derivatives as store-operated calcium channel (SOC) inhibitors.

The Ca2+ entry from store-operated Ca2+ channel (SOC) is involved in regulating colorectal cancer progression, such as cell migration. SOC activation is due to STIM1 translocation and interaction with Orai1 upon Ca2+ depletion in the ER. Numerous SOC inhibitors, like 2-APB, have been developed and demonstrated their inhibition effects in the preclinical stage. However, most currently used SOC inhibitors have higher cytotoxicity or opposite effects at different doses, and the drugs to target SOC in the clinic are lacking. In this study, a total of 13 difluorobenzamide compounds had been synthesized and examined the inhibitory effects on SOC with Ca2+ imaging and wound-healing migration assay. Among them, 2,6-Difluoro-N-(5-(4-fluorophenyl)pyridine-2-yl)benzamide (MPT0M004, 8a) demonstrated a prominent inhibitory ability on SOC. Furthermore, the cell proliferation assay results showed that MPT0M004 (8a) had lower cytotoxicity than 2-APB, the reference compound. In the pharmacokinetic study, MPT0M004 (8a) has a long half-life (T1/2 = 24 h) and lower daily dose administered intravenously with an oral bioavailability (F = 34%). Therefore, MPT0M004 (8a) has the potential to be a lead compound as a SOC inhibitor and further develop into a potential drug to treat colorectal cancer.

Aspirin and Sulindac act via different mechanisms to inhibit store-operated calcium channel: Implications for colorectal cancer metastasis.

Store-operated Ca2+ channel (SOC)-regulated Ca2+ entry is involved in inflammation and colorectal cancer (CRC) progression, but clinically applicable treatments targeting this mechanism are lacking. Recent studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) not only inhibit inflammation but they also suppress Ca2+ entry via SOC (SOCE). Therefore, delineating the mechanisms of SOCE inhibition by NSAIDs may lead to new CRC treatments. In this study, we tested eight candidate NSAIDs in Ca2+ imaging experiments and found that Aspirin and Sulindac were the most effective at suppressing SOCE. Furthermore, time-lapse FRET imaging using TIRF microscopy and ground state depletion (GSD) super-resolution (SR) imaging revealed that SOC was inhibited by Aspirin and Sulindac via different mechanisms. Aspirin quickly interrupted the STIM1-Orai1 interaction, whereas Sulindac mainly suppressed STIM1 translocation. Additionally, Aspirin and Sulindac both inhibited metastasis-related endpoints in CRC cells. Both drugs were used throughout the study at doses that suppressed CRC cell migration and invasion without altering cell survival. This is the first study to reveal the differential inhibitory mechanisms of Aspirin and Sulindac on SOC activity. Thus, our results shed new light on the therapeutic potential of Aspirin for CRC and SOCE-related diseases.

Genetic Association of the Functional WDR4 Gene in Male Fertility.

Infertility is one of the important problems in the modern world. Male infertility is characterized by several clinical manifestations, including low sperm production (oligozoospermia), reduced sperm motility (asthenozoospermia), and abnormal sperm morphology (teratozoospermia). WDR4, known as Wuho, controls fertility in Drosophila. However, it is unclear whether WDR4 is associated with clinical manifestations of male fertility in human. Here, we attempted to determine the physiological functions of WDR4 gene. Two cohorts were applied to address this question. The first cohort was the general population from Taiwan Biobank. Genomic profiles from 68,948 individuals and 87 common physiological traits were applied for phenome-wide association studies (PheWAS). The second cohort comprised patients with male infertility from Wan Fang Hospital, Taipei Medical University. In total, 81 male participants were recruited for the genetic association study. Clinical records including gender, age, total testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), total sperm number, sperm motility, and sperm morphology were collected. In the first cohort, results from PheWAS exhibited no associations between WDR4 genetic variants and 87 common physiological traits. In the second cohort, a total of four tagging single-nucleotide polymorphisms (tSNPs) from WDR4 gene (rs2298666, rs465663, rs2248490, and rs3746939) were selected for genotyping. We found that SNP rs465663 solely associated with asthenozoospermia. Functional annotations through the GTEx portal revealed the correlation between TT or TC genotype and low expression of WDR4. Furthermore, we used mouse embryonic fibroblasts cells from mwdr4 heterozygous (+/‒) mice for functional validation by western blotting. Indeed, low expression of WDR4 contributed to ROS-induced DNA fragmentation. In conclusion, our results suggest a critical role of WDR4 gene variant as well as protein expression in asthenozoospermia.

Evaluation for the Genetic Association between Store-Operated Calcium Influx Pathway (STIM1 and ORAI1) and Human Hepatocellular Carcinoma in Patients with Chronic Hepatitis B Infection.

Hepatocellular carcinoma (HCC) often develops from chronic hepatitis B (CHB) through replication of hepatitis B virus (HBV) infection. Calcium (Ca2+) signaling plays an essential role in HBV replication. Store-operated calcium (SOC) channels are a major pathway of Ca2+ entry into non-excitable cells such as immune cells and cancer cells. The basic components of SOC signaling include the STIM1 and ORAI1 genes. However, the roles of STIM1 and ORAI1 in HBV-mediated HCC are still unclear. Thus, long-term follow-up of HBV cohort was carried out in this study. This study recruited 3631 patients with chronic hepatitis (345 patients with HCC, 3286 patients without HCC) in a Taiwanese population. Genetic variants of the STIM1 and ORAI1 genes were detected using an Axiom CHB1 genome-wide array. Clinical associations of 40 polymorphisms were analyzed. Three of the STIM1 single-nucleotide polymorphisms (SNPs) (rs6578418, rs7116520, and rs11030472) and one SNP of ORAI1 (rs6486795) showed a trend of being associated with HCC disease (p < 0.05). However, after correction for multiple testing, none of the SNPs reached a significant level (q > 0.05); in contrast, neither STIM1 nor ORAI1 showed a significant association with HCC progression in CHB patients. Functional studies by both total internal reflection fluorescence images and transwell migration assay indicated the critical roles of SOC-mediated signaling in HCC migration. In conclusion, we reported a weak correlation between STIM1/ORAI1 polymorphisms and the risk of HCC progression in CHB patients.

MiR-22 as a metabolic silencer and liver tumor suppressor.

With obesity rate consistently increasing, a strong relationship between obesity and fatty liver disease has been discovered. More than 90% of bariatric surgery patients also have non-alcoholic fatty liver diseases (NAFLDs). NAFLD and non-alcoholic steatohepatitis (NASH), which are the hepatic manifestations of metabolic syndrome, can lead to liver carcinogenesis. Unfortunately, there is no effective medicine that can be used to treat NASH or liver cancer. Thus, it is critically important to understand the mechanism underlying the development of these diseases. Extensive evidence suggests that microRNA 22 (miR-22) can be a diagnostic marker for liver diseases as well as a treatment target. This review paper focuses on the roles of miR-22 in metabolism, steatosis, and liver carcinogenesis. Literature search is limited based on the publications included in the PubMed database in the recent 10 years.

Phthalate plasticizer di(2-ethyl-hexyl) phthalate induces cyclooxygenase-2 expression in gastric adenocarcinoma cells.

The phthalate plasticizer, di(2-ethyl-hexyl) phthalate (DEHP), and its derived metabolites are common anthropogenic environmental toxins, which are known to act as endocrine disruptors. Numerous studies have associated DEHP with disruption of sex hormones, abnormal development of reproductive organs, allergies, and inflammation. Its role in promoting inflammation has been reported by both human epidemiological and animal studies. In stomach tissue, chronic inflammation is known to accompany mucosal damage, and pave the way to gastritis, stomach ulcers, and ultimately gastric cancer. Eastern Asian populations possess the highest gastric cancer incidences in the world. Coincidentally, East Asia is one of the world's major sites for plastics manufacture and export. Thus, possible correlations between DEHP, a common plasticizer, and gastric cancer are of great interest. Our study revealed several critical findings. First, even at very low dosage, mimicking the residual plasticizer exposure, detrimental effects of DEHP on gastric cells can be detected. Second, gastric cells treated with DEHP increased cyclooxygenase-2 (COX-2) in a time-dependent manner. Third, promoter deletion studies revealed a critical role of nuclear factor-kappa B (NF-κB) for COX-2 gene responses. Finally, our results indicated that a low concentration of DEHP is able to trigger COX-2 activation via the extracellular signal-regulated kinase (ERK1/2) and NF-κB signaling pathway. Taken together, we demonstrate that very low doses of DEHP enhance the expression of the prototypical inflammatory gene, COX-2, in gastric cancer cells via ERK1/2 and NF-κB activation. This study provides important insights into the inflammatory process and damages associated with phthalate plasticizers exposure.

VAMP8, a vesicle-SNARE required for RAB37-mediated exocytosis, possesses a tumor metastasis suppressor function.

We previously identified a metastasis suppressor RAB37 small GTPase that regulated exocytosis of tissue inhibitor of metalloproteinases 1 (TIMP1) to suppress lung cancer metastasis. Here, we show that vesicle-associated membrane protein 8 (VAMP8), a v-SNARE (vesicle soluble N-ethylmaleimide-sensitive factor activating protein receptor), interacts with RAB37 and drives the secretion of TIMP1 to inhibit tumor metastases. Confocal and total internal reflection fluorescence microscopic images demonstrated that VAMP8 co-localized with RAB37 and facilitated trafficking of RAB37-TIMP1 vesicles. Reconstitution experiments using tail-vein injection and lung-to-lung metastasis in mice showed that VAMP8 was essential for RAB37-regulated vesicle trafficking of TIMP1 to suppress cancer metastasis. Lung cancer patients with low VAMP8 showed distant metastasis, poor overall survival and progression-free survival. Importantly, multivariate Cox regression analysis indicated that patients with low VAMP8/low RAB37 expression profile showed significantly high risk of death (hazard ratio = 3.42, P < 0.001) even after adjusting for tumor metastasis parameter. Our findings reveal that VAMP8 is a novel v-SNARE crucial for RAB37-mediated exocytic transport of TIMP1 to suppress lung tumor metastasis. VAMP8 possesses a tumor metastasis suppressor function with a prognostic value in lung cancer.

Dysregulation of Rab37-Mediated Cross-talk between Cancer Cells and Endothelial Cells via Thrombospondin-1 Promotes Tumor Neovasculature and Metastasis.

Purpose: Accumulating evidence indicates that factors secreted by cancer epithelial cells shape the tumor microenvironment to promote cancer invasion and metastasis. Recent studies also shed light on alterations of Rab small GTPase-mediated exocytosis in tumorigenesis. However, the mechanisms for Rab-mediated exocytosis in tumor microenvironment remain elusive. We aimed to investigate the interplay between Rab37-mediated exocytosis and tumor microenvironment, focusing on endothelial cell motility and angiogenesis.Experimental Design: We performed fluorescence IHC for Rab37, thrombospondin-1 (TSP1, an antiangiogenesis factor), and angiogenesis marker CD31 in 183 surgically resected esophageal squamous cell carcinoma (ESCC) patient samples. Cell migration, invasion, angiogenesis, and tumor metastasis were measured.Results: ESCC patients with low expression of Rab37 or TSP1 significantly correlated with high CD31 expression and were associated with worse progression-free survival. The multivariate Cox regression analysis showed that concordant low expression of both Rab37 and TSP1 was an independent prognostic factor of ESCC patients. Rab37-mediated exocytosis of TSP1 led to the inhibition of neovasculature in vitro and in vivo Secreted TSP1 from cancer cells with Rab37 exocytic function inhibited the p-FAK/p-paxillin/p-ERK migration signaling in both cancer epithelial cells and their surrounding endothelial cells. Dysfunction of Rab37 or loss of TSP1 abrogated the suppressive effects on angiogenesis and metastasis.Conclusions: Our findings suggest that Rab37-mediated TSP1 secretion in cancer cells suppresses metastasis and angiogenesis via a cross-talk with endothelial cells and reveal a novel component of the vesicular exocytic machinery in tumor microenvironment and tumor progression. Dysregulation of Rab37/TSP1 axis has clinical implications for prognosis prediction. Clin Cancer Res; 23(9); 2335-45. ©2016 AACR.

Small GTPase Rab37 targets tissue inhibitor of metalloproteinase 1 for exocytosis and thus suppresses tumour metastasis.

Rab small GTPases are master regulators of membrane trafficking and guide vesicle targeting. Recent publications show that Rab-controlled trafficking pathways are altered during tumorigenesis. However, whether any of the Rabs plays a metastasis suppressor role is least explored. Here we address the metastasis suppressive function of human Rab37 (hRAB37) using secretomics, cell, animal and clinical analyses. We show that tissue inhibitor of metalloproteinase 1 (TIMP1), a secreted glycoprotein that inhibits extracellular matrix turnover, is a novel cargo of hRAB37. hRAB37 regulates the exocytosis of TIMP1 in a nucleotide-dependent manner to inactivate matrix metalloproteinase 9 (MMP9) migration axis in vitro and in vivo. Dysfunction of hRAB37 or TIMP1 abrogates metastasis suppression. Lung cancer patients with metastasis and poor survival show low hRAB37 protein expression coinciding with low TIMP1 in tumours. Our findings identify hRAB37 as a novel metastasis suppressor Rab that functions through the TIMP1-MMP9 pathway and has significant prognostic power.

Cox-2 gene overexpression in ureteral stump urothelial carcinoma following nephrectomy for renal cell carcinoma: a case report.

INTRODUCTION: A primary ureteral stump tumor after a nephrectomy is rare; urothelial carcinoma of the ureteral stump after a nephrectomy for renal cell carcinoma is even rarer. A thorough review of the literature indicated that only seven cases have previously been reported. In this study, we report the first Taiwanese case of urothelial carcinoma of the ureteral stump after a nephrectomy. It is also the first female case in the literature. The relationship between inflammatory genes, medication history and ureteral stump carcinoma after a nephrectomy for renal cell carcinoma has not been reported. CASE PRESENTATION: A 72-year-old Asian Taiwanese women with chronic hepatitis C, liver cirrhosis and chronic kidney disease underwent a hand-assisted laparoscopic radical nephrectomy in 2001 due to renal cell carcinoma. Nine years later, she was diagnosed with ureteral stump urothelial carcinoma. Genetic and medication surveys were performed. Importantly, our patient had taken Chinese herbal drugs for more than 10 years and the inflammatory gene, Cox-2, was highly expressed in this patient. This is the first report to study the relationship between the Cox-2 gene and ureteral stump carcinoma after a nephrectomy for renal cell carcinoma. CONCLUSION: Long-term multiple use of Chinese herbal drugs could be one of the important risk factors for developing urothelial cancer. Close functional coupling between Chinese herbal drugs, Cox-2 gene activation and urothelial cancer should be further investigated.

Involvement of store-operated calcium signaling in EGF-mediated COX-2 gene activation in cancer cells.

Growing evidence shows that chronic inflammation drives the progression of colorectal cancer (CRC). Cyclooxygenase-2 (COX-2) is one of the most important inflammatory genes involved in solid tumor metastasis. Epidermal growth factor receptor (EGFR) also plays a key role in cancer cell development. We compared the expression levels of EGFR and COX-2 between tumor and normal tissues from 20 CRC patients and studied the molecular mechanism of EGFR-mediated COX-2 gene expression in cancer cells. Our results indicated that COX-2 expression was markedly increased after EGF stimulation. COX-2 promoter analysis indicated the involvement of cyclic AMP-responsive element (CRE) and nuclear factor of activated T cells/nuclear factor interleukin-6 (NFAT/NF-IL6)-binding sites in EGF-mediated signaling pathways. Furthermore, EGF-mediated COX-2 activation was prevented by 2-aminoethoxydiphenyl borate (2-APB), a store-operated Ca(2+) channel inhibitor. Transfection of siRNA against ORAI1 or STIM1, the key regulators of store-operated Ca(2+) channels, showed significant inhibitory effects on EGF-mediated COX-2 expression. In conclusion, store-operated Ca(2+) entry is involved in the activation of transcription factors (CREB/NFAT) that are responsible for delivering EGF-mediated signals to evoke inflammatory cascades and is eventually related to CRC tumorigenesis.

Orai1/CRACM1 overexpression suppresses cell proliferation via attenuation of the store-operated calcium influx-mediated signalling pathway in A549 lung cancer cells.

BACKGROUND: Orai1/CRACM1 is a principal component of the store-operated calcium channels. Store-operated calcium influx is highly correlated with inflammatory reactions, immunological regulation, and cell proliferation. Epidermal growth factor (EGF), which plays an important role in the regulation of cell proliferation, can activate store-operated calcium channels. However, the consequences of Orai1/CRACM1 overexpression in EGF-mediated lung cancer cells growth are not known. METHODS: To investigate the role of Orai1/CRACM1 in EGF-mediated lung cancer cell proliferation, Orai1/CRACM1 plasmids were transfected into cells by lipofection. A cell proliferation assay, immunofluorescence staining, flow cytometry, and real-time polymerase chain reaction were employed to monitor cell proliferation. The calcium influx signals were investigated using a fluorescent-based calcium assay. RESULTS: Transfection of Orai1/CRACM1 plasmids resulted in the inhibition of EGF-mediated cell proliferation. ERK1/2 and Akt phosphorylation were inhibited by Orai1/CRACM1 overexpression. Expression of the cell cycle modulator p21 was induced in the Orai1/CRACM1-overexpressing cells, whereas the expression of cyclin D3 was reduced. Flow cytometry revealed that overexpression of Orai1/CRACM1 resulted in G0/G1 cell cycle arrest. Importantly, Orai1/CRACM1 overexpression significantly attenuated EGF-mediated store-operated calcium influx. In addition, application of 2-APB, a store-operated calcium channel inhibitor, resulted in the inhibition of EGF-mediated cancer cell proliferation. CONCLUSIONS: We conclude that Orai1/CRACM1 overexpression attenuates store-operated Ca(2+) influx that in turn blocks EGF-mediated proliferative signaling and drives cell cycle arrest.

Association of ORAI1 haplotypes with the risk of HLA-B27 positive ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammation of the sacroiliac joints, spine and peripheral joints. The aetiology of ankylosing spondylitis is still unclear. Previous studies have indicated that genetics factors such as human leukocyte antigen HLA-B27 associates to AS susceptibility. We carried out a case-control study to determine whether the genetic polymorphisms of ORAI1 gene, a major component of store-operated calcium channels that involved the regulation of immune system, is a susceptibility factor to AS in a Taiwanese population. We enrolled 361 AS patients fulfilled the modified New York criteria and 379 controls from community. Five tagging single nucleotides polymorphisms (tSNPs) at ORAI1 were selected from the data of Han Chinese population in HapMap project. Clinical statuses of AS were assessed by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), and Bath Ankylosing Spondylitis Global Index (BAS-G). Our results indicated that subjects carrying the minor allele homozygote (CC) of the promoter SNP rs12313273 or TT homozygote of the SNP rs7135617 had an increased risk of HLA-B27 positive AS. The minor allele C of 3'UTR SNP rs712853 exerted a protective effect to HLA-B27 positive AS. Furthermore, the rs12313273/rs7135617 pairwise allele analysis found that C-G (OR 1.69, 95% CI 1.27, 2.25; p = 0.0003) and T-T (OR 1.75, 95% CI 1.36, 2.27; p<0.0001) haplotypes had a significantly association with the risk of HLA-B27-positive AS in comparison with the T-G carriers. This is the first study that indicate haplotypes of ORAI1 (rs12313273 and rs7135617) are associated with the risk of HLA-B27 positive AS.

ITPKC single nucleotide polymorphism associated with the Kawasaki disease in a Taiwanese population.

Kawasaki disease (KD) is characterized by systemic vasculitis with unknown etiology. Previous studies from Japan indicated that a gene polymorphism of ITPKC (rs28493229) is responsible for susceptibility to KD. We collected DNA samples from 1,531 Taiwanese subjects (341 KD patients and 1,190 controls) for genotyping ITPKC. In this study, no significant association was noted for the ITPKC polymorphism (rs28493229) between the controls and KD patients, although the CC genotype was overrepresented. We further combined our data with previously published case/control KD studies in the Taiwanese population and performed a meta-analysis. A significant association between rs28493229 and KD was found (Odds Ratio:1.36, 95% Confidence Interval 1.12-1.66). Importantly, a significant association was obtained between rs28493229 and KD patients with aneurysm formation (P = 0.001, under the recessive model). Taken together, our results indicated that C-allele of ITPKC SNP rs28493229 is associated with the susceptibility and aneurysm formation in KD patients in a Taiwanese population.

Histamine regulates cyclooxygenase 2 gene activation through Orai1-mediated NFκB activation in lung cancer cells.

Histamine, an important chemical mediator, has been shown to regulate inflammation and allergic responses. Stimulation of histamine receptors results in a significant increase in cytoplasmic Ca(2+), which could be mediated by inositol trisphosphate (IP(3))-dependent store-operated Ca(2+) channels (SOC). However, the link between histamine-mediated signaling and activation of inflammatory genes such as cyclooxygenase 2 (COX-2) is still unknown. Our study indicated that the COX-2 protein was highly expressed in human lung cancer cells. Following stimulation with 10 µM of histamine, both store-operated Ca(2+) entry (SOCE) and COX-2 gene expression were evoked. Histamine-mediated COX-2 activation can be prevented by 2-APB and SKF-96365, SOC channel inhibitors. In addition, deletion analysis of the COX-2 promoter suggested that the region between -80 bp and -250 bp, which contains NFκB binding sites, is the key element for histamine-mediated signaling. Knocking down ORAI1, one of the essential molecules of store-operated calcium channels, attenuated histamine-mediated COX-2 expression and NFκB activation. These results indicated that ORAI1-mediated NFκB activation was an important signaling pathway, responsible for transmitting histamine signals that trigger inflammatory reactions.

Divalent lead cations induce cyclooxygenase-2 gene expression by epidermal growth factor receptor/nuclear factor-kappa B signaling in A431carcinoma cells.

Divalent lead cations (Pb²+) are toxic metal pollutants that may contribute to inflammatory diseases in people and animals. Human vascular smooth muscle cells in culture respond to low concentrations of Pb²+ ions by activating mediators of inflammation via the plasma membrane epidermal growth factor receptor (EGFR). These include cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 as well as the hormone-like lipid compound prostaglandin E2. To further clarify the mechanism by which Pb²+ induces such mediators of inflammation, we tested human epidermoid carcinoma cell line A431 that expresses high levels of EGFR. Reverse transcription PCR and western blots confirmed A431 cells treated with a low concentration (1 µM) of Pb²+ in the form of lead (II) nitrate increased expression of COX-2 mRNA and its encoded protein in a time-dependent manner. Promoter deletion analysis revealed the transcription factor known as nuclear factor-kappa B (NF-κB) was a necessary component of the COX-2 gene response. NF-κB inhibitor BAY 11-7082 suppressed Pb²+-induced COX-2 mRNA expression, and EGFR inhibitors AG1478 and PD153035 as well as EGFR small interfering RNA reduced the coincident nuclear translocation of NF-κB. Our findings support the hypothesis that low concentrations of Pb²+ ions incite inflammation by inducing COX-2 gene expression via the EGFR/NF-κB signal transduction pathway.

Involvement of the epidermal growth factor receptor in Pb²+-induced activation of cPLA2/COX-2 genes and PGE2 production in vascular smooth muscle cells.

Lead (Pb²+) is one of the most common heavy metal pollutants, which can cause chronic cardiovascular diseases. To clarify the mechanism by which Pb²+ induces inflammatory reactions, we examined the expression of inflammatory genes including encoding cyclooxygenase-2 (COX-2), cytosolic phospholipase A2 (cPLA2), and their down stream product prostaglandin E2 (PGE2) in CRL1999 cells that is a vascular smooth muscle cell line from human aorta. The expression of COX-2/cPLA2 genes and PGE2 secretion was increased markedly after cells were exposed to 1 µM Pb²+. PD098059, a MEK inhibitor, suppressed Pb²+-mediated inflammatory reactions; this indicates the involvement of the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Furthermore, Pb²+-induced activation of the COX-2/cPLA2 genes was inhibited by both epidermal growth factor receptor (EGFR) inhibitors (AG1478 and PD153035) and EGFR siRNA. Short-term stimulation with Pb²+ induced EGFR phosphorylation at the Tyr residue (position, 1173). Importantly, overexpression of EGFR resulted in a significant potentiation effect on Pb²+-induced gene expression. Taken together, our results indicate that 1 µM Pb²+ can induce PGE2 secretion by upregulating the transcription of COX-2/cPLA2 genes. EGFR is the key target in the plasma membrane responsible for transmitting Pb²+ signals in order to trigger downstream inflammatory cascades.