Exogenous LL-37 but not homogenates of desquamated oral epithelial cells shows activity against Streptococcus mutans.

OBJECTIVE: The antimicrobial peptide hCAP18/LL-37 is detected in desquamated epithelial cells of human whole saliva, but the functional importance of this pool of hCAP18/LL-37 is not understood. Here, we assess the impact of homogenates of desquamated oral epithelial cells and exogenous, synthetic LL-37 on two oral bacteria: S. mutans and S. gordonii. MATERIAL AND METHODS: Desquamated epithelial cells of unstimulated whole saliva were isolated and cellular and extracellular levels of hCAP18/LL-37 analyzed by ELISA. Bacterial viability was determined by BacLight Live/Dead staining and confocal laser scanning microscopy. RESULTS: Desquamated oral epithelial cells harboured hCAP18/LL-37, and they spontaneously released/leaked the peptide to their medium. Exogenous, synthetic LL-37 showed cytotoxic activity against S. mutans but not S gordonii, suggesting that LL-37 acts differentially on these two types of oral bacteria. Homogenates of desquamated oral epithelial cells had no effect on S. mutans viability. Treatment with exogenous, synthetic LL-37 (8 and 10 µM) reduced S. mutans viability, whereas lower concentrations (0.1 and 1 µM) of the peptide lacked effect. CONCLUSIONS: Desquamated oral epithelial cells contain hCAP18/LL-37, but their cellular levels of hCAP18/LL-37 are too low to affect S. mutans viability, whereas exogenous, synthetic LL-37 has a strong effect on these bacteria.

What can we learn about functional importance of human antimicrobial peptide LL-37 in the oral environment from severe congenital neutropenia (Kostmann disease)?

The human antimicrobial peptide LL-37 is produced by neutrophils and epithelial cells, and the peptide can be detected in plasma as well as saliva. LL-37 is active against both gram-positive and gram-negative bacteria including oral pathogens such as Porphyromonas gingivalis and Streptococcus mutans. Besides its antimicrobial properties, LL-37 modulates the innate immune system, and furthermore, it also affects host cell viability. Although, both structural and functional properties of LL-37 have been extensively investigated, its physiological/pathophysiological importance in-vivo is not completely understood. In this review, Kostmann disease (morbus Kostmann) is highlighted since it may represent a LL-37 knockdown model which can provide new important information and insights about the functional role of LL-37 in the human in-vivo setting. Patients with Kostmann disease suffer from neutropenia, and although they are treated with recombinant granulocyte colony-stimulating factor (G-CSF) to normalize their levels of neutrophils, they lack or have very low levels of LL-37 in plasma, saliva and neutrophils. Interestingly, these patients suffer from severe periodontal disease, linking LL-37-deficiency to oral infections. Thus, LL-37 seems to play an important pathophysiological role in the oral environment antagonizing oral pathogens and thereby prevents oral infections.

Human host defense peptide LL-37 facilitates double-stranded RNA pro-inflammatory signaling through up-regulation of TLR3 expression in vascular smooth muscle cells.

OBJECTIVE: The importance of human host defense peptide LL-37 in vascular innate immunity is not understood. Here, we assess the impact of LL-37 on double-stranded RNA (dsRNA) signaling in human vascular smooth muscle cells. MATERIALS AND METHODS: Cellular import of LL-37 and synthetic dsRNA (poly I:C) were investigated by immunocytochemistry and fluorescence imaging. Transcript and protein expression were determined by qPCR, ELISA and Western blot. Knockdown of TLR3 was performed by siRNA. RESULTS: LL-37 was rapidly internalized, suggesting that it has intracellular actions. Co-stimulation with poly I:C and LL-37 enhanced pro-inflammatory IL-6 and MCP-1 transcripts several fold compared to treatment with poly I:C or LL-37 alone. Poly I:C increased IL-6 and MCP-1 protein production, and this effect was potentiated by LL-37. LL-37-induced stimulation of poly I:C signaling was not associated with enhanced import of poly I:C. Treatment with poly I:C and LL-37 in combination increased expression of dsRNA receptor TLR3 compared to stimulation with poly I:C or LL-37 alone. In TLR3 knockdown cells, treatment with poly I:C and LL-37 in combination had no effect on IL-6 and MCP-1 expression, showing loss of function. CONCLUSIONS: LL-37 potentiates dsRNA-induced cytokine production through up-regulation of TLR3 expression representing a novel pro-inflammatory mechanism.

Antimicrobial peptide LL-37 and its pro-form, hCAP18, in desquamated epithelial cells of human whole saliva.

The antimicrobial peptide LL-37 is active against oral bacteria and has been demonstrated to be present in human saliva, but its distribution in different fractions of saliva is not known. LL-37 is formed from its intracellular pro-form, hCAP18, in an extracellular enzymatic reaction catalyzed by proteinase 3 and kallikrein 5. Here, we prepared cell-containing and cell-free fractions of unstimulated human whole saliva by centrifugation after depolymerization of mucins with dithiothreitol, and measured the levels of hCAP18/LL-37 in these fractions using ELISA. Cellular expression of hCAP18/LL-37 was determined by western blotting and immunocytochemistry. The ELISA analyses demonstrated that both cells and cell-free saliva contained hCAP18/LL-37. Western blot analysis of cell-pellet homogenates showed a strong band corresponding to hCAP18 at the correct molecular weight and a weak band corresponding to LL-37. Phase-contrast and light microscopy revealed that the cells consisted of desquamated epithelial cells. These cells expressed cytoplasmic immunoreactivity for hCAP18/LL-37. The peripheral part of the cytoplasm, corresponding to the plasma membrane, was particularly rich in hCAP18/LL-37 immunoreactivity. No immunoreactivity was observed after omission of the primary antibody. We conclude that desquamated epithelial cells of human whole saliva contain antimicrobial hCAP18/LL-37, suggesting that these cells may take part in the innate immune system by harboring and releasing these peptides.

Odontoblast-like MDPC-23 cells produce pro-inflammatory IL-6 in response to lipoteichoic acid and express the antimicrobial peptide CRAMP.

Objective: Odontoblasts are thought to be involved in innate immunity but their precise role in this process is not fully understood. Here, we assess effects of lipopolysaccharide (LPS) and lipoteichoic acid (LTA), produced by Gram-negative and Gram-positive bacteria, respectively, on matrix metalloproteinase-8 (MMP-8), interleukin-6 (IL-6) and cathelin-related antimicrobial peptide (CRAMP) expression in odontoblast-like MDPC-23 cells.Material and methods: Gene activity and protein production was determined by quantitative real-time RT-PCR and ELISA, respectively. Cellular expression of CRAMP was determined by immunocytochemistry.Results: Stimulation with LTA (5 and 25 µg/ml) but not LPS (1 and 5 µg/ml) for 24 h enhanced IL-6 mRNA expression. The LTA-induced up-regulation of IL-6 mRNA levels was associated with increased IL-6 protein levels. Stimulation with either LPS or LTA for 24 h lacked effect on both MMP-8 transcript and protein expression. Immunocytochemistry disclosed that MDPC-23 cells expressed immunoreactivity for CRAMP. MDPC-23 cells showed mRNA expression for CRAMP, but stimulation with either LPS or LTA did not modulate CRAMP transcript expression.Conclusions: We show that MDPC-23 cells possess immune-like cell properties such as LTA-induced IL-6 production and expression of the antimicrobial peptide CRAMP, suggesting that odontoblasts may modulate innate immunity via these mechanisms.

Sesquiterpene lactones from Ambrosia arborescens Mill. inhibit pro-inflammatory cytokine expression and modulate NF-κB signaling in human skin cells.

BACKGROUND: Ambrosia arborescens has been used in Andean traditional medicine to reduce problems associated with various inflammatory diseases and conditions, although the underlying mechanism is unknown. HYPOTHESIS/PURPOSE: The sesquiterpene lactones (SLs) coronopilin and damsin, which are major secondary metabolites of A. arborescens, have anti-inflammatory activity by attenuation of IL-6 and MCP-1 expression and inhibition of NF-κB in human dermal fibroblasts (HDFa) and human keratinocytes (HaCaT). STUDY DESIGN: In order to confirm a high concentration of damsin and coronopilin in the plant material, a quantitative method was developed. The effect of the pure compounds on cytokine and NF-κB expression was examined, as well as their effects on HDFa and HaCaT cell morphology and viability. METHODS: Coronopilin and damsin were quantified by HPLC-DAD analysis, from EtOAc extracts of the aerial parts of A. arborescens. Cell morphology was investigated by phase-contrast microscopy and cell viability by the MTT assay. IL-6 and MCP-1 cytokine gene expression was assessed by quantitative real-time RT-PCR in LPS stimulated cells. The NF-κB pathway was studied through western blotting of the phosphorylated forms of p65 and p50/p105, as well as the non-phosphorylated IκB. Dexamethasone was used as positive control. RESULTS: Dry aerial parts contained 12.3  mg/g and 13.4  mg/g of coronopilin and damsin, respectively. Treatment with either compound (1-10 µM) for 24  h attenuated LPS-induced mRNA expression of the pro-inflammatory cytokine IL-6 and the chemokine MCP-1 in HDFa cells. The down-regulation of MCP-1 mRNA induced by coronopilin and damsin was confirmed on the protein level. Damsin reduced phosphorylated p65 and p105 subunits in HDFa cells. Neither coronopilin nor damsin affected HDFa cell morphology and viability within the used concentration range (1-10 µM). Also, in HaCaT cells, treatment with damsin (1-10 µM) for 24  h inhibited the MCP-1 expression, and damsin thereby attenuated cytokine expression both in HDFa and HaCaT cells. CONCLUSION: We show that coronopilin and damsin from A. arborescens inhibit pro-inflammatory IL-6 and MCP-1 expression in human skin cells via NF-κB inhibition, suggesting that they may be useful for antagonizing inflammatory conditions of the human skin.

Array profiling reveals contribution of Cthrc1 to growth of the denervated rat urinary bladder.

Bladder denervation and bladder outlet obstruction are urological conditions that cause bladder growth. Transcriptomic surveys in outlet obstruction have identified differentially expressed genes, but similar studies following denervation have not been done. This was addressed using a rat model in which the pelvic ganglia were cryo-ablated followed by bladder microarray analyses. At 10 days following denervation, bladder weight had increased 5.6-fold, and 2,890 mRNAs and 135 micro-RNAs (miRNAs) were differentially expressed. Comparison with array data from obstructed bladders demonstrated overlap between the conditions, and 10% of mRNAs changed significantly and in the same direction. Many mRNAs, including collagen triple helix repeat containing 1 ( Cthrc1), Prc1, Plod2, and Dkk3, and miRNAs, such as miR-212 and miR-29, resided in the shared signature. Discordantly regulated transcripts in the two models were rare, making up for <0.07% of all changes, and the gene products in this category localized to the urothelium of normal bladders. These transcripts may potentially be used to diagnose sensory denervation. Western blotting demonstrated directionally consistent changes at the protein level, with increases of, e.g., Cthrc1, Prc1, Plod2, and Dkk3. We chose Cthrc1 for further studies and found that Cthrc1 was induced in the smooth muscle cell (SMC) layer following denervation. TGF-ß1 stimulation and miR-30d-5p inhibition increased Cthrc1 in bladder SMCs, and knockdown and overexpression of Cthrc1 reduced and increased SMC proliferation. This work defines common and distinguishing features of bladder denervation and obstruction and suggests a role for Cthrc1 in bladder growth following denervation.

Secretory leukocyte protease inhibitor regulates human periodontal ligament cell production of pro-inflammatory cytokines.

OBJECTIVE: Regulation of immune-like cell properties of periodontal ligament (PDL) cells is not understood. We investigate the importance of secretory leukocyte protease inhibitor (SLPI) for production of pro-inflammatory cytokines in human PDL cells. MATERIALS AND METHODS: PDL cells were isolated from teeth extracted for orthodontic reasons. Cellular location of SLPI was investigated by immunocytochemistry. Cytokine transcript and protein expression were assessed by quantitative real-time RT-PCR and Western blotting. SLPI gene activity was knocked-down by siRNA. NF-κB signaling was assessed by measuring IκBα, and phosphorylated p65 and p105 protein expression. RESULTS: PDL cells showed cytoplasmic expression of SLPI. Cellular expression level of SLPI negatively correlated to LPS-induced stimulation of IL-6 and MCP-1. Both SLPI gene activity and protein were reduced by about 70% in PDL cells treated with SLPI siRNA compared to cells treated with non-coding construct. Treatment with SLPI siRNA was associated with up-regulation of both basal and LPS-stimulated IL-6, MCP-1 and TLRs mRNA expression. The up-regulation of MCP-1 transcript in SLPI siRNA-treated cells was confirmed on protein level. SLPI siRNA-treatment enhanced the phosphorylated NF-κB p105 protein expression. CONCLUSIONS: SLPI regulates PDL cell pro-inflammatory cytokine expression and modulates NF-κB signaling, suggesting that SLPI governs the immune cell-like properties of PDL cells.

Pyk2 inhibition promotes contractile differentiation in arterial smooth muscle.

Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage-dependent toward voltage-independent regulation of the intracellular calcium level, and inhibition of non-voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium-dependent signaling is the FAK-family non-receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium-dependent manner. We used the Pyk2 inhibitor PF-4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF-BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long-term (24-96 hr) treatment with PF-4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L-type calcium channel and large-conductance calcium-activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store-operated calcium influx were reduced after Pyk2 inhibition of growth-stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions.

Inhibition of microRNA-125a promotes human endothelial cell proliferation and viability through an antiapoptotic mechanism.

The microRNA-125a (miR-125a) is highly expressed in endothelial cells, but its role in vascular biology is not known. Endothelial cell proliferation and viability play an important role in endothelial healing, and we hypothesize that miR-125a regulates this process. The aim of the present study was to investigate if miR-125a controls human endothelial cell proliferation, viability and endothelial healing, and to assess the mechanisms involved. We showed that overexpression of miR-125a by transfection with miR-125a mimic reduced human umbilical vein endothelial cell (HUVEC) proliferation and viability, and stimulated apoptosis as demonstrated by a miR-125a-induced increase of the proportion of annexin V-positive cells monitored by flow cytometry. Moreover, we showed that the miR-125a mimic downregulated the antiapoptotic Bcl2 protein and upregulated caspase 3, suggesting that these two proteins represent molecular targets for miR-125a. Accordingly, transfection with miR-125a inhibitor, downregulating miR-125a expression, promoted HUVEC proliferation and viability, and reduced apoptosis. Importantly, transfection with miR-125a inhibitor promoted HUVEC tube formation in Matrigel, suggesting that reduction of miR-125a has a proangiogenic effect. In conclusion, downregulation of miR-125a through local transfection with miR-125a inhibitor might be a new way to enhance endothelial cell proliferation and viability, thereby promoting the reendothelialization observed in response to intimal injury.

The G protein-coupled estrogen receptor 1 (GPER1/GPR30) agonist G-1 regulates vascular smooth muscle cell Ca²âº handling.

The G protein-coupled estrogen receptor GPER1/GPR30 is implicated in blood pressure regulation but the mechanisms are not identified. Here, we hypothesize that GPER1 controls blood pressure by regulating vascular smooth muscle cell Ca(2+) handling. Treatment with the GPER1 agonist G-1 (in the µM concentration range) acutely reduced spontaneous and synchronous Ca(2+) spike activity in A7r5 vascular smooth muscle cells expressing mRNA for GPER1. Furthermore, G-1 (1 µM) attenuated the thromboxane A2 analogue U46619-stimulated Ca(2+) spike activity but had no effect on the U46619-induced increase in the basal level of Ca(2+). The voltage-sensitive L-type Ca(2+) channel blocker nifedipine (100 nM) reduced Ca(2+) spike activity similar to G-1. Pharmacological, but not physiological, concentrations of the estrogen 17ß-estradiol reduced Ca(2+) spike activity. The GPER1 antagonist G-15 blocked G-1-induced downregulation of Ca(2+) spike activity, supporting a GPER1-dependent mechanism. G-1 (1 µM) and nifedipine (100 nM) attenuated the 30-mM KCl-evoked rise in intracellular Ca(2+) concentration, suggesting that G-1 blocks inflow of Ca(2+) via voltage-sensitive Ca(2+) channels. In conclusion, we demonstrate that the GPER1 agonist G-1 regulates vascular smooth muscle cell Ca(2+) handling by lowering Ca(2+) spike activity, suggesting a role for this mechanism in GPER1-mediated control of blood pressure. © 2013 S. Karger AG, Basel.

Identification and characterization of new mechanisms in vascular oestrogen signalling.

Oestrogen exerts vasculoprotective effects in different experimental settings through inhibition of vascular smooth muscle cell proliferation, stimulation of nitric oxide production and attenuation of inflammation. Although these oestrogen-evoked beneficial effects have been attributed to oestrogen receptor alpha (ERα), also ER beta (ERß) and the novel ER G protein-coupled receptor 30 (GPR30)/G protein-coupled ER1 probably play significant roles in vascular oestrogen signalling. Oestrogen-evoked vasculoprotective effects are well documented in various experimental models, but the underlying mechanisms are still incompletely understood. The age hypothesis represents an interesting and promising model to explain the discrepancy between experimental data showing beneficial vascular effects of oestrogen treatment and the clinical findings on hormone replacement therapy obtained in big epidemiology surveys, where no protective effect from supplementation with oestrogen is observed. Identification of novel ERs expressed also in the vascular system offers exciting opportunities for the future to find and characterize the mechanisms behind oestrogen-evoked beneficial effects in vascular health and disease. Importantly, some vascular effects of pharmacological concentrations of oestrogen are ER-independent, suggesting that oestrogen besides its specific effects through ERα, ERß and GPR30 also affects vascular function via ER-independent mechanisms probably reflecting interaction of the hydrophobic oestrogen molecule with cell membrane properties. In this MiniReview, we focus on the importance of these different vascular ER subtypes in health and disease.

Differential effects of LPS from Escherichia coli and Porphyromonas gingivalis on IL-6 production in human periodontal ligament cells.

OBJECTIVE: Periodontal ligament (PDL) cells produce IL-6 upon stimulation with inflammation promoters, but the signaling pathways involved have not been characterized. This study investigates underlying mechanisms behind regulation of PDL cell IL-6 production by E. coli and P. gingivalis LPS. MATERIALS AND METHODS: Human PDL cells, endothelial cells and monocytes were stimulated with E. coli or P. gingivalis LPS in the presence or absence of pharmacological agents in order to disclose pathways involved in LPS signaling. Gene expression and cellular protein levels were assessed by quantitative real-time PCR and ELISA, respectively. RESULTS: Stimulation with LPS from E. coli (1 µg/ml) for 24 h enhanced PDL cell IL-6 expression several fold, demonstrated both on transcript and protein levels, but P. gingivalis LPS (1-5 µg/ml) had no effect. TLR2 mRNA was more highly expressed than TLR4 transcript in PDL cells. Treatment with the non-selective nitric oxide synthase inhibitor L-NAME (100 µM) reduced E. coli LPS-induced PDL cell IL-6 by 30%, while neither aminoguanidine (10 µM), an inhibitor of inducible nitric oxide synthase, nor estrogen (17ß-estradiol, 100 nM) influenced IL-6. Treatment with the glucocorticoid dexamethasone (1 µM) totally prevented the E. coli LPS-induced PDL cell IL-6. In endothelial cells, neither E. coli LPS nor P. gingivalis LPS promoted IL-6 production. In monocytes, serving as positive control, both E. coli and P. gingivalis LPS stimulated IL-6. CONCLUSIONS: E. coli LPS but not P. gingivalis LPS stimulates PDL cell IL-6 production through a glucocorticoid-sensitive mechanism involving nitric oxide formation, probably via endothelial nitric oxide synthase.

Beneficial effects of hormone replacement therapy on periodontitis are vitamin D associated.

BACKGROUND: Possible synergism between female sex hormones and vitamin D on periodontitis pathology has not been assessed. Here, the authors investigate effects of estrogen, progesterone, and vitamin D on periodontitis in a population-based sample and use cell studies to explore mechanistic explanations of the population-based findings. METHODS: The epidemiologic analysis uses cross-sectional data from the continuous National Health and Nutrition Examination Survey 2001 to 2004. The cross sections include 1,230 women aged 40 to 85 years who received a periodontal examination, responded to questions regarding hormone replacement therapy (HRT), and provided a blood sample for serum vitamin D assessments. For mechanistic cell culture studies, human monocytes were cultured with or without lipopolysaccharide (LPS), estradiol, progesterone, and/or 1,25-dihydroxyvitamin D3; and transcriptional activity of interleukin (IL)-6, IL-1ß, B lymphocyte chemoattractant (BLC), and regulated on activation normal T-cell expressed and secreted (RANTES) was assessed. RESULTS: HRT use (versus none) was associated with higher attachment levels and more teeth only among participants who were vitamin D sufficient (>20 ng/mL). The odds ratio for having moderate/severe periodontitis among users of HRT versus participants who did not use HRT was 0.69 among participants who were vitamin D sufficient and 1.19 in participants who were vitamin D deficient. LPS-induced IL-6, IL-1ß, and BLC expression was attenuated in human monocytes treated with estrogen and progesterone. Downregulation of IL-6 expression by estrogen and progesterone was potentiated when vitamin D was included. LPS-induced IL-6 and RANTES expression was decreased, and BLC expression was totally reversed, by vitamin D treatment. CONCLUSIONS: The association between HRT and clinical periodontal measures was strongest among women with high vitamin D levels. This association is plausibly mediated via an anti-inflammatory transcriptional mechanism.

The G protein-coupled oestrogen receptor 1 agonist G-1 disrupts endothelial cell microtubule structure in a receptor-independent manner.

The G protein-coupled oestrogen receptor GPER1, also known as GPR30, has been implicated in oestrogen signalling, but the physiological importance of GPER1 is not fully understood. The GPER1 agonist G-1 has become an important tool to assess GPER1-mediated cellular effects. Here, we report that this substance, besides acting via GPER1, affects the microtubule network in endothelial cells. Treatment with G-1 (3 µM) for 24 h reduced DNA synthesis by about 60 % in mouse microvascular endothelial bEnd.3 cells. Treatment with 3 µM G-1 prevented outgrowth of primary endothelial cells from mouse aortic explants embedded in Matrigel. Treatment with G-1 (0.3-3 µM) for 24 h disrupted bEnd.3 cell and HUVEC microtubule structure in a concentration-dependent manner as assessed by laser-scanning confocal immunofluorescence microscopy. G-1-induced (3 µM) disruption of microtubule was observed also after acute (3 and 6 h) treatment and in the presence of the protein synthesis inhibitor cycloheximide. Disruption of microtubules by 3 µM G-1 was observed in aortic smooth muscle cells obtained from both GPER1 knockout and wild-type mice, suggesting that G-1 influences microtubules through a mechanism independent of GPER1. G-1 dose dependently (10-50 µM) stimulated microtubule assembly in vitro. On the other hand, microtubules appeared normal in the presence of 10-50 µM G-1 as determined by electron microscopy. We suggest that G-1-promoted endothelial cell anti-proliferation is due in part to alteration of microtubule organization through a mechanism independent of GPER1. This G-1-promoted mechanism may be used to block unwanted endothelial cell proliferation and angiogenesis such as that observed in, e.g. cancer.

G protein-coupled oestrogen receptor 1 (GPER1)/GPR30: a new player in cardiovascular and metabolic oestrogenic signalling.

Oestrogens are important sex hormones central to health and disease in both genders that have protective effects on the cardiovascular and metabolic systems. These hormones act in complex ways via both genomic and non-genomic mechanisms. The genomic mechanisms are relatively well characterized, whereas the non-genomic ones are only beginning to be explored. Two oestrogen receptors (ER), ERα and ERß, have been described that act as nuclear transcription factors but can also associate with the plasma membrane and influence cytosolic signalling. ERα has been shown to mediate both anti-atherogenic effects and pro-survival effects in pancreatic ß-cells. In recent years, a third membrane-bound ER has emerged, G protein-coupled receptor 30 or G protein-coupled oestrogen receptor 1 (GPER1), which mediates oestrogenic responses in cardiovascular and metabolic regulation. Both GPER1 knock-out models and pharmacological agents are now available to study GPER1 function. These tools have revealed that GPER1 activation may have several beneficial effects in the cardiovascular system including vasorelaxation, inhibition of smooth muscle cell proliferation, and protection of the myocardium against ischaemia/reperfusion injury, and in the metabolic system including stimulation of insulin release and protection against pancreatic ß-cell apoptosis. Thus, GPER1 is emerging as a candidate therapeutic target in both cardiovascular and metabolic disease.

The GPER1 agonist G-1 attenuates endothelial cell proliferation by inhibiting DNA synthesis and accumulating cells in the S and G2 phases of the cell cycle.

G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor 1 (GPER1) is expressed in the vasculature, but the importance of vascular GPER1 remains to be clarified. Here we investigate effects of the GPER1 agonist G-1 on endothelial cell proliferation using mouse microvascular endothelial bEnd.3 cells. The bEnd.3 cells express mRNA for GPER1. The bEnd.3 cells expressed both ERα and ERß immunoreactivities. Treatment with G-1 reduced DNA synthesis and cell number with IC(50) values of about 2 µM. GPER1 siRNA prevented G-1-induced attenuation of DNA synthesis. G-1 accumulated cells in S and G2 phases of the cell cycle, suggesting that G-1 blocks transition between G2 and M. G-1 had no effect on DNA synthesis in COS-7 cells only weakly expressing GPER1 mRNA. 17ß-Estradiol had no effect on DNA synthesis in physiological concentrations (nM). The ER blocker ICI182780 reduced DNA synthesis with similar potency as G-1. Treatment with the ERK/MAP kinase inhibitor PD98059 had no effect on G-1-induced attenuation of DNA synthesis. G-1- induced antiproliferation was observed not only in bEnd.3 cells but also in human umbilical vein endothelial cells and HMEC-1 endothelial cells. We conclude that the GPER1 agonist G-1 attenuates endothelial cell proliferation via inhibition of DNA synthesis and by accumulation of cells in S and G2.

Down-regulation of endothelial cell estrogen receptor expression by the inflammation promoter LPS.

Endothelial cells express both estrogen receptor (ER) alpha and beta. The objective of this study was to investigate if and how mediators of inflammation regulate endothelial cell ERalpha and ERbeta expression. ERalpha and ERbeta transcript and protein expression were determined by real-time quantitative PCR and Western blotting, respectively, in endothelial cell line bEnd.3 cells stimulated with the inflammation promoter lipopolysaccharide (E. coli LPS). Stimulation with LPS (500 ng/ml and 10 microg/ml) for 4 days reduced both ERalpha and ERbeta mRNA levels. The glucocorticoid dexamethasone (1 microM) had no effect on LPS-induced attenuation of ERalpha and beta transcript expression. Full-length 66-67 kDa ERalpha protein was unaffected by 4 days stimulation with LPS, while the 46-kDa ERalpha isoform was reduced by about 20%. ERbeta protein was reduced by about 40% by LPS at 4 days. Treatment with 17beta-estradiol (E(2), 100 nM) for 4 days increased ERbeta mRNA by about 8 times but had no effect on ERalpha mRNA level. The E(2)-induced increase in ERbeta transcript was not associated with increased ERbeta protein. E(2) increased ERbeta mRNA expression also in the presence of LPS, suggesting that inflammation-induced impairment of ERbeta signalling is rescued by estrogen.

LPS induces GROalpha chemokine production via NF-kappaB in oral fibroblasts.

OBJECTIVE AND DESIGN: Chemotaxis of neutrophils from blood to the inflammation process plays an important role in development of periodontal inflammation. The novel chemokine GROalpha, also named CXCL1, is a strong chemoattractant for neutrophils. Data on production and regulation of GROalpha by oral fibroblasts have not previously been presented. MATERIALS AND METHODS: GROalpha mRNA and protein levels were determined in human periodontal ligament cells and mouse gingival fibroblasts by quantitative real-time PCR and ELISA. RESULTS: We disclose that both human periodontal ligament cells and mouse gingival fibroblasts produce GROalpha in response to LPS stimulation. Stimulation with LPS for 24 h increased both mRNA for GROalpha and GROalpha protein. The steroid hormone estrogen had no effect on LPS-induced GROalpha mRNA expression. Treatment with the glucocorticoid dexamethasone attenuated LPS-induced GROalpha production, and the NF-kappaB blocker MG 132 fully prevented LPS-induced GROalpha. CONCLUSIONS: Oral fibroblasts respond to LPS stimulation by increasing GROalpha production via the transcription factor NF-kappaB, suggesting that this mechanism may be involved in development of periodontal inflammation.

Deletion of the G protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice.

In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.