Takehiro Kaida

Interleukin-6 Synthesis Induced by Prostaglandin E2: Cross-Talk Regulation by Protein Kinase C

We previously showed that prostaglandin E2 (PGE2) stimulates multiple intracellular signaling pathways as follows: by activation of adenylate cyclase; phosphoinositide (PI)-hydrolyzing phospholipase C and phosphatidylcholine (PC)-hydrolyzing phospholipase D; and by induction of Ca2+ influx in osteoblast-like MC3T3-E1 cells. In this study, we investigated the effect of PGE2 on the synthesis of interleukin-6 (IL-6) and its regulatory mechanism in MC3T3-E1 cells. PGE2 significantly stimulated IL-6 secretion in a dose-dependent manner in the range between 1 nmol/L and 10 micromol/L. A23187, a calcium ionophore, or dibutyryl-cAMP significantly induced IL-6 secretion. The effect of a combination of A23187 and dibutyryl-cAMP on IL-6 secretion was additive. The depletion of extracellular Ca2+ by EGTA reduced the PGE2-induced IL-6 secretion. EP1 receptor antagonist inhibited the PGE2-induced IL-6 secretion. H-89, an inhibitor of cAMP-dependent protein kinase, decreased the PGE2-induced IL-6 secretion. EP2 receptor agonist alone stimulated IL-6 secretion. However, EP4 receptor antagonist had little effect on IL-6 secretion. Calphostin C, a specific inhibitor of protein kinase C (PKC), enhanced the secretion of IL-6 induced by PGE2. The stimulative effect of PGE2 on IL-6 secretion was significantly enhanced in PKC downregulated MC3T3-E1 cells. Pertussis toxin enhanced PGE2-induced IL-6 secretion. These results strongly suggest that PGE2 stimulates IL-6 synthesis through both Ca2+ mobilization from extracellular space via EP1 receptor and cAMP production via EP2 receptor in osteoblast-like cells, and that the PKC activation by PGE2 itself regulates oversynthesis of IL-6.

Inhibitory effects of propofol on intracellular signaling by endothelin-1 in aortic smooth muscle cells.

Background Blood pressure decreases when propofol is administered. However, the exact mechanism underlying the vascular effects of propofol has not yet been elucidated. Endothelin produced by vascular endothelial cells is a potent vasoactive peptide that elicits prolonged contraction of vascular smooth muscle cells. The effects of propofol on endothelin‐1‐induced intracellular signaling in an aortic smooth muscle cell line, A10 cells, were examined. Methods Cultured A10 cells were pretreated with propofol for 20 min and then stimulated with endothelin‐1. The effect of propofol on the endothelin‐1‐induced Ca2+ influx into A10 cells was evaluated by measuring intracellular45 Ca2+. The effects of propofol on the endothelin‐1‐induced activation of phosphatidylinositol‐hydrolyzing phospholipase C and phosphatidylcholine‐hydrolyzing phospholipase D were evaluated by measuring the formation of inositol phosphates and choline, respectively. The effect of propofol on endothelin‐1 binding to its receptor was determined by an [sup 125 I] endothelin‐1‐binding assay. Results Propofol inhibited the the endothelin‐1‐induced Ca2+ influx, but this was significant only at supuraclinical concentrations. The endothelin‐1‐stimulated formation of inositol phosphates was significantly suppressed by propofol. However, propofol had no effect on the formation of inositol phosphates induced by NaF, an activator of heterotrimeric guanosine triphosphate (GTP)‐binding proteins. Propofol inhibited the endothelin‐1‐induced formation of choline. Propofol had no effect on the binding of endothelin‐1 to its receptor. Conclusions These results suggest that propofol inhibits endothelin‐1‐induced intracellular signaling in vascular smooth muscle cells. The inhibitory effect of propofol might be exerted at a point between the endothelin‐1 receptor and its GTP‐binding protein. However, because all significant effects are observed at high concentrations, clinical relevance is unclear.

Involvement of protein kinase C activation in endothelin-1-induced secretion of interleukin-6 in osteoblast-like cells.

We previously reported that endothelin-1 (ET)-1 stimulates phospholipase D (PLD) independently of phosphoinositide hydrolysis in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of ET-1 on the secretion of interleukin-6 (IL-6) and the involvement of protein kinase C (PKC) activation in the IL-6 secretion in these cells. ET-1 significantly stimulated IL-6 secretion time-dependently up to 72 h. The stimulative effect was dose-dependent in the range between 1 nM and 1 microM. BQ123, a selective antagonist of endothelinA (ETA) receptor, inhibited the ET-1-induced IL-6 secretion. On the contrary, BQ788, a selective antagonist of endothelinB (ETB) receptor, had no effect. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, significantly stimulated IL-6 secretion. However, 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, did not affect IL-6 secretion. The effect of a combination of ET-1 and TPA on IL-6 secretion was not additive. Calphostin C, a specific PKC inhibitor, significantly inhibited the ET-1-induced IL-6 secretion. Both ET-1- and TPA-induced IL-6 secretion were reduced in PKC downregulated MC3T3-E1 cells. These results strongly suggest that ET-1 stimulates IL-6 secretion via ETA receptor in osteoblast-like cells and that PKC activation is involved in the ET-1-induced IL-6 secretion.

Effects of several agents on UVB- and UVA plus systemic fluoroquinolone-induced erythema of guinea pig skin evaluated by reflectance colorimetry.

The aim of this study was to clarify the mechanisms underlying the erythema of guinea pig skin induced by ultraviolet (UV) irradiation alone and in combination with a systemic fluoroquinolone (FQ). The effects of several drugs which may modify the actions of some inflammatory mediators and radicals possibly released in the inflamed site on the erythema were examined and compared in an objective and quantitative way by measuring the change in color of the irradiated skin, determined as the change in chroma (C*) with use of reflectance colorimetry. After confirming that the C* value increased in an irradiation dose-dependent manner and reached a plateau 1-2 h after irradiation of UVB alone or UVA coadministered with an FQ, Y-26611 (10 mg/kg, i.p.), guinea pigs were pretreated with indomethacin, butylated hydroxytoluene (BHT) or beta-carotene before, or treated with H1- or H2-receptor antagonist, superoxide dismutase or N omega-nitro-L- arginine methyl ester after UV irradiation, and their inhibitory effects against erythema were evaluated. It was suggested that there are some substantial differences between UVB- and UVA plus FQ-induced erythemas. Although histamine makes little contribution to both types of erythema, metabolites of arachidonic acid catalyzed by cyclooxygenase contribute more to UVB-induced erythema, whereas superoxides take more part in UVA plus FQ-induced erythema. Furthermore, nitric oxide seems to participate in both types of erythema; however, the pretreatment with BHT or beta-carotene was ineffective against both erythemas. From these results, interventions should be directed to powerfully scavenging radicals for prevention and treatment of UV plus FQ-induced phototoxicity.

GR144053, a fibrinogen receptor antagonist, enhances the suppression of neointima formation by losartan, an angiotensin II receptor antagonist, in the injured carotid artery of hamster

1 The present study investigated the inhibitory effect of losartan, a type 1 angiotensin II receptor (AT1) antagonist, and of combined treatment with losartan and GR144053, a fibrinogen receptor (GPIIb/IIIa) antagonist, on neointima formation subsequent to vascular injury in the hamster carotid artery. Vascular injury was achieved by a roughened‐tip 2F catheter and the neointimal area was measured up to 2 weeeks inducing the injury. 2 Compared to non‐treated hamsters (intimal area (IA)/internal elastic laminal area (IELA) ratio = 60.3±5.9%, n=12), losartan dissolved in drinking water (1, 3 and 10 mg kg−1 per day, n=8 each) reduced neointimal area dose‐dependently, a significant decrease (IA/IELA=39.7±5.6%) being attained with the highest dose when it was administered from 1 day before injury. However, neointima formation was not prevented even with the highest dose of losartan when the administration was started after injury. 3 When the administration of GR144053 (1.0 mg kg−1 per hour) via an implanted osmotic pump was started 30 min before the injury and continued for the next 2 weeks, no suppression of neointima formation was observed, although platelet aggregation evoked ex vivo by adenosine diphosphate (ADP) at the end of treatment period was efficiently inhibited. 4 In separate experiments in which 5‐bromo‐2‐deoxy‐Uridine (BrdU) was used to test smooth muscle cell (SMC) proliferation 1 and 7 days after injury, the ratio of SMC proliferation in the injured area was only slightly decreased by losartan when its administration was started after the injury, despite the marked reduction of SMC proliferation when treatment was started before the injury. Treatment with GR144053 as indicated above also significantly decreased the SMC proliferating index 1 day after the injury. 5 To examine the potential benefit of the coadministration of the GPIIb/IIIa antagonist with the AT1 receptor antagonist, GR144053 (1.0 mg kg−1 per hour) was combined with post‐injury treatment with losartan (10 mg kg−1 per day). This markedly reduced the proliferation of SMCs and significantly decreased the neointimal area (IA/IELA=31.2±4.6%) measured 2 weeks following the catheterization. 6 According to the results of a time‐dependent study in which GR144053 was given in combination with post injury treatment with losartan for 1, 3, 7 or 14 days, neointima formation could be reduced by treatment with GR144053 for just 7 days. 7 In conclusion, GR144053, a fibrinogen receptor antagonist, enhanced the inhibitory effect of losartan, an AT1 receptor antagonist, on neointima formation in the damaged carotid artery of hamsters.

Protein kinase C activation by interleukin (IL)‐1 limits IL‐1‐induced IL‐6 synthesis in osteoblast‐like cells: Involvement of phosphatidylcholine‐specific phospholipase C

We investigated the regulatory mechanism of interleukin‐6 (IL‐6) synthesis induced by interleukin‐1 (IL‐1) in osteoblast‐like MC3T3‐E1 cells. IL‐1 stimulated the secretion of IL‐6 in a dose‐dependent manner in the range between 0.1 and 100 ng/ml. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), significantly enhanced the IL‐1‐induced secretion of IL‐6. The stimulative effect of IL‐1 was markedly amplified in PKC down‐regulated MC3T3‐E1 cells. IL‐1 produced diacylglycerol in MC3T3‐E1 cells. IL‐1 had little effect on the formation of inositol phosphates and choline. On the contrary, IL‐1 significantly stimulated the formation of phosphocholine dose‐dependently. D‐609, an inhibitor of phosphatidylcholine‐specific phospholipase C, suppressed the IL‐1‐induced diacylglycerol production. The IL‐1‐induced IL‐6 secretion was significantly enhanced by D‐609. These results indicate that IL‐1 activates PKC via phosphatidylcholine‐specific phospholipase C in osteoblast‐like cells, and the PKC activation then limits IL‐6 synthesis induced by IL‐1 itself. J. Cell. Biochem. 67:103–111, 1997.

Effect of vitamin D3 on interleukin-6 synthesis induced by prostaglandins in osteoblasts

In previous studies, we have shown that prostaglandin F2alpha (PGF2alpha) stimulates interleukin-6 (IL-6) synthesis via activation of protein kinase C in osteoblast-like MC3T3-E1 cells, and that prostaglandin E1 (PGE1) induces the synthesis of IL-6 through protein kinase A activation. In the present study, we investigated the effect of vitamin D3 on IL-6 synthesis in MC3T3-E1 cells. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), an active form of vitamin D3, inhibited the IL-6 synthesis induced by PGF2alpha or PGE1. On the contrary, 24,25-dihydroxyvitamin D3, an inactive form of vitamin D3, had no effect. 1,25-(OH)2D3 did not affect the IL-6 synthesis stimulated by 12-O-tetradecanoyl-phorbol-13-acetate, an activator of protein kinase C. The IL-6 synthesis induced by cholera toxin or forskolin was significantly inhibited by 1,25-(OH)2D3. However, 1,25-(OH)2D3 had little effect on the IL-6 synthesis induced by dibutyryl cAMP. These results strongly suggest that 1,25-(OH)2D3, an active form of vitamin D3, inhibits IL-6 synthesis at both the protein kinase C pathway and the protein kinase A pathway in osteoblasts.

Retinoic acid suppresses interleukin-6 synthesis induced by prostaglandins in osteoblasts

We previously reported that prostaglandin E1 (PGE1) induces the synthesis of interleukin-6 (IL-6) via cAMP production in osteoblast-like MC3T3-E1 cells, and that, on the other hand, prostaglandin F2alpha (PGF2alpha) stimulates IL-6 synthesis via activation of protein kinase C. In the present study, we examined the effect of retinoic acid on IL-6 synthesis induced by these two prostaglandins in MC3T3-E1 cells. Retinoic acid inhibited the IL-6 synthesis induced by PGF2alpha or PGE1 in a dose-dependent manner in the range between 0.1 and 10 nM. Retinoic acid also suppressed the IL-6 synthesis stimulated by 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C. The IL-6 synthesis induced by cholera toxin, forskolin or dibutyryl cAMP was inhibited by retinoic acid. However, retinoic acid had little effect on the IL-6 synthesis induced by interleukin-1. These results indicate that retinoic acid inhibits IL-6 synthesis induced by prostaglandins in osteoblasts as follows: the inhibitory effect on the PGE1-induced IL-6 synthesis is exerted at a point downstream from cAMP, and the inhibitory effect on the PGF2alpha-induced IL-6 synthesis is exerted at a point downstream from protein kinase C.