Effect of gammalinolenic acid, dihomogammalinolenic acid, ascorbyl-6-gammalinolenic acid, and ascorbyl-6-dihomo gammalinolenic acid on histamine- and methacholine-induced contraction of the isolated guinea pig tracheal chain

Prostaglandin E2 (PGE2) is known to autoamplify its production in the osteoblasts through the induction of prostaglandin G/H synthase-2 (PGHS-2), which is the inducible form of the rate-limiting enzyme in PG synthesis, PGHS. To elucidate the cellular mechanism mediating this process, we have employed the PGE2 analogs, which are specific agonists for four subtypes of PGE receptor, and studied the potency of these analogs to induce PGHS-2 mRNA in mouse osteoblastic MC3T3-E1 cells. The induction was mainly observed by 17-phenyl-omega-trinor PGE2 (EP1 agonist) and sulprostone (EP3/EP1 agonist), but not by butaprost (EP2 agonist) or 11-deoxy PGE1 (EP4/EP2 agonist). Since EP3 subtype was undetectable in MC3T3-E1 cells, these data indicate that PGHS-2 mRNA induction is mediated through EP1 subtype of PGE receptor in MC3T3-E1 cells. PGE2 production determined by radioimmunoassay was also increased by 17-phenyl-omega-trinor PGE2 and sulprostone. The autoamplification of PGE2 production is considered to be important in elongating the otherwise short-lived PGE2 action in certain physiological conditions such as mechanical stress and fracture healing, as well as the pathological inflammatory bone loss. The observations in the present study provide us with the better understanding of these processes.     

Prostaglandin E receptor subtypes in cultured rat microglia and their role in reducing lipopolysaccharide-induced interleukin-1beta production

Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. Recent work has shown that PGE2, while generally considered a proinflammatory molecule, reduces microglial activation and thus has an antiinflammatory effect on these cells. To gain further insight to the mechanisms by which PGE2 influences the activation of microglia, we investigated PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, expression and function in cultured rat microglia. RT-PCR showed the presence of the EP1 and EP2 but not EP3 and EP4 receptor subtypes. Sequencing confirmed their identity with previously published receptor subtypes. PGE2 and the EP1 agonist 17-phenyl trinor PGE2 but not the EP3 agonist sulprostone elicited reversible intracellular [Ca2+] increases in microglia as measured by fura-2. PGE2 and the EP2/EP4-specific agonists 11-deoxy-PGE1 and 19-hydroxy-PGE2 but not the EP4-selective agonist 1-hydroxy-PGE1 induced dose-dependent production of cyclic AMP (cAMP). Interleukin (IL)-1beta production, a marker of activated microglia, was also measured following lipopolysaccharide exposure in the presence or absence of the receptor subtype agonists. PGE2 and the EP2 agonists reduced IL-1beta production. IL-1beta production was unchanged by EP1, EP3, and EP4 agonists. The adenylyl cyclase activator forskolin and the cAMP analogue dibutyryl cAMP also reduced IL-1beta production. Thus, the inhibitory effects of PGE2 on microglia are mediated by the EP2 receptor subtype, and the signaling mechanism of this effect is likely via cAMP. These results show that the effects of PGE2 on microglia are receptor subtype-specific. Furthermore, they suggest that specific and selective manipulation of the effects of PGs on microglia and, as a result, brain function may be possible.     

Body/self awareness and interpersonal communications: fundamental components of reproductive health awareness

We investigated the role of Sonic hedgehog (SHH) in osteoblast differentiation and bone formation. The numbers of ALP-positive cells in the mouse fibroblastic cell line C3H10T1/2 and the mouse osteoblastic cell line MC3T3-E1 were increased by co-culture with chicken fibroblasts transfected with chicken Shh cDNA encoding amino-terminal peptide (Shh-N). The conditioned medium of Shh-N-RCAS-transfected chicken fibroblast cultures also significantly increased ALP activity in both C3H10T1/2 and MC3T3-E1 cells. Intramuscular transplantation of Shh-N-RCAS-transfected chicken fibroblasts into athymic mice induced ectopic bone formation. These results indicate that SHH induces osteoblast differentiation and ectopic bone formation.     

Technical features of a CCD video camera system to record cardiac fluorescence data

Prostaglandin E2 (PGE2) is a potent local mediator of cell growth and differentiation in various tissues. The receptors for PGE2 have been classified into four pharmacological subtypes, EP1, EP2, EP3, and EP4, based on the responses to selective agonists and antagonists. We have cloned a functional cDNA for the rat EP2 receptor subtype from a rat lung cDNA library. The rat EP2 receptor cDNA encodes 357 amino acid residues having high homology with the human and mouse EP2 receptors and containing seven putative transmembrane domains. In COS-7 cells transfected with rat EP2 cDNA, specific [3H]PGE2 binding was found with a dissociation constant of 14.9 nM, and this binding was inhibited by unlabeled PGE2 and PGE2 alpha. PGE2 and butaprost, an EP2 selective agonist, were effective in increasing the cAMP level in the COS-7 cell transfectants. Northern blot and RT-PCR analysis showed widespread distribution of the EP2 receptor in various tissues. Higher EP2 expression was found in fetal long bones and calvariae than in adult by RT-PCR and in situ hybridization, suggesting a role for this receptor in rapidly growing skeletal tissue.     

Potentiation of retinoic acid-induced differentiation of HL-60 cells by prostaglandin EP2 receptor

Human promyeloid HL-60 cells are differentiated by all-trans retinoic acid (RA) to granulocytes, and prostaglandin (PG) E2 potentiates the RA-induced differentiation. Here we examined which subtype of PGE receptors was involved in this potentiating activity of PGE2. Northern blot analysis demonstrated that HL-60 cells expressed three subtypes of PGE receptor, EP2, EP3, and EP4. Among various EP agonists, and EP2-selective agonist, butaprost, preferentially potentiated the RA-induced differentiation of HL-60 cells. Butaprost not only decreased the half-maximal concentration of RA but also increased the maximal level of the differentiation. Butaprost concentration-dependently stimulated the cAMP formation, and 8-Br-cAMP strongly potentiated the RA-induced differentiation. These results demonstrate that the EP2 receptor enhances the RA-induced differentiation of HL-60 cells via stimulation of adenylate cyclase.     

Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes

We examined the contribution of specific EP receptors in regulating cell growth. By RT-PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE2 receptor subtypes associated with cAMP signaling (EP2, EP3, and small amounts of EP4). In actively growing, non-confluent primary keratinocyte cultures, the EP2 and EP4 selective agonists, 11-deoxy PGE1 and 1-OH PGE1, caused complete reversal of indomethacin-induced growth inhibition. The EP3/EP2 agonist (misoprostol), and the EP1/EP2 agonist (17-phenyl trinor PGE2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP2 and/or EP4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP3 receptors by sulprostone, which is virtually devoid of agonist activity at EP2 or EP4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanisms, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.     

Cloning of mouse diastrophic dysplasia sulfate transporter gene induced during osteoblast differentiation by bone morphogenetic protein-2

Although intensive studies have been directed at understanding osteoblastic differentiation, the molecular mechanisms are still unclear. In this study, we describe a cDNA that encodes a sulfate transporter that was cloned as a gene induced in osteoblast precursor cells in association with osteoblastic differentiation. Based on the fact that bone morphogenetic protein-2 (BMP-2) induces osteoblastic phenotypes in immature mouse fibroblastic C3H10T1/2 cells, we performed a subtraction hybridization between BMP-2-treated and untreated cells, and have isolated one clone (designated as st-ob for sulfate transporter in osteoblast) induced by BMP-2 that is constantly expressed in osteoblastic cells. The deduced amino acid sequence and proposed structure of st-ob are mostly identical to those of the human diastrophic dysplasia sulfate transporter gene product (DTDST). St-ob mRNA was abundantly expressed in the thymus, testis, calvaria and osteoblastic MC3T3-E1 cells, whereas its expression was faint in C3H10T1/2 cells. Expression of st-ob in C3H10T1/2 cells was increased by transforming growth factor-beta1 (TGF-beta1), retinoic acid and dexamethasone as well as BMP-2. Furthermore, BMP-2 increased sulfate incorporation in C3H10T1/2 cells about twice as high as the baseline level. Osteoblasts actively take up sulfate to synthesize proteoglycans, which are one of the major components of the extracellular matrix of bone and cartilage. The present study demonstrates that st-ob induced during osteoblastic differentiation is an important phenotype of osteoblasts for characterizing their function.     

Roles of prostaglandin E-receptor subtypes in gastric and duodenal bicarbonate secretion in rats

BACKGROUND & AIMS: Receptors activated by prostaglandin (PG) E2 are pharmacologically subdivided into four subtypes (EP1-EP4). The EP-receptor subtype(s) involved in stimulation of gastroduodenal HCO3- secretion in rats were investigated. METHODS: Under urethane anesthesia, a stomach mounted in an ex vivo chamber or a proximal duodenal loop was perfused with saline, and HCO3- secretion was measured using a pH-stat method. RESULTS: Intravenous PGE2 increased HCO3- secretion by the gastroduodenal mucosa; this action was verapamil sensitive and, only in the duodenum, potentiated by isobutylmethyl xanthine (IBMX). Duodenal HCO3- secretion was stimulated by enprostil, sulprostone (EP1/EP3 agonist), misoprostol (EP2/EP3 agonist), and ONO-NT012 (EP3 agonist) but was not affected by butaprost (EP2 agonist) or 17-phenyl-PGE2 (EP1 agonist). Gastric HCO3- secretion was stimulated by sulprostone, enprostil, and 17-phenyl-PGE2 but not by misoprostol, butaprost, or ONO-NT012. SC-51089 (EP1 antagonist) inhibited the HCO3--stimulatory action of sulprostone only in the stomach. IBMX potentiated the HCO3- response to sulprostone in the duodenum, whereas verapamil reduced the response in both the stomach and duodenum. CONCLUSIONS: PGE stimulates HCO3- secretion via different EP-receptor subtypes in the stomach and duodenum: in the stomach, EP1 receptors are linked to Ca2+; in the duodenum, EP3 receptors are coupled with both adenosine 3', 5'-cyclic monophosphate and Ca2+.     

Viscosupplementation: a new concept in the treatment of sacroiliac joint syndrome: a preliminary report of four cases

OBJECTIVE: To evaluate the modulatory effects of interleukin (IL)-1beta and prostaglandin (PG)E2 on the PGE2 receptor subtype EP1 in amnion cell cultures. METHODS: Amnion cell cultures were incubated in increasing concentrations of (IL)-1beta or PGE2. Cultures were also incubated in high concentrations of IL-1beta and PGE2 in combination. Changes in EP1 receptor levels were evaluated by western and northern blot analysis. Culture fluid PGE2 levels were measured by enzyme-linked immunosorbent assay. RESULTS: EP1 receptor protein levels decreased with increasing levels of PGE2 (r = -0.82, P < .05). EP1 receptor protein (r = 0.95, P < .05), EP1 mRNA (r = 0.95, P < .01), and culture fluid PGE2 levels (P < .01) were all increased after IL-1beta administration. EP1 receptor levels also increased approximately fourfold in response to IL-1beta incubation even in the presence of high agonist (PGE2) concentrations (P < .01). CONCLUSION: The results of this study show that IL-1beta might be involved in infection-induced preterm labor by interfering with the normal regulation of EP1 receptor levels and with the promotion of increased PGE2 production in amnion tissue.     

Analysis of genes implicated in osteogenic signaling by osteogenic protein-1 (OP-1) using differential display method

Bone morphogenetic proteins (BMPs), members of a transforming growth factor-beta (TGF-beta) superfamily, are growth and differentiation factors which induce ectopic bone formation in vivo. Although many studies on osteoinductive properties of BMPs have been conducted, little is known about the downstream components in the signal transduction machinery, beyond the mechanism of BMP receptor activation. In this study, the osteogenic effects by osteogenic protein-1 (OP-1, BMP-7) on osteoblastic cell line MC3T3-E1 and murine stromal cell line ST2 were investigated, especially focusing on differentially expressed genes induced by OP-1 using the differential display method. The major findings were as follows: 1) Alkaline phosphatase specific activities of both MC3T3-E1 and ST2 increased in a dose-dependent manner by OP-1 stimulation. 2) Northern analysis showed a significant increase of osteocalcin mRNA after 7 days of OP-1 treatment. 3) 77 genes, which were differentially expressed in MC 3 T 3-E1 and ST 2 cells, were detected on differential display fingerprints after 16-hour treatment of OP-1. 4) Some of these clones showed high levels of identical to known genes. 5) One clone called ST3v, down-regulated in ST2 cells by OP-1 stimulation, was confirmed with quantitative RT-PCR.     

Okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells

Alkaline phosphatase activity is regulated by various hormones and growth factors at least in part through the phosphorylation of target proteins during the bone cell differentiation. To investigate the role of protein phosphorylation in alkaline phosphatase activity in MC3T3-E1 osteoblast, we used okadaic acid which is a potent specific inhibitor of serine/threonine protein phosphatases to type 1 and 2A. Alkaline phosphatase activity in cellular layer was measured by spectrophotometer using p-nitrophenyl phosphate as substrate and data were expressed as p-nitrophenyl of nmol/min/mg of protein. Okadaic acid (1-50 ng/ml) caused the inhibition of alkaline phosphatase activity in MC3TC-E1 cells. At 50 ng/ml of okadaic acid showed the maximal inhibitory effect on alkaline phosphatase activity. Okadaic acid (50 ng/ml) also inhibited alkaline phosphatase activity in all differentiation stages. These results indicate that okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells.     

Effects of ionizing radiation on proliferation and differentiation of osteoblast-like cells

Diagnostic radiation for immediate post-surgical assessment of osseointegrated dental implants has been discouraged, due to the possibility of detrimental effects of ionizing radiation on healing and remodeling of bone. To assess this possibility, we investigated the effects of ionizing radiation on proliferation and differentiation of osteoblasts using osteoblast-like cells isolated from the calvariae of newborn rats (ROB) and a clonal osteoblastic cell line (MC3T3-E1). The cells were exposed on day 3 to a single dose of x-rays at either 40, 100, 400, or 4000 mGy, respectively, from a linear accelerator radiotherapeutic machine (Linac) or a 40-mGy dose from a diagnostic chest x-ray machine. The effects of radiation on cell growth and alkaline-phosphatase-specific (ALP) activity were evaluated at three-day intervals after irradiation up to day 12 in ROB cells, and evaluated at day 12 in MC3T3-E1 cells. At the culture end-point, the effects on formation of bone-like nodules were also evaluated in both ROB and MC3T3-E1 cells. Exposure of 4000 mGy differentially affected the two cell types. It inhibited cell growth and alkaline phosphatase activity, and inhibited DNA content in MC3T3-E1 cells. This irradiation also strongly inhibited the formation of bone-like nodules in ROB cells. On the other hand, exposure of 40-, 100-, and 400-mGy (Linac) and 40-mGy (diagnostic quality) irradiation induced no significant changes in cell growth, alkaline phosphatase activity, and formation of bone-like nodules in ROB cells. These doses also induced no significant changes in DNA content and ALP activity in MC3T3-E1 cells. These results indicate that ionizing radiation at a single dose of up to 400 mGy induces no significant changes in cell growth and differentiation of osteoblast-like cells, at least in vitro. Higher radiation doses (4000 mGy) may exert different effects on cell proliferation and cell differentiation of osteoblasts, depending on the cell types affected. Thus, diagnostic radiation seems to have less effect on proliferation and differentiation of osteoblasts.     

Human endometrial stromal cells generate uncombined alpha-subunit from human chorionic gonadotropin, which can synergize with progesterone to induce decidualization

Prostaglandin E2 (PGE2) is an endogenous hormone of adrenal zona glomerulosa cells and is released in response to stimulation by agonists such as angiotensin II (Ang II). It stimulates the release of aldosterone from cultured bovine adrenal zona glomerulosa cells. These studies were designed to determine whether this steroidogenic effect of PGE2 was mediated by an EP1, EP2, or EP3 receptor. Prostaglandin E2 and 11-deoxy PGE1, an EP2-selective agonist, stimulated aldosterone release in a concentration-related manner with an ED50 of 300 nmol/L for PGE2 and 2 micromol/L for 11-deoxy PGE1. The maximal effect of PGE2 was less than that of angiotensin II. 17-Phenyl trinor PGE2, an EP1-selective agonist, required concentrations of 100 micromol/L to stimulate aldosterone release and sulprostone, an EP3/EP1-selective agonist, failed to alter aldosterone release. The EP1-selective antagonist SC19220 failed to alter basal or PGE2-stimulated aldosterone release over a range of concentrations. PGE2 and 11-deoxy PGE1 also stimulated an increase in both intracellular and extracellular cAMP. This increase was time- and concentration-related. The ED50 for PGE2 was 9.8 micromol/L. 17-Phenyl trinor PGE2 and sulprostone were without effect. Using fura-2 loaded cells, PGE2 (2 micromol/L), dibutyryl cAMP (2 mmol/L), and Ang 11 (2 micromol/L) increased intracellular calcium over basal concentrations by 5.5-fold, 3-fold, and 6.2-fold, respectively. Like PGE2, dibutyryl cAMP also stimulated aldosterone release. PGE2- and dibutyryl cAMP-induced aldosterone release were blocked by the calcium channel inhibitor diltiazem. These studies indicate that PGE2 is a potent stimulus for aldosterone release and that the effect is mediated by EP2 receptors. Both cAMP and calcium appear to mediate the steroidogenic effect of PGE2 and calcium seems to be distal to cAMP.     

Regulation of Zn-alpha2-glycoprotein-mediated cell adhesion by kininogens and their derivatives

MC3T3-E1 (mouse osteoblast-like) cells adhered to a tissue culture plate coated with human Zn-alpha2-glycoprotein (Znalpha2gp). The adhesion of MC3T3-E1 cells to Znalpha2gp was inhibited by synthetic peptides such as RGDS and ELRGDV, and by antibody against vitronectin receptor. These findings suggested that the RGD region of Znalpha2gp interacts with the vitronectin receptor (alphavbeta3) on the MC3T3-E1 cell surface. Furthermore, we found that the common heavy chain of both HMW- and LMW-kininogens accelerated the Znalpha2gp-mediated MC3T3-E1 cell adhesion. Among the three domains of the common heavy chain of both kininogens, domain 3 promoted the cell adhesion by up to 200%. Among the nine synthetic peptides covering domain 3, the peptide, N334AEVYVVPWEKKIYPTVN351 accelerated in a dose-dependent manner the Znalpha2gp- and vitronectin (VN)-mediated MC3T3-E1 cell adhesion. These findings suggested that a defined region of domain 3 is responsible for the acceleration of cell adhesion.     

Molecular biology of prostaglandin E receptors--expression of multi-function by PGE receptor subtypes and isoforms

We reviewed the recent advance in the molecular characterization of prostaglandin E(PGE) receptor. PGE exerts versatile actions in diverse tissues and cells through specific cell surface receptors. PGE receptors(EP) consist of 4 subtypes(EP1, EP2, EP3 and EP4), which differ in the couple of signal transduction. In addition, at least three isoforms of EP3 are produced through alternative RNA splicing from a single gene and differ only in the efficiency of G protein activation and in the specificity of coupling to G proteins. These subtypes and isoforms express and function in specific tissues and cells in various organs, in where they mediate a variety of PGE2-induced physiological responses.     

Fluoroaluminate induces activation and association of Src and Pyk2 tyrosine kinases in osteoblastic MC3T3-E1 cells

Fluoride is known to increase bone mass in vivo, probably through stimulation of osteoblast proliferation; however, the mechanisms of fluoroaluminate action in osteoblasts have not yet been elucidated. We have previously shown that in osteoblastic MC3T3-E1 cells, fluoroaluminate stimulates G protein-mediated protein tyrosine phosphorylation (Scaronuscarona, M., Standke, G. J. R., Jeschke, M., and Rohner, D. (1997) Biochem. Biophys. Res. Commun. 235, 680-684). Although the Ser/Thr kinases Erk1, Erk2, and p70(S6K) were activated in response to fluoroaluminate, the identity of fluoroaluminate-activated tyrosine kinase(s) remained elusive. In this study, we show that in MC3T3-E1 cells, fluoroaluminate induces a 110-kDa tyrosine-phosphorylated protein that we identify as Pyk2, a cytoplasmic tyrosine kinase related to Fak (focal adhesion kinase). The tyrosine phosphorylation of Pyk2 increased in a dose- and time-dependent manner. The autophosphorylation activity of Pyk2 increased 3-fold and reached its maximum within 10 min of fluoroaluminate treatment. Fluoroaluminate also induced activation of Src and the association of Pyk2 with Src. The phosphorylation of Src-associated Pyk2 increased >20-fold in in vitro kinase assays, suggesting that Pyk2 is phosphorylated by Src. Although MC3T3-E1 cells express much more Fak than Pyk2, Src preferentially associated with Pyk2. In vitro, Pyk2 bound to the Src SH2 domain, suggesting that this interaction mediates the Src-Pyk2 association in cells. These data indicate that osteoblastic cells express Pyk2, which is tyrosine-phosphorylated and activated in response to G protein activation by fluoroaluminate, and that the mechanism of Pyk2 activation most likely involves Src. Thus, Src and Pyk2 are tyrosine kinases involved in G protein-mediated tyrosine phosphorylation in osteoblastic cells and may be important for the osteogenic action of fluoroaluminate.